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Sample records for detect bovine tuberculosis

  1. Detection of lipomannan in cattle infected with bovine tuberculosis

    Science.gov (United States)

    Early and rapid detection of bovine tuberculosis (bTB) is critical to controlling the spread of this disease in cattle and other animals. In this study, we demonstrate the development of an immunoassay for the direct detection of the bovine bTB biomarker, lipomannan (LM) in serum using a waveguide-...

  2. Bovine Tuberculosis

    Science.gov (United States)

    Tuberculosis (TB) in animals and humans may result from exposure to bacilli within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedii, M. microti, M. caprae, or M. canetti). Mycobacterium bovis is the species most often isolated from tuberculous catt...

  3. Bovine tuberculosis

    Science.gov (United States)

    Tuberculosis (TB) in animals and humans may result from exposure to bacilli within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedii, M. microti, M. caprae, or M. canetti) . Mycobacterium bovis is the species most often isolated from tuberculous cat...

  4. Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

    Directory of Open Access Journals (Sweden)

    Cristina P. Araújo

    2014-06-01

    Full Text Available Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

  5. Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR.

    Science.gov (United States)

    Araújo, Cristina P; Osório, Ana Luiza A R; Jorge, Klaudia S G; Ramos, Carlos A N; Souza Filho, Antonio F; Vidal, Carlos E S; Vargas, Agueda P C; Roxo, Eliana; Rocha, Adalgiza S; Suffys, Philip N; Fonseca, Antônio A; Silva, Marcio R; Barbosa Neto, José D; Cerqueira, Valíria D; Araújo, Flábio R

    2014-01-01

    Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

  6. Use of PCR for detection of milk naturally infected with bacilli of bovine tuberculosis

    Directory of Open Access Journals (Sweden)

    Ricardo César Tavares Carvalho

    2014-08-01

    Full Text Available The causative agent of bovine tuberculosis (BTB is Mycobacterium bovis, a bacteria belonging to M. tuberculosis complex (MTC. The definitive diagnosis is realized by isolation and identification of the M. bovis from clinical samples, using a combination of traditional culture and biochemical methods, which is considered the “gold standard”. This procedure is cumbersome and time-consuming. We evaluated a PCR assay for the direct detection of MTC DNA in naturally contaminated milk, using primers that were previously tested and proven reliable to target the IS6110 element. Milk previously seeded with M. bovis was used as the starting material, for padronization of the technique. The procedure involved extracting the DNA by enzymatic lysis (proteinase K and lysozyme, phenol, chloroform, isoamyl alcohol, followed by ethanol precipitation and PCR. The PCR assay allowed us to detect BTB in artificially contaminated milk, with a detection limit of 100 CFU/mL, and was also able to detect the bacillus in 50% (75/150 of samples from naturally infected animals. This procedure could be used to assist the in vivo diagnosis for BTB, complementing the sorological or microbiological tests and becomes an alternative option for implementation in epidemiological studies of BTB transmission and to prevent contaminated milk from entering the food supply.

  7. Fasciola hepatica is associated with failure to detect bovine tuberculosis in dairy cattle

    Science.gov (United States)

    Claridge, Jen; Diggle, Peter; McCann, Catherine M.; Mulcahy, Grace; Flynn, Rob; McNair, Jim; Strain, Sam; Welsh, Michael

    2014-01-01

    Bovine tuberculosis (BTB) is a significant and intractable disease of cattle caused by Mycobacterium bovis. In the UK, despite an aggressive eradication programme, the prevalence of BTB is increasing with an unexplained, exponential rise in cases year on year. Here we show in a study involving 3026 dairy herds in England and Wales that there is a significant negative association between exposure to the common, ubiquitous helminth parasite, Fasciola hepatica and diagnosis of BTB. The magnitude of the single intradermal comparative cervical tuberculin test used to diagnose BTB is reduced in cattle experimentally co-infected with M. bovis and F. hepatica. We estimate an under-ascertainment rate of about one-third (95% Confidence Intervals 27-38%) among our study farms, in the hypothetical situation of no exposure to F. hepatica. This finding may in part explain the continuing spread of BTB and the failure of the current eradication programme in the UK. PMID:22617293

  8. Detection of Mycobacterium tuberculosis and Mycobacterium avium Complexes by Real-Time PCR in Bovine Milk from Brazilian Dairy Farms.

    Science.gov (United States)

    Bezerra, André Vinícius Andrade; Dos Reis, Emily Marques; Rodrigues, Rogério Oliveira; Cenci, Alexander; Cerva, Cristine; Mayer, Fabiana Quoos

    2015-05-01

    Foodborne diseases are a public health problem worldwide. The consumption of contaminated raw milk has been recognized as a major cause of transmission of bovine tuberculosis to humans. Other mycobacteria that may be present in raw milk and may cause diseases are those belonging to the Mycobacterium avium complex. In this study, molecular biology tools were applied to investigate raw milk contamination with Mycobacterium spp. in family dairy farms from Rio Grande do Sul, southern Brazil. Furthermore, different variables related to the source of the milk, herd characteristics, and management were evaluated for their effect on milk contamination. Five hundred and two samples were analyzed, of which 354 were from the Northwest region (102 farms with samples from 93 bulk tanks and 261 animals) and 148 from the South region of the state (22 farms with samples from 23 bulk tanks and 125 animals). Among them, 10 (1.99%) and 7 (1.39%) were positive for Mycobacterium tuberculosis (9 confirmed as Mycobacterium bovis) and M. avium complexes, respectively. There was no difference in the frequencies of positive samples between the regions or the sample sources. Of the positive samples, 4 were collected from a bulk tank (1 positive for M. avium and 3 for M. tuberculosis). Moreover, 1 sample was positive concomitantly for M. tuberculosis and M. avium complexes. On risk analysis, no variable was associated with raw milk contamination by M. tuberculosis complex species. However, washing the udders of all animals and drying them with paper towels were weakly classified as risk factors for M. avium contamination. Positive samples were obtained from both animals and bulk tanks, which emphasizes the importance of tuberculosis control programs and provides evidence that milk monitoring can be used as a control practice. Moreover, the findings of this study reinforce the need for awareness of the problems of raw milk consumption among the general population.

  9. 76 FR 26239 - Bovine Tuberculosis and Brucellosis; Public Meetings

    Science.gov (United States)

    2011-05-06

    ...; ] DEPARTMENT OF AGRICULTURE Animal and Plant Health Inspection Service Bovine Tuberculosis and Brucellosis... framework being developed for the bovine tuberculosis and brucellosis programs in the United States. The... tuberculosis (TB) and bovine brucellosis in the United States. In keeping with its commitment to...

  10. Evaluation of the efficiency of nested q-PCR in the detection of Mycobacterium tuberculosis complex directly from tuberculosis-suspected lesions in post-mortem macroscopic inspections of bovine carcasses slaughtered in the state of Mato Grosso, Brazil.

    Science.gov (United States)

    Carvalho, Ricardo César Tavares; Furlanetto, Leone Vinícius; Maruyama, Fernanda Harumy; Araújo, Cristina Pires de; Barros, Sílvia Letícia Bomfim; Ramos, Carlos Alberto do Nascimento; Dutra, Valéria; Araújo, Flábio Ribeiro de; Paschoalin, Vânia Margaret Flosi; Nakazato, Luciano; Figueiredo, Eduardo Eustáquio de Souza

    2015-08-01

    Bovine tuberculosis (BTB) is a zoonotic disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTC). The quick and specific detection of this species is of extreme importance, since BTB may cause economic impacts, in addition to presenting imminent risks to human health. In the present study a nested real-time PCR test (nested q-PCR) was used in post-mortem evaluations to assess cattle carcasses with BTB-suspected lesions. A total of 41,193 cattle slaughtered in slaughterhouses located in the state of Mato Grosso, were examined. Of the examined animals, 198 (0.48%) showed BTB-suspected lesions. M. bovis was isolated in 1.5% (3/198) of the samples. Multiplex-PCR detected MTC in 7% (14/198) of the samples. The nested q-PCR test detected MTC in 28% (56/198) of the BTB-suspected lesions, demonstrating higher efficiency when compared to the multiplex-PCR and conventional microbiology. Nested q-PCR can therefore be used as a complementary test in the national program for control and eradication of bovine tuberculosis.

  11. 76 FR 38602 - Bovine Tuberculosis and Brucellosis; Program Framework

    Science.gov (United States)

    2011-07-01

    ... Animal and Plant Health Inspection Service Bovine Tuberculosis and Brucellosis; Program Framework AGENCY... extending the comment period on a new framework being developed for the bovine tuberculosis and brucellosis... bovine brucellosis in the United States. The notice stated that USDA would hold four public...

  12. Parallel Detection of Bovine Tuberculosis with Different Assays%对奶牛结核病平行检测的不同方法

    Institute of Scientific and Technical Information of China (English)

    路广计; 曹瑞; 张军; 刘乃强; 陶茂晖; 韩庆安; 董维亚; 刘志勇; 轩秋燕

    2014-01-01

    115 cattle in a dairy cattle farm in Tangshan,Hebei province were detected with pure bovine tuberculin (PPD)skin allergy test(TST,using domestic PPD as domestic TST,with imported PPD as imported TST), single intradermal comparative cervical tuberculin test(SICCT)and gamma interferon enzyme-linked immune-sorbent assay(IFN-γ-ELISA)at the same time to analyze the cattle health status,evaluate the advantages and disadvantages of different assays and put forward applicable suggestions. The results showed there were 15 TB positive cattle(including 5 mixed infected with bovine and avian tuberculosis),100 negative cattle(including 1 avian tuberculosis positive). The TB positive rate was 13.04%,the avian tuberculosis positive rate was 5.22%,mixed infection rate was 4.35%. Tests showed that for cattle herd with non-mycobacterium tuberculosis infection ,it was dififcult to conifrm the bovine tuberculosis infection only by one test. Therefore,it was recommend ifrst to use the imported TST and then to retest the positive and suspicious ones by IFN-γ-ELISA to eliminate the false positives. For early infected herds,IFN-γ-ELISA test can be directly used for detection.%[目的]综合分析奶牛的健康状况,评价不同检测方法的优缺点,提出实际应用建议。[方法]同时采用单纯牛结核菌素(PPD)皮内变态反应试验(TST,使用国产PPD简称国产TST、使用进口PPD简称进口TST)、欧盟比较变态反应试验(SICTT)和牛结核γ-干扰素酶联免疫吸附试验(IFN-γ-ELISA),对河北省唐山市某奶牛场存栏的115头牛进行结核病平行检测。[结果]经综合判定牛型结核阳性15头(其中5头为牛型、禽型双阳性)、阴性100头(其中1头为禽型阳性),牛型结核阳性率13.04%、禽型结核阳性率5.22%、二者混合感染率为4.35%。试验表明,对于伴有非结核分枝杆菌感染的牛群,仅靠一种活体检测方法难以做出牛结核病准确诊断。[结论

  13. Human bovine tuberculosis - remains in the differential.

    LENUS (Irish Health Repository)

    Bilal, Shaukat

    2010-11-01

    Mycobacterium bovis is a pathogen of cattle. The unpasteurized milk of affected cattle is a source of infection in humans. Despite the screening of cattle and the pasteurization of milk, M bovis has not been eradicated. A high index of clinical suspicion is needed in symptomatic patients with a history of possible exposure. At risk groups include animal workers, farmers, meat packers, vets and zoo keepers. Humans are usually infected by the aerosol route. We present two cases of human bovine tuberculosis. One was a presumptive case and the second was a confirmed case. Both responded well to antituberculous therapy. In the confirmed case, there was evidence of transmission to the partner living in the same house. Rifampicin prophylaxis was given to the exposed case. The M. bovis from the confirmed case was isoniazid resistant, in addition to having the well known resistance to pyrazinamide. Isoniazid resistance has been described before in those who are immunocompromised. We describe it in an immunocompetent patient.

  14. Spatial epidemiology of bovine tuberculosis in Mexico.

    Science.gov (United States)

    Martínez, Horacio Zendejas; Suazo, Feliciano Milián; Cuador Gil, José Quintín; Bello, Gustavo Cruz; Anaya Escalera, Ana María; Márquez, Gabriel Huitrón; Casanova, Leticia García

    2007-01-01

    The purpose of this study was to use geographic information systems (GIS) and geo-statistical methods of ordinary kriging to predict the prevalence and distribution of bovine tuberculosis (TB) in Jalisco, Mexico. A random sample of 2 287 herds selected from a set of 48 766 was used for the analysis. Spatial location of herds was obtained by either a personal global positioning system (GPS), a database from the Instituto Nacional de Estadìstica Geografìa e Informàtica (INEGI) or Google Earth. Information on TB prevalence was provided by the Jalisco Commission for the Control and Eradication of Tuberculosis (COEETB). Prediction of TB was obtained using ordinary kriging in the geostatistical analyst module in ArcView8. A predicted high prevalence area of TB matching the distribution of dairy cattle was observed. This prediction was in agreement with the prevalence calculated on the total 48 766 herds. Validation was performed taking estimated values of TB prevalence at each municipality, extracted from the kriging surface and then compared with the real prevalence values using a correlation test, giving a value of 0.78, indicating that GIS and kriging are reliable tools for the estimation of TB distribution based on a random sample. This resulted in a significant savings of resources.

  15. Bovine tuberculosis in a wild boar (Sus scrofa) in Poland.

    Science.gov (United States)

    Krajewska, Monika; Lipiec, Marek; Zabost, Anna; Augustynowicz-Kopeć, Ewa; Szulowski, Krzysztof

    2014-10-01

    Poland is officially tuberculosis free and bovine tuberculosis (BTB) cases are rarely found except in bovids. We found BTB in a wild boar (Sus scrofa) in the Bieszczady Mountains, southeastern Poland. Studies suggest possible transmission of infection between free-living European bison (Bison bonasus caucasicus) and wild boar in this area.

  16. First approach to molecular epidemiology of bovine tuberculosis in Colombia

    Directory of Open Access Journals (Sweden)

    Jimena Jojoa-Jojoa

    2015-12-01

    Full Text Available Objective. To investigate the presence of Mycobacterium bovis and other Mycobacterium species in samples of cattle and buffalo in Colombia, to start the molecular characterization of M. bovis in the country. Material and methods. 492 samples were collected from herds identified with the presence of infected animals through the PPD, by the Group of Bovine Tuberculosis ICA Colombian Agricultural Institute in eight departments of Colombia. Lymph nodes of head, thorax and abdomen, gross lesions of tissues with tuberculosis, nasal swabs, milk, blood and fresh cheeses were included. Samples were subjected to detection of Mycobacterium bovis and other mycobacteria by conventional microbiological analysis and PCR-6110 and spoligotyping molecular assays. Results. In the samples analyzed especially in lymph nodes, Mycobacterium bovis was demonstrated with genotypes reported and not previously reported in the world, as well as M. tuberculosis in Antioquia, Cundinamarca, Boyacá and Magdalena departments. Conclusions. In Colombia there are at least 7 genotypes of M. bovis that are infected and sick cattle and buffalo from four different departments becoming serious threat to public health.

  17. Interferon Gamma Assay for the Diagnosis of Bovine Tuberculosis

    Science.gov (United States)

    Contact Irene Schiller Prionics AG Wagistrasse 27A CH-8952 Schlieren Switzerland irene.schiller@prionics.com Introduction Bovine tuberculosis (bTB), a zoonotic disease with a major economic impact, continues to be a significant problem with a global perspective and increasing prevalence in vario...

  18. Diagnosis of bovine tuberculosis: review of main techniques

    Directory of Open Access Journals (Sweden)

    D. F. Ramos

    Full Text Available Abstract Bovine tuberculosis (BTB remains an important economic and zoonotic problem in Latin America. Traditionally, the fight against BTB is initiated by the implementation of routine diagnostic tests for certification of free properties. The diagnosis of BTB can be made by direct and indirect methods, in which we can mention clinical, post mortem, histopathological, immunological, bacteriological and molecular methods. The renewal of scientific interest in tuberculosis in recent year has led to develop and improve methods of diagnosis, prevention, control and eradication of BTB. The aim of this review is to present and discuss different diagnosis methods of BTB.

  19. Diagnosis of bovine tuberculosis: review of main techniques.

    Science.gov (United States)

    Ramos, D F; Silva, P E A; Dellagostin, O A

    2015-11-01

    Bovine tuberculosis (BTB) remains an important economic and zoonotic problem in Latin America. Traditionally, the fight against BTB is initiated by the implementation of routine diagnostic tests for certification of free properties. The diagnosis of BTB can be made by direct and indirect methods, in which we can mention clinical, post mortem, histopathological, immunological, bacteriological and molecular methods. The renewal of scientific interest in tuberculosis in recent year has led to develop and improve methods of diagnosis, prevention, control and eradication of BTB. The aim of this review is to present and discuss different diagnosis methods of BTB.

  20. Scientific Opinion on field trials for bovine tuberculosis vaccination

    Directory of Open Access Journals (Sweden)

    EFSA Panel on Animal Health and Welfare (AHAW

    2013-12-01

    Full Text Available The opinion provides advice relating to the design of field trials to test the performance of a vaccine for bovine tuberculosis (bTB, along with a test to Detect Infected among Vaccinated Animals (DIVA. The objective of cattle vaccination is to use the vaccine in combination with presently applied control measures within the EU as an aid towards bTB eradication. The ideal field trials for the DIVA test will follow the OIE guidelines for test validation. Any deviations from the ideal trial design in relation to DIVA test performance should be justified, and the bias that may subsequently be introduced should be accounted for. The ideal field trial design for vaccination performance should implement a double-blind randomised test scenario, and allow for known risk factors in the field situation. Any deviations from the ideal trial design in relation to vaccine performance should also be justified and bias that may subsequently be introduced should be accounted for. Relevant risk factors and possible confounders that should be taken into consideration in the design of field trials are described in this opinion. The safety of a candidate vaccine is guaranteed in the registration of a vaccine medication by a competent authority. The field trials will need to fulfil these requirements to prove that the use of this vaccine in the field is safe for both public health and the environment. Some additional remarks regarding the safety of this specific vaccine are included in this opinion.

  1. Re-activation of bovine tuberculosis in a patient treated with infliximab

    DEFF Research Database (Denmark)

    Larsen, Mette Vang; Thomsen, V Ø; Sørensen, Inge Juul

    2008-01-01

    Treatment with tumour necrosis factor-alpha inhibitors increases the risk of tuberculosis (TB). Screening for latent TB infection (LTBI) and prophylactic treatment has become mandatory. A 79-yr-old female with a history of severe erosive sero-positive rheumatoid arthritis was screened for LTBI......-infected cattle. Re-activation of bovine tuberculosis is a risk in people with recent or previous exposure to unpasteurised dairy products. The QuantiFERON-TB test has the potential to detect Mycobacterium bovis infection. Indeterminate test results reflect either anergy, due to poor immunity, or technical...... problems and should be cautiously interpreted and as a minimum be repeated. Studies are ongoing to determine the role of QuantiFERON-TB testing in the screening for latent tuberculosis infection....

  2. Re-activation of bovine tuberculosis in a patient treated with infliximab

    DEFF Research Database (Denmark)

    Larsen, Mette Vang; Thomsen, V Ø; Sørensen, Inge Juul

    2008-01-01

    Treatment with tumour necrosis factor-alpha inhibitors increases the risk of tuberculosis (TB). Screening for latent TB infection (LTBI) and prophylactic treatment has become mandatory. A 79-yr-old female with a history of severe erosive sero-positive rheumatoid arthritis was screened for LTBI......-infected cattle. Re-activation of bovine tuberculosis is a risk in people with recent or previous exposure to unpasteurised dairy products. The QuantiFERON-TB test has the potential to detect Mycobacterium bovis infection. Indeterminate test results reflect either anergy, due to poor immunity, or technical...... problems and should be cautiously interpreted and as a minimum be repeated. Studies are ongoing to determine the role of QuantiFERON-TB testing in the screening for latent tuberculosis infection....

  3. Purification in antigen and application in diagnosis of IFN-γ-ELISA detection of bovine tuberculosis%诊断抗原的纯化在牛γ-干扰素ELISA法中的应用

    Institute of Scientific and Technical Information of China (English)

    邱美珍; 周望平; 肖兵南; 杜丽飞; 胡述光; 刘毅; 陈江

    2011-01-01

    比较皮内变态反应(TST)、抗酸染色、牛结核ELISA(PPD-ELISA)和γ-干扰素ELISA法(IFN-γ-ELISA)对牛结核的检测结果,并通过比较牛PPD、禽PPD、亲和层析牛PPD(牛PPD')的IFN-γ-ELISA检测结果,探索诊断抗原的纯化在牛IFN-γ-ELISA法中的应用.结果表明,与抗酸染色相比,IFN-γ-ELISA的敏感性、特异性和符合率都比较高,分别为83.33%(5/6),98.14%(53/54),98.33%(58/60);牛PPD、禽PPD、牛PPD'3种特异性抗原刺激产生IFN-γ释放反应的结果表明,1例抗酸染色阳性的牛,未经亲和层析处理牛PPD的IFN-γ-ELISA检测OD值偏高,判为牛结核阳性,亲和层析纯化牛PPD的IFN-γ-ELISA检测OD值较低,判为牛结核阴性,表明亲和层析处理能排除环境分支杆菌对试验的干扰,IFN-γ-ELISA法有望替代TST法在中国推广使用.%The test results of the bovine tuberculin skin test (TST), acid-fast stain test, ELISA test and bovine IFN-γELISA diagnosis test respectively were compared for detection of tuberculosis of them, three stimulators, detected by IFN-γ-ELISA including bovine PPD, avian PPD and bovine PPD at affinity chromatography were compared in exploration for application of purification of diagnostic antigen in the IFN-Υ-ELISA method.Using acid-fast staining as the standard, the sensitivity of IFN-Υ-ELISA detection reached 83.33%(5/6), and its specificity reached 98.14%(53/54), the consistency reached 98.33% (58/60).As the other result, acid-fast stain detected a positive sera with high OD value and produced a positive result using bovine IFN-γ-ELISA in which the antigen was not purified at affinity chromatography, but the result was negative with low OD value using bovine IFN-γ-ELIS in which the antigen was purified at affinity chromatography.In conclusion, the miscarriage of justice caused by infected with Mycobacterium bovis and environmental Mycobacterium simultaneouly could be prevented using bovine IFN-γ-ELISA in which the antigen was

  4. New skin test for detection of bovine tuberculosis on the basis of antigen-displaying polyester inclusions produced by recombinant Escherichia coli.

    Science.gov (United States)

    Chen, Shuxiong; Parlane, Natalie A; Lee, Jason; Wedlock, D Neil; Buddle, Bryce M; Rehm, Bernd H A

    2014-04-01

    The tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria, as some antigens in purified protein derivative (PPD) prepared from Mycobacterium bovis are present in nonpathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10, and Rv3615c, are present in members of the pathogenic Mycobacterium tuberculosis complex but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity, these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen-displaying inclusions in recombinant Escherichia coli. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The surface exposure and accessibility of antigens were assessed by enzyme-linked immunosorbent assay (ELISA). Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than did beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple-antigen-displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally infected with M. bovis with no false-positive reactions observed in those sensitized to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from noninfected animals.

  5. Differential Gene Expression Segregates Cattle Confirmed Positive for Bovine Tuberculosis from Antemortem Tuberculosis Test-False Positive Cattle Originating from Herds Free of Bovine Tuberculosis

    Directory of Open Access Journals (Sweden)

    Ailam Lim

    2012-01-01

    Full Text Available Antemortem tests for bovine tuberculosis (bTB currently used in the US measure cell-mediated immune responses against Mycobacterium bovis. Postmortem tests for bTB rely on observation of gross and histologic lesions of bTB, followed by bacterial isolation or molecular diagnostics. Cumulative data from the state of Michigan indicates that 98 to 99% of cattle that react positively in antemortem tests are not confirmed positive for bTB at postmortem examination. Understanding the fundamental differences in gene regulation between antemortem test-false positive cattle and cattle that have bTB may allow identification of molecular markers that can be exploited to better separate infected from noninfected cattle. An immunospecific cDNA microarray was used to identify altered gene expression (≤0.01 of 122 gene features between antemortem test-false positive cattle and bTB-infected cattle following a 4-hour stimulation of whole blood with tuberculin. Further analysis using quantitative real-time PCR assays validated altered expression of 8 genes that had differential power (adj  ≤0.05 to segregate cattle confirmed positive for bovine tuberculosis from antemortem tuberculosis test-false positive cattle originating from herds free of bovine tuberculosis.

  6. Bovine Tuberculosis and Brucellosis in Cattle and African Buffalo in the Limpopo National Park, Mozambique.

    Science.gov (United States)

    Tanner, M; Inlameia, O; Michel, A; Maxlhuza, G; Pondja, A; Fafetine, J; Macucule, B; Zacarias, M; Manguele, J; Moiane, I C; Marranangumbe, A S; Mulandane, F; Schönfeld, C; Moser, I; van Helden, P; Machado, A

    2015-12-01

    Bovine tuberculosis (BTB) and brucellosis are prevalent in buffaloes of the Kruger National Park (KNP, South Africa). Both diseases were considered to have no or a very low prevalence in wildlife and livestock in and around the Limpopo National Park (LNP, Mozambique). The same applies for tuberculosis in Gonarezhou National Park (GNP, Zimbabwe), but just recently, BTB was detected in buffaloes in the GNP and fears arose that the disease might also spread to the LNP as a result of the partial removal of the fences between the three parks to form the Great Limpopo Transfrontier Park. To assess the status of both diseases in and around LNP, 62 buffaloes were tested for bovine tuberculosis (BTB) and bovine brucellosis. The percentage of positive BTB reactors in buffalo was 8.06% using BovidTB Stat-Pak® and 0% with BOVIGAM® IFN-γ test and IDEXX ELISA. The brucellosis seroprevalence in buffalo was found to be 17.72% and 27.42% using Rose Bengal Test (RBT) and ELISA, respectively. In addition, 2445 cattle in and around the LNP were examined for BTB using the single intradermal cervical comparative tuberculin test (SICCT), and an apparent prevalence of 0.98% was found with no significant difference inside (0.5%) and outside (1.3%) the park. This is the first published report on the presence of positive reactors to BTB and bovine brucellosis in buffalo and cattle in and outside the LNP. Monitoring the wildlife-livestock-human interface of zoonotic high-impact diseases such as BTB and brucellosis is of outmost importance for the successful implementation and management of any transfrontier park that aims to improve the livelihoods of the local communities.

  7. Comparison of nine DNA extraction methods for the diagnosis of bovine tuberculosis by real time PCR

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    André Moura

    2016-07-01

    Full Text Available ABSTRACT: Bovine tuberculosis is an infectious disease with a high impact on the cattle industry, particularly in developing countries. PCR is a very sensitive method for detection of infectious agents, but the sensitivity of molecular diagnosis is largely dependent on the efficiency of the DNA extraction methods. The objective of this study was to evaluate DNA extraction methods for direct detection of Mycobacterium bovis in bovine tissue. Nine commercial kits for DNA extraction were evaluated when combined with two real time PCRs. The DNeasy Blood & Tissue Kit from QIAGEN showed better performance and sensitivity followed by the DNA Mini Kit RBC and FTA Elute Micro Card. Results suggested that, even when the analytical sensitivity of the qPCR is very high, the extraction method can influence the diagnostic sensitivity.

  8. Potential application of new diagnostic methods for controlling bovine Tuberculosis in Brazil

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    Luciana dos Santos Medeiros

    2010-10-01

    Full Text Available Bovine tuberculosis, a chronic infection in cattle caused by Mycobacterium bovis, remains an economic and public health problem for several countries. Due to its economic impact on international trade, contagious nature, and implications for human health, global programs to eradicate the disease were implemented worldwide. Those programs are based on slaughtering PPD-reactive animals. Despite the National Programs in Brazil, complete eradication has not been achieved, and the disease remains, albeit at a lower prevalence. The central purpose of this review is to address diagnostic tests for tuberculosis. Considering the course of the infection in cattle, at least two tests, ideally complementary to one another, may be necessary for an adequate diagnosis: the first based on the cellular response, and the second capable of identifying anergic animals by detection of specific anti-M.bovis antibodies.

  9. Progress in the control of bovine tuberculosis in Spanish wildlife.

    Science.gov (United States)

    Gortazar, Christian; Vicente, Joaquín; Boadella, Mariana; Ballesteros, Cristina; Galindo, Ruth C; Garrido, Joseba; Aranaz, Alicia; de la Fuente, José

    2011-07-01

    Despite the compulsory test and slaughter campaigns in cattle, bovine tuberculosis (bTB) is still present in Spain, and the role of wildlife reservoirs is increasingly recognized. We provide an update on recent progress made in bTB control in Spanish wildlife, including aspects of epidemiology, surveillance, host-pathogen interaction and wildlife vaccination. At the high densities and in the particular circumstances of Mediterranean environments, wild ungulates, mainly Eurasian wild boar and red deer, are able to maintain Mycobacterium bovis circulation even in absence of domestic livestock. Infection is widespread among wild ungulates in the south of the country, local infection prevalence being as high as 52% in wild boar and 27% in red deer. Risk factors identified include host genetic susceptibility, abundance, spatial aggregation at feeders and waterholes, scavenging, and social behaviour. An increasing trend of bTB compatible lesions was reported among wild boar and red deer inspected between 1992 and 2004 in Southwestern Spain. Sporadic cases of badger TB have been detected, further complicating the picture. Gene expression profiles were characterized in European wild boar and Iberian red deer naturally infected with M. bovis. The comparative analysis of gene expression profiles in wildlife hosts in response to infection advanced our understanding of the molecular mechanisms of infection and pathogenesis, revealed common and distinctive host responses to infection and identified candidate genes associated with resistance to bTB and for the characterization of host response to infection and vaccination. Ongoing research is producing valuable knowledge on vaccine delivery, safety and efficacy issues. Baits for the oral delivery of BCG vaccine preparations to wild boar piglets were developed and evaluated. The use of selective feeders during the summer was found to be a potentially reliable bait-deployment strategy. Safety experiments yielded no isolation of M

  10. Estimating the hidden burden of bovine tuberculosis in Great Britain.

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    Andrew J K Conlan

    Full Text Available The number of cattle herds placed under movement restrictions in Great Britain (GB due to the suspected presence of bovine tuberculosis (bTB has progressively increased over the past 25 years despite an intensive and costly test-and-slaughter control program. Around 38% of herds that clear movement restrictions experience a recurrent incident (breakdown within 24 months, suggesting that infection may be persisting within herds. Reactivity to tuberculin, the basis of diagnostic testing, is dependent on the time from infection. Thus, testing efficiency varies between outbreaks, depending on weight of transmission and cannot be directly estimated. In this paper, we use Approximate Bayesian Computation (ABC to parameterize two within-herd transmission models within a rigorous inferential framework. Previous within-herd models of bTB have relied on ad-hoc methods of parameterization and used a single model structure (SORI where animals are assumed to become detectable by testing before they become infectious. We study such a conventional within-herd model of bTB and an alternative model, motivated by recent animal challenge studies, where there is no period of epidemiological latency before animals become infectious (SOR. Under both models we estimate that cattle-to-cattle transmission rates are non-linearly density dependent. The basic reproductive ratio for our conventional within-herd model, estimated for scenarios with no statutory controls, increases from 1.5 (0.26-4.9; 95% CI in a herd of 30 cattle up to 4.9 (0.99-14.0 in a herd of 400. Under this model we estimate that 50% (33-67 of recurrent breakdowns in Britain can be attributed to infection missed by tuberculin testing. However this figure falls to 24% (11-42 of recurrent breakdowns under our alternative model. Under both models the estimated extrinsic force of infection increases with the burden of missed infection. Hence, improved herd-level testing is unlikely to reduce recurrence

  11. Prevalence of bovine tuberculosis in beef feedlot of Borena cattle by ...

    African Journals Online (AJOL)

    ... Immunology and Veterinary Public Health, College of Veterinary. Medicine ... prevalence of bovine tuberculosis and its associated risk factors in beef animals ... and the sample size was increased to 501 cattle considering the dropout and.

  12. Epidemiology caught in the causal web of bovine tuberculosis.

    Science.gov (United States)

    Pfeiffer, D U

    2013-11-01

    Bovine tuberculosis in domestic cattle in the presence of significant infection levels in wild animal species represents a major challenge for disease control. The use of wild animal population density reduction as part of risk management policies is highly controversial from the perspectives of scientific effectiveness and societal acceptability. The experience in Great Britain in dealing with this problem over the last 20 years demonstrates the need to engage in an integrated approach towards risk governance to more effectively deal with such a complex and contentious multifactorial animal disease problem. As part of this process, the traditional emphasis on bioscientific, in particular epidemiological, research needs to be complemented by relevant social science approaches. In addition, the risk assessment as well as the risk management should have effective participatory elements. © 2013 Blackwell Verlag GmbH.

  13. 牛结核γ-干扰素ELISA检测方法在检疫工作中的应用%The Application of Gamma Interferon ELISA in Bovine Tuberculosis Detection

    Institute of Scientific and Technical Information of China (English)

    周培校; 李静; 张鲁安; 李杰; 曹瑞; 张喜悦; 李岩

    2014-01-01

    为评价牛γ-干扰素ELISA检测方法检测牛结核的效果及国产试剂盒的检测效果,本试验首先将国产试剂盒与Prionics试剂盒对42份相对阳性的样品和105份相对阴性的样品进行对比研究。然后对5个规模化牛场的3000头奶牛首先进行国产单纯结核菌素颈部皮内变态反应试验,3天后选取皮内变态反应阳性和可疑及部分阴性牛共418头,进行牛γ-干扰素试验。结果国产试剂盒对阳性和阴性样品的检测敏感性和特异性分别为95.2%和100%,与Priobics试剂盒的符合率为99.3%。表明国产试剂盒与进口试剂盒的检测能力一致,牛γ-干扰素检测方法准确可靠。5个牛场的3000头奶牛单纯结核菌素颈部皮内变态反应试验阳性为138头,可疑105头。γ-干扰素试验对418头奶牛的检测,其中阳性74头,与颈部皮内变态反应(可疑牛暂时视为阴性)的符合率为60.5%。%In order to evaluate the effectiveness of gamma interferon test to detect bovine tuberculosis and the detection effect of a domestic kit, a comparative study was done between Prionics kits and the domestic kits using 42 positive samples and 105 negative samples. Then 3000 cattle from five intensive farms were first tested with single skin test, and 3 days later, 418 cattle including skin test positive and suspicious ones and some negative ones were tested with gamma interferon test. Results showed that the sensitivity and specificity of the domestic kit were 95.2%and 100%respectively. and the coincidence was 99.3%with the Prionics kit,suggesting that the gamma interferon test was accurate and reli-able for detection of bovine tuberculosis, and the domestic kit was as good as the Prionics kit. There were 138 skin test positive cattle and 105 suspicious cattle from 3000 tested cattle. In gamma interferon test, 74/418 cattle were detected positive, and the coincidence rate was 60.5%with skin test (suspicious cattle considered as

  14. National control and eradication program of bovine tuberculosis in Chile.

    Science.gov (United States)

    Max, Vanessa; Paredes, Luis; Rivera, Alejandro; Ternicier, Claudio

    2011-07-05

    There have been reports of the presence of bovine tuberculosis (TB) in Chile for more than 100 years. Several prevalence studies have revealed that there is a wide spectrum of disease across the country with certain geographic areas where the disease is endemic through to other geographic areas where infection is sporadic and at very low prevalence. In 2009, this information was used to divide Chile into different geographic zones based on prevalence rates. This will enable the correct actions to be undertaken to reduce the prevalence of TB. Thus the northern part of Chile which has a medium to high prevalence of TB will be categorized as a control zone. In contrast, the southern part of Chile which has a high proportion of the bovine population, has a low prevalence of TB and will be classified as an eradication zone (Paredes, 2008). Although there have been several past attempts to create a national control and eradication program in Chile, none has been successful. A national program is proposed, and outlined in this paper. Progress toward program initiation in 2009 has been difficult, mostly because of the global economic crisis, difficulties in the milk and meat industry, and social and political issues. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. Vaccination of white-tailed deer (Odocoileus virginianus) for protection against bovine tuberculosis

    Science.gov (United States)

    Bovine tuberculosis (bTB), caused by Mycobacterium bovis and other related species in the M. tuberculosis complex, pose a serious continual threat to the health and economic wellbeing of wildlife, livestock, and humans worldwide. Wildlife reservoirs of bTB play a very important role in the epidemio...

  16. Bovine Tuberculosis: Analyzing the Parameters of the Interferon Gamma Assay and Improved Diagnosis with New Antigens

    Science.gov (United States)

    Bovine tuberculosis (TB), a zoonotic disease with a major economic impact, continues to be a significant problem with a global perspective. The BOVIGAM® interferon gamma (IFN-gamma) assay constitutes a laboratory-based tuberculosis test and is widely used complementary to the tuberculin skin test....

  17. Bovine tuberculosis in Canadian wildlife: an updated history.

    Science.gov (United States)

    Wobeser, Gary

    2009-11-01

    Mycobacterium bovis infection in wild animals attracted little attention in Canada until the disease was almost eliminated from domestic livestock. Tuberculosis was endemic in plains bison and occurred in elk, moose, and mule deer in Buffalo National Park (BNP), Alberta during the 1920s and 1930s. Bison were moved from BNP to Wood Buffalo National Park (WBNP), where tuberculosis became, and remains, endemic in bison, posing a risk to efforts to restore bison in northern Canada. Tuberculosis was found in a white-tailed deer in Ontario in 1959, and in an infected elk near Riding Mountain National Park (RMNP), Manitoba in 1992. Intense surveillance has resulted in detection of 40 elk, 8 white-tailed deer, and 7 cattle herds infected between 1997 and 2008 in the RMNP area. The strains of M. bovis in the RMNP area are different from strains tested from cattle and bison elsewhere in Canada. Management of tuberculosis in cattle and wild animals is challenging because of uncertainty about the ecology of the disease in various species, difficulty in obtaining samples and population data from wildlife, lack of validated tests, overlapping jurisdictions and authority, and conflicting values and opinions among stakeholders.

  18. Bovine tuberculosis at the human-livestock-wildlife interface: Is it a public health problem in Tanzania? A review

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    Bugwesa Z. Katale

    2012-06-01

    Full Text Available Despite the apparent public health concern about Bovine tuberculosis (BTB in Tanzania, little has been done regarding the zoonotic importance of the disease and raising awareness of the community to prevent the disease. Bovine tuberculosis is a potential zoonotic disease that can infect a variety of hosts, including humans. The presence of multiple hosts including wild animals, inefficient diagnostic techniques, absence of defined national controls and eradication programs could impede the control of bovine TB. In Tanzania, the diagnosis of Mycobacterium bovis in animals is mostly carried out by tuberculin skin testing, meat inspection in abattoirs and only rarely using bacteriological techniques. The estimated prevalence of BTB in animals in Tanzania varies and ranges across regions from 0.2% to 13.3%, which is likely to be an underestimate if not confirmed by bacteriology or molecular techniques. Mycobacterium bovis has been detected and isolated from different animal species and has been recovered in 10% of apparently healthy wildebeest that did not show lesions at post-mortem. The transmission of the disease from animals to humans can occur directly through the aerosol route and indirectly by consumption of raw milk. This poses an emerging disease threat in the current era of HIV confection in Tanzania and elsewhere. Mycobacterium bovis is one of the causative agents of human extra pulmonary tuberculosis. In Tanzania there was a significant increase (116.6% of extrapulmonary cases reported between 1995 and 2009, suggesting the possibility of widespread M. bovis and Mycobacterium tuberculosis infection due to general rise of Human Immunodeficiency virus (HIV. This paper aims to review the potential health and economic impact of bovine tuberculosis and challenges to its control in order to safeguard human and animal population in Tanzania.

  19. Control of bovine tuberculosis in a farmed red deer herd in England

    Science.gov (United States)

    Busch, F.; Bannerman, F.; Liggett, S.; Griffin, F.; Clarke, J.; Lyashchenko, K. P.; Rhodes, S.

    2017-01-01

    This report describes how Mycobacterium bovis infection was controlled and eventually eradicated in a farmed red deer herd in the north of England, following sustained tuberculin skin testing supplemented with serological (antibody) tests over a period of approximately two years. By taking advantage of the anamnestic antibody response produced by the skin test to detect skin test-negative, antibody-positive infected individuals, a total of 35 additional animals were identified, including 2 with gross visible lesions typical of bovine tuberculosis (BTB). Without detection and removal, these animals would have posed a continued risk of BTB persistence within the herd and potentially contributed to the spread of infection from deer into wildlife and surrounding cattle farms in an area of low BTB incidence. This case supports the use of ancillary diagnostic serological tests to speed up the resolution of incidents of BTB caused by M bovis in captive deer herds. PMID:28100768

  20. Assessment of Mycobacterium tuberculosis OmpATb as a Novel Antigen for the Diagnosis of Bovine Tuberculosis

    Science.gov (United States)

    In search for improved tools to control bovine tuberculosis, the development of diagnostic tests with improved specificity and sensitivity has a high priority. Such tests require the identification of enhanced immunodiagnostic reagents. In this study, we evaluated the potential of Rv0899 (Outer memb...

  1. Evaluation of Surveillance for Documentation of Freedom from Bovine Tuberculosis

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    Liza R. Nielsen

    2013-06-01

    Full Text Available The objective was to study how surveillance for bovine tuberculosis (bTB could be made more resource-effective in a bTB free country. A stochastic scenario tree model was developed to: (1 evaluate the sensitivity (CSe of four surveillance system components (SSC (i.e., meat inspection of slaughtered domestic cattle, farmed deer and pigs, and tuberculin testing of adult export cattle given that bTB would enter one of these components, (2 estimate the probability of freedom (PFree from bTB over time, and (3 evaluate how future alternative programmes based on visual meat inspection would affect the confidence in freedom from bTB at the very low animal-level design prevalence 0.0002% and a low probabilities of introduction (1%. All, except the export cattle component reached a PFree above 96% within five years. The PFree was slightly reduced if surveillance was changed to visual inspection, e.g., PFree was reduced from 96.5% to 94.3% in the cattle component, and from 98.5% to 97.7% in the pig component after 24 years. In conclusion, visual meat inspection of pigs and cattle will only reduce the confidence in freedom from bTB slightly. However, with negligible probability of introduction (0.1% the PFree could be maintained well above 99% in the cattle, pigs and deer components, which highlights the importance of rigid testing and quarantine procedures in trade of livestock.

  2. Farmers' confidence in vaccinating badgers against bovine tuberculosis.

    Science.gov (United States)

    Enticott, G; Maye, D; Ilbery, B; Fisher, R; Kirwan, J

    2012-02-25

    This paper examines UK farmers' levels of confidence in vaccinating badgers against bovine tuberculosis (bTB) and their trust in the Government's ability to deal with bTB. In 2010, a badger vaccine based on the BCG vaccine was licensed following field trials and used as part of the UK Government's Badger Vaccination Deployment Project. A stratified random sample of cattle farmers in five different locations of England was surveyed using a telephone survey to elicit their views of badger vaccination. The survey provided a total of 341 responses with a response rate of 80 per cent. Results suggest that the farmers are cautious about badger vaccination, appearing to be neither overly confident nor unconfident in it. However, the farmers did not reveal high levels of trust in the Government to manage bTB policy or badger vaccination. There were no differences in the levels of confidence or trust between farms that were under bTB restrictions at the time of the survey and those that were not or between farms with historically high levels of bTB. Analysis of principal components suggests that 33 per cent of the farmers accepted badger vaccination, but that acceptance is dependent on the wider social and political environment.

  3. A history of bovine tuberculosis eradication policy in Northern Ireland.

    Science.gov (United States)

    Robinson, P A

    2015-11-01

    Despite many years of state-sponsored efforts to eradicate the disease from cattle through testing and slaughter, bovine tuberculosis (bTB) is still regarded as the most important and complex of animal health challenges facing the British livestock agricultural industry. This paper provides a historical analysis of the ongoing bTB statutory eradication programme in one part of the UK - Northern Ireland (NI) - which began in 1949 as a voluntary scheme, but between 1959 and 1960 became compulsory for all cattle herd-owners. Tracing bTB back through time sets the eradication efforts of the present day within a deeper context, and provides signposts for what developed in subsequent decades. The findings are based primarily on empirical research using historical published reports of the Ministry of Agriculture and state documents held in the public archives in NI, and they emphasize the need to consider the economic, social and political contexts of disease eradication efforts and their influences on both the past and the present.

  4. Use of genomics to track bovine tuberculosis transmission.

    Science.gov (United States)

    Kao, R R; Price-Carter, M; Robbe-Austerman, S

    2016-04-01

    The control of any infectious disease of livestock is made more difficult by the presence of a wildlife reservoir, as the reservoir is often poorly observed and difficult to manage. This problem is particularly acute for bovine tuberculosis (bTB) because the long duration of infection and low levels of infectiousness make tracing the sources of infection difficult. For over 30 years, the process of contact tracing has been aided by the exploitation of molecular markers in the pathogen, but this has largely only been capable of characterising broad associations between large communities of similar types. However, the recent advent of mass high-throughput 'whole-genome' sequencing (WGS) has revolutionised forensic epidemiology for other diseases, and now it has the potential to do so for bTB. In this review, the authors consider the historical context of WGS use and look at what prior molecular techniques have already achieved. They outline the key approaches to interpreting WGS data and consider both the role of advanced analytical techniques that exploit the evolutionary and epidemiological properties of the system and the problems associated with quantifying the role of hidden reservoirs of disease. Finally, they consider the particular difficulties associated with developing this technology for routine diagnostics and its potential for mass use.

  5. Development and optimization of an ESAT6-ELISA-based detection system of Mycobacterium tuberculosis complex, suitable for bovine TB eradication

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    Rouhollah Keshavarz

    2015-01-01

    Conclusion: Performing the ELISA test based on ESAT-6 antigen shows that this test can be a suitable way for screening beside the tuberculin test for accurate detection. It is noticed that the specificity of the ELISA test was determined more than the tuberculin test, especially since the culture and PCR are gold standards. Therefore, incorrect sampling can change the specificity of the tuberculin test. The results of this kit can encourage designing of an ELISA kit for the detection of human TB.

  6. Opportunities for Improved Serodiagnosis of Human Tuberculosis, Bovine Tuberculosis, and Paratuberculosis

    Directory of Open Access Journals (Sweden)

    Ashutosh Wadhwa

    2012-01-01

    Full Text Available Mycobacterial infections—tuberculosis (TB, bovine tuberculosis (bTB, and Johne’s disease (JD—are major infectious diseases of both human and animals. Methods presently in use for diagnosis of mycobacterial infections include bacterial culture, nucleic acid amplification, tuberculin skin test, interferon-γ assay, and serology. Serological tests have several advantages over other methods, including short turn-around time, relatively simple procedures, and low cost. However, current serodiagnostic methods for TB, bTB and JD exhibit low sensitivity and/or specificity. Recent studies that have aimed to develop improved serodiagnostic tests have mostly focused on identifying useful species-specific protein antigens. A review of recent attempts to improve diagnostic test performance indicates that the use of multiple antigens can improve the accuracy of serodiagnosis of these mycobacterial diseases. Mycobacteria also produce a variety of species-specific nonprotein molecules; however, only a few such molecules (e.g., cord factor and lipoarabinomannan have so far been evaluated for their effectiveness as diagnostic antigens. For TB and bTB, there has been recent progress in developing laboratory-free diagnostic methods. New technologies such as microfluidics and “Lab-on-Chip” are examples of promising new technologies that can underpin development of laboratory-free diagnostic devices for these mycobacterial infections.

  7. Prevalence of bovine tuberculosis in African buffalo at Kruger National Park.

    Science.gov (United States)

    Rodwell, T C; Kriek, N P; Bengis, R G; Whyte, I J; Viljoen, P C; de Vos, V; Boyce, W M

    2001-04-01

    Bovine tuberculosis (BTB) was first detected in Kruger National Park (KNP) in a single African buffalo (Syncerus caffer) in 1990. In 1991/1992, 2,071 African buffalo were examined for BTB as part of a culling program that removed animals from all known herds in KNP. The prevalence of BTB in 1991/1992 was estimated to be 0%, 4.4% (+/-0.6%), and 27.1% (+/-1.4%), in the north, central, and south zones of KNP, respectively. In 1998, a stratified, two-stage cluster sampling method was used to estimate that the prevalence of BTB was 1.5% (+/-2.5%), 16% (+/-5.3%), and 38.2% (+/-6.3%), in the north, central, and south zones, respectively. This represented a significant increase in prevalence (P management strategies. The methodology and sample sizes used in 1998 are appropriate for future BTB monitoring in KNP.

  8. Bovine tuberculosis at the wildlife-livestock-human interface in Hamer Woreda, South Omo, Southern Ethiopia.

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    Rea Tschopp

    Full Text Available Bovine tuberculosis (BTB is endemic in cattle in the Ethiopian Highlands but no studies have been done so far in pastoralists in South Omo. This study assessed the prevalence of bovine tuberculosis (BTB at an intensive interface of livestock, wildlife and pastoralists in Hamer Woreda (South Omo, Ethiopia. A cross-sectional survey including a comparative intradermal skin testing (CIDT was conducted in 499 zebu cattle and 186 goats in 12 settlements. Sputum samples from 26 symptomatic livestock owners were cultured for TB. Fifty-one wildlife samples from 13 different species were also collected in the same area and tested with serological (lateral flow assay and bacteriological (culture of lymph nodes techniques. Individual BTB prevalence in cattle was 0.8% (CI: 0.3%-2% with the >4 mm cut-off and 3.4% (CI: 2.1%-5.4% with the >2 mm cut-off. Herd prevalence was 33.3% and 83% when using the >4 and the >2 mm cut-off respectively. There was no correlation between age, sex, body condition and positive reactors upon univariate analysis. None of the goats were reactors for BTB. Acid fast bacilli (AFB were detected in 50% of the wildlife cultures, 79.2% of which were identified as Mycobacterium terrae complex. No M. bovis was detected. Twenty-seven percent of tested wildlife were sero-positive. Four sputum cultures (15.4% yielded AFB positive colonies among which one was M. tuberculosis and 3 non-tuberculous mycobacteria (NTM. The prevalence of M. avium-complex (MAC was 4.2% in wildlife, 2.5% in cattle and 0.5% in goats. In conclusion, individual BTB prevalence was low, but herd prevalence high in cattle and BTB was not detected in goats, wildlife and humans despite an intensive contact interface. On the contrary, NTMs were highly prevalent and some Mycobacterium spp were more prevalent in specific species. The role of NTMs in livestock and co-infection with BTB need further research.

  9. Bovine tuberculosis at the wildlife-livestock-human interface in Hamer Woreda, South Omo, Southern Ethiopia.

    Science.gov (United States)

    Tschopp, Rea; Aseffa, Abraham; Schelling, Esther; Berg, Stefan; Hailu, Elena; Gadisa, Endalamaw; Habtamu, Meseret; Argaw, Kifle; Zinsstag, Jakob

    2010-08-17

    Bovine tuberculosis (BTB) is endemic in cattle in the Ethiopian Highlands but no studies have been done so far in pastoralists in South Omo. This study assessed the prevalence of bovine tuberculosis (BTB) at an intensive interface of livestock, wildlife and pastoralists in Hamer Woreda (South Omo), Ethiopia. A cross-sectional survey including a comparative intradermal skin testing (CIDT) was conducted in 499 zebu cattle and 186 goats in 12 settlements. Sputum samples from 26 symptomatic livestock owners were cultured for TB. Fifty-one wildlife samples from 13 different species were also collected in the same area and tested with serological (lateral flow assay) and bacteriological (culture of lymph nodes) techniques. Individual BTB prevalence in cattle was 0.8% (CI: 0.3%-2%) with the >4 mm cut-off and 3.4% (CI: 2.1%-5.4%) with the >2 mm cut-off. Herd prevalence was 33.3% and 83% when using the >4 and the >2 mm cut-off respectively. There was no correlation between age, sex, body condition and positive reactors upon univariate analysis. None of the goats were reactors for BTB. Acid fast bacilli (AFB) were detected in 50% of the wildlife cultures, 79.2% of which were identified as Mycobacterium terrae complex. No M. bovis was detected. Twenty-seven percent of tested wildlife were sero-positive. Four sputum cultures (15.4%) yielded AFB positive colonies among which one was M. tuberculosis and 3 non-tuberculous mycobacteria (NTM). The prevalence of M. avium-complex (MAC) was 4.2% in wildlife, 2.5% in cattle and 0.5% in goats. In conclusion, individual BTB prevalence was low, but herd prevalence high in cattle and BTB was not detected in goats, wildlife and humans despite an intensive contact interface. On the contrary, NTMs were highly prevalent and some Mycobacterium spp were more prevalent in specific species. The role of NTMs in livestock and co-infection with BTB need further research.

  10. Potential Benefits of Cattle Vaccination as a Supplementary Control for Bovine Tuberculosis

    Science.gov (United States)

    Conlan, Andrew J. K.; Brooks Pollock, Ellen; McKinley, Trevelyan J.; Mitchell, Andrew P.; Jones, Gareth J.; Vordermeier, Martin; Wood, James L. N.

    2015-01-01

    Vaccination for the control of bovine tuberculosis (bTB) in cattle is not currently used within any international control program, and is illegal within the EU. Candidate vaccines, based upon Mycobacterium bovis bacillus Calmette-Guérin (BCG) all interfere with the action of the tuberculin skin test, which is used to determine if animals, herds and countries are officially bTB-free. New diagnostic tests that Differentiate Infected from Vaccinated Animals (DIVA) offer the potential to introduce vaccination within existing eradication programs. We use within-herd transmission models estimated from historical data from Great Britain (GB) to explore the feasibility of such supplemental use of vaccination. The economic impact of bovine Tuberculosis for farmers is dominated by the costs associated with testing, and associated restrictions on animal movements. Farmers’ willingness to adopt vaccination will require vaccination to not only reduce the burden of infection, but also the risk of restrictions being imposed. We find that, under the intensive sequence of testing in GB, it is the specificity of the DIVA test, rather than the sensitivity, that is the greatest barrier to see a herd level benefit of vaccination. The potential negative effects of vaccination could be mitigated through relaxation of testing. However, this could potentially increase the hidden burden of infection within Officially TB Free herds. Using our models, we explore the range of the DIVA test characteristics necessary to see a protective herd level benefit of vaccination. We estimate that a DIVA specificity of at least 99.85% and sensitivity of >40% is required to see a protective benefit of vaccination with no increase in the risk of missed infection. Data from experimentally infected animals suggest that this target specificity could be achieved in vaccinates using a cocktail of three DIVA antigens while maintaining a sensitivity of 73.3% (95%CI: 61.9, 82.9%) relative to post

  11. Epidemiologic characterization of bovine tuberculosis in the State of Rondônia, Brazil

    Directory of Open Access Journals (Sweden)

    Fabiano Benitez Vendrame

    2016-11-01

    Full Text Available A cross sectional study was performed between June 2009 and March 2010 to determine the situation of bovine tuberculosis (bTB in Rondônia. The state was divided into three regions and, in each of them, 300 farms with reproductive activity were randomly chosen and considered as primary sample units. In the selected farms, an epidemiologic questionnaire was applied. A fixed number of bovine females older than two years of age was randomly selected and tested through comparative cervical tuberculin test. Considering the State of Rondônia, the apparent prevalence of bTB positive farms was 2.3% (95% CI = 1.5–3.5%. The prevalence in the regions varied from 1.7% (95% CI = 0.7 – 4% to 3% (95% CI = 1.6–5.7%. The apparent prevalence of bTB positive animals in the State of Rondônia was 0.12% (95% CI = 0.06–0.25% and varied from 0.08% (95% CI = 0.04–0.18% to 0.15% (95% CI = 0.07–0.33% in the regions. The risk factor associated to tuberculosis in the State of Rondônia was the acquisition of animals (OR = 7.1; 95% CI = 1.6–31.1. The State of Rondônia should implement a surveillance system to detect bTB-infected herds to certify them as bTB-free. Moreover, an efficient health education program to inform farmers to test replacement animals for bTB prior to introduction in their herds should also be implemented.

  12. Badgers prefer cattle pasture but avoid cattle: implications for bovine tuberculosis control.

    Science.gov (United States)

    Woodroffe, Rosie; Donnelly, Christl A; Ham, Cally; Jackson, Seth Y B; Moyes, Kelly; Chapman, Kayna; Stratton, Naomi G; Cartwright, Samantha J

    2016-10-01

    Effective management of infectious disease relies upon understanding mechanisms of pathogen transmission. In particular, while models of disease dynamics usually assume transmission through direct contact, transmission through environmental contamination can cause different dynamics. We used Global Positioning System (GPS) collars and proximity-sensing contact-collars to explore opportunities for transmission of Mycobacterium bovis [causal agent of bovine tuberculosis] between cattle and badgers (Meles meles). Cattle pasture was badgers' most preferred habitat. Nevertheless, although collared cattle spent 2914 collar-nights in the home ranges of contact-collared badgers, and 5380 collar-nights in the home ranges of GPS-collared badgers, we detected no direct contacts between the two species. Simultaneous GPS-tracking revealed that badgers preferred land > 50 m from cattle. Very infrequent direct contact indicates that badger-to-cattle and cattle-to-badger M. bovis transmission may typically occur through contamination of the two species' shared environment. This information should help to inform tuberculosis control by guiding both modelling and farm management.

  13. Bovine tuberculosis in Canadian wildlife: An updated history

    OpenAIRE

    Wobeser, Gary

    2009-01-01

    Mycobacterium bovis infection in wild animals attracted little attention in Canada until the disease was almost eliminated from domestic livestock. Tuberculosis was endemic in plains bison and occurred in elk, moose, and mule deer in Buffalo National Park (BNP), Alberta during the 1920s and 1930s. Bison were moved from BNP to Wood Buffalo National Park (WBNP), where tuberculosis became, and remains, endemic in bison, posing a risk to efforts to restore bison in northern Canada. Tuberculosis w...

  14. The consequences of vaccination with the Johne's disease vaccine, Gudair, on diagnosis of bovine tuberculosis.

    Science.gov (United States)

    Coad, M; Clifford, D J; Vordermeier, H M; Whelan, A O

    2013-03-09

    The single intradermal comparative cervical tuberculin skin-test (SICCT) remains the primary surveillance tool to diagnose bovine tuberculosis (BTB) in the UK. Therefore, understanding the potential confounding influences on this test is important. This study investigated the effects of vaccination against Johne's disease (JD) on the immunodiagnosis of BTB using a Mycobacterium bovis BCG vaccination model as a surrogate of M bovis infection. Calves were vaccinated with either BCG (an attenuated live vaccine) or the JD vaccine, Gudair (a heat-inactivated suspension of Mycobacterium avium subspecies paratuberculosis), or a combination of both, and SICCT responses were measured approximately six and 12 weeks postvaccination. Animals vaccinated with Gudair only were negative to the SICCT test, thus supporting the specificity of the SICCT test following Gudair vaccination. However, while animals vaccinated with BCG-only demonstrated a bovine tuberculin-biased response as expected, covaccination with Gudair resulted in a bias towards avian tuberculin in the SICCT test. Therefore, our model demonstrates the potential of the Gudair vaccine to reduce the sensitivity of the SICCT. In addition, while we also demonstrate that Gudair vaccination can compromise the specificity of serological tests to detect JD, the specificity of defined M bovis antigens in serological or interferon gamma-based blood assays was not compromised by the vaccine.

  15. Bovine Tuberculosis: Effect of the Tuberculin Skin Test on the Interferon Gamma Assay

    Science.gov (United States)

    Introduction Bovine tuberculosis (bTB) is a disease of zoonotic and economic importance. Despite intensive eradication efforts over decades, bTB continues to be a problem with global perspective. The control of bTB is based on a test and slaughter policy and/or abattoir surveillance. Two types of tu...

  16. Bovine Tuberculosis: Effect of the Tuberculin Skin Test on In vitro Interferon gamma Responses

    Science.gov (United States)

    Bovine tuberculosis (bTB) is a disease of zoonotic and economic importance. In many countries, control is based on test and slaughter policies and/or abattoir surveillance. For testing, cell mediated immune- (CMI-) based assays (i.e., Tuberculin skin test (TST) supplemented by the interferon gamma (...

  17. Comparison of Tuberculin Activity in the Interferon-gamma Assay for the Diagnosis of Bovine Tuberculosis

    Science.gov (United States)

    Cattle infected with bovine tuberculosis still represent a serious regulatory and health concern in a variety of countries. Early diagnosis using the in vitro interferon gamma (IFN-gamma) assay has been applied for more than a decade. Briefly, IFN-gamma responses in whole blood cultures stimulated w...

  18. The importance of ‘neighbourhood’ in the persistence of bovine tuberculosis in Irish cattle herds

    NARCIS (Netherlands)

    White, P.W.; Martin, S.W.; Jong, de M.C.M.; O'Keeffe, J.J.; More, S.J.; Frankena, K.

    2013-01-01

    Local persistence of infection is a key feature of bovine tuberculosis (bTB) among cattle herds in the Republic of Ireland. The aim of this study was to determine the relative importance of ‘neighbourhood’, specifically farm-to-farm spread and spread from wildlife, in the persistence of bTB by

  19. Absence of bovine tuberculosis in feral swine (Sus scrofa) from the Southern Texas border region

    Science.gov (United States)

    Free-ranging wildlife, like feral swine (Sus scrofa), harbor a variety of diseases that are infectious to livestock and could negatively impact agricultural production. Information is lacking regarding the exposure and infection rates for bovine tuberculosis (Mycobacterium bovis; bTB), and many othe...

  20. Risk factors for bovine tuberculosis (bTB) in cattle in Ethiopia

    NARCIS (Netherlands)

    Dejene, Sintayehu W.; Heitkonig, Ignas; Prins, Herbert H.T.; Lemma, Fitsum A.; Mekonnen, Daniel A.; Alemu, Zelalem E.; Kelkay, Tessema Z.; Boer, de Fred

    2016-01-01

    Bovine tuberculosis (bTB) infection is generally correlated with individual cattle's age, sex, body condition, and with husbandry practices such as herd composition, cattle movement, herd size, production system and proximity to wildlife - including bTB maintenance hosts. We tested the

  1. Prevalence and risk factors of bovine tuberculosis in dairy cattle in Eritrea

    NARCIS (Netherlands)

    Ghebremariam, Michael K; Rutten, V P M G|info:eu-repo/dai/nl/092848028; Vernooij, J C M|info:eu-repo/dai/nl/340304596; Uqbazghi, K; Tesfaalem, T; Butsuamlak, T; Idris, A M; Nielen, M|info:eu-repo/dai/nl/123535298; Michel, A L

    2016-01-01

    BACKGROUND: The prevalence of bovine tuberculosis (BTB) in dairy cattle in the three major milk producing regions of Eritrea was assessed by subjecting 15,354 dairy cattle, 50 % of Eritrea's dairy cattle population, to the single intradermal comparative tuberculin test (SICTT). Skin test results wer

  2. Monitoring and Analysis of Bovine Tuberculosis%牛结核病监测与分析研究

    Institute of Scientific and Technical Information of China (English)

    雷程红; 冉多良; 余佳琳; 蒋龙; 刘瑜; 张寅生

    2012-01-01

    [目的]掌握牛结核病的感染情况及其感染原因,逐步控制包括人在内的结核病的发病率,帮助奶牛养殖业持续健康发展.[方法]2010年下半年对乌鲁木齐周边地区部分牛用“提纯结核菌素皮内变态反应”方法进行了结核病监测.[结果]对监测结果数据进行统计分析显示:此次共监测牛1 052头,其中检出结核阳性牛5头,阳性率为0.47%.[结论]牛结核病零星散发,应做好检疫和扑杀工作.%[ Objective ] To have a good knowledge of the infection of bovine tuberculosis and its causes in order to gradually control the tuberculosis rate including human being's and help cow industry go into a continuous and healthy development. [ Method ]" Purification of cattle tuberculin skin allergy" method was used to monitor the tuberculosis of cattle in those areas around Urumqi in the second half of 2010. [Result] The result shows; among the 1,052 cattle detected, five heads of cattle showed positive tuberculosis signs. The rate is 0.47%. [ Conclusion] Bovine tuberculosis occurred sporadically, and the quarantine and culling work should be done well.

  3. Understanding the Mechanisms of Immunopathogenesis of Human and Bovine Tuberculosis

    Science.gov (United States)

    Extensive investigations have revealed that zoonotic pathogens in the Mycobacterium tuberculosis complex (MTBC) evolved from a common ancestor. Although all the members can cause disease in one or more species of mammals, Mycobacterium tuberculosis (Mtb) and M. bovis (Mbv) are the major pathogens ...

  4. Widespread Environmental Contamination with Mycobacterium tuberculosis Complex Revealed by a Molecular Detection Protocol.

    Science.gov (United States)

    Santos, Nuno; Santos, Catarina; Valente, Teresa; Gortázar, Christian; Almeida, Virgílio; Correia-Neves, Margarida

    2015-01-01

    Environmental contamination with Mycobacterium tuberculosis complex (MTC) has been considered crucial for bovine tuberculosis persistence in multi-host-pathogen systems. However, MTC contamination has been difficult to detect due to methodological issues. In an attempt to overcome this limitation we developed an improved protocol for the detection of MTC DNA. MTC DNA concentration was estimated by the Most Probable Number (MPN) method. Making use of this protocol we showed that MTC contamination is widespread in different types of environmental samples from the Iberian Peninsula, which supports indirect transmission as a contributing mechanism for the maintenance of bovine tuberculosis in this multi-host-pathogen system. The proportion of MTC DNA positive samples was higher in the bovine tuberculosis-infected than in presumed negative area (0.32 and 0.18, respectively). Detection varied with the type of environmental sample and was more frequent in sediment from dams and less frequent in water also from dams (0.22 and 0.05, respectively). The proportion of MTC-positive samples was significantly higher in spring (p<0.001), but MTC DNA concentration per sample was higher in autumn and lower in summer. The average MTC DNA concentration in positive samples was 0.82 MPN/g (CI95 0.70-0.98 MPN/g). We were further able to amplify a DNA sequence specific of Mycobacterium bovis/caprae in 4 environmental samples from the bTB-infected area.

  5. Prevalence and risk factors of bovine tuberculosis in Nili Ravi buffaloes in the Punjab, Pakistan

    Directory of Open Access Journals (Sweden)

    A. Khan

    2010-02-01

    Full Text Available The present study was executed to determine the magnitude of bovine tuberculosis (BTB in buffaloes in native type of husbandry practices and impact of certain factors in the prevalence of bovine tuberculosis in buffaloes in the Punjab, Pakistan. Three year cross sectional study was carried out on female population of Nili Ravi buffaloes (n = 2526 maintained at 10 Government Livestock Experimental Stations, and peri urban areas of the three major cites i.e., Lahore, Faisalabad and Okara. These animals were screened with comparative intradermal tuberculin test (CIDT by using two types of tuberculins i.e., mammalian and avian. The reaction of tuberculins injected was interpreted after 72 hours post injection. The data were analyzed by Chi-square test and Pearson correlation. Relative risk and other associated factors were calculated to describe the association with prevalence of tuberculosis in buffaloes. The prevalence of bovine tuberculosis on the basis of CIDT was 12.72%. The BTB among different livestock farms varied significantly (P8 years old age, body weight >550 kg, 3-6 parity, pregnant animals, and animals with >7 liters milk yield. The husbandry factors which greatly influence the prevalence was poor feeding (RR=2.615, high fly density (RR= 1.3474, poor management (RR=1.315, contact with wildlife (RR=1.4507, poor farm conditions (RR=1.4708, quarantine measures (RR=1.1557 and poor sanitation of farm (RR= 1.3701.

  6. Tuberculosis Detection by Giant African Pouched Rats

    Science.gov (United States)

    Poling, Alan; Weetjens, Bart; Cox, Christophe; Beyene, Negussie; Durgin, Amy; Mahoney, Amanda

    2011-01-01

    In recent years, operant discrimination training procedures have been used to teach giant African pouched rats to detect tuberculosis (TB) in human sputum samples. This article summarizes how the rats are trained and used operationally, as well as their performance in studies published to date. Available data suggest that pouched rats, which can…

  7. Tuberculosis Detection by Giant African Pouched Rats

    Science.gov (United States)

    Poling, Alan; Weetjens, Bart; Cox, Christophe; Beyene, Negussie; Durgin, Amy; Mahoney, Amanda

    2011-01-01

    In recent years, operant discrimination training procedures have been used to teach giant African pouched rats to detect tuberculosis (TB) in human sputum samples. This article summarizes how the rats are trained and used operationally, as well as their performance in studies published to date. Available data suggest that pouched rats, which can…

  8. Development of an antibody to bovine IL-2 reveals multifunctional CD4 T(EM cells in cattle naturally infected with bovine tuberculosis.

    Directory of Open Access Journals (Sweden)

    Adam O Whelan

    Full Text Available Gaining a better understanding of the T cell mechanisms underlying natural immunity to bovine tuberculosis would help to identify immune correlates of disease progression and facilitate the rational design of improved vaccine and diagnostic strategies. CD4 T cells play an established central role in immunity to TB, and recent interest has focussed on the potential role of multifunctional CD4 T cells expressing IFN-γ, IL-2 and TNF-α. Until now, it has not been possible to assess the contribution of these multifunctional CD4 T cells in cattle due to the lack of reagents to detect bovine IL-2 (bIL-2. Using recombinant phage display technology, we have identified an antibody that recognises biologically active bIL-2. Using this antibody, we have developed a polychromatic flow cytometric staining panel that has allowed the investigation of multifunctional CD4 T-cells responses in cattle naturally infected with M. bovis. Assessment of the frequency of antigen specific CD4 T cell subsets reveals a dominant IFN-γ(+IL-2(+TNF-α(+ and IFN-γ(+ TNF-α(+ response in naturally infected cattle. These multifunctional CD4 T cells express a CD44(hiCD45RO(+CD62L(lo T-effector memory (T(EM phenotype and display higher cytokine median fluorescence intensities than single cytokine producers, consistent with an enhanced 'quality of response' as reported for multifunctional cells in human and murine systems. Through our development of these novel immunological bovine tools, we provide the first description of multifunctional T(EM cells in cattle. Application of these tools will improve our understanding of protective immunity in bovine TB and allow more direct comparisons of the complex T cell mediated immune responses between murine models, human clinical studies and bovine TB models in the future.

  9. Systemic bovine tuberculosis: a case report/
    Tuberculose bovina sistêmica: um relato de caso

    OpenAIRE

    Selwyn Arlington Headley

    2002-01-01

    Gross and histopathological lesions associated with Mycobacterium bovis in an ox are described. Differential diagnoses of frequently occurring gross lesions in cattle that could be easily confused with bovine tuberculosis are discussed, and a comparative analysis of M. bovis induced tuberculosis lesions in wildlife is made.Alterações macroscópicas e histopatológicas associadas a Mycobacterium bovis são descritas em um bovino. O diagnóstico diferencial de lesões macroscópicas mais freqüentemen...

  10. Evaluation of Cocktails with Recombinant Proteins of Mycobacterium bovis for a Specific Diagnosis of Bovine Tuberculosis

    Directory of Open Access Journals (Sweden)

    María Laura Mon

    2014-01-01

    Full Text Available The Delayed type hypersensitivity skin test (DTH and interferon-gamma assay are used for the diagnosis of bovine tuberculosis (TBB. The specificity of these diagnoses, however, is compromised because both are based on the response against purified protein derivative of Mycobacterium bovis (PPD-B. In this study, we assessed the potential of two cocktails containing M. bovis recombinant proteins: cocktail 1 (C1: ESAT-6, CFP-10 and MPB83 and cocktail 2 (C2: ESAT-6, CFP-10, MPB83, HspX, TB10.3, and MPB70. C1, C2, and PPD-B showed similar response by DTH in M. bovis-sensitized guinea pigs. Importantly, C1 induced a lower response than PPD-B in M. avium-sensitized guinea pigs. In cattle, C1 displayed better performance than PPD-B and C2; indeed, C1 showed the least detection of animals either vaccinated or Map-infected. To optimize the composition of the cocktails, we obtained protein fractions from PPD-B and tested their immunogenicity in experimentally M. bovis-infected cattle. In one highly reactive fraction, seven proteins were identified. The inclusion of FixB in C1 enhanced the recognition of M. bovis-infected cattle without compromising specificity. Our data provide a promising basis for the future development of a cocktail for TBB detection without interference by the presence of sensitized or infected animals with other mycobacteria.

  11. Evaluation of Cocktails with Recombinant Proteins of Mycobacterium bovis for a Specific Diagnosis of Bovine Tuberculosis

    Science.gov (United States)

    Mon, María Laura; Moyano, Roberto Damián; Viale, Mariana Noelia; Colombatti Olivieri, María Alejandra; Gamietea, Ignacio José; Montenegro, Valeria Noely; Alonso, Bernardo; Santangelo, María de la Paz; Singh, Mahavir; Duran, Rosario; Romano, María Isabel

    2014-01-01

    The Delayed type hypersensitivity skin test (DTH) and interferon-gamma assay are used for the diagnosis of bovine tuberculosis (TBB). The specificity of these diagnoses, however, is compromised because both are based on the response against purified protein derivative of Mycobacterium bovis (PPD-B). In this study, we assessed the potential of two cocktails containing M. bovis recombinant proteins: cocktail 1 (C1): ESAT-6, CFP-10 and MPB83 and cocktail 2 (C2): ESAT-6, CFP-10, MPB83, HspX, TB10.3, and MPB70. C1, C2, and PPD-B showed similar response by DTH in M. bovis-sensitized guinea pigs. Importantly, C1 induced a lower response than PPD-B in M. avium-sensitized guinea pigs. In cattle, C1 displayed better performance than PPD-B and C2; indeed, C1 showed the least detection of animals either vaccinated or Map-infected. To optimize the composition of the cocktails, we obtained protein fractions from PPD-B and tested their immunogenicity in experimentally M. bovis-infected cattle. In one highly reactive fraction, seven proteins were identified. The inclusion of FixB in C1 enhanced the recognition of M. bovis-infected cattle without compromising specificity. Our data provide a promising basis for the future development of a cocktail for TBB detection without interference by the presence of sensitized or infected animals with other mycobacteria. PMID:25110654

  12. Bayesian receiver operating characteristic estimation of multiple tests for diagnosis of bovine tuberculosis in Chadian cattle.

    Directory of Open Access Journals (Sweden)

    Borna Müller

    Full Text Available BACKGROUND: Bovine tuberculosis (BTB today primarily affects developing countries. In Africa, the disease is present essentially on the whole continent; however, little accurate information on its distribution and prevalence is available. Also, attempts to evaluate diagnostic tests for BTB in naturally infected cattle are scarce and mostly complicated by the absence of knowledge of the true disease status of the tested animals. However, diagnostic test evaluation in a given setting is a prerequisite for the implementation of local surveillance schemes and control measures. METHODOLOGY/PRINCIPAL FINDINGS: We subjected a slaughterhouse population of 954 Chadian cattle to single intra-dermal comparative cervical tuberculin (SICCT testing and two recently developed fluorescence polarization assays (FPA. Using a Bayesian modeling approach we computed the receiver operating characteristic (ROC curve of each diagnostic test, the true disease prevalence in the sampled population and the disease status of all sampled animals in the absence of knowledge of the true disease status of the sampled animals. In our Chadian setting, SICCT performed better if the cut-off for positive test interpretation was lowered from >4 mm (OIE standard cut-off to >2 mm. Using this cut-off, SICCT showed a sensitivity and specificity of 66% and 89%, respectively. Both FPA tests showed sensitivities below 50% but specificities above 90%. The true disease prevalence was estimated at 8%. Altogether, 11% of the sampled animals showed gross visible tuberculous lesions. However, modeling of the BTB disease status of the sampled animals indicated that 72% of the suspected tuberculosis lesions detected during standard meat inspections were due to other pathogens than Mycobacterium bovis. CONCLUSIONS/SIGNIFICANCE: Our results have important implications for BTB diagnosis in a high incidence sub-Saharan African setting and demonstrate the practicability of our Bayesian approach for

  13. Bovine tuberculosis in Europe from the perspective of an officially tuberculosis free country: trade, surveillance and diagnostics.

    Science.gov (United States)

    Schiller, Irene; Waters, W Ray; RayWaters, W; Vordermeier, H Martin; Jemmi, Thomas; Welsh, Michael; Keck, Nicolas; Whelan, Adam; Gormley, Eamonn; Boschiroli, Maria Laura; Moyen, Jean Louis; Vela, Carmen; Cagiola, Monica; Buddle, Bryce M; Palmer, Mitchell; Thacker, Tyler; Oesch, Bruno

    2011-07-05

    Switzerland has been officially free of bovine tuberculosis (OTF) since 1960. Since 1980 the control of bovine tuberculosis (bTB) has been reduced to passive abattoir surveillance. Isolated cases of bTB, partly due to reactivation of human Mycobacterium bovis infections with subsequent transmission to cattle, have been noticed in the last years. In Europe, the overall prevalence of bTB is slightly increasing. Both OTF and non-OTF countries report increases in the proportion of bTB positive cattle herds. Current bTB eradication and control programs in Europe are facing a range of challenges. Whole herd depopulation is becoming a less attractive option for economic reasons and due to animal welfare concerns. Live animal trade is increasing both at national and international levels. Regarding these tendencies and taking into account the chronicity of bTB infection, pre-movement testing is becoming increasingly important as a central tool for eradication and for protection against re-introduction of bTB. Pre-movement testing, however specifically focuses on the infection status in individuals, requiring a high level of diagnostic accuracy to correctly diagnose infected animals. Current screening tests for bTB, however, have been designed to meet demands as herd tests. This illustrates that the modification of existing and/or the development of new diagnostics for bTB might be needed. The tuberculin skin test (TST), the primary screening test for bTB may in certain situations have low sensitivity. The interferon gamma (IFN-γ) assay is accepted to be more sensitive compared to TST. Reduced specificity, however, especially in areas of low bTB prevalence raises concerns. New antigen combinations including Rv3615c, OmpATb and others have been shown to complement ESAT-6 and CFP-10 in the whole blood IFN-γ assay and resulted in improved sensitivity (compared to ESAT-6 and CFP-10) and specificity (compared to tuberculins). Lesion detection after slaughter represents a cost

  14. Long-term assessment of wild boar harvesting and cattle removal for bovine tuberculosis control in free ranging populations.

    Directory of Open Access Journals (Sweden)

    Gregorio Mentaberre

    Full Text Available Wild boar is a recognized reservoir of bovine tuberculosis (TB in the Mediterranean ecosystems, but information is scarce outside of hotspots in southern Spain. We describe the first high-prevalence focus of TB in a non-managed wild boar population in northern Spain and the result of eight years of TB management. Measures implemented for disease control included the control of the local wild boar population through culling and stamping out of a sympatric infected cattle herd. Post-mortem inspection for detection of tuberculosis-like lesions as well as cultures from selected head and cervical lymph nodes was done in 745 wild boar, 355 Iberian ibexes and five cattle between 2004 and 2012. The seasonal prevalence of TB reached 70% amongst adult wild boar and ten different spoligotypes and 13 MIRU-VNTR profiles were detected, although more than half of the isolates were included in the same clonal complex. Only 11% of infected boars had generalized lesions. None of the ibexes were affected, supporting their irrelevance in the epidemiology of TB. An infected cattle herd grazed the zone where 168 of the 197 infected boars were harvested. Cattle removal and wild boar culling together contributed to a decrease in TB prevalence. The need for holistic, sustained over time, intensive and adapted TB control strategies taking into account the multi-host nature of the disease is highlighted. The potential risk for tuberculosis emergence in wildlife scenarios where the risk is assumed to be low should be addressed.

  15. Long-term assessment of wild boar harvesting and cattle removal for bovine tuberculosis control in free ranging populations.

    Science.gov (United States)

    Mentaberre, Gregorio; Romero, Beatriz; de Juan, Lucía; Navarro-González, Nora; Velarde, Roser; Mateos, Ana; Marco, Ignasi; Olivé-Boix, Xavier; Domínguez, Lucas; Lavín, Santiago; Serrano, Emmanuel

    2014-01-01

    Wild boar is a recognized reservoir of bovine tuberculosis (TB) in the Mediterranean ecosystems, but information is scarce outside of hotspots in southern Spain. We describe the first high-prevalence focus of TB in a non-managed wild boar population in northern Spain and the result of eight years of TB management. Measures implemented for disease control included the control of the local wild boar population through culling and stamping out of a sympatric infected cattle herd. Post-mortem inspection for detection of tuberculosis-like lesions as well as cultures from selected head and cervical lymph nodes was done in 745 wild boar, 355 Iberian ibexes and five cattle between 2004 and 2012. The seasonal prevalence of TB reached 70% amongst adult wild boar and ten different spoligotypes and 13 MIRU-VNTR profiles were detected, although more than half of the isolates were included in the same clonal complex. Only 11% of infected boars had generalized lesions. None of the ibexes were affected, supporting their irrelevance in the epidemiology of TB. An infected cattle herd grazed the zone where 168 of the 197 infected boars were harvested. Cattle removal and wild boar culling together contributed to a decrease in TB prevalence. The need for holistic, sustained over time, intensive and adapted TB control strategies taking into account the multi-host nature of the disease is highlighted. The potential risk for tuberculosis emergence in wildlife scenarios where the risk is assumed to be low should be addressed.

  16. Interleukin-17A as a biomarker for bovine tuberculosis

    Science.gov (United States)

    T helper (Th) 17-associated cytokines are integral in the immune response to tuberculosis, initiating both protective and harmful inflammatory responses. The aim of the present study was to evaluate applied aspects of IL-17 biology in the context of Mycobacterium bovis infection of cattle. Using RNA...

  17. Bovine tuberculosis research: Immune mechanisms relevant to biomedical applications

    Science.gov (United States)

    Pioneer studies on infectious disease and immunology by Jenner, Pasteur, Koch, Von Behring, Nocard, Roux, and Ehrlich forged a path for the dual-purpose with dual benefit approach, clearly demonstrating the relevance of veterinary studies for biomedical applications. Tuberculosis (TB), primarily due...

  18. Vaccine approaches for bovine tuberculosis: Correlates of protection and relevance to human tuberculosis

    Science.gov (United States)

    Tuberculosis (TB), primarily due to Mycobacterium tuberculosis in humans and Mycobacterium bovis in cattle, is a classic model of the One Health Concept. M. bovis Bacillus Calmette Guerin (BCG) was first proven effective in cattle prior to use in humans. Recent experimental trials with cattle have d...

  19. The immune response to bovine tuberculosis: Correlates of protection and relevance to human tuberculosis

    Science.gov (United States)

    Tuberculosis (TB), primarily due to Mycobacterium tuberculosis in humans and Mycobacterium bovis in cattle, is a classic model for demonstration of the One Health Concept. Early studies with cattle were instrumental in the development of the use of Koch’s tuberculin as an in vivo measure of cell-med...

  20. Evidence of presence of Mycobacterium tuberculosis in bovine tissue samples by multiplex PCR: possible relevance to reverse zoonosis.

    Science.gov (United States)

    Mittal, M; Chakravarti, S; Sharma, V; Sanjeeth, B S; Churamani, C P; Kanwar, N S

    2014-04-01

    Bovine tuberculosis, caused by Mycobacterium bovis, remains one of the most important zoonotic health concerns worldwide. The transmission of Mycobacterium tuberculosis from humans to animals also occurs especially in countries where there is close interaction of humans with the animals. In the present study, thirty bovine lung tissue autopsy samples from an organized dairy farm located in North India were screened for the presence of Mycobacterium tuberculosis complex by smear microscopy, histopathological findings and PCR. Differential diagnosis of M. tuberculosis and M. bovis was made based on the deletion of mce-3 operon in M. bovis. The present study found eight of these samples positive for M. tuberculosis by multiplex PCR. Sequencing was performed on two PCR-positive representative samples and on annotation, and BLAST analysis confirmed the presence of gene fragment specific to Mycobacterium tuberculosis. The presence of M. tuberculosis in all the positive samples raises the possibility of human-to-cattle transmission and possible adaptation of this organism in bovine tissues. This study accentuates the importance of screening and differential diagnosis of Mycobacterium tuberculosis complex in humans and livestock for adopting effective TB control and eradication programmes.

  1. Prevalence of bovine tuberculosis in Ethiopian slaughter cattle based on post-mortem examination.

    Science.gov (United States)

    Demelash, B; Inangolet, F; Oloya, J; Asseged, B; Badaso, M; Yilkal, A; Skjerve, E

    2009-06-01

    A study aimed at describing the magnitude and distribution of gross lesions compatible with bovine tuberculosis (BTB) in Ethiiopian slaughter cattle in five abattoirs (four municipal and one export) located in various cattle husbandry systems in Ethiopia was carried out from July 2006 to January 2007 using detailed meat inspection procedure. Five representative abattoirs (four municipal and one export) located in distinct livestock management systems were selected. A total of 3322 cattle; 2876 (86.6%) male, 446 (13.4%) female; 3094 (93.1%) indigenous zebu, 140 (4.2%) crossbred and 88 (2.7%) pure exotic cattle were included in the study. A nine-year meat inspection record was also analyzed to elucidate the trend of BTB in the local cattle population.Of the carcasses inspected, 337 (10.2%, 95%CI= [9.2-11.2]) had lesions suggestive of tuberculosis, 69 (20.5%) generalized and 268 (79.5%) localized.TB prevalence showed a marked variation between categories of age, breed, class of animals, abattoir, geographic origin and husbandry system. It was higher in old and young animals than middle age group; in exotic than local breed; in calves than other classes of animals. The highest and lowest prevalence of TB was recorded in Adama (24.7%, 95%CI= [21.1-28.7]) and Yabello abattoirs (4.2%, 95%CI= [2.6-6.6]), respectively. Cattle whose origin was from Addis Ababa and its surrounding areas had higher prevalence of TB infection (23.9%, 95%CI= [17.6-31.5]).Cattle maintained in dairy farms had high degree of exposure (23.9%, 95%CI= [16.7-32.9]) to the infection than those maintained in other types of management system. Analysis of meat inspection records also revealed an increasing incidence of TB over the years. Our study demonstrated a high prevalence of tuberculosis in Ethiopian slaughter cattle and this could infer to similar scenario in a reference cattle population in the country. In view of Ethiopia's increasing involvement in livestock export trade, the reported high

  2. Epidemiological investigation of bovine tuberculosis herd breakdowns in Spain 2009/2011.

    Science.gov (United States)

    Guta, Sintayehu; Casal, Jordi; Napp, Sebastian; Saez, Jose Luis; Garcia-Saenz, Ariadna; Perez de Val, Bernat; Romero, Beatriz; Alvarez, Julio; Allepuz, Alberto

    2014-01-01

    We analyzed the most likely cause of 687 bovine tuberculosis (bTB) breakdowns detected in Spain between 2009 and 2011 (i.e., 22% of the total number of breakdowns detected during this period). Seven possible causes were considered: i) residual infection; ii) introduction of infected cattle from other herds; iii) sharing of pastures with infected herds; iv) contiguous spread from infected neighbor herds; v) presence of infected goats in the farm; vi) interaction with wildlife reservoirs and vii) contact with an infected human. For each possible cause a decision tree was developed and key questions were included in each of them. Answers to these key questions lead to different events within each decision tree. In order to assess the likelihood of occurrence of the different events a qualitative risk assessment approach was used. For this purpose, an expert opinion workshop was organized and ordinal values, ranging from 0 to 9 (i.e., null to very high likelihood of occurrence) were assigned. The analysis identified residual infection as the most frequent cause of bTB breakdowns (22.3%; 95%CI: 19.4-25.6), followed by interaction with wildlife reservoirs (13.1%; 95%CI: 10.8-15.8). The introduction of infected cattle, sharing of pastures and contiguous spread from infected neighbour herds were also identified as relevant causes. In 41.6% (95%CI: 38.0-45.4) of the breakdowns the origin of infection remained unknown. Veterinary officers conducting bTB breakdown investigations have to state their opinion about the possible cause of each breakdown. Comparison between the results of our analysis and the opinion from veterinary officers revealed a slight concordance. This slight agreement might reflect a lack of harmonized criteria to assess the most likely cause of bTB breakdowns as well as different perceptions about the importance of the possible causes. This is especially relevant in the case of the role of wildlife reservoirs.

  3. Epidemiological investigation of bovine tuberculosis herd breakdowns in Spain 2009/2011.

    Directory of Open Access Journals (Sweden)

    Sintayehu Guta

    Full Text Available We analyzed the most likely cause of 687 bovine tuberculosis (bTB breakdowns detected in Spain between 2009 and 2011 (i.e., 22% of the total number of breakdowns detected during this period. Seven possible causes were considered: i residual infection; ii introduction of infected cattle from other herds; iii sharing of pastures with infected herds; iv contiguous spread from infected neighbor herds; v presence of infected goats in the farm; vi interaction with wildlife reservoirs and vii contact with an infected human. For each possible cause a decision tree was developed and key questions were included in each of them. Answers to these key questions lead to different events within each decision tree. In order to assess the likelihood of occurrence of the different events a qualitative risk assessment approach was used. For this purpose, an expert opinion workshop was organized and ordinal values, ranging from 0 to 9 (i.e., null to very high likelihood of occurrence were assigned. The analysis identified residual infection as the most frequent cause of bTB breakdowns (22.3%; 95%CI: 19.4-25.6, followed by interaction with wildlife reservoirs (13.1%; 95%CI: 10.8-15.8. The introduction of infected cattle, sharing of pastures and contiguous spread from infected neighbour herds were also identified as relevant causes. In 41.6% (95%CI: 38.0-45.4 of the breakdowns the origin of infection remained unknown. Veterinary officers conducting bTB breakdown investigations have to state their opinion about the possible cause of each breakdown. Comparison between the results of our analysis and the opinion from veterinary officers revealed a slight concordance. This slight agreement might reflect a lack of harmonized criteria to assess the most likely cause of bTB breakdowns as well as different perceptions about the importance of the possible causes. This is especially relevant in the case of the role of wildlife reservoirs.

  4. Epidemiological investigation of bovine tuberculosis outbreaks in Uruguay (2011-2013).

    Science.gov (United States)

    Picasso, Catalina; Alvarez, Julio; VanderWaal, Kimberly L; Fernandez, Federico; Gil, Andres; Wells, Scott J; Perez, Andres

    2017-03-01

    Bovine tuberculosis (bTB) is a chronic disease of cattle caused by infection with the Mycobacterium bovis. While bTB prevalence in Uruguay has been low (<11 outbreaks/year) for the past 50 years as a consequence of a national control program, annual incidence increased in 2011 through 2013-15, 26 and 16 infected herds each year, raising concerns from livestock stakeholders and the government. The goal of this study was to assess the spatial dynamics of bTB in Uruguay from 2011 to 2013 and the association between bTB and potential demographic and movement risk factors at the herd level using data provided by the Uruguayan Ministry of Livestock, Agriculture, and Fisheries. Clustering of incident outbreaks was assessed using the Cuzick-Edwards' test and the Bernoulli model of the spatial scan statistic, and a conditional multivariable logistic regression model was used to assess risk factors associated with bTB in a subset of Uruguayan dairy farms. Significant (P<0.05) global clustering was detected in 2012, while high-risk local clusters were detected in southwestern (2011, 2012, 2013), northwestern (2012), and southeastern (2012) Uruguay. Increased risk of bTB in different regions of Uruguay suggests a potential role of animal movements in disease dissemination. Larger herds, higher numbers of animals purchased, and incoming steers to the farm were associated with increased odds of breaking with bTB, in agreement with previous studies but also suggesting other additional sources of risk. These results will contribute to enhanced effectiveness of bTB control programs in Uruguay with the ultimate objective of preventing or mitigating the impact of the disease in the human and animal populations of the country.

  5. Spatial Dynamics of Bovine Tuberculosis in the Autonomous Community of Madrid, Spain (2010–2012)

    Science.gov (United States)

    de la Cruz, Maria Luisa; Perez, Andres; Bezos, Javier; Pages, Enrique; Casal, Carmen; Carpintero, Jesus; Romero, Beatriz; Dominguez, Lucas; Barker, Christopher M.; Diaz, Rosa; Alvarez, Julio

    2014-01-01

    Progress in control of bovine tuberculosis (bTB) is often not uniform, usually due to the effect of one or more sometimes unknown epidemiological factors impairing the success of eradication programs. Use of spatial analysis can help to identify clusters of persistence of disease, leading to the identification of these factors thus allowing the implementation of targeted control measures, and may provide some insights of disease transmission, particularly when combined with molecular typing techniques. Here, the spatial dynamics of bTB in a high prevalence region of Spain were assessed during a three year period (2010–2012) using data from the eradication campaigns to detect clusters of positive bTB herds and of those infected with certain Mycobacterium bovis strains (characterized using spoligotyping and VNTR typing). In addition, the within-herd transmission coefficient (β) was estimated in infected herds and its spatial distribution and association with other potential outbreak and herd variables was evaluated. Significant clustering of positive herds was identified in the three years of the study in the same location (“high risk area”). Three spoligotypes (SB0339, SB0121 and SB1142) accounted for >70% of the outbreaks detected in the three years. VNTR subtyping revealed the presence of few but highly prevalent strains within the high risk area, suggesting maintained transmission in the area. The spatial autocorrelation found in the distribution of the estimated within-herd transmission coefficients in herds located within distances <14 km and the results of the spatial regression analysis, support the hypothesis of shared local factors affecting disease transmission in farms located at a close proximity. PMID:25536514

  6. Sensitivity of Bovine Tuberculosis Surveillance in Wildlife in France: A Scenario Tree Approach.

    Directory of Open Access Journals (Sweden)

    Julie Rivière

    Full Text Available Bovine tuberculosis (bTB is a common disease in cattle and wildlife, with an impact on animal and human health, and economic implications. Infected wild animals have been detected in some European countries, and bTB reservoirs in wildlife have been identified, potentially hindering the eradication of bTB from cattle populations. However, the surveillance of bTB in wildlife involves several practical difficulties and is not currently covered by EU legislation. We report here the first assessment of the sensitivity of the bTB surveillance system for free-ranging wildlife launched in France in 2011 (the Sylvatub system, based on scenario tree modelling. Three surveillance system components were identified: (i passive scanning surveillance for hunted wild boar, red deer and roe deer, based on carcass examination, (ii passive surveillance on animals found dead, moribund or with abnormal behaviour, for wild boar, red deer, roe deer and badger and (iii active surveillance for wild boar and badger. The application of these three surveillance system components depends on the geographic risk of bTB infection in wildlife, which in turn depends on the prevalence of bTB in cattle. We estimated the effectiveness of the three components of the Sylvatub surveillance system quantitatively, for each species separately. Active surveillance and passive scanning surveillance by carcass examination were the approaches most likely to detect at least one infected animal in a population with a given design prevalence, regardless of the local risk level and species considered. The awareness of hunters, which depends on their training and the geographic risk, was found to affect surveillance sensitivity. The results obtained are relevant for hunters and veterinary authorities wishing to determine the actual efficacy of wildlife bTB surveillance as a function of geographic area and species, and could provide support for decision-making processes concerning the enhancement

  7. Spatial dynamics of bovine tuberculosis in the Autonomous Community of Madrid, Spain (2010-2012.

    Directory of Open Access Journals (Sweden)

    Maria Luisa de la Cruz

    Full Text Available Progress in control of bovine tuberculosis (bTB is often not uniform, usually due to the effect of one or more sometimes unknown epidemiological factors impairing the success of eradication programs. Use of spatial analysis can help to identify clusters of persistence of disease, leading to the identification of these factors thus allowing the implementation of targeted control measures, and may provide some insights of disease transmission, particularly when combined with molecular typing techniques. Here, the spatial dynamics of bTB in a high prevalence region of Spain were assessed during a three year period (2010-2012 using data from the eradication campaigns to detect clusters of positive bTB herds and of those infected with certain Mycobacterium bovis strains (characterized using spoligotyping and VNTR typing. In addition, the within-herd transmission coefficient (β was estimated in infected herds and its spatial distribution and association with other potential outbreak and herd variables was evaluated. Significant clustering of positive herds was identified in the three years of the study in the same location ("high risk area". Three spoligotypes (SB0339, SB0121 and SB1142 accounted for >70% of the outbreaks detected in the three years. VNTR subtyping revealed the presence of few but highly prevalent strains within the high risk area, suggesting maintained transmission in the area. The spatial autocorrelation found in the distribution of the estimated within-herd transmission coefficients in herds located within distances <14 km and the results of the spatial regression analysis, support the hypothesis of shared local factors affecting disease transmission in farms located at a close proximity.

  8. 牛结核病研究进展%Advances on Bovine Tuberculosis

    Institute of Scientific and Technical Information of China (English)

    付建; 王敏兰; 温荣辉

    2012-01-01

    Bovine tuberculosis (TB) is a zoonotic chronic infectious disease caused by Mycobacterium tuberculosis in cattle,and the epidemic has been taking advantage of the spread in recent years, so it not only has caused significant economic losses to China' s aquaculture industry, but also has affected the public health and safety. The epidemiology, etiology, pathogenesis, diagnostics and integrated control of bovine tuberculosis were reviewed in order to provide reference for related studies.%牛结核病是由牛分支杆菌引起的一种人畜共患慢性传染病,近年来该病的发病率有增长的趋势,不仅给我国的养殖业造成重大经济损失,而且影响到公共卫生安全。主要对牛结核病的流行病学、病原学、发病机制、诊断方法、综合防控等方面的研究进展作一介绍,以期为相关研究提供参考。

  9. Farm characteristics and farmer perceptions associated with bovine tuberculosis incidents in areas of emerging endemic spread.

    Science.gov (United States)

    Broughan, J M; Maye, D; Carmody, P; Brunton, L A; Ashton, A; Wint, W; Alexander, N; Naylor, R; Ward, K; Goodchild, A V; Hinchliffe, S; Eglin, R D; Upton, P; Nicholson, R; Enticott, G

    2016-07-01

    While much is known about the risk factors for bovine tuberculosis (bTB) in herds located in high incidence areas, the drivers of bTB spread in areas of emerging endemicity are less well established. Epidemiological analysis and intensive social research identified natural and social risk factors that may prevent or encourage the spread of disease. These were investigated using a case-control study design to survey farmers in areas defined as recently having become endemic for bTB (from or after 2006). Telephone surveys were conducted for 113 farms with a recent history of a bTB incident where their officially tuberculosis free status had been withdrawn (OTFW) (cases) and 224 controls with no history of a bTB incident, matched on location, production type and the rate of endemic bTB spread. Farmers were questioned about a range of farm management strategies, farm characteristics, herd health, wildlife and biosecurity measures with a focus on farmer attitudes and behaviours such as farmers' perception of endemicity and feelings of control, openness and social cohesion. Data generated in the telephone surveys was supplemented with existing herd-level data and analysed using conditional logistic regression. Overall, herd size (OR 1.07), purchasing an animal at a cattle market compared to purchasing outside of markets (OR 2.6), the number of contiguous bTB incidents (2.30) and the number of inconclusive reactors detected in the 2 years prior to the case incident (OR 1.95) significantly increased the odds of a bTB incident. Beef herds using a field parcel more than 3.2km away from the main farm and dairy herds reporting Johne's disease in the previous 12 months were 3.0 and 4.7 times more likely to have a recent history of a bTB incident, respectively. Beef herds reporting maize growing near, but not on, their farm were less likely to be case herds. Operating a closed farm in the two years prior to the case breakdown did not reduce the odds of a bTB incident. Farmers

  10. Systemic bovine tuberculosis: a case report/ Tuberculose bovina sistêmica: um relato de caso

    Directory of Open Access Journals (Sweden)

    Selwyn Arlington Headley

    2002-05-01

    Full Text Available Gross and histopathological lesions associated with Mycobacterium bovis in an ox are described. Differential diagnoses of frequently occurring gross lesions in cattle that could be easily confused with bovine tuberculosis are discussed, and a comparative analysis of M. bovis induced tuberculosis lesions in wildlife is made.Alterações macroscópicas e histopatológicas associadas a Mycobacterium bovis são descritas em um bovino. O diagnóstico diferencial de lesões macroscópicas mais freqüentemente encontradas em bovinos que podem ser confundidas com a tuberculose bovina é discutida. Uma análise comparativa de lesões induzidas por M. bovis em animais silvestres foi realizada.

  11. A preliminary study of genetic factors that influence susceptibility to bovine tuberculosis in the British cattle herd.

    Directory of Open Access Journals (Sweden)

    Erin E Driscoll

    Full Text Available Associations between specific host genes and susceptibility to Mycobacterial infections such as tuberculosis have been reported in several species. Bovine tuberculosis (bTB impacts greatly the UK cattle industry, yet genetic predispositions have yet to be identified. We therefore used a candidate gene approach to study 384 cattle of which 160 had reacted positively to an antigenic skin test ('reactors'. Our approach was unusual in that it used microsatellite markers, embraced high breed diversity and focused particularly on detecting genes showing heterozygote advantage, a mode of action often overlooked in SNP-based studies. A panel of neutral markers was used to control for population substructure and using a general linear model-based approach we were also able to control for age. We found that substructure was surprisingly weak and identified two genomic regions that were strongly associated with reactor status, identified by markers INRA111 and BMS2753. In general the strength of association detected tended to vary depending on whether age was included in the model. At INRA111 a single genotype appears strongly protective with an overall odds ratio of 2.2, the effect being consistent across nine diverse breeds. Our results suggest that breeding strategies could be devised that would appreciably increase genetic resistance of cattle to bTB (strictly, reduce the frequency of incidence of reactors with implications for the current debate concerning badger-culling.

  12. Continuous Age-Structured Model for Bovine Tuberculosis in African buffalo

    Science.gov (United States)

    Anguelov, R.; Kojouharov, H.

    2009-10-01

    The paper deals with a model of the spread of bovine tuberculosis in the buffalo population in the Kruger National Park in South Africa. The model uses continuous age structure and it is formulated in terms of partial differential equations using eight epidemiological classes (compartments). More precisely, the age density for each class at time t satisfies a one way wave equation, where the age is the space variable. The continuous age model discussed here is derived from a 2006 age groups model by P. C. Cross and W. M. Getz.

  13. Evaluation of adenosine deaminase seric activity in the diagnosis of bovine tuberculosis

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    Márcio Roberto Silva

    2006-06-01

    Full Text Available Determination of seric levels of adenosine deaminase (ADA, an enzyme produced by monocytes/macrophages and lymphocytes, has been used in the diagnosis of human tuberculosis (TB. In the present study, ADA seric activity was evaluated comparatively to the comparative tuberculin test in the diagnosis of bovine tuberculosis. Two hundred fifty-six cattle were classified by origin and by the comparative tuberculin test as TB-positive animals (n = 52, from herds where the Mycobacterium bovis had previously been isolated, and TB-negative animals (n = 204, TB-free herds. The mean ADA seric value from the TB-positive group (4.45 ± 2.33 U/L was significantly lower (p = 0.008 than that observed in sera from the TB-negative group (6.12 ± 4.47 U/L. When animals from a herd with clinical cases of enzootic bovine leukosis of TB-negative group were withdrawn from analysis, the mean ADA seric values of TB-negative group (5.12 ± 3.75 U/L was not significantly different anymore from that of the TB-positive group (p = 0.28. There was no agreement in the diagnosis of bovine TB between comparative tuberculin test and determination of ADA seric values, using two different cutoff points, being 6.12 U/L and 15.0 U/L, (kappa = -0.086 and kappa = -0.082, respectively. In conclusion, the determination of ADA seric activity was not a good auxiliary test for bovine TB, because it was not able to distinguish between TB-positive and TB-negative animals.

  14. Herd-Level Risk Factors for Bovine Tuberculosis: A Literature Review

    Directory of Open Access Journals (Sweden)

    Robin A. Skuce

    2012-01-01

    Full Text Available Bovine tuberculosis (TB, caused by Mycobacterium bovis, is one of the most challenging endemic diseases currently facing government, the veterinary profession, and the farming industry in the United Kingdom and Ireland and in several other countries. The disease has a notoriously complex epidemiology; the scientific evidence supports both cattle-cattle and wildlife-cattle transmission routes. To produce more effective ways of reducing such transmission, it is important to understand those risk factors which influence the presence or absence of bovine TB in cattle herds. Here we review the literature on herd-level risk factor studies. Whilst risk factors operate at different scales and may vary across regions, epidemiological studies have identified a number of risk factors associated with bovine TB herd breakdowns, including the purchase of cattle, the occurrence of bovine TB in contiguous herds, and/or the surrounding area as well as herd size. Other factors identified in some studies include farm and herd management practices, such as, the spreading of slurry, the use of certain housing types, farms having multiple premises, and the use of silage clamps. In general, the most consistently identified risk factors are biologically plausible and consistent with known transmission routes involving cattle-cattle and wildlife-cattle pathways.

  15. Rapid Reagentless Detection of M. tuberculosis H37Ra in Respiratory Effluents

    Energy Technology Data Exchange (ETDEWEB)

    Adams, K L; Steele, P T; Bogan, M J; Sadler, N M; Martin, S; Martin, A N; Frank, M

    2008-01-29

    Two similar mycobacteria, Mycobacteria tuberculosis H37Ra and Mycobacteria smegmatis are rapidly detected and identified within samples containing a complex background of respiratory effluents using Single Particle Aerosol Mass Spectrometry (SPAMS). M. tuberculosis H37Ra (TBa), an avirulent strain, is used as a surrogate for virulent tuberculosis (TBv); M. smegmatis (MSm) is utilized as a near neighbor confounder for TBa. Bovine lung surfactant and human exhaled breath condensate are used as first-order surrogates for infected human lung expirations from patients with pulmonary tuberculosis. This simulated background sputum is mixed with TBa or MSm and nebulized to produce conglomerate aerosol particles, single particles that contain a bacterium embedded within a background respiratory matrix. Mass spectra of single conglomerate particles exhibit ions associated with both respiratory effluents and mycobacteria. Spectral features distinguishing TBa from MSm in pure and conglomerate particles are shown. SPAMS pattern matching alarm algorithms are able to distinguish TBa containing particles from background matrix and MSm for >50% of the test particles, which is sufficient to enable a high probability of detection and a low false alarm rate if an adequate number of such particles are present. These results indicate the potential usefulness of SPAMS for rapid, reagentless tuberculosis screening.

  16. Validity of intradermal tuberculin testing for the screening of bovine tuberculosis in Madagascar.

    Science.gov (United States)

    Quirin, R; Rasolofo, V; Andriambololona, R; Ramboasolo, A; Rasolonavalona, T; Raharisolo, C; Rakotoaritahina, H; Chanteau, S; Boisier, P

    2001-09-01

    A sample survey with the objective of determining the prevalence of bovine tuberculosis by means of an intradermal tuberculin test was conducted in Madagascar and it was found that the prevalence rate varied from 0-30% by veterinary district. In order to estimate the true prevalence, the validity of the test was investigated by assessing its sensitivity and specificity in two groups of animals from two different regions, which were destined for slaughter. In the first group where the probability of non-infected animals should have been the highest, sensitivity was estimated at 0.52 (n = 21) and specificity at 0.99 (n = 79). In the second group selected on the basis of apparent ill health of the animals in a high-prevalence bovine tuberculosis area, sensitivity was estimated at 0.8 (n = 10) and specificity at 1 (n = 12). The results obtained from both groups of cattle were not combined for statistical purposes because the sensitivity of the skin test seemed to fluctuate in relation to the chronicity of the disease. These fluctuations are discussed. However, since the first group of zebu cattle was more representative of the cattle population across the country as a whole, its results were retained as operational parameters for further screening.

  17. Tuberculosis Detection in Paratuberculosis Vaccinated Calves: New Alternatives against Interference

    Science.gov (United States)

    Serrano, Miriam; Elguezabal, Natalia; Sevilla, Iker A.; Geijo, María V.; Molina, Elena; Arrazuria, Rakel; Urkitza, Alfonso; Jones, Gareth J.; Vordermeier, Martin; Garrido, Joseba M.; Juste, Ramón A.

    2017-01-01

    Paratuberculosis vaccination in cattle has been restricted due to its possible interference with the official diagnostic methods used in tuberculosis eradication programs. To overcome this drawback, new possibilities to detect Mycobacterium bovis infected cattle in paratuberculosis vaccinated animals were studied under experimental conditions. Three groups of 5 calves each were included in the experiment: one paratuberculosis vaccinated group, one paratuberculosis vaccinated and M. bovis infected group and one M. bovis infected group. The performance of the IFN-gamma release assay (IGRA) and the skin test using conventional avian and bovine tuberculins (A- and B-PPD) but also other more specific antigens (ESAT-6/CFP10 and Rv3615c) was studied under official and new diagnostic criteria. Regarding the IGRA of vaccinated groups, when A- and B-PPD were used the sensitivity reached 100% at the first post-challenge sampling, dropping down to 40–80% in subsequent samplings. The sensitivity for the specific antigens was 80–100% and the specificity was also improved. After adapting the diagnostic criteria for the conventional antigens in the skin test, the ability to differentiate between M. bovis infected and non-infected animals included in paratuberculosis vaccinated groups was enhanced. Taking for positive a relative skin thickness increase of at least 100%, the single intradermal test specificity and sensitivity yielded 100%. The comparative intradermal test was equally accurate considering a B-PPD relative skin increase of at least 100% and greater than or equal to that produced by A-PPD. Using the specific antigens as a proteic cocktail, the specificity and sensitivity reached 100% considering the new relative and absolute cut-offs in all experimental groups (Δ≥30% and Δmm ≥ 2, respectively). Results suggest that the interference caused by paratuberculosis vaccination in cattle could be completely overcome by applying new approaches to the official

  18. A Summary of Research on Bovine Tuberculosis Vaccines%牛结核病疫苗研究概述

    Institute of Scientific and Technical Information of China (English)

    谭伟; 谢志勤; 谢芝勋

    2014-01-01

    It is almost one hundred years since the Bacille Calmette Guerin (BCG)vaccine strain were isolated by Calmette and Guerin. BCG vaccine can be used for prevention of bovine tuberculosis. Research and development of bovine tuberculosis vaccines have been continuously conducted as the currently used BCG vaccines can only provide partial protection for cattle. In this review, we will introduce bovine tuberculosis vaccines that are currently in use, and major types, challenges and future directions of candidate bovine tuberculosis vaccines that are under development.%BCG疫苗株自Calmette和Guerin俩人分离出至今已有近百年的历史。BCG疫苗可用于预防牛结核病,但对牛的保护效果不完全,所以人们仍在不断地研发新型的牛结核病疫苗。本文将介绍目前正在使用及研发的牛结核病疫苗的主要类型、面临的挑战及今后的发展方向。

  19. Effect of culling and vaccination on bovine tuberculosis infection in a European badger (Meles meles) population by spatial simulation modelling

    NARCIS (Netherlands)

    Abdou, Marwa; Frankena, Klaas; O'Keeffe, James; Byrne, Andrew W.

    2016-01-01

    The control of bovine tuberculosis (bTB) in cattle herds in the Republic of Ireland (ROI) is partially hindered by spill-back infection from wild badgers (Meles meles). The aim of this study was to determine the relative effects of interventions (combinations of culling and/or vaccination) on bTB

  20. Prevalence of bovine tuberculosis in a dairy cattle farm and a research farm in Ghana

    Directory of Open Access Journals (Sweden)

    Adwoa Asante-Poku

    2014-04-01

    Full Text Available The aim of the study was to estimate the prevalence of bovine tuberculosis (BTB and to identify the mycobacterial species causing BTB in a dairy farm and research farm. Six hundred and eighty-five cattle were screened for BTB by using the Comparative intradermal tuberculin test (CTT. Positive reactors were slaughtered and carcasses were taken for isolation of mycobacterial species. This was followed by speciation of isolates using both standard conventional and molecular assays. Seventeen of the cattle were positive by CTT, giving a crude BTB prevalence of 2.48% among cattle from the two farms. Six of the 17 samples (35.30% yielded positive acid-fast bacilli cultures and three of the isolates were identified as Mycobacterium tuberculosis complex (MTBC, which were sub-divided into two Mycobacterium tuberculosis sensu scrito (Mtb and one Mycobacterium africanum; the remaining three were Mycobacterium other than tuberculoisis (MOTT. Spoligotyping further characterised the two Mtb isolates as Ghana (spoligotype Data Base 4 number 53 and Latin American Mediterranean (LAM, whilst spoligotyping and Single Nucleotide Polymorphism (SNP analysis typed the M. africanum as West African 1. Microseq 500 analysis identified two of the MOTT as Mycobacterium flavescens and Mycobacterium Moriokaense respectively, whilst the remaining one could not be identified. This study observed the prevalence of bovine TB among cattle from two farms in Ghana as 2.48% and confirms the public health importance of M. africanum as a pathogen in Ghana. 

  1. Epidemiological Investigation of Bovine Tuberculosis Herd Breakdowns in Spain 2009/2011

    Science.gov (United States)

    Guta, Sintayehu; Casal, Jordi; Napp, Sebastian; Saez, Jose Luis; Garcia-Saenz, Ariadna; Perez de Val, Bernat; Romero, Beatriz; Alvarez, Julio; Allepuz, Alberto

    2014-01-01

    We analyzed the most likely cause of 687 bovine tuberculosis (bTB) breakdowns detected in Spain between 2009 and 2011 (i.e., 22% of the total number of breakdowns detected during this period). Seven possible causes were considered: i) residual infection; ii) introduction of infected cattle from other herds; iii) sharing of pastures with infected herds; iv) contiguous spread from infected neighbor herds; v) presence of infected goats in the farm; vi) interaction with wildlife reservoirs and vii) contact with an infected human. For each possible cause a decision tree was developed and key questions were included in each of them. Answers to these key questions lead to different events within each decision tree. In order to assess the likelihood of occurrence of the different events a qualitative risk assessment approach was used. For this purpose, an expert opinion workshop was organized and ordinal values, ranging from 0 to 9 (i.e., null to very high likelihood of occurrence) were assigned. The analysis identified residual infection as the most frequent cause of bTB breakdowns (22.3%; 95%CI: 19.4–25.6), followed by interaction with wildlife reservoirs (13.1%; 95%CI: 10.8–15.8). The introduction of infected cattle, sharing of pastures and contiguous spread from infected neighbour herds were also identified as relevant causes. In 41.6% (95%CI: 38.0–45.4) of the breakdowns the origin of infection remained unknown. Veterinary officers conducting bTB breakdown investigations have to state their opinion about the possible cause of each breakdown. Comparison between the results of our analysis and the opinion from veterinary officers revealed a slight concordance. This slight agreement might reflect a lack of harmonized criteria to assess the most likely cause of bTB breakdowns as well as different perceptions about the importance of the possible causes. This is especially relevant in the case of the role of wildlife reservoirs. PMID:25127254

  2. Epidemiological tracing of bovine tuberculosis in Switzerland, multilocus variable number of tandem repeat analysis of Mycobacterium bovis and Mycobacterium caprae

    Science.gov (United States)

    Scherrer, Simone; Friedel, Ute; Frei, Daniel; Suter, Dominique; Perler, Lukas; Wittenbrink, Max M.

    2017-01-01

    Background After 15 years of absence, in 2013 bovine tuberculosis (bTB), caused by Mycobacterium (M.) bovis and M. caprae, reemerged in the Swiss dairy cattle population. In order to identify the sources of infection as well as the spread of the agents, molecular-epidemiologic tracing by MIRU-VNTR analysis in combination with spoligotyping was performed. A total of 17 M. bovis and 7 M. caprae isolates were cultured from tuberculous bovine lymph nodes and analyzed with a set of 49 genetic markers by using automated capillary electrophoresis. Results The outbreak in the western part of Switzerland was caused by M. bovis spoligotype SB0120. With the exception of four single-locus variations observed in MIRU 20, the MIRU-VNTR profiles of the 17 M. bovis isolates were identical, indicating a single source of infection. M. bovis detected in one archival bovine specimen from the outbreak region showed an identical MIRU-VNTR profile, suggesting persistence of the agent in a dairy herd for nearly fifteen years. The outbreak in the eastern part of Switzerland was caused by M. caprae spoligotype SB0418. All Swiss M. caprae isolates showed the Lechtal-type MIRU-VNTR profile, described as endemic in wild ruminants and in dairy cattle in Austrian bordering regions. This suggests the agent was most likely introduced by Swiss dairy cattle summering on Austrian pastures. Conclusions The present study is the first MIRU-VNTR analysis of Swiss bTB mycobacterial isolates. The genotyping assay was found to be highly discriminating and suitable for the epidemiological tracing of further outbreaks. These findings will contribute to the development of an international MIRU-VNTR database aiming to improve bTB surveillance. PMID:28222182

  3. Severity of bovine tuberculosis is associated with co-infection with common pathogens in wild boar.

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    David Risco

    Full Text Available Co-infections with parasites or viruses drive tuberculosis dynamics in humans, but little is known about their effects in other non-human hosts. This work aims to investigate the relationship between Mycobacterium bovis infection and other pathogens in wild boar (Sus scrofa, a recognized reservoir of bovine tuberculosis (bTB in Mediterranean ecosystems. For this purpose, it has been assessed whether contacts with common concomitant pathogens are associated with the development of severe bTB lesions in 165 wild boar from mid-western Spain. The presence of bTB lesions affecting only one anatomic location (cervical lymph nodes, or more severe patterns affecting more than one location (mainly cervical lymph nodes and lungs, was assessed in infected animals. In addition, the existence of contacts with other pathogens such as porcine circovirus type 2 (PCV2, Aujeszky's disease virus (ADV, swine influenza virus, porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and Metastrongylus spp, was evaluated by means of serological, microbiological and parasitological techniques. The existence of contacts with a structured community of pathogens in wild boar infected by M. bovis was statistically investigated by null models. Association between this community of pathogens and bTB severity was examined using a Partial Least Squares regression approach. Results showed that adult wild boar infected by M. bovis had contacted with some specific, non-random pathogen combinations. Contact with PCV2, ADV and infection by Metastrongylus spp, was positively correlated to tuberculosis severity. Therefore, measures against these concomitant pathogens such as vaccination or deworming, might be useful in tuberculosis control programmes in the wild boar. However, given the unexpected consequences of altering any community of organisms, further research should evaluate the impact of such measures

  4. Severity of Bovine Tuberculosis Is Associated with Co-Infection with Common Pathogens in Wild Boar

    Science.gov (United States)

    Risco, David; Serrano, Emmanuel; Fernández-Llario, Pedro; Cuesta, Jesús M.; Gonçalves, Pilar; García-Jiménez, Waldo L.; Martínez, Remigio; Cerrato, Rosario; Velarde, Roser; Gómez, Luis; Segalés, Joaquím; Hermoso de Mendoza, Javier

    2014-01-01

    Co-infections with parasites or viruses drive tuberculosis dynamics in humans, but little is known about their effects in other non-human hosts. This work aims to investigate the relationship between Mycobacterium bovis infection and other pathogens in wild boar (Sus scrofa), a recognized reservoir of bovine tuberculosis (bTB) in Mediterranean ecosystems. For this purpose, it has been assessed whether contacts with common concomitant pathogens are associated with the development of severe bTB lesions in 165 wild boar from mid-western Spain. The presence of bTB lesions affecting only one anatomic location (cervical lymph nodes), or more severe patterns affecting more than one location (mainly cervical lymph nodes and lungs), was assessed in infected animals. In addition, the existence of contacts with other pathogens such as porcine circovirus type 2 (PCV2), Aujeszky's disease virus (ADV), swine influenza virus, porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and Metastrongylus spp, was evaluated by means of serological, microbiological and parasitological techniques. The existence of contacts with a structured community of pathogens in wild boar infected by M. bovis was statistically investigated by null models. Association between this community of pathogens and bTB severity was examined using a Partial Least Squares regression approach. Results showed that adult wild boar infected by M. bovis had contacted with some specific, non-random pathogen combinations. Contact with PCV2, ADV and infection by Metastrongylus spp, was positively correlated to tuberculosis severity. Therefore, measures against these concomitant pathogens such as vaccination or deworming, might be useful in tuberculosis control programmes in the wild boar. However, given the unexpected consequences of altering any community of organisms, further research should evaluate the impact of such measures under

  5. Cultural and molecular detection of zoonotic tuberculosis and its ...

    African Journals Online (AJOL)

    Cultural and molecular detection of zoonotic tuberculosis and its public health impacts in selected districts of Tigray region, Ethiopia. ... Besides, livestock owners were interviewed for the evaluation of the zoonotic potential of BTB. On the basis ...

  6. DETECTION OF THE BOVINE VIRAL DIARRHEA ANTIBODIES

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    I. V. Goraichuk

    2013-06-01

    Full Text Available Bovine viral diarrhea is a widespread infection of cattle that has a wide range of clinical symptoms in domestic and wild ruminants. It is a major problem in cattle and causes significant economic losses in the cattle industry. The virus infects bovines of all ages and causes both immunosuppression and reproductive, respiratory and digestive disorders. Persistently infected cattle are the main factor in transmission of the disease between and among herds. Comparative results of antibodies presence received by two methods of enzymoimmunoassay and virus neutralization test are given in the paper. During the work, 1010 samples of blood serum of cattle from three farms in the Kharkiv region were selected and analyzed. Bovine viral diarrhea virus concerning antibodies were found by enzymoimmunoassay in 704 samples (69.7% using commercial kit and in 690 samples (68.3% using in house method. After results clarification by virus neutralization test, bovine viral diarrhea antibodies were found in 712 samples (70.5%. Immunoenzyme analysis is recommended for mass screening of cattle for viral diarrhea occurrence. The results confirm that the sensitivity immunoenzyme analysis satisfies the requirements of the diagnostic methods. Using the neutralization reaction of viruses as the «gold standard» of serological methods, it is appropriate to clarify the results of immunoenzyme analysis. Since the results contain a signi ficant number of false positive results, it is necessary to carry out comprehensive studies using both serological and molecular genetics methods.

  7. Biosensor-based detection of tuberculosis

    NARCIS (Netherlands)

    Srivastava, Saurabh K.; Rijn, Van Cees J.M.; Jongsma, Maarten A.

    2016-01-01

    Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb.), is one of the most prevalent and serious infectious diseases worldwide with an estimated annual global mortality of 1.4 million in 2010. Diagnosis of TB in the developing world is very challenging due to the limited suitability of c

  8. Non-tuberculous Mycobacteria in South African Wildlife: Neglected Pathogens and Potential Impediments for Bovine Tuberculosis Diagnosis

    Science.gov (United States)

    Gcebe, Nomakorinte; Hlokwe, Tiny M.

    2017-01-01

    Non-tuberculous mycobacteria (NTM) are not only emerging and opportunistic pathogens of both humans and animals, but from a veterinary point of view some species induce cross-reactive immune responses that hamper the diagnosis of bovine tuberculosis (bTB) in both livestock and wildlife. Little information is available about NTM species circulating in wildlife species of South Africa. In this study, we determined the diversity of NTM isolated from wildlife species from South Africa as well as Botswana. Thirty known NTM species and subspecies, as well as unidentified NTM, and NTM closely related to Mycobacterium goodii/Mycobacterium smegmatis were identified from 102 isolates cultured between the years 1998 and 2010, using a combination of molecular assays viz PCR and sequencing of different Mycobacterial house-keeping genes as well as single nucleotide polymorphism (SNP) analysis. The NTM identified in this study include the following species which were isolated from tissue with tuberculosis- like lesions in the absence of Mycobacterium tuberculosis complex (MTBC) implying their potential role as pathogens of animals: Mycobacterium abscessus subsp. bolletii, Mycobacterium gastri, Mycobacterium species closely related to Mycobacterium goodii/Mycobacterium smegmatis, Mycobacterium brasiliensis, Mycobacterium sinense JMD 601, Mycobacterium avium subsp. avium, Mycobacterium sp. GR-2007, Mycobacterium bouchedurhonense, and Mycobacterium septicum/M. peregrinum. Mycobaterium brasiliensis, Mycobacterium gastri, Mycobacterium sp. GR-2007, and a potential novel Mycobacterium species closely related to Mycobacterium goodii were found for the first time in this study to be potential pathogens of animals. Mycobacterium simiae was isolated from a sample originating from a tuberculin skin test positive reactor, demonstrating its potential to elicit inappropriate immune responses in animals that may interfere with diagnosis of tuberculosis by immunology. Mycobacterium abscessus

  9. Bovine tuberculosis in livestock and wild boar on the Mediterranean island, Corsica.

    Science.gov (United States)

    Richomme, Céline; Boschiroli, María Laura; Hars, Jean; Casabianca, François; Ducrot, Christian

    2010-04-01

    The zoonotic agent of bovine tuberculosis (bTB), Mycobacterium bovis, can be transmitted between domestic and wild animals, threatening wildlife populations and control programs for bTB in cattle. In Corsica, a French Mediterranean island where domestic and wild species have close interactions, bTB cases have been reported in cattle, pigs, and wild boar. Moreover, genotypes of M. bovis found in wild and domestic animals from the same area were identical. These data strongly suggest that wild and domestic animals are associated in an epidemiologic bTB-transmission cycle. More investigations are needed, not only to understand the role played by each species in order to implement appropriate control measures, but also to assess the risk of transmission to humans.

  10. Descriptive Epidemiology of Bovine Tuberculosis in Michigan (1975–2010: Lessons Learned

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    Chika C. Okafor

    2011-01-01

    Full Text Available Despite ongoing eradication efforts, bovine tuberculosis (BTB remains a challenge in Michigan livestock and wildlife. The objectives of this study were to (1 review the epidemiology of BTB in Michigan cattle, privately owned cervids, and wildlife between 1975 and 2010 and (2 identify important lessons learned from the review and eradication strategies. BTB information was accessed from the Michigan BTB Eradication Project agencies. Cattle herds (49, privately owned deer herds (4, and wild white-tailed deer (668 were found infected with BTB during the review period. BTB has occurred primarily in counties located at the northern portion of the state's Lower Peninsula. Currently used BTB eradication strategies have successfully controlled BTB spread. However additional changes in BTB surveillance, prevention, and eradication strategies could improve eradication efforts.

  11. The Case for Live Attenuated Vaccines against the Neglected Zoonotic Diseases Brucellosis and Bovine Tuberculosis

    Science.gov (United States)

    Pandey, Aseem; Cabello, Ana; Akoolo, Lavoisier; Rice-Ficht, Allison; Arenas-Gamboa, Angela; McMurray, David; Ficht, Thomas A.; de Figueiredo, Paul

    2016-01-01

    Vaccination of humans and animals with live attenuated organisms has proven to be an effective means of combatting some important infectious diseases. In fact, the 20th century witnessed tremendous improvements in human and animal health worldwide as a consequence of large-scale vaccination programs with live attenuated vaccines (LAVs). Here, we use the neglected zoonotic diseases brucellosis and bovine tuberculosis (BTb) caused by Brucella spp. and Mycobacterium bovis (M. bovis), respectively, as comparative models to outline the merits of LAV platforms with emphasis on molecular strategies that have been pursued to generate LAVs with enhanced vaccine safety and efficacy profiles. Finally, we discuss the prospects of LAV platforms in the fight against brucellosis and BTb and outline new avenues for future research towards developing effective vaccines using LAV platforms. PMID:27537413

  12. Descriptive epidemiology of bovine tuberculosis in michigan (1975-2010): lessons learned.

    Science.gov (United States)

    Okafor, Chika C; Grooms, Daniel L; Bruning-Fann, Colleen S; Averill, James J; Kaneene, John B

    2011-01-01

    Despite ongoing eradication efforts, bovine tuberculosis (BTB) remains a challenge in Michigan livestock and wildlife. The objectives of this study were to (1) review the epidemiology of BTB in Michigan cattle, privately owned cervids, and wildlife between 1975 and 2010 and (2) identify important lessons learned from the review and eradication strategies. BTB information was accessed from the Michigan BTB Eradication Project agencies. Cattle herds (49), privately owned deer herds (4), and wild white-tailed deer (668) were found infected with BTB during the review period. BTB has occurred primarily in counties located at the northern portion of the state's Lower Peninsula. Currently used BTB eradication strategies have successfully controlled BTB spread. However additional changes in BTB surveillance, prevention, and eradication strategies could improve eradication efforts.

  13. Bovine tuberculosis: prevalence and diagnostic efficacy of routine meat inspection procedure in Woldiya municipality abattoir north Wollo zone, Ethiopia.

    Science.gov (United States)

    Aylate, Alemu; Shah, Shahid Nazir; Aleme, Haileluel; Gizaw, Tarkegn Tintagu

    2013-03-01

    Bovine tuberculosis (BTB) is a widespread and endemic disease of cattle in Ethiopia posing a significant threat to public health. Regular surveillance by skin test, bacteriology, and molecular methods is not feasible due to lack of resources. Thus, routine abattoir (RA) inspection will continue to play a key role for national surveillance. A cross-sectional study was conducted at Woldiya municipal abattoir from April 1, 2009 to April 5, 2010 to estimate the prevalence of BTB in slaughtered cattle on the basis of detailed abattoir inspection and to compare efficacy of RA inspection with respect to detailed abattoir inspection and isolation and identification of Mycobacterium. Diagnostic accuracies (with corresponding measures of statistical uncertainty) were determined by computing test property statistics (sensitivity and specificity). Agreement between RA and detailed abattoir inspections was measured using kappa statistics. Out of 1,029 slaughtered heads of cattle examined during the study period, 63 (6.12 %) and 15 (1.45 %) were diagnosed with gross tuberculous lesions by detailed abattoir meat inspections and RA meat inspections, respectively, making a prevalence of 6.12 % (95 % CI: 5.2-7.1) on the basis of detailed abattoir inspection. About 59.45 % of tuberculous lesions were observed in the lungs and associated lymph nodes, whereas 35.13 % lesions were from the lymph nodes of the head. From 63 cattle suspected with tuberculosis (TB) based on detailed abattoir meat inspection, nine (19.05 %) were identified as Mycobacterium bovis, while three (4.8 %) as Mycobacterium tuberculosis. The sensitivity of RA meat inspection was 23.8 % in comparison to the detailed abattoir meat inspection and 25 % in comparison to culture, respectively. Poor agreement (k = 0.37) was seen between RA meat examination and detailed abattoir meat examination methods. Similarly, poor agreement (k = 0.013) was seen between RA meat examination and culture results. In

  14. Surveillance of bovine tuberculosis and risk estimation of a future reservoir formation in wildlife in Switzerland and Liechtenstein.

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    Janne Marie Schöning

    Full Text Available Bovine tuberculosis (bTB caused by Mycobacterium bovis or M. caprae has recently (re- emerged in livestock and wildlife in all countries bordering Switzerland (CH and the Principality of Liechtenstein (FL. Comprehensive data for Swiss and Liechtenstein wildlife are not available so far, although two native species, wild boar (Sus scrofa and red deer (Cervus elaphus elaphus, act as bTB reservoirs elsewhere in continental Europe. Our aims were (1 to assess the occurrence of bTB in these wild ungulates in CH/FL and to reinforce scanning surveillance in all wild mammals; (2 to evaluate the risk of a future bTB reservoir formation in wild boar and red deer in CH/FL. Tissue samples collected from 2009 to 2011 from 434 hunted red deer and wild boar and from eight diseased ungulates with tuberculosis-like lesions were tested by direct real-time PCR and culture to detect mycobacteria of the Mycobacterium tuberculosis complex (MTBC. Identification of suspicious colonies was attempted by real-time PCR, genotyping and spoligotyping. Information on risk factors for bTB maintenance within wildlife populations was retrieved from the literature and the situation regarding identified factors was assessed for our study areas. Mycobacteria of the MTBC were detected in six out of 165 wild boar (3.6%; 95% CI: 1.4-7.8 but none of the 269 red deer (0%; 0-1.4. M. microti was identified in two MTBC-positive wild boar, while species identification remained unsuccessful in four cases. Main risk factors for bTB maintenance worldwide, including different causes of aggregation often resulting from intensive wildlife management, are largely absent in CH and FL. In conclusion, M. bovis and M. caprae were not detected but we report for the first time MTBC mycobacteria in Swiss wild boar. Present conditions seem unfavorable for a reservoir emergence, nevertheless increasing population numbers of wild ungulates and offal consumption may represent a risk.

  15. Lessons learned during the successful eradication of bovine tuberculosis from Australia

    Science.gov (United States)

    More, S. J.; Radunz, B.; Glanville, R. J.

    2015-01-01

    There are very few international examples of the successful eradication of bovine tuberculosis (TB, caused by infection with Mycobacterium bovis) from a national cattle population. This paper presents a brief overview of the successful TB eradication programme in Australia from 1970, with primary emphasis on lessons of international relevance that were learned from the Australian experience. The national brucellosis and tuberculosis eradication campaign ran for 27 years from 1970 to 1997 and has been followed by ongoing abattoir surveillance. Rapid progress towards eradication was made in southern Australia, but proved much more challenging in extensive pastoral areas of northern Australia. Declaration of TB freedom was made on December 31, 1997. A range of factors were critical to this success, including a compelling rationale for eradication, an agreed final outcome, industry commitment and financial support, a business model for programme planning, implementation and review, consistent and transparent technical standards underpinned by a strict regulatory regime and applied research, the critical role of abattoir surveillance, effective elimination of residual infection and objective measures of programme progress. Although direct translation of some of these experiences may not be possible, many of the lessons learned from the Australian experience may be relevant to other countries. PMID:26338937

  16. Analysis of breath volatile organic compounds as a screening tool for detection of Tuberculosis in cattle

    Science.gov (United States)

    • Keywords: bovine tuberculosis; Mycobacterium bovis; breath analysis; volatile organic compound; gas chromatography; mass spectrometry; NaNose • Introduction: This presentation describes two studies exploring the use of breath VOCs to identify Mycobacterium bovis infection in cattle. • Methods: ...

  17. Local cattle movements in response to ongoing bovine tuberculosis zonation and regulations in Michigan, USA.

    Science.gov (United States)

    Grear, Daniel A; Kaneene, John B; Averill, James J; Webb, Colleen T

    2014-06-01

    Bovine tuberculosis (Mycobacterium bovis) is an ongoing management issue in the state of Michigan with eradication from livestock as the ultimate goal. Eradication has been a challenge owing to the presence of a wildlife reservoir; competing interests in managing the livestock and wildlife hosts; and many uncertainties in transmission dynamics of M. bovis. One of the cornerstones of the eradication effort has been to stop movement of infected cattle among farms by imposing strict pre-movement testing on cattle being moved within, into and out of the Modified Accredited Zone (MAZ) in northeastern Michigan. In addition to pre-movement tuberculosis testing, detailed information about the origin and destination premises of all movements within the MAZ has been recorded in Michigan. The aim of this study was to describe the farm-to-farm movements of cattle within the MAZ, report changes in the network of movements during a 6-year period when the MAZ was a constant size (2004-2009), and examine changes in cattle movement patterns when the MAZ was reduced from 11 to 5 counties in 2010. Non-slaughter cattle movement within the MAZ was characterized by predominantly local movements at a sub-county scale. Premises that shipped cattle were primarily senders or receivers, but rarely both. From 2004 to 2009, the number of cattle shipped, size of shipments, number of shipments and distance of shipments decreased; there was little change in the network patterns of interaction among individual premises; and interactions among all premises became more disconnected. After accounting for MAZ size, there were also no changes in cattle movement network patterns following the reduction of the MAZ in 2010. The movement of cattle was likely not a key risk factor in bTB spread among premises in the MAZ during the study period and the effect of zonation and movement regulations appeared to further reduce the risk of tuberculosis spread via cattle movements among farms in Michigan's MAZ.

  18. DETECTION OF MYCOBACTERIUM TUBERCULOSIS IN BLOOD FOR DIAGNOSIS OF GENERALISED TUBERCULOSIS IN HIV-POSITIVE PATIENTS

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    V. N. Zimina

    2017-01-01

    Full Text Available Objective: To study the informative value of the detection of mycobacteria in blood with the cultural method in patients with suspected tuberculous sepsis and to determine the most significant clinical and laboratory criteria for testing. Materials and methods: The investigation to detect M.tuberculosis was fulfilled in 159 HIV-positive patients with suspected tuberculosis sepsis. Blood culture was completed with culture medium Myco/F Lytic Culture Vials and analyzer BACTEC 9050. Results: Mycobacteria were detected in blood of 19 patients (11,9% of all patients: in 18 patients the growth of М. tuberculosis complex was detected (25,3% of all patients with diagnosed tuberculosis and in 1 patient it was Mycobacterium avium complex (0,6% of all patients. It was shown, that the probability of M.tuberculosis detection was especially associated with the severity of the disease, immunosupression (less than 100 cells/mkl, hemoglobin quantity less than 90 g/l (levels were determined through the seeking for the most significant cutoffs. It was not proofed, that meningoencephalitis develops more often in patients with proven bacteremia. There were no evident differences in detection frequency of mycobacteria in sputum between patients with tuberculous sepsis and without it.

  19. Disease, predation and demography: Assessing the impacts of bovine tuberculosis on African buffalo by monitoring at individual and population levels

    Science.gov (United States)

    Cross, P.C.; Heisey, D.M.; Bowers, J.A.; Hay, C.T.; Wolhuter, J.; Buss, P.; Hofmeyr, M.; Michel, A.L.; Bengis, Roy G.; Bird, T.L.F.; du Toit, Johan T.; Getz, W.M.

    2009-01-01

    1. Understanding the effects of disease is critical to determining appropriate management responses, but estimating those effects in wildlife species is challenging. We used bovine tuberculosis (BTB) in the African buffalo Syncerus caffer population of Kruger National Park, South Africa, as a case study to highlight the issues associated with estimating chronic disease effects in a long-lived host. 2. We used known and radiocollared buffalo, aerial census data, and a natural gradient in pathogen prevalence to investigate if: (i) at the individual level, BTB infection reduces reproduction; (ii) BTB infection increases vulnerability to predation; and (iii) at the population level, increased BTB prevalence causes reduced population growth. 3. There was only a marginal reduction in calving success associated with BTB infection, as indexed by the probability of sighting a known adult female with or without a calf (P = 0??065). 4. Since 1991, BTB prevalence increased from 27 to 45% in the southern region and from 4 to 28% in the central region of Kruger National Park. The prevalence in the northern regions was only 1??5% in 1998. Buffalo population growth rates, however, were neither statistically different among regions nor declining over time. 5. Lions Panthera leo did not appear to preferentially kill test-positive buffalo. The best (Akaike's Information Criterion corrected for small sample size) AICc model with BTB as a covariate [exp(??) = 0??49; 95% CI = (0??24-1??02)] suggested that the mortality hazard for positive individuals was no greater than for test-negative individuals. 6. Synthesis and applications. Test accuracy, time-varying disease status, and movement among populations are some of the issues that make the detection of chronic disease impacts challenging. For these reasons, the demographic impacts of bovine tuberculosis in the Kruger National Park remain undetectable despite 6 years of study on known individuals and 40 years of population counts

  20. The bovine tuberculosis burden in cattle herds in zones with low dose radiation pollution in Ukraine

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    Svitlana Pozmogova

    2009-06-01

    Full Text Available The authors describe a study of the tuberculosis (TB incidence in cattle exposed to low doses of radiation resulting from the Chernobyl (pronounced ‘Chornobyl’ in Ukrainian nuclear plant catastrophe in 1986. The purpose of the study was to determine if ionising radiation influences the number of outbreaks of bovine TB and their severity on farms in the Kyiv, Cherkasy and Chernigiv regions of Ukraine. These farms are all located within a 200 km radius of Chernobyl and have had low-dose radiation pollution. Pathological and blood samples were taken from cattle in those regions that had positive TB skin tests. Mycobacterium spp. were isolated, differentiated by PCR, analysed and tested in guinea-pigs and rabbits. Species differentiation showed a significant percentage of atypical mycobacteria, which resulted in the allergic reactions to tuberculin antigen in the skin test. Mixed infection of M. bovis and M. avium subsp. hominissuis was found in three cases. The results concluded that low-dose radiation plays a major role in the occurrence of bovine TB in regions affected by the Chernobyl nuclear disaster.

  1. Assessing the histopathology to depict the different stages of bovine tuberculosis infection in a naturally infected herd

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    Luciana S. Medeiros

    2012-02-01

    Full Text Available The standard method for detection of bovine tuberculosis (TB is the single intradermal tuberculin test (SITT. Nevertheless, current studies suggest that a single test is not enough to detect all cattle infected by TB, particularly when animals present different stages of infection. A dairy herd comprised of 270 cows was studied and 15 were reactive to SITT plus nine inconclusive animals. Blood samples (for IFN and ELISA were collected from these 24 cows. At 30 days after injection of PPD, all the cows that were reactive to any of the employed tests were slaughtered, and tissues were processed by Bacteriology, Histopathology (HP and PCR. According to HP 33.4% of the animals were positive, 45.8% inconclusive and 20.8% were negative. The inconclusive samples came from IFN positive animals, signalizing recent infection. Regarding the animals that were negative to HP, all of them were identified by IFN while ELISA was negative. Immune responses are different in recent and advanced infections, what supports the identification between chronically or recently infected animals. This multidisciplinary approach is mandatory for the interpretation of the various tools that are frequently employed for the diagnosis of TB and mainly to identify all infected animals.

  2. Tuberculosis

    Directory of Open Access Journals (Sweden)

    Elena Morán López

    2001-04-01

    infested individual to a healthy subject mainly by means of saliva containing these microorganisms, or indirectly by inhaling the bacillus which may be present in daily used objects for months due to its high resistance. Myobacteria causing tuberculosis in the immunocompetent man are tuberculosis and bovis whereas other types may produce tuberculosis in immunocompromised individuals. The pathogenecity of this bacillus is related to its capacity of escaping from macrophage-induced destruction and provoking retarded hypersensitivity. This disease has very few oral manifestations; in general, a sore mainly located in the back of the tongue is the only observed sign. Tuberculosis threatens to become an incurable disease because of the poor administration of anti-tuberculosis programs, that is why, WHO proposes DOTS (directly observed treatment of short duration for its detection and treatment. This programs begins to achieve satisfactory results, although in the last five-year period, 88% of the patients estimated to be tuberculosis-infested was not covered by DOTS.

  3. Display of Antigens on Polyester Inclusions Lowers the Antigen Concentration Required for a Bovine Tuberculosis Skin Test.

    Science.gov (United States)

    Parlane, Natalie A; Chen, Shuxiong; Jones, Gareth J; Vordermeier, H Martin; Wedlock, D Neil; Rehm, Bernd H A; Buddle, Bryce M

    2015-10-28

    The tuberculin skin test is the primary screening test for the diagnosis of bovine tuberculosis (TB), and use of this test has been very valuable in the control of this disease in many countries. However, the test lacks specificity when cattle have been exposed to environmental mycobacteria or vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG). Recent studies showed that the use of three or four recombinant mycobacterial proteins, including 6-kDa early secretory antigenic target (ESAT6), 10-kDa culture filtrate protein (CFP10), Rv3615c, and Rv3020c, or a peptide cocktail derived from those proteins, in the skin test greatly enhanced test specificity, with minimal loss of test sensitivity. The proteins are present in members of the pathogenic Mycobacterium tuberculosis complex but are absent in or not expressed by the majority of environmental mycobacteria and the BCG vaccine strain. To produce a low-cost skin test reagent, the proteins were displayed at high density on polyester beads through translational fusion to a polyhydroxyalkanoate synthase that mediates the formation of antigen-displaying inclusions in recombinant Escherichia coli. Display of the proteins on the polyester beads greatly increased their immunogenicity, allowing for the use of very low concentrations of proteins (0.1 to 3 μg of mycobacterial protein/inoculum) in the skin test. Polyester beads simultaneously displaying all four proteins were produced in a single fermentation process. The polyester beads displaying three or four mycobacterial proteins were shown to have high sensitivity for detection of M. bovis-infected cattle and induced minimal responses in animals exposed to environmental mycobacteria or vaccinated with BCG.

  4. [Detection of mycobacteria tuberculosis in patients with urogenital tuberculosis by PCR method].

    Science.gov (United States)

    Dochviri, T Z; Katsitadze, V A; Khosiashvili, G Z; Chigogidze, T G

    2005-02-01

    The study was carried out in hospital patients as well as in outpatients at the National Centre of Tuberculosis and Lung Diseases of Georgia (2002-2004). The group consisting of 32 patients with tuberculosis of urogenital system has been studied (newly detected forms). Except clinical laboratory, culture and X-ray contrast methods, two additional methods were used in testing of this group of patients. The examination of their urine, at the same time, was carried out by the Polymerase Chain Reaction method in order to detect Kochi bacillus and by three-time bacterioscopy of urine for acid resistant bacteria. Mycobacterium tuberculosis in urine has been detected in 26 (81,25%) patients by PCR method, and by urine bacterioscopy--acid fast bacilli (AFB+) in 18 (56,25%) patients. The histo-morphological investigation of specimens obtained by surgery confirmed the TB diagnosis in all patients. This study on patients suspected of Tuberculosis of genital-urinary system gives us an opportunity to update the diagnostic algorithm by including the modern molecular methods. This algorithm will help in timely detection of Tuberculosis, in selection of adequate therapy and in prevention of the further progression of the disease.

  5. Exploring the use of molecular epidemiology to track bovine tuberculosis in Nigeria: an overview from 2002 to 2004.

    Science.gov (United States)

    Cadmus, S I B; Gordon, S V; Hewinson, R G; Smith, N H

    2011-07-05

    Tuberculosis remains a major public health problem in Nigeria. While human to human transmission of Mycobacterium tuberculosis is clearly of major importance in driving the tuberculosis epidemic in Nigeria, the impact of Mycobacterium bovis transmission from infected cattle is largely unknown. Molecular epidemiology of M. bovis in Nigeria will increase our understanding of this endemic disease and provide tools to assess cattle-to-human transmission. Between 2002 and 2004, molecular techniques including spoligotyping, variable number of tandem repeats (VNTR) typing and deletion typing were used to track and analyze a sample of strains of the M. tuberculosis complex circulating in the cattle population in Ibadan, Southwestern Nigeria. In all, 180 isolates were typed with a view to elucidating epidemiological information on circulating strains, occurrence of transborder transmission and molecular diversity of the M. bovis strains. Results obtained showed that 99% (178/180) of the isolates were M. bovis, while the remaining were M. tuberculosis and M. africanum. In all, strains of M. bovis had 34 different spoligotypes: strains with spoligotype pattern SB0944 (as designated by www.mbovis.org) were the most common (46% of strains). This molecular type is also common in countries neighbouring Nigeria. Strains with this spoligotype pattern could be further divided into 40 different VNTR types. This analysis shows the value of simple molecular epidemiological techniques applied to strains of M. bovis and suggests that further epidemiological studies will shed more light on the transmission dynamics of bovine tuberculosis locally and across neighbouring African countries.

  6. Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk

    NARCIS (Netherlands)

    Wellenberg, G.J.; Verstraten, E.; Belak, S.; Verschuren, S.B.E.; Rijsewijk, F.A.M.; Peshev, R.; Oirschot, van J.T.

    2001-01-01

    A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were

  7. Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk

    NARCIS (Netherlands)

    Wellenberg, G.J.; Verstraten, E.; Belak, S.; Verschuren, S.B.E.; Rijsewijk, F.A.M.; Peshev, R.; Oirschot, van J.T.

    2001-01-01

    A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were

  8. An immunochemical assay to detect DNA damage in bovine sperm

    NARCIS (Netherlands)

    Schans, G.P. van der; Haring, R.; Dijk- Knijnenburg, H.C.M. van; Bruijnzeel, P.L.B.; Daas, N.H.G. den

    2000-01-01

    An immunochemical assay has been developed to detect oxidative damage in bovine sperm DNA. Sperm DNA contains a large amount of oxidative damage as a result of exposure to exogenous agents, but damage also can caused by normal metabolic processes and the absence of DNA repair in the later stages of

  9. La Tuberculosis Bovina en Venezuela: patogénesis, epidemiología, respuesta inmunitaria y nuevas alternativas para el diagnóstico - Bovine Tuberculosis in Venezuela: pathogenesis, epidemiology, immune response and new alternative for the diagnosis

    Directory of Open Access Journals (Sweden)

    Rivera P., Sergio

    2010-09-01

    Full Text Available ResumenLa tuberculosis bovina, enfermedad infectocontagiosa causada porMycobacterium bovis, se encuentra difundida por el mundo entero, es unproblema de salud pública (Zoonosis, debido a que el M. bovis puedeinfectar al hombre produciendo un cuadro de tuberculosis clínicamenteindistinguible al causado por M. tuberculosis, el cual es causa de latuberculosis humana.SummaryBovine tuberculosis, an infectious disease caused by Mycobacterium bovis, is widespread throughout the world, is a public health problem (Zoonosis, because M. bovis can infect humans producing a sindrome of tuberculosis clinically indistinguishable to that caused by M. tuberculosis, which causes human tuberculosis. M. bovis is an intracellular pathogen that infects cells of the host immune system mainly macrophages, once inside the macrophage the mycobacteria may be destroyed those of the different microbicides mechanisms and used the macrophage for survival, multiply and travel within the body and gain access to other tissues via the bloodstream or lymphatic system.

  10. Carcinoma de células escamosas en linfonódulo mandibular diagnosticado a la inspección en matadero como tuberculosis bovina Squamous cell carcinoma in a mandibular lymph node diagnosed as bovine tuberculosis during slaughterhouse inspection

    Directory of Open Access Journals (Sweden)

    C Lecocq

    2012-01-01

    Full Text Available El presente trabajo establece que el diagnóstico de tuberculosis (TBC bovina comprende múltiples técnicas que en conjunto llegan a un diagnóstico certero, no obstante, la inspección en matadero es quizás una de las más relevantes en la vigilancia de la TBC bovina, por cuanto ésta debe abocarse a lo que indica el manual de inspección del Servicio Agrícola y Ganadero (SAG. Aún así, es posible encontrar interpretaciones erróneas de los hallazgos en matadero, que confunden al inspector y que instan a aumentar la rigurosidad de la inspección.The diagnosis of bovine tuberculosis (TBC involves the combination of several techniques to obtain an accurate diagnosis. Among them, the inspection of the animal body at the slaughterhouse is, perhaps, one of the most important for the surveillance of bovine TBC. In Chile, the guidelines for this practice appear in the Official Inspection Manual of the Agricultural and Livestock Service (SAG. Nevertheless, there are wrong interpretations of findings at the slaughterhouse. This study describes a squamous cell carcinoma in the submandibular lymph node of a cow that was initially diagnosed as tuberculosis, based on the presence of caseous necrosis and mineralization detected during postmortem gross examination. Squamous cell carcinoma is a common tumor of white-faced cows such as Hereford, which tend to metastasize to regional lymph nodes like the mandibular lymph node.

  11. Risk factors for visible lesions or positive laboratory tests in bovine tuberculosis reactor cattle in Northern Ireland.

    Science.gov (United States)

    O'Hagan, M J H; Courcier, E A; Drewe, J A; Gordon, A W; McNair, J; Abernethy, D A

    2015-07-01

    An observational case-control study was conducted to investigate risk factors for confirmed bovine tuberculosis (bTB) infection in cattle reacting positively to the single intradermal comparative cervical test (SICCT) in Northern Ireland in the years 1998, 2002 and 2006. Macroscopic lesions were detected at slaughter (positive visible lesion (VL) status) in 43.0% of reactor cattle, whilst 45.3% of those sampled were confirmed as bTB positive due to the presence of lesions or positive histopathology/mycobacterial culture (positive bTB status). In 97.5% of the reactors, the VL status and bTB status were either both negative or both positive. Generalized linear mixed model analyses were conducted on data of 24,923 reactor cattle with the variables herd identifier, local veterinary office (DVO) and abattoir being used as random effects within all the models generated at univariable and multivariable level. The other variables within the dataset were used as fixed effects. Significant risk factors associated with VL status and bTB status at multivariable level (pbovine tuberculin injection site, epidemiological status of skin test, total number of reactors at the disclosure test, mean herd size and prior response to the skin test. These risk factors are likely related to the time since infection, the strength of the challenge of infection and the susceptibility of the animal. These findings are important as the detection of visible lesions and the confirmation of bTB are an integral part of the overall bTB control programme in Northern Ireland and the veterinary meat inspection and hygiene programme. The visible lesion status and bTB status of an animal can affect the way in which bTB breakdowns are managed, since failure to detect visible lesions and recovery of Mycobacterium bovis can lead to a less stringent follow-up after other risk factors have been taken into account.

  12. Fast and efficient detection of tuberculosis antigens using liposome encapsulated secretory proteins of Mycobacterium tuberculosis.

    Science.gov (United States)

    Tiwari, Dileep; Haque, Shafiul; Tiwari, Ram P; Jawed, Arshad; Govender, Thavendran; Kruger, Hendrik G

    2017-04-01

    A rapid and efficient diagnostic test was developed for the detection of Mycobacterium tuberculosis antigens in serum samples of active tuberculosis (TB) and extrapulmonary TB patients via a liposomal agglutination-based method. A rapid card test has been developed to facilitate the recognition of high-affinity binding rabbit raised purified culture filtrate protein antibodies coupled on the surface of activated liposomal preparation. In the presence of TB antigens, the polyclonal antibodies bound to the liposomal particles demonstrate a visible agglutination reaction. The developed assay was simple, rapid, reliable, sensitive, and specific as a diagnostic test for the detection of antigens in serum samples of clinically confirmed cases of TB within 4-5 minutes' duration. The test was evaluated at different hospitals, medical colleges, and pathology centers, and involved 1483 participants. This investigation was conducted to detect the presence of these antigens during the period of active growth of the microorganism in serum samples for pulmonary TB and processed tissue biopsy for other extrapulmonary TB. Results obtained using this test were compared with acid-fast bacilli smear and culture results. Our study demonstrated that the newly developed liposome tuberculosis antigen card test detected antigens in our study population with approximately 97.48% sensitivity and 95.79% specificity. This is the first study to report the liposomal encapsulation of culture filtrate proteins from M. tuberculosis for diagnostic application. Copyright © 2015. Published by Elsevier B.V.

  13. Prevalence and herd-level risk factors for bovine tuberculosis in the State of Paraná, Brazil

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    Maria do Carmo Pessôa Silva

    2016-11-01

    Full Text Available Bovine tuberculosis is a zoonosis with worldwide distribution. Its control has a direct impact on public health and livestock production. This study estimated the prevalence of infected herds and adult bovines and evaluated risk factors associated with the presence of tuberculosis within herds in the state of Paraná. The state was divided in seven livestock regions and independent sampling was performed. A total of 1,419 farms were sampled and 16,045 animals were tested using the intradermal comparative cervical tuberculin diagnostic test. The apparent and estimated prevalence rates in farms and adult bovine animals were 2.15% (95% CI: 1.31-3.00 and 0.42% (95% CI: 0.04-0.81, respectively. It was not possible to state with 95% confidence that the disease prevalence in any region was significantly different from that in other regions. There were no positive animals in the western region, and the prevalence of positive herds and animals in the other regions ranged from 1.03% to 3.89% and 0.17% to 1.08%, respectively. The logistic regression model identified larger herd size (OR = 2.4 and mechanical cmilking (OR = 5.18 as risk factors associated with the presence of bovine tuberculosis. The combination of low prevalence with risk factors associated to larger herds and more intensive dairy farming, renders the state of Paraná a good candidate for the implementation of industry-based free-herd accreditation schemes and makes a case for planning risk-based surveillance targeted at major dairy basins.

  14. Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): bovine tuberculosis

    DEFF Research Database (Denmark)

    EFSA Panel on Animal Health and Welfare; More, Simon J.; Bøtner, Anette

    2017-01-01

    Bovine tuberculosis has been assessed according to the criteria of the Animal Health Law (AHL), in particular criteria of Article 7 on disease profile and impacts, Article 5 on the eligibility of bovine tuberculosis to be listed, Article 9 for the categorisation of bovine tuberculosis according t...... referred to in points (b), (c), (d) and (e) of Article 9(1). The main animal species to be listed for bovine tuberculosis according to Article 8(3) criteria are several mammal species, as indicated in the present opinion....... to disease prevention and control rules as in Annex IV and Article 8 on the list of animal species related to bovine tuberculosis. The assessment has been performed following a methodology composed of information collection and compilation, expert judgement on each criterion at individual and......, if no consensus was reached before, also at collective level. The output is composed of the categorical answer, and for the questions where no consensus was reached, the different supporting views are reported. Details on the methodology used for this assessment are explained in a separate opinion. According...

  15. Farmer attitudes to vaccination and culling of badgers in controlling bovine tuberculosis.

    Science.gov (United States)

    Warren, M; Lobley, M; Winter, M

    2013-07-13

    Controversy persists in England, Wales and Northern Ireland concerning methods of controlling the transmission of bovine tuberculosis (bTB) between badgers and cattle. The National Trust, a major land-owning heritage organisation, in 2011, began a programme of vaccinating badgers against bTB on its Killerton Estate in Devon. Most of the estate is farmed by 18 tenant farmers, who thus have a strong interest in the Trust's approach, particularly as all have felt the effects of the disease. This article reports on a study of the attitudes to vaccination of badgers and to the alternative of a culling programme, using face-to-face interviews with 14 of the tenants. The results indicated first that the views of the respondents were more nuanced than the contemporary public debate about badger control would suggest. Secondly, the attitude of the interviewees to vaccination of badgers against bTB was generally one of resigned acceptance. Thirdly, most respondents would prefer a combination of an effective vaccination programme with an effective culling programme, the latter reducing population of density sufficiently (and preferably targeting the badgers most likely to be diseased) for vaccination to have a reasonable chance of success. While based on a small sample, these results will contribute to the vigorous debate concerning contrasting policy approaches to bTB control in England, Wales and Northern Ireland.

  16. Farming on the edge: farmer attitudes to bovine tuberculosis in newly endemic areas.

    Science.gov (United States)

    Enticott, G; Maye, D; Carmody, P; Naylor, R; Ward, K; Hinchliffe, S; Wint, W; Alexander, N; Elgin, R; Ashton, A; Upton, P; Nicholson, R; Goodchild, T; Brunton, L; Broughan, J

    2015-10-31

    Defra's recent strategy to eradicate bovine tuberculosis (bTB) establishes three spatial zones: high-risk areas (HRAs) and low-risk areas, and an area referred to as 'the edge', which marks the areas where infection is spreading outwards from the HRA. Little is known about farmers in the edge area, their attitudes towards bTB and their farming practices. This paper examines farmers' practices and attitudes towards bTB in standardised epidemiologically defined areas. A survey was developed to collect data on farmer attitudes, behaviours, practices and environmental conditions as part of an interdisciplinary analysis of bTB risk factors. Survey items were developed from a literature review and focus groups with vets and farmers in different locations within the edge area. A case-control sampling framework was adopted with farms sampled from areas identified as recently endemic for bTB. 347 farmers participated in the survey including 117 with bTB, representing a 70per cent response rate. Results show that farmers believe they are unable to do anything about bTB but are keen for the government intervention to help control the spread of bTB.

  17. Eliminating bovine tuberculosis in cattle and badgers: insight from a dynamic model.

    Science.gov (United States)

    Brooks-Pollock, Ellen; Wood, James L N

    2015-06-01

    Bovine tuberculosis (BTB) is a multi-species infection that commonly affects cattle and badgers in Great Britain. Despite years of study, the impact of badgers on BTB incidence in cattle is poorly understood. Using a two-host transmission model of BTB in cattle and badgers, we find that published data and parameter estimates are most consistent with a system at the threshold of control. The most consistent explanation for data obtained from cattle and badger populations includes within-host reproduction numbers close to 1 and between-host reproduction numbers of approximately 0.05. In terms of controlling infection in cattle, reducing cattle-to-cattle transmission is essential. In some regions, even large reductions in badger prevalence can have a modest impact on cattle infection and a multi-stranded approach is necessary that also targets badger-to-cattle transmission directly. The new perspective highlighted by this two-host approach provides insight into the control of BTB in Great Britain.

  18. Estimating epidemiological parameters for bovine tuberculosis in British cattle using a Bayesian partial-likelihood approach.

    Science.gov (United States)

    O'Hare, A; Orton, R J; Bessell, P R; Kao, R R

    2014-05-22

    Fitting models with Bayesian likelihood-based parameter inference is becoming increasingly important in infectious disease epidemiology. Detailed datasets present the opportunity to identify subsets of these data that capture important characteristics of the underlying epidemiology. One such dataset describes the epidemic of bovine tuberculosis (bTB) in British cattle, which is also an important exemplar of a disease with a wildlife reservoir (the Eurasian badger). Here, we evaluate a set of nested dynamic models of bTB transmission, including individual- and herd-level transmission heterogeneity and assuming minimal prior knowledge of the transmission and diagnostic test parameters. We performed a likelihood-based bootstrapping operation on the model to infer parameters based only on the recorded numbers of cattle testing positive for bTB at the start of each herd outbreak considering high- and low-risk areas separately. Models without herd heterogeneity are preferred in both areas though there is some evidence for super-spreading cattle. Similar to previous studies, we found low test sensitivities and high within-herd basic reproduction numbers (R0), suggesting that there may be many unobserved infections in cattle, even though the current testing regime is sufficient to control within-herd epidemics in most cases. Compared with other, more data-heavy approaches, the summary data used in our approach are easily collected, making our approach attractive for other systems.

  19. Use of bacterial whole-genome sequencing to investigate local persistence and spread in bovine tuberculosis

    Directory of Open Access Journals (Sweden)

    Hannah Trewby

    2016-03-01

    Full Text Available Mycobacterium bovis is the causal agent of bovine tuberculosis, one of the most important diseases currently facing the UK cattle industry. Here, we use high-density whole genome sequencing (WGS in a defined sub-population of M. bovis in 145 cattle across 66 herd breakdowns to gain insights into local spread and persistence. We show that despite low divergence among isolates, WGS can in principle expose contributions of under-sampled host populations to M. bovis transmission. However, we demonstrate that in our data such a signal is due to molecular type switching, which had been previously undocumented for M. bovis. Isolates from farms with a known history of direct cattle movement between them did not show a statistical signal of higher genetic similarity. Despite an overall signal of genetic isolation by distance, genetic distances also showed no apparent relationship with spatial distance among affected farms over distances <5 km. Using simulations, we find that even over the brief evolutionary timescale covered by our data, Bayesian phylogeographic approaches are feasible. Applying such approaches showed that M. bovis dispersal in this system is heterogeneous but slow overall, averaging 2 km/year. These results confirm that widespread application of WGS to M. bovis will bring novel and important insights into the dynamics of M. bovis spread and persistence, but that the current questions most pertinent to control will be best addressed using approaches that more directly integrate WGS with additional epidemiological data.

  20. Detection of Mycobacterium bovis in bovine and bubaline tissues using nested-PCR for TbD1.

    Directory of Open Access Journals (Sweden)

    Cristina P Araújo

    Full Text Available In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR, 97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analytical specificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-related Actinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positive results in the comparative intradermal tuberculin test (CITT, as well as from those with lesions compatible with tuberculosis (LCT that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals with CITT- results, with no visible lesions (NVL and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in the detection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals than the culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay to detect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

  1. Bovine Tuberculosis in Cattle in the Highlands of Cameroon: Seroprevalence Estimates and Rates of Tuberculin Skin Test Reactors at Modified Cut-Offs

    Directory of Open Access Journals (Sweden)

    J. Awah-Ndukum

    2012-01-01

    Full Text Available The aim of this study was to obtain epidemiological estimates of bovine tuberculosis (TB prevalence in cattle in the highlands of Cameroon using two population-based tuberculin skin test (TST surveys in the years 2009 and 2010. However, prior to the TST survey in 2010, blood was collected from already chosen cattle for serological assay. Anti-bovine TB antibodies was detected in 37.17% of tested animals and bovine TB prevalence estimates were 3.59%–7.48%, 8.92%–13.25%, 11.77%–17.26% and 13.14%–18.35% for comparative TST at ≥4 mm, ≥3 mm and ≥2 mm cut-off points and single TST, respectively. The agreement between TST and lateral flow was generally higher in TST positive than in TST negative subjects. The K coefficients were 0.119, 0.234, 0.251 and 0.254 for comparative TST at ≥4 mm, ≥3 mm and ≥2 mm cut-off points and the single TST groups, respectively. Chi square statistics revealed that strong (P48 associations existed between seroprevalence rates and TST reactors. The study suggested that using lateral flow assay and TST at severe interpretations could improve the perception of bovine TB in Cameroon. The importance of defining TST at modified cut-offs and disease status by post-mortem detection and mycobacterial culture of TB lesions in local environments cannot be overemphasised.

  2. Post-mortem examination and laboratory-based analysis for the diagnosis of bovine tuberculosis among dairy cattle in Ecuador.

    Science.gov (United States)

    Proaño-Pérez, Freddy; Benitez-Ortiz, Washington; Desmecht, Daniel; Coral, Marco; Ortiz, Julio; Ron, Lenin; Portaels, Françoise; Rigouts, Leen; Linden, Annick

    2011-08-01

    Veterinary inspection in slaughterhouses allows for the detection of macroscopic lesions reminiscent of bovine tuberculosis, but the presence of Mycobacterium bovis must be confirmed by laboratory methods. This study aimed at comparing the performances of the standard diagnostic tools used to identify M. bovis in tissue specimens sampled from suspicious animals. During a two years period, 1390 cattle were inspected at the Machachi abattoir in the Mejia canton - Ecuador. A total of 33 animals with granulomatous lesions were detected, representing 2.33% (16/687) and 2.42% (17/703) animals examined in 2007 and 2008, respectively. Ninety-four tissue specimens were sampled and screened for the presence of mycobacteria. Acid-fast bacilli were identified in one third of the suspicious cattle (11/33) and suggestive microscopic lesions in 27.3% (9/33) of the samples examined by direct microscopy and histopathology, respectively. Culturing on Stonebrink medium and 16S-rRNA-based polymerase chain reaction (PCR) yielded 36.4% (12/33) and 27.3% (9/33) of positives, respectively. Compared to culture, other diagnostic procedures displayed a lower sensitivity, with 56.5% for PCR, and 43.5% for direct microscopy and histopathology; however, the specificity was higher (94.4% for PCR and microscopy, and 97.2% for histopathology). We conclude that reliable post-mortem laboratory testing either requires the combination of a set of available diagnostic tools or necessitates the development of improved new-generation tools with better sensitivity and specificity characteristics.

  3. THE RELATIVE PREVALENCE OF HUMAN AND BOVINE TYPES OF TUBERCLE BACILLI IN BONE AND JOINT TUBERCULOSIS OCCURRING IN CHILDREN.

    Science.gov (United States)

    Fraser, J

    1912-10-01

    The all important point revealed by the investigation is the fact that a large proportion of bone and joint tuberculosis occurring in children in Edinburgh owes its origin to infection by the bovine bacillus. The bovine bacillus is introduced into the system practically by one route only, that of ingestion, and the medium with which it is ingested is cow's milk. It is not my intention to criticize in any way the existing conditions of milk supply. I have furnished proof of what is actually occurring and no one will deny that the evil is a remediable one. In those cases in which the human bacillus was present, a considerable proportion showed a definite history of pulmonary tuberculosis affecting a co-resident, and every fact went to prove that the infection had been a direct one from patient to child. A complete distinction can be drawn between human and bovine bacilli, and the distinction is best secured by subjecting the organism to a series of tests such as I have detailed. The subject is one which ought to be investigated in a series of different localities. It is possible that the locus may be a factor in the explanation of the difference between the above results and those of other observers.

  4. Zirconia based nucleic acid sensor for Mycobacterium tuberculosis detection

    Science.gov (United States)

    Das, Maumita; Sumana, Gajjala; Nagarajan, R.; Malhotra, B. D.

    2010-03-01

    Nanostructured zirconium oxide (ZrO2) film (particle size˜35 nm), electrochemically deposited onto gold(Au) surface, has been used to immobilize 21-mer oligonucleotide probe (ssDNA) specific to Mycobacterium tuberculosis by utilizing affinity between oxygen atom of phosphoric group and zirconium to fabricate DNA biosensor. This DNA-ZrO2/Au bioelectrode, characterized using x-ray diffraction, Fourier transform infrared spectroscopy, cyclic voltammetry, and scanning electron microscopy techniques, can be used for early and rapid diagnosis of M. tuberculosis with detection limit of 0.065 ng/μL within 60s.

  5. Bovine Tuberculosis Vaccine Efficacy Studies: Neonatal Calves and White-tailed Deer

    Science.gov (United States)

    Introduction Tuberculosis (TB) in humans and animals may result from exposure to bacilli within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedi, M. microti, M. caprae, or M. canetti)(#1). Mycobacterium bovis is the species most often isolated from ...

  6. Generation of Monoclonal Antibodies against Ag85A Antigen of Mycobacterium tuberculosis and Application in a Competitive ELISA for Serodiagnosis of Bovine Tuberculosis

    Directory of Open Access Journals (Sweden)

    Zhengzhong Xu

    2017-06-01

    Full Text Available The Ag85 complex functions as the main secretory protein of Mycobacterium tuberculosis (M. tuberculosis and BCG. This complex is composed of the proteins, Ag85A, Ag85B, and Ag85C, with Ag85A thought to play the largest role within the complex. However, the lack of commercially available monoclonal antibodies (mAbs against Ag85A still hinders the biological and applicative research on this protein. In this study, we developed and identified anti-Ag85A mAbs, and five hybridoma cells were established. Using the indirect immunofluorescence test, we found that two anti-Ag85A mAbs did not cross-react with Ag85B and/or Ag85C. In addition, we showed that all of the mAbs tested in this study are able to react with endogenous Ag85A protein in BCG and rBCG:Ag85A using indirect ELISA and Western blot analyses. A competitive ELISA (cELISA based on mAb 3B8 was developed, the analyses of clinic serum samples from cattle with bovine tuberculosis (TB and healthy cattle demonstrated that the sensitivity of the cELISA was 54.2% (26/48 and the specificity was 83.5% (167/200. This study demonstrated that the mAbs against Ag85A will provide useful reagents for further investigation into the function of the Ag85 complex and can be used for serodiagnosis of bovine TB.

  7. Gene polymorphisms in African buffalo associated with susceptibility to bovine tuberculosis infection.

    Directory of Open Access Journals (Sweden)

    Nikki le Roex

    Full Text Available Bovine tuberculosis (BTB is a chronic, highly infectious disease that affects humans, cattle and numerous species of wildlife. In developing countries such as South Africa, the existence of extensive wildlife-human-livestock interfaces poses a significant risk of Mycobacterium bovis transmission between these groups, and has far-reaching ecological, economic and public health impacts. The African buffalo (Syncerus caffer, acts as a maintenance host for Mycobacterium bovis, and maintains and transmits the disease within the buffalo and to other species. In this study we aimed to investigate genetic susceptibility of buffalo for Mycobacterium bovis infection. Samples from 868 African buffalo of the Cape buffalo subspecies were used in this study. SNPs (n = 69, with predicted functional consequences in genes related to the immune system, were genotyped in this buffalo population by competitive allele-specific SNP genotyping. Case-control association testing and statistical analyses identified three SNPs associated with BTB status in buffalo. These SNPs, SNP41, SNP137 and SNP144, are located in the SLC7A13, DMBT1 and IL1α genes, respectively. SNP137 remained significantly associated after permutation testing. The three genetic polymorphisms identified are located in promising candidate genes for further exploration into genetic susceptibility to BTB in buffalo and other bovids, such as the domestic cow. These polymorphisms/genes may also hold potential for marker-assisted breeding programmes, with the aim of breeding more BTB-resistant animals and herds within both the national parks and the private sector.

  8. Disease Control in Wildlife: Evaluating a Test and Cull Programme for Bovine Tuberculosis in African Buffalo.

    Science.gov (United States)

    le Roex, N; Cooper, D; van Helden, P D; Hoal, E G; Jolles, A E

    2016-12-01

    Providing an evidence base for wildlife population management is difficult, due to limited opportunities for experimentation and study replication at the population level. We utilized an opportunity to assess the outcome of a test and cull programme aimed at limiting the spread of Mycobacterium bovis in African buffalo. Buffalo act as reservoirs of M. bovis, the causative agent of bovine tuberculosis (BTB), which can have major economic, ecological and public health impacts through the risk of infection to other wildlife species, livestock and surrounding communities. BTB prevalence data were collected in conjunction with disease control operations in Hluhluwe-iMfolozi Park, South Africa, from 1999 to 2006. A total of 4733 buffalo (250-950 per year) were tested for BTB using the single comparative intradermal tuberculin (SCIT) test, with BTB-positive animals culled, and negative animals released. BTB prevalence was spatially and temporally variable, ranging from 2.3% to 54.7%. Geographic area was a strong predictor of BTB transmission in HiP, owing to relatively stable herds and home ranges. Herds experiencing more intensive and frequent captures showed reduced per capita disease transmission risk and less increase in herd prevalence over time. Disease hot spots did not expand spatially over time, and BTB prevalence in all but the hot spot areas was maintained between 10% and 15% throughout the study period. Our data suggest that HiP's test and cull programme was effective at reducing BTB transmission in buffalo, with capture effort and interval found to be the crucial components of the programme. The programme was thus successful with respect to the original goals; however, there are additional factors that should be considered in future cost/benefit analyses and decision-making. These findings may be utilized and expanded in future collaborative work between wildlife managers, veterinarians and scientists, to optimize wildlife disease control programmes and

  9. Brucellosis and bovine tuberculosis prevalence in livestock from pastoralist communities adjacent to Awash National Park, Ethiopia.

    Science.gov (United States)

    Tschopp, Rea; Bekele, Shiferaw; Moti, Tesfaye; Young, Douglas; Aseffa, Abraham

    2015-06-15

    This cross-sectional study investigated the prevalence of brucellosis and bovine tuberculosis (BTB) in local cattle and goat breeds of Oromo and Afar pastoralist communities living in two distinct parts around the Awash National Park. A questionnaire survey was carried out to assess information on husbandry, milk consumption habits, and on knowledge-attitude-practice regarding both diseases. Among a total of 771 animals from all sites tested by comparative intradermal tuberculin test (CIDT) none were BTB reactors with the >4mm cut-off. Using the >2mm cut-off, individual apparent prevalence was 0.9% (95%CI: 0.23-3.56%) in cattle and 0.7% (95%CI: 0.12-3.45%) in goats. Herd prevalence in Oromia and Afar sites was 0% and 66.7% respectively in goats and 16.7% and 50% in cattle. Among the 327 animals tested by enzyme linked immunoassay for brucellosis, 4.8% (95%CI: 1.2-17.1%) of cattle and 22.8% (95%CI: 5.98-29.5%) of goats were reactors. Highest individual prevalence of both diseases was found in Afar settlements with brucellosis being as high as 50%. Respondent ethnicity was the only risk factor for brucellosis positivity in goats in the univariable risk factor analysis. Knowledge about the diseases was poor. Raw goat milk was regularly consumed by women and children, putting them at risk for brucellosis. This study highlighted an increased prevalence gradient of BTB and brucellosis from West to East along the study sites with high brucellosis individual prevalence and abortion rates among Afar settlements in particular.

  10. The importance of 'neighbourhood' in the persistence of bovine tuberculosis in Irish cattle herds.

    Science.gov (United States)

    White, Paul W; Martin, S Wayne; De Jong, Mart C M; O'Keeffe, James J; More, Simon J; Frankena, Klaas

    2013-07-01

    Local persistence of infection is a key feature of bovine tuberculosis (bTB) among cattle herds in the Republic of Ireland. The aim of this study was to determine the relative importance of 'neighbourhood', specifically farm-to-farm spread and spread from wildlife, in the persistence of bTB by investigating herds having a bTB episode in 2006. A case-control study was conducted on the association between the occurrence of a bTB episode in 2006 and the occurrence of bTB in previous years among neighbouring herd(s) within 1 km, while controlling for each herd's bTB history and other risk factors. Neighbouring herds were grouped into three zones, based on distance, and bTB incidence measures summarised within each zone and by calendar year (2001-2005). The incidence of bTB was associated with an increased animal incidence in two subsets of neighbouring herds: (i) herds directly contiguous during the previous 2 years (attributable fraction=0.20), and (ii) herds at a distance of >25 m in the previous year (attributable fraction=0.19). Other predictors of bTB in a herd in 2006 included the occurrence of a bTB episode within that herd in any of the previous 5 years, herd size, and the number of animals purchased at age greater than 12 months. An infected wildlife source best explains the existence of a "neighbouring herd risk" for bTB at distances greater than 25 m. Further studies will be necessary to determine to what extent neighbouring herd risk within 25 m may be confounded by the same wildlife (badger) source.

  11. Assessing vaccination as a control strategy in an ongoing epidemic: Bovine tuberculosis in African buffalo

    Science.gov (United States)

    Cross, Paul C.; Getz, W.M.

    2006-01-01

    Bovine tuberculosis (BTB) is an exotic disease invading the buffalo population (Syncerus caffer) of the Kruger National Park (KNP), South Africa. We used a sex and age-structured epidemiological model to assess the effectiveness of a vaccination program and define important research directions. The model allows for dispersal between a focal herd and background population and was parameterized with a combination of published data and analyses of over 130 radio-collared buffalo in the central region of the KNP. Radio-tracking data indicated that all sex and age categories move between mixed herds, and males over 8 years old had higher mortality and dispersal rates than any other sex or age category. In part due to the high dispersal rates of buffalo, sensitivity analyses indicate that disease prevalence in the background population accounts for the most variability in the BTB prevalence and quasi-eradication within the focal herd. Vaccination rate and the transmission coefficient were the second and third most important parameters of the sensitivity analyses. Further analyses of the model without dispersal suggest that the amount of vaccination necessary for quasi-eradication (i.e. prevalence 70% of the calf population would have to be vaccinated every year to reduce the prevalence to less than 1%. If the half-life of the vaccine is less than 5 years, even vaccinating every calf for 50 years may not eradicate BTB. Thus, although vaccination provides a means of controlling BTB prevalence it should be combined with other control measures if eradication is the objective.

  12. 牛结核病的诊断研究进展%The Research Progress in Diagnosis for Bovine Tuberculosis

    Institute of Scientific and Technical Information of China (English)

    李蓉

    2014-01-01

    牛结核病是由结核分支杆菌引起的牛的一种慢性消耗性传染病,不仅严重危害养牛业的发展,也对人的身体健康造成威胁。做好牛结核病的诊断,是实现该病净化的一种重要手段和前提。本文从病原学、血清学、分子生物学等方面对该病的诊断进行了阐述。%Bovine tuberculosis is a serious zoonotic disease which caused by mycobacterium bo-vis, it not only cause losses to the dairy industry, but also closely related to people’s health. The diagnosis of bovine tuberculosis is an important method in the control of the epidemic. The methods of diagnostic techniques such as aetiology, serum and molecular biology techniques were re-viewed in this paper.

  13. Development of a test for bovine tuberculosis in cattle based on measurement of gamma interferon mRNA by real-time PCR.

    Science.gov (United States)

    Gan, W; Zhou, X; Yang, H; Chen, H; Qiao, J; Khan, S H; Yang, L; Yin, X; Zhao, D

    2013-08-01

    The infection status of cattle for bovine tuberculosis (bTB) was determined by real-time PCR, comparing the levels of IFN-γ mRNA in blood cultures stimulated with either bovine or avian tuberculin with non-stimulated control (phosphate buffer saline, PBS) blood culture. Totally, 137 cattle were tested to validate the assay, in which 54 were IFN-γ real-time quantitative PCR (RT-qPCR) positive, while the remaining 83 were found negative. Meanwhile, the IFN-γ ELISA test was carried out using the Bovigam IFN-γ detection ELISA kit and these results were used as a standard. The results of the single intradermal tuberculin tests (SIDT) and IFN-γ RT-qPCR tests were compared and revealed that the RT-qPCR correlated better with the ELISA and its accuracy was higher than SIDT. This indicates the RT-qPCR is a useful diagnostic method for bTB in cattle. However, several limitations remain for our approach, such as lack of a TB lesions or postmortem test results as a gold standard. Further improvements should be made in the future to increase accuracy of diagnosis of bTB in cattle.

  14. Tuberculosis case detection in a state prison system.

    OpenAIRE

    Brock, N N; Reeves, M; LaMarre, M; DeVoe, B

    1998-01-01

    OBJECTIVE: This study was designed to describe the epidemiology of tuberculosis ((TB) among inmates in the Georgia state prison system; to evaluate the effectiveness of the TB case detection methods used; to evaluate the use of contact tracing for inmate TB cases; and to determine rates of completion of therapy. METHODS: Using a standardized form, the authors abstracted data from reports to the Centers for Disease Control and Prevention, prison hospital medical charts, and county health depar...

  15. Update on Comparative Studies for Diagnosis of Bovine Tuberculosis Using The Interferon Gamma (IFN-gamma) Assay with Tuberculins or Alternative Antigens for Whole Blood Stimulation

    Science.gov (United States)

    Bovine Tuberculosis is a respiratory disease caused by Mycobacterium bovis (M. bovis). It is a major infectious disease found worldwide in domestic animals, particularly cattle, as well as in certain wildlife populations. Although the disease has been effectively eliminated in many countries and reg...

  16. The use of PCR technique in the identification of Mycobacterium species responsible for bovine tuberculosis in cattle and buffaloes in Pakistan.

    Science.gov (United States)

    Akhtar, Farah; Javed, Muhammad Tariq; Aziz-ur-Rehman; Khan, Muhammad Nisar; Akhtar, Pervez; Hussain, Sayed Misdaq; Aslam, Muhammad Sohaib; Kausar, Razia; Qamar, Mehwish; Cagiola, Monica

    2015-08-01

    Bovine tuberculosis is one of the important diseases of dairy and wild animals. The disease is prevalent all over the world, though developed countries have tremendously reduced the prevalence through eradication campaigns. The prevalence of disease in Pakistan on the basis of tuberculin testing or culture isolation of the organism has been reported previously. It is, however, important to use the latest diagnostic tools, i.e. PCR to confirm the type of Mycobacterium infecting the animals in Pakistan. Therefore, the present study was carried out to assess the utility of direct PCR on milk samples and nasal swabs to confirm the type of Mycobacterium infecting the animals. This study was carried out on 215 cattle and buffaloes of more than 2 years of age present at two livestock farms. The tuberculin results showed 22.5% prevalence at one farm and 25.9% at the other with an overall prevalence of 24.7%. The 92.5% of milk samples and/or nasal swabs showed positive PCR for Mycobacterium genus, 86.8% for Mycobacterium tuberculosis complex and 77.4% for Mycobacterium bovis. The M. bovis by PCR was detected in 13.2% of milk samples, 24.5% of nasal swabs and 39.6% of both milk samples + nasal swabs. The results suggested that there are 60% higher chance for a nasal swab to yield a positive PCR for M. bovis than the milk sample. It can be concluded from the present study that tuberculin testing is a useful method in studying the prevalence of disease as the PCR for Mycobacterium genus was positive in 92.5%, M. tuberculosis complex in 86.8% and Mycobacterium bovis in 77.4% cases.

  17. Wildlife-livestock interactions and risk areas for cross-species spread of bovine tuberculosis.

    Science.gov (United States)

    Meunier, Natascha V; Sebulime, Peregrine; White, Richard G; Kock, Richard

    2017-01-23

    The transmission of diseases between livestock and wildlife can be a hindrance to effective disease control. Maintenance hosts and contact rates should be explored to further understand the transmission dynamics at the wildlife-livestock interface. Bovine tuberculosis (BTB) has been shown to have wildlife maintenance hosts and has been confirmed as present in the African buffalo (Syncerus caffer) in the Queen Elizabeth National Park (QENP) in Uganda since the 1960s. The first aim of this study was to explore the spatio-temporal spread of cattle illegally grazing within the QENP recorded by the Uganda Wildlife Authority (UWA) rangers in a wildlife crime database. Secondly, we aimed to quantify wildlife-livestock interactions and cattle movements, on the border of QENP, using a longitudinal questionnaire completed by 30 livestock owners. From this database, 426 cattle sightings were recorded within QENP in 8 years. Thirteen (3.1%) of these came within a 300 m-4 week space-time window of a buffalo herd, using the recorded GPS data. Livestock owners reported an average of 1.04 (95% CI 0.97-1.11) sightings of Uganda kob, waterbuck, buffalo or warthog per day over a 3-month period, with a rate of 0.22 (95% CI 0.20-0.25) sightings of buffalo per farmer per day. Reports placed 85.3% of the ungulate sightings and 88.0% of the buffalo sightings as further than 50 m away. Ungulate sightings were more likely to be closer to cattle at the homestead (OR 2.0, 95% CI 1.1-3.6) compared with the grazing area. Each cattle herd mixed with an average of five other cattle herds at both the communal grazing and watering points on a daily basis. Although wildlife and cattle regularly shared grazing and watering areas, they seldom came into contact close enough for aerosol transmission. Between species infection transmission is therefore likely to be by indirect or non-respiratory routes, which is suspected to be an infrequent mechanism of transmission of BTB. Occasional cross

  18. Wildlife Interactions on Baited Places and Waterholes in a French Area Infected by Bovine Tuberculosis

    Science.gov (United States)

    Payne, Ariane; Philipon, Sixtine; Hars, Jean; Dufour, Barbara; Gilot-Fromont, Emmanuelle

    2017-01-01

    Interactions among wildlife species are major drivers for the transmission of multi-host pathogens, such as Mycobacterium bovis, which also affect livestock. Although France is officially free from bovine tuberculosis (bTB), some areas are still harboring infection in cattle and wildlife. We aimed at characterizing the visits of susceptible wild species (badger, red deer, and wild boar) at baited places and waterholes, considered as possible hotspots for contacts. We described the visits in terms of frequency, duration, and number of individuals and studied the influence of the season. Then, we estimated the frequency of intraspecies and interspecies interactions occurring at baited places and waterholes which may lead to bTB transmission, including direct and indirect contacts through the soil or water. We used camera traps placed on baited places and waterholes on 13 locations monitored during 21 months. The number of visits, their duration, and the number of individuals per visit were analyzed by generalized linear mixed models for each targeted species. The frequency of the interspecies and intraspecies interactions was also analyzed separately. The season, the type of site (baited place or waterhole), and the location were the explanatory variables. Badgers’ visits and interactions were more frequent than for other species (mean: 0.60 visit/day and 5.42 interactions/day) especially on baited places. Red deer only visited waterholes. Wild boars visited most often baited places in spring–summer and waterholes in autumn–winter. They came in higher number than other species, especially on baited places. Direct interactions were uncommon. The most frequent interspecies interactions occurred between red deer and wild boar (mean: 4.02 interactions/day). Baited places and waterholes are important interfaces between the different wild species involved in the bTB multi-host system in this area. They can thus promote intraspecies and interspecies b

  19. Recent advances in the management of bovine tuberculosis in free-ranging wildlife.

    Science.gov (United States)

    O'Brien, Daniel J; Schmitt, Stephen M; Rudolph, Brent A; Nugent, Graham

    2011-07-05

    Established foci of Mycobacterium bovis (the causative agent of bovine tuberculosis [bTB]) in free-ranging wildlife are currently under various stages of management on three continents (Africa, Europe and North America) and in New Zealand. Other, as yet undiagnosed, foci seem likely to exist elsewhere. The complex roles that these wildlife foci play in the ecology of bTB remain among the greatest challenges facing bTB control globally. Conceptually, management of bTB in free-ranging wildlife can be thought of as progressing from the discovery of an outbreak through frequently overlapping stages of epidemiological characterization, initial control, simulation and forecasting, focused control, and verification of eradication. Surveillance in its various forms remains a critical component of assessment throughout. Since the Fourth International M. bovis Conference in 2005, research on management of bTB in free-ranging wildlife has encompassed such areas as the human dimensions of wildlife management, mitigation of bTB risks from wildlife on cattle farms, vaccine biology, and epidemiology, with a major contribution from simulation modeling. In order to advance the actual field management of bTB, however, research must be sufficiently grounded to aid development of practical, affordable and politically defensible management interventions which stand a reasonable chance of being implemented. The current management of two wildlife reservoirs of bTB, brushtail possums (Trichosurus vulpecula) in New Zealand, and white-tailed deer (Odocoileus virginianus) in Michigan, USA, serve as contrasting examples of different wildlife management strategies aimed at achieving a common goal. In New Zealand, the importance of agricultural export markets and the status of the possum as a non-native pest have facilitated direct, aggressive management of the disease reservoir, resulting in considerable progress towards bTB freedom since 1994. In Michigan, the relative importance of the

  20. Role of Cattle Movements in Bovine Tuberculosis Spread in France between 2005 and 2014.

    Science.gov (United States)

    Palisson, Aurore; Courcoul, Aurélie; Durand, Benoit

    2016-01-01

    Live animal movements are a major transmission route for the spread of infectious agents such as Mycobacterium bovis, the main agent of bovine Tuberculosis (bTB). France became officially bTB-free in 2001, but M. bovis is still circulating in the cattle population, with about a hundred of outbreaks per year, most located in a few geographic areas. The aim of this study was to analyse the role of cattle movements in bTB spread in France between 2005 and 2014, using social network analysis and logistic regression models. At a global scale, the trade network was studied to assess the association between several centrality measures and bTB infection though a case-control analysis. The bTB infection status was associated with a higher in-degree (odds-ratio [OR] = 2.4 [1.1-5.4]) and with a higher ingoing contact chain (OR = 2.2 [1.0-4.7]). At a more local scale, a second case-control analysis was conducted to estimate the relative importance of cattle movements and spatial neighbourhood. Only direct purchase from infected herds was shown to be associated with bTB infection (OR = 2.9 [1.7-5.2]), spatial proximity to infected herds being the predominant risk factor, with decreasing ORs when distance increases. Indeed, the population attributable fraction was 12% [5%-18%] for cattle movements and 73% [68%-78%] for spatial neighbourhood. Based on these results, networks of potential effective contacts between herds were built and analysed for the three major spoligotypes reported in France. In these networks, the links representing cattle movements were associated with higher edge betweenness than those representing the spatial proximity between infected herds. They were often links connecting distinct communities and sometimes distinct geographical areas. Therefore, although their role was quantitatively lower than the one of spatial neighbourhood, cattle movements appear to have been essential in the French bTB dynamics between 2005 and 2014.

  1. Risk Factors for Bovine Tuberculosis (bTB in Cattle in Ethiopia.

    Directory of Open Access Journals (Sweden)

    Sintayehu W Dejene

    Full Text Available Bovine tuberculosis (bTB infection is generally correlated with individual cattle's age, sex, body condition, and with husbandry practices such as herd composition, cattle movement, herd size, production system and proximity to wildlife-including bTB maintenance hosts. We tested the correlation between those factors and the prevalence of bTB, which is endemic in Ethiopia's highland cattle, in the Afar Region and Awash National Park between November 2013 and April 2015. A total of 2550 cattle from 102 herds were tested for bTB presence using the comparative intradermal tuberculin test (CITT. Data on herd structure, herd movement, management and production system, livestock transfer, and contact with wildlife were collected using semi-structured interviews with cattle herders and herd owners. The individual overall prevalence of cattle bTB was 5.5%, with a herd prevalence of 46%. Generalized Linear Mixed Models with a random herd-effect were used to analyse risk factors of cattle reactors within each herd. The older the age of the cattle and the lower the body condition the higher the chance of a positive bTB test result, but sex, lactation status and reproductive status were not correlated with bTB status. At herd level, General Linear Models showed that pastoral production systems with transhumant herds had a higher bTB prevalence than sedentary herds. A model averaging analysis identified herd size, contact with wildlife, and the interaction of herd size and contact with wildlife as significant risk factors for bTB prevalence in cattle. A subsequent Structural Equation Model showed that the probability of contact with wildlife was influenced by herd size, through herd movement. Larger herds moved more and grazed in larger areas, hence the probability of grazing in an area with wildlife and contact with either infected cattle or infected wildlife hosts increased, enhancing the chances for bTB infection. Therefore, future bTB control strategies

  2. Wildlife-livestock interactions and risk areas for cross-species spread of bovine tuberculosis

    Directory of Open Access Journals (Sweden)

    Natascha V. Meunier

    2017-01-01

    Full Text Available The transmission of diseases between livestock and wildlife can be a hindrance to effective disease control. Maintenance hosts and contact rates should be explored to further understand the transmission dynamics at the wildlife-livestock interface. Bovine tuberculosis (BTB has been shown to have wildlife maintenance hosts and has been confirmed as present in the African buffalo (Syncerus caffer in the Queen Elizabeth National Park (QENP in Uganda since the 1960s. The first aim of this study was to explore the spatio-temporal spread of cattle illegally grazing within the QENP recorded by the Uganda Wildlife Authority (UWA rangers in a wildlife crime database. Secondly, we aimed to quantify wildlife-livestock interactions and cattle movements, on the border of QENP, using a longitudinal questionnaire completed by 30 livestock owners. From this database, 426 cattle sightings were recorded within QENP in 8 years. Thirteen (3.1% of these came within a 300 m–4 week space-time window of a buffalo herd, using the recorded GPS data. Livestock owners reported an average of 1.04 (95% CI 0.97–1.11 sightings of Uganda kob, waterbuck, buffalo or warthog per day over a 3-month period, with a rate of 0.22 (95% CI 0.20–0.25 sightings of buffalo per farmer per day. Reports placed 85.3% of the ungulate sightings and 88.0% of the buffalo sightings as further than 50 m away. Ungulate sightings were more likely to be closer to cattle at the homestead (OR 2.0, 95% CI 1.1–3.6 compared with the grazing area. Each cattle herd mixed with an average of five other cattle herds at both the communal grazing and watering points on a daily basis. Although wildlife and cattle regularly shared grazing and watering areas, they seldom came into contact close enough for aerosol transmission. Between species infection transmission is therefore likely to be by indirect or non-respiratory routes, which is suspected to be an infrequent mechanism of transmission of BTB

  3. Effect of the inoculation site of bovine purified protein derivative (PPD) on the skin fold thickness increase in cattle from officially tuberculosis free and tuberculosis-infected herds.

    Science.gov (United States)

    Casal, Carmen; Alvarez, Julio; Bezos, Javier; Quick, Harrison; Díez-Guerrier, Alberto; Romero, Beatriz; Saez, Jose L; Liandris, Emmanouil; Navarro, Alejandro; Perez, Andrés; Domínguez, Lucas; de Juan, Lucía

    2015-09-01

    The official technique for diagnosis of bovine tuberculosis (bTB) worldwide is the tuberculin skin test, based on the evaluation of the skin thickness increase after the intradermal inoculation of a purified protein derivative (PPD) in cattle. A number of studies performed on experimentally infected or sensitized cattle have suggested that the relative sensitivity of the cervical test (performed in the neck) may vary depending on the exact location in which the PPD is injected. However, quantitative evidence on the variation of the test accuracy associated to changes in the site of inoculation in naturally infected animals (the population in which performance of the test is most critical for disease eradication) is lacking. Here, the probability of obtaining a positive reaction (>2 or 4 millimeters and/or presence of local clinical signs) after multiple inoculations of bovine PPD in different cervical and scapular locations was assessed in animals from five bTB-infected herds (818 cattle receiving eight inoculations) using a hierarchical Bayesian logistic regression model and adjusting for the potential effect of age and sex. The effect of the inoculation site was also assessed qualitatively in animals from four officially tuberculosis free (OTF) herds (two inoculations in 210 animals and eight inoculations in 38 cattle). Although no differences in the qualitative outcome of the test were observed in cattle from OTF herds, a statistically important association between the test outcome and the inoculation site in animals from infected herds was observed, with higher probabilities of positive results when the test was performed in the neck anterior area. Our results suggest that test sensitivity may be maximized by considering the area of the neck in which the test is applied, although lack of effect of the inoculation site in the specificity of the test should be confirmed in a larger sample.

  4. Tuberculosis

    Directory of Open Access Journals (Sweden)

    C. Robert Horsburgh, Jr

    2014-03-01

    Full Text Available This article reviews the published literature on tuberculosis from September 2012 to August 2013 and describes important advances in tuberculosis epidemiology, microbiology, pathology, clinical pharmacology, genetics, treatment and prevention.

  5. Tuberculosis

    OpenAIRE

    C. Robert Horsburgh, Jr

    2014-01-01

    This article reviews the published literature on tuberculosis from September 2012 to August 2013 and describes important advances in tuberculosis epidemiology, microbiology, pathology, clinical pharmacology, genetics, treatment and prevention.

  6. Detection of tuberculosis using hybrid features from chest radiographs

    Science.gov (United States)

    Fatima, Ayesha; Akram, M. Usman; Akhtar, Mahmood; Shafique, Irrum

    2017-02-01

    Tuberculosis is an infectious disease and becomes a major threat all over the world but still diagnosis of tuberculosis is a challenging task. In literature, chest radiographs are considered as most commonly used medical images in under developed countries for the diagnosis of TB. Different methods have been proposed but they are not helpful for radiologists due to cost and accuracy issues. Our paper presents a methodology in which different combinations of features are extracted based on intensities, shape and texture of chest radiograph and given to classifier for the detection of TB. The performance of our methodology is evaluated using publically available standard dataset Montgomery Country (MC) which contains 138 CXRs among which 80 CXRs are normal and 58 CXRs are abnormal including effusion and miliary patterns etc. The accuracy of 81.16% was achieved and the results show that proposed method have outperformed existing state of the art methods on MC dataset.

  7. ROLE OF CBNAAT IN RAPID DETECTION OF MYCOBACTERIUM TUBERCULOSIS IN PLHIV IN A HIGHLY PREVALENT STATE

    Directory of Open Access Journals (Sweden)

    Pragati Rao

    2016-05-01

    Full Text Available Tuberculosis can occur at any stage of HIV disease and it presents differently according to level of immunosuppression. Patients are paucibacillary, involve hilar and mediastinal lymph nodes, lack cavitation and are smear negative. Sputum culture takes 4-8 weeks for mycobacteria to grow, hence a newly launched cartridge based nucleic acid amplification test by WHO offers a promising solution to these challenges in detecting presumptive pulmonary tuberculosis. AIM OF STUDY Comparing efficacy of fluorescent microscopy and CBNAAT for diagnosing pulmonary tuberculosis in PLHIV and detecting Rifampicin resistance. MATERIAL AND METHODS This study included all HIV infected patients suspected to have tuberculosis, including drug-resistant tuberculosis, coming to our district tuberculosis centre. The patients were enrolled and provided Xpert MTB/RIF. Simultaneously, smear for AFB was done in same patients. RESULTS The study was done for 2 months from February to march 2016. Out of 231 HIV positive patients, 59 cases (25.54% had tuberculosis. Sputum smear for AFB negative and GeneXpert positive were 45(76.27%. 8(13.55% cases were Rifampicin resistance and 51 (86.44% were sensitive out of all tuberculosis patients. CONCLUSION This study demonstrates the limitations of conventional sputum microscopy. CBNAAT detects more tuberculosis cases in lesser time. Rifampicin resistance is also detected. Hence drug-resistant tuberculosis can be screened. Early treatment of tuberculosis can be addressed with Xpert MTB/RIF testing.

  8. Detección de la expresión génica in vivo de Mycobacterium tuberculosis durante la tuberculosis pulmonar activa Mycobacterium tuberculosis in vivo-expressed genes detection during active pulmonary tuberculosis

    Directory of Open Access Journals (Sweden)

    Alejandra Otazo M

    2012-12-01

    Full Text Available El estudio de la expresión génica de Mycobacterium tuberculosis ha involucrado la experimentación "in vitro ", "ex vivo " e "in vivo " (modelos animales, pero aún sin el éxito esperado. Proponemos que revelar los factores clave de la tuberculosis humana requiere investigar la expresión génica de M. tuberculosis dentro del ser humano ("in vivo ". Para ello, aislamos el mRNA total de M. tuberculosis, desde muestras clínicas respiratorias de pacientes con diagnóstico de tuberculosis pulmonar; posteriormente, sintetizamos el dscDNA y lo analizamos mediante RT-PCR cualitativo. Detectamos la expresión de la secuencia de inserción IS6110 y de los genes "housekeeping " 16SrRNA y sigA en M. tuberculosis creciendo in vivo (tuberculosis pulmonar así como cultivado in vitro. La expresión de los genes mprA y mprB, que codifican el sistema de transducción de señales MprAB, sólo se detectó en M. tuberculosis crecido in vitro. Con nuestros resultados damos el primer paso hacia la implementación de un método no invasivo para el estudio del transcriptoma de M. tuberculosis, dentro de su único hospedero natural, con el fin de analizar la regulación "in vivo" de los determinantes genéticos requeridos para su virulencia y patogénesis.Mycobacterium tuberculosis gene expression studies have involved "in vitro", "ex vivo" and "in vivo" experiments (animal models, but without the expected success. We propose that key features of human tuberculosis could be discovered by studying the M. tuberculosis gene expression within the human host. Therefore, we isolated totalM. tuberculosis mRNA from human clinical respiratory specimens of patients diagnosed with pulmonary tuberculosis; after this, we synthesized the dscDNA and tested it by qualitative RT-PCR assays. We detected the expression of IS6110 insertion sequence and of the "housekeeping" genes 16SrRNA andsigA in M. tuberculosis grown in vivo (pulmonary tuberculosis as well as grown in vitro M

  9. Determining fertility in a bovine subject comprises detecting in a sample from the bovine subject the presence or absence of genetic marker alleles associated with a trait indicative of fertility of the bovine subject and/or off-spring

    DEFF Research Database (Denmark)

    2009-01-01

    for determining fertility in a bovine subject; and selecting bovine subjects for breeding purposes (all claimed). DETAILED DESCRIPTION - Determining fertility in a bovine subject comprises detecting in a sample from the bovine subject the presence or absence of two or more genetic marker alleles......NOVELTY - Determining fertility in a bovine subject comprises detecting in a sample from the bovine subject the presence or absence of two or more genetic marker alleles that are associated with a trait indicative of fertility of the bovine subject and/or off-spring. USE - The methods are useful...... purposes; and (2) a diagnostic kit for detecting the presence or absence in a bovine subject of two or more genetic marker alleles comprising a detection member....

  10. Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

    Science.gov (United States)

    Hiraiwa, Morgan; Kim, Jong-Hoon; Lee, Hyun-Boo; Inoue, Shinnosuke; Becker, Annie L.; Weigel, Kris M.; Cangelosi, Gerard A.; Lee, Kyong-Hoon; Chung, Jae-Hyun

    2015-05-01

    Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low-cost detection of Mycobacterium tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on the microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee-ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing and rinsing steps are presented with the use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU mL-1, comparable to a more labor-intensive fluorescence detection method reported previously.

  11. Bovine tuberculosis in African buffaloes: observations regarding Mycobacterium bovis shedding into water and exposure to environmental mycobacteria

    Directory of Open Access Journals (Sweden)

    van Helden Paul D

    2007-09-01

    Full Text Available Abstract Background African buffaloes are the maintenance host for Mycobacterium bovis in the endemically infected Kruger National Park (KNP. The infection is primarily spread between buffaloes via the respiratory route, but it is not known whether shedding of M. bovis in nasal and oral excretions may lead to contamination of ground and surface water and facilitate the transmission to other animal species. A study to investigate the possibility of water contamination with M. bovis was conducted in association with a BCG vaccination trial in African buffalo. Groups of vaccinated and nonvaccinated buffaloes were kept together with known infected in-contact buffalo cows to allow natural M. bovis transmission under semi-free ranging conditions. In the absence of horizontal transmission vaccinated and control buffaloes were experimentally challenged with M. bovis. Hence, all study buffaloes in the vaccination trial could be considered potential shedders and provided a suitable setting for investigating questions relating to the tenacity of M. bovis shed in water. Results Serial water samples were collected from the drinking troughs of the buffaloes once per season over an eleven-month period and cultured for presence of mycobacteria. All water samples were found to be negative for M. bovis, but 16 non-tuberculous Mycobacterium spp. isolates were cultured. The non-tuberculous Mycobacterium species were further characterised using 5'-16S rDNA PCR-sequencing, resulting in the identification of M. terrae, M. vaccae (or vanbaalenii, M. engbaekii, M. thermoresistibile as well as at least two species which have not yet been classified. Conclusion The absence of detectable levels of Mycobacterium bovis in the trough water suggests that diseased buffalo do not commonly shed the organism in high quantities in nasal and oral discharges. Surface water may therefore not be likely to play an important role in the transmission of bovine tuberculosis from buffalo

  12. Comparison of microscopy and PCR in detection of Mycobacterium tuberculosis

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    Pooja Saxena

    2014-12-01

    Full Text Available Objective: Tuberculosis (TB one of the most important major causes of mortality and morbidity around the world. Early detection of TB affords to prevent transmission of TB and better treatment options. The aim of this study is to evaluate sensitivity of fluorescent, Ziehl Neelsen microscopy and Polymerase Chain Reaction (PCR method. Methods: Fifty samples of clinically suspected cases of pulmonary tuberculosis were collected and processed for Myco­bacteria by Fluorescent and ZN staining. Negative samples by staining methods were processed by PCR method using primer IS6110 of M. tuberculosis complex. Results: 33/50 (66.0% samples were positive by Fluorescent and 23/50 (46% were positive by ZN method. All micro­scopically negative samples (n=17 were processed to PCR and only seven of samples (41.1% showed positive results. Conclusion: We concluded that PCR is best but it required lot of investment. J Microbiol Infect Dis 2014; 4(4: 141-144

  13. Occurrence and Distribution of bovine tuberculosis (Mycobacterium bovis in Slaughtered cattle in the abattoirs of Bauchi State, Nigeria

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    Adamu Saleh Saidu

    2015-03-01

    Full Text Available Aim: This study was aimed to determine the prevalence of bovine tuberculosis (bTB in slaughtered cattle in Bauchi State, Nigeria. The cause (s of grossly suspected bTB lesions encountered at the abattoirs during post-mortem (PM, as whether due to Mycobacterium bovis alone or together with other acid fast bacilli (AFB. Materials and Methods: A cross-sectional abattoir based study was conducted on 800 cattle slaughtered in the Northern, Central and Southern zonal abattoirs of Bauchi State, Nigeria, from June to August 2013; using PM meat inspection, Ziehl- Neelsen staining (ZN and confirmatory polymerase chain reaction (PCR techniques. Results: The occurrence of bTB lesions from the organs of slaughtered cattle in Bauchi State, showed that the lungs had the highest number of suspected tissues 65 (54.20%, followed by the lymph nodes 28 (23.30% while the heart, liver, spleen, intestines and mammary glands had the other 8.3%, 6.7%, 5.0%, 1.7%, and 0.8%, suspected tissues respectively. By ZN microscopic staining all 100% (2/2 of the intestines were positive for bTB, followed by the heart with 50% (5/10, then the lungs 29.23% (19/65; while the liver, lymph nodes, and spleen had 25%, 21.43% and 16.67% respectively were tested positive for bTB. It was only the mammary gland that tested negative for bTB in all the suspected tissues sampled. By PCR, the intestines had the highest positive bTB with 100% (2/2, followed by the liver with 12.5% (1/8, and then the lungs with 7.8% (5/65. The lymph nodes had 7.14% (2/28 tissues that tested positive for bTB. However, the spleen, heart and mammary gland were all tested negative with 0%; indicating that the false positive for bTB detected by ZN were confirmed by PCR. While based on the location of the abattoirs in the three senatorial zones of Bauchi State, Bauchi zonal abattoir had the highest number of suspected bTB cases 75 (62.50%, followed by Katagum zonal slaughter house with 32 (26.7% and then Misau with 13

  14. A restatement of the natural science evidence base relevant to the control of bovine tuberculosis in Great Britain.

    Science.gov (United States)

    Godfray, H Charles J; Donnelly, Christl A; Kao, Rowland R; Macdonald, David W; McDonald, Robbie A; Petrokofsky, Gillian; Wood, James L N; Woodroffe, Rosie; Young, Douglas B; McLean, Angela R

    2013-10-07

    Bovine tuberculosis (bTB) is a very important disease of cattle in Great Britain, where it has been increasing in incidence and geographical distribution. In addition to cattle, it infects other species of domestic and wild animals, in particular the European badger (Meles meles). Policy to control bTB is vigorously debated and contentious because of its implications for the livestock industry and because some policy options involve culling badgers, the most important wildlife reservoir. This paper describes a project to provide a succinct summary of the natural science evidence base relevant to the control of bTB, couched in terms that are as policy-neutral as possible. Each evidence statement is placed into one of four categories describing the nature of the underlying information. The evidence summary forms the appendix to this paper and an annotated bibliography is provided in the electronic supplementary material.

  15. Evidence of Mycobacterium tuberculosis complex bacteraemia in intradermal skin test positive cattle detected using phage-RPA.

    Science.gov (United States)

    Swift, Benjamin M C; Convery, Thomas W; Rees, Catherine E D

    2016-10-02

    Bovine tuberculosis is a zoonotic infectious disease caused by Mycobacterium bovis that affects cattle and can cause tuberculosis in a range of wildlife animals. A bacteriophage-based method combined with PCR (phage-PCR) has been recently used to detect and identify viable pathogenic mycobacteria in the peripheral blood mononuclear cells (PBMCs) of animals suffering from paratuberculosis. To adapt this method for the detection of M. bovis in blood, a new isothermal DNA amplification protocol using Recombinase Polymerase Amplification (RPA) was developed and was found to be able to detect M. bovis BCG within 48 h, with a limit of detection of approximately 10 cells per ml of blood for artificially inoculated blood samples. When blood samples (2 ml) from a Single Comparative Cervical Intradermal Tuberculin (SCCIT)- negative beef herd were tested, Mycobacterium tuberculosis complex (MTC) cells were not detected from any (45) of the blood samples. However when blood samples from SCCIT-positive animals were tested, viable MTC bacteria were detected in 66 % (27/41) of samples. Of these 41 animals sampled, 32 % (13) had visible lesions. In the visible lesion (VL) group, 85 % (11/13) had detectable levels of MTC whereas only 57 % (16/28) of animals which had no visible lesions (NVL) were found to have detectable mycobacteraemia. These results indicated that this simple, rapid method can be applied for the study of M. bovis infections. The frequency with which viable mycobacteria were detected in the peripheral blood of SCCIT-positive animals changes the paradigm of this disease.

  16. Bovine tuberculosis at a cattle-small ruminant-human interface in Meskan, Gurage region, Central Ethiopia

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    Tschopp Rea

    2011-11-01

    Full Text Available Abstract Background Bovine tuberculosis (BTB is endemic in Ethiopian cattle. The aim of this study was to assess BTB prevalence at an intensive contact interface in Meskan Woreda (district in cattle, small ruminants and suspected TB-lymphadenitis (TBLN human patients. Methods The comparative intradermal test (CIDT was carried out for all animals involved in the cross-sectional study and results interpreted using a > 4 mm and a > 2 mm cut-off. One PPD positive goat was slaughtered and lymph nodes subjected to culture and molecular typing. In the same villages, people with lymphadenitis were subjected to clinical examination. Fine needle aspirates (FNA were taken from suspected TBLN and analyzed by smear microscopy and molecular typing. Results A total of 1214 cattle and 406 small ruminants were tested for BTB. In cattle, overall individual prevalence (> 2 mm cut-off was 6.8% (CI: 5.4-8.5% with 100% herd prevalence. Only three small ruminants (2 sheep and 1 goat were reactors. The overall individual prevalence in small ruminants (> 2 mm cut-off was 0.4% (CI: 0.03-5.1% with 25% herd prevalence. Cattle from owners with PPD positive small ruminants were all PPD negative. 83% of the owners kept their sheep and goats inside their house at night and 5% drank regularly goat milk. FNAs were taken from 33 TBLN suspected cases out of a total of 127 screened individuals with lymph node swellings. Based on cytology results, 12 were confirmed TBLN cases. Nine out of 33 cultures were AFB positive. Culture positive samples were subjected to molecular typing and they all yielded M. tuberculosis. M. tuberculosis was also isolated from the goat that was slaughtered. Conclusions This study highlighted a low BTB prevalence in sheep and goats despite intensive contact with cattle reactors. TBLN in humans was caused entirely by M. tuberculosis, the human pathogen. M. tuberculosis seems to circulate also in livestock but their role at the interface is unknown.

  17. The ligase chain reaction as a primary screening tool for the detection of culture positive tuberculosis.

    LENUS (Irish Health Repository)

    O'Connor, T M

    2012-02-03

    BACKGROUND: The ligase chain reaction Mycobacterium tuberculosis assay uses ligase chain reaction technology to detect tuberculous DNA sequences in clinical specimens. A study was undertaken to determine its sensitivity and specificity as a primary screening tool for the detection of culture positive tuberculosis. METHODS: The study was conducted on 2420 clinical specimens (sputum, bronchoalveolar lavage fluid, pleural fluid, urine) submitted for primary screening for Mycobacterium tuberculosis to a regional medical microbiology laboratory. Specimens were tested in parallel with smear, ligase chain reaction, and culture. RESULTS: Thirty nine patients had specimens testing positive by the ligase chain reaction assay. Thirty two patients had newly diagnosed tuberculosis, one had a tuberculosis relapse, three had tuberculosis (on antituberculous therapy when tested), and three had healed tuberculosis. In the newly diagnosed group specimens were smear positive in 21 cases (66%), ligase chain reaction positive in 30 cases (94%), and culture positive in 32 cases (100%). Using a positive culture to diagnose active tuberculosis, the ligase chain reaction assay had a sensitivity of 93.9%, a specificity of 99.8%, a positive predictive value of 83.8%, and a negative predictive value of 99.9%. CONCLUSIONS: This study is the largest clinical trial to date to report the efficacy of the ligase chain reaction as a primary screening tool to detect Mycobacterium tuberculosis infection. The authors conclude that ligase chain reaction is a useful primary screening test for tuberculosis, offering speed and discrimination in the early stages of diagnosis and complementing traditional smear and culture techniques.

  18. The bovine tuberculosis burden in cattle herds in zones with low dose radiation pollution in the Ukraine

    Energy Technology Data Exchange (ETDEWEB)

    Weller, Richard E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Skrypnyk, Artem [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Zavgorodniy, Andriy [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Stegniy, Borys [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Gerilovych, Anton [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Kutsan, Oleksandr [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Pozmogova, Svitlana [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Sapko, Svitlana [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2009-02-01

    The authors describe a study of the tuberculosis (TB) incidence in cattle exposed to low doses of radiation resulting from the Chernobyl (pronounced ‘Chornobyl’ in Ukrainian) nuclear plant catastrophe in 1986. The purpose of the study was to determine if ionising radiation influences the number of outbreaks of bovine TB and their severity on farms in the Kyiv, Cherkasy and Chernigiv regions of the Ukraine. These farms are all located within a 200 km radius of Chernobyl and have had low-dose radiation pollution. Pathological and blood samples were taken from cattle in those regions that had positive TB skin tests. Mycobacterium spp. were isolated, differentiated by PCR, analysed and tested in guinea pigs and rabbits. Species differentiation showed a significant percentage of atypical mycobacteria, which resulted in the allergic reactions to tuberculin antigen in the skin test. Mixed infection of M. bovis and M. avium subsp. hominissuis was found in three cases. The results concluded that low-dose radiation plays a major role in the occurrence of bovine TB in regions affected by the Chernobyl nuclear disaster.

  19. Estimation of sensitivity and specificity of bacteriology, histopathology and PCR for the confirmatory diagnosis of bovine tuberculosis using latent class analysis.

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    Aurélie Courcoul

    Full Text Available Bacteriology and histopathology are the most commonly used tests used for official confirmatory diagnosis of bovine tuberculosis (bTB in cattle in most countries. PCR is also being used increasingly because it allows a fast diagnosis. This test could be applied as a supplement to or replacement for current bTB confirmatory diagnostic tests but its characteristics have first to be evaluated. The aim of this study was to estimate and compare sensitivities and specificities of bacteriology, histopathology and PCR under French field conditions, in the absence of a gold standard using latent class analysis. The studied population consisted of 5,211 animals from which samples were subjected to bacteriology and PCR (LSI VetMAX™ Mycobacterium tuberculosis Complex PCR Kit, Life Technologies as their herd of origin was either suspected or confirmed infected with bTB or because bTB-like lesions were detected during slaughterhouse inspection. Samples from 697 of these animals (all with bTB-like lesions were subjected to histopathology. Bayesian models were developed, allowing for dependence between bacteriology and PCR, while assuming independence from histopathology. The sensitivity of PCR was higher than that of bacteriology (on average 87.7% [82.5-92.3%] versus 78.1% [72.9-82.8%] while specificity of both tests was very good (on average 97.0% for PCR [94.3-99.0%] and 99.1% for bacteriology [97.1-100.0%]. Histopathology was at least as sensitive as PCR (on average 93.6% [89.9-96.9%] but less specific than the two other tests (on average 83.3% [78.7-87.6%]. These results suggest that PCR has the potential to replace bacteriology to confirm bTB in samples submitted from suspect cattle.

  20. Responses to diagnostic tests for bovine tuberculosis in dairy and non-dairy cattle naturally exposed to Mycobacterium bovis in Great Britain.

    Science.gov (United States)

    Downs, S H; Broughan, J M; Goodchild, A V; Upton, P A; Durr, P A

    2016-10-01

    Field surveillance of British cattle using the single intradermal comparative cervical tuberculin (SICCT) test shows a higher incidence rate of bovine tuberculosis (bTB) in dairy compared to beef herds, but a lower probability of post-mortem examination confirmed (PMC) Mycobacterium bovis infection in dairy herds. A cross-sectional study was conducted to compare animal level differences in bTB detection between dairy and non-dairy cattle in Great Britain. During the period from 2002 to 2005, 200 (41% dairy) reactors in the SICCT test (standard interpretation) were randomly selected, and 200 in-contact cattle (43% dairy) were purposively selected from bTB-infected herds. Interferon (IFN)-γ responses in blood to bovine and avian purified protein derivative (PPD), and early secretory antigen target 6 kDa and culture filtrate protein 10 (ESAT-6/CFP10), were measured. The post-mortem examination included gross pathological examination, mycobacterial culture and histopathology. The proportions of cattle positive to ESAT6/CFP10 were 26% (95% confidence interval, CI, 15-39%) in dairy reactors and 62% (95% CI 51-72%) in non-dairy reactors (P dairy reactors and 69% (95% CI 60-78%) in non-dairy reactors (P dairy reactors compared to non-dairy reactors, after controlling for bTB prevalence, herd size and SICCT test response, was 0.27 (95% CI 0.14-0.53; P dairy reactors than for beef reactors aged >2 years (P <0.001).

  1. Risk, knowledge and preventive measures of smallholder dairy farmers in northern Malawi with regard to zoonotic brucellosis and bovine tuberculosis

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    Stanly Fon Tebug

    2014-02-01

    Full Text Available Milk production using local cattle breed-types is an age-old practice in Malawi. Although dairy farming is becoming more common as a result of the increasing population and demand for milk and milk products, there is limited knowledge of the farmers’ awareness of zoonotic disease risks, their preventative practices and the disease burden in animals. This study determined dairy farmers’ general knowledge of zoonoses, assessed their risks for infection with zoonotic bovine tuberculosis (bTB and brucellosis, and evaluated farm practices to prevent disease transmission. A questionnaire was drawn up and administered by the authors. It was used to collect information about the knowledge and preventive practices of 140 out of 684 registered dairy farmers at Mzuzu Agricultural Development Division, northern Malawi. During a second visit to 60 out of the 140 farms, a total of 156 and 95 cattle were tested for brucellosis and tuberculosis, respectively. Most farmers (77.1% knew or had heard of zoonotic diseases, whilst 75.0% correctly named at least one zoonotic disease. More survey participants named tuberculosis as a zoonotic disease compared to brucellosis (74.3% versus 2.9%. The most commonly named means of transmission were milk (67.0% and meat (56.0%. Almost all survey participants (96.4% practised at least one farm activity that could lead to potential transmission of brucellosis or bTB, including sale (67.0% and consumption (34.0% of unpasteurised milk. Antibodies against brucellosis were found in 12 cattle (7.7%, whilst one animal (1.1% reacted to the tuberculin skin test. General knowledge about possible transmission of diseases between humans and animals was high, although most farmers practised risk behaviours that could potentially expose the public to milk-borne zoonotic diseases such as brucellosis and bTB. Furthermore, some animals had positive results for brucellosis and tuberculosis tests. Therefore, improvement of zoonotic disease

  2. Risk, knowledge and preventive measures of smallholder dairy farmers in northern Malawi with regard to zoonotic brucellosis and bovine tuberculosis.

    Science.gov (United States)

    Tebug, Stanly Fon; Njunga, Gilson R; Chagunda, Mizeck G G; Mapemba, Jacob P; Awah-Ndukum, Julius; Wiedemann, Steffi

    2014-02-28

    Milk production using local cattle breed-types is an age-old practice in Malawi. Although dairy farming is becoming more common as a result of the increasing population and demand for milk and milk products, there is limited knowledge of the farmers' awareness of zoonotic disease risks, their preventative practices and the disease burden in animals. This study determined dairy farmers' general knowledge of zoonoses, assessed their risks for infection with zoonotic bovine tuberculosis (bTB) and brucellosis, and evaluated farm practices to prevent disease transmission. A questionnaire was drawn up and administered by the authors. It was used to collect information about the knowledge and preventive practices of 140 out of 684 registered dairy farmers at Mzuzu Agricultural Development Division, northern Malawi. During a second visit to 60 out of the 140 farms, a total of 156 and 95 cattle were tested for brucellosis and tuberculosis, respectively. Most farmers (77.1%) knew or had heard of zoonotic diseases, whilst 75.0% correctly named at least one zoonotic disease. More survey participants named tuberculosis as a zoonotic disease compared to brucellosis (74.3% versus 2.9%). The most commonly named means of transmission were milk (67.0%) and meat (56.0%). Almost all survey participants (96.4%) practised at least one farm activity that could lead to potential transmission of brucellosis or bTB, including sale (67.0%) and consumption (34.0%) of unpasteurised milk. Antibodies against brucellosis were found in 12 cattle (7.7%), whilst one animal (1.1%) reacted to the tuberculin skin test. General knowledge about possible transmission of diseases between humans and animals was high, although most farmers practised risk behaviours that could potentially expose the public to milk-borne zoonotic diseases such as brucellosis and bTB. Furthermore, some animals had positive results for brucellosis and tuberculosis tests. Therefore, improvement of zoonotic disease prevention

  3. Polyfunctional cytokine responses by central memory CD4*T cells in response to bovine tuberculosis

    Science.gov (United States)

    CD4 T cells are crucial in immunity to tuberculosis (TB). Polyfunctional CD4 T cells simultaneously produce interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) and play relevant roles in several chronic infections, including human TB. Mycobacterium bovis in...

  4. Polyfunctional cytokine responses by central memory CD4+T cells in response to bovine tuberculosis

    Science.gov (United States)

    CD4 T cells are crucial in immunity to tuberculosis (TB). Polyfunctional CD4 T cells simultaneously produce interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) and play relevant roles in several chronic infections, including human TB and HIV. Mycobacterium ...

  5. Bovine Tuberculosis and Brucellosis in Traditionally Managed Livestock in Selected Districts of Southern Province of Zambia

    Directory of Open Access Journals (Sweden)

    J. B. Muma

    2013-01-01

    Full Text Available A study was performed in 2008 to estimate the prevalence of tuberculosis and brucellosis in traditionally reared cattle of Southern Province in Zambia in four districts. The single comparative intradermal tuberculin test (SCITT was used to identify TB reactors, and the Rose Bengal test (RBT, followed by confirmation with competitive enzyme-linked immunosorbent assay (c-ELISA, was used to test for brucellosis. A total of 459 animals were tested for tuberculosis and 395 for brucellosis. The overall prevalence of BTB based on the 4 mm and 3 mm cutoff criteria was 4.8% (95% CI: 2.6–7.0% and 6.3% (95% CI: 3.8–8.8%, respectively. Change in skin thickness on SCITT was influenced by initial skin-fold thickness at the inoculation site, where animals with thinner skin had a tendency to give a larger tuberculin response. Brucellosis seroprevalence was estimated at 20.7% (95% CI: 17.0–24.4%. Comparison between results from RBT and c-ELISA showed good agreement (84.1% and revealed subjectivity in RBT test results. Differences in brucellosis and tuberculosis prevalence across districts were attributed to type of husbandry practices and ecological factors. High prevalence of tuberculosis and brucellosis suggests that control control programmes are necessary for improved cattle productivity and reduced public health risk.

  6. Rapid detection of tuberculosis using droplet-based microfluidics

    Science.gov (United States)

    Rosenfeld, Liat; Cheng, Yunfeng; Rao, Jianghong; Tang, Sindy K. Y.

    2014-03-01

    Tuberculosis is one of the most deadly diseases that kills over one million people each year and infects one-third of the world's population. The disease is spread by infection with Mycobacterium tuberculosis (Mtb). Owing to its airborne transmission, early diagnosis is critical to the prevention and control of TB. Standard diagnostic methods, acid-fast smear from sputum, often do not become positive until after transmission occurs, which allows the spread of the disease. Culture-based techniques are more sensitive, but take weeks to obtain results because of the extremely slow growth rate of Mtb. In this study a new method to detect indicator enzyme based on the isolation of tubercle bacillus in a large number of picoliter droplets combined with a fluorescent probe has been developed. We use BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and a designed BlaC-specific fluorogenic substrates as probes for Mtb detection. We present here a new method to detect the indicator enzyme based on the isolation, digitization and concentration of bacteria samples in a large number of picoliter drops. We show that by controlling the size of the droplets we can control the rate of conversion. Hence rapid increase in signal has been observed as the size of the drops has been decreased. Our vision is that this tool will be able to detect tubercle bacilli in a sensitive, rapid, specific and quantitative manner in vitro at a low cost, particularly in resource limited settings where TB is the most prevalent.

  7. Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.

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    David S Boyle

    Full Text Available Improved access to effective tests for diagnosing tuberculosis (TB has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110 and 20 fg (IS1081were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9 and 86.1% (95%CI: 78.1, 94.1 respectively (n = 71. Specificities were 100% and 88.6% (95% CI: 80.8, 96.1 respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2 and 70.8% (95%CI: 62.9, 78.7 were obtained (n = 90. Specificities were 95.4 (95% CI: 92.3,98.1 and 88% (95% CI: 83.6, 92.4 respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB

  8. Detection of anti-tuberculosis activity in some folklore plants by radiometric BACTEC assay.

    Science.gov (United States)

    Gupta, V K; Shukla, C; Bisht, G R S; Saikia, D; Kumar, S; Thakur, R L

    2011-01-01

    The anti-tubercular drugs are less effective because of the emergence of multi-drug resistant (MDR) and extensively drug resistant (XDR) strains of M. tuberculosis, so plants being an alternative source of anti-microbial compounds. The aim of this study was to investigate anti-tuberculosis potential of the plants using Mycobacterium smegmatis as a rapid screening model for detection of anti-mycobacterial activity and further to evaluate the active plants for anti-tuberculosis activity against M. tuberculosis using radiometric BACTEC assay. The 15 plants were screened for anti-mycobacterial activity against M. smegmatis by the disk diffusion assay. The ethanolic extracts of Mallotus philippensis, Vitex negundo, Colebrookea oppositifolia, Rumex hastatus, Mimosa pudica, Kalanchoe integra and Flacourtia ramontchii were active against M. smegmatis in primary screening. The anti-tuberculosis potential was identified in the leaves extracts of Mallotus philippensis by radiometric BACTEC assay. The ethanolic extract of M. philippensis showed anti-tuberculosis activity against virulent and avirulent strains of M. tuberculosis H(37) Rv and M. tuberculosis H(37) Ra with minimum inhibitory concentration 0·25 and 0·125 mg ml(-1), respectively. The inhibition in growth index values of M. tuberculosis was observed in the presence of ethyl acetate fraction at a minimum concentration of 0·05 mg ml(-1). We found that BACTEC radiometric assay is a valuable method for detection of anti-tuberculosis activity of the plant extracts. The results indicate that ethanolic extract and ethyl acetate fraction of M. philippensis exhibited significant anti-mycobacterial activity against M. tuberculosis. These findings provide scientific evidence to support the traditional medicinal uses of M. philippensis and indicate a promising potential of this plant for the development of anti-tuberculosis agent. © 2010 The Authors. Letters in Applied Microbiology © 2010 The Society for Applied

  9. Effect of culling and vaccination on bovine tuberculosis infection in a European badger (Meles meles) population by spatial simulation modelling.

    Science.gov (United States)

    Abdou, Marwa; Frankena, Klaas; O'Keeffe, James; Byrne, Andrew W

    2016-03-01

    The control of bovine tuberculosis (bTB) in cattle herds in the Republic of Ireland (ROI) is partially hindered by spill-back infection from wild badgers (Meles meles). The aim of this study was to determine the relative effects of interventions (combinations of culling and/or vaccination) on bTB dynamics in an Irish badger population. A spatial agent-based stochastic simulation model was developed to evaluate the effect of various control strategies for bovine tuberculosis in badgers: single control strategies (culling, selective culling, vaccination, and vaccine baits), and combined strategies (Test vaccinate/cull (TVC)), split area approaches using culling and vaccination, or selective culling and vaccination, and mixed scenarios where culling was conducted for five years and followed by vaccination or by a TVC strategy. The effect of each control strategy was evaluated over a 20-year period. Badger control was simulated in 25%, 50%, and 75% area (limited area strategy) or in the entire area (100%, wide area strategy). For endemic bTB, a culling strategy was successful in eradicating bTB from the population only if applied as an area-wide strategy. However, this was achieved only by risking the extinction of the badger population. Selective culling strategies (selective culling or TVC) mitigated this negative impact on the badger population's viability. Furthermore, both strategies (selective culling and TVC) allowed the badger population to recover gradually, in compensation for the population reduction following the initial use of removal strategies. The model predicted that vaccination can be effective in reducing bTB prevalence in badgers, when used in combination with culling strategies (i.e. TVC or other strategies). If fecundity was reduced below its natural levels (e.g. by using wildlife contraceptives), the effectiveness of vaccination strategies improved. Split-area simulations highlighted that interventions can have indirect effects (e.g. on

  10. Microbiological analysis of oral samples for detection of Mycobacterium tuberculosis by nested polymerase chain reaction in tuberculosis patients with periodontitis

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    Sunil Kumar Palakuru

    2012-01-01

    Conclusion: The TB status of the patient did not influence the periodontal status. However, the presence of M. tuberculosis in plaque and saliva as detected in this study might pose a grave risk of transmission of the disease through aerosols during dental procedures.

  11. Spatial Targeting for Bovine Tuberculosis Control: Can the Locations of Infected Cattle Be Used to Find Infected Badgers?

    Science.gov (United States)

    Smith, Catherine M; Downs, Sara H; Mitchell, Andy; Hayward, Andrew C; Fry, Hannah; Le Comber, Steven C

    2015-01-01

    Bovine tuberculosis is a disease of historical importance to human health in the UK that remains a major animal health and economic issue. Control of the disease in cattle is complicated by the presence of a reservoir species, the Eurasian badger. In spite of uncertainty in the degree to which cattle disease results from transmission from badgers, and opposition from environmental groups, culling of badgers has been licenced in two large areas in England. Methods to limit culls to smaller areas that target badgers infected with TB whilst minimising the number of uninfected badgers culled is therefore of considerable interest. Here, we use historical data from a large-scale field trial of badger culling to assess two alternative hypothetical methods of targeting TB-infected badgers based on the distribution of cattle TB incidents: (i) a simple circular 'ring cull'; and (ii) geographic profiling, a novel technique for spatial targeting of infectious disease control that predicts the locations of sources of infection based on the distribution of linked cases. Our results showed that both methods required coverage of very large areas to ensure a substantial proportion of infected badgers were removed, and would result in many uninfected badgers being culled. Geographic profiling, which accounts for clustering of infections in badger and cattle populations, produced a small but non-significant increase in the proportion of setts with TB-infected compared to uninfected badgers included in a cull. It also provided no overall improvement at targeting setts with infected badgers compared to the ring cull. Cattle TB incidents in this study were therefore insufficiently clustered around TB-infected badger setts to design an efficient spatially targeted cull; and this analysis provided no evidence to support a move towards spatially targeted badger culling policies for bovine TB control.

  12. [Particular features of detection of patients with urogenital tuberculosis and their management].

    Science.gov (United States)

    Zhuravlev, V N; Golubev, D N; Novikov, B I; Skorniakov, S N; Medvinskiĭ, I D; Arkanov, L V; Cherniaev, I A; Borodin, É P; Verbetskiĭ, A F; Bobykin, E N

    2012-01-01

    The rate and trend in extrapulmonary tuberculosis (TB) incidence including urogenital tuberculosis (UTB) were estimated in population of the Sverdlovsk region for the last 25 years. Long-term results of treatment of 591 patients with different forms of UTB (renal parenchyma TB, tuberculosis papillitis, monocavernous and polycavernous renal TB, male genital TB) were studied. Ureter was involved in tuberculosis process in 24.7% of UTB cases, urinary bladder--in 20.1%, renal TB combined with male genital TB. Early (non-destructive) forms incidence increased 2.8-fold while advanced forms incidence decreased 1.7-fold. This shows an increased level of detection. Total number of patients operated in state hospitals with undetected, mostly complicated urogenital male tuberculosis remains high--from 7.3 to 16% from all newly detected patients.

  13. Tuberculosis

    OpenAIRE

    Latorre Tortello, Pablo

    2011-01-01

    Por definición, la tuberculosis pulmonar es la localizaci6n del M. tuberculosis en el tracto respiratorio, la forma más común y principal de la afección y la única capaz de contagiar a otras personas. El M. tuberculosis, descubierto por Robert Koch en 1882, el bacilo de Koch, es un bacilo delgado, inmóvil, de 4 micras de longitud media, aerobio obligado, que se tiñe de rajo por la tinción de Ziehl-Neelsen. Debido a la coraza lipídica de su pared, lo hace resistente a la decoloración con ácido...

  14. Tuberculosis

    OpenAIRE

    Latorre Tortello, Pablo

    2011-01-01

    Por definición, la tuberculosis pulmonar es la localizaci6n del M. tuberculosis en el tracto respiratorio, la forma más común y principal de la afección y la única capaz de contagiar a otras personas. El M. tuberculosis, descubierto por Robert Koch en 1882, el bacilo de Koch, es un bacilo delgado, inmóvil, de 4 micras de longitud media, aerobio obligado, que se tiñe de rajo por la tinción de Ziehl-Neelsen. Debido a la coraza lipídica de su pared, lo hace resistente a la decoloración con ácido...

  15. Evaluating wildlife-cattle contact rates to improve the understanding of dynamics of bovine tuberculosis transmission in Michigan, USA.

    Science.gov (United States)

    Lavelle, Michael J; Kay, Shannon L; Pepin, Kim M; Grear, Daniel A; Campa, Henry; VerCauteren, Kurt C

    2016-12-01

    Direct and indirect contacts among individuals drive transmission of infectious disease. When multiple interacting species are susceptible to the same pathogen, risk assessment must include all potential host species. Bovine tuberculosis (bTB) is an example of a disease that can be transmitted among several wildlife species and to cattle, although the potential role of several wildlife species in spillback to cattle remains unclear. To better understand the complex network of contacts and factors driving disease transmission, we fitted proximity logger collars to beef and dairy cattle (n=37), white-tailed deer (Odocoileus virginianus; n=29), raccoon (Procyon lotor; n=53), and Virginia opossum (Didelphis virginiana; n=79) for 16 months in Michigan's Lower Peninsula, USA. We determined inter- and intra-species direct and indirect contact rates. Data on indirect contact was calculated when collared animals visited stationary proximity loggers placed at cattle feed and water resources. Most contact between wildlife species and cattle was indirect, with the highest contact rates occurring between raccoons and cattle during summer and fall. Nearly all visits (>99%) to cattle feed and water sources were by cattle, whereas visitation to stored cattle feed was dominated by deer and raccoon (46% and 38%, respectively). Our results suggest that indirect contact resulting from wildlife species visiting cattle-related resources could pose a risk of disease transmission to cattle and deserves continued attention with active mitigation.

  16. Preventing the Establishment of a Wildlife Disease Reservoir: A Case Study of Bovine Tuberculosis in Wild Deer in Minnesota, USA

    Directory of Open Access Journals (Sweden)

    Michelle Carstensen

    2011-01-01

    Full Text Available Bovine tuberculosis (bTB has been found in 12 cattle operations and 27 free-ranging white-tailed deer (Odocoileus virginianus in northwestern Minnesota, following the state's most recent outbreak of the disease in 2005 in the northwest part of the state. Both deer and cattle have the same strain of bTB. The Minnesota Board of Animal Health has been leading efforts to eradicate the disease in Minnesota's cattle, which have included the depopulation of all infected herds, a cattle buy-out program, and mandatory fencing of stored feeds. The Minnesota Department of Natural Resources began surveillance efforts in free-ranging white-tailed deer in fall 2005. All bTB-infected deer have been found within a 16 km2 area in direct association with infected cattle farms. Aggressive efforts to reduce deer densities through liberalized hunting and sharpshooting have resulted in a 55% decline in deer densities. Also, recreational feeding of wild deer has been banned. Disease prevalence in deer has decreased from 1.2% in 2005 to an undetectable level in 2010.

  17. A review of bovine tuberculosis at the wildlife-livestock-human interface in sub-Saharan Africa.

    Science.gov (United States)

    De Garine-Wichatitsky, M; Caron, A; Kock, R; Tschopp, R; Munyeme, M; Hofmeyr, M; Michel, A

    2013-07-01

    Infection of wild animals by bovine tuberculosis (bTB) is raising concern worldwide. This article reviews the current epidemiological situation, risk of emergence and control options at the wildlife–livestock–human interface in sub-Saharan Africa. In livestock, bTB has been confirmed in the majority of countries from all parts of the continent. Wildlife infection is confirmed in seven countries from southern and eastern Africa, apparently spreading in the southern Africa region. Mycobacterium bovis has been isolated from 17 wild mammal species, although only four are suspected to play a role as maintenance host. Zoonotic risks are a concern, but no direct spillover from wildlife to humans has been documented, and no case of bTB spillback from wildlife to livestock has been confirmed. In this paper we assess the main risk factors of bTB spillover at the wildlife–livestock–human interface and suggest several research themes which could improve the control of the disease in the African context.

  18. Incorporating farmer observations in efforts to manage bovine tuberculosis using barrier fencing at the wildlife-livestock interface.

    Science.gov (United States)

    Brook, Ryan K

    2010-05-01

    A federal and provincial cost-shared program was initiated in 2001 around Riding Mountain National Park in southwestern Manitoba, Canada to provide free game wire barrier fences for baled hay storage areas to prevent transmission of TB among cattle (Bos taurus), wild elk (Cervus elaphus), and deer (Odocoileus virginianus). Farmer observations of cervids on their farms were evaluated by interviewing 50 farmers that owned a game wire fence for >1 year. Of those interviewed, 82% reported some type of elk or deer damage to hay bales in the field or in their yard prior to fencing. After fencing, 23% of respondents reported some annual damage to stored hay bales that were not inside the fence, but there was a 100% decrease in the estimated annual value of hay losses. Incursions of deer inside the barrier fence were reported by 18% of respondents and most of these were due to leaving gates open. No incursions of elk inside a barrier fence were reported. Despite the important successes achieved, barrier fencing of hay bales alone does not completely protect cattle from bovine tuberculosis.

  19. Effect of host diversity and species assemblage composition on bovine tuberculosis (bTB) risk in Ethiopian cattle.

    Science.gov (United States)

    Sintayehu, Dejene W; Heitkönig, Ignas M A; Prins, Herbert H T; Tessema, Zewdu K; DE Boer, Willem F

    2017-05-01

    Current theories on diversity-disease relationships describe host species diversity and species identity as important factors influencing disease risk, either diluting or amplifying disease prevalence in a community. Whereas the simple term 'diversity' embodies a set of animal community characteristics, it is not clear how different measures of species diversity are correlated with disease risk. We therefore tested the effects of species richness, Pielou's evenness and Shannon's diversity on bovine tuberculosis (bTB) risk in cattle in the Afar Region and Awash National Park between November 2013 and April 2015. We also analysed the identity effect of a particular species and the effect of host habitat use overlap on bTB risk. We used the comparative intradermal tuberculin test to assess the number of bTB-infected cattle. Our results suggested a dilution effect through species evenness. We found that the identity effect of greater kudu - a maintenance host - confounded the dilution effect of species diversity on bTB risk. bTB infection was positively correlated with habitat use overlap between greater kudu and cattle. Different diversity indices have to be considered together for assessing diversity-disease relationships, for understanding the underlying causal mechanisms. We posit that unpacking diversity metrics is also relevant for formulating disease control strategies to manage cattle in ecosystems characterized by seasonally limited resources and intense wildlife-livestock interactions.

  20. Developing a risk-based trading scheme for cattle in England: farmer perspectives on managing trading risk for bovine tuberculosis.

    Science.gov (United States)

    Little, R; Wheeler, K; Edge, S

    2017-02-11

    This paper examines farmer attitudes towards the development of a voluntary risk-based trading scheme for cattle in England as a risk mitigation measure for bovine tuberculosis (bTB). The research reported here was commissioned to gather evidence on the type of scheme that would have a good chance of success in improving the information farmers receive about the bTB risk of cattle they buy. Telephone interviews were conducted with a stratified random sample of 203 cattle farmers in England, splitting the interviews equally between respondents in the high-risk area and low-risk area for bTB. Supplementary interviews and focus groups with farmers were also carried out across the risk areas. Results suggest a greater enthusiasm for a risk-based trading scheme in low-risk areas compared with high-risk areas and among members of breed societies and cattle health schemes. Third-party certification of herds by private vets or the Animal and Plant Health Agency were regarded as the most credible source, with farmer self-certification being favoured by sellers, but being regarded as least credible by buyers. Understanding farmers' attitudes towards voluntary risk-based trading is important to gauge likely uptake, understand preferences for information provision and to assist in monitoring, evaluating and refining the scheme once established.

  1. Mycobacterium genotypes in pulmonary tuberculosis infections and their detection by trained African giant pouched rats.

    Science.gov (United States)

    Mgode, Georgies F; Cohen-Bacrie, Stéphan; Bedotto, Marielle; Weetjens, Bart J; Cox, Christophe; Jubitana, Maureen; Kuipers, Dian; Machang'u, Robert S; Kazwala, Rudovick; Mfinanga, Sayoki G; Kaufmann, Stefan H E; Drancourt, Michel

    2015-02-01

    Tuberculosis (TB) diagnosis in low-income countries is mainly done by microscopy. Hence, little is known about the diversity of Mycobacterium spp. in TB infections. Different genotypes or lineages of Mycobacterium tuberculosis vary in virulence and induce different inflammatory and immune responses. Trained Cricetomys rats show a potential for rapid diagnosis of TB. They detect over 28 % of smear-negative, culture-positive TB. However, it is unknown whether these rats can equally detect sputa from patients infected with different genotypes of M. tuberculosis. A 4-month prospective study on diversity of Mycobacterium spp. was conducted in Dar es Salaam, Tanzania. 252 sputa from 161 subjects were cultured on Lowenstein-Jensen medium and thereafter tested by rats. Mycobacterial isolates were subjected to molecular identification and multispacer sequence typing (MST) to determine species and genotypes. A total of 34 Mycobacterium spp. isolates consisting of 32 M. tuberculosis, 1 M. avium subsp. hominissuis and 1 M. intracellulare were obtained. MST analyses of 26 M. tuberculosis isolates yielded 10 distinct MST genotypes, including 3 new genotypes with two clusters of related patterns not grouped by geographic areas. Genotype MST-67, shared by one-third of M. tuberculosis isolates, was associated with the Mwananyamala clinic. This study shows that diverse M. tuberculosis genotypes (n = 10) occur in Dar es Salaam and trained rats detect 80 % of the genotypes. Sputa with two M. tuberculosis genotypes (20 %), M. avium hominissuis and M. intracellulare were not detected. Therefore, rats detect sputa with different M. tuberculosis genotypes and can be used to detect TB in resource-poor countries.

  2. Prevalência da tuberculose em bovinos abatidos em Minas Gerais Prevalence of tuberculosis among bovines slaughtered in Minas Gerais, Brazil

    Directory of Open Access Journals (Sweden)

    F. Baptista

    2004-10-01

    Full Text Available A prevalência da tuberculose em bovinos abatidos em Minas Gerais, de 1993 a 1997, em 10 matadouros sujeitos à Inspeção Federal, foi de 0,7‰. Os bovinos procederam principalmente deste Estado (74% e de Goiás (25%. A prevalência variou, temporal e espacialmente, entre zero e 8,7‰ e é idêntica à de outros levantamentos parciais feitos no Brasil desde 1924. Contribuíram para ela o local de abate e o perfil dos bovinos abatidos. Em Minas Gerais foi de 0,8‰ e em Goiás 0,4‰. A tuberculose foi diagnosticada em 90 municípios de Minas Gerais e em 17 de Goiás, correspondendo a 16,8% e 12,7% dos municípios mineiros e goianos, respectivamente. A prevalência da tuberculose em Minas Gerais foi maior nos bovinos abatidos na Região Sudoeste (1,7‰.The prevalence of tuberculosis among bovines slaughtered in Minas Gerais State, Brazil, from 1993 to 1997, in 10 slaughterhouses under Federal Inspection, was 0.7‰. The cattle was native mainly from Minas Gerais and Goiás states, respectively 74% and 25%. Prevalence of tuberculosis varied, temporal and spatially, from zero to 8.7‰. This variation was also observed in other partial studies in Brazil since 1924. Place of slaughter and the kind of bovines slaughtered contributed to this variation. The prevalence of tuberculosis in Minas Gerais cattle was 0.8‰ and that of Goiás was 0.4‰. Of investigated municipalities in Minas Gerais, 16.8% were affected by tuberculosis. The occurrence in cattle of municipalities of Goiás was 12.7%. In Minas Gerais, the Southwest Region presented the highest prevalence of tuberculosis (1.7‰.

  3. Detection of Mycobacterium tuberculosis Complex in New World Monkeys in Peru.

    Science.gov (United States)

    Rosenbaum, Marieke; Mendoza, Patricia; Ghersi, Bruno M; Wilbur, Alicia K; Perez-Brumer, Amaya; Cavero Yong, Nancy; Kasper, Matthew R; Montano, Silvia; Zunt, Joseph R; Jones-Engel, Lisa

    2015-06-01

    The Mycobacterium tuberculosis complex causes tuberculosis in humans and nonhuman primates and is a global public health concern. Standard diagnostics rely upon host immune responses to detect infection in nonhuman primates and lack sensitivity and specificity across the spectrum of mycobacterial infection in these species. We have previously shown that the Oral Swab PCR (OSP) assay, a direct pathogen detection method, can identify the presence of M. tuberculosis complex in laboratory and free-ranging Old World monkeys. Addressing the current limitations in tuberculosis diagnostics in primates, including sample acquisition and pathogen detection, this paper furthers our understanding of the presence of the tuberculosis-causing bacteria among New World monkeys in close contact with humans. Here we use the minimally invasive OSP assay, which includes buccal swab collection followed by amplification of the IS6110 repetitive nucleic acid sequence specific to M. tuberculosis complex subspecies, to detect the bacteria in the mouths of Peruvian New World monkeys. A total of 220 buccal swabs from 16 species were obtained and positive amplification of the IS6110 sequence was observed in 30 (13.6%) of the samples. To our knowledge, this is the first documentation of M. tuberculosis complex DNA in a diverse sample of Peruvian Neotropical primates.

  4. Evaluation of DNA microarray for detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates

    Institute of Scientific and Technical Information of China (English)

    王峰

    2013-01-01

    Objective To evaluate the performance of DNA microarray for rapid detection resistance to rifampin and isoniazid in Mycobacterium tuberculosis clinical isolates and identify suitable target sites for molecular genetic test. Methods Twenty-four clinical Mycobacterium

  5. Bovine tuberculosis in Iran: The past, present and future of a national disease control program

    Directory of Open Access Journals (Sweden)

    Keyvan Tadayon

    2015-01-01

    Bovine TB was initially reported in Iran by a French veterinarian in local breeds of cattle. An official attempt to control the disease was started in the 1940s, which runs today on a national scale. This mini-review addresses a variety of different epidemiological issues in bTb control in the world and in Iran from an immunologist's eye to find the cure for human cases infected with M. bovis. In addition, the benefits and drawbacks of this control scheme are discussed.

  6. One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3

    Directory of Open Access Journals (Sweden)

    Thonur Leenadevi

    2012-03-01

    Full Text Available Abstract Background Detection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR to detect the viruses commonly implicated in BRD. Results A mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV, bovine herpes virus type 1 (BoHV-1 and bovine parainfluenza virus type 3 (BPI3 i & ii nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using in vitro transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI and/or indirect fluorescent antibody test (IFAT. Conclusions The mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 102 copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA, minor groove binding (MGB and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD.

  7. Early and increased tuberculosis case detection: Implementation, measurement, and evaluation

    NARCIS (Netherlands)

    Creswell, J.H.

    2015-01-01

    Despite substantial millions of people being treated by National Tuberculosis Control Programs (NTPs) and their partners over the last 25 years and millions of lives being saved, tuberculosis (TB) continues to be a leading cause of morbidity and mortality in many low- and middle-income countries and

  8. Early and increased tuberculosis case detection: Implementation, measurement, and evaluation

    NARCIS (Netherlands)

    Creswell, J.H.

    2015-01-01

    Despite substantial millions of people being treated by National Tuberculosis Control Programs (NTPs) and their partners over the last 25 years and millions of lives being saved, tuberculosis (TB) continues to be a leading cause of morbidity and mortality in many low- and middle-income countries and

  9. Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates

    OpenAIRE

    Maningi, Nontuthuko E.; Daum, Luke T.; Rodriguez, John D.; Mphahlele, Matsie; Peters, Remco P. H.; Fischer, Gerald W.; Chambers, James P.; Fourie, P. Bernard

    2015-01-01

    The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolat...

  10. Early and efficient detection of Mycobacterium tuberculosis in sputum by microscopic observation of broth cultures.

    Directory of Open Access Journals (Sweden)

    Benson R Kidenya

    Full Text Available Early, efficient and inexpensive methods for the detection of pulmonary tuberculosis are urgently needed for effective patient management as well as to interrupt transmission. These methods to detect M. tuberculosis in a timely and affordable way are not yet widely available in resource-limited settings. In a developing-country setting, we prospectively evaluated two methods for culturing and detecting M. tuberculosis in sputum. Sputum samples were cultured in liquid assay (micro broth culture in microplate wells and growth was detected by microscopic observation, or in Löwenstein-Jensen (LJ solid media where growth was detected by visual inspection for colonies. Sputum samples were collected from 321 tuberculosis (TB suspects attending Bugando Medical Centre, in Mwanza, Tanzania, and were cultured in parallel. Pulmonary tuberculosis cases were diagnosed using the American Thoracic Society diagnostic standards. There were a total of 200 (62.3% pulmonary tuberculosis cases. Liquid assay with microscopic detection detected a significantly higher proportion of cases than LJ solid culture: 89.0% (95% confidence interval [CI], 84.7% to 93.3% versus 77.0% (95% CI, 71.2% to 82.8% (p = 0.0007. The median turn around time to diagnose tuberculosis was significantly shorter for micro broth culture than for the LJ solid culture, 9 days (interquartile range [IQR] 7-13, versus 21 days (IQR 14-28 (p<0.0001. The cost for micro broth culture (labor inclusive in our study was US $4.56 per sample, versus US $11.35 per sample for the LJ solid culture. The liquid assay (micro broth culture is an early, feasible, and inexpensive method for detection of pulmonary tuberculosis in resource limited settings.

  11. From mosques to classrooms: mobilizing the community to enhance case detection of tuberculosis.

    Science.gov (United States)

    Rifat, Mahfuza; Rusen, I D; Mahmud, Mohammad Hasan; Nayer, Israt; Islam, Akramul; Ahmed, Faruque

    2008-09-01

    In response to the global challenge of inadequate case detection of tuberculosis (TB), the Fund for Innovative DOTS Expansion through Local Initiatives to Stop Tuberculosis (FIDELIS) was developed in 2003 to rapidly assess and implement innovative approaches to increase the detection of new smear-positive TB cases. As previously reported, a wide range of target populations and interventions has been incorporated into successful FIDELIS projects.

  12. Detection of streptomycin residues in local meat of bovine and ovine

    Directory of Open Access Journals (Sweden)

    O. A. Abdullah

    2012-01-01

    Full Text Available From meat retails in Mosul province, forty-five meat samples of local ovine and bovine (23 bovine samples and 22 ovine samples were collected. The period of collection was during November 2010 to May 2011, by means of multistage random sampling for detection of streptomycin residues. Enzyme linked immunosorbent assay (ELISA was used for detection of streptomycin residues. The results revealed that eleven ovine meat samples (50% were positive to streptomycin residue, with a mean value 35.06 µg kg-1, while 14 bovine meat samples (60.86% were positive to residual streptomycin with a mean value 59.56 µg kg-1. From the results, it is clear that all tested meat samples (ovine and bovine were safe enough for human consumption.

  13. Increasing tuberculosis case detection: lessons from the Republic of Moldova.

    Science.gov (United States)

    Soltan, Viorel; Henry, Asma Khalid; Crudu, Valeriu; Zatusevski, Irina

    2008-01-01

    The Republic of Moldova undertook reforms in tuberculosis (TB) control and health care consistent with international recommendations and advanced towards the global target for case detection. The number of TB cases notified increased overall by 50% during 2001-2005. Expansion of the DOTS strategy and full coverage coincided with a greater role for primary health care (PHC) in TB control and the advent of national insurance for TB diagnosis and treatment. These developments and improvements in laboratories, surveillance, medical personnel skills, and public awareness contributed to increased case detection. The Republic of Moldova addressed both demand and supply sides in these efforts. It increased effective demand for TB services by dispersing diagnostic capability, instituting financing mechanisms and saturating the public with information on symptoms, transmission and treatment. It increased the supply of TB services by upgrading the laboratory network, revamping surveillance and training practitioners. The Republic of Moldova's experience offers lessons for other countries: TB-PHC integration allowed more suspect cases to be diagnosed at nearby PHC clinics, contributing to more cases being notified. Innovative TB communications reached the general public, vulnerable groups, practitioners and the media. TB control projects built on each other and national coordination mechanisms served to identify funding for the most pressing needs. There are challenges remaining for TB control in the Republic of Moldova, not least the stable treatment success rate, but the country can list valuable lessons and achievements.

  14. Isolation and identification of bovine tuberculosis in a Brazilian herd (São Paulo

    Directory of Open Access Journals (Sweden)

    AFC Nassar

    2007-08-01

    Full Text Available Mycobacterium was verified in animals from a Brazilian dairy herd, a total of 42 samples from 30 cows were submitted to culture and the isolated strains were analyzed by two polymerase chain reaction (PCR, the first specific for species belonging to the Mycobacterium complex (MTBC and the other for differentiating M. tuberculosis from M. bovis. Twenty seven samples (64.3% from 18 animals (60% were positive for mycobacteria by culture, including samples from 15 retrofaryngeal lymphnodes (55.5%, 9 prescapular lymphnodes (33.3%, 2 lungs (7.4%, and 1 liver (3.7%. All isolated colonies were confirmed by PCR to contain MTBC organisms, and were identified as M. bovis by the same methodology.

  15. Molecular Detection of Mixed Infections of Mycobacterium tuberculosis Strains in Sputum Samples from Patients in Karonga District, Malawi ▿ †

    OpenAIRE

    Mallard, Kim; McNerney, Ruth; Crampin, Amelia C; Houben, Rein; Ndlovu, Richard; Munthali, Lumbani; Warren, Robin M.; French, Neil; Judith R Glynn

    2010-01-01

    The occurrence of mixed infections of Mycobacterium tuberculosis is no longer disputed. However, their frequency, and the impact they may have on our understanding of tuberculosis (TB) pathogenesis and epidemiology, remains undetermined. Most previous studies of frequency applied genotyping techniques to cultured M. tuberculosis isolates and found mixed infections to be rare. PCR-based techniques may be more sensitive for detecting multiple M. tuberculosis strains and can be applied to sputum...

  16. Tuberculosis.

    Science.gov (United States)

    Jacobson, Karen R

    2017-02-07

    This issue provides a clinical overview of tuberculosis, focusing on screening, prevention, diagnosis, and treatment. The content of In the Clinic is drawn from the clinical information and education resources of the American College of Physicians (ACP), including MKSAP (Medical Knowledge and Self-Assessment Program). Annals of Internal Medicine editors develop In the Clinic in collaboration with the ACP's Medical Education and Publishing divisions and with the assistance of additional science writers and physician writers.

  17. Evaluation of the interferon-γ assay on blood collected at exsanguination of cattle under field conditions for surveillance of bovine tuberculosis.

    Science.gov (United States)

    Okafor, C C; Grooms, D L; Bolin, S R; Averill, J J; Kaneene, J B

    2014-12-01

    Development of point of concentration (POC) surveillance strategies for bovine tuberculosis (bTB) would facilitate global efforts to eradicate bTB. The interferon-gamma (IFNγ) assay can detect IFNγ responses to Mycobacterium bovis in blood collected at commencement of exsanguination (COE) of experimentally challenged cattle but has not been evaluated under field conditions. The current study was aimed at determining (i) whether blood collected at COE of cattle at slaughter, under field conditions, is practical to obtain and useful for identifying cattle as IFNγ positive for bTB, (ii) whether the results of the IFNγ assay obtained at COE reliably compare with results obtained from live animals in the field, and (iii) whether the identified animal(s) originated from bTB-infected or bTB-exposed herds. Cattle from three risk groups were used: the highest risk group consisted of 49 cattle from 3 bTB-infected herds; the medium risk group consisted of 24 cattle from a potentially exposed herd; and the lowest risk group consisted of 60 cattle from herds with no known history of bTB exposure. The IFNγ assay was performed on blood collected both before stunning and at COE of cattle at slaughter. An enhanced slaughter inspection for gross lesions consistent with bTB was performed on all cattle. In addition, lymph nodes were cultured for M. bovis for cattle that tested positive for bTB via the IFNγ assay and for most cattle that tested negative for bTB. Cattle, both with and without lesions consistent with bTB, were identified as positive for bTB by the IFNγ assay using blood collected at COE, but none of the positive cattle originated from the lowest risk group. The current study demonstrates that blood collected at COE of cattle is both a practical and moderately reliable sample for accessing bTB infection using the IFNγ assay.

  18. Identification and evaluation of new Mycobacterium bovis antigens in the in vitro interferon gamma release assay for bovine tuberculosis diagnosis.

    Science.gov (United States)

    Eirin, María E; Macias, Analia; Magnano, Gabriel; Morsella, Claudia; Mendez, Laura; Blanco, Federico C; Bianco, María V; Severina, Walter; Alito, Alicia; Pando, Maria de Los Angeles; Singh, Mahavir; Spallek, Ralph; Paolicchi, Fernando A; Bigi, Fabiana; Cataldi, Angel A

    2015-12-01

    Bovine tuberculosis (bTB) is a common zoonotic disease, caused by Mycobacterium bovis (M. bovis), responsible for significant economic losses worldwide. Its diagnosis is based on the detection of cell mediated immunity under the exposure to protein purified derivative tuberculin (PPD), a complex and poorly characterized reagent. The cross-reactivity to non-tuberculous mycobacterium species (false-positive results) has been crucial to develop a more proper antigen. In the present study, we selected six M. bovis Open Reading Frames (Mb1992, Mb2031c, Mb2319, Mb2843c, Mb2845c and Mb3212c) by in-silico analysis and evaluated them in experimental and natural infection; none of these antigens had been previously assessed as diagnostic antigens for bTB. The reactivity performance was tested in animals with both positive and negative Tuberculin Skin Test (TST) results as well as in cattle infected with Mycobacterium avium subesp. paratuberculosis (MAP). The six recombinant antigens individually induced an IFN-γ response, with overall responder frequency ranging from 18.3 to 31%. Mb2845c was the most valuable antigen with the potential to discriminate TST-positive cattle from either TST-negative or MAP infected animals. Mb2845c showed similar performance to that observed with ESAT-6 and PPD-B among TST and MTC specific-PCR positive animals, although this result needs to be proven in further studies with a higher sample size. Our data confirm the feacibility to implement bioinformatic screening tools and suggest Mb2845c as a potential diagnostic antigen to be tested in protein cocktails to evaluate their contribution to bTB diagnosis.

  19. Early and increased tuberculosis case detection: Implementation, measurement, and evaluation

    OpenAIRE

    Creswell, J.H.

    2015-01-01

    Despite substantial millions of people being treated by National Tuberculosis Control Programs (NTPs) and their partners over the last 25 years and millions of lives being saved, tuberculosis (TB) continues to be a leading cause of morbidity and mortality in many low- and middle-income countries and is currently estimated to kill 1.5 million people a year. Globally, the number of people diagnosed and notified to NTPs has hardly changed since 2007, meaning that more than three million people a...

  20. Culling-induced changes in badger (Meles meles behaviour, social organisation and the epidemiology of bovine tuberculosis.

    Directory of Open Access Journals (Sweden)

    Philip Riordan

    Full Text Available In the UK, attempts since the 1970s to control the incidence of bovine tuberculosis (bTB in cattle by culling a wildlife host, the European badger (Meles meles, have produced equivocal results. Culling-induced social perturbation of badger populations may lead to unexpected outcomes. We test predictions from the 'perturbation hypothesis', determining the impact of culling operations on badger populations, movement of surviving individuals and the influence on the epidemiology of bTB in badgers using data dervied from two study areas within the UK Government's Randomised Badger Culling Trial (RBCT. Culling operations did not remove all individuals from setts, with between 34-43% of badgers removed from targeted social groups. After culling, bTB prevalence increased in badger social groups neighbouring removals, particularly amongst cubs. Seventy individual adult badgers were fitted with radio-collars, yielding 8,311 locational fixes from both sites between November 2001 and December 2003. Home range areas of animals surviving within removed groups increased by 43.5% in response to culling. Overlap between summer ranges of individuals from Neighbouring social groups in the treatment population increased by 73.3% in response to culling. The movement rate of individuals between social groups was low, but increased after culling, in Removed and Neighbouring social groups. Increased bTB prevalence in Neighbouring groups was associated with badger movements both into and out of these groups, although none of the moving individuals themselves tested positive for bTB. Significant increases in both the frequency of individual badger movements between groups and the emergence of bTB were observed in response to culling. However, no direct evidence was found to link the two phenomena. We hypothesise that the social disruption caused by culling may not only increase direct contact and thus disease transmission between surviving badgers, but may also increase

  1. Stochastic simulation modeling to determine time to detect Bovine Viral Diarrhea antibodies in bulk tank milk

    DEFF Research Database (Denmark)

    Foddai, Alessandro; Enøe, Claes; Krogh, Kaspar

    2014-01-01

    A stochastic simulation model was developed to estimate the time from introduction ofBovine Viral Diarrhea Virus (BVDV) in a herd to detection of antibodies in bulk tank milk(BTM) samples using three ELISAs. We assumed that antibodies could be detected, after afixed threshold prevalence...

  2. Parameter estimation and use of gamma interferon assay for the diagnosis of bovine tuberculosis in Brazil

    Directory of Open Access Journals (Sweden)

    Luciano B. Lopes

    2012-04-01

    Full Text Available This study aimed to evaluate the interference of tuberculin test on the gamma-interferon (INFg assay, to estimate the sensitivity and specificity of the INFg assay in Brazilian conditions, and to simulate multiple testing using the comparative tuberculin test and the INFg assay. Three hundred-fifty cattle from two TB-free and two TB-infected herds were submitted to the comparative tuberculin test and the INFg assay. The comparative tuberculin test was performed using avian and bovine PPD. The INFg assay was performed by the BovigamTM kit (CSL Veterinary, Australia, according to the manufacturer's specifications. Sensitivity and specificity of the INFg assay were assessed by a Bayesian latent class model. These diagnostic parameters were also estimate for multiple testing. The results of INFg assay on D0 and D3 after the comparative tuberculin test were compared by the McNemar's test and kappa statistics. Results of mean optical density from INFg assay on both days were similar. Sensitivity and specificity of the INFg assay showed results varying (95% confidence intervals from 72 to 100% and 74 to 100% respectively. Sensitivity of parallel testing was over 97.5%, while specificity of serial testing was over 99.7%. The INFg assay proved to be a very useful diagnostic method.

  3. Detection of monoclonal integration of bovine leukemia virus proviral DNA as a malignant marker in two enzootic bovine leukosis cases with difficult clinical diagnosis

    OpenAIRE

    Miura, Saori; HORIUCHI, Noriyuki; Matsumoto, Kotaro; KOBAYASHI, Yoshiyasu; Kawazu, Shin-ichiro; INOKUMA, Hisashi

    2015-01-01

    Monoclonal integration of bovine leukemia virus (BLV) proviral DNA into bovine genomes was detected in peripheral blood from two clinical cases of enzootic bovine leukosis (EBL) without enlargement of superficial lymph nodes. A BLV-specific probe hybridized with 1 to 3 EcoRI and HindIII fragments in these 2 atypical EBL cattle by Southern blotting and hybridization, as well as in 3 typical EBL cattle. The probe also hybridized to a large number of EcoRI and HindIII fragments in 5 cattle with ...

  4. Zoonotic tuberculosis in human beings caused by Mycobacterium bovis-a call for action.

    Science.gov (United States)

    Olea-Popelka, Francisco; Muwonge, Adrian; Perera, Alejandro; Dean, Anna S; Mumford, Elizabeth; Erlacher-Vindel, Elisabeth; Forcella, Simona; Silk, Benjamin J; Ditiu, Lucica; El Idrissi, Ahmed; Raviglione, Mario; Cosivi, Ottorino; LoBue, Philip; Fujiwara, Paula I

    2017-01-01

    Mycobacterium tuberculosis is recognised as the primary cause of human tuberculosis worldwide. However, substantial evidence suggests that the burden of Mycobacterium bovis, the cause of bovine tuberculosis, might be underestimated in human beings as the cause of zoonotic tuberculosis. In 2013, results from a systematic review and meta-analysis of global zoonotic tuberculosis showed that the same challenges and concerns expressed 15 years ago remain valid. These challenges faced by people with zoonotic tuberculosis might not be proportional to the scientific attention and resources allocated in recent years to other diseases. The burden of zoonotic tuberculosis in people needs important reassessment, especially in areas where bovine tuberculosis is endemic and where people live in conditions that favour direct contact with infected animals or animal products. As countries move towards detecting the 3 million tuberculosis cases estimated to be missed annually, and in view of WHO's end TB strategy endorsed by the health authorities of WHO Member States in 2014 to achieve a world free of tuberculosis by 2035, we call on all tuberculosis stakeholders to act to accurately diagnose and treat tuberculosis caused by M bovis in human beings. Copyright © 2017 World Health Organization. Published by Elsevier Ltd/Inc/BV. All rights reserved. Published by Elsevier Ltd.. All rights reserved.

  5. Impact of external sources of infection on the dynamics of bovine tuberculosis in modelled badger populations

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    Hardstaff Joanne L

    2012-06-01

    Full Text Available Abstract Background The persistence of bovine TB (bTB in various countries throughout the world is enhanced by the existence of wildlife hosts for the infection. In Britain and Ireland, the principal wildlife host for bTB is the badger (Meles meles. The objective of our study was to examine the dynamics of bTB in badgers in relation to both badger-derived infection from within the population and externally-derived, trickle-type, infection, such as could occur from other species or environmental sources, using a spatial stochastic simulation model. Results The presence of external sources of infection can increase mean prevalence and reduce the threshold group size for disease persistence. Above the threshold equilibrium group size of 6–8 individuals predicted by the model for bTB persistence in badgers based on internal infection alone, external sources of infection have relatively little impact on the persistence or level of disease. However, within a critical range of group sizes just below this threshold level, external infection becomes much more important in determining disease dynamics. Within this critical range, external infection increases the ratio of intra- to inter-group infections due to the greater probability of external infections entering fully-susceptible groups. The effect is to enable bTB persistence and increase bTB prevalence in badger populations which would not be able to maintain bTB based on internal infection alone. Conclusions External sources of bTB infection can contribute to the persistence of bTB in badger populations. In high-density badger populations, internal badger-derived infections occur at a sufficient rate that the additional effect of external sources in exacerbating disease is minimal. However, in lower-density populations, external sources of infection are much more important in enhancing bTB prevalence and persistence. In such circumstances, it is particularly important that control strategies to

  6. Detection of Mycobacterium tuberculosis complex by nested polymerase chain reaction in pulmonary and extrapulmonary specimens

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    Adriana Antônia da Cruz Furini

    2013-12-01

    Full Text Available OBJECTIVE: To compare the performance of nested polymerase chain reaction (NPCR with that of cultures in the detection of the Mycobacterium tuberculosis complex in pulmonary and extrapulmonary specimens.METHODS: We analyzed 20 and 78 pulmonary and extrapulmonary specimens, respectively, of 67 hospitalized patients suspected of having tuberculosis. An automated microbial system was used for the identification of Mycobacterium spp. cultures, and M. tuberculosis IS6110 was used as the target sequence in the NPCR. The kappa statistic was used in order to assess the level of agreement among the results.RESULTS: Among the 67 patients, 6 and 5, respectively, were diagnosed with pulmonary and extrapulmonary tuberculosis, and the NPCR was positive in all of the cases. Among the 98 clinical specimens, smear microscopy, culture, and NPCR were positive in 6.00%, 8.16%, and 13.26%, respectively. Comparing the results of NPCR with those of cultures (the gold standard, we found that NPCR had a sensitivity and specificity of 100% and 83%, respectively, in pulmonary specimens, compared with 83% and 96%, respectively, in extrapulmonary specimens, with good concordance between the tests (kappa, 0.50 and 0.6867, respectively.CONCLUSIONS: Although NPCR proved to be a very useful tool for the detection of M. tuberculosis complex, clinical, epidemiological, and other laboratory data should also be considered in the diagnosis and treatment of pulmonary and extrapulmonary tuberculosis.

  7. An appraisal of PCR-based technology in the detection of Mycobacterium tuberculosis.

    Science.gov (United States)

    Sankar, Sathish; Ramamurthy, Mageshbabu; Nandagopal, Balaji; Sridharan, Gopalan

    2011-02-01

    Tuberculosis is an under-recognized yet catastrophic health problem, particularly in developing countries. The HIV pandemic has served to increase the number of susceptible individuals, and multidrug-resistance and poor socioeconomic conditions also augment the prevalence and the consequences of the disease. To control the disease and its spread, it is vital that tuberculosis diagnostics are accurate and rapid. Whereas microscopy and culture have several limitations (low sensitivity is a problem for the former, while the latter has a delayed turnaround time), PCR-based techniques targeting regions of the Mycobacterium tuberculosis genome such as IS6110 have proved to be useful. The purpose of this review is to assess the use of PCR-RFLP, nested PCR and real-time PCR protocols and the choice of target regions for the detection of M. tuberculosis. Real-time PCR for the detection of M. tuberculosis target genes in clinical specimens has contributed to improving diagnosis and epidemiologic surveillance in the past decade. However, targeting one genome sequence such as IS6110 may not by itself be sufficiently sensitive to reach 100% diagnosis, especially in the case of pulmonary tuberculosis. Additional testing for target genome sequences such as hsp65 seems encouraging. An interesting approach would be a multiplex real-time PCR targeting both IS6110 and hsp65 to achieve comprehensive and specific molecular diagnosis. This technology needs development and adequate field testing before it becomes the acceptable gold standard for diagnosis.

  8. Mycobacterium tuberculosis complex detected by modified fluorescent in situ hybridization in lymph nodes of clinical samples.

    Science.gov (United States)

    Rodriguez-Nuñez, Juan; Avelar, Francisco J; Marquez, Francisco; Rivas-Santiago, Bruno; Quiñones, Cesar; Guerrero-Barrera, Alma L

    2012-01-12

    Lymph node tuberculosis (TB) is the leading cause of extrapulmonary tuberculosis and is the most frequently identified type in Aguascalientes, Mexico. Conventional diagnosis has serious limitations for rapid detection of extrapulmonary tuberculosis in clinical samples. Here PCR and modified FISH have been tested as complementary diagnosis methods for extrapulmonary tuberculosis. The specific insertion sequence IS6110 for Mycobacterium tuberculosis complex was used to perform PCR and build DNA and PNA FISH probes (20bp). PCR and modified DNA and PNA FISH assays were performed to evaluate 41 lymph node paraffin-embedded tissue samples, in comparison with the histopathology diagnosis, which was considered the gold standard (22 positive and 19 negative). In comparison with histopathology diagnosis PCR showed 62.5 % sensitivity and 77.8 % specificity (χ(2) = 4.583 p 0.05). Ziehl Neelsen stain was positive in only four cases of 22 lymph node samples positive to histopathology.  In contrast, PCR and modified DNA FISH were positive in 20 cases of the same group. The negative cases were coincident in all tests. PCR and DNA FISH showed a significant increase in the number of cases detected and also showed higher sensitivity and specificity compared with data reported by traditional methodology. In developing countries, these techniques could help to complement the early diagnosis and timely treatment of extrapulmonary tuberculosis.

  9. Effectivity of PCR and AGID methods to detect of enzootic bovine leukosis in Indonesia

    Directory of Open Access Journals (Sweden)

    Saepulloh M

    2015-03-01

    Full Text Available Enzootic Bovine Leucosis (EBL is one of viral diseases in cattle caused by bovine leukemia virus (BLV, from Retroviridae. The virus can be detected using severals methods such as Polymerase Chain Reaction (PCR, while antibody can be detected using Agar Gel Immunodifussion (AGID. The aim of this experiment was to study the effectivity of PCR and AGID methods to detect enzootic bovine leukosis virus in Indonesia. Samples of peripheral blood leukocyte (PBL were collected from cattles those with and without showing clinical signs. A total of 307 blood and serum samples were tested against BLV using PCR and AGID tests, while 21 semen samples which were from similar animals for blood collection were collected only for PCR test. The results indicated that twelve cattles have positive results with PCR test in PBL, but from those cattles only seven were positive with AGID. On the other hand, the PCR did not detect EBL in 21 bovine semen samples tested, although one sample gave positive result with PCR in PBL. This results indicated that PCR method from blood samples was more sensitive than that AGID method. The PCR detection was also more sensitive for PBL than that for semen samples

  10. General and advanced diagnostic tools to detect Mycobacterium tuberculosis and their drug susceptibility: a review

    NARCIS (Netherlands)

    Gazi, M.A.; Islam, M.R.; Kibria, M.; Mahmud, Z.

    2015-01-01

    The global control of tuberculosis remains a great challenge from the standpoint of diagnosis, detection of drug resistance, and treatment, because treatment can only be initiated when infection is detected, and is guided by the results of antimicrobial susceptibility testing. To a large extent, non

  11. Increased TNF-alpha/IFN-gamma/IL-2 and decreased TNF-alpha/IFN-gamma production by central memory T cells are associated with protective responses against bovine tuberculosis following BCG vaccination

    Science.gov (United States)

    Central memory T cells (Tcm’s) and polyfunctional CD4 T responses contribute to vaccine-elicited protection with both human and bovine tuberculosis (TB); however, their combined role in protective immunity to TB is unclear. To address this question, we evaluated polyfunctional cytokine responses by ...

  12. FIDELIS--innovative approaches to increasing global case detection of tuberculosis.

    Science.gov (United States)

    Rusen, I D; Enarson, Donald A

    2006-01-01

    Tuberculosis was declared a global public health emergency in 1993. In 2003, only 45% of the world's estimated new smear-positive tuberculosis cases were detected-well below the 70% global case detection target set by the World Health Organization. The FIDELIS (Fund for Innovative DOTS Expansion Through Local Initiatives to Stop TB) initiative is a new global disease control initiative that has been developed to rapidly assess and implement innovative approaches to increase tuberculosis case detection. To date, 32 projects have been approved-covering approximately 378 million people in 13 countries-24 (75%) of which are in the world's 6 highest-burden countries. A wide range of target populations and interventions have been incorporated into successful FIDELIS projects. The FIDELIS initiative may serve as a model to discover best practices to address other urgent global public health problems.

  13. Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution

    Science.gov (United States)

    Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described cow feces-spec...

  14. Sensitive detection of Myobacterium avium subsp paratuberculosis in bovine semen by real-time PCR

    NARCIS (Netherlands)

    Herthnek, D.; Englund, S.; Willemsen, P.T.J.; Bolske, G.

    2006-01-01

    Aims: To develop a fast and sensitive protocol for detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine semen and to make a critical evaluation of the analytical sensitivity. Methods and Results: Processed semen was spiked with known amounts of MAP. Semen from different bulls as

  15. Characterization of effector and memory T cell subsets in the immune response to bovine tuberculosis in cattle.

    Directory of Open Access Journals (Sweden)

    Mayara F Maggioli

    Full Text Available Cultured IFN-γ ELISPOT assays are primarily a measure of central memory T cell (Tcm responses with humans; however, this important subset of lymphocytes is poorly characterized in cattle. Vaccine-elicited cultured IFN-γ ELISPOT responses correlate with protection against bovine tuberculosis in cattle. However, whether this assay measures cattle Tcm responses or not is uncertain. The objective of the present study was to characterize the relative contribution of Tcm (CCR7+, CD62Lhi, CD45RO+, T effector memory (Tem, defined as: CCR7-, CD62Llow/int, CD45RO+, and T effector cells (CCR7-, CD62L-/low, CD45RO-, in the immune response to Mycobacterium bovis. Peripheral blood mononuclear cells (PBMC from infected cattle were stimulated with a cocktail of M. bovis purified protein derivative, rTb10.4 and rAg85A for 13 days with periodic addition of fresh media and rIL-2. On day 13, cultured PBMC were re-stimulated with medium alone, rESAT-6:CFP10 or PPDb with fresh autologous adherent cells for antigen presentation. Cultured cells (13 days or fresh PBMCs (ex vivo response from the same calves were analyzed for IFN-γ production, proliferation, and CD4, CD45RO, CD62L, CD44, and CCR7 expression via flow cytometry after overnight stimulation. In response to mycobacterial antigens, ~75% of CD4+ IFN-γ+ cells in long-term cultures expressed a Tcm phenotype while less than 10% of the ex vivo response consisted of Tcm cells. Upon re-exposure to antigen, long-term cultured cells were highly proliferative, a distinctive characteristic of Tcm, and the predominant phenotype within the long-term cultures switched from Tcm to Tem. These findings suggest that proliferative responses of Tcm cells to some extent occurs simultaneously with reversion to effector phenotypes (mostly Tem. The present study characterizes Tcm cells of cattle and their participation in the response to M. bovis infection.

  16. The role of multiple wildlife hosts in the persistence and spread of bovine tuberculosis in New Zealand

    Science.gov (United States)

    Barron, MC; Tompkins, DM; Ramsey, DSL; Bosson, MAJ

    2015-01-01

    Abstract AIM: To explore how the inclusion of multi-host dynamics affects the predicted prevalence of bovine tuberculosis (TB) in possums and other host species following the current best practice for control of TB in large difficult and remote areas, to identify which host species are responsible for changes in predicted prevalence, and whether TB can persist in possum-free host communities. METHODS: Multi-host TB models were constructed, comprising three host species with density-dependent population growth, density-dependent disease transmission and susceptible and infected classes. Models were parameterised for two case studies of current concern in New Zealand, namely chronic TB persistence in a possum-deer-pig complex in extensive forest, and in a possum-pig-ferret complex in unforested semi-arid shrub and grasslands. Persistence of TB in the face of best practice possum control was evaluated from model simulations, and the contribution of different hosts to persistence of TB was assessed by removing each host species in turn from the simulations. A sensitivity test explored how different parameter values affected modelled persistence of TB. RESULTS: The forest multi-host model-predicted amplification of TB prevalence due to the presence of pigs. The presence of pigs and/or deer did not jeopardise the success of best practice possum control in eradicating TB from the system, as pigs and deer are effectively end-hosts for TB. Sensitivity analyses indicated these interpretations were robust to uncertainty in model parameter values. The grassland system model predicted that the multi-host species complex could potentially lead to failure of eradication of TB under possum-only control, due to TB persisting in ferret and pig populations in the absence of possum hosts through reciprocal scavenging, resulting in spillback transmission to possums once their populations had started to recover from control. CONCLUSIONS: With respect to management of TB, for modelled

  17. Modeling and mitigating winter hay bale damage by elk in a low prevalence bovine tuberculosis endemic zone.

    Science.gov (United States)

    Gooding, R M; Brook, R K

    2014-05-01

    Wildlife causes extensive crop damage throughout much of North America and these shared feeds are a key risk factor in the transmission of diseases between wildlife and livestock, including bovine tuberculosis (TB). Predicting wildlife use of agricultural crops can provide insight directed toward targeted disease mitigation at areas of potential indirect interaction. In this study, we quantified use of hay bales by elk (Cervus canadensis) during the winter in southwestern Manitoba, Canada using a database of 952 damage claims paid compensation from 1994 to 2012. We evaluated environmental factors predicted to determine risk of hay bale damage on each quarter section by elk using a Resource Selection Probability Function (RSPF) model. The most important variables (as measured for each quarter section and based on cumulative Akaike weights that scale from 0 to 1) were distance to protected areas (1.00), forest including a buffer around the quarter section (1.00), forage crop including a buffer around the quarter section (1.00), distance from streams (0.99), forage crop (0.92), cereal and oilseed crop cover including a buffer (0.85), and forest cover (0.82). We then developed an RSPF-based predictive map of damage to hay bales by elk that identified key areas with high probability of damage (RSPF≥0.6), accounting for 3.5% of the study area. We then multiplied the RSPF values by the inverse of the proximity to known cases of TB positive elk and determined that 0.51% of the study area had an overall high combined probability of hay bale damage and proximity to TB positive elk (i.e. adjusted probability of ≥0.6). In the southern half of the study area where 164 hay yard barrier fences have been implemented since 2002, there has been a significant decrease in the number of annual claims. Barrier fencing around Riding Mountain National Park has been successful at reducing elk damage where it has been implemented. In our study area, prevalence of TB in both cattle (0

  18. Comparison of output-based approaches used to substantiate bovine tuberculosis free status in Danish cattle herds.

    Science.gov (United States)

    Foddai, Alessandro; Nielsen, Liza Rosenbaum; Willeberg, Preben; Alban, Lis

    2015-09-01

    We compared two published studies based on different output-based surveillance models, which were used for evaluating the performance of two meat inspection systems in cattle and to substantiate freedom from bovine tuberculosis (bTB) in Denmark. The systems were the current meat inspection methods (CMI) vs. the visual-only inspection (VOI). In one study, the surveillance system sensitivity (SSe) was estimated to substantiate the bTB free status. The other study used SSe in the estimation of the probability of freedom (PFree), based on the epidemiological concept of negative predictive value to substantiate the bTB free status. Both studies found that changing from CMI to VOI would markedly decrease the SSe. However, the two studies reported diverging conclusions regarding the effect on the substantiation of Denmark as a bTB free country, if VOI were to be introduced. The objectives of this work were: (a) to investigate the reasons why conclusions based on the two models differed, and (b) to create a hybrid model based on elements from both studies to evaluate the impact of a change from CMI to VOI. The hybrid model was based on the PFree approach to substantiate freedom from bTB and was parametrized with inputs according to the newest available information. The PFree was updated on an annual basis for each of 42 years of test-negative surveillance data (1995-2037), while assuming a low (cattle herds. The most important reasons for the difference between the study conclusions were: the approach chosen to substantiate the bTB free status (SSe vs. PFree) and the number of years of surveillance data considered. With the hybrid model, the PFree reached a level >95% after the first year of surveillance and remained ≥96% with both the CMI and VOI systems until the end of the analyzed period. It is appropriate to use the PFree of the surveillance system to substantiate confidence in bTB free status, when test-negative surveillance results can be documented over an

  19. Linking bovine tuberculosis on cattle farms to white-tailed deer and environmental variables using Bayesian hierarchical analysis

    Science.gov (United States)

    Walter, William D.; Smith, Rick; Vanderklok, Mike; VerCauterren, Kurt C.

    2014-01-01

    Bovine tuberculosis is a bacterial disease caused by Mycobacterium bovis in livestock and wildlife with hosts that include Eurasian badgers (Meles meles), brushtail possum (Trichosurus vulpecula), and white-tailed deer (Odocoileus virginianus). Risk-assessment efforts in Michigan have been initiated on farms to minimize interactions of cattle with wildlife hosts but research onM. bovis on cattle farms has not investigated the spatial context of disease epidemiology. To incorporate spatially explicit data, initial likelihood of infection probabilities for cattle farms tested for M. bovis, prevalence of M. bovis in white-tailed deer, deer density, and environmental variables for each farm were modeled in a Bayesian hierarchical framework. We used geo-referenced locations of 762 cattle farms that have been tested for M. bovis, white-tailed deer prevalence, and several environmental variables that may lead to long-term survival and viability of M. bovis on farms and surrounding habitats (i.e., soil type, habitat type). Bayesian hierarchical analyses identified deer prevalence and proportion of sandy soil within our sampling grid as the most supported model. Analysis of cattle farms tested for M. bovisidentified that for every 1% increase in sandy soil resulted in an increase in odds of infection by 4%. Our analysis revealed that the influence of prevalence of M. bovis in white-tailed deer was still a concern even after considerable efforts to prevent cattle interactions with white-tailed deer through on-farm mitigation and reduction in the deer population. Cattle farms test positive for M. bovis annually in our study area suggesting that the potential for an environmental source either on farms or in the surrounding landscape may contributing to new or re-infections with M. bovis. Our research provides an initial assessment of potential environmental factors that could be incorporated into additional modeling efforts as more knowledge of deer herd

  20. Bovine coronavirus detection in a collection of diarrheic stool samples positive for group a bovine rotavirus

    Directory of Open Access Journals (Sweden)

    Aline Fernandes Barry

    2009-11-01

    Full Text Available Neonatal diarrhea is an important cause of economic losses for cattle farmers. The main viral etiologies of enteric diseases are group A rotaviruses (GARV and the bovine coronavirus (BCoV. Although both viruses infect calves of the same age, the occurrence of mixed infections is still under studied. The present study describes the co-infection of BCoV and GARV in stool samples. Forty-four diarrheic fecal samples from calves up to 60 days old that had previously tested positive for GARV by SS-PAGE were analyzed using semi-nested PCR for BCoV. A product with 251 bp of the BCoV nucleoprotein gene was amplified in 15.9% (7/44 of the samples, demonstrating that co-infection is not an unusual event. These results reinforce the need for testing for both GARV and BCoV, even in fecal samples that previously tested positive for one virus.A diarreia neonatal é uma importante causa de perdas econômicas para a criação de bovinos. Os principais agentes etiológicos virais das doenças entéricas são o rotavírus bovino grupo A (GARV e o coronavírus bovino (BCoV. Embora ambos os vírus infectem bezerros na mesma faixa etária, infecções mistas ainda são pouco estudadas. O presente trabalho descreve a identificação do BCoV em amostras de fezes positivas para o GARV, caracterizando a ocorrência de infecções mistas. Quarenta e quatro amostras de fezes diarreicas de bezerros com até 60 dias de idade, previamente identificadas como positivas para o GARV bovino por meio da técnica de SS-PAGE, foram avaliadas quanto a presença do BCoV pela técnica de semi-nested PCR. Um produto com 251 pb do gene da nucleoproteína do BCoV foi amplificado em 15,9% (7/44 das amostras de fezes demonstrando que a co-infecção não é um evento raro. Esse resultado enfatizada a importância da realização simultânea do diagnóstico para esses dois importantes vírus entéricos de bezerros em surtos de diarreia neonatal tanto em rebanhos bovinos leiteiros quanto de

  1. Detection of Mycobacterium tuberculosis and Mycobacterium bovis from human sputum samples through multiplex PCR.

    Science.gov (United States)

    Jabbar, Abdul; Khan, Jafar; Ullah, Aman; Rehman, Hazir; Ali, Ijaz

    2015-07-01

    Tuberculosis (TB) has a long history and being present even before the start of recording history. It has left detrimental effects on all aspect of the life and geared the developments in the science of health. TB is caused by Mycobacterium tuberculosis complex (MTBC) including five species M. tuberculosis, M. bovis, M. africanum, M. canetti, and M. microti. M. tuberculosis and M. bovis infect both animals and humans. Therefore, differentiation of these two closely related species is very important for epidemiological and management purpose. We undertook the present study to characterize mycobacteria isolated from sputum of known TB patients by conventional methods and further, by multiplex PCR (mPCR) to detect the prevalence of Zoonotic TB (TB caused by M. bovis). Sputum samples from TB patient were collected from two tertiary care hospitals in Peshawar i.e. Lady Reading Hospital and Hayatabad Medical Complex. All the samples were subjected to Ziehl Neelsen (ZN) stain, culture on Lowenstein Jensen (LJ) and Stone Brink medium, Nitrate reduction test and multiplex PCR. A total of hundred mycobacterial strains were isolated from these samples on the basis of ZN staining, cultural and biochemical methods. Later on, these isolates were subjected to multiplex PCR by using pncATB-1.2 and pncAMT-2 primers specific to M. tuberculosis and JB21, JB22 primers specific to M. bovis. By means of conventional method, these hundred cultures isolates were differentiated into M. tuberculosis (ninety six) and M. bovis (four). Furthermore, by mPCR, it was determined that out of hundred isolates, ninety-eight were identified as M. tuberculosis and two isolates as M. bovis. This molecular method enables to differentiate M. bovis from M. tuberculosis in human sputum.

  2. Real-time PCR with internal amplification control for detecting tuberculosis: method design and validation.

    Science.gov (United States)

    Flores, E; Rodríguez, J C; Garcia-Pachón, E; Soto, J L; Ruiz, M; Escribano, I; Royo, G

    2009-08-01

    Real-time PCR has been a major development in the diagnosis of tuberculosis. However, most tests do not include an internal amplification control (IAC), which therefore limits it clinical application. In this study a new, easy to perform real-time PCR test with IAC was designed and validated in clinical samples. The primers amplified a 163-bp fragment of IS6110 of Mycobacterium tuberculosis and the IAC was designed with a fragment of a different microorganism (Chlamydia trachomatis). The interassay and intraassay variation of this test were very low (0.45-1.65% and 0.18-1.80%, respectively). The detection accuracy was validated in 50 samples (25 urine, 25 sputum) with different concentrations of M. tuberculosis, 18 clinical isolates of non-tuberculous mycobacteria and 148 samples with clinical suspicion of pulmonary tuberculosis. The specificity was 100%. The detection limit of this PCR test without IAC was approximately 15 bacteria and with IAC approximately 32 bacteria. This real-time PCR with IAC assay can improve the detection of M. tuberculosis and contribute to standardization of this diagnostic technique.

  3. Increased TNF-alpha/IFN-gamma/IL-2 and decreased TNF-alpha/IFN-gamma production by central memory T cells are associated with protective responses against bovine tuberculosis following BCG vaccination

    Directory of Open Access Journals (Sweden)

    Mayara Fernanda Maggioli

    2016-10-01

    Full Text Available Central memory T cells (Tcm and polyfunctional CD4 T cell responses contribute to vaccine-elicited protection with both human and bovine tuberculosis (TB; however, their combined role in protective immunity to TB is unclear. To address this question, we evaluated polyfunctional cytokine responses by CD4 T cell effector / memory populations from bacille Calmette Guerin (BCG vaccinated and non-vaccinated calves prior to and after aerosol challenge with virulent Mycobacterium bovis. Polyfunctional cytokine expression patterns in the response by Tcm, effector memory, and effector T cell subsets were similar between BCG-vaccinated and M. bovis-infected calves; only differing in magnitude (i.e., infected > vaccinated. BCG vaccination, however, did alter the kinetics of the ensuing response to virulent M. bovis infection. Early after challenge (three weeks post-infection, non-vaccinates had greater antigen-specific IFN-γ/TNF-α and lesser IFN-γ/TNF-α/IL-2 responses by Tcm cells than did vaccinated animals. Importantly, these differences were also associated with mycobacterial burden upon necropsy. Polyfunctional responses to ESAT-6:CFP10 (antigens not synthesized by BCG strains were detected in memory subsets, as well as in effector cells, as early as three weeks after challenge. These findings suggest that cell fate divergence may occur early after antigen priming in the response to bovine TB and that memory and effector T cells may expand concurrently during the initial phase of the immune response. In summary, robust IFN-γ/TNF-α response by Tcm cells is associated with greater mycobacterial burden while IFN-γ/TNF-α/IL-2 response by Tcm cells are indicative of a protective response to bovine TB.

  4. Comparison of bronchial brushing and sputum in detection of pediatric pulmonary tuberculosis.

    Science.gov (United States)

    Chen, Qiao-Pei; Ren, Shi-Feng; Wang, Xin-Feng; Wang, Mao-Shui

    2016-01-27

    The retrospective study aimed to evaluate the diagnostic value of bronchial brushing and sputum using acid fast bacilli smear, mycobacterial culture and real-time PCR in detection of pediatric pulmonary tuberculosis, sensitivity and specificity of bronchial brushing and sputum examined by the three methods were calculated and compared to each other. Data showed there were no significant difference in sensitivity between bronchial brushing and matched sputum using each method. But the specificity of real-time PCR on bronchial brushing was lower than on sputum. Compared with bronchial brushing, sputum was better specimen in detection of pediatric pulmonary tuberculosis.

  5. MTB-DR-RIF 9G test: Detection and discrimination of tuberculosis and multi-drug resistant tuberculosis strains.

    Science.gov (United States)

    Song, Keum-Soo; Nimse, Satish Balasaheb; Cho, Nam Hoon; Sung, Nackmoon; Kim, Hee-Jin; Yang, Jeongseong; Kim, Taisun

    2015-12-01

    This report describes the evaluation of the novel MTB-DR-RIF 9G test for the accurate detection and discrimination of Mycobacterium tuberculosis (MTB) and rifampicin-resistant M. tuberculosis (MTB-DR-RIF) in the clinical samples. The procedure included the amplification of a nucleotide fragment of the rpoB gene of the MTB and MTB-DR-RIF strains and their hybridization with the immobilized probes. The MTB-DR-RIF 9G test was evaluated for its ability to detect and discriminate MTB and MTB-DR-RIF strains in 113 known clinical samples. The accuracy of the MTB-DR-RIF 9G test was determined by comparing its results with sequencing analysis and drug susceptibility testing. The sensitivity and specificity of the MTB-DR-RIF 9G test at 95% confidence interval were found to be 95.4% (89.5-98.5) and 100% (69.2-100), respectively. The positive predictive value and negative predictive value of the MTB-DR-RIF 9G test at 95% confidence interval were found to be 100% (85.0-95.9) and 66.7% (38.4-88.18), respectively. Sequencing analysis of all samples indicated that the mutations present in the regions identified with the MTB-DR-RIF 9G assay can be detected accurately.

  6. FIDELIS—Innovative Approaches to Increasing Global Case Detection of Tuberculosis

    Science.gov (United States)

    Rusen, I. D.; Enarson, Donald A.

    2006-01-01

    Tuberculosis was declared a global public health emergency in 1993. In 2003, only 45% of the world’s estimated new smear-positive tuberculosis cases were detected—well below the 70% global case detection target set by the World Health Organization. The FIDELIS (Fund for Innovative DOTS Expansion Through Local Initiatives to Stop TB) initiative is a new global disease control initiative that has been developed to rapidly assess and implement innovative approaches to increase tuberculosis case detection. To date, 32 projects have been approved—covering approximately 378 million people in 13 countries—24 (75%) of which are in the world’s 6 highest-burden countries. A wide range of target populations and interventions have been incorporated into successful FIDELIS projects. The FIDELIS initiative may serve as a model to discover best practices to address other urgent global public health problems. PMID:16317206

  7. Growth inhibitory factors in bovine faeces impairs detection of Salmonella Dublin by conventional culture procedure

    DEFF Research Database (Denmark)

    Baggesen, Dorte Lau; Nielsen, L.R.; Sørensen, Gitte

    2007-01-01

    Aims: To analyse the relative importance of different biological and technical factors on the analytical sensitivity of conventional culture methods for detection of Salmonella Dublin in cattle faeces. Methods and Results: Faeces samples collected from six adult bovines from different salmonella-...... by focusing on the strain variations and the ecology of the faecal sample. Detailed investigation of the faecal flora (pathogens and normal flora) and the interaction with chemical factors may result in developing an improved method for detection of S. Dublin....

  8. Rapid Detection of Rifampin-resistant Clinical Isolates of Mycobacterium tuberculosis by Reverse Dot Blot Hybridization

    Institute of Scientific and Technical Information of China (English)

    GUO Qian; WAN Kang Lin; YU Yan; ZHU Yan Ling; ZHAO Xiu Qin; LIU Zhi Guang; ZHANG Yuan Yuan; LI Gui Lian; WEI Jian Hao; WU Yi Mou

    2015-01-01

    Objective A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in‘hot mutation region’ of Mycobacterium tuberculosis (M. tuberculosis). Methods 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. Results The sensitivity and specificity of the RDBH assay were 91.2%(165/181) and 98.3%(117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7%(293/300), 98.2%(164/167), and 97.0%(129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. Conclusion Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.

  9. Application of Deletion- Targeted Multiplex PCR technique for detection of Mycobacterium tuberculosis Beijing strains in samples from tuberculosis patients.

    Directory of Open Access Journals (Sweden)

    Azar Dokht Khosravi

    2014-10-01

    Full Text Available Molecular epidemiological studies have shown that certain genotypes of Mycobacterium tuberculosis (MTB are over-represented in limited geographical regions, suggesting of evolution of certain genotypes with increasing virulence and pathogenicity. Beijing strain of MTB was initially described by its potential to cause outbreaks worldwide and its association with drug resistance. Due to tuberculosis (TB-related mortality which is associated with Beijing genotype, this study was designed with the aim to detect the MTB Beijing genotype in the region of study.A total of 170 clinical isolates of MTB were collected from the TB reference laboratory of Khuzestan province, Iran, over one year period from February 2010 to February 2011. Phenotypic tests were used for preliminary detection of MTB. Culture positive MTB isolates were confirmed by multiplex PCR based on IS6110 gene with subsequent screening for resistance to isoniazid (INH, and rifampin (RIF by PCR using relevant primers. Three set of primers were used to differentiate Beijing from non-Beijing strains by using Deletion- Targeted Multiplex (DTM PCR.From 160 PCR-confirmed MTB isolates, 18 (11.25% showed mutation in katG gene related to INH resistance and 20 (12.5%, associated with mutation in rpoB gene related to RIF resistance, and 8 (5% were detected as Beijing strain using multiplex PCR. The majority of detected Beijing strains (6/8[75%] comprised mutation in katG gene with the prevalent mutation specifically in codon 315. In 4 Beijing strains (2.5%, mutation in rpoB gene were also detected.Using DTM- PCR, the rate of Beijing strains in the region of study was determined as 5%. Although for detection of MTB antimicrobial resistance, it is advised to use a combination of conventional antimicrobial susceptibility testing and molecular techniques, however for time saving, it seems that DTM-PCR, is a simple technique for use in areas of the world where Beijing strains are highly prevalent.

  10. Comparison of the performance of five different immunoassays to detect specific antibodies against emerging atypical bovine pestivirus

    DEFF Research Database (Denmark)

    Larska, Magdalena; Polak, Mirosław P.; Liu, Lihong

    2013-01-01

    Bovine pestiviruses represent a considerably variable group. In addition to the two accepted species BVDV-1 and BVDV-2, a number of atypical bovine pestiviruses have been detected both in foetal calf sera and in field samples. The sera collected during the initial six weeks of experimental infect...

  11. Immunochromatographic Lateral-flow test strip for the rapid detection of added bovine rennet whey in milk and milk powder

    NARCIS (Netherlands)

    Martin-Hernandez, C.; Munoz, M.; Daury, C.; Weymuth, H.; Kemmers-Voncken, A.; Corbation, V.; Toribo, T.; Bremer, M.G.E.G.

    2009-01-01

    An immunochromatographic lateral-flow test dipstick test was developed for the fast detection of bovine rennet whey in liquid milk and milk powder. The test is based on the binding of casein glycomacropeptide (cGMP) by two specific anti-bovine ¿-casein monoclonal antibodies and has a visual detectio

  12. Gold nanoparticle aggregation-based colorimetric assay for β-casein detection in bovine milk samples.

    Science.gov (United States)

    Li, Y S; Zhou, Y; Meng, X Y; Zhang, Y Y; Song, F; Lu, S Y; Ren, H L; Hu, P; Liu, Z S; Zhang, J H

    2014-11-01

    Traditional Kjeldahl method, used for quality evaluation of bovine milk, has intrinsic defects of time-consuming sample preparation and two analyses to determine the difference between non-protein nitrogen content and total protein nitrogen content. Herein, based upon antibody functionalized gold nanoparticles (AuNPs), we described a colorimetric method for β-casein (β-CN) detection in bovine milk samples. The linear dynamic range and the LOD were 0.08-250 μg mL(-1), and 0.03 μg mL(-1) respectively. In addition, the real content of β-CN in bovine milk was measured by using the developed assay. The results are closely correlated with those from Kjeldahl method. The advantages of β-CN triggered AuNP aggregation-based colorimetric assay are simple signal generation, the high sensitivity and specificity as well as no need of complicated sample preparation, which make it for on-site detection of β-CN in bovine milk samples.

  13. Detecting β-Casein Variation in Bovine Milk

    Directory of Open Access Journals (Sweden)

    Anna Maria Caroli

    2016-01-01

    Full Text Available In bovine species, β-casein (β-CN is characterized by genetic polymorphism. The two most common protein variants are β-CN A2 (the original one and A1, differing from A2 for one amino acid substitution (Pro67 to His67. Several bioactive peptides affecting milk nutritional properties can originate from β-CN. Among them, β-casomorphin-7 (BCM7 ranging from amino acid 60 to 66 can be released more easily from β-CN variants carrying His67 (A1 type instead of Pro67 (A2 type. Nowadays, “A2 milk” is produced in different countries claiming its potential benefits in human health. The aim of this study was to further develop and apply an isoelectric focusing electrophoresis (IEF method to bulk and individual milk samples in order to improve its use for β-CN studies. We succeeded in identifying A2 milk samples correctly and quantifying the percentage of A2, A1, and B variants in bulk samples not derived from A2 milk as well as in individual milk samples. The method allows us to quantify the relative proportion of β-CN variants in whole milk without eliminating whey protein by acid or enzymatic precipitation of caseins. The aim of this study was also to study the different behavior of β-CN and β-lactoglobulin (β-LG in the presence of trichloroacetic acid (TCA. The higher sensitivity of β-CN to TCA allows quantifying β-CN variants after TCA fixation because β-LG is not visible. Monitoring β-CN variation in cattle breeds is important in order to maintain a certain balance between Pro67 and His67 in dairy products. Overall, the debate between A1 and A2 milk needs further investigation.

  14. Double-antibody based immunoassay for the detection of β-casein in bovine milk samples.

    Science.gov (United States)

    Zhou, Y; Song, F; Li, Y S; Liu, J Q; Lu, S Y; Ren, H L; Liu, Z S; Zhang, Y Y; Yang, L; Li, Z H; Zhang, J H; Wang, X R

    2013-11-01

    The concentration of casein (CN) is one of the most important parameters for measuring the quality of bovine milk. Traditional approach to CN concentration determination is Kjeldahl, which is an indirect method for determination of total nitrogen content. Here, we described a double-antibody based direct immunoassay for the detection of β-CN in bovine milk samples. Monoclonal antibody (McAb) was used as capture antibody and polyclonal antibody (PcAb) labelled with horseradish peroxidase (HRP) as detection antibody. With the direct immunoassay format, the linear range of the detection was 0.1-10.0 μg mL(-1). The detection limit was 0.04 μg mL(-1). In addition, the concentration of β-CN in real bovine milk samples has been detected by the developed immunoassay. There was a good correlation between the results obtained by the developed technique and Kjeldahl method from commercial samples. Compared to the traditional approach, the advantage of the assay is no need of time-consuming sample pretreatment.

  15. Assessment of terbium (III) as a luminescent probe for the detection of tuberculosis biomarkers

    Energy Technology Data Exchange (ETDEWEB)

    Bamogo, W. [CNRS, IRAMIS, UMR 3685 NIMBE/LEDNA, F-91191 Gif-sur-Yvette (France); Mugherli, L. [CEA, IRAMIS, UMR 3685 NIMBE/LEDNA, F-91191 Gif-sur-Yvette (France); Banyasz, A. [CNRS, IRAMIS, LIDyL/Laboratoire Francis Perrin, URA 2453, F-91191 Gif-sur-Yvette (France); Novelli-Rousseau, A.; Mallard, F. [BioMérieux SA, F-38000 Grenoble (France); Tran-Thi, T.-H., E-mail: thu-hoa.tran-thi@cea.fr [CNRS, IRAMIS, UMR 3685 NIMBE/LEDNA, F-91191 Gif-sur-Yvette (France)

    2015-10-08

    A detection method for nicotinic acid, a specific metabolite marker of Mycobacterium tuberculosis present in cultures and patients' breath, is studied in complex solutions containing other metabolites and in biological media such as urine, saliva and breath condensate. The method is based on the analysis of the luminescence increase of Tb{sup 3+} complexes in the presence of nicotinic acid due to the energy transfer from the excited ligand to the lanthanide ion. It is shown that other potential markers found in M. tuberculosis culture supernatant, such as methyl phenylacetate, p-methyl anisate, methyl nicotinate and 2-methoxy biphenyl, can interfere with nicotinic acid via a competitive absorption of the excitation photons. A new strategy to circumvent these interferences is proposed with an upstream trapping of volatile markers preceding the detection of nicotinic acid in the liquid phase via the luminescence of Tb{sup 3+} complexes. The cost of the method is evaluated and compared with the Xpert MTB/RIF test endorsed by the World Health Organization. - Highlights: • Nicotinic acid, a specific marker of M. tuberculosis, can be detected via luminescence. • The detection limit with a commercial phosphorimeter is 0.4 µmol·L{sup -1}. • Other metabolites of M. tuberculosis can interfere via absorbed excitation light. • The interference can be removed via trapping of the most volatile metabolites. • A breath analysis procedure's cost is compared with the Xpert TBM/RIF test.

  16. Detection of tuberculosis with digital chest radiography: automatic reading versus interpretation by clinical officers

    NARCIS (Netherlands)

    Maduskar, P.; Muyoyeta, M.; Ayles, H.; Hogeweg, L.E.; Peters-Bax, L.; Ginneken, B. van

    2013-01-01

    SETTING: A busy urban health centre in Lusaka, Zambia. OBJECTIVE: To compare the accuracy of automated reading (CAD4TB) with the interpretation of digital chest radiograph (CXR) by clinical officers for the detection of tuberculosis (TB). DESIGN: A retrospective analysis was performed on 161

  17. [Revisiting the causes of low detection of M. tuberculosis in urine].

    Science.gov (United States)

    Kul'chavenia, E V; Al'khovik, O I; Cherednichenko, A G

    2014-01-01

    The evaluation of potential anti-TB activity and bactericidal activity against opportunistic enterobacteria in urine of healthy people using the automated BACTEC MGIT 960 system for cultivation and determination of drug resistance of mycobacteria and culture on solid media was performed. It has been established that the urine of healthy people do not have bactericidal activity against M. tuberculosis and E. coli in vitro. The one of the possible reasons for the low detection of the pathogen in urogenital tuberculosis--asymptomatic bacteriuria--was identified.

  18. Analytical and Clinical Evaluation of the Epistem Genedrive Assay for Detection of Mycobacterium tuberculosis

    Science.gov (United States)

    Shenai, Shubhada; Armstrong, Derek T.; Valli, Eloise; Dolinger, David L.; Nakiyingi, Lydia; Dietze, Reynaldo; Dalcolmo, Margareth Pretti; Nicol, Mark P.; Zemanay, Widaad; Manabe, Yuka; Hadad, David Jamil; Marques-Rodrigues, Patricia; Palaci, Moises; Peres, Renata L.; Gaeddert, Mary; Armakovitch, Sandra; Nonyane, Bareng A. S.; Denkinger, Claudia M.; Banada, Padmapriya; Joloba, Moses L.; Ellner, Jerrold; Boehme, Catharina; Alland, David

    2016-01-01

    The Epistem Genedrive assay rapidly detects the Mycobacterium tuberculosis complex from sputum and is currently available for clinical use. However, the analytical and clinical performance of this test has not been fully evaluated. The analytical limit of detection (LOD) of the Genedrive PCR amplification was tested with genomic DNA; the performance of the complete (sample processing plus amplification) system was tested by spiking M. tuberculosis mc26030 cells into distilled water and M. tuberculosis-negative sputum. Specificity was tested using common respiratory pathogens and nontuberculosis mycobacteria. A clinical evaluation enrolled adults with suspected pulmonary tuberculosis, obtained three sputum samples from each participant, and compared the accuracy of the Genedrive to that of the Xpert MTB/RIF assay using M. tuberculosis cultures as the reference standard. The Genedrive assay had an LOD of 1 pg/μl (100 genomic DNA copies/reaction). The LODs of the system were 2.5 × 104 CFU/ml and 2.5 × 105 CFU/ml for cells spiked into water and sputum, respectively. False-positive rpoB probe signals were observed in 3/32 (9.4%) of the negative controls and also in few samples containing Mycobacterium abscessus, Mycobacterium gordonae, or Mycobacterium thermoresistibile. In the clinical study, among 336 analyzed participants, the overall sensitivities for the tuberculosis case detection of Genedrive, Xpert, and smear microscopy were 45.4% (95% confidence interval [CI], 35.2% to 55.8%), 91.8% (95% CI, 84.4% to 96.4%), and 77.3% (95% CI, 67.7% to 85.2%), respectively. The sensitivities of Genedrive and Xpert for the detection of smear-microscopy-negative tuberculosis were 0% (95% CI, 0% to 15.4%) and 68.2% (95% CI, 45.1% to 86.1%), respectively. The Genedrive assay did not meet performance standards recommended by the World Health Organization for a smear microscopy replacement tuberculosis test. Epistem is working on modifications to improve the assay. PMID:26865685

  19. Detection of monoclonal integration of bovine leukemia virus proviral DNA as a malignant marker in two enzootic bovine leukosis cases with difficult clinical diagnosis.

    Science.gov (United States)

    Miura, Saori; Horiuchi, Noriyuki; Matsumoto, Kotaro; Kobayashi, Yoshiyasu; Kawazu, Shin-Ichiro; Inokuma, Hisashi

    2015-07-01

    Monoclonal integration of bovine leukemia virus (BLV) proviral DNA into bovine genomes was detected in peripheral blood from two clinical cases of enzootic bovine leukosis (EBL) without enlargement of superficial lymph nodes. A BLV-specific probe hybridized with 1 to 3 EcoRI and HindIII fragments in these 2 atypical EBL cattle by Southern blotting and hybridization, as well as in 3 typical EBL cattle. The probe also hybridized to a large number of EcoRI and HindIII fragments in 5 cattle with persistent leukosis. These results suggest that the detection of monoclonal integration of BLV provirus into the host genome may serve as a marker of monoclonal proliferation and malignancy in difficult to diagnose EBL cattle.

  20. Descriptive Epidemiology and Whole Genome Sequencing Analysis for an Outbreak of Bovine Tuberculosis in Beef Cattle and White-Tailed Deer in Northwestern Minnesota.

    Science.gov (United States)

    Glaser, Linda; Carstensen, Michelle; Shaw, Sheryl; Robbe-Austerman, Suelee; Wunschmann, Arno; Grear, Dan; Stuber, Tod; Thomsen, Bruce

    2016-01-01

    Bovine tuberculosis (bTB) was discovered in a Minnesota cow through routine slaughter surveillance in 2005 and the resulting epidemiological investigation led to the discovery of infection in both cattle and white-tailed deer in the state. From 2005 through 2009, a total of 12 beef cattle herds and 27 free-ranging white-tailed deer (Odocoileus virginianus) were found infected in a small geographic region of northwestern Minnesota. Genotyping of isolates determined both cattle and deer shared the same strain of bTB, and it was similar to types found in cattle in the southwestern United States and Mexico. Whole genomic sequencing confirmed the introduction of this infection into Minnesota was recent, with little genetic divergence. Aggressive surveillance and management efforts in both cattle and deer continued from 2010-2012; no additional infections were discovered. Over 10,000 deer were tested and 705 whole herd cattle tests performed in the investigation of this outbreak.

  1. Bovine tuberculosis in Rwanda: Prevalence and economic impact evaluation by meat inspection at Société des Abattoirs de Nyabugogo-Nyabugogo Abattoir, Kigali.

    Science.gov (United States)

    Habarugira, Gervais; Rukelibuga, Joseph; Nanyingi, Mark O; Mushonga, Borden

    2014-11-05

    Despite the significant public health burden of bovine tuberculosis (bTB) in Rwanda, the prevalence of bTB is poorly documented. This study was conducted to estimate the prevalence of bTB in cattle using gross examination of granulomatous lesions, to identify mycobacteria species in suspected samples, and to evaluate the economic impact of meat condemnation based on bTB-like lesions in the meat industry in Rwanda. Routine meat inspection was conducted at Société des Abattoirs de Nyabugogo (SABAN)-Nyabugogo Abattoir. Tissue samples including 31 lymph nodes, 3 lungs and 2 livers were obtained from cattle of different ages with gross tuberculous lesions. Mycobacterium bovis was identified using microscopy with Kinyoun staining and isolation of mycobacterial species in culture on Löwenstein-Jensen and Colestos media, further identified using biochemical tests. Our findings, based on culture and postmortem results, show that the prevalence of bTB is 0.5%(0.587*148/16753), with an overall gross tuberculous lesion prevalence of 0.9% (148/16753). The presence of lesions were higher in cattle aged 2 years and older (1.6% vs. 0.6%, p Bovine tuberculosis caused condemnation of 1683.5 kg of meat, resulting in an estimated loss of $4810. Our findings indicate that the prevalence of bTB in Rwanda is significant, and that bTB is a major cause of meat condemnation requiring continued implementation of surveillance and control measures. Furthermore, the results from this study also show important variations in sensitivity of the different tests that were used to determine the prevalence of bTB in cattle in Rwanda.

  2. Molecular detection of multidrug-resistant tuberculosis among smear-positive pulmonary tuberculosis patients in Jigjiga town, Ethiopia

    Science.gov (United States)

    Brhane, Mussie; Kebede, Ameha; Petros, Yohannes

    2017-01-01

    Background Molecular methods that target drug resistance mutations are suitable approaches for rapid drug susceptibility testing to detect multidrug-resistant tuberculosis (MDR-TB). The aim of the study was to determine MDR-TB cases and to analyze the frequency of gene mutations associated with rifampicin (RIF) and/or isoniazid (INH) resistance of Mycobacterium tuberculosis among smear-positive pulmonary tuberculosis patients. Methods Institution-based cross-sectional study design was employed. Sputum specimens were collected, and using a pretested questionnaire, data for associated risk factors for drug resistance were collected from 105 consecutive smear-positive pulmonary tuberculosis patients in Karamara General Hospital. Specimens were transported to Harar Health Research and Regional Laboratory, Harar, where molecular drug susceptibility testing was performed using GenoType® MTBDRplus assay. Results Of the total 105 sputum specimens, 98 (93.3%) gave interpretable results, in which 67 (68.4%) were new cases and 31 (31.6%) were previously treated cases. Of these, 80 (81.6%) were sensitive to both drugs and 18 (18.4%) were resistant to RIF and/or INH. The prevalences of MDR-TB in total cases, new, and previously treated cases were 10 (10.2%), 3 (4.5%), and 7 (22.6%), respectively. Among the ten total RIF-resistant specimens, eight (80%) had resulted because of absence of rpoB WT8 and presence of MUT3 and in all specimens, the amino acids changed were Ser531Lue. Of the 18 total INH-resistant specimens, 15 (83.3%) had mutations in the katG gene (katG MUT1, Ser315Thr1), indicating high-level resistance, while 3 (14.7%) had mutations in the inhA promoter gene (Cys15Thr), indicating low-level resistance. Conclusion Among the mutations associated with resistance to RIF and INH, the majority were in codon 531 of the rpoB gene and codon 315 of the katG gene. Relatively high prevalence of MDR-TB was observed in the study.

  3. Detection of Rifampin Resistance in Mycobacterium tuberculosis by Double Gradient-Denaturing Gradient Gel Electrophoresis

    Science.gov (United States)

    Scarpellini, Paolo; Braglia, Sergio; Carrera, Paola; Cedri, Maura; Cichero, Paola; Colombo, Alessia; Crucianelli, Rosella; Gori, Andrea; Ferrari, Maurizio; Lazzarin, Adriano

    1999-01-01

    We applied double gradient-denaturing gradient gel electrophoresis (DG-DGGE) for the rapid detection of rifampin (RMP) resistance from rpoB PCR products of Mycobacterium tuberculosis isolates and clinical samples. The results of this method were fully concordant with those of DNA sequencing and susceptibility testing analyses. DG-DGGE is a valid alternative to the other methods of detecting mutations for predicting RMP resistance. PMID:10508043

  4. The gamma-interferon test: its usefulness in a bovine tuberculosis survey in African buffaloes (Syncerus caffer) in the Kruger National Park.

    Science.gov (United States)

    Grobler, D G; Michel, Anita L; De Klerk, Lin-Mari; Bengis, R G

    2002-09-01

    A survey to determine the bovine tuberculosis status of buffalo herds north of the Olifants River in the Kruger National Park was conducted, using a new diagnostic approach. Diagnosis of Mycobacterium bovis infection was accomplished using the gamma-interferon assay technique in 608 adult buffaloes out of a total of 29 discreet herds. The animals were immobilized in groups of 10-15, bled, individually marked and then revived and released on site. As soon as test results were available (after 26-36 h), the same buffalo herd was relocated by tracking the frequency of a radio-collar previously fitted to one adult cow per group during the initial operation. Bovine reactors were identified, darted and euthanased from the helicopter. Necropsy and culture findings of all culled buffaloes showed excellent correlation with the results of the ante-mortem gamma-interferon test. The survey revealed that over and above the two positive herds that had been identified during a previous survey carried out in 1996, there were three additional, but previously unidentified, infected herds in the region north of the Olifants River.

  5. DETECTION OF ANTIBODIES AGAINST BOVINE HERPES VIRUS 1, BOVINE VIRAL DIARRHEA VIRUS AND BOVINE RESPIRATORY SYNCYTIAL VIRUS IN EARLY AND ULTRA-EARLY WEANED BEEF CALVES

    Directory of Open Access Journals (Sweden)

    Diego Daniel Gonzalez

    2013-01-01

    Full Text Available Bovine respiratory disease is the leading cause of morbidity and mortality in weaned calves. In Argentina, two weaning practices have been implemented. In the early weaning, the calf is removed from the cow at 60-70 days of age while in ultra-early weaning the calf is weaned at 30-45 days of age. The purposes of both systems is to improve cow body condition, calf performance, conception rates and forage availability for the cow. In this study we evaluated the antibody response against BVDV and BoHV1 in early and ultra-early weaned calves that had received a conventional vaccination schedule (first dose at weaning and a booster 21 days post-weaning. Passively acquired immunity may provide protection against disease caused by these viruses. The presence of antibodies against BRSV, a virus that was not present in the vaccines used, was also evaluated as an indirect indicator of viral circulation in the herd. At the time of vaccination, calves presented a wide range of maternally-derived antibody titers. Vaccination against BoHV-1 did not evoke seroconvertion and antibody titers continued to decay throughout the experience. After vaccination, seroconversion to BVDV could be detected in calves with low antibody titers, while higher antibody titers exerted an inhibitory effect of the active humoral response.

  6. Detection of bovine meat and bone meal in animal feed at a level of 0.1%.

    Science.gov (United States)

    Aarts, Henk J M; Bouw, El M; Buntjer, Jaap B; Lenstra, Johannes A; Van Raamsdonk, Leo W D

    2006-01-01

    For the control of the transmission of bovine spongiform encephalopathy in cattle via feedstuff, a real-time polymerase chain reaction assay was developed with ruminant-specific Bov-B SINE primers, SYBR Green fluorescence detection, and melting curve analysis. In formulated cattle and chicken feed samples spiked with pure bovine and sheep meat and bone meal heated at 133 degrees C for 20 min, a contamination level of 0.1% was detected.

  7. Au-nanoprobes for detection of SNPs associated with antibiotic resistance in Mycobacterium tuberculosis

    Science.gov (United States)

    Veigas, Bruno; Machado, Diana; Perdigão, João; Portugal, Isabel; Couto, Isabel; Viveiros, Miguel; Baptista, Pedro V.

    2010-10-01

    Tuberculosis (TB) is one of the leading causes of infection in humans, causing high morbility and mortality all over the world. The rate of new cases of multidrug resistant tuberculosis (MDRTB) continues to increase, and since these infections are very difficult to manage, they constitute a serious health problem. In most cases, drug resistance in Mycobacterium tuberculosis has been related to mutations in several loci within the pathogen's genome. The development of fast, cheap and simple screening methodologies would be of paramount relevance for the early detection of these mutations, essential for the timely and effective diagnosis and management of MDRTB patients. The use of gold nanoparticles derivatized with thiol-modified oligonucleotides (Au-nanoprobes) has led to new approaches in molecular diagnostics. Based on the differential non-cross-linking aggregation of Au-nanoprobes, we were able to develop a colorimetric method for the detection of specific sequences and to apply this approach to pathogen identification and single base mutations/single nucleotide polymorphisms (SNP) discrimination. Here we report on the development of Au-nanoprobes for the specific identification of SNPs within the beta subunit of the RNA polymerase (rpoB locus), responsible for resistance to rifampicin in over 95% of rifampicin resistant M. tuberculosis strains.

  8. Au-nanoprobes for detection of SNPs associated with antibiotic resistance in Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Veigas, Bruno; Baptista, Pedro V [CIGMH, Departamento de Ciencias da Vida, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica (Portugal); Machado, Diana; Couto, Isabel; Viveiros, Miguel [Unidade de Micobacterias, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL) (Portugal); Perdigao, Joao; Portugal, Isabel, E-mail: pmvb@fct.unl.pt [Centro de Patogenese Molecular/URIA, Faculdade de Farmacia, Universidade de Lisboa, Lisboa (Portugal)

    2010-10-15

    Tuberculosis (TB) is one of the leading causes of infection in humans, causing high morbility and mortality all over the world. The rate of new cases of multidrug resistant tuberculosis (MDRTB) continues to increase, and since these infections are very difficult to manage, they constitute a serious health problem. In most cases, drug resistance in Mycobacterium tuberculosis has been related to mutations in several loci within the pathogen's genome. The development of fast, cheap and simple screening methodologies would be of paramount relevance for the early detection of these mutations, essential for the timely and effective diagnosis and management of MDRTB patients. The use of gold nanoparticles derivatized with thiol-modified oligonucleotides (Au-nanoprobes) has led to new approaches in molecular diagnostics. Based on the differential non-cross-linking aggregation of Au-nanoprobes, we were able to develop a colorimetric method for the detection of specific sequences and to apply this approach to pathogen identification and single base mutations/single nucleotide polymorphisms (SNP) discrimination. Here we report on the development of Au-nanoprobes for the specific identification of SNPs within the beta subunit of the RNA polymerase (rpoB locus), responsible for resistance to rifampicin in over 95% of rifampicin resistant M. tuberculosis strains.

  9. Pouched Rats’ Detection of Tuberculosis in Human Sputum: Comparison to Culturing and Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Amanda Mahoney

    2012-01-01

    Full Text Available Setting. Tanzania. Objective. To compare microscopy as conducted in direct observation of treatment, short course centers to pouched rats as detectors of Mycobacterium tuberculosis. Design. Ten pouched rats were trained to detect tuberculosis in sputum using operant conditioning techniques. The rats evaluated 910 samples previously evaluated by smear microscopy. All samples were also evaluated through culturing and multiplex polymerase chain reaction was performed on culture growths to classify the bacteria. Results. The patientwise sensitivity of microscopy was 58.0%, and the patient-wise specificity was 97.3%. Used as a group of 10 with a cutoff (defined as the number of rat indications to classify a sample as positive for Mycobacterium tuberculosis of 1, the rats increased new case detection by 46.8% relative to microscopy alone. The average samplewise sensitivity of the individual rats was 68.4% (range 61.1–73.8%, and the mean specificity was 87.3% (range 84.7–90.3%. Conclusion. These results suggest that pouched rats are a valuable adjunct to, and may be a viable substitute for, sputum smear microscopy as a tuberculosis diagnostic in resource-poor countries.

  10. Detection of bovine herpesvirus 2 and bovine herpesvirus 4 DNA in trigeminal ganglia of naturally infected cattle by polymerase chain reaction.

    Science.gov (United States)

    Campos, F S; Franco, A C; Oliveira, M T; Firpo, R; Strelczuk, G; Fontoura, F E; Kulmann, M I R; Maidana, S; Romera, S A; Spilki, F R; Silva, A D; Hübner, S O; Roehe, P M

    2014-06-25

    Establishment of latent infection within specific tissues in the host is a common biological feature of the herpesviruses. In the case of bovine herpesvirus 2 (BoHV-2), latency is established in neuronal tissues, while bovine herpesvirus 4 (BoHV-4) and ovine herpesvirus 2 (OvHV-2) latent virus targets on cells of the monocytic lineage. This study was conducted in quest of BoHV-2, BoHV-4 and OvHV-2 DNA in two hundred trigeminal ganglia (TG) specimens, derived from one hundred clinically healthy cattle, majority of them naturally infected with bovine herpesvirus 1 (BoHV-1) and bovine herpesvirus 5 (BoHV-5). Total DNA extracted from ganglia was analyzed by polymerase chain reaction (PCR) designed to amplify part of the genes coding for BoHV-2, and BoHV-4 glycoprotein B and, for OvHV-2, the gene coding for phosphoribosylformylglycinamidine synthase-like protein. BoHV-2 DNA was detected in TG samples of two (2%) and BoHV-4 DNA in nine (9%) of the animals, whereas OvHV-2 DNA could not be detected in any of the TG DNA. The two animals in which BoHV-2 DNA was identified were also co-infected with BoHV-1 and BoHV-5. Within the nine animals in which BoHV-4 DNA was detected, six were also co-infected with BoHV-1 and BoHV-5. This report provides for the first time evidence that viral DNA from BoHV-2 and BoHV-4 can be occasionally detected in TG of naturally infected cattle. Likewise, in this report we provided for the first time evidence that the co-infection of cattle with three distinct bovine herpesviruses might be a naturally occurring phenomenon.

  11. Detecting endotoxin activity in bovine serum using an automated testing system.

    Science.gov (United States)

    Suzuki, Kazuyuki; Shimamori, Toshio; Sato, Ayano; Tsukano, Kenji; Tsuchiya, Masakazu; Lakritz, Jeffrey

    2015-08-01

    The aim of the present study was to compare the ability of the commercially available portable test system (PTS(TM)) to detect endotoxin activity in bovine serum, with that of the traditional LAL-kinetic turbidimetric (KT) and chromogenic (KC) assays. Prior to testing, serum samples, which were obtained from endotoxin-challenged cattle, were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. The performance of the PTS(TM) was not significantly different from that of the traditional LAL-based assays. The results using PTS(TM) correlated with those using KT (r(2)=0.963, PPTS(TM) could be applied as a simplified system to assess endotoxin activity in bovine serum.

  12. Intraspecies transmission of L-type-like Bovine Spongiform Encephalopathy detected in Japan.

    Science.gov (United States)

    Fukuda, Shigeo; Iwamaru, Yoshifumi; Imamura, Morikazu; Masujin, Kentarou; Shimizu, Yoshihisa; Matsuura, Yuichi; Shu, Yujing; Kurachi, Megumi; Kasai, Kazuo; Murayama, Yuichi; Onoe, Sadao; Hagiwara, Ken'ichi; Sata, Tetsutaro; Mohri, Shirou; Yokoyama, Takashi; Okada, Hiroyuki

    2009-12-01

    It has been assumed that the agent causing BSE in cattle is a uniform strain (classical BSE); however, different neuropathological and molecular phenotypes of BSE (atypical BSE) have been recently reported. We demonstrated the successful transmission of L-type-like atypical BSE detected in Japan (BSE/JP24 isolate) to cattle. Based on the incubation period, neuropathological hallmarks, and molecular properties of the abnormal host prion protein, the characteristics of BSE/JP24 prion were apparently distinguishable from the classical BSE prion and closely resemble those of bovine amyloidotic spongiform encephalopathy prion detected in Italy.

  13. Rapid Detection of rpoB Gene Mutations in Rif-resistant M.tuberculosis Isolates by Obligonucleotide Microarray

    Institute of Scientific and Technical Information of China (English)

    AI-HUA SUN; XING-LI FAN; LI-WEI LI; LI-FANG WANG; WEN-YING AN; JIE YAN

    2009-01-01

    Objective To detect the specific mutations in rpoB gene of Mycobacterium tuberculosis by oligonucleotide microarray.Methods Four wild-type and 8 mutant probes were used to detect rifampin resistant strains.Target DNA of M.tuberculosis was amplified by PCR,hybridized and scanned.Direct sequencing was performed to verify the results of oligonuclcotide microarray.Results of the 102 rifampin-resistant strains 98 (96.1%) had mutations in the rpoB genes. Conclusion Oligonucleotide microarray with mutation-specific probes is a reliable and useful tool for the rapid and accurate diagnosis of rifampin resistance in M.tuberculosis isolates.

  14. Detection of Mycobacterium tuberculosis resistance mutations to rifampin and isoniazid by real-time PCR

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    Hristea A

    2010-01-01

    Full Text Available Objective: The objective of our study was to evaluate the use of a real-time polymerase chain reaction (PCR-based technique for the prediction of phenotypic resistance of Mycobacterium tuberculosis. Materials and Methods: We tested 67 M tuberculosis strains (26 drug resistant and 41 drug susceptible using a method recommended for the LightCycler platform. The susceptibility testing was performed by the absolute concentration method. For rifampin resistance, two regions of the rpoB gene were targeted, while for identification of isoniazid resistance, we searched for mutations in katG and inhA genes. Results: The sensitivity and specificity of this method for rapid detection of mutations for isoniazid resistance were 96% (95% CI: 88% to 100% and 95% (95% CI: 89% to 100%, respectively. For detection of rifampin resistance, the sensitivity and specificity were 92% (95% CI: 81% to 100% and 74% (95% CI: 61% to 87%, respectively. The main isoniazid resistance mechanism identified in our isolates is related to changes in the katG gene that encodes catalase. We found that for rifampin resistance the concordance between the predicted and observed phenotype was less than satisfactory. Conclusions: Using this method, the best accuracy for genotyping compared with phenotypic resistance testing was obtained for detecting isoniazid resistance mutations. Although real-time PCR assay may be a valuable diagnostic tool, it is not yet completely satisfactory for detection of drug resistance mutations in M tuberculosis.

  15. A pragmatic approach to measuring, monitoring and evaluating interventions for improved tuberculosis case detection.

    Science.gov (United States)

    Blok, Lucie; Creswell, Jacob; Stevens, Robert; Brouwer, Miranda; Ramis, Oriol; Weil, Olivier; Klatser, Paul; Sahu, Suvanand; Bakker, Mirjam I

    2014-09-01

    The inability to detect all individuals with active tuberculosis has led to a growing interest in new approaches to improve case detection. Policy makers and program staff face important challenges measuring effectiveness of newly introduced interventions and reviewing feasibility of scaling-up successful approaches. While robust research will continue to be needed to document impact and influence policy, it may not always be feasible for all interventions and programmatic evidence is also critical to understand what can be expected in routine settings. The effects of interventions on early and improved tuberculosis detection can be documented through well-designed program evaluations. We present a pragmatic framework for evaluating and measuring the effect of improved case detection strategies using systematically collected intervention data in combination with routine tuberculosis notification data applying historical and contemporary controls. Standardized process evaluation and systematic documentation of program implementation design, cost and context will contribute to explaining observed levels of success and may help to identify conditions needed for success. Findings can then guide decisions on scale-up and replication in different target populations and settings. © The Author 2014. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.

  16. Genotype MTBDR plus assay for molecular detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis

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    Soniya Sharma

    2014-01-01

    Full Text Available Aim: This study was performed for the rapid identification of Mycobacterium tuberculosis complex and its resistance to rifampicin and isoniazid, directly from the sputum samples of pulmonary tuberculosis patients. Materials and Methods: A commercially available genotype MTBDR plus assay was used for the identification and detection of mutations in Mycobacterial isolates. A total of 100 sputum samples of pulmonary tuberculosis patients were analyzed by using the genotype MTBDR plus assay. The MTBDR plus assay is designed to detect the mutations in the hotspot region of rpoB gene, katG and regulatory region of inhA gene. Results: The genotype MTBDR plus assay detected 22% multidrug resistant (MDR, 2% rifampicin (RMP monoresistant and 1% isoniazid (INH monoresistant isolates. In 22 MDR isolates, the codons most frequently involved in RMP-associated mutations were codon 531 (54.55%, 516 (31.82% and 526 (13.63%, and 90.90% of MDR isolates showed KatG S315T mutations and 9.1% showed inhA C-15T mutations associated with INH resistance. Conclusion: The new genotype MTBDR plus assay represents a rapid, reliable tool for the detection of MDR-TB, wherein results are obtained in 5 h allowing early and appropriate treatment, which is essential to cut the transmission path and reduce the spread of MDR-TB. The genotype MTBDR plus assay can readily be included in a routine laboratory work for the early diagnosis and control of MDR-TB.

  17. Detection of Mycobacterium tuberculosis DNA in clinical samples by using a simple lysis method and polymerase chain reaction.

    Science.gov (United States)

    Folgueira, L; Delgado, R; Palenque, E; Noriega, A R

    1993-01-01

    We have evaluated the polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples from patients with tuberculous infection. Two simple methods for mycobacterial DNA release have been compared: sonication and lysis with nonionic detergents and proteinase K. The more effective method was the enzymatic technique. By using this protocol with 75 specimens we detected M. tuberculosis DNA in all of the samples, whereas only 48 and 71 samples were positive by acid-fast staining and culture, respectively. Images PMID:8463383

  18. Evaluation of milk cathelicidin for detection of bovine mastitis.

    Science.gov (United States)

    Addis, M F; Tedde, V; Puggioni, G M G; Pisanu, S; Casula, A; Locatelli, C; Rota, N; Bronzo, V; Moroni, P; Uzzau, S

    2016-10-01

    with SCC >200,000 cells/mL (94.9% Sp for cathelicidin vs. 96.3% for SCC >200,000). The limited Se of BC (38.8%) was also confirmed in this study, and BC showed a slightly lower Sp than both cathelicidin and SCC for most of threshold combinations. This study confirmed that cathelicidin is released in the milk of cows with mastitis and that its presence is highly correlated with SCC. The measurement of cathelicidin by ELISA may hold significant potential for improving the sensitivity of mastitis detection in dairy cows while maintaining high specificity. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  19. Clinical value of polymerase chain reaction in the diagnosis of joint tuberculosis by detecting the DNA of Mycobacterium tuberculosis.

    Science.gov (United States)

    Sun, Yong-sheng; Lou, Si-quan; Wen, Jian-min; Lv, Wei-xin; Jiao, Chang-geng; Yang, Su-min; Xu, Hai-bin

    2011-02-01

    To assess the clinical value of polymerase chain reaction (PCR) in the diagnosis and differential diagnosis of joint tuberculosis (TB). PCR was used blindly to detect the DNA of Mycobacterium tuberculosis (M.TB) in five specimens of M.TB, 5 of BCG, and 10 of other bacteria. Then, M. TB in 98 samples from patients with joint TB and 100 samples from patients with non-tubercular joint disorders were detected by PCR, acid-fast staining and culture,. The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of PCR were calculated. The χ2 test was used for statistical analysis of the frequency of various factors. At the same time, some problems with PCR were also systematically analyzed. (1) In the "standard samples", both M. TB and BCG showed positive while other bacteria were negative. (2) In 98 cases from patients with joint TB, 81 were positive by PCR, 6 by acid-fast staining, and 17 by culture. In 100 cases from patients with non-tuberculous joint disorders, 9 were positive by PCR, and none by either acid-fast staining or culture. Sensitivity, specificity, accuracy, positive and negative predictive value of PCR were 82.65% (81/98), 91.00% (91/100), 86.87% (172/198), 90.00% (81/90) and 84.26% (91/108), respectively. (3) The positive rates for PCR, acid-fast staining and culture in detection of M. TB were 82.65% (81/98), 6.12% (6/98), and 17.34% (17/98), respectively. There were statistically significant differences between the three methods (P < 0.001). (4) The process of PCR is automatic, and can be completed within 3 to 6 hours, whereas 4 to 8 weeks are required for the conventional culture of M. TB. PCR is a sensitive, specific, rapid, simple and minimally invasive method for detection of M. TB in samples from joint TB, and can play an important role in early and rapid diagnosis and differential diagnosis of joint TB. But it also has some limitations, such as false positivity and false negativity. © 2011 Tianjin Hospital

  20. Development of low-cost inverted microscope to detect early growth of Mycobacterium tuberculosis in MODS culture.

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    Mirko Zimic

    Full Text Available BACKGROUND: The microscopic observation drug susceptibility (MODS assay for rapid, low-cost detection of tuberculosis and multidrug resistant tuberculosis depends upon visualization of the characteristic cording colonies of Mycobacterium tuberculosis in liquid media. This has conventionally required an inverted light microscope in order to inspect the MODS culture plates from below. Few tuberculosis laboratories have this item and the capital cost of $5,000 for a high-end microscope could be a significant obstacle to MODS roll-out. METHODOLOGY: We hypothesized that the precise definition provided by costly high-specification inverted light microscopes might not be necessary for pattern recognition. SIGNIFICANCE: In this work we describe the development of a low-cost artesenal inverted microscope that can operate in both a standard or digital mode to effectively replace the expensive commercial inverted light microscope, and an integrated system that could permit a local and remote diagnosis of tuberculosis.

  1. Analytical and Clinical Evaluation of the Epistem Genedrive Assay for Detection of Mycobacterium tuberculosis.

    Science.gov (United States)

    Shenai, Shubhada; Armstrong, Derek T; Valli, Eloise; Dolinger, David L; Nakiyingi, Lydia; Dietze, Reynaldo; Dalcolmo, Margareth Pretti; Nicol, Mark P; Zemanay, Widaad; Manabe, Yuka; Hadad, David Jamil; Marques-Rodrigues, Patricia; Palaci, Moises; Peres, Renata L; Gaeddert, Mary; Armakovitch, Sandra; Nonyane, Bareng A S; Denkinger, Claudia M; Banada, Padmapriya; Joloba, Moses L; Ellner, Jerrold; Boehme, Catharina; Alland, David; Dorman, Susan E

    2016-04-01

    The Epistem Genedrive assay rapidly detects the Mycobacterium tuberculosis omplex from sputum and is currently available for clinical use. However, the analytical and clinical performance of this test has not been fully evaluated. The analytical limit of detection (LOD) of the Genedrive PCR amplification was tested with genomic DNA; the performance of the complete (sample processing plus amplification) system was tested by spiking M. tuberculosismc(2)6030 cells into distilled water andM. tuberculosis-negative sputum. Specificity was tested using common respiratory pathogens and nontuberculosis mycobacteria. A clinical evaluation enrolled adults with suspected pulmonary tuberculosis, obtained three sputum samples from each participant, and compared the accuracy of the Gene drive to that of the Xpert MTB/RIF assay using M. tuberculosiscultures as the reference standard. The Genedrive assay had an LOD of 1 pg/μl (100 genomic DNA copies/reaction). The LODs of the system were 2.5 × 10(4)CFU/ml and 2.5 × 10(5)CFU/ml for cells spiked into water and sputum, respectively. False-positiverpoBprobe signals were observed in 3/32 (9.4%) of the negative controls and also in few samples containing Mycobacterium abscessus,Mycobacterium gordonae, o rMycobacterium thermoresistibile In the clinical study, among 336 analyzed participants, the overall sensitivities for the tuberculosis case detection of Gene drive, Xpert, and smear microscopy were 45.4% (95% confidence interval [CI], 35.2% to 55.8%), 91.8% (95% CI, 84.4% to 96.4%), and 77.3% (95% CI, 67.7% to 85.2%), respectively. The sensitivities of Gene drive and Xpert for the detection of smear-microscopy-negative tuberculosis were 0% (95% CI, 0% to 15.4%) and 68.2% (95% CI, 45.1% to 86.1%), respectively. The Genedrive assay did not meet performance standards recommended by the World Health Organization for a smear microscopy replacement tuberculosis test. Epistem is working on modifications to improve the assay.

  2. Evaluation of Cobas TaqMan MTB PCR for detection of Mycobacterium tuberculosis.

    Science.gov (United States)

    Kim, Jeong Hyun; Kim, Young Jae; Ki, Chang-Seok; Kim, Ji-Youn; Lee, Nam Yong

    2011-01-01

    Nucleic acid-based amplification tests allow the rapid detection of Mycobacterium tuberculosis. Recently, a real-time PCR assay for M. tuberculosis complex, the Cobas TaqMan MTB test (Roche Diagnostics, Basel, Switzerland), was introduced. We performed a prospective study to evaluate the diagnostic performance of the Cobas TaqMan MTB test system. A total of 406 specimens collected from 247 patients were simultaneously tested by conventional culture, Cobas Amplicor MTB PCR, and TaqMan MTB PCR. The cross-reactivity with other Mycobacterium species and the detection limit were also evaluated. Among 406 specimens, a total of 24 specimens (5.9%) were culture positive: 14 specimens were positive by both TaqMan and Amplicor MTB PCRs, while 5 specimens were positive by only TaqMan PCR. The remaining five specimens were negative by both PCR methods. Seven specimens with negative culture results were positive by TaqMan PCR, but five of these were negative by Amplicor MTB PCR. The sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values were 79.1%, 98.2%, 73.1%, and 98.7% for TaqMan and 58.3%, 99.5%, 87.5%, and 97.4% for the Amplicor MTB PCR test, respectively. There was no cross-reactivity with M. tuberculosis and nontuberculous mycobacterial species. The detection limit for the Cobas TaqMan MTB PCR test was 4.0 copies/μl. The Cobas TaqMan MTB PCR test showed higher sensitivity for detection of the M. tuberculosis complex without disturbing the specificity and NPV than the Amplicor MTB PCR test.

  3. Detection of a mecC-positive Staphylococcus saprophyticus from bovine mastitis in Argentina.

    Science.gov (United States)

    Srednik, Mariela E; Archambault, Marie; Jacques, Mario; Gentilini, Elida R

    2017-07-18

    Bovine mastitis causes important economic losses in the dairy industry. Coagulase-negative staphylococci (CNS) are a group of bacteria commonly isolated from bovine mastitis and can display resistance to a wide range of antimicrobial agents. The objective of this study was to determine staphylococcal resistance towards β-lactam, macrolide and lincosamide antimicrobials in quarters previously treated with third-generation cephalosporin and after lincosamide intramammary therapy. Sick quarters of eighteen cows from Villaguay, Entre Ríos (Argentina) with clinical mastitis were studied. All staphylococcal isolates were tested by disk diffusion for their antimicrobial susceptibilities. Cefoxitin resistance was investigated by PCR and sequencing for both the mecA and mecC genes. Resistances to penicillin, oxacillin and cefoxitin were observed, whereas no resistance to macrolide and lincosamide was detected. A cefoxitin-resistant Staphylococcus saprophyticus was found to be mecA-negative but mecC-positive. This study reports for the first time the mecC gene from a CNS in bovine mastitis in South America. Because CNS may act as reservoirs of antimicrobial resistance genes, they can be seen as a potential public health threat with respect to antimicrobial resistance and the development of multiple resistance. Also, the emergence of methicillin-resistant phenotypes will limit therapeutic options. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

  4. Detection of bovine respiratory syncytial virus infections in young dairy and beef cattle in Poland.

    Science.gov (United States)

    Urban-Chmiel, Renata; Wernicki, Andrzej; Puchalski, Andrzej; Dec, Marta; Stęgierska, Diana; Grooms, Daniel L; Barbu, Nicolas I

    2015-03-01

    Bovine respiratory syncytial virus (BRSV) is a major contributor to bovine respiratory disease complex in dairy and beef calves, especially during the first year of life. There is a lack of comprehensive information about the prevalence of infection in cattle herds in Poland as well as in European countries outside the European Union. The aim of this study was to estimate the prevalence of BRSV infections in young beef and dairy cattle in southeastern Poland, a region that has direct contact with non-EU countries. Animals & methods: Nasal swabs and sera (n = 120) were obtained from young cattle aged 6-12 months from 45 farms in eastern and southeastern Poland. BRSV antigen detection in the nasal swabs was carried out using a rapid immunomigration assay used in diagnosing human respiratory syncytial virus (hRSV) infections in humans, while antibodies to BRSV were detected in the sera by ELISA antibody detection. The study confirmed the presence of BRSV infections in young cattle under 12 months of age from both dairy and beef herds. BRSV was detected in 27 of the 45 herds (60%) sampled. Findings from this study indicate a high prevalence of BRSV infections in cattle in Poland, which may have a significant influence on health status and animal performance. The prevalence of infection is similar to that in other parts of Poland and other countries in Europe. Development of strategies to reduce BRSV infections is needed to improve health and productivity.

  5. Detection of Helicobacter pylori in raw bovine milk by fluorescence in situ hybridization (FISH).

    Science.gov (United States)

    Angelidis, Apostolos S; Tirodimos, Ilias; Bobos, Mattheos; Kalamaki, Mary S; Papageorgiou, Demetrios K; Arvanitidou, Malamatenia

    2011-12-02

    The transmission pathways of Helicobacter pylori in humans have not been fully elucidated. Research in the last decade has proposed that foodborne transmission, among others, may be a plausible route of human infection. Owing to the organism's fastidious growth characteristics and its ability to convert to viable, yet unculturable states upon exposure to stress conditions, the detection of H. pylori in foods via culture-dependent methods has been proven to be laborious, difficult and in most cases unsuccessful. Hence, nucleic acid-based methods have been proposed as alternative methods but, to date, only PCR-based methods have been reported in the literature. In the current study, fluorescence in situ hybridization (FISH) was used for the detection of H. pylori in raw, bulk-tank bovine milk. After repeated milk centrifugation and washing steps, the bacterial flora of raw milk was subjected to fixation and permeabilization and H. pylori detection was conducted by FISH after hybridization with an H. pylori-specific 16S rRNA-directed fluorescent oligonucleotide probe. Using this protocol, H. pylori was detected in four out of the twenty (20%) raw milk samples examined. The data presented in this manuscript indicate that FISH can serve as an alternative molecular method for screening raw bovine milk for the presence of H. pylori.

  6. Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for Halal authentication.

    Science.gov (United States)

    Nikzad, Jafar; Shahhosseini, Soraya; Tabarzad, Maryam; Nafissi-Varcheh, Nastaran; Torshabi, Maryam

    2017-02-14

    In the pharmaceutical industry, hard- and soft-shelled capsules are typically made from gelatin, commonly derived from bovine and porcine sources. To ensure that pharmaceutical products comply with halal regulations in Muslim countries (no porcine products allowed), development of a valid, reliable, quick, and most importantly, cost-effective tests are of utmost importance. We developed a species-specific duplex polymerase chain reaction (PCR) assay targeting 149 bp porcine and 271 bp bovine mitochondrial DNA (mtDNA) to simultaneously detect both porcine and bovine DNA (in one reaction at the same time) in gelatin. Some additional simplex PCR tests (targeting 126 bp bovine and 212 bp porcine mtDNA) and real-time PCR using a commercially available kit (for identification of porcine DNA) were used to verify the selectivity and sensitivity of our duplex PCR. After optimization of DNA extraction and PCR methods, hard/soft pharmaceutical gelatin capsules (containing drug) were tested for the presence of porcine and/or bovine DNA. Duplex PCR detected the presence of as little as 0.1% porcine DNA, which was more accurate than the commercially available kit. Of all gelatin capsules tested (n = 24), 50% contained porcine DNA (pure porcine gelatin alone or in combination with bovine gelatin). Duplex PCR presents an easy-to-follow, quick, low-cost and reliable method to simultaneously detect porcine and bovine DNAs (>100 bp) in minute amounts in highly processed gelatin-containing pharmaceutical products (with a 0.1% sensitivity for porcine DNA) which may be used for halal authentication. Simultaneous detection of porcine and bovine DNA in gelatin capsules by duplex PCR.

  7. Detection of Mycobacterium isolates with different methods and their resistance ratios against anti-tuberculosis drugs

    Directory of Open Access Journals (Sweden)

    Mustafa Altındiş, Zafer Çetinkaya, Raike Kalaycı, Ihsan H Ciftçi, Alpaslan Arslan, Orhan C. Aktepe

    2011-06-01

    Full Text Available Objectives: The aim of the present study was to evaluate the efficacy (recovery rate, time to detection and Drug SusceptibilityTests –DST- of Mycobacteria-only B460 of new colorimetric medium, Dio-TK and to compare it with routinely used conventional media, Lowenstein Jensen (LJ and Bactec 460 TB culture system.Materials and methods: Totally 901 clinic specimens were investigated for assignment of tuberculosis by Ehrlich-Ziehl-Nielsen smear strain method, Lowenstein-Jensen, BACTEC 460TB and Dio-TK medium culture systems.Results: Nineteen of 901 clinic specimens (2.1% were positive by any of these methods. 17 (89.5% of these specimens positive found by smear strain method, 17 (89.5% by Lowenstein-Jensen, 19 (100% by BACTEC 460TB and 14 (73.7% by Dio-TK medium. NAP and Niacin identification tests were applied to Mycobacterium strains. 12 (63.1% of 19 isolates were identified as M.tuberculosis complex and 7 (36.9% were identified as Mycobacterium other than tuberculosis (MOTT bacilli. 10 (83.3% of 12 M.tuberculosis complex strains were not resistant to any major drug. But one of 2 isolate was resistant to streptomycin and the other one isolate was resistant to both streptomycin and isoniazid.Conclusion: Our data suggest that some advantages (such as an early detection and differentiation mycobacterium growth from contamination of the Dio-TK CS over other mycobacterial culture systems make it a practical and rapid system for daily use, and a suitable alternative to other currently available solid media, such as LJ, for detection time of mycobacteria and DST. J Microbiol Infect Dis 2011;1 (1 :5-9.

  8. Detection of Tuberculosis Infection Hotspots Using Activity Spaces Based Spatial Approach in an Urban Tokyo, from 2003 to 2011.

    Directory of Open Access Journals (Sweden)

    Kiyohiko Izumi

    Full Text Available Identifying ongoing tuberculosis infection sites is crucial for breaking chains of transmission in tuberculosis-prevalent urban areas. Previous studies have pointed out that detection of local accumulation of tuberculosis patients based on their residential addresses may be limited by a lack of matching between residences and tuberculosis infection sites. This study aimed to identify possible tuberculosis hotspots using TB genotype clustering statuses and a concept of "activity space", a place where patients spend most of their waking hours. We further compared the spatial distribution by different residential statuses and describe urban environmental features of the detected hotspots.Culture-positive tuberculosis patients notified to Shinjuku city from 2003 to 2011 were enrolled in this case-based cross-sectional study, and their demographic and clinical information, TB genotype clustering statuses, and activity space were collected. Spatial statistics (Global Moran's I and Getis-Ord Gi* statistics identified significant hotspots in 152 census tracts, and urban environmental features and tuberculosis patients' characteristics in these hotspots were assessed.Of the enrolled 643 culture-positive tuberculosis patients, 416 (64.2% were general inhabitants, 42 (6.5% were foreign-born people, and 184 were homeless people (28.6%. The percentage of overall genotype clustering was 43.7%. Genotype-clustered general inhabitants and homeless people formed significant hotspots around a major railway station, whereas the non-clustered general inhabitants formed no hotspots. This suggested the detected hotspots of activity spaces may reflect ongoing tuberculosis transmission sites and were characterized by smaller residential floor size and a higher proportion of non-working households.Activity space-based spatial analysis suggested possible TB transmission sites around the major railway station and it can assist in further comprehension of TB transmission

  9. Performance assessment of the GenoType MTBDRplus test and DNA sequencing in detection of multidrug-resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Huang, Wei-Lun; Chen, Huang-Yau; Kuo, Yuh-Min; Jou, Ruwen

    2009-08-01

    To facilitate the management of multidrug-resistant (MDR) tuberculosis, two nucleic acid sequence-based methods, the GenoType MTBDRplus test and DNA sequencing, were assessed for the rapid detection of drug-resistant Mycobacterium tuberculosis for the first time in the Asia-Pacific region. The performances of these two assays in detecting the presence of rifampin (rifampicin) (RIF) and isoniazid (INH) resistance-associated mutations in the rpoB, katG, inhA regulatory region, inhA, and oxyR-ahpC genes were compared to that of a conventional agar proportion drug susceptibility test. A total of 242 MDR and 30 pansusceptible M. tuberculosis isolates were evaluated in this study. The sensitivities obtained for RIF-resistant detection by the GenoType MTBDRplus test and by resistance gene sequencing were 95.5% and 97.9%, respectively. The sensitivities for INH resistance detection by the GenoType MTBDRplus test and by resistance gene sequencing were 81.8% and 93.4%, respectively. Together, the sensitivity for MDR tuberculosis detection was 78.5% with the GenoType MTBDRplus test and 91.3% by resistance gene sequencing. The specificity for RIF resistance, INH resistance, and MDR detection was 100% by both methods. The GenoType MTBDRplus test has the advantage of a short turnaround time for drug-resistant M. tuberculosis detection. Overall, the two assays performed equally well in detecting RIF resistance (P = 0.13). However, DNA sequencing demonstrated superior performance in detecting INH resistance (P tuberculosis (P GenoType MTBDRplus test, especially for different geographic areas with genetically diverse M. tuberculosis strains.

  10. The Nexus between Bovine Tuberculosis and Fasciolosis Infections in Cattle of the Kafue Basin Ecosystem in Zambia: Implications on Abattoir Surveillance

    Directory of Open Access Journals (Sweden)

    Musso Munyeme

    2012-01-01

    Full Text Available Bovine tuberculosis (bTB and fasciolosis are important but neglected diseases that result in chronic infections in cattle. However, in Zambia, these diseases are mainly diagnosed at abattoirs during routine meat inspection. Albeit the coinfection status, these diseases have been reported as nothing more than normal separate findings without an explanatory phenomena. Forthwith, we formulated this study to assess the possible association of the two diseases in a known high prevalence area on the Kafue basin ecosystem. Of the 1,680 animals screened, 600 (35.7%; 95% CI 33.4%–38% and 124 (7.4%; 95% CI 6.1%–8.6% had fasciolosis and tuberculous lesions; respectively, whilst 72 had both fasciola and tuberculous lesions representing 12% (95% CI 9.4%–14.6% and 58.1% (95% CI; 49.3%–66.7% of the total positives for fasciola and tuberculosis, respectively. Jaundice was seen in 304 animals, 18.1% (95% CI; 16.3%–19.9% and was significantly correlated to fasciolosis (r=0.59, P<0.0001. A significant association (χ2=76.2, df=1, and P<0.0001 was found between fasciolosis and tuberculous lesions. Simple logistic regression intimated fasciolosis as a strong predictor for tuberculous lesions with animals that had fasciola being five times more likely to have tuberculous lesions (odds ratio = 4.8, 95% CI: 3.3–7.0. This study indicates that transmission and spatial risk factors of communicable and noncommunicable diseases such as bTB and fasciolosis can be correlated in an ecosystem such as the Kafue flats.

  11. Mode of binding of the tuberculosis prodrug isoniazid to heme peroxidases: binding studies and crystal structure of bovine lactoperoxidase with isoniazid at 2.7 A resolution.

    Science.gov (United States)

    Singh, Amit K; Kumar, Ramasamy P; Pandey, Nisha; Singh, Nagendra; Sinha, Mau; Bhushan, Asha; Kaur, Punit; Sharma, Sujata; Singh, Tej P

    2010-01-01

    Isoniazid (INH) is an anti-tuberculosis prodrug that is activated by mammalian lactoperoxidase and Mycobacterium tuberculosis catalase peroxidase (MtCP). We report here binding studies, an enzyme assay involving INH, and the crystal structure of the complex of bovine lactoperoxidase (LPO) with INH to illuminate binding properties and INH activation as well as the mode of diffusion and interactions together with a detailed structural and functional comparison with MtCP. The structure determination shows that isoniazid binds to LPO at the substrate binding site on the distal heme side. The substrate binding site is connected to the protein surface through a long hydrophobic channel. The acyl hydrazide moiety of isoniazid interacts with Phe(422) O, Gln(423) O(epsilon1), and Phe(254) O. In this arrangement, pyridinyl nitrogen forms a hydrogen bond with a water molecule, W-1, which in turn forms three hydrogen bonds with Fe(3+), His(109) N(epsilon2), and Gln(105) N(epsilon2). The remaining two sides of isoniazid form hydrophobic interactions with the atoms of heme pyrrole ring A, C(beta) and C(gamma) atoms of Glu(258), and C(gamma) and C(delta) atoms of Arg(255). The binding studies indicate that INH binds to LPO with a value of 0.9 x 10(-6) m for the dissociation constant. The nitro blue tetrazolium reduction assay shows that INH is activated by the reaction of LPO-H(2)O(2) with INH. This suggests that LPO can be used for INH activation. It also indicates that the conversion of INH into isonicotinoyl radical by LPO may be the cause of INH toxicity.

  12. Bovine tuberculosis in Rwanda: Prevalence and economic impact evaluation by meat inspection at Société des Abattoirs de Nyabugogo-Nyabugogo Abattoir, Kigali

    Directory of Open Access Journals (Sweden)

    Gervais Habarugira

    2014-02-01

    Full Text Available Despite the significant public health burden of bovine tuberculosis (bTB in Rwanda, the prevalence of bTB is poorly documented. This study was conducted to estimate the prevalence of bTB in cattle using gross examination of granulomatous lesions, to identify mycobacteria species in suspected samples, and to evaluate the economic impact of meat condemnation based on bTB-like lesions in the meat industry in Rwanda. Routine meat inspection was conducted at Société des Abattoirs de Nyabugogo (SABAN-Nyabugogo Abattoir. Tissue samples including 31 lymph nodes, 3 lungs and 2 livers were obtained from cattle of different ages with gross tuberculous lesions. Mycobacterium bovis was identified using microscopy with Kinyoun staining and isolation of mycobacterial species in culture on Löwenstein–Jensen and Colestos media, further identified using biochemical tests. Our findings, based on culture and postmortem results, show that the prevalence of bTB is 0.5%(0.587*148/16753, with an overall gross tuberculous lesion prevalence of 0.9% (148/16753. The presence of lesions were higher in cattle aged 2 years and older (1.6% vs. 0.6%, p < 0.05 and higher in females than in males (1.4% vs. 0.6%, p < 0.05. Of the 36 samples tested, 26 (72.2% were positive by microscopic examination with Kinyoun staining while M. bovis was culture-confirmed in 21 (58.7% cases. Bovine tuberculosis caused condemnation of 1683.5 kg of meat, resulting in an estimated loss of $4810. Our findings indicate that the prevalence of bTB in Rwanda is significant, and that bTB is a major cause of meat condemnation requiring continued implementation of surveillance and control measures. Furthermore, the results from this study also show important variations in sensitivity of the different tests that were used to determine the prevalence of bTB in cattle in Rwanda.

  13. Evaluating use of cattle winter feeding areas by elk and white-tailed deer: implications for managing bovine tuberculosis transmission risk from the ground up.

    Science.gov (United States)

    Brook, Ryan K; Wal, Eric Vander; van Beest, Floris M; McLachlan, Stéphane M

    2013-02-01

    Transmission of bovine tuberculosis (Mycobacterium bovis) among wildlife and livestock has created important risks for conservation and agriculture. Management strategies aimed at controlling TB have typically been top-down, regionally focused, and government-led programs that were at best only partially successful. The purpose of this study was to quantify co-mingling of elk and white-tailed deer (WTD) with cattle at multiple spatial scales (i.e., the regional farm scale and winter cattle feeding area patch) in southwestern Manitoba, Canada, to assess the potential for bovine tuberculosis transmission and identify alternative management strategies. For each spatial scale we quantified use of cattle farms by elk and white-tailed deer. We mailed questionnaires to rural households and then conducted personal interviews with 86 cattle farmers to map the spatial distribution of their cattle winter feeding areas at a fine scale. We deployed Global Positioning System (GPS) collars on 48 wild elk and 16 wild white-tailed deer from 2003 to 2011. Elk were observed on farms by 66% of cattle producers, including 5% and 20% who observed direct and indirect contact, respectively, between elk and cattle. Cattle producers consistently (≈100%) observed white-tailed deer on their farms, including 11% and 47% whom observed direct and indirect contact, respectively, between white-tailed deer and cattle. A higher probability of white-tailed deer-cattle contact at the regional scale occurs on farms that (1) left crop residues specifically for wildlife, (2) had larger cattle herds, (3) used round bale feeders, and (4) were farther away from protected areas. None of the GPS-collared elk locations overlapped with cattle winter feeding areas. In contrast, 21% of GPS-collared white-tailed deer locations overlapped with winter cattle winter feeding areas (22% of these were from male WTD and 78% were from female WTD). White-tailed deer selected cattle winter feeding areas with higher (1

  14. Duplex detection of the Mycobacterium tuberculosis complex and medically important non-tuberculosis mycobacteria by real-time PCR based on the rnpB gene.

    Science.gov (United States)

    Abdeldaim, Guma; Svensson, Erik; Blomberg, Jonas; Herrmann, Björn

    2016-11-01

    A duplex real-time PCR based on the rnpB gene was developed for Mycobacterium spp. The assay was specific for the Mycobacterium tuberculosis complex (MTB) and also detected all 19 tested species of non-tuberculous mycobacteria (NTM). The assay was evaluated on 404 clinical samples: 290 respiratory samples and 114 from tissue and other non-respiratory body sites. M. tuberculosis was detected by culture in 40 samples and in 30 samples by the assay. The MTB assay showed a sensitivity similar to Roche Cobas Amplicor MTB-PCR (Roche Molecular Systems, Pleasanton, CA, USA). There were only nine samples with non-tuberculous mycobacteria detected by culture. Six of them were detected by the PCR assay.

  15. Detection of Ethambutol - resistant Mycobacterium tuberculosis strains by MAS-PCR method and comparison with Proportion

    Directory of Open Access Journals (Sweden)

    M. Asgharzadeh

    2007-01-01

    Full Text Available Abstract Background and purpose: Ethambutol (EMB is one of the first - line drugs used for anti-tubercular therapy but resistance to this medicine is developed in many parts of the world. EMB resistant strains commonly have embB mutations. Purpose of this research was detection of EMB-resistant Mycobactercium tuberculosis strains isolated from patients by MAS-PCR method and comparison with Proportion procedure.Materials and Methods: One hundred and twenty M. tuberculosis strains were isolated from patients with tulerculosis in Tabriz TB research center. Susceptibility testing to EMB was performed by the Proportion method. DNA was isolated from cultivated cells by SDS-proteinase K modified method. Isolated DNA was used as the template for PCR reaction.Results: One hundred and sixteen strains were susceptible to EMB and 4 (3.33% strains were resistant to EMB. All EMB resistant strains were multidrug-resistant. The MAS-PCR method was used to evaluate of mutation in the embB306 codon. Mutation was seen at the embB306 codon in all resistant strains to ethambutol.Conclusion: The results showed that MAS-PCR method can be used as a simple and rapid procedure for detecting EMB-resistance in Mycobacterium tuberculosis strains.

  16. Rapid detection of MDR-Mycobacterium tuberculosis using modified PCR-SSCP from clinical Specimens

    Institute of Scientific and Technical Information of China (English)

    Imani Fooladi Abbas Ali; Farzam Babak; Mousavi Seyed Fazlollah; Jonaidi Jafari Nematollah

    2014-01-01

    Objective: To design a rapid test to detect the rifampin (RIF) and isoniazid (INH) resistant mutant based on polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique that analyzes the katG, rpoB genes.Methods:tuberculosis. To determine the susceptibility of isolates to anti TB drugs, the proportional method was used. Mutations presented within the amplified products of the katG, rpoB genes were evaluated by SSCP.Results:Using proportional method, 12 (11.6%) and 9 (8.7%) isolates were resistant respectively Biochemical test as well as IS6110 targeting PCR revealed 103 clinical samples were to INH and RIF and 9 (8.7%) isolates showed resistance to both drug (multi-drug resistant tuberculosis). Three (2.9%) multi-drug resistant tuberculosis and two INH resistant isolates were detected by the PCR-SSCP and sequencing. The sensitivity and specificity of PCR-SSCP for multi-drug resistant isolates were 33% and 100%, respectively.Conclusions:Complete agreement between SSCP and sequencing can indicate that resistance-associated mutations have occurred in other genes except our considered genes.

  17. Biomarkers for the detection, prognois and evaluation of active tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Shinimukundan, Harshini [Los Alamos National Laboratory

    2010-12-08

    The global TS surveillance workshop aims to address the problems with current methods for the detection of TB, and tracking emergence of resistant strains. The purpose of the attached presentation is to review the current methods in the detection of pathogen biomarkers for TB and if that technology has promise for diagnosis of TB. A summary of three biomarkers and some data on their detection strategies is presented. Some of the work is from LANL work but much of it is derived from literature references on the subject.

  18. Validation of a real-time PCR assay for the molecular identification of Mycobacterium tuberculosis.

    Science.gov (United States)

    Sales, Mariana L; Fonseca Júnior, Antônio Augusto; Orzil, Lívia; Alencar, Andrea Padilha; Silva, Marcio Roberto; Issa, Marina Azevedo; Soares Filho, Paulo Martins; Lage, Andrey Pereira; Heinemann, Marcos Bryan

    2014-01-01

    Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method.

  19. Comparison of levels and duration of detection of antibodies to bovine viral diarrhea virus 1, bovine viral diarrhea virus 2, bovine respiratory syncytial virus, bovine herpesvirus 1, and bovine parainfluenza virus 3 in calves fed maternal colostrum or a colostrum-replacement product.

    Science.gov (United States)

    Chamorro, Manuel F; Walz, Paul H; Haines, Deborah M; Passler, Thomas; Earleywine, Thomas; Palomares, Roberto A; Riddell, Kay P; Galik, Patricia; Zhang, Yijing; Givens, M Daniel

    2014-04-01

    Colostrum-replacement products are an alternative to provide passive immunity to neonatal calves; however, their ability to provide adequate levels of antibodies recognizing respiratory viruses has not been described. The objective of this study was to compare the serum levels of IgG at 2 d of age and the duration of detection of antibodies to bovine viral diarrhea virus 1 (BVDV-1), bovine viral diarrhea virus 2 (BVDV-2), bovine respiratory syncytial virus (BRSV), bovine herpesvirus 1 (BHV-1), and bovine parainfluenza virus 3 (BPIV-3) in calves fed maternal colostrum (MC) or a colostrum replacement (CR) at birth. Forty newborn male Holstein calves were assigned to the CR or the MC group. Group CR (n = 20) received 2 packets of colostrum replacement (100 g of IgG per 470-g packet), while group MC (n = 20) received 3.8 L of maternal colostrum. Blood samples for detection of IgG and virus antibodies were collected from each calf at birth, at 2 and 7 d, and monthly until the calves became seronegative. Calves in the MC group had greater IgG concentrations at 2 d of age. The apparent efficiency of absorption of IgG was greater in the MC group than in the CR group, although the difference was not significant. Calves in the CR group had greater concentrations of BVDV neutralizing antibodies during the first 4 mo of life. The levels of antibodies to BRSV, BHV-1, and BPIV-3 were similar in the 2 groups. The mean time to seronegativity was similar for each virus in the 2 groups; however, greater variation was observed in the antibody levels and in the duration of detection of immunity in the MC group than in the CR group. Thus, the CR product provided calves with more uniform levels and duration of antibodies to common bovine respiratory viruses.

  20. Selected Pool of Peptides from ESAT-6 and CFP-10 Proteins for Detection of Mycobacterium tuberculosis Infection

    Science.gov (United States)

    Scarpellini, Paolo; Tasca, Silvana; Galli, Laura; Beretta, Alberto; Lazzarin, Adriano; Fortis, Claudio

    2004-01-01

    We have validated a new test for detecting Mycobacterium tuberculosis infection. A pool of synthetic peptides derived from ESAT-6 and CFP-10 proteins was used to detect the number of specific gamma interferon-producing T cells by means of an enzyme-linked immunospot assay. Sixty-eight individuals positive for M. tuberculosis infection, either human immunodeficiency virus-seropositive or -seronegative, were studied. The test results were highly specific (87.5%) and sensitive (93.1%), more so than a classical lymphoproliferative assay (specificity and sensitivity of 77.27%), opening new possibilities for diagnosis and screening of tuberculosis. Moreover, the test allowed us to distinguish individuals infected with M. tuberculosis from those vaccinated with BCG. PMID:15297485

  1. Rapid detection of Mycobacterium tuberculosis complex in sputum Samples using PURE TB-LAMP assay.

    Science.gov (United States)

    N'guessan, K; Horo, K; Coulibaly, I; Adegbele, J; Kouame-Adjei, N; Seck-Angu, H; Guei, A; Kouakou, J; Dosso, M

    2016-12-01

    Lack of rapid and accurate diagnostic testing is a critical obstacle to global tuberculosis (TB) control. Sensitivity of sputum smear microscopy (SSM) is not optimal; however, it remains the most prevalent tool for TB confirmation in poor countries. As a part of passive case finding of TB detection, this study was conducted to determine the clinical performance of PURE TB-LAMP assay using liquid culture medium as the gold standard. Centre Antituberculeux de Yopougon is one of the 17 intermediate Tuberculosis centers in Côte d'Ivoire. A standardized questionnaire was submitted to patients with signs and symptoms consistent with tuberculosis by a trained caregiver. After obtaining signed consent forms, sputum samples were collected according to National TB Control Programme guidelines (spot-morning). SSM after Ziehl-Neelsen staining and TB-LAMP assay were blindly performed on the first sample. Samples transported to Institut Pasteur de Côte d'Ivoire were decontaminated according to the N-acetyl-L-Cystein method. In Mycobacteria Growth Indicator Tube (MGIT), 500mL of pellets were inoculated and incubated in the MGIT 960 system. MPT64 antigen was detected in positive cultures. Of the 500 patients enrolled, 469 (232men and 239 women) patients were included. The mean ages of men and women were 36.9 (15-86) and 37.3 (15-37.3) years, respectively. There were 56 (12.2%) HIV-infected patients, including 14 women. Clinical isolates of M. tuberculosis complex were detected for 157 (33.5%) patients. Compared with culturing, the overall sensitivity and specificity of SSM were 86% (95% confidence interval [CI]=81-91) and 96% (95% CI=94-98), respectively. The overall sensitivity and specificity for TB-LAMP was 92% (95% CI=0.88-0.96) and 94% (95% CI=0.91-0.97), respectively. Positive likelihood ratios for TB-LAMP and SSM were 15.3 and 21.5, respectively, and negative likelihood ratios for TB-LAMP and SSM were 0.09 and 0.15, respectively. Among the 469 patients, active

  2. New tools for detecting latent tuberculosis infection: evaluation of RD1-specific long-term response.

    Science.gov (United States)

    Butera, Ornella; Chiacchio, Teresa; Carrara, Stefania; Casetti, Rita; Vanini, Valentina; Meraviglia, Serena; Guggino, Giuliana; Dieli, Francesco; Vecchi, Marco; Lauria, Francesco N; Marruchella, Almerico; Laurenti, Patrizia; Singh, Mahavir; Caccamo, Nadia; Girardi, Enrico; Goletti, Delia

    2009-11-21

    Interferon-gamma (IFN-gamma) release assays (IGRAs) were designed to detect latent tuberculosis infection (LTBI). However, discrepancies were found between the tuberculin skin test (TST) and IGRAs results that cannot be attributed to prior Bacille Calmètte Guerin vaccinations. The aim of this study was to evaluate tools for improving LTBI diagnosis by analyzing the IFN-gamma response to RD1 proteins in prolonged (long-term response) whole blood tests in those subjects resulting negative to assays such as QuantiFERON-TB Gold In tube (QFT-IT). The study population included 106 healthy TST+ individuals with suspected LTBI (recent contact of smear-positive TB and homeless) consecutively enrolled. As controls, 13 healthy subjects unexposed to M. tuberculosis (TST-, QFT-IT-) and 29 subjects with cured pulmonary TB were enrolled. IFN-gamma whole blood response to RD1 proteins and QFT-IT were evaluated at day 1 post-culture. A prolonged test evaluating long-term IFN-gamma response (7-day) to RD1 proteins in diluted whole blood was performed. Among the enrolled TST+ subjects with suspected LTBI, 70/106 (66.0%) responded to QFT-IT and 64/106 (60.3%) to RD1 proteins at day 1. To evaluate whether a prolonged test could improve the detection of LTBI, we set up the test using cured TB patients (with a microbiologically diagnosed past pulmonary disease) who resulted QFT-IT-negative and healthy controls as comparator groups. Using this assay, a statistically significant difference was found between IFN-gamma levels in cured TB patients compared to healthy controls (p < 0.006). Based on these data, we constructed a receiver operating characteristic (ROC) curve and we calculated a cut-off. Based on the cut-off value, we found that among the 36 enrolled TST+ subjects with suspected LTBI not responding to QFT-IT, a long term response to RD1 proteins was detected in 11 subjects (30.6%). These results indicate that IFN-gamma long-term response to M. tuberculosis RD1 antigens may be

  3. Application of hyperbranched rolling circle amplification for direct detection of mycobacterium tuberculosis in clinical sputum specimens.

    Directory of Open Access Journals (Sweden)

    Yang Liu

    Full Text Available BACKGROUND: Global tuberculosis (TB control is encumbered by the lack of a rapid and simple detection method for diagnosis, especially in low-resource areas. An isothermal amplification method, hyperbranched rolling circle amplification (HRCA, was optimized to detect Mycobacterium tuberculosis (Mtb in clinical sputum specimens. METHODS: A clinical validation study was performed to assess the diagnostic accuracy of HRCA. In order to analyze the detection limit of HRCA under optimal conditions, the method was initially used to detect purified H37Rv strain DNA and culture suspensions. Next, three strains of Mycobacterium tuberculosis complex (MTC and eight strains of non-tuberculosis mycobacterium (NTM were analyzed in order to evaluate specificity. Sputum specimens from 136 patients with diagnosed pulmonary TB, 38 lung cancer patients, and 34 healthy donors were tested by HRCA to validate the clinical application of HRCA for the rapid detection of Mtb. RESULTS: The detection limit of HRCA for purified H37Rv DNA and culture suspensions was 740 aM and 200cfu/ml, respectively. The results of all MTC strains were positive in contrast to the NTM specimens which were all negative. The detection sensitivity for the 136 sputum specimens from TB patients was 77.2% (105/136, which was slightly lower than that of quantitative real-time PCR(79.4%, 108/136 and culture (80.9%,110/136. The sensitivity of all three methods was statistically higher than smear microscopy (44.9%, 61/136. The overall specificity of HRCA was 98.6% (71/72 which was similar to that of quantitative real-time PCR (qRT-PCR and smear/culture methods (100%, 72/72. CONCLUSIONS: Use of the HRCA assay for detection of Mtb within clinical sputum specimens was demonstrated to be highly sensitive and specific. Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture. Therefore, HRCA is a promising TB diagnostic tool that can be

  4. Detecting katG Drug Resistant Genetic Mutation among pneumoconiosis patients complicated with tuberculosis in Mycobacterium tuberculosis L-forms by PCR-SSCP

    Institute of Scientific and Technical Information of China (English)

    Jun Lu; Zhao Zheng; Song Ye; Chaopin Li

    2006-01-01

    Objective: To study the relationship between mutation in the katG gene and drug resistance of INH in Mycobacterium tuberculosis L-forms among patients of pneumoconiosis complicated with pulmonary tuberculosis, and to explore the clinical application of PCR-SSCP. Methods:A total of 52 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected. Mutation in the katG genes was detected by PCR-SSCP and traditional antimicrobial susceptibility test (AST). Results:The results by AST showed that there were 40 persisters in 52 clinical isolated strains. The drug resistant rate was 76.92%(40/52),and the gene mutation rate of katG was 57.70% (30/52)by.PCR-SSCP, the difference was no quite significance (χ2 = 2.8507, P >0.05). The coincidence rate of two methods was 75.00% (30/40). Conclusion: The detectionrate of katG resistant strains in Mycobacterium tuberculosis L-forms was high by PCR-SSCP. The combined application of PCR-SSCP and traditional antimicrobial susceptibility test can improve the detecting rate.

  5. Early detection of Mycobacterium tuberculosis complex in BACTEC MGIT cultures using nucleic acid amplification.

    Science.gov (United States)

    Lin, S Y; Hwang, S C; Yang, Y C; Wang, C F; Chen, Y H; Chen, T C; Lu, P L

    2016-06-01

    We evaluated the application of nucleic acid amplification (NAA) in liquid cultures for the early detection of Mycobacterium tuberculosis. The Cobas TaqMan MTB test, IS6110 real-time PCR, and hsp65 PCR-restriction fragment length polymorphism (RFLP) analysis were used to detect BACTEC MGIT 960 (MGIT) cultures on days 3, 5, 7, and 14. The procedure was initially tested with a reference strain, H37Rv (ATCC 27294). Subsequently, 200 clinical specimens, including 150 Acid Fast bacillus (AFB) smear-positive and 50 AFB smear-negative samples, were examined. The Cobas TaqMan MTB test and IS6110-based PCR analysis were able to detect M. tuberculosis after 1 day when the inoculum of H37Rv was >3 x 10(-2) CFU/ml. After a 5-day incubation in the MGIT system, all three NAA assays had a positive detection regardless of the inoculum size. After a 1-day incubation of the clinical specimens in the MGIT system, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the Cobas TaqMan MTB assay were 70.2%, 100%, 100%, and 82.3% respectively. For IS6110-based PCR analysis, these values were 63.1%, 100%, 100%, and 78.9%, and were 88.1%, 100%, 100%, and 92.1% respectively for hsp65 PCR-RFLP analysis. After a 3-day incubation, the specificity and PPV were 100% for all three NAA tests; the Cobas TaqMan MTB assay had the best sensitivity (97.6%) and NPV (98.3%). The sensitivity, specificity, PPV, and NPV for conventional culture analysis were 98.8%, 100%, 100%, and 99.1%. Thus, NAA may be useful for the early detection of M. tuberculosis after 3 days in MGIT.

  6. Proposed model to study the economic impact of bovine brucellosis and tuberculosis: Case study of Pirassununga, SP, Brazil

    Directory of Open Access Journals (Sweden)

    Valéria Stacchini Ferreira Homem

    2016-11-01

    Full Text Available Following the recent implementation of the National Program for the Control and Eradication of Brucellosis and Tuberculosis (PNCEBT, and the lack of economic research on animal health in Brazil, we created a model to estimate the economic impact of brucellosis and tuberculosis on livestock production, using the municipality of Pirassununga/SP as a case study. The first part of our study consisted of a literature review of the estimates of losses caused by the two diseases. The impact on production was converted into economic losses, also represented by monetary values, using secondary references from 2003. The losses were calculated for the entire municipality and took into account the actual prevalence of disease in the animals, and the type of disease in each of the affected properties. The results revealed the annual losses in Pirassununga, which, at the time of the study, were as high as R$ 132,676.23 for brucellosis (updated value by the IGP-M: R$273,407.43, and between R$192,500.00 and R$ 430,252.00 for tuberculosis (values updated by the IGP-M: R$ 396,686.97 and R$ 886,825.25. These results suggest that these diseases had a significant impact on livestock production in the municipality of Pirassununga. The impacts they have on agriculture, and the economy, should be rapidly be communicated throughout Brazil because this information will significantly improve the management of veterinary sanitary processes.

  7. Determining resistance to mastitis in a bovine subject involves detecting presence or absence of genetic marker associated with trait indicative of mastitis resistance of the bovine subject and/or off-spring from it

    DEFF Research Database (Denmark)

    2010-01-01

    subject. The bovine subject is a member of Finnish Ayrshire, Swedish Red and White, or Danish Red cattle breed (claimed). ADVANTAGE - The method efficiently selects bovine subjects with increased resistance to mastitis and thereby avoiding economic losses in connection with animals suffering from mastitis......NOVELTY - Determining (m1) resistance to mastitis in a bovine subject, involves detecting in a sample from the bovine subject the presence or absence of at least one genetic marker that is associated with at least one trait indicative of mastitis resistance of the bovine subject and/or off......-spring from it, where the genetic marker is located on the bovine chromosome BTA11 in the region flanked by and including the zeta-chain associated protein 70kD (ZAP70) and CD8B genes, where the presence or absence of the genetic marker is indicative of mastitis resistance. USE - For determining resistance...

  8. Comparison of different diagnostic techniques for the detection of cryptosporidiosis in bovines

    Directory of Open Access Journals (Sweden)

    H. K. M. Rekha

    2016-02-01

    Full Text Available Aim: Aim of the present study was to compare different methods, viz., Sheather’s sugar flotation (SSF, Ziehl-Neelsen (ZN, Kinyoun’s acid-fast method (KAF, safranin-methylene blue staining (SMB, and negative staining techniques such as nigrosin staining, light green staining, and malachite green staining for the detection of Cryptosporidium spp. oocysts in bovines. Materials and Methods: A total of 455 fecal samples from bovines were collected from private, government farms and from the clinical cases presented to Department of Medicine, Veterinary College, Bengaluru. They were subjected for SSF, ZN, KAF, SMB and negative staining methods. Results: Out of 455 animal fecal samples screened 5.71% were found positive for Cryptosporidium spp. oocysts. The species were identified as Cryptosporidium parvum in calves and Cryptosporidium andersoni in adults based on the morphological characterization and micrometry of the oocysts. Conclusions: Of all the techniques, fecal flotation with sheather’s was found to be more specific and sensitive method for the detection of Cryptosporidium spp. oocysts. Among the conventional staining methods, the SMB gives better differentiation between oocysts and yeast. Among the three negative staining methods, malachite green was found sensitive over the other methods.

  9. Picobirnavirus detection in bovine and buffalo calves from foothills of Himalaya and Central India.

    Science.gov (United States)

    Malik, Yashpal Singh; Chandrashekar, K M; Sharma, Kuldeep; Haq, Adil A; Vaid, Nirupama; Chakravarti, Somendu; Batra, Munish; Singh, Rashmi; Pandey, A B

    2011-12-01

    The present study describes detection of picobirnavirus (PBV) in faecal samples from bovine and buffalo calves employing the polyacrylamide gel electrophoresis (PAGE). A total of 136 faecal samples from buffalo (n = 122) and cow calves (n = 14) exhibiting clinical signs of diarrhoea and from healthy calves were collected during 2007-2010 from subtropical (central India) and tarai area of western temperate Himalayan foothills (Uttarakhand). The dsRNA nature of the virus was confirmed by nuclease treatment (RNase A, RNaseT1 and DNase 1). PAGE results confirmed 3.67% (5/136) positivity for PBV, showing a typical genomic migration pattern with two discrete bands with size of approximately 2.4 and 1.7 kbps for the larger and smaller segments, respectively. Among the five PBV samples identified, three were from buffalo calves and one from cow calf exhibiting clinical signs of acute diarrhoea, while one sample from non-diarrhoeic buffalo calf also showed the presence of PBV. None of the samples showed dual infection of rotavirus and PBV. The preliminary findings indicate sporadic incidences of PBV in bovine calves and emphasize the need for the development of better diagnostics for early detection and genetic characterization of these emerging isolates of farm animals of economic significance.

  10. Sperm morphometry: a tool for detecting biophysical changes associated with viability in cryopreserved bovine spermatozoa.

    Science.gov (United States)

    García-Herreros, M; Leal, C L V

    2014-09-01

    The aim of this study was to determine whether computerised sperm head morphometric analysis can be used as a diagnostic tool for detecting biophysical changes associated with sperm viability in frozen-thawed bovine spermatozoa. Ejaculates from five bulls (4 ejaculates/bull) were pooled and processed for computerised morphometric analysis, and SYBR-14 green/ethidium homodimer-1 fluorescence-based live/dead viability assay was used simultaneously to confirm the viability index of frozen-thawed spermatozoa. Sperm samples were assigned to three experimental groups. The first group was enriched in live spermatozoa (after a double Percoll selection), the second group was enriched in dead spermatozoa (after a refreeze-thaw procedure), and the last group was a 50 : 50 pool of live/dead spermatozoa (from first and second group samples). There were significant differences (P sperm morphometric dimensional parameters among the three groups analysed, being the lowest overall sperm head dimension found in the second (dead spermatozoa) group. In conclusion, sperm head morphometry can be used as a potential diagnostic tool for detecting biophysical changes associated with sperm viability in frozen-thawed bovine spermatozoa.

  11. Serological and molecular detection of bovine leukemia virus in cattle in Iraq.

    Science.gov (United States)

    Khudhair, Yahia Ismail; Hasso, Saleem Amin; Yaseen, Nahi Y; Al-Shammari, Ahmed Majeed

    2016-06-08

    Bovine leukemia virus (BLV) is highly endemic in many countries, including Iraq, and it impacts the beef and dairy industries. The current study sought to determine the percentage of BLV infection and persistent lymphocytosis (PL) in cattle in central Iraq. Hematological, serological, and molecular observations in cross breeds and local breeds of Iraqi cattle naturally infected with BLV were conducted in the peripheral blood mononuclear cells of 400 cattle (340 cross breed and 60 local breed) using enzyme-linked immunosorbent assay and polymerase chain reaction (PCR). On the basis of the absolute number of lymphocytes, five of the 31 positive PCR cases had PL. Among these leukemic cattle, one case exhibited overt neutrophilia. Serum samples were used to detect BLV antibodies, which were observed in 28 (7%) samples. PCR detected BLV provirus in 31 samples (7.75%). All 28 of the seropositive samples and the 3 seronegative samples were positive using PCR. Associations were observed between bovine leukosis and cattle breed, age and sex. Age-specific analysis showed that the BLV percentage increased with age in both breeds. Female cattle (29 animals; 7.34%) exhibited significantly higher infectivity than male cattle (two animals; 4.34%). In conclusion, comprehensive screening for all affected animals is needed in Iraq; programs that segregate cattle can be an effective and important method to control and/or eliminate the BLV.

  12. Field test of a novel detection device for Mycobacterium tuberculosis antigen in cough

    Directory of Open Access Journals (Sweden)

    Tesfaye Ato

    2010-06-01

    Full Text Available Abstract Background Tuberculosis is a highly infectious disease that is spread from person to person by infected aerosols emitted by patients with respiratory forms of the disease. We describe a novel device that utilizes immunosensor and bio-optical technology to detect M. tuberculosis antigen (Ag85B in cough and demonstrate its use under field conditions during a pilot study in an area of high TB incidence. Methods The TB Breathalyzer device (Rapid Biosensor Systems Ltd was field tested in the outpatient clinic of Adama Hospital, Ethiopia. Adults seeking diagnosis for respiratory complaints were tested. Following nebulization with 0.9% saline patients were asked to cough into a disposable collection device where cough aerosols were deposited. Devices were then inserted into a portable instrument to assess whether antigen was present in the sample. Demographic and clinical data were recorded and all patients were subjected to chest radiogram and examination of sputum by Ziehl-Nielsen microscopy. In the absence of culture treatment decisions were based on smear microscopy, chest x-ray and clinical assessment. Breathalyzer testing was undertaken by a separate physician to triage and diagnostic assessment. Results Sixty individuals were each subjected to a breathalyzer test. The procedure was well tolerated and for each patient the testing was completed in less than 10 min. Positive breath test results were recorded for 29 (48% patients. Of 31 patients with a diagnosis of tuberculosis 23 (74%; 95% CI 55-87 were found positive for antigen in their breath and 20 (64%; 95% CI 45-80 were smear positive for acid fast bacilli in their sputum. Six patients provided apparent false positive breathalyzer results that did not correlate with a diagnosis of tuberculosis. Conclusions We propose that the breathalyzer device described warrants further investigation as a tool for studying exhalation of M. tuberculosis. The portability, simplicity of use and speed

  13. Valuing conservation benefits of disease control in wildlife: A choice experiment approach to bovine tuberculosis management in New Zealand's native forests.

    Science.gov (United States)

    Tait, Peter; Saunders, Caroline; Nugent, Graham; Rutherford, Paul

    2017-03-15

    We assess the non-monetary environmental benefits that accrue incidentally in New Zealand (NZ) from pest management conducted primarily to control an animal disease, bovine tuberculosis (TB). TB is an infectious disease that is one of the world's most serious animal health problems and, in many parts of the developing world, still a major mortality risk for humans. The incidence of TB in New Zealand (NZ) farmed livestock has been reduced progressively over the last 20 years, largely due to extensive and sustained population control of the main wildlife reservoir of disease, the introduced brushtail possum. Possums are also major pests that threaten indigenous forest biodiversity, and so extensive possum control for TB mitigation also incidental benefits conservation, but the extent and public value of this benefit has yet to be quantified. We conducted a choice experiment survey of the NZ public in an effort to value the native forest biodiversity benefits of TB-related possum control. We find strong public support for conservation outcomes consequent to TB-possum control in public native forests. The public place substantial value on the most observable biodiversity benefits of TB possum control, such as improved forest canopies and presence of native birds. The benefits, costs and values of TB-possum control are discussed in relation to the future directives of NZ's TB control programme, which is headed toward first regional and then national level disease eradication. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. New tools for detecting latent tuberculosis infection: evaluation of RD1-specific long-term response

    Directory of Open Access Journals (Sweden)

    Laurenti Patrizia

    2009-11-01

    Full Text Available Abstract Background Interferon-gamma (IFN-γ release assays (IGRAs were designed to detect latent tuberculosis infection (LTBI. However, discrepancies were found between the tuberculin skin test (TST and IGRAs results that cannot be attributed to prior Bacille Calmètte Guerin vaccinations. The aim of this study was to evaluate tools for improving LTBI diagnosis by analyzing the IFN-γ response to RD1 proteins in prolonged (long-term response whole blood tests in those subjects resulting negative to assays such as QuantiFERON-TB Gold In tube (QFT-IT. Methods The study population included 106 healthy TST+ individuals with suspected LTBI (recent contact of smear-positive TB and homeless consecutively enrolled. As controls, 13 healthy subjects unexposed to M. tuberculosis (TST-, QFT-IT- and 29 subjects with cured pulmonary TB were enrolled. IFN-γ whole blood response to RD1 proteins and QFT-IT were evaluated at day 1 post-culture. A prolonged test evaluating long-term IFN-γ response (7-day to RD1 proteins in diluted whole blood was performed. Results Among the enrolled TST+ subjects with suspected LTBI, 70/106 (66.0% responded to QFT-IT and 64/106 (60.3% to RD1 proteins at day 1. To evaluate whether a prolonged test could improve the detection of LTBI, we set up the test using cured TB patients (with a microbiologically diagnosed past pulmonary disease who resulted QFT-IT-negative and healthy controls as comparator groups. Using this assay, a statistically significant difference was found between IFN-γ levels in cured TB patients compared to healthy controls (p Conclusion These results indicate that IFN-γ long-term response to M. tuberculosis RD1 antigens may be used to detect past infection with M. tuberculosis and may help to identify additional individuals with LTBI who resulted negative in the short-term tests. These data may provide useful information for improving immunodiagnostic tests for tuberculosis infection, especially in

  15. Whole-genome sequencing to detect recent transmission of Mycobacterium tuberculosis in settings with a high burden of tuberculosis.

    Science.gov (United States)

    Luo, Tao; Yang, Chongguang; Peng, Ying; Lu, Liping; Sun, Guomei; Wu, Jie; Jin, Xiaoping; Hong, Jianjun; Li, Fabin; Mei, Jian; DeRiemer, Kathryn; Gao, Qian

    2014-07-01

    Whole genome sequencing (WGS) of Mycobacterium tuberculosis has been used to trace the transmission of M. tuberculosis, the causative agent of tuberculosis (TB). Previously published studies using WGS were conducted in developed countries with a low TB burden. We sought to evaluate the relative usefulness of traditional VNTR and SNP typing methods, WGS and epidemiological investigations to study the recent transmission of M. tuberculosis in a high TB burden country. We conducted epidemiological investigations of 42 TB patients whose M. tuberculosis isolates were classified into three clusters based on variable-number tandem repeat (VNTR) typing. We applied WGS to 32 (76.2%) of the 42 strains and calculated the pairwise genomic distances between strains within each cluster. Eighteen (56.3%) of the 32 strains had genomic differences ≥100 SNPs with every other strain, suggesting that direct transmission did not likely occurred. Ten strains were grouped into four WGS-based clusters with genomic distances ≤5 SNPs within each cluster, and confirmed epidemiological links were identified in two of these clusters. Our results indicate that WGS provides reliable resolution for tracing the transmission of M. tuberculosis in high TB burden settings. The high resolution of WGS is particularly useful to confirm or exclude the possibility of direct transmission events defined by traditional typing methods.

  16. Rapid determination of ampicillin in bovine milk by liquid chromatography with fluorescence detection

    Energy Technology Data Exchange (ETDEWEB)

    Ang, C.Y.W.; Luo, Wenhong [National Center for Toxicological Research, Jefferson, AR (United States)

    1997-01-01

    A rapid and sensitive liquid chromatographic (LC) method was developed for the determination of ampicillin residues in raw bovine milk, processed skim milk, and pasteurized, homogenized whole milk with vitamin D. Milk samples were deproteinized with trichloroacetic acid (TCA) and acetonictrile. After centrifugation, the clear supernatant was reacted with formaldehyde and TCA under heat. The major fluorescent derivative of ampicillin was then determined by reversed-phase LC with fluorescence detection. Average recoveries of ampicillin fortified at 5, 10, and 20 ppb (ng/mL) were all >85% with coefficients of variation <10%. Limits of detection ranged from 0.31 to 0.51 ppb and limits of quantitation, from 0.66 to 1.2 ppb. After appropriate validation, this method should be suitable for rapid analysis of milk for ampicillin residues at the tolerance level of 10 ppb. 16 refs., 4 figs., 3 tabs.

  17. An IMS/ATP Assay for the Detection of Mycobacterium tuberculosis in Urine

    Directory of Open Access Journals (Sweden)

    Dawn M. Hunter

    2012-01-01

    Full Text Available Background. Although sputum smears are the gold standard for diagnosis of tuberculosis, sensitivity in HIV/TB coinfection cases is low, indicating a need for alternative methods. Urine is being increasingly evaluated. Materials and Methods. A novel method for detecting Mycobacterium tuberculosis (MTB in synthetic urine using a combined IMS/ATP assay was evaluated. Preliminary work established standard ATP conditions and the sensitivity and specificity of the MTB antibody. Eighty-four blinded samples in four replicate assays were evaluated for the presence of MTB using labeled immunomagnetic beads for capture. Beads were separated, washed, and resuspended in broth and added to a microtiter plate. Bioluminescent output was measured and signal-to-noise ratios were calculated. All samples were plated on Middlebrook 7H10 agar or trypticase soy agar to determine limit of detection and recoveries. Results and Conclusions. MTB was distinguished from common bacteriuria isolates and other nontarget bacteria by its ATP results. IMS/ATP successfully detected 19 of 28 samples of MTB in synthetic urine with a limit of detection of 104 CFU/ml. Sensitivity and specificity were 67.9% and 82.1%, respectively. This assay offers a possible rapid screening method for HIV-positive patients with suspected coinfection to improve MTB diagnosis.

  18. Saliva as an alternative specimen for detection of Schmallenberg virus-specific antibodies in bovines.

    Science.gov (United States)

    Lazutka, Justas; Spakova, Aliona; Sereika, Vilimas; Lelesius, Raimundas; Sasnauskas, Kestutis; Petraityte-Burneikiene, Rasa

    2015-09-15

    Schmallenberg virus (SBV), discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. Enzyme-linked immunosorbent assays (ELISA) are commercially available for detection of SBV-specific antibodies in bovine sera and milk. Here we describe the development and evaluation of an indirect ELISA based on a yeast derived recombinant SBV nucleocapsid protein (N) for the detection of SBV-specific antibodies in bovine saliva. Development of a non-invasive test to detect antibodies in individual bovine saliva samples could potentially provide a test suitable for calves and adult cattle. The aim of this study was to investigate the agreement between the levels of antibodies (IgG) measured in milk and sera, and the level of antibodies (IgG and IgA) in saliva, in comparison with the antibody levels detected in sera and milk with commercially available test. Serum, milk and saliva samples from 58 cows were collected from three dairy herds in Lithuania and tested for the presence of SBV-specific antibodies. The presence of IgG antibodies was tested in parallel serum and milk samples, while the presence of IgA and IgG antibodies was tested in saliva samples. The presence of SBV-specific IgG and IgA in saliva was tested using an indirect ELISA based on a yeast-derived recombinant N protein. The presence of SBV-specific IgG in milk and sera was tested in parallel using a commercial recombinant protein based test. The sensitivities of the newly developed tests were as follows: 96 % for the IgG serum assay and 94 % for the IgG milk assay and 85 % and 98 % for IgG and IgA in saliva tests, when compared with data generated by a commercial IgG assay. Data from testing the saliva IgG and IgA and also the milk and serum IgG with indirect SBV-specific ELISAs showed close agreement with the commercial serum and milk IgG assay data

  19. Multidrug-resistant tuberculosis: rapid detection of resistance to rifampin and high or low levels of isoniazid in clinical specimens and isolates

    DEFF Research Database (Denmark)

    Vijdea, R.; Stegger, M.; Sosnovskaja, A.

    2008-01-01

    The aim of the present study was to evaluate a new improved multiplex polymerase chain reaction (PCR) hybridisation assay to detect multidrug-resistant tuberculosis. The assay, developed to detect rifampin (rpoB) and isoniazid (katG) gene mutations causing Mycobacterium tuberculosis resistance, w...... tool to combat and prevent new cases of multi- and extensively drug-resistant tuberculosis Udgivelsesdato: 2008/11...

  20. Array microscopy technology and its application to digital detection of Mycobacterium tuberculosis

    Science.gov (United States)

    McCall, Brian P.

    Tuberculosis causes more deaths worldwide than any other curable infectious disease. This is the case despite tuberculosis appearing to be on the verge of eradication midway through the last century. Efforts at reversing the spread of tuberculosis have intensified since the early 1990s. Since then, microscopy has been the primary frontline diagnostic. In this dissertation, advances in clinical microscopy towards array microscopy for digital detection of Mycobacterium tuberculosis are presented. Digital array microscopy separates the tasks of microscope operation and pathogen detection and will reduce the specialization needed in order to operate the microscope. Distributing the work and reducing specialization will allow this technology to be deployed at the point of care, taking the front-line diagnostic for tuberculosis from the microscopy center to the community health center. By improving access to microscopy centers, hundreds of thousands of lives can be saved. For this dissertation, a lens was designed that can be manufactured as 4x6 array of microscopes. This lens design is diffraction limited, having less than 0.071 waves of aberration (root mean square) over the entire field of view. A total area imaged onto a full-frame digital image sensor is expected to be 3.94 mm2, which according to tuberculosis microscopy guidelines is more than sufficient for a sensitive diagnosis. The design is tolerant to single point diamond turning manufacturing errors, as found by tolerance analysis and by fabricating a prototype. Diamond micro-milling, a fabrication technique for lens array molds, was applied to plastic plano-concave and plano-convex lens arrays, and found to produce high quality optical surfaces. The micro-milling technique did not prove robust enough to produce bi-convex and meniscus lens arrays in a variety of lens shapes, however, and it required lengthy fabrication times. In order to rapidly prototype new lenses, a new diamond machining technique was

  1. Application of PCR-SSCP in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis

    Institute of Scientific and Technical Information of China (English)

    LU Jun; JIANG Shan; ZHENG Zhao

    2006-01-01

    Objective: To study the relationship between the polymorphism of drug resistant gene rpoB and drug resistance against rifampicin(RFP) of M. tuberculosis L-forms, and to evaluate its clinical application. Methods: A total of 52 clinical isolated strains of M. tuberculosis L-forms were collected. rpoB gene polymorphism was analyzed by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) and conventional antimicrobial susceptibility test (AST). Their results were compared. Results: AST results showed that 38 of 52 clinical isolated strains were drug resistance (73.08%),while PCR-SSCP indicated 65.38% (32/52) rpoB gene polymorphism. There was no statistic significance(χ2= 2.4914) between the 2 methods. Conclusion:Combined the application of PCR-SSCP with AST in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis may have advantages at earlier diagnosis and guidance of clinical medications.

  2. Fasciola hepatica is associated with the failure to detect bovine tuberculosis in dairy cattle

    National Research Council Canada - National Science Library

    Claridge, Jen; Diggle, Peter; McCann, Catherine M; Mulcahy, Grace; Flynn, Rob; McNair, Jim; Strain, Sam; Welsh, Michael; Baylis, Matthew; Williams, Diana J L

    2012-01-01

    .... Here we show in a study involving 3,026 dairy herds in England and Wales that there is a significant negative association between exposure to the common, ubiquitous helminth parasite, Fasciola...

  3. Face mask sampling for the detection of Mycobacterium tuberculosis in expelled aerosols.

    Directory of Open Access Journals (Sweden)

    Caroline M L Williams

    Full Text Available Although tuberculosis is transmitted by the airborne route, direct information on the natural output of bacilli into air by source cases is very limited. We sought to address this through sampling of expelled aerosols in face masks that were subsequently analyzed for mycobacterial contamination.In series 1, 17 smear microscopy positive patients wore standard surgical face masks once or twice for periods between 10 minutes and 5 hours; mycobacterial contamination was detected using a bacteriophage assay. In series 2, 19 patients with suspected tuberculosis were studied in Leicester UK and 10 patients with at least one positive smear were studied in The Gambia. These subjects wore one FFP30 mask modified to contain a gelatin filter for one hour; this was subsequently analyzed by the Xpert MTB/RIF system.In series 1, the bacteriophage assay detected live mycobacteria in 11/17 patients with wearing times between 10 and 120 minutes. Variation was seen in mask positivity and the level of contamination detected in multiple samples from the same patient. Two patients had non-tuberculous mycobacterial infections. In series 2, 13/20 patients with pulmonary tuberculosis produced positive masks and 0/9 patients with extrapulmonary or non-tuberculous diagnoses were mask positive. Overall, 65% of patients with confirmed pulmonary mycobacterial infection gave positive masks and this included 3/6 patients who received diagnostic bronchoalveolar lavages.Mask sampling provides a simple means of assessing mycobacterial output in non-sputum expectorant. The approach shows potential for application to the study of airborne transmission and to diagnosis.

  4. Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial

    Directory of Open Access Journals (Sweden)

    El mashad Noha

    2009-01-01

    Full Text Available Abstract Background Tuberculosis is a growing international health concern. It is the biggest killer among the infectious diseases in the world today. Early detection of drug resistance allows starting of an appropriate treatment. Resistance to drugs is due to particular genomic mutations in specific genes of Mycobacterium tuberculosis(MTB. The aim of this study was to identify the presence of Isoniazid (INH and Rifampicin(RIF drug resistance in new and previously treated tuberculosis (TB cases using DNA sequencing. Methods This study was carried out on 153 tuberculous patients with positive Bactec 460 culture for acid fast bacilli. Results Of the 153 patients, 105 (68.6% were new cases and 48 (31.4% were previously treated cases. Drug susceptibility testing on Bactec revealed 50 resistant cases for one or more of the first line antituberculous. Genotypic analysis was done only for rifampicin resistant specimens (23 cases and INH resistant specimens (26 cases to detect mutations responsible for drug resistance by PCR amplification of rpoB gene for rifampicin resistant cases and KatG gene for isoniazid resistant cases. Finally, DNA sequencing was done for detection of mutation within rpoB and KatG genes. Genotypic analysis of RIF resistant cases revealed that 20/23 cases (86.9% of RIF resistance were having rpoB gene mutation versus 3 cases (13.1% having no mutation with a high statistical significant difference between them (P Conclusion We can conclude that rifampicin resistance could be used as a useful surrogate marker for estimation of multidrug resistance. In addition, Genotypic method was superior to that of the traditional phenotypic method which is time-consuming taking several weeks or longer.

  5. Utility of line probe assay for the early detection of multidrug-resistant pulmonary tuberculosis

    Directory of Open Access Journals (Sweden)

    K Madhuri

    2015-01-01

    Full Text Available Background: Despite endorsement of the line probe assay (LPA for the diagnosis of drug-resistant pulmonary tuberculosis patients, there is limited data available on the performance of LPAs in India, especially from high burden states like Maharashtra, for the early diagnosis and detection of drug resistance, in order to initiate timely and appropriate treatment. Objective: To evaluate the utility of the line probe assay (LPA for the early diagnosis of drug-resistant pulmonary tuberculosis as compared to the ′Gold standard′ 1% proportion method (PM. Materials and Methods: A total of 687 patients suspected of pulmonary tuberculosis were screened. One hundred samples (95 sputum and 5 BAL, positive for Acid Fast Bacilli (AFB by Ziehl Neelson (ZN smears, were included in the study. Digested and decontaminated specimens were subjected directly to the LPA (Genotype MTBDR@ plus assay and were processed in parallel using the conventional culture on the Lowenstein-Jensen (LJ medium followed by drug susceptibility testing (DST using the PM. Results: All the 100 samples gave interpretable results on LPA with a turnaround time of 24-48 hours as opposed to six to eight weeks taken by the 1% proportion method. Sensitivity for the detection of rifampicin, isoniazid, and multidrug resistance (MDR was 98.1, 92.1, and 95%, respectively, with a specificity of 97.8% for rifampicin and 98.33% for MDR detection. It also had the additional advantage of allowing a study of mutation patterns. Conclusions: High performance characteristics and a short turnaround time makes LPA an excellent diagnostic tool, for an early and accurate diagnosis, in a high MDR- TB-prevalent region, as reflected from our data.

  6. Whole genome sequencing of Mycobacterium tuberculosis for detection of drug resistance: a systematic review.

    Science.gov (United States)

    Papaventsis, D; Casali, N; Kontsevaya, I; Drobniewski, F; Cirillo, D M; Nikolayevskyy, V

    2017-02-01

    We conducted a systematic review to determine the diagnostic accuracy of whole genome sequencing (WGS) of Mycobacterium tuberculosis for the detection of resistance to first- and second-line anti-tuberculosis (TB) drugs. The study was conducted according to the criteria of the Preferred Reporting Items for Systematic Reviews group. A total of 20 publications were included. The sensitivity, specificity, positive-predictive value and negative-predictive value of WGS using phenotypic drug susceptibility testing methods as a reference standard were determined. Anti-TB agents tested included all first-line drugs, a variety of reserve drugs, as well as new drugs. Polymorphisms in a total of 53 genes were tested for associations with drug resistance. Pooled sensitivity and specificity values for detection of resistance to selected first-line drugs were 0.98 (95% CI 0.93-0.98) and 0.98 (95% CI 0.98-1.00) for rifampicin and 0.97 (95% CI 0.94-0.99) and 0.93 (95% CI 0.91-0.96) for isoniazid, respectively. Due to high heterogeneity in study designs, lack of data, knowledge of resistance mechanisms and clarity on exclusion of phylogenetic markers, there was a significant variation in analytical performance of WGS for the remaining first-line, reserved drugs and new drugs. Whole genome sequencing could be considered a promising alternative to existing phenotypic and molecular drug susceptibility testing methods for rifampicin and isoniazid pending standardization of analytical pipelines. To ensure clinical relevance of WGS for detection of M. tuberculosis complex drug resistance, future studies should include information on clinical outcomes. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  7. Detection of antibiotic residues in bovine milk by a voltammetric electronic tongue system.

    Science.gov (United States)

    Wei, Zhenbo; Wang, Jun

    2011-05-23

    A voltammetric electronic tongue (VE-tongue) was developed to detect antibiotic residues in bovine milk. Six antibiotics (Chloramphenicol, Erythromycin, Kanamycin sulfate, Neomycin sulfate, Streptomycin sulfate and Tetracycline HCl) spiked at four different concentration levels (0.5, 1, 1.5 and 2 maximum residue limits (MRLs)) were classified based on VE-tongue by two pattern recognition methods: principal component analysis (PCA) and discriminant function analysis (DFA). The VE-tongue was composed of five working electrodes (gold, silver, platinum, palladium, and titanium) positioned in a standard three-electrode configuration. The Multi-frequency large amplitude pulse voltammetry (MLAPV) which consisted of four segments (1 Hz, 10 Hz, 100 Hz and 1000 Hz) was applied as potential waveform. The six antibiotics at the MRLs could not be separated from bovine milk completely by PCA, but all the samples were demarcated clearly by DFA. Three regression models: Principal Component Regression Analysis (PCR), Partial Least Squares Regression (PLSR), and Least Squares-Support Vector Machines (LS-SVM) were used for concentrations of antibiotics prediction. All the regression models performed well, and PCR had the most stable results.

  8. Detection of antibiotic residues in bovine milk by a voltammetric electronic tongue system

    Energy Technology Data Exchange (ETDEWEB)

    Wei Zhenbo [Zhejiang University, Department of Bio-Systems Engineering, 268 Kaixuan Road, Hangzhou, Zhejiang 310029 (China); Wang Jun, E-mail: jwang@zju.edu.cn [Zhejiang University, Department of Bio-Systems Engineering, 268 Kaixuan Road, Hangzhou, Zhejiang 310029 (China)

    2011-05-23

    A voltammetric electronic tongue (VE-tongue) was developed to detect antibiotic residues in bovine milk. Six antibiotics (Chloramphenicol, Erythromycin, Kanamycin sulfate, Neomycin sulfate, Streptomycin sulfate and Tetracycline HCl) spiked at four different concentration levels (0.5, 1, 1.5 and 2 maximum residue limits (MRLs)) were classified based on VE-tongue by two pattern recognition methods: principal component analysis (PCA) and discriminant function analysis (DFA). The VE-tongue was composed of five working electrodes (gold, silver, platinum, palladium, and titanium) positioned in a standard three-electrode configuration. The Multi-frequency large amplitude pulse voltammetry (MLAPV) which consisted of four segments (1 Hz, 10 Hz, 100 Hz and 1000 Hz) was applied as potential waveform. The six antibiotics at the MRLs could not be separated from bovine milk completely by PCA, but all the samples were demarcated clearly by DFA. Three regression models: Principal Component Regression Analysis (PCR), Partial Least Squares Regression (PLSR), and Least Squares-Support Vector Machines (LS-SVM) were used for concentrations of antibiotics prediction. All the regression models performed well, and PCR had the most stable results.

  9. Molecular detection of bovine immunodeficiency virus in water buffaloes (Bubalus bubalis) from the Amazon region, Brazil.

    Science.gov (United States)

    Albernaz, Tatiane Teles; Leite, Rômulo Cerqueira; Reis, Jenner Karlison Pimenta; de Sousa Rodrigues, Ana Paula; da Cunha Kassar, Telissa; Resende, Claudia Fideles; de Oliveira, Cairo Henrique Sousa; Silva, Rafaela das Mercês; Salvarani, Felipe Masiero; Barbosa, José Diomedes

    2015-12-01

    Bovine immunodeficiency is a chronic progressive disease caused by a lentivirus that affects cattle and buffaloes. Although the infection has been described in cattle in some countries, including in Brazil, there are only two reports of infection in buffaloes: one in Pakistan and one in Cambodia. The aim of the present study was to survey the occurrence of bovine immunodeficiency virus (BIV) in water buffaloes from the Amazon region, Pará state, Brazil. BIV proviral DNA was surveyed in 607 whole blood samples of water buffaloes from 10 farms located in the state of Pará using semi-nested polymerase chain reaction (PCR) (PCR-SN) to amplify the pol region of the viral genome. Of the 607 samples tested, 27 (4.4 %) were positive for BIV proviral DNA. The amplified fragments were confirmed by sequence analysis after cloning and nucleotide sequencing. The sequence obtained had 99 % similarity to the reference strain (R-29). The present study provides important epidemiological data because BIV was detected for the first time in water buffaloes in Brazil. Further, the results suggest the possibility of the virus being a risk factor for herd health because it may be a potential causal agent of chronic disease and, also may be associated to other infectious diseases.

  10. [Detection of bovine leukaemia virus (BLV) in tissue samples of naturally and experimentally infected cattle].

    Science.gov (United States)

    Teifke, Jens P; Vahlenkamp, Thomas W

    2008-01-01

    Enzootic bovine leukaemia (EBL) which is caused by the bovine leukaemia virus (BLV) still plays a remarkable role despite a significant success in sanitation programmes. In the Federal Republic of Germany it was not possible to eradicate the disease until today. Sporadically during slaughter or necropsy of cattle neoplastic lesions of the lymphatic tissues are observed that need to be clarified with regard to BLV as etiological agent. Due to the fact that in most instances no serological data are available from the respective animals and blood drawings from the original holdings are not easy to obtain the polymerase chain reaction (PCR) opens new avenues as supplementary diagnostic tool to test unfixed lymphatic tissues for the presence of BLV proviral DNA. Lymph node tissues from 10 naturally or experimentally BLV-infected cattle, which have been monitored virologically and serologically, and tissues from 4 negative animals were processed, DNA was extracted and subjected to PCR to amplify BLV env gene specific sequences. The results show that in cattle with BLV-induced leukosis as well as in cattle, which were clinically healthy and unsuspicious at slaughter or at post-mortem, either with persistent lymphocytosis (PL) or without, BLV proviral DNA could be detected easily in samples of lymphatic tissues and in high concordance with serological data. In this article data from the National and OIE reference laboratory for EBL at the Friedrich-Loeffler-Institut (FLI, Germany) are presented. Elaborated laboratory protocols for processing of tissue samples and performing of BLV-PCR are recommended.

  11. Usefulness of serological ELISA assay forTaenia saginata to detect naturally infected bovines

    Directory of Open Access Journals (Sweden)

    Silvana de Cássia Paulan

    Full Text Available Bovine cysticercosis, a cosmopolitan disease caused by Taenia saginata, leads to economic losses due to carcass devaluation at slaughter. Sanitary inspection at slaughterhouses, the routine diagnostic method in Brazil, lacks the necessary sensitivity to detect the mildly infected cattle that are typically encoutered in Brazil. In this study we have tested cattle sera from animals diagnosed as positive and negative by veterianry inspection for (1 anti-parasite antibodies using metacestodes antigens (T. solium vesicular fluid and T. saginata secretions and (2 the HP10 secreted antigen of viable metacestodes. The cut-off values were calculated by ROC curve for intense and mild infections conditions, and by the classical method ( for negative samples. The sensitivity and specificity of these diagnostic tests were different depending on the assumed cut-off value and, importantly, whether the infection was mild or intense. In spite of these observations, however, such ELISA assays for serum antibodies and parasite antigens constitute an important tool for epidemiological porposes, and in establishing priorities for the control of bovine cysticercosis.

  12. Development of a highly sensitive one-tube nested real-time PCR for detecting Mycobacterium tuberculosis.

    Science.gov (United States)

    Choi, Yeonim; Jeon, Bo-Young; Shim, Tae Sun; Jin, Hyunwoo; Cho, Sang-Nae; Lee, Hyeyoung

    2014-12-01

    Rapid, accurate detection of Mycobacterium tuberculosis is crucial in the diagnosis of tuberculosis (TB), but conventional diagnostic methods have limited sensitivity and specificity or are time consuming. A new highly sensitive nucleic acid amplification test, combined nested and real-time polymerase chain reaction (PCR) in a single tube (one-tube nested real-time PCR), was developed for detecting M. tuberculosis, which takes advantage of two PCR techniques, i.e., nested PCR and real-time PCR. One-tube nested real-time PCR was designed to have two sequential reactions with two sets of primers and dual probes for the insertion sequence (IS) 6110 sequence of M. tuberculosis in a single closed tube. The minimum limits of detection of IS6110 real-time PCR and IS6110 one-tube nested real-time PCR were 100 fg/μL and 1 fg/μL of M. tuberculosis DNA, respectively. AdvanSure TB/non-tuberculous mycobacteria (NTM) real-time PCR, IS6110 real-time PCR, and two-tube nested real-time PCR showed 100% sensitivity and 100% specificity for clinical M. tuberculosis isolates and NTM isolates. In comparison, the sensitivities of AdvanSure TB/NTM real-time PCR, single IS6110 real-time PCR, and one-tube nested real-time PCR were 91% (152/167), 94.6% (158/167), and 100% (167/167) for sputum specimens, respectively. In conclusion, IS6110 one-tube nested real-time PCR is useful for detecting M. tuberculosis due to its high sensitivity and simple manipulation.

  13. Limit of detection of Toxocara canis larvae in experimentally contaminated bovine milk

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    Edlayne Larissa Gretter Machado Pereira

    2016-02-01

    Full Text Available This study aimed to evaluate the limit of detection of Toxocara canis larvae in experimentally contaminated commercial bovine milk samples, based on a centrifuge-sedimentation technique. Firstly, bovine milk (whole and skim samples were contaminated with 50 T. canis larvae in order to evaluate the interference of milk fat with the recovery of the larvae. Next, the effects of 10% formalin (100 ?L, ether (100 ?L, and a combination of both solutions on the recovery of the larvae was examined. Thereafter, the limit of detection of the larvae was determined using the solution (from step 2 considered optimal for degreasing the milk sample. Samples were contaminated with aliquots of 1, 5, 10, 25, and 50 larvae. For each milk sample (1.0 mL, 15 repetitions were analysed. The recovery of the larvae from the skim milk samples was higher (p = 0.0031 than that from the whole milk samples. No significant difference (p = 0.5681 was observed with regard to the percentage of recovered larvae when comparing the degreasing solutions. Nevertheless, the formalin-ether combination was more efficient for recovering the larvae (73.1% than ether (71.9%, formalin (67.6%, and pure whole milk (70.0%. Concerning the limit of detection (using formalin-ether, all the samples contaminated with 5, 10, 25, and 50 larvae tested positive (minimum: 62.7%. Of the samples contaminated with a single larva, 66.7% tested positive. These results suggest that the centrifugation-sedimentation technique may be useful for recovering larvae of Toxocara spp. in naturally or experimentally contaminated milk samples obtained from a wide range of animal species.

  14. Detection of chloramphenicol residue in bovine meat using Liquid Chromatography Mass Spectrometry

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    Widiastuti R

    2014-03-01

    Full Text Available Chloramphenicol (CAP is a broad spectrum antibiotic that has been banned in many countries due to its serius side effect to human. Detection of CAP residue in food has been determined to a minimum required performance limit (MRPL of 0.3 ng/g. The purpose of this research was to conduct the analysis of CAP residue in bovine meat by using LCMS and to study the presence of CAP residue in marketed bovine meat samples. LC separation was done on a Shimpack column C18 with ammonium acetate 10 mM/water as mobile phase, and ESI-MS analysis in negative ion mode. The coefficient of determination, R2 = 0.9981 at concentration of 0.125, 0.25, 0.63, 1,00 and 2.00 ng/g. Recovery at three fortification levels (0.25, 0.50 and 1.00 ng/g was in the range 77.5, 97.3 and 83.4%. The decision limit and the detection capability were 0.15 ng/g and 0.17 ng/g respectively. Analysis results of 52 marketed samples showed that CAP residue were detected in 9 samples in the concentration range of 0.14 to 2.70 ng/g and 6 among those positive samples were above the MRPL value. Therefore, it is important to increase the awareness and also to monitor regularly CAP residues in food originated from animal to provide safe food for the consumers.

  15. Culture and molecular method for detection of Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis in milk and dairy products.

    Science.gov (United States)

    Messelhäusser, U; Kämpf, P; Hörmansdorfer, S; Wagner, B; Schalch, B; Busch, U; Höller, C; Wallner, P; Barth, G; Rampp, A

    2012-01-01

    A combined molecular and cultural method for the detection of the Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium subsp. paratuberculosis was developed and tested with artificially contaminated milk and dairy products. Results indicate that the method can be used for a reliable detection as a basis for first risk assessments.

  16. Evaluation of a miniature microscope objective designed for fluorescence array microscopy detection of Mycobacterium tuberculosis.

    Science.gov (United States)

    McCall, Brian; Olsen, Randall J; Nelles, Nicole J; Williams, Dawn L; Jackson, Kevin; Richards-Kortum, Rebecca; Graviss, Edward A; Tkaczyk, Tomasz S

    2014-03-01

    A prototype miniature objective that was designed for a point-of-care diagnostic array microscope for detection of Mycobacterium tuberculosis and previously fabricated and presented in a proof of concept is evaluated for its effectiveness in detecting acid-fast bacteria. To evaluate the ability of the microscope to resolve submicron features and details in the image of acid-fast microorganisms stained with a fluorescent dye, and to evaluate the accuracy of clinical diagnoses made with digital images acquired with the objective. The lens prescription data for the microscope design are presented. A test platform is built by combining parts of a standard microscope, a prototype objective, and a digital single-lens reflex camera. Counts of acid-fast bacteria made with the prototype objective are compared to counts obtained with a standard microscope over matched fields of view. Two sets of 20 smears, positive and negative, are diagnosed by 2 pathologists as sputum smear positive or sputum smear negative, using both a standard clinical microscope and the prototype objective under evaluation. The results are compared to a reference diagnosis of the same sample. More bacteria are counted in matched fields of view in digital images taken with the prototype objective than with the standard clinical microscope. All diagnostic results are found to be highly concordant. An array microscope built with this miniature lens design will be able to detect M tuberculosis with high sensitivity and specificity.

  17. Low-cost rapid detection of rifampicin resistant tuberculosis using bacteriophage in Kampala, Uganda

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    Smith Peter G

    2007-01-01

    Full Text Available Abstract Background Resistance to anti-tuberculosis drugs is a serious public health problem. Multi-drug resistant tuberculosis (MDR-TB, defined as resistance to at least rifampicin and isoniazid, has been reported in all regions of the world. Current phenotypic methods of assessing drug susceptibility of M. tuberculosis are slow. Rapid molecular methods to detect resistance to rifampicin have been developed but they are not affordable in some high prevalence countries such as those in sub Saharan Africa. A simple multi-well plate assay using mycobacteriophage D29 has been developed to test M. tuberculosis isolates for resistance to rifampicin. The purpose of this study was to investigate the performance of this technology in Kampala, Uganda. Methods In a blinded study 149 M. tuberculosis isolates were tested for resistance to rifampicin by the phage assay and results compared to those from routine phenotypic testing in BACTEC 460. Three concentrations of drug were used 2, 4 and 10 μg/ml. Isolates found resistant by either assay were subjected to sequence analysis of a 81 bp fragment of the rpoB gene to identify mutations predictive of resistance. Four isolates with discrepant phage and BACTEC results were tested in a second phenotypic assay to determine minimal inhibitory concentrations. Results Initial analysis suggested a sensitivity and specificity of 100% and 96.5% respectively for the phage assay used at 4 and 10 μg/ml when compared to the BACTEC 460. However, further analysis revealed 4 false negative results from the BACTEC 460 and the phage assay proved the more sensitive and specific of the two tests. Of the 39 isolates found resistant by the phage assay 38 (97.4% were found to have mutations predictive of resistance in the 81 bp region of the rpoB gene. When used at 2 μg/ml false resistant results were observed from the phage assay. The cost of reagents for testing each isolate was estimated to be 1.3US$ when testing a batch of 20

  18. Protection against bovine tuberculosis induced by oral vaccination of cattle with Mycobacterium bovis BCG is not enhanced by co-administration of mycobacterial protein vaccines.

    Science.gov (United States)

    Wedlock, D Neil; Aldwell, Frank E; Vordermeier, H Martin; Hewinson, R Glyn; Buddle, Bryce M

    2011-12-15

    Mycobacterium bovis bacille Calmette-Guérin (BCG) delivered to calves by the oral route in a formulated lipid matrix has been previously shown to induce protection against bovine tuberculosis. A study was conducted in cattle to determine if a combination of a low dose of oral BCG and a protein vaccine could induce protective immunity to tuberculosis while not sensitising animals to tuberculin. Groups of calves (10 per group) were vaccinated by administering 2 × 10(7)colony forming units (CFU) of BCG orally or a combination of 2 × 10(7)CFU oral BCG and a protein vaccine comprised of M. bovis culture filtrate proteins (CFP) formulated with the adjuvants Chitin and Gel 01 and delivered by the intranasal route, or CFP formulated with Emulsigen and the TLR2 agonist Pam(3)CSK(4) and administered by the subcutaneous (s.c.) route. Two further groups were vaccinated with the CFP/Chitin/Gel 01 or CFP/Emulsigen/Pam(3)CSK(4) vaccines alone. Positive control groups were given 10(8)CFU oral BCG or 10(6)CFU s.c. BCG while a negative control group was non-vaccinated. All animals were challenged with M. bovis 15 weeks after vaccination and euthanized and necropsied at 16 weeks following challenge. Groups of cattle vaccinated with s.c. BCG, 10(8)CFU or 2 × 10(7)CFU oral BCG showed significant reductions in seven, three and four pathological or microbiological disease parameters, respectively, compared to the results for the non-vaccinated group. There was no evidence of protection in calves vaccinated with the combination of oral BCG and CFP/Emulsigen/Pam(3)CSK(4) or oral BCG and CFP/Chitin/Gel 01 or vaccinated with the protein vaccines alone. Positive responses in the comparative cervical skin test at 12 weeks after vaccination were only observed in animals vaccinated with s.c. BCG, 10(8)CFU oral BCG or a combination of 2 × 10(7)CFU oral BCG and CFP/Chitin/Gel 01. In conclusion, co-administration of a protein vaccine, administered by either systemic or mucosal routes with oral

  19. Prevalence of bovine tuberculosis and risk factor assessment in cattle in rural livestock areas of Govuro District in the Southeast of Mozambique.

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    Ivânia Moiane

    Full Text Available BACKGROUND: Bovine tuberculosis (bTB, caused by Mycobacterium bovis, is an infectious disease of cattle that also affects other domestic animals, free-ranging and farmed wildlife, and also humans. In Mozambique, scattered surveys have reported a wide variation of bTB prevalence rates in cattle from different regions. Due to direct economic repercussions on livestock and indirect consequences for human health and wildlife, knowing the prevalence rates of the disease is essential to define an effective control strategy. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional study was conducted in Govuro district to determine bTB prevalence in cattle and identify associated risk factors. A representative sample of the cattle population was defined, stratified by livestock areas (n = 14. A total of 1136 cattle from 289 farmers were tested using the single comparative intradermal tuberculin test. The overall apparent prevalence was estimated at 39.6% (95% CI 36.8-42.5 using a diagnostic threshold cut-off according to the World Organization for Animal Health. bTB reactors were found in 13 livestock areas, with prevalence rates ranging from 8.1 to 65.8%. Age was the main risk factor; animals older than 4 years were more likely to be positive reactors (OR = 3.2, 95% CI: 2.2-4.7. Landim local breed showed a lower prevalence than crossbred animals (Landim × Brahman (OR = 0.6, 95% CI: 0.4-0.8. CONCLUSIONS/SIGNIFICANCE: The findings reveal an urgent need for intervention with effective, area-based, control measures in order to reduce bTB prevalence and prevent its spread to the human population. In addition to the high prevalence, population habits in Govuro, particularly the consumption of raw milk, clearly may potentiate the transmission to humans. Thus, further studies on human tuberculosis and the molecular characterization of the predominant strain lineages that cause bTB in cattle and humans are urgently required to evaluate the impact on human health in

  20. Detection and Quantification of Mycobacterium tuberculosis in the Sputum of Culture-Negative HIV-infected Pulmonary Tuberculosis Suspects: A Proof-of-Concept Study

    Science.gov (United States)

    Madico, Guillermo; Mpeirwe, Moses; White, Laura; Vinhas, Solange; Orr, Beverley; Orikiriza, Patrick; Miller, Nancy S.; Gaeddert, Mary; Mwanga-Amumpaire, Juliet; Palaci, Moises; Kreiswirth, Barry; Straight, Joe; Dietze, Reynaldo; Boum, Yap; Jones-López, Edward C.

    2016-01-01

    Rationale Rapid diagnosis of pulmonary tuberculosis (TB) is critical for timely initiation of treatment and interruption of transmission. Yet, despite recent advances, many patients remain undiagnosed. Culture, usually considered the most sensitive diagnostic method, is sub-optimal for paucibacillary disease. Methods We evaluated the Totally Optimized PCR (TOP) TB assay, a new molecular test that we hypothesize is more sensitive than culture. After pre-clinical studies, we estimated TOP’s per-patient sensitivity and specificity in a convenience sample of 261 HIV-infected pulmonary TB suspects enrolled into a TB diagnostic study in Mbarara, Uganda against MGIT culture, Xpert MTB/RIF and a composite reference standard. We validated results with a confirmatory PCR used for sequencing M. tuberculosis. Measurements and Results Using culture as reference, TOP had 100% sensitivity but 35% specificity. Against a composite reference standard, the sensitivity of culture (27%) and Xpert MTB/RIF (27%) was lower than TOP (99%), with similar specificity (100%, 98% and 87%, respectively). In unadjusted analyses, culture-negative/TOP-positive patients were more likely to be older (P<0·001), female (P<0·001), have salivary sputum (P = 0·05), sputum smear-negative (P<0.001) and less advanced disease on chest radiograph (P = 0.05). M. tuberculosis genotypes identified in sputum by DNA sequencing exhibit differential growth in culture. Conclusions These findings suggest that the TOP TB assay is accurately detecting M. tuberculosis DNA in the sputum of culture-negative tuberculosis suspects. Our results require prospective validation with clinical outcomes. If the operating characteristics of the TOP assay are confirmed in future studies, it will be justified as a “TB rule out” test. PMID:27391604

  1. Combination of multiplex PCR with denaturing high-performance liquid chromatography for rapid detection of Mycobacterium genus and simultaneous identification of the Mycobacterium tuberculosis complex.

    Science.gov (United States)

    Chen, Ru; Gao, Xiao-Bo; Liu, Zhi-Hui; Shen, Xiao-Bing; Guo, Ai-Zhen; Duan, Yan-Yu; Liu, Zhi-Ling; Wu, Xiao-Wei; Zhu, Dao-Zhong

    2013-09-01

    A new assay with the combination of multiplex polymerase chain reaction and denaturing high-performance liquid chromatography analysis was developed for simultaneous detection of Mycobacterium genus and identification of the Mycobacterium tuberculosis complex (MTC). Targeting at genus-specific 16S rRNA sequence of Mycobacterium and specific insertion elements IS6110 and IS1081 of MTC, the assay was validated with 84 strains covering 23 mycobacteria species and 30 strains of non-mycobacteria species. No cross reactivity was observed. Clinical application was carried out on 198 specimens (155 human sputum and 43 bovine tissue samples) and compared with culture. The multiplex assay detected all culture-positive (36 in number) and 35.2% (57/162) culture-negative specimens. The molecular assay was fast that could be completed within 1 h on purified DNA, with the limit of detection as 0.8-1.6 pg per reaction on DNA template. This work provided a useful laboratory tool for rapid identification of Mycobacterium and differentiation of MTC and nontuberculous mycobacteria.

  2. A rapid and sensitive method to detect mycobacterium tuberculosis DNA by fluorescence polarization

    Institute of Scientific and Technical Information of China (English)

    白玉杰; 赵锦荣; 薛丽; 刘爱翔; 张文红; 郭晏海; 闫小君

    2004-01-01

    Objective: To develop a new high sensitivity, rapid and simple mycobacterium tuberculosis DNA detection method using fluorescence polarization technology. Methods: In our asymmetric PCR protocol, 100 times mole of TB-R primer was added than in the usual symmetric PCR to get enough single strands PCR product. The probe TB-5'-TAMRA and PCR products were mixed in a tube and incubate for 5 - 15 min at 46 C. The polarization (mp) was measured using victor2Multilabel Plate Reader. Results: Asymmetric and symmetric PCR products were analyzed by the FP method. Asymmetric PCR products are detected more sensitively than symmetric ones. The polarization values of probe associated with asymmetric products were much higher than with symmetric products. Conclusion: This fluorescence polarization assay in conjunction with asymmetric PCR is a powerful and widely applicable method for the rapid and sensitive detection of micro-organisms in clinical laboratories.

  3. Detection of Bovine Leukaemia Virus Antibodies and Proviral DNA in Colostrum Replacers.

    Science.gov (United States)

    Choudhury, B; Finnegan, C; Phillips, A; Horigan, M; Pollard, T; Steinbach, F

    2015-10-01

    Great Britain has been bovine leukaemia virus (BLV) disease free since 1999. We recently reported three separate incidents of BLV seropositivity on farms with home-reared cattle due to the use of colostrum replacer rather than infection with BLV (Emerg. Infect. Dis., 19, 2013, 1027). These cases were all linked via the use of the same brand of colostrum replacer. Here, we investigate further by examining multiple brands of colostrum replacer for proviral DNA and BLV antibodies. BLV antibodies were detected in 7 of the colostrum replacers tested, with PCR concurring in two cases. Thus, the use of these BLV antibody-positive colostrum replacers may also lead to false-positive serological diagnostics.

  4. Detecting a low prevalence of latent tuberculosis among health care workers in Denmark detected by M. tuberculosis specific IFN-gamma whole-blood test

    DEFF Research Database (Denmark)

    Soborg, Bolette; Andersen, Aase B; Larsen, Helle K

    2007-01-01

    The study was designed to estimate prevalence of tuberculosis infection among health care workers, using the tuberculin skin test (TST) and the new M. tuberculosis specific diagnostic whole-blood test and to identify possible risk factors. Employees at 2 departments of infectious diseases...

  5. Investigation of polymerase chain reaction assays to improve detection of bacterial involvement in bovine respiratory disease.

    Science.gov (United States)

    Bell, Colin J; Blackburn, Paul; Elliott, Mark; Patterson, Tony I A P; Ellison, Sean; Lahuerta-Marin, Angela; Ball, Hywel J

    2014-09-01

    Bovine respiratory disease (BRD) causes severe economic losses to the cattle farming industry worldwide. The major bacterial organisms contributing to the BRD complex are Mannheimia haemolytica, Histophilus somni, Mycoplasma bovis, Pasteurella multocida, and Trueperella pyogenes. The postmortem detection of these organisms in pneumonic lung tissue is generally conducted using standard culture-based techniques where the presence of therapeutic antibiotics in the tissue can inhibit bacterial isolation. In the current study, conventional and real-time polymerase chain reaction (PCR) assays were used to assess the prevalence of these 5 organisms in grossly pneumonic lung samples from 150 animals submitted for postmortem examination, and the results were compared with those obtained using culture techniques. Mannheimia haemolytica was detected in 51 cases (34%) by PCR and in 33 cases (22%) by culture, H. somni was detected in 35 cases (23.3%) by PCR and in 6 cases (4%) by culture, Myc. bovis was detected in 53 cases (35.3%) by PCR and in 29 cases (19.3%) by culture, P. multocida was detected in 50 cases (33.3%) by PCR and in 31 cases (20.7%) by culture, and T. pyogenes was detected in 42 cases (28%) by PCR and in 31 cases (20.7%) by culture, with all differences being statistically significant. The PCR assays indicated positive results for 111 cases (74%) whereas 82 cases (54.6%) were culture positive. The PCR assays have demonstrated a significantly higher rate of detection of all 5 organisms in cases of pneumonia in cattle in Northern Ireland than was detected by current standard procedures.

  6. Stochastic simulation modeling to determine time to detect Bovine Viral Diarrhea antibodies in bulk tank milk.

    Science.gov (United States)

    Foddai, Alessandro; Enøe, Claes; Krogh, Kaspar; Stockmarr, Anders; Halasa, Tariq

    2014-11-01

    A stochastic simulation model was developed to estimate the time from introduction of Bovine Viral Diarrhea Virus (BVDV) in a herd to detection of antibodies in bulk tank milk (BTM) samples using three ELISAs. We assumed that antibodies could be detected, after a fixed threshold prevalence of seroconverted milking cows was reached in the herd. Different thresholds were set for each ELISA, according to previous studies. For each test, antibody detection was simulated in small (70 cows), medium (150 cows) and large (320 cows) herds. The assays included were: (1) the Danish blocking ELISA, (2) the SVANOVIR(®)BVDV-Ab ELISA, and (3) the ELISA BVD/MD p80 Institute Pourquier. The validation of the model was mainly carried out by comparing the predicted incidence of persistently infected (PI) calves and the predicted detection time, with records from a BVD infected herd. Results showed that the SVANOVIR, which was the most efficient ELISA, could detect antibodies in the BTM of a large herd 280 days (95% prediction interval: 218; 568) after a transiently infected (TI) milking cow has been introduced into the herd. The estimated time to detection after introduction of one PI calf was 111 days (44; 605). With SVANOVIR ELISA the incidence of PIs and dead born calves could be limited and the impact of the disease on the animal welfare and income of farmers (before detection) could be minimized. The results from the simulation modeling can be used to improve the current Danish BVD surveillance program in detecting early infected herds. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Rapid identification of mycobacteria and rapid detection of drug resistance in Mycobacterium tuberculosis in cultured isolates and in respiratory specimens.

    Science.gov (United States)

    Yam, Wing-Cheong; Siu, Kit-Hang Gilman

    2013-01-01

    Recent advances in molecular biology and better understanding of the genetic basis of drug resistance have allowed rapid identification of mycobacteria and rapid detection of drug resistance of Mycobacterium tuberculosis present in cultured isolates or in respiratory specimens. In this chapter, several simple nucleic acid amplification-based techniques are introduced as molecular approach for clinical diagnosis of tuberculosis. A one-tube nested IS6110-based polymerase chain reaction (PCR) is used for M. tuberculosis complex identification; the use of a multiplex allele-specific PCR is demonstrated to detect the isoniazid resistance; PCR-sequencing assays are applied for rifampicin and ofloxacin resistance detection and 16S rDNA sequencing is utilized for identification of mycobacterial species from cultures of acid fast bacilli (AFB). Despite the high specificity and sensitivity of the molecular techniques, mycobacterial culture remains the "Gold Standard" for tuberculosis diagnosis. Negative results of molecular tests never preclude the infection or the presence of drug resistance. These technological advancements are, therefore, not intended to replace the conventional tests, but rather have major complementary roles in tuberculosis diagnosis.

  8. EU-Approved Rapid Tests for Bovine Spongform Encephalopathy Detect Atypical Forms: A Study for Their Sensitivities

    NARCIS (Netherlands)

    Meloni, D.; Davidse, A.; Langeveld, J.P.M.; varello, K.; Casalone, C.; Corona, C.; Balkema-Buschmann, A.; Groschup, M.; Ingravalle, F.; Bozzetta, E.

    2012-01-01

    Since 2004 it become clear that atypical bovine spongiform encephalopthies (BSEs) exist in cattle. Whenever their detection has relied on active surveillance plans implemented in Europe since 2001 by rapid tests, the overall and inter-laboratory performance of these diagnostic systems in the

  9. EU-Approved Rapid Tests for Bovine Spongform Encephalopathy Detect Atypical Forms: A Study for Their Sensitivities

    NARCIS (Netherlands)

    Meloni, D.; Davidse, A.; Langeveld, J.P.M.; varello, K.; Casalone, C.; Corona, C.; Balkema-Buschmann, A.; Groschup, M.; Ingravalle, F.; Bozzetta, E.

    2012-01-01

    Since 2004 it become clear that atypical bovine spongiform encephalopthies (BSEs) exist in cattle. Whenever their detection has relied on active surveillance plans implemented in Europe since 2001 by rapid tests, the overall and inter-laboratory performance of these diagnostic systems in the detecti

  10. Detection of bovine meat and bone meal in animal feed at a level of 0.1%

    NARCIS (Netherlands)

    Aarts, H.J.M.; Bouw, E.M.; Buntjer, J.B.; Lenstra, J.A.; Raamsdonk, van L.W.D.

    2006-01-01

    For the control of the transmission of bovine spongiform encephalopathy in cattle via feedstuff, a real-time polymerase chain reaction assay was developed with ruminant-specific Bov-B SINE primers, SYBR® Green fluorescence detection, and melting curve analysis. In formulated cattle and chicken feed

  11. Detection of bovine meat and bone meal in animal feed at a level of 0.1%

    NARCIS (Netherlands)

    Aarts, H.J.M.; Bouw, E.M.; Buntjer, J.B.; Lenstra, J.A.; Raamsdonk, van L.W.D.

    2006-01-01

    For the control of the transmission of bovine spongiform encephalopathy in cattle via feedstuff, a real-time polymerase chain reaction assay was developed with ruminant-specific Bov-B SINE primers, SYBR® Green fluorescence detection, and melting curve analysis. In formulated cattle and chicken feed

  12. A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition

    Directory of Open Access Journals (Sweden)

    Saegerman Claude

    2008-08-01

    Full Text Available Abstract Background Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and clinical signs and lesions of gastroenteritis were reported in bovines. Due to their genetic proximity, potential zoonotic transmission or animal reservoir can be hypothesized for noroviruses. RT-PCR has become the "gold standard" for the detection of noroviruses in faecal and environmental samples. With such samples, the control for inhibition of the reaction during amplification and detection is crucial to avoid false negative results, which might otherwise not be detected. The aim of the reported method is to detect, with a SYBR Green technology, a broad range of noroviruses with a control for inhibition. Results A SYBR Green real-time RT-PCR assay was developed making use of a foreign internal RNA control added in the same tube. This assay is able to detect human and bovine noroviruses belonging to genogroups I, II and III and to distinguish between norovirus and internal control amplicons using melting curve analysis. A 10-fold dilution of samples appears to be the method of choice to remove inhibition. This assay was validated with human and bovine stool samples previously tested for norovirus by conventional RT-PCR. Conclusion This SYBR Green real-time RT-PCR assay allows the detection of the most important human and bovine noroviruses in the same assay, and avoids false negative results making use of an internal control. Melting curves allow the discrimination between the internal control and norovirus amplicons. It gives preliminary information about the species of origin. The sensitivity of the developed assay is higher than conventional RT-PCR and a 10-fold dilution of samples showed a better efficiency and reproducibility to remove RT-PCR inhibition than addition of bovine serum albumin.

  13. Detection and molecular characterization of bovine leukemia virus in Philippine cattle.

    Science.gov (United States)

    Polat, Meripet; Ohno, Ayumu; Takeshima, Shin-Nosuke; Kim, Jiyun; Kikuya, Mari; Matsumoto, Yuki; Mingala, Claro Niegos; Onuma, Misao; Aida, Yoko

    2015-01-01

    Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide, imposing a severe economic impact on the dairy cattle industry. However, there are no comprehensive studies on the distribution of BLV in the Philippines, and the genetic characteristics of Philippine BLV strains are unknown. Therefore, the aim of this study was to detect BLV infections in the Philippines and determined their genetic variability. Blood samples were obtained from 1116 cattle from different farms on five Philippine islands, and BLV provirus was detected by BLV-CoCoMo-qPCR-2 and nested PCR targeting BLV long terminal repeats. Out of 1116 samples, 108 (9.7 %) and 54 (4.8 %) were positive for BLV provirus, as determined by BLV-CoCoMo-qPCR-2 and nested PCR, respectively. Of the five islands, Luzon Island showed the highest prevalence of BLV infection (23.1 %). Partial env gp51 genes from 43 samples, which were positive for BLV provirus by both methods, were sequenced for phylogenetic analysis. Phylogenetic analysis based on a 423-bp fragment of the env gene revealed that Philippine BLV strains clustered into either genotype 1 or genotype 6. Substitutions were mainly found in antigenic determinants, such as the CD4(+) T-cell epitope, the CD8(+) T-cell epitope, the second neutralizing domain, B and E epitopes, and these substitutions varied according to genotype. This study provides comprehensive information regarding BLV infection levels in the Philippines and documents the presence of two BLV genotypes, genotypes 1 and 6, in this population.

  14. Evaluation of the Genotype MTBDR Assay for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Isolates

    OpenAIRE

    2006-01-01

    A novel PCR-based reverse hybridization method Genotype MTBDR assay (Hain Lifescience GmbH, Nehren, Germany) was evaluated for rapid detection of rifampin (RIF) and isoniazid (INH) resistance in Turkish Mycobacterium tuberculosis isolates. The Genotype MTBDR assay is designed to detect mutations within the 81-bp hotspot region of rpoB and mutations at katG codon 315. A total of 41 RIF-resistant M. tuberculosis isolates with rpoB mutations that were previously tested by the INNO-LiPA Rif.TB ki...

  15. Detection of Atypical H-Type Bovine Spongiform Encephalopathy and Discrimination of Bovine Prion Strains by Real-Time Quaking-Induced Conversion.

    Science.gov (United States)

    Masujin, Kentaro; Orrú, Christina D; Miyazawa, Kohtaro; Groveman, Bradley R; Raymond, Lynne D; Hughson, Andrew G; Caughey, Byron

    2016-03-01

    Prion diseases of cattle include the classical bovine spongiform encephalopathy (C-BSE) and the atypical H-type BSE (H-BSE) and L-type BSE (L-BSE) strains. Although the C- and L-BSE strains can be detected and discriminated by ultrasensitive real-time quaking-induced conversion (RT-QuIC) assays, no such test has yet been described for the detection of H-BSE or the discrimination of each of the major bovine prion strains. Here, we demonstrate an RT-QuIC assay for H-BSE that can detect as little as 10(-9) dilutions of brain tissue and neat cerebrospinal fluid samples from clinically affected cattle. Moreover, comparisons of the reactivities with different recombinant prion protein substrates and/or immunoblot band profiles of proteinase K-treated RT-QuIC reaction products indicated that H-, L-, and C-BSE have distinctive prion seeding activities and can be discriminated by RT-QuIC on this basis.

  16. Detection of synthetic corticosteroids in bovine urine by chemiluminescence high-performance liquid chromatography.

    Science.gov (United States)

    Vázquez, B I; Feás, X; Lolo, M; Fente, C A; Franco, C M; Cepeda, A

    2005-01-01

    The development of a black market of chemical cocktails for illegal growth promotion in food-producing animals includes substances that are potentially dangerous for human health, such as synthetic corticosteroids. The potential presence of these residues in food makes it necessary to develop rapid and sensitive analytical methodologies to detect such substances, preferably in live animals before they arrive at the market. A chemiluminescence (CL) detection method for the determination of four synthetic corticosteroids (prednisolone, betamethasone, dexamethasone and flumethasone) in bovine urine has been developed. The proposed system, which does not need any derivatization procedure, offers an easy method well suited for routine research. Urine samples were homogenized with methanol:water (50:50, v/v) and centrifuged. The upper layer was collected and Strata X cartridges were used for cleaning up. The purified residues were evaporated to dryness and then redissolved in the mobile phase. Analysis of the extracts was performed using high-performance liquid chromatography with chemiluminescence detection, employing luminol as the CL reagent. The recovery curves, obtained at four spiking levels (different for each corticosteroid), showed that recoveries of at least 70% could be obtained for urine. The chemiluminescence detection procedure afforded satisfactory results with respect to sensitivity and the LOD and LOQ, taken as the first point of the regression curve, ranged from 4 ppb to 65 ppb. The maximum mean RSD was below 13% and below 15% for intra- and inter-day assay, respectively, in all cases.

  17. Biological and biochemical characterization of L-type-like bovine spongiform encephalopathy (BSE) detected in Japanese black beef cattle

    OpenAIRE

    2008-01-01

    A case of L-type-like atypical bovine spongiform encephalopathy was detected in 14-year-old Japanese black beef cattle (BSE/JP24). To clarify the biological and biochemical properties of the prion in BSE/JP24, we performed a transmission study with wild-type mice and bovinized transgenic mice (TgBoPrP). The BSE/JP24 prion was transmitted to TgBoPrP mice with the incubation period of 199.7 ± 3.4 days, which was shorter than that of classical BSE (C-BSE) (223.5 ± 13.5 days). Further, C-BSE was ...

  18. Diagnostic accuracy of xpert test in tuberculosis detection: A systematic review and meta-analysis

    Directory of Open Access Journals (Sweden)

    Ravdeep Kaur

    2016-01-01

    Full Text Available Background: World Health Organization (WHO recommends the use of Xpert MTB/RIF assay for rapid diagnosis of tuberculosis (TB and detection of rifampicin resistance. This systematic review was done to know about the diagnostic accuracy and cost-effectiveness of the Xpert MTB/RIF assay. Methods: A systematic literature search was conducted in following databases: Cochrane Central Register of Controlled Trials and Cochrane Database of Systematic Reviews, MEDLINE, PUBMED, Scopus, Science Direct and Google Scholar for relevant studies for studies published between 2010 and December 2014. Studies given in the systematic reviews were accessed separately and used for analysis. Selection of studies, data extraction and assessment of quality of included studies was performed independently by two reviewers. Studies evaluating the diagnostic accuracy of Xpert MTB/RIF assay among adult or predominantly adult patients (≥14 years, presumed to have pulmonary TB with or without HIV infection were included in the review. Also, studies that had assessed the diagnostic accuracy of Xpert MTB/RIF assay using sputum and other respiratory specimens were included. Results: The included studies had a low risk of any form of bias, showing that findings are of high scientific validity and credibility. Quantitative analysis of 37 included studies shows that Xpert MTB/RIF is an accurate diagnostic test for TB and detection of rifampicin resistance. Conclusion: Xpert MTB/RIF assay is a robust, sensitive and specific test for accurate diagnosis of tuberculosis as compared to conventional tests like culture and microscopic examination.

  19. Diagnostic Accuracy of Xpert Test in Tuberculosis Detection: A Systematic Review and Meta-analysis

    Science.gov (United States)

    Kaur, Ravdeep; Kachroo, Kavita; Sharma, Jitendar Kumar; Vatturi, Satyanarayana Murthy; Dang, Amit

    2016-01-01

    Background: World Health Organization (WHO) recommends the use of Xpert MTB/RIF assay for rapid diagnosis of tuberculosis (TB) and detection of rifampicin resistance. This systematic review was done to know about the diagnostic accuracy and cost-effectiveness of the Xpert MTB/RIF assay. Methods: A systematic literature search was conducted in following databases: Cochrane Central Register of Controlled Trials and Cochrane Database of Systematic Reviews, MEDLINE, PUBMED, Scopus, Science Direct and Google Scholar for relevant studies for studies published between 2010 and December 2014. Studies given in the systematic reviews were accessed separately and used for analysis. Selection of studies, data extraction and assessment of quality of included studies was performed independently by two reviewers. Studies evaluating the diagnostic accuracy of Xpert MTB/RIF assay among adult or predominantly adult patients (≥14 years), presumed to have pulmonary TB with or without HIV infection were included in the review. Also, studies that had assessed the diagnostic accuracy of Xpert MTB/RIF assay using sputum and other respiratory specimens were included. Results: The included studies had a low risk of any form of bias, showing that findings are of high scientific validity and credibility. Quantitative analysis of 37 included studies shows that Xpert MTB/RIF is an accurate diagnostic test for TB and detection of rifampicin resistance. Conclusion: Xpert MTB/RIF assay is a robust, sensitive and specific test for accurate diagnosis of tuberculosis as compared to conventional tests like culture and microscopic examination. PMID:27013842

  20. Assessment of terbium (III) as a luminescent probe for the detection of tuberculosis biomarkers.

    Science.gov (United States)

    Bamogo, W; Mugherli, L; Banyasz, A; Novelli-Rousseau, A; Mallard, F; Tran-Thi, T-H

    2015-10-08

    A detection method for nicotinic acid, a specific metabolite marker of Mycobacterium tuberculosis present in cultures and patients' breath, is studied in complex solutions containing other metabolites and in biological media such as urine, saliva and breath condensate. The method is based on the analysis of the luminescence increase of Tb(3+) complexes in the presence of nicotinic acid due to the energy transfer from the excited ligand to the lanthanide ion. It is shown that other potential markers found in M. tuberculosis culture supernatant, such as methyl phenylacetate, p-methyl anisate, methyl nicotinate and 2-methoxy biphenyl, can interfere with nicotinic acid via a competitive absorption of the excitation photons. A new strategy to circumvent these interferences is proposed with an upstream trapping of volatile markers preceding the detection of nicotinic acid in the liquid phase via the luminescence of Tb(3+) complexes. The cost of the method is evaluated and compared with the Xpert MTB/RIF test endorsed by the World Health Organization.

  1. Husbandry practices, badger sett density and habitat composition as risk factors for transient and persistent bovine tuberculosis on UK cattle farms.

    Science.gov (United States)

    Reilly, L A; Courtenay, O

    2007-07-16

    Bovine tuberculosis (bTB) is a persistent problem in cattle herds in Great Britain and Ireland. Farm management and cattle husbandry practices can influence the risk of transmission of bTB and hence the likelihood of bTB breakdown (>or=1 reactor to the tuberculin skin test). Biological differences are expected in the transmission dynamics, and hence risk factors for bTB breakdown, on farms where infection persists in the herd compared to farms where infection is more sporadic or short-lived. Comparative case-control studies were performed to test farm management practices as potential risk factors for transient (under breakdown restrictions for 6 months) bTB breakdown over 5 years (1995-1999) on 179 and 171 UK cattle farms, respectively. Farms were characterised for badger sett density and farm habitat composition by ground survey, farmers were questioned retrospectively on management practices, and cases and controls were identified from national tuberculin test records. Controlling for routine tuberculin testing interval, log-transformed herd size, regional location, badger sett density and farm habitat complexity, multivariable logistic regression identified increased odds of both transient and persistent breakdown on farms that bought-in cows (odds ratio (OR)>or=4.9; 95% confidence interval (CI)>or=1.1;22.8). In addition, the purchase of >50 head of cattle (OR=4.0, 95% CI=1.0;16.0) and the storage of manure for >or/=6 months (OR=4.4; 95% CI=1.3;15.4) were risk factors for transient breakdown, whereas the use of silage clamps (OR=9.1; 95% CI=2.0;40.8) increased the risk of persistent breakdown. Decreased odds of both transient and persistent breakdown were associated with higher stocking densities (>3cattle/ha) (ORsilage storage in addition to the relative density of badgers. Implications for bTB management are discussed.

  2. Eradication of bovine tuberculosis at a herd-level in Madrid, Spain: study of within-herd transmission dynamics over a 12 year period

    Directory of Open Access Journals (Sweden)

    Alvarez Julio

    2012-06-01

    Full Text Available Abstract Background Eradication of bovine tuberculosis (bTB through the application of test-and-cull programs is a declared goal of developed countries in which the disease is still endemic. Here, longitudinal data from more than 1,700 cattle herds tested during a 12 year-period in the eradication program in the region of Madrid, Spain, were analyzed to quantify the within-herd transmission coefficient (β depending on the herd-type (beef/dairy/bullfighting. In addition, the probability to recover the officially bTB free (OTF status in infected herds depending on the type of herd and the diagnostic strategy implemented was assessed using Cox proportional hazard models. Results Overall, dairy herds showed higher β (median 4.7 than beef or bullfighting herds (2.3 and 2.2 respectively. Introduction of interferon-gamma (IFN-γ as an ancillary test produced an apparent increase in the β coefficient regardless of production type, likely due to an increase in diagnostic sensitivity. Time to recover OTF status was also significantly lower in dairy herds, and length of bTB episodes was significantly reduced when the IFN-γ was implemented to manage the outbreak. Conclusions Our results suggest that bTB spreads more rapidly in dairy herds compared to other herd types, a likely cause being management and demographic-related factors. However, outbreaks in dairy herds can be controlled more rapidly than in typically extensive herd types. Finally, IFN-γ proved its usefulness to rapidly eradicate bTB at a herd-level.

  3. Development of risk-based trading farm scoring system to assist with the control of bovine tuberculosis in cattle in England and Wales.

    Science.gov (United States)

    Adkin, A; Brouwer, A; Simons, R R L; Smith, R P; Arnold, M E; Broughan, J; Kosmider, R; Downs, S H

    2016-01-01

    Identifying and ranking cattle herds with a higher risk of being or becoming infected on known risk factors can help target farm biosecurity, surveillance schemes and reduce spread through animal trading. This paper describes a quantitative approach to develop risk scores, based on the probability of infection in a herd with bovine tuberculosis (bTB), to be used in a risk-based trading (RBT) scheme in England and Wales. To produce a practical scoring system the risk factors included need to be simple and quick to understand, sufficiently informative and derived from centralised national databases to enable verification and assess compliance. A logistic regression identified herd history of bTB, local bTB prevalence, herd size and movements of animals onto farms in batches from high risk areas as being significantly associated with the probability of bTB infection on farm. Risk factors were assigned points using the estimated odds ratios to weight them. The farm risk score was defined as the sum of these individual points yielding a range from 1 to 5 and was calculated for each cattle farm that was trading animals in England and Wales at the start of a year. Within 12 months, of those farms tested, 30.3% of score 5 farms had a breakdown (sensitivity). Of farms scoring 1-4 only 5.4% incurred a breakdown (1-specificity). The use of this risk scoring system within RBT has the potential to reduce infected cattle movements; however, there are cost implications in ensuring that the information underpinning any system is accurate and up to date.

  4. Influence of the 2009 financial crisis on detection of advanced pulmonary tuberculosis in Osaka city, Japan: a cross-sectional study

    OpenAIRE

    Danno, Katsura; Komukai, Jun; Yoshida, Hideki; Matsumoto, Kenji; Koda, Shinichi; Terakawa, Kazuhiko; Iso, Hiroyasu

    2013-01-01

    Objective To investigate the association between the economic recession and the detection of advanced cases of pulmonary tuberculosis in Osaka city from 2007 to 2009. Design A repeated cross-sectional study. Setting Osaka city has been the highest tuberculosis burden area in Japan. After the previous global financial crisis, the unemployment rate in Osaka prefecture has deteriorated from 5.3% in 2008 to 6.6% in 2009. Participants During the study period, 3406 pulmonary tuberculosis cases were...

  5. Development of a TIRF-based biosensor for sensitive detection of progesterone in bovine milk.

    Science.gov (United States)

    Käppel, Nina D; Pröll, Florian; Gauglitz, Guenter

    2007-04-15

    A total internal reflectance fluorescence (TIRF)-based biosensor for progesterone in bovine milk was developed and tested by measuring the progesterone level in daily milk samples for 25 days, covering a whole estrus cycle. The detection is based on total internal reflectance fluorescence. The assay has been designed as a binding-inhibition test with a progesterone derivative covalently immobilized on the sensor surface and a monoclonal anti-progesterone antibody as biological recognition element. First an existing progesterone assay was optimized by reducing the assay time per measurement, resulting in an assay time of about 5 min and reaching a limit of detection (LOD) of 0.04 ng mL(-1) and a quantification limit (LOQ) of 0.34 ng mL(-1). After calibration the assay was tested by measuring the progesterone level in daily milk samples over several weeks. An estrus cycle of a cow could be measured. As results become available within minutes without any preparation or pre-concentration of the milk samples the fully automated TIRF-based biosensor for progesterone can be used in-line in the milking parlor and thus could be an important tool for reproductive management of dairy cattle detecting heat and predicting pregnancy, which are critical parameters in milk production.

  6. Extra pulmonary tuberculosis: Rapid identification of Mycobacterium tuberculosis grown in Mycobacterium growth indicator tube 960 and Lowenstein-Jensen media, employing Standard diagnostics Bioline Mycobacterium tuberculosis protein 64 antigen detection kit

    Directory of Open Access Journals (Sweden)

    G Kandhakumari

    2015-01-01

    Full Text Available Background: Investigation of extra pulmonary tuberculosis (EPTB in and around Pondicherry is being carried out since August 2011 in our tertiary care super specialty hospital. Objectives: To compare the rapid Kit SD Bio-Line MPT 64 Ag with conventional and time consuming biochemical tests. Confirmation of Mycobacterium tuberculosis at a reasonable time frame is the main thrust. Materials and Methods: Thirty three Mycobacterium tuberculosis and four Non-Tuberculous Mycobacteria (NTM grown in MGIT960 system/Lowenstein-Jensen media (LJ were examined by the rapid MPT 64 antigen detection as well as a battery of conventional tests like niacin, nitrate reduction, paraminobenzoic acid susceptibility and cord formation. Results and Conclusion: . Both the rapid kit and conventional tests correctly identified 33 M.tuberculosis isolates. Keeping conventional identification as reference, sensitivity and specificity for rapid kit was 100%. Rapid kit which takes only 15 minutes is accurate, cost effective, and facilitates early treatment for these EPTB patients, whose clinical specimens are paucibacillary.

  7. Detection of ESAT-6 by a label free miniature immuno-electrochemical biosensor as a diagnostic tool for tuberculosis.

    Science.gov (United States)

    Diouani, Mohamed Fethi; Ouerghi, Oussama; Refai, Amira; Belgacem, Kamel; Tlili, Chaker; Laouini, Dhafer; Essafi, Makram

    2017-05-01

    Tuberculosis is a worldwide disease considered as a major health problem with high morbidity and mortality rates. Poor detection of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis remains a major obstacle to the global control of this disease. Here we report the development of a new test based on the detection of the major virulent factor of Mtb, namely the early secreted antigenic target 6-kDa protein or ESAT-6. A label free electrochemical immunosensor using an anti-ESAT-6 monoclonal antibody as a bio-receptor is described herein. Anti-ESAT-6 antibodies were first covalently immobilized on the surface of a gold screen-printed electrode functionalized via a self-assembled thiol monolayer. Interaction between the bio-receptor and ESAT-6 antigen was evaluated by square wave voltammetry method using [Fe(CN)6](3-/4-) as redox probe. The detection limit of ESAT-6 antigen was 7ng/ml. The immunosensor has also been able to detect native ESAT-6 antigen secreted in cell culture filtrates of three pathogenic strains of Mtb (CDC1551, H37RV and H8N8). Overall, this work describes an immune-electrochemical biosensor, based on ESAT-6 antigen detection, as a useful diagnostic tool for tuberculosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates.

    Science.gov (United States)

    Maningi, Nontuthuko E; Daum, Luke T; Rodriguez, John D; Mphahlele, Matsie; Peters, Remco P H; Fischer, Gerald W; Chambers, James P; Fourie, P Bernard

    2015-12-01

    The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncA gene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n = 88), and concordance values between the Bactec 460, the Wayne test, or pncA gene Sanger sequencing and NGS results were 82% (n = 88), 83% (n = 88), and 89% (n = 88), respectively. NGS confirmed the majority of pncA mutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS of pncA improved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.

  9. Assessment of the N-PCR assay in diagnosis of pleural tuberculosis: detection of M. tuberculosis in pleural fluid and sputum collected in tandem.

    Directory of Open Access Journals (Sweden)

    Parameet Kumar

    Full Text Available BACKGROUND: The nonspecific clinical presentation and paucibacillary nature of tuberculous pleuritis remains a challenge for diagnosis. Diagnosis of tuberculous pleural effusion depends on the demonstration of the presence of tubercle bacilli in the sputum, pleural fluid, or pleural biopsy specimen, or demonstration of granuloma in pleura by histological examination. We examined the clinical utility of the diagnosis of pleural tuberculosis using the in house N-PCR assay, AFB smear microscopy and culture. Besides pleural fluid the inclusion of sputum in the efficacy of diagnosis of pleural tuberculosis was scrutinized. METHODOLOGY/PRINCIPAL FINDINGS: Pleural fluid and sputum samples of 58 tuberculous and 42 non-tuberculous pleural effusion patients were processed for AFB smear microscopy, culture and the N-PCR assay. Mycobacteria were detected exclusively in tuberculous pleural effusion samples. None of the non-tuberculous pleural effusion samples were positive for mycobacteria. Comparative analysis showed that the N-PCR assay had the highest sensitivity. Inclusion of sputum along with pleural fluid increased N-PCR sensitivity from 51.7 to 70.6% (p<0.0001.This improved sensitivity was reflected in AFB smear microscopy and isolation by culture. The sensitivity enhanced on inclusion of sputum from 3.4 (p = 0.50 to 10.3% (p = 0.038 for AFB smear microscopy and for isolation of mycobacteria from 10.3(p = 0.03 to 22.4% (p = 0.0005. Thirteen isolates were obtained from 58 pleural tuberculosis patients. Eleven mycobacterial isolates were identified as M. tuberculosis and two as M. fortuitum and M. chelonae. Complete concordance was seen between the biochemical identification of isolates and the N-PCR identification of mycobacterial species prior to isolation. CONCLUSIONS/SIGNIFICANCE: To the best of our knowledge this is the first PCR based report on utility of sputum for diagnosis of pleural tuberculosis. The present study demonstrates that a

  10. Detecting drug resistant genetic mutation among pneumoconiosis patients complicated with tuberculosis in Mycobacterium tuberculosis L-forms application of PCR-SSCP technique in Huainan mining district

    Institute of Scientific and Technical Information of China (English)

    Jun Lu; Shan Jiang; Song Ye; Chaopin Li

    2007-01-01

    Objective: To study the relationship between drug resistant genetic mutation and drug resistance in Mycobacterium tuberculosis L-form, discuss the internal relationship between drug resistances and drug-resistant related genes and explore the value of PCRSSCP to clinical application. Methods: A total of 52 clinically isolated strains of tuberculosis L-form were collected among 97pneumoconiosis patients complicated with tuberculosis. The gene mutations of katG, rpoB and rpsL were detected by PCR-SSCP,and the results were compared with those analyzed by traditional antimicrobial susceptibility test(AST). Results: The gene mutation rates of katG, rpoB and rpsL by PCR-SSCP were respectively 57.70% (30/52), 65.38% (32/52) and 40.38% (21/52). The rate of reversion was 78.85%(41/52) and the result of drug-resistant genes was invariable. The results of AST showed that there were 40 (76.92%) multi-drug resistant strains in 52 clinically isolated strains. The number for three-drug resistant strain was 21(40.38%) and that of two-drug resistant was 19(36.54%), but only 12 (23.08%) strains were one drug resistant. The rate of total drug-resistance was 100%, but there were 15 strains of allied mutation of three genes, 16 of two mutations and 6 of only one by PCR-SSCP. The coincidences were respectively 71.43%, 84.12% and 50.00%. Then there was no significant difference between the allied mutations of multi-drug resistant gene and the mutations of only one drug resistant gene (P > 0.05). Conclusion: PCRSSCP technique has a higher sensibility and specificity to detect the genes of katG, rpoB and rpsL in tuberculosis L-form among pneumoconiosis complicated with tuberculosis,and the detecting rate of two drug resistant strains and three drug resistant strains was higher. The combined application of PCR-SSCP and AST has advantages at earlier diagnosis and guidance of clinical medications.

  11. ASSESSMENT OF DETECTION EFFICACY OF MYCOBACTERIUM TUBERCULOSIS IN SPUTUM SAMPLES BY REAL TIME PCR BASED METHOD.

    Science.gov (United States)

    Upadhyaya, S K; Dixit, S M; Shrestha, S; Dangol, S D; Pokhrel, D; Banjara, S; Shrestha, K S; Hengoju, S; Dahal, B K

    2013-07-01

    Tuberculosis (TB) is a major public health problem in Nepal and ranks as one of the most prevalent communicable diseases throughout the country. In Nepal, 45% of total population is infected with TB and 40,000 people get TB every year. Twenty thousand new sputum positive cases are seen every year and 5000-7000 people die each year from TB. Thirty sputum samples were collected from Sukraraj Tropical and Infectious Disease Hospital, Teku, Kathmandu, Nepal and the comparative study of Acid-fast Bacilli (AFB) test and Real time PCR were conducted separately with the culture test which is regarded as gold standard by WHO. Detection of Mycobacterium tuberculosis through use of Real time PCR was found to be higher as compared to AFB and culture. Real-time PCR test showed higher sensitivity (100%) and specificity (94.11%) as compared to AFB test with sensitivity of 84.61% and specificity of 88.24%. Positive predictive value was found to be 84.61% and 92.86% for AFB and Q-PCR respectively. Negative predictive value was found to be 88.24%, and for Q-PCR, it was found to be 100%. Our statistics clearly show that TB diagnosis by Q-PCR is highly efficient and reliableover conventional methods of diagnosis and here we recommend its use in the hospitals and clinics of Nepal.

  12. Pyrosequencing for rapid detection of Mycobacterium tuberculosis second-line drugs and ethambutol resistance.

    Science.gov (United States)

    Lacoma, Alicia; Molina-Moya, Barbara; Prat, Cristina; Pimkina, Edita; Diaz, Jessica; Dudnyk, Andriy; García-Sierra, Nerea; Haba, Lucía; Maldonado, Jose; Samper, Sofia; Ruiz-Manzano, Juan; Ausina, Vicente; Dominguez, Jose

    2015-11-01

    The aim of this work was to study the diagnostic accuracy of pyrosequencing to detect resistance to fluoroquinolones, kanamycin, amikacin, capreomycin, and ethambutol (EMB) in Mycobacterium tuberculosis clinical strains. One hundred four clinical isolates previously characterized by BACTEC 460TB/MGIT 960 were included. Specific mutations were targeted in gyrA, rrs, eis promoter, and embB. When there was a discordant result between BACTEC and pyrosequencing, Genotype MTBDRsl (Hain Lifescience, Nehren, Germany) was performed. Sensitivity and specificity of pyrosequencing were 70.6% and 100%, respectively, for fluoroquinolones; 93.3% and 81.7%, respectively, for kanamycin; 94.1% and 95.9%, respectively, for amikacin; 90.0% and 100%, respectively, for capreomycin; and 64.8% and 87.8%, respectively, for EMB. This study shows that pyrosequencing may be a useful tool for making early decisions regarding second-line drugs and EMB resistance. However, for a correct management of patients with suspected extensively drug-resistant tuberculosis, susceptibility results obtained by molecular methods should be confirmed by a phenotypic method.

  13. Cost-effectiveness of a new strategy to detect pulmonary tuberculosis in household contacts in Myanmar.

    Science.gov (United States)

    Htet, K K K; Liabsuetrakul, T; Thein, S

    2017-02-01

    Guidelines regarding household contact tracing for pulmonary tuberculosis (TB) in different countries vary according to case detection methods. To compare costs spent on detecting one TB case among household contacts between different contact tracing strategies in Mandalay City, Myanmar. Cost estimation of case detection and diagnostic procedures using two different strategies were calculated. A modified conventional model included screening for TB signs and symptoms, sputum examination for those with positive signs and symptoms and chest X-ray (CXR) for those with negative sputum results. An interventional model included CXR, sputum examination if CXR was abnormal and Xpert® MTB/RIF assay for those with negative sputum results. Estimated costs in each model were stratified by age <15 and 15 years. The additional cost per TB case detected using the interventional model was US$35.41 compared to the modified conventional model. The probability that the interventional model was cost-effective using a threshold of US$100 per case detected was 81% (83% for those aged 15 years and 65% for those aged <15 years). The interventional model was more cost-effective in detecting one more pulmonary TB case among household contacts than the modified conventional model.

  14. Comparison of the COBAS AMPLICOR MTB and BDProbeTec ET assays for detection of Mycobacterium tuberculosis in respiratory specimens.

    NARCIS (Netherlands)

    W.H.F. Goessens (Wil); P. de Man (Peter); J.G. Koeleman; A. Luijendijk (Ad); R. te Witt (René); H.P. Endtz (Hubert); A.F. van Belkum (Alex)

    2005-01-01

    textabstractThe performances of the BDProbeTec ET (Becton Dickinson) and COBAS AMPLICOR MTB (Roche) were retrospectively evaluated for detecting Mycobacterium tuberculosis complex in various respiratory specimens. The BACTEC and MGIT liquid culture system (Becton Dickinson) was used as a reference m

  15. Detection by radioimmunoassay and enzyme-linked immunosorbent assay of coronavirus antibodies in bovine serum and lacteal secretions.

    Science.gov (United States)

    Rodak, L; Babiuk, L A; Acres, S D

    1982-07-01

    The sensitivity of a radioimmunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA), and a serum neutralization assay (SN) for detecting antibodies to bovine coronavirus in serum and colostrum were compared. Although there proved to be a good correlation among all three assays (r = 0.915 and 0.964 for RIA with SN and ELISA, respectively), RIA and ELISA proved to be at least 10 times more sensitive than neutralization tests. By using these techniques, it was possible to detect a time-dependent decrease in antibody levels in bovine colostrum after parturition. Using ELISA, we demonstrated that 12 of 12 herds in Saskatchewan, and 109 of 110 animals tested, and antibody to bovine coronavirus. There was no elevated antibody response in serum or lacteal secretions of cows vaccinated once or twice with a commercially available modified live rota-coronavirus vaccine. In addition to being more sensitive than SN, ELISA and RIA proved to have other advantages for measuring antibody levels to bovine coronavirus and therefore warrant wider use as tools in diagnostic virology.

  16. Molecular detection of bovine coronavirus in a diarrhea outbreak in pasture-feeding Nellore steers in southern Brazil.

    Science.gov (United States)

    Ribeiro, Juliane; Lorenzetti, Elis; Alfieri, Alice Fernandes; Alfieri, Amauri Alcindo

    2016-03-01

    Worldwide diarrhea outbreaks in cattle herds are more frequently detected in calves being that diarrhea outbreaks in adult cattle are not common. Winter dysentery (WD) is a bovine coronavirus (BCoV) enteric infection that is more reported in Northern hemisphere. Seasonal outbreaks of WD in adult cattle occur mainly in dairy cows. WD has not been described in beef cattle herds of tropical countries. This study describes the molecular detection of BCoV in a diarrhea outbreak in beef cattle steers (Nellore) raised on pasture in Parana, southern Brazil. During the outbreak, the farm had about 600 fattening steers. Watery and bloody diarrhea unresponsive to systemic broad-spectrum antibiotic therapy reveals a morbidity rate of approximately 15 %. The BCoV N gene was identified in 42.9 % (6/14) of the diarrheic fecal samples evaluated by semi-nested polymerase chain reaction (SN-PCR) technique. Other enteric microorganisms occasionally identified in adult cattle and evaluated in this study such as bovine groups A, B, and C rotavirus, bovine viral diarrhea virus, bovine torovirus, aichivirus B, and Eimeria sp. were not identified in the fecal samples. To the best knowledge of the authors, this is the first description of the BCoV diagnosis in fecal samples collected in a diarrhea outbreak in adult beef cattle grazing in the grass in a tropical country.

  17. Bovine Papillomavirus in Brazil: Detection of Coinfection of Unusual Types by a PCR-RFLP Method

    Directory of Open Access Journals (Sweden)

    R. F. Carvalho

    2013-01-01

    Full Text Available Bovine papillomavirus (BPV is recognized as a causal agent of benign and malignant tumors in cattle. Thirteen types of BPV are currently characterized and classified into three distinct genera, associated with different pathological outcomes. The described BPV types as well as other putative ones have been demonstrated by molecular biology methods, mainly by the employment of degenerated PCR primers. Specifically, divergences in the nucleotide sequence of the L1 gene are useful for the identification and classification of new papillomavirus types. On the present work, a method based on the PCR-RFLP technique and DNA sequencing was evaluated as a screening tool, allowing for the detection of two relatively rare types of BPV in lesions samples from a six-year-old Holstein dairy cow, chronically affected with cutaneous papillomatosis. These findings point to the dissemination of BPVs with unclear pathogenic potential, since two relatively rare, new described BPV types, which were first characterized in Japan, were also detected in Brazil.

  18. Molecular characterisation of a bovine-like rotavirus detected from a giraffe

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    O'Shea Helen

    2008-11-01

    Full Text Available Abstract Background Rotavirus (RV, is a member of the Reoviridae family and an important etiological agent of acute viral gastroenteritis in the young. Rotaviruses have a wide host range infecting a broad range of animal species, however little is known about rotavirus infection in exotic animals. In this paper we report the first characterisation of a RV strain from a giraffe calf. Results This report describes the identification and detailed molecular characterisation of a rotavirus strain detected from a 14-day-old Giraffe (Giraffa camelopardalis, presenting with acute diarrhea. The RV strain detected from the giraffe was characterized molecularly as G10P[11]. Detailed sequence analysis of VP4 and VP7 revealed significant identity at the amino acid sequence level to Bovine RV (BoRV. Conclusion This study demonstrates the need for continuous surveillance of RV strains in various animal populations, which will facilitate the identification of rotavirus hosts not previously reported. Furthermore, extending typical epidemiology studies to a broader host range will contribute to the timely identification of new emerging strain types.

  19. Influences on the sensitivity of real-time PCR for the detection of bovine DNA in heat-sterilised feedstuffs.

    Science.gov (United States)

    Abdel-Fattah, Fathy; Gaede, Wolfgang

    2011-06-01

    The present study evaluated two previously developed methods for amplification of bovine mtDNA segments of 109 and 271 base pairs (bp) by real-time polymerase chain reaction (PCR). Beef samples were sterilised experimentally at different temperatures (126 degrees C, 129 degrees C, 132 degrees C and 135 degrees C). These experimentally sterilised beef samples and nine commercial meat and bone meals (MBM) were mixed to a reference plant concentrate in strengths of 50%, 10%, 5%, and 1%. The results of the following PCR showed that the Bos-109 real-time PCR assay was able to detect all the experimental beef samples with exception of the mixtures of beef heated experimentally to 135 degrees C. In mixtures of industrial MBM bovine DNA were always found. Comparatively, the beef sterilised at 135 degrees C and 132 degrees C (and their respective mixtures) and the mixture containing 1% of beef sterilised at 129 degrees C were not detectable with the PCR assay amplifying a target of 271 bp. Using this PCR mixtures of industrial MBM were only weakly detected. The low concentrated mixtures of the extremely processed MBM-1 and MBM-2 even reported negative. These results indicate that the detectability of bovine DNA is strongly influenced by the degree of the thermal treatment. Only the PCR assay amplifying relatively short fragments of a multi-copy mitochondrial target was reliable for the detection of correctly heated MBM in mixed feed.

  20. DETECÇÃO DO COMPLEXO Mycobacterium tuberculosis NO LEITE PELA REAÇÃO EM CADEIA DA POLIMERASE SEGUIDA DE ANÁLISE DE RESTRIÇÃO DO FRAGMENTO AMPLIFICADO (PRA DETECTION OF Mycobacterium tuberculosis COMPLEX BY PCR-RESTRICTION FRAGMENT LENGTH POLYMORFISM ANALYSIS OF THE HSP65 GENE

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    Joab Trajano Silva

    2008-12-01

    Full Text Available Mycobacterium bovis é membro do complexo Mycobacterium tuberculosis (MTBC, grupo este composto por espécies com grande homologia genética. É o agente etiológico da tuberculose bovina, importante zoonose transmissível ao homem, principalmente através da inalação do bacilo e/ou pelo consumo de leite e derivados não-pasteurizados provenientes de vacas tuberculosas. O objetivo deste estudo foi padronizar a identificação de micobactérias do complexo M. tuberculosis presentes no leite, por metodologia molecular. Fez-se a extração de DNA diretamente do leite contaminado e realizou-se a identificação molecular pela reação em cadeia da polimerase seguida de análise de restrição do fragmento amplificado (PRA. Utilizaram-se inhagens de referência e leite cru artificialmente contaminado com M. bovis IP. Um fragmento de 441pb do gene hsp65 foi amplificado, tratado com BstEII e HaeIII e empregou-se o perfil de restrição enzimática obtido para identificar o complexo M. tuberculosis no leite. Com a PRA foi possível detectar com especificidade e sensibilidade a presença de M. bovis em até 10 UFC/mL de leite. A metodologia padronizada poderá auxiliar os métodos microbiológicos e bioquímicos tradicionalmente usados na identificação do bacilo em alimentos suspeitos de contaminação, como, por exemplo, o leite proveniente de animais suspeitos de infecção por M. bovis.

    Palavras-chaves: Análise de perfil de restrição enzimática (PRA, complexo Mycobacterium tuberculosis, leite, Mycobacterium bovis, limite de detecção (PCR. Mycobacterium bovis is a member of the M. tuberculosis complex, a group composed by species with high genetic homology. The pathogen is the etiological agent of bovine tuberculosis, an important zoonosis that is mainly transmitted by inhalation of infectious droplet nuclei or by ingestion of milk and crude milk derivative products from tuberculosis cows. The definitive identification of M. bovis

  1. Deteksi Bovine Herpesvirus-1 Secara Immunohistokimia pada Membran Korioallantois Telur Ayam Berembrio (IMMUNOHISTOCHEMISTRY DETECTION OF BOVINE HERPESVIRUS-1 IN CORIOALLANTOIC MEMBRANE OF CHICKEN EMBRYONATED EGG

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    Yuli Purwandari Kristianingrum

    2016-01-01

    Full Text Available Infectious Bovine Rhinotracheitis (IBR is caused by Bovine Herpes virus-1 in the cattle. The clinicalsigns demonstrate depression, anorexia, swelling of the vulva, redness of the vestibule, pustule and ulceron the vaginal mucosal. Based on previous research, IBR virus from the nasal swab could be grown inchorio-allantoic membrane of embryonated chicken eggs. This study aim was to confirm whether IBR virusin cattle could be grown in embryonated chicken eggs as a substitute for cell culture. A total of five nasalswab samples from the cows that were positive for IBR infection (diagnosed by Polymerase Chain Reactionand cell culture were inoculated on the chorio-allantois membrane of embryonated chicken eggs.Observation of lesions performed at 3-5 days after inoculation. Re-inoculation (passage was done threetimes. Pock characteristic lesions were observed on the corioallantoic membrane with the size of 5-7 mm,rounded shape, opaque edge, with necrosis in the central area. Furthermore, pock lesions were processedfor hematoxylin and eosin staining and immuno-histochemistry. The result of hematoxylin and eosinstaining showed that the formation of intranuclear inclusion bodies and vacuolization of the epithelial cellof membrane was observed. Immuno-histochemistry staining showed positive reaction for antibodiesagainst BHV-1 in the epithelial cells membrane. In conclusion, embryonated chicken eggs could be usedas a medium for detection of IBR.

  2. Detecção de ácidos nucléicos de Brucella spp., Leptospira spp., herpesvirus bovino e vírus da diarréia viral bovina, em fetos bovinos abortados e em animais mortos no perinatal Detection of Brucella spp., Leptospira spp., bovine herpesvirus and bovine viral diarrhea virus nucleic acids in aborted fetuses and bovines dead perinatal

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    A. Cortez

    2006-12-01

    Full Text Available Samples of 114 bovine fetuses and 10 calves, which dead in perinatal period, were examined for detection of DNA. The most common detected agent was Brucella spp. in 17 samples (13.7% followed by Leptospira spp. in 4 cases (3.2%,bovine herpesvirus (BHV and bovine viral diarrhea (BVDV in 3 animals (2.4% each, and 1 for the association of BVDV and BHV. In 77.4 % (96/124 of the samples it was not possible to detect any agent.

  3. Reliability of Cobas Amplicor PCR test in detection of Mycobacterium tuberculosis in respiratory and nonorespiratory specimens

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    Lepšanović Zorica

    2009-01-01

    Full Text Available Background/Aim. Traditional methods for detection of mycobacteria, such as microscopic examination for the presence of acid-fast bacilli and isolation of the organism by culture, have either a low sensitivity and/or specificity, or take weeks before a definite result is available. Molecular methods, especially those based on nucleic acid amplification, are rapid diagnostic methods which combine high sensitivity and high specificity. The aim of this study was to determine the usefulness of the Cobas Amplicor Mycobacterium tuberculosis polymerase chain reaction (CAPCR assay in detecting the tuberculosis cause in respiratory and nonrespiratory specimens (compared to culture. Methods. Specimens were decontaminated by the N-acetyl-L-cystein- NaOH method. A 500 μL aliquot of the processed specimen were used for inoculation of Löwenstein-Jensen (L-J slants, a drop for acid-fast staining, and 100 μL for PCR. The Cobas Amplicor PCR was performed according to the manufacturer's instructions. Results. A total of 110 respiratory and 355 nonrespiratory specimens were investigated. After resolving discrepancies by reviewing medical history, overall sensitivity, specificity, and positive and negative predictive values for CA-PCR assay compared to culture, were 83%, 100%, 100%, and 96.8%, respectively. In comparison, they were 50%, 99.7%, 87.5%, and 98%, respectively, for the nonrespiratory specimens. The inhibition rate was 2.8% for respiratory, and 7.6% for nonrespiratory specimens. Conclusion. CA-PCR is a reliable assay that enables specialists to start treatment promptly on a positive test result. Lower value for specificity in a group of nonrespiratory specimens is a consequence of an extremely small number of mycobacteria in some of them.

  4. Epidemiological modelling for the assessment of bovine tuberculosis surveillance in the dairy farm network in Emilia-Romagna (Italy

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    Gianluigi Rossi

    2015-06-01

    Our analysis showed that slaughterhouse inspection is the most effective surveillance component in reducing the time for disease detection, while routine surveillance in reducing the number of multi-farms epidemics. On the other hand, testing exchanged cattle improved the performance of the surveillance system only marginally.

  5. Two alternative DNA extraction methods to improve the detection of Mycobacterium-tuberculosis-complex members in cattle and red deer tissue samples.

    Science.gov (United States)

    Fell, Shari; Bröckl, Stephanie; Büttner, Mathias; Rettinger, Anna; Zimmermann, Pia; Straubinger, Reinhard K

    2016-09-15

    Bovine tuberculosis (bTB), which is caused by Mycobacterium bovis and M. caprae, is a notifiable animal disease in Germany. Diagnostic procedure is based on a prescribed protocol that is published in the framework of German bTB legislation. In this protocol small sample volumes are used for DNA extraction followed by real-time PCR analyses. As mycobacteria tend to concentrate in granuloma and the infected tissue in early stages of infection does not necessarily show any visible lesions, it is likely that DNA extraction from only small tissue samples (20-40 mg) of a randomly chosen spot from the organ and following PCR testing may result in false negative results. In this study two DNA extraction methods were developed to process larger sample volumes to increase the detection sensitivity of mycobacterial DNA in animal tissue. The first extraction method is based on magnetic capture, in which specific capture oligonucleotides were utilized. These nucleotides are linked to magnetic particles and capture Mycobacterium-tuberculosis-complex (MTC) DNA released from 10 to 15 g of tissue material. In a second approach remaining sediments from the magnetic capture protocol were further processed with a less complex extraction protocol that can be used in daily routine diagnostics. A total number of 100 tissue samples from 34 cattle (n = 74) and 18 red deer (n = 26) were analyzed with the developed protocols and results were compared to the prescribed protocol. All three extraction methods yield reliable results by the real-time PCR analysis. The use of larger sample volume led to a sensitivity increase of DNA detection which was shown by the decrease of Ct-values. Furthermore five samples which were tested negative or questionable by the official extraction protocol were detected positive by real time PCR when the alternative extraction methods were used. By calculating the kappa index, the three extraction protocols resulted in a moderate (0.52; protocol 1 vs 3

  6. Computer-aided detection and quantification of cavitary tuberculosis from CT scans

    Science.gov (United States)

    Xu, Ziyue; Bagci, Ulas; Kubler, Andre; Luna, Brian; Jain, Sanjay; Bishai, William R.; Mollura, Daniel J.

    2013-01-01

    Purpose: To present a computer-aided detection tool for identifying, quantifying, and evaluating tuberculosis (TB) cavities in the infected lungs from computed tomography (CT) scans. Methods: The authors’ proposed method is based on a novel shape-based automated detection algorithm on CT scans followed by a fuzzy connectedness (FC) delineation procedure. In order to assess interaction between cavities and airways, the authors first roughly identified air-filled structures (airway, cavities, esophagus, etc.) by thresholding over Hounsfield unit of CT image. Then, airway and cavity structure detection was conducted within the support vector machine classification algorithm. Once airway and cavities were detected automatically, the authors extracted airway tree using a hybrid multiscale approach based on novel affinity relations within the FC framework and segmented cavities using intensity-based FC algorithm. At final step, the authors refined airway structures within the local regions of FC with finer control. Cavity segmentation results were compared to the reference truths provided by expert radiologists and cavity formation was tracked longitudinally from serial CT scans through shape and volume information automatically determined through the authors’ proposed system. Morphological evolution of the cavitary TB were analyzed accordingly with this process. Finally, the authors computed the minimum distance between cavity surface and nearby airway structures by using the linear time distance transform algorithm to explore potential role of airways in cavity formation and morphological evolution. Results: The proposed methodology was qualitatively and quantitatively evaluated on pulmonary CT images of rabbits experimentally infected with TB, and multiple markers such as cavity volume, cavity surface area, minimum distance from cavity surface to the nearest bronchial-tree, and longitudinal change of these markers (namely, morphological evolution of cavities) were

  7. Molecular detection of mixed infections of Mycobacterium tuberculosis strains in sputum samples from patients in Karonga District, Malawi.

    Science.gov (United States)

    Mallard, Kim; McNerney, Ruth; Crampin, Amelia C; Houben, Rein; Ndlovu, Richard; Munthali, Lumbani; Warren, Robin M; French, Neil; Glynn, Judith R

    2010-12-01

    The occurrence of mixed infections of Mycobacterium tuberculosis is no longer disputed. However, their frequency, and the impact they may have on our understanding of tuberculosis (TB) pathogenesis and epidemiology, remains undetermined. Most previous studies of frequency applied genotyping techniques to cultured M. tuberculosis isolates and found mixed infections to be rare. PCR-based techniques may be more sensitive for detecting multiple M. tuberculosis strains and can be applied to sputum. To date, one study in South Africa has used a PCR approach and suggested that mixed infection could be common. We investigated mixed infections in northern Malawi using two lineage-specific PCR assays targeting the Latin American-Mediterranean (LAM) and Beijing lineages. Compared with spoligotyping, the specificity and sensitivity of both assays was 100%. From 160 culture-positive sputa, mixed LAM and non-LAM strains were detected in 4 sputa belonging to 2 (2.8%) patients. Both patients were HIV positive, with no history of TB. Cultured isolates from both patients showed only LAM by PCR and spoligotyping. In a set of 377 cultured isolates, 4 were mixed LAM and non-LAM. Only one showed evidence of more than one M. tuberculosis strain using IS6110-based restriction fragment length polymorphism (IS6110-RFLP) and spoligotyping analyses. Corresponding sputa for the 4 isolates were unavailable. Mixed Beijing and non-Beijing strains were not detected in this study. Mixed infections appear to be rare in our setting and are unlikely to affect findings based on DNA fingerprinting data. Molecular methods, which avoid the selective nature of culture and target distinct strains, are well suited to detection of mixed infections.

  8. Tuberculose bovina no Estado da Paraíba: estudo retrospectivo Bovine tuberculosis in the state of Paraíba: retrospective survey

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    Salomão M. Figueiredo

    2010-09-01

    Full Text Available O objetivo do presente trabalho foi determinar a frequência de propriedades positivas (focos e de animais positivos para a tuberculose bovina no Estado da Paraíba. Foram utilizados dados da Agência de Defesa Agropecuária do Estado, coletados de suas 23 microrregiões, durante o período de janeiro de 2008 a julho de 2009. Durante esse período, foram examinadas 10.963 propriedades e 54.472 bovinos foram submetidos ao teste de tuberculinização. Para o diagnóstico foi utilizada, como prova de triagem, a tuberculinização cervical simples para gado de leite e a tuberculinização na prega caudal para gado de corte; como prova confirmatória foi utilizada a tuberculinização cervical comparativa. Uma propriedade foi considerada foco quando apresentou pelo menos um animal soropositivo. Das propriedades investigadas, 62 (0,57% apresentaram pelo menos um animal positivo e dos animais analisados, 136 (0,25% foram positivos. Houve diferença significativa (pThe aim of this study was to determine the frequency of positive herds (foci and positive animals for bovine tuberculosis in the state of Paraíba, Northeast region of Brazil. Data from the Agency of Agricultural Protection in the state, collected from its 23 microregions, during the January 2008 to July 2009 period, were used. During this period, 10,963 herds were examined and 54,472 cattle were submitted to the tuberculin test. For diagnosis the cervical and caudal-fold tuberculin tests were used as screening tests in dairy and beef cattle, respectively; as confirmatory test, comparative cervical test was used. A herd was considered focus when presented at least one positive animal. Of the herds investigated, 62 (0.57% had at least one positive animal, and of the animals examined, 136 (0.25% were positive. There was significant difference (p<0.001 in the proportion of positivity for females (0.32% and males (0.04%. Despite low frequency of foci of brucellosis and seropositive animals, it is

  9. Assessing the impact of a cattle risk-based trading scheme on the movement of bovine tuberculosis infected animals in England and Wales.

    Science.gov (United States)

    Adkin, A; Brouwer, A; Downs, S H; Kelly, L

    2016-01-01

    The adoption of bovine tuberculosis (bTB) risk-based trading (RBT) schemes has the potential to reduce the risk of bTB spread. However, any scheme will have cost implications that need to be balanced against its likely success in reducing bTB. This paper describes the first stochastic quantitative model assessing the impact of the implementation of a cattle risk-based trading scheme to inform policy makers and contribute to cost-benefit analyses. A risk assessment for England and Wales was developed to estimate the number of infected cattle traded using historic movement data recorded between July 2010 and June 2011. Three scenarios were implemented: cattle traded with no RBT scheme in place, voluntary provision of the score and a compulsory, statutory scheme applying a bTB risk score to each farm. For each scenario, changes in trade were estimated due to provision of the risk score to potential purchasers. An estimated mean of 3981 bTB infected animals were sold to purchasers with no RBT scheme in place in one year, with 90% confidence the true value was between 2775 and 5288. This result is dependent on the estimated between herd prevalence used in the risk assessment which is uncertain. With the voluntary provision of the risk score by farmers, on average, 17% of movements was affected (purchaser did not wish to buy once the risk score was available), with a reduction of 23% in infected animals being purchased initially. The compulsory provision of the risk score in a statutory scheme resulted in an estimated mean change to 26% of movements, with a reduction of 37% in infected animals being purchased initially, increasing to a 53% reduction in infected movements from higher risk sellers (score 4 and 5). The estimated mean reduction in infected animals being purchased could be improved to 45% given a 10% reduction in risky purchase behaviour by farmers which may be achieved through education programmes, or to an estimated mean of 49% if a rule was implemented

  10. Herd-level risk factors for bovine tuberculosis and adoption of related biosecurity measures in Northern Ireland: A case-control study.

    Science.gov (United States)

    O'Hagan, M J H; Matthews, D I; Laird, C; McDowell, S W J

    2016-07-01

    Bovine tuberculosis (bTB) is a zoonotic disease which is endemic in Northern Ireland. As it has proven difficult to eradicate this disease, partly due to a wildlife reservoir being present in the European badger (Meles meles), a case-control study was conducted in a high incidence area in 2010-2011. The aim was to identify risk factors for bTB breakdown relating to cattle and badgers, and to assess the adoption of bTB related biosecurity measures on farms. Face-to-face questionnaires with farmers and surveys of badger setts and farm boundaries were conducted on 117 farms with a recent bTB breakdown (cases) and 75 farms without a recent breakdown (controls). On logistic regression at univariable and multivariable levels, significant risk factors associated with being a case herd included having an accessible badger sett within the farm boundaries in a field grazed in the last year (odds ratio, OR, 4.14; 95% confidence interval, CI, 1.79, 9.55), observation of live badgers (OR 4.14; 95% CI 1.79, 9.55), purchase of beef cattle (OR 4.60; 95% CI 1.61, 13.13), use of contractors to spread slurry (OR 2.83; 95% CI 1.24, 6.49), feeding meal on top of silage (OR 3.55; 95% CI 1.53, 8.23) and feeding magnesium supplement (OR = 3.77; 95% CI 1.39, 10.17). The majority of setts within the farm boundary were stated to be accessible by cattle (77.1%; 95% CI 71.2, 83.0%) and 66.8% (95% CI 63.8, 69.7%) of farm boundaries provided opportunities for nose-to-nose contact between cattle. Adoption of bTB related biosecurity measures, especially with regards to purchasing cattle and badger-related measures, was lower than measures related to disinfection and washing.

  11. Assessment of the probability of introduction of bovine tuberculosis to Danish cattle farms via imports of live cattle from abroad and immigrant workers.

    Science.gov (United States)

    Foddai, Alessandro; Nielsen, Liza Rosenbaum; Krogh, Kaspar; Alban, Lis

    2015-12-01

    Denmark has been recognized as officially free (OTF) from bovine tuberculosis (bTB) since 1980. In this study, we estimated the annual probability (PIntro) of introducing Mycobacterium bovis into the Danish cattle population, through (a) imports of cattle and (b) foreign personnel working in Danish cattle herds. Data from 2000 to 2013 with date, number and origin of imported live cattle were obtained from the Danish Cattle Federation. Information on immigrants working in Danish cattle herds was obtained through a questionnaire sent by email to a sample of Danish cattle farmers (N=460). Inputs obtained from data analysis, expert opinion, the questionnaire and literature were fed into three stochastic scenario tree models used to simulate the effect of import trade patterns, and contact between immigrant workers and cattle. We also investigated the opportunity of testing animals imported from OTF countries by tuberculin skin test and animals from non-OTF countries by interferon-γ test (IFN-γ), exemplified by using year 2009 where the number of imported animals was higher than usual. Results showed that PIntro is driven mainly by importation of live cattle. The combined median annual probability of introducing M. bovis into the Danish cattle population by either imported live cattle or infectious immigrant workers, ranged from 0.3% (90% prediction interval (P.I.): 0.04%:1.4%) in 2001 to 4.9% (90% P.I.: 0.6%; 19.2%) in 2009. The median of the median PIntro estimates from the 14 years was 0.7% (median of 90% P.I.: 0.08%; 3.5%). Hence, on average, at least one introduction each 143 years could be expected, if the annual number of imported animals does not change remarkably in the future. If the number of imported animals increases, compared to the years we analyzed, additional testing of imported cattle might be considered. For example, in 2009, PIntro would have been reduced from 4.9% to 0.8% (90% P.I.: 0.1%; 4.7%) if animals from OTF countries had been tested with

  12. Bovine viral diarrhea virus antigen detection across whole cattle hides using two antigen-capture enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Vander Ley, Brian L; Ridpath, Julia F; Sweiger, Shaun H

    2012-05-01

    Bovine viral diarrhea virus is a costly disease of cattle that can be controlled by vaccination, biosecurity, and removal of persistently infected cattle. Development and proficiency testing of assays to identify persistently infected cattle requires substantial quantities of known positive- and negative-sample material. The objective of this study was to determine what sections of bovine skin contained Bovine viral diarrhea virus antigen. Two commercially available antigen-capture enzyme-linked immunoassays were used to test subsamples representing the entire skin of 3 persistently infected calves. Both assays detected Bovine viral diarrhea virus antigen in the samples indicated for use by assay protocol. However, one assay identified all subsamples as positive, while the second assay identified 64.4% of subsamples as positive. These results show that use of samples other than those specified by the assay protocol must be validated for each individual assay. In this study, alternative sample sites and use of the entire hide for proficiency testing would be acceptable for only one of the assays tested.

  13. Application of a multiplex PCR assay for Campylobacter fetus detection and subspecies differentiation in uncultured samples of aborted bovine fetuses.

    Science.gov (United States)

    Iraola, Gregorio; Hernández, Martín; Calleros, Lucía; Paolicchi, Fernando; Silveyra, Silvia; Velilla, Alejandra; Carretto, Luis; Rodríguez, Eliana; Pérez, Ruben

    2012-12-01

    Campylobacter (C.) fetus (epsilonproteobacteria) is an important veterinary pathogen. This species is currently divided into C. fetus subspecies (subsp.) fetus (Cff) and C. fetus subsp. venerealis (Cfv). Cfv is the causative agent of bovine genital Campylobacteriosis, an infectious disease that leads to severe reproductive problems in cattle worldwide. Cff is a more general pathogen that causes reproductive problems mainly in sheep although cattle can also be affected. Here we describe a multiplex PCR method to detect C. fetus and differentiate between subspecies in a single step. The assay was standardized using cultured strains and successfully used to analyze the abomasal liquid of aborted bovine fetuses without any pre-enrichment step. Results of our assay were completely consistent with those of traditional bacteriological diagnostic methods. Furthermore, the multiplex PCR technique we developed may be easily adopted by any molecular diagnostic laboratory as a complementary tool for detecting C. fetus subspecies and obtaining epidemiological information about abortion events in cattle.

  14. Peroxidase-linked assay for detection of antibodies against bovine leukosis virus.

    Science.gov (United States)

    de Castro, Clarissa C; Nunes, Cristina F; Finger, Paula F; Siedler, Bianca S; Dummer, Luana; de Lima, Marcelo; Leite, Fábio P L; Fischer, Geferson; Vargas, Gilberto D'A; Hübner, Silvia de O

    2013-01-01

    A peroxidase linked assay (PLA) was designed to screen bovine sera for the presence of specific antibodies against bovine leukosis virus (BLV). Out of 201 samples of bovine sera analyzed, 52.2% were considered positive by PLA, 26.4% by AGID, and 38.9% by ELISA. Western blotting analyses excluded 27 samples found to be positive by PLA. PLA showed 100% of sensitivity when compared with AGID and ELISA. Specificity was 64.8% and 78%, respectively (kappa coefficients were 0.70 and 0.83). These findings indicate that PLA can be used as an alternative method for the diagnosis of BLV infection in cattle.

  15. Development of magnetic nanoparticle based calorimetric assay for the detection of bovine mastitis in cow milk.

    Science.gov (United States)

    Chinnappan, Raja; Al Attas, Sana; Kaman, Wendy E; Bikker, Floris J; Zourob, Mohammed

    2017-04-15

    Mastitis in dairy cattle is an inflammatory reaction of the udder tissue. Mastitis increases plasmin levels, leading to an increased proteolysis of milk proteins such as casein, resulting in a significant decrease in milk quality and related dairy products. Due to its key-role in mastitis, we used plasmin proteolytic activity as a biomarker for the detection of mastitis in bovine mastitic milk. Inspired by earlier studies on protease activity using mastitic milk samples, we developed a simple colorimetric assay to distinguish mastitic milk from milk derived from healthy animals. The plasmin substrate coupled to magnetic nanoparticles form a black self-assembled monolayer on a gold sensor surface. In the presence of increased levels of plasmin, the substrate is cleaved and the peptide fragment attached to the magnetic beads, will be attracted by the magnet which is present under the sensor strips revealing the golden surface. We found the area of the golden color surface proportional to plasmin activity. The sensitivity of this method was determined to be 1 ng/ml of plasmin in vitro. Next, we tested the biosensor using mastitis positive milk of which infection is confirmed by bacterial cultures. This newly developed colorimetric biosensor has high potential in applications for the diagnosis of mastitis with potential spin offs to health, food and environmental sectors. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Molecular detection of bovine leukemia virus in peripheral blood of Iranian cattle, camel and sheep.

    Science.gov (United States)

    Nekoei, S; Hafshejani, T Taktaz; Doosti, A; Khamesipour, F

    2015-01-01

    Bovine leukemia virus (BLV) is a deltaretrovirus which infects and induces proliferation of B-lymphocytes in the peripheral blood circulation and in lymphoid organs primarily of cattle, leading to leukemia/lymphoma. This study was carried out to investigate the presence of BLV in cattle, sheep and camels from the Chaharmahal va Bakhtiary and Isfahan provinces in Iran. A total of 874 blood samples collected from cattle, sheep and camels were used in this study to detect BLV using a nested-PCR. The results from this study indicated that 17.2% (n=874) of all blood samples collected were positive for BLV. The percentages of blood samples positive for BLV from cattle, sheep and camels were 22.1 (n=657), 5.3 (n=95) and 0 (n=122) respectively. The results from this study showed that BLV infected cattle and sheep. Camels seemed to be resistant to BLV infection. This study contributes to the nationwide effort to obtain baseline information on the prevalence of BLV, which will assist in planning the control strategy for the disease in Iran.

  17. Detection of Different Bovine Papillomavirus Types and Co-infection in Bloodstream of Cattle.

    Science.gov (United States)

    Santos, E U D; Silva, M A R; Pontes, N E; Coutinho, L C A; Paiva, S S L; Castro, R S; Freitas, A C

    2016-02-01

    Bovine papillomavirus (BPV) is a diverse group of double-stranded DNA oncogenic viruses. BPVs are classically described as epitheliotropic, however, they have been detected in body fluids, such as blood and semen. The presence of BPV in these sites can have implications for the dissemination of BPV. The aim of this study was to verify the prevalence of BPV types in cattle blood. A total of 57 blood samples were analyzed by PCR using BPV type-specific primers to BPVs 1-6 and 8-10, and subsequent sequencing. Sequencing quality was determined using Staden package with Phred 20. Similarity analysis was performed with BioEdit and BLAST programs to assess the identity with known BPV types. Statistical analysis was performed by Fisher's exact test. The results showed seven different types of BPVs in the blood, with the exception of BPV 5 and 9. This is the first study that demonstrates BPVs 3, 6, 8 and 10 DNA in cattle blood. BPVs 1 and 2 were the viral types most frequent in blood, while BPVs 4 and 10 were the least frequent types. All the samples showed co-infection by at least two BPV types. These data suggest that several BPV types may infect blood cells at the same time and demonstrate the possibility that the BPV infection in non-epithelial tissue can occur without restriction to one or two viral types. These results can contribute to future studies aimed at the control and prevention of papillomaviruses.

  18. The bovine 5' AMPK gene family: mapping and single nucleotide polymorphism detection.

    Science.gov (United States)

    McKay, Stephanie D; White, Stephen N; Kata, Srinivas R; Loan, Raymond; Womack, James E

    2003-12-01

    The 5'-AMP-activated protein kinase (AMPK) family is an ancient stress response system whose primary function is regulation of cellular ATP. Activation of AMPK, which is instigated by environmental and nutritional stresses, initiates energy-conserving measures that protect the cell by inhibition and phosphorylation of key enzymes in energy-consuming biochemical pathways. The seven genes that comprise the bovine AMPK family were mapped in cattle by using a radiation hybrid panel. The seven genes mapped to six different cattle chromosomes, each with a LOD score greater than 10.0. PRKAA1 mapped to BTA 20, PRKAA2 and PRKAB2 to BTA 3, PRKAB1 to BTA 17, PRKAG1 to BTA 5, PRKAG2 to BTA 4, and PRKAG3 to BTA 2. Five of the seven genes mapped to regions expected from human/cattle comparative maps. PRKAB2 and PRKAG3, however, have not been mapped in humans. We predict these genes to be located on HSA 1 and 2, respectively. Additionally, one synonymous and one non-synonymous single nucleotide polymorphism (SNP) were detected in PRKAG3 in Bos taurus cattle. In an effort to determine ancestral origins, various herds of mixed breed cattle as well as other ruminant species were characterized for sequence variation in this region of PRKAG3. Owing to the physiological importance of this gene family, we believe that its individual genes are candidate genes for conferring resistance to diseases in cattle.

  19. Detection of an untyped strain of bovine respiratory syncytial virus in a dairy herd

    Directory of Open Access Journals (Sweden)

    Ingrid Bortolin Affonso

    2014-10-01

    Full Text Available Bovine respiratory syncytial virus (BRSV causes important lower respiratory tract illness in calves. According to F and G proteins genetic sequences, three BRSV subgroups have been reported and characterized in several countries, showing differences in its distribution. In Brazil, the virus is widely disseminated throughout the herds and the few characterized isolates revealed the solely occurrence of the subgroup B. This study describes the detection and characterization of an untyped BRSV strain from a twenty-days-old calf from a herd without clinical respiratory disease. Nasal swabs were analyzed by RT-nested PCR for the F and G proteins genes. One sample has amplified the F protein gene. Sequencing and subsequent phylogenetic reconstruction were accomplished, revealing that the strain could not be grouped with any other BRSV subgroups reported. This result may suggest that the BRSV is in constantly evolution, even in Brazil, where the vaccination is not a common practice. More detailed studies about BRSV characterization are necessary to know the virus subgroups distribution among the Brazilian herds to recommend appropriated immunoprophylaxis.

  20. Evaluation of the detection of Mycobacterium tuberculosis with metabolic activity in culture-negative human clinical samples.

    Science.gov (United States)

    Cubero, N; Esteban, J; Palenque, E; Rosell, A; Garcia, M J

    2013-03-01

    Mycobacterium tuberculosis is assumed to remain in a quiescent state during latent infection, being unable to grow in culture. The aim of this study was to evaluate the detection of viable but non-cultivable bacilli with metabolic activity in human clinical samples using a procedure that is independent of the immunological status of the patient. The study was performed on 66 human clinical samples, from patients subjected to routine diagnosis to rule out a mycobacterial infection. Specimens from pulmonary and extra-pulmonary origins were verified to contain human DNA before testing for M. tuberculosis DNA, rRNA and transient RNA by real-time quantitative PCR. Clinical records of 55 patients were also reviewed. We were able to detect viable but non-cultivable bacilli with a metabolic activity in both pulmonary and extra-pulmonary samples. Mycobacterium tuberculosis RNA was detected in the majority of culture-positive samples whereas it was detected in one-third of culture-negative samples, 20% of them showed metabolic activity. Amplifications of the ftsZ gene and particularly of the main promoter of the ribosomal operon rrnA, namely PCL1, seem to be good targets to detect active bacilli putatively involved in latent infection. Moreover, this last target would provide information on the basal metabolic activity of the bacilli detected. © 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

  1. Bovine papillomavirus type 2 detection in the urinary bladder of cattle with chronic enzootic haematuria

    Directory of Open Access Journals (Sweden)

    Sheila R Wosiacki

    2006-09-01

    Full Text Available The bovine papillomavirus type 2 (BPV-2 involvement in the aetiology of chronic enzootic haematuria associated to bracken fern ingestion has been suggested for a long time. However, a few reports have shown the presence of the BPV-2 in urinary bladder tumors of cattle. The aim of this study was to investigate the presence of the BPV-2 infection in the urinary bladder of cattle with chronic enzootic haematuria in Brazilian cattle herds. Sixty-two urinary bladders were collected from adult cattle in beef herds from the north region of the state of Paraná, Brazil. According to clinical and pathological finds the specimens were distributed in three groups: the group A was constituted by 22 urinary bladders with macroscopic lesions collected at necropsy of cattle with clinical signs of chronic enzootic haematuria; the group B by 30 urinary bladders with macroscopic lesions collected in a slaughterhouse of cows coming from bracken fern-endemic geographical region; and the group C (control by 10 urinary bladders without macroscopic lesions collected from asymptomatic cattle in a bracken fern-free geographical region. By a semi-nested polymerase chain reaction (PCR assay, with an internal control, a fragment of the BPV-2 L1 gene with 386 bp length was amplified in 36 (58% urinary bladder. The rate of BPV-2 positive urinary bladders was 50% (11/22 for group A, 80% (24/30 for group B, and 10% (1/10 for group C (control. The rate of the positive results found in groups A and B that included urinary bladder samples with macroscopic lesions was 67% (35/52 and the detection of the BPV-2 in both groups was significantly higher (P < 0.05 than in the control group. RFLP with Rsa I and Hae III enzymes evaluated the specificity of the BPV-2 amplicons. The PCR internal control that amplified a 626 bp fragment of the ND5 gene of the bovine mitochondrial genome was amplified in all analyzed samples and excluded false-negatives or invalid results in the semi

  2. Comparison of rapid tests for detection of rifampicin-resistant Mycobacterium tuberculosis in Kampala, Uganda

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    McNerney Ruth

    2009-08-01

    Full Text Available Abstract Background Drug resistant tuberculosis (TB is a growing concern worldwide. Rapid detection of resistance expedites appropriate intervention to control the disease. Several technologies have recently been reported to detect rifampicin resistant Mycobacterium tuberculosis directly in sputum samples. These include phenotypic culture based methods, tests for gene mutations and tests based on bacteriophage replication. The aim of the present study was to assess the feasibility of implementing technology for rapid detection of rifampicin resistance in a high disease burden setting in Africa. Methods Sputum specimens from re-treatment TB patients presenting to the Mulago Hospital National TB Treatment Centre in Kampala, Uganda, were examined by conventional methods and simultaneously used in one of the four direct susceptibility tests, namely direct BACTEC 460, Etest, "in-house" phage test, and INNO- Rif.TB. The reference method was the BACTEC 460 indirect culture drug susceptibility testing. Test performance, cost and turn around times were assessed. Results In comparison with indirect BACTEC 460, the respective sensitivities and specificities for detecting rifampicin resistance were 100% and 100% for direct BACTEC and the Etest, 94% and 95% for the phage test, and 87% and 87% for the Inno-LiPA assay. Turn around times ranged from an average of 3 days for the INNO-LiPA and phage tests, 8 days for the direct BACTEC 460 and 20 days for the Etest. All methods were faster than the indirect BACTEC 460 which had a mean turn around time of 24 days. The cost per test, including labour ranged from $18.60 to $41.92 (USD. Conclusion All four rapid technologies were shown capable of detecting rifampicin resistance directly from sputum. The LiPA proved rapid, but was the most expensive. It was noted, however, that the LiPA test allows sterilization of samples prior to testing thereby reducing the risk of accidental laboratory transmission. In contrast the

  3. A volumetric meter chip for point-of-care quantitative detection of bovine catalase for food safety control.

    Science.gov (United States)

    Cui, Xingye; Hu, Jie; Choi, Jane Ru; Huang, Yalin; Wang, Xuemin; Lu, Tian Jian; Xu, Feng

    2016-09-07

    A volumetric meter chip was developed for quantitative point-of-care (POC) analysis of bovine catalase, a bioindicator of bovine mastitis, in milk samples. The meter chip displays multiplexed quantitative results by presenting the distance of ink bar advancement that is detectable by the naked eye. The meter chip comprises a poly(methyl methacrylate) (PMMA) layer, a double-sided adhesive (DSA) layer and a glass slide layer fabricated by the laser-etching method, which is typically simple, rapid (∼3 min per chip), and cost effective (∼$0.2 per chip). Specially designed "U shape" reaction cells are covered by an adhesive tape that serves as an on-off switch, enabling the simple operation of the assay. As a proof of concept, we employed the developed meter chip for the quantification of bovine catalase in raw milk samples to detect catalase concentrations as low as 20 μg/mL. The meter chip has great potential to detect various target analytes for a wide range of POC applications.

  4. Detecting a low prevalence of latent tuberculosis among health care workers in Denmark detected by M. tuberculosis specific IFN-gamma whole-blood test

    DEFF Research Database (Denmark)

    Soborg, Bolette; Andersen, Aase B; Larsen, Helle K;

    2007-01-01

    The study was designed to estimate prevalence of tuberculosis infection among health care workers, using the tuberculin skin test (TST) and the new M. tuberculosis specific diagnostic whole-blood test and to identify possible risk factors. Employees at 2 departments of infectious diseases...... TST whereas only 2 of 139 (1%) had a positive QuantiFERON TB-Gold test (QFT-TB). 42 of 106 (40%) BCG vaccinated had positive TST (> or =12 mm) compared with 2 of 27 (7%) unvaccinated persons. Among 47 persons with positive TST, 42 (89%) were BCG- vaccinated. The 2 QFT-TB positive participants as well...... and not to infection with M. tuberculosis. The overall transmission rate determined by QFT-TB was found to be very low. The QFT-TB may be useful in distinguishing persons with latent TB infection from persons with positive TST due to BCG vaccination and its use may reduce anxiety....

  5. Detection and characterization of viruses as field and vaccine strains in feedlot cattle with bovine respiratory disease

    Science.gov (United States)

    This study investigated viruses in bovine respiratory disease (BRD) cases in feedlots, including bovine herpesvirus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine coronaviruses (BoCV) and parainfluenza-3 virus (PI3V). Nasal swabs were collected fro...

  6. Success of active tuberculosis case detection among high-risk groups in urban slums in Pakistan.

    Science.gov (United States)

    Fatima, R; Qadeer, E; Enarson, D A; Creswell, J; Stevens, R H; Stevens, R; Hinderaker, S G; Anwar, K; ul Haq, M

    2014-09-01

    In Pakistan, patients with symptoms suggestive of tuberculosis (TB) seek care from a wide array of health care providers, many of whom do not notify cases to the National TB Programme (NTP). We evaluated an active case detection intervention in five randomly selected districts in urban slums of Sindh Province, Pakistan. To evaluate the increase in case notification of smear-positive TB by active case finding at community-based chest camps by engaging the private providers. A cross-sectional study of TB case detection associated with a project using integrated intervention and chest camps. From April 2011 to September 2012, the total number of clients seen in the camps was 165 280. Of all the attendees, 13 481 (12.7%) were examined by sputum smear microscopy. The proportion of smear-positive results was significantly higher among those from engaged private providers than among those referred from camps (OR 1.54, 95%CI 1.42-1.66). During the project, the total number of smear-positive TB notifications increased over the intervention period from 5158 to 8275. Active case detection by engaging private providers and chest camps can significantly increase the number of smear-positive TB case notifications.

  7. A dual molecular beacon approach for fast detection of Mycobacterium tuberculosis.

    Science.gov (United States)

    Yu, Chuan-Xing; Zhao, Zi-Yun; Lv, Jian-Xin; Zhu, Ling

    2013-02-01

    The main objectives of this study were to assess a dual molecular beacon approach for fast detection of Mycobacterium tuberculosis (MT). MT beacon (Tb-B) was designed to target the unique IS6110 (114 bp) and rpoB (215 bp) fragment of the MT (H37Ra) genome, and the two fragments were inserted into the PMD-19T vector after purification, by PCR and sequencing, to construct plasmids. Different dilutions of positive plasmid standards were used for dual molecular beacon RT-PCR of rpoB and IS6110, and standard curves were established.The results show that the dual molecular beacon of rpoB and IS6110 detecting MT was stable (CV is 1.91-2.68 %) with a high amplification efficiency (95.6 %). In addition, the strains of non MT did not generate fluorescence signals, while strains of MT did, indicating that the primers and molecular beacons were specific, and only MT complex was amplified. The linear range was wide (10(3)-10(11) copies/mL), and clinical specimens presenting different bacterial counts can be detected.

  8. Multiple-instance learning for computer-aided detection of tuberculosis

    Science.gov (United States)

    Melendez, J.; Sánchez, C. I.; Philipsen, R. H. H. M.; Maduskar, P.; van Ginneken, B.

    2014-03-01

    Detection of tuberculosis (TB) on chest radiographs (CXRs) is a hard problem. Therefore, to help radiologists or even take their place when they are not available, computer-aided detection (CAD) systems are being developed. In order to reach a performance comparable to that of human experts, the pattern recognition algorithms of these systems are typically trained on large CXR databases that have been manually annotated to indicate the abnormal lung regions. However, manually outlining those regions constitutes a time-consuming process that, besides, is prone to inconsistencies and errors introduced by interobserver variability and the absence of an external reference standard. In this paper, we investigate an alternative pattern classi cation method, namely multiple-instance learning (MIL), that does not require such detailed information for a CAD system to be trained. We have applied this alternative approach to a CAD system aimed at detecting textural lesions associated with TB. Only the case (or image) condition (normal or abnormal) was provided in the training stage. We compared the resulting performance with those achieved by several variations of a conventional system trained with detailed annotations. A database of 917 CXRs was constructed for experimentation. It was divided into two roughly equal parts that were used as training and test sets. The area under the receiver operating characteristic curve was utilized as a performance measure. Our experiments show that, by applying the investigated MIL approach, comparable results as with the aforementioned conventional systems are obtained in most cases, without requiring condition information at the lesion level.

  9. Molecular approaches for detection of the multi-drug resistant tuberculosis (MDR-TB in Bangladesh.

    Directory of Open Access Journals (Sweden)

    Tafsina Haque Aurin

    Full Text Available The principal obstacles in the treatment of tuberculosis (TB are delayed and inaccurate diagnosis which often leads to the onset of the drug resistant TB cases. To avail the appropriate treatment of the patients and to hinder the transmission of drug-resistant TB, accurate and rapid detection of resistant isolates is critical. Present study was designed to demonstrate the efficacy of molecular techniques inclusive of line probe assay (LPA and GeneXpert MTB/RIF methods for the detection of multi-drug resistant (MDR TB. Sputum samples from 300 different categories of treated and new TB cases were tested for the detection of possible mutation in the resistance specific genes (rpoB, inhA and katG through Genotype MTBDRplus assay or LPA and GeneXpert MTB/RIF tests. Culture based conventional drug susceptibility test (DST was also carried out to measure the efficacy of the molecular methods employed. Among 300 samples, 191 (63.7% and 193 (64.3% cases were found to be resistant against rifampicin in LPA and GeneXpert methods, respectively; while 189 (63% cases of rifampicin resistance were detected by conventional DST methods. On the other hand, 196 (65.3% and 191 (63.7% isolates showed isoniazid resistance as detected by LPA and conventional drug susceptibility test (DST, respectively. Among the drug resistant isolates (collectively 198 in LPA and 193 in conventional DST, 189 (95.6% and 187 (96.9% were considered to be MDR as examined by LPA and conventional DST, respectively. Category-II and -IV patients encountered higher frequency of drug resistance compared to those from category-I and new cases. Considering the higher sensitivity, specificity and accuracy along with the required time to results significantly shorter, our study supports the adoption of LPA and GeneXpert assay as efficient tools in detecting drug resistant TB in Bangladesh.

  10. Detecting and characterizing cellular responses to Mycobacterium tuberculosis from histology slides.

    Science.gov (United States)

    Niazi, M Khalid Khan; Beamer, Gillian; Gurcan, Metin N

    2014-02-01

    Infection with Mycobacterium tuberculosis (M.tb) results in immune cell recruitment to the lungs, forming macrophage-rich regions (granulomas) and lymphocyte-rich regions (lymphocytic cuffs). The objective of this study was to accurately identify and characterize these regions from hematoxylin and eosin (H&E)-stained tissue slides. The two target regions (granulomas and lymphocytic cuffs) can be identified by their morphological characteristics. Their most differentiating characteristic on H&E slides is cell density. We developed a computational framework, called DeHiDe, to detect and classify high cell-density regions in histology slides. DeHiDe employed a novel internuclei geodesic distance calculation and Dulmange Mendelsohn permutation to detect and classify high cell-density regions. Lung tissue slides of mice experimentally infected with M.tb were stained with H&E and digitized. A total of 21 digital slides were used to develop and train the computational framework. The performance of the framework was evaluated using two main outcome measures: correct detection of potential regions, and correct classification of potential regions into granulomas and lymphocytic cuffs. DeHiDe provided a detection accuracy of 99.39% while it correctly classified 90.87% of the detected regions for the images where the expert pathologist produced the same ground truth during the first and second round of annotations. We showed that DeHiDe could detect high cell-density regions in a heterogeneous cell environment with non-convex tissue shapes. © 2013 International Society for Advancement of Cytometry.

  11. Mycobacterium tuberculosis DosR Regulon Gene Rv2004c Encodes a Novel Antigen with Pro-inflammatory Functions and Potential Diagnostic Application for Detection of Latent Tuberculosis

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    Sankara Narayana Doddam

    2017-06-01

    Full Text Available Approximately 1.7 billion people in the world harbor latent Mycobacterium tuberculosis (Mtb with a substantial risk of progression to clinical outcome. Containment of these seed beds of Mtb is essential to eliminate tuberculosis completely in high burden settings such as India. Hence, there is an urgent need for the identification of new serological markers for detection or vaccine candidates to prevent latent tuberculosis infection (LTBI. DosR regulon antigens of Mtb might serve as attractive targets for LTBI diagnosis or vaccine development as they are specifically expressed and are upregulated during latent phase. In this study, we investigated the role of Rv2004c, a member of DosR regulon (exclusive to Mtb complex, in host–pathogen interaction and its immunogenic potential in LTBI, active TB, and healthy control cohorts. Rv2004c elicited strong antibody response in individuals with LTBI compared to active TB patients and healthy cohorts. Recombinant Rv2004c induced pro-inflammatory cytokine response in human peripheral blood mononuclear cells and THP-1 cells via NF-κB phosphorylation. Interaction of Rv2004c with toll-like receptor (TLR-2 was confirmed using HEK-Blue hTLR-2 and pull-down assays. Rv2004c enhanced the surface expression of TLR-2 at mRNA and protein levels in THP-1 cells. Our findings revealed that Rv2004c induces strong humoral and cell mediated immune responses. Given these observations, we propose Rv2004c to be a potential diagnostic marker or an attractive vaccine candidate that can be useful against LTBI.

  12. Tuberculosis in African lions

    NARCIS (Netherlands)

    Maas, M.

    2013-01-01

    Lions (Panthera leo) are susceptible to Mycobacterium bovis (M. bovis) infection, resulting in bovine tuberculosis (BTB). This chronic, debilitating disease can affect multiple organs, particularly the lungs, and may ultimately lead to death of the infected animal. Cases of lion BTB have been descri

  13. Determination of 11 quinolones in bovine milk using immunoaffinity stir bar sorptive microextraction and liquid chromatography with fluorescence detection.

    Science.gov (United States)

    Yao, Kai; Zhang, Wei; Yang, Linyan; Gong, Jianfang; Li, Liuan; Jin, Tianming; Li, Cun

    2015-10-15

    A sensitive, selective and reproducible immunoaffinity stir bar sorptive microextraction (SBSME) coupled with liquid chromatography-fluorescence method for determination of 11 quinolones (QNs) in bovine milk was developed and validated. It is first report of a broad-specificity monoclonal antibody to QNs that has been immobilized to glass bar for preparation of a re-usable immunoaffinity stir bar. Analytes were extracted by placing stir bar in milk and shaking on a rotary shaker for 30min at 30rpm, followed by liquid chromatography and fluorescence detection. The newly developed method has limits of detection for each QN from 0.05 to 0.1ng/g with intra-day and inter-day precision ranging from 3.2 to 11.9% and from 5.2 to 12.5%, respectively. This allowed us to quantitatively analyze drugs in bovine milk with the advantage of significantly simplified sample preparation. The proposed method was successfully applied to the bovine milk samples analyses with QNs, demonstrating its rare application in animal food safety analysis.

  14. Sulfated dextrans enhance in vitro amplification of bovine spongiform encephalopathy PrP(Sc and enable ultrasensitive detection of bovine PrP(Sc.

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    Yuichi Murayama

    Full Text Available BACKGROUND: Prions, infectious agents associated with prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE in cattle, and scrapie in sheep and goats, are primarily comprised of PrP(Sc, a protease-resistant misfolded isoform of the cellular prion protein PrP(C. Protein misfolding cyclic amplification (PMCA is a highly sensitive technique used to detect minute amounts of scrapie PrP(Sc. However, the current PMCA technique has been unsuccessful in achieving good amplification in cattle. The detailed distribution of PrP(Sc in BSE-affected cattle therefore remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: We report here that PrP(Sc derived from BSE-affected cattle can be amplified ultra-efficiently by PMCA in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrP(Sc from the saliva, palatine tonsils, lymph nodes, ileocecal region, and muscular tissues of BSE-affected cattle. Individual differences in the distribution of PrP(Sc in spleen and cerebrospinal fluid samples were observed in terminal-stage animals. However, the presence of PrP(Sc in blood was not substantiated in the BSE-affected cattle examined. CONCLUSIONS/SIGNIFICANCE: The distribution of PrP(Sc is not restricted to the nervous system and can spread to peripheral tissues in the terminal disease stage. The finding that PrP(Sc could be amplified in the saliva of an asymptomatic animal suggests a potential usefulness of this technique for BSE diagnosis. This highly sensitive method also has other practical applications, including safety evaluation or safety assurance of products and byproducts manufactured from bovine source materials.

  15. Highly Accurate Antibody Assays for Early and Rapid Detection of Tuberculosis in African and Asian Elephants

    Science.gov (United States)

    Tuberculosis (TB) in elephants is a re-emerging zoonotic disease caused primarily by Mycobacterium tuberculosis. Current methods for screening and diagnosis rely on trunk wash culture, which has serious limitations due to low test sensitivity, slow turn-around time, and variable sample quality. Inn...

  16. Determination of oxolinic acid, danofloxacin, ciprofloxacin, and enrofloxacin in porcine and bovine meat by micellar liquid chromatography with fluorescence detection.

    Science.gov (United States)

    Terrado-Campos, David; Tayeb-Cherif, Khaled; Peris-Vicente, Juan; Carda-Broch, Samuel; Esteve-Romero, Josep

    2017-04-15

    A method was developed for the determination of oxolinic acid, danofloxacin, ciprofloxacin and enrofloxacin by micellar liquid chromatography - fluorescence detection in commercial porcine and bovine meat. The samples were ultrasonicated in a micellar solution, free of organic solvent, to extract the analytes, and the supernatant was directly injected. The quinolones were resolved in 0.9998), trueness (89.3-105.1%), precision (<8.3%), decision limit (<12% over the maximum residue limit), detection capability (<21% over the maximum residue limit), ruggedness (<5.6%) and stability. The procedure was rapid, eco-friendly, safe and easy-to-handle.

  17. Gold nanoisland structures integrated in a lab-on-a-chip for plasmonic detection of bovine growth hormone

    Science.gov (United States)

    Ozhikandathil, Jayan; Badilescu, Simona; Packirisamy, Muthukumaran

    2012-07-01

    Three-dimensional gold nanostructures fabricated through a novel convective assembly method are treated thermally to obtain a nanoisland morphology. The new structure is proved to be adequate for the detection of bovine growth hormone, by using an immunoassay method based on the localized surface plasmon resonance band of gold. The nanoisland structures are integrated into a microfluidic device and the spectral measurements are carried out by introducing the device directly in the light beam of a ultraviolet-visible spectrophotometer. The principal motivation for this work is the need for a simple and rapid method of detection of hormone levels in milk and milk products.

  18. Comparison of Sputum Smear Microscopy and Rapid Tuberculosisantibody Detection Test Kits for Diagnosis of Pulmonary Tuberculosis in Abia State, Nigeria

    Institute of Scientific and Technical Information of China (English)

    Emmanuel Olufemi Ekundayo[1; Sam D. Abbey[2; Onuka Okorie[3

    2014-01-01

    The SSM (sputum smear microscopy) and five immunochromatographic tuberculosis antibody detection tests (DiaSpot TB, Spodex TB, SD Rapid TB, Clinotech TB Screen and Precious One-step TB) were compared for diagnosis of active TB at the Leprosy and Tuberculosis Referral Hospital, Uzuakoli, Abia State, Nigeria. Sputum specimens from 150 study participants (male/female ratio, 0.81) were cultured on Lowenstein-Jensen slopes and direct smears were stained by Ziehl-Neelsen technique and examined by light microscopy. Sera were tested for anti-TB antibodies using the rapid TB tests. A total of 91 participants were culture positive, 79 (86.8%) for M. tuberculosis and 12 (13.2%) for nontuberculous mycobacteria. The sensitivity of SSM was 50% (95% CI: 39.0-61.0) and specificity was 92.3% (95% CI: 86.4-98.2) in those culture positive for M. tuberculosis. The sensitivity and specificity of the Rapid TB tests ranged from 24.1-39.2% and 78.4-87.8%, respectively. None of the five rapid TB tests had acceptable level of accuracy for diagnosis of active TB. The sensitivity of SSM though moderate is inadequate for long term TB control in this setting.

  19. Highly sensitive detection of bovine serum albumin based on the aggregation of triangular silver nanoplates

    Science.gov (United States)

    Zhang, Ling Ling; Ma, Fang Fang; Kuang, Yang Fang; Cheng, Shu; Long, Yun Fei; Xiao, Qiu Guo

    2016-02-01

    A simple, fast and highly sensitive spectrophotometric method for the determination of bovine serum albumin (BSA) has been developed based on the interactions between triangular silver nanoplates (TAgNPs) and BSA in the presence of Britton-Robison buffer solution (BR). Particularly, the wavelength of absorption maximum (λmax) of TAgNPs is red shifted in the presence of BSA together with Britton-Robinson buffer solution (BR, pH = 2.56), and the color of the solution changed from blue to light blue. This may be due to the interactions between BSA molecules on the surface of TAgNPs through electrostatic forces, hydrogen bonds, hydrophobic effects and van der Waals forces at pH 2.56, which leads to the aggregation of TAgNPs. The determination of BSA was achieved by measuring the change of λmax corresponding to localized surface plasmon resonance (LSPR) from UV-visible spectrophotometry. It was found that the shift value in the wavelength of absorption maximum (Δλ, the difference in absorption maxima of the TAgNPs/BSA/BR mixture and the TAgNPs/BR mixture) was proportionate to the concentration of BSA in the range of 1.0 ng mL- 1 to 100.0 ng mL- 1 with the correlation coefficient of r = 0.9969. The detection limit (3 σ/k) for BSA was found to be as low as 0.5 ng mL- 1.

  20. Electrochemical biosensor for Mycobacterium tuberculosis DNA detection based on gold nanotubes array electrode platform.

    Science.gov (United States)

    Torati, Sri Ramulu; Reddy, Venu; Yoon, Seok Soo; Kim, CheolGi

    2016-04-15

    The template assisted electrochemical deposition technique was used for the synthesis of gold nanotubes array (AuNTsA). The morphological structure of the synthesized AuNTsA was observed by scanning electron microscopy and found that the individual nanotubes are around 1.5 μm in length with a diameter of 200 nm. Nanotubes are vertically aligned to the Au thick film, which is formed during the synthesis process of nanotubes. The electrochemical performance of the AuNTsA was compared with the bare Au electrode and found that AuNTsA has better electron transfer surface than bare Au electrode which is due to the high surface area. Hence, the AuNTsA was used as an electrode for the fabrication of DNA hybridization biosensor for detection of Mycobacterium Tuberculosis DNA. The DNA hybridization biosensor constructed by AuNTsA electrode was characterized by cyclic voltammetry technique with Fe(CN)6(3-/4-) as an electrochemical redox indicator. The selectivity of the fabricated biosensor was illustrated by hybridization with complementary DNA and non-complementary DNA with probe DNA immobilized AuNTsA electrode using methylene blue as a hybridization indicator. The developed electrochemical DNA biosensor shows good linear range of complementary DNA concentration from 0.01 ng/μL to 100 ng/μL with high detection limit.

  1. Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples.

    Science.gov (United States)

    Cai, Hugh Y; Bell-Rogers, Patricia; Parker, Lois; Prescott, John F

    2005-11-01

    A real-time polymerase chain reaction (PCR) assay using hybridization probes on a LightCycler platform was developed for detection of Mycoplasma bovis from individual bovine mastitis milk and pneumonic lung tissues. The detection limit was 550 colony forming units (cfu)/ml of milk and 650 cfu/25 mg of lung tissue. A panel of bovine Mycoplasma and of other bovine-origin bacteria were tested; only M. bovis strains were positive, with a melting peak of 66.6 degrees C. Mycoplasma agalactiae PG2 was also positive and could be distinguished because it had a melting peak of 63.1 degrees C. In validation testing of clinical samples, the relative sensitivity and specificity were 100% and 99.3% for individual milks and 96.6% and 100% for the lung tissue. Using M. bovis real-time PCR, the M. bovis culture-positive milk samples were estimated to contain between 5 x 10(4) and 7.7 x 10(8) cfu/ml and the M. bovis culture-positive lungs between 1 x 10(3) and 1 x 10(9) cfu/25 mg. Isolation, confirmed with the real-time PCR and colony fluorescent antibody test, showed that at the herd level, the proportion of samples positive for M. bovis isolation in mastitis milk samples submitted to the Mastitis Laboratory, Animal Health Laboratory, University of Guelph, Ontario, Canada, was 2.4% (5/201). We conclude that this probe-based real-time PCR assay is a sensitive, specific, and rapid method to identify M. bovis infection in bovine milk and pneumonic lungs.

  2. Detection of Mycobacterium tuberculosis complex in formalin-fixed, paraffin-embedded tissue specimens with necrotizing granulomatous inflammation by strand displacement amplification

    DEFF Research Database (Denmark)

    Johansen, Isik Somuncu; Thomsen, Vibeke Østergaard; Forsgren, Arne;

    2004-01-01

    Rapid, reliable diagnosis of tuberculosis is essential to initiate correct treatment, avoid severe complications, and prevent transmission. Conventional microbiological methods may not be an option if samples are formalin-fixed and paraffin-embedded (FFPE) for histopathological examination....... With the demonstration of necrotizing granulomatous inflammation, tuberculosis becomes an important differential diagnosis, although it was not initially suspected. Following paraffin extraction, BDProbeTec ET strand displacement amplification for detection of Mycobacterium tuberculosis complex (MTC) was applied to 47...... prospectively and 19 retrospectively collected FFPE samples from various sources with granulomatous inflammation and results were compared to tuberculosis notification. Of the prospective samples, 20 were from patients who were notified as having tuberculosis and the assay was positive in 18 (90%). Specificity...

  3. Detection of Mycobacterium tuberculosis complex in formalin-fixed, paraffin-embedded tissue specimens with necrotizing granulomatous inflammation by strand displacement amplification

    DEFF Research Database (Denmark)

    Johansen, Isik Somuncu; Thomsen, Vibeke Østergaard; Forsgren, Arne;

    2004-01-01

    . With the demonstration of necrotizing granulomatous inflammation, tuberculosis becomes an important differential diagnosis, although it was not initially suspected. Following paraffin extraction, BDProbeTec ET strand displacement amplification for detection of Mycobacterium tuberculosis complex (MTC) was applied to 47......Rapid, reliable diagnosis of tuberculosis is essential to initiate correct treatment, avoid severe complications, and prevent transmission. Conventional microbiological methods may not be an option if samples are formalin-fixed and paraffin-embedded (FFPE) for histopathological examination...... prospectively and 19 retrospectively collected FFPE samples from various sources with granulomatous inflammation and results were compared to tuberculosis notification. Of the prospective samples, 20 were from patients who were notified as having tuberculosis and the assay was positive in 18 (90%). Specificity...

  4. Standardization of a TaqMan-based real-time PCR for the detection of Mycobacterium tuberculosis-complex in human sputum.

    Science.gov (United States)

    Barletta, Francesca; Vandelannoote, Koen; Collantes, Jimena; Evans, Carlton A; Arévalo, Jorge; Rigouts, Leen

    2014-10-01

    Real-time polymerase chain reaction (qPCR) was optimized for detecting Mycobacterium tuberculosis in sputum. Sputum was collected from patients (N = 112) with suspected pulmonary tuberculosis, tested by smear microscopy, decontaminated, and split into equal aliquots that were cultured in Löwenstein-Jensen medium and tested by qPCR for the small mobile genetic element IS6110. The human ERV3 sequence was used as an internal control. 3 of 112 (3%) qPCR failed. For the remaining 109 samples, qPCR diagnosed tuberculosis in 79 of 84 patients with culture-proven tuberculosis, and sensitivity was greater than microscopy (94% versus 76%, respectively, P tuberculosis mycobacteria. The qPCR cost ∼5US$ per sample and provided same-day results compared with 2-6 weeks for culture.

  5. Detection and evaluation of the mutations of embB gene in ethambutol-susceptible and resistant Mycobacterium tuberculosis isolates from China

    Institute of Scientific and Technical Information of China (English)

    WU Xue-qiong; LIANG Jian-qin; ZHANG Jun-xian; LU Yang; LI Hong-min; ZHANG Guang-yu; YAN Guo-rui; DING Bei-chuan

    2005-01-01

    @@ The situation of tuberculosis (TB) and Mycobacterium tuberculosis (M. tuberculosis) drug resistance is still very serious in China. Improper treatment for TB may lead to a prolonged course of antimicrobial therapy and spreading of drug resistant strains. Thus, rapid detection of drug resistant strains will allow prompt initiation and adjustment of effective chemotherapeutic treatment of TB. Ethambutol (EMB) is a first line anti-TB drug. embB Met306 is located in a cytoplasmic loop that forms an EMB resistance determining region,1 Lety et al2 have demonstrated that a missense mutation in the M. smegmatis embB gene confers EMB resistance. The identification of mutations in embB gene may offer a means to rapidly screen M. tuberculosis isolates for EMB resistance. Thus, in this study, we analysed 197 isolates of M. tuberculosis from Chinese people and characterized mutations in the 258 bp region of the embB gene.

  6. Do Instructional Videos on Sputum Submission Result in Increased Tuberculosis Case Detection? A Randomized Controlled Trial.

    Directory of Open Access Journals (Sweden)

    Grace Mhalu

    Full Text Available We examined the effect of an instructional video about the production of diagnostic sputum on case detection of tuberculosis (TB, and evaluated the acceptance of the video.Randomized controlled trial.We prepared a culturally adapted instructional video for sputum submission. We analyzed 200 presumptive TB cases coughing for more than two weeks who attended the outpatient department of the governmental Municipal Hospital in Mwananyamala (Dar es Salaam, Tanzania. They were randomly assigned to either receive instructions on sputum submission using the video before submission (intervention group, n = 100 or standard of care (control group, n = 100. Sputum samples were examined for volume, quality and presence of acid-fast bacilli by experienced laboratory technicians blinded to study groups.Median age was 39.1 years (interquartile range 37.0-50.0; 94 (47% were females, 106 (53% were males, and 49 (24.5% were HIV-infected. We found that the instructional video intervention was associated with detection of a higher proportion of microscopically confirmed cases (56%, 95% confidence interval [95% CI] 45.7-65.9%, sputum smear positive patients in the intervention group versus 23%, 95% CI 15.2-32.5%, in the control group, p <0.0001, an increase in volume of specimen defined as a volume ≥3ml (78%, 95% CI 68.6-85.7%, versus 45%, 95% CI 35.0-55.3%, p <0.0001, and specimens less likely to be salivary (14%, 95% CI 7.9-22.4%, versus 39%, 95% CI 29.4-49.3%, p = 0.0001. Older age, but not the HIV status or sex, modified the effectiveness of the intervention by improving it positively. When asked how well the video instructions were understood, the majority of patients in the intervention group reported to have understood the video instructions well (97%. Most of the patients thought the video would be useful in the cultural setting of Tanzania (92%.Sputum submission instructional videos increased the yield of tuberculosis cases through better quality of sputum

  7. Detection and molecular characterization of 9,000-year-old Mycobacterium tuberculosis from a Neolithic settlement in the Eastern Mediterranean.

    Directory of Open Access Journals (Sweden)

    Israel Hershkovitz

    Full Text Available BACKGROUND: Mycobacterium tuberculosis is the principal etiologic agent of human tuberculosis. It has no environmental reservoir and is believed to have co-evolved with its host over millennia. This is supported by skeletal evidence of the disease in early humans, and inferred from M. tuberculosis genomic analysis. Direct examination of ancient human remains for M. tuberculosis biomarkers should aid our understanding of the nature of prehistoric tuberculosis and the host/pathogen relationship. METHODOLOGY/PRINCIPAL FINDINGS: We used conventional PCR to examine bone samples with typical tuberculosis lesions from a woman and infant, who were buried together in the now submerged site of Atlit-Yam in the Eastern Mediterranean, dating from 9,250-8,160 years ago. Rigorous precautions were taken to prevent contamination, and independent centers were used to confirm authenticity of findings. DNA from five M tuberculosis genetic loci was detected and had characteristics consistent with extant genetic lineages. High performance liquid chromatography was used as an independent method of verification and it directly detected mycolic acid lipid biomarkers, specific for the M. tuberculosis complex. CONCLUSIONS/SIGNIFICANCE: Human tuberculosis was confirmed by morphological and molecular methods in a population living in one of the first villages with evidence of agriculture and animal domestication. The widespread use of animals was not a source of infection but may have supported a denser human population that facilitated transmission of the tubercle bacillus. The similarity of the M. tuberculosis genetic signature with those of today gives support to the theory of a long-term co-existence of host and pathogen.

  8. Molecular detection and characterization of bovine viral diarrhea virus in Mongolian cattle and yaks.

    Science.gov (United States)

    Ochirkhuu, Nyamsuren; Konnai, Satoru; Odbileg, Raadan; Odzaya, Battogtokh; Gansukh, Shura; Murata, Shiro; Ohashi, Kazuhiko

    2016-08-01

    Bovine viral diarrhea virus (BVDV) is classified into two species, namely, Bovine viral diarrhea virus 1 and Bovine viral diarrhea virus 2, and affects cattle worldwide, resulting in significant economic loss. The prevalence of BVDV-1 and BVDV-2 infections and its genotypes in Mongolian animals has not been studied. In this study, we surveyed BVDV infection in dairy cattle and yaks from Bornuur and Bulgan counties by RT-PCR, and the average infection rate in the sampling sites was 15.8 % and 20.0 %, respectively. In addition, molecular features of the 5'-UTR region of the BVDV genome in Mongolian cattle and yaks were identified as belonging to the subtypes BVDV-1a and BVDV-2a, respectively. Determining the prevalence, geographical distribution, and molecular diversity of BVDV-1 and BVDV-2 in various host species in Mongolia is important for further studies and process control programs.

  9. Detection and characterisation of Complement protein activity in bovine milk by bactericidal sequestration assay.

    Science.gov (United States)

    Maye, Susan; Stanton, Catherine; Fitzgerald, Gerald F; Kelly, Philip M

    2015-08-01

    While the Complement protein system in human milk is well characterised, there is little information on its presence and activity in bovine milk. Complement forms part of the innate immune system, hence the importance of its contribution during milk ingestion to the overall defences of the neonate. A bactericidal sequestration assay, featuring a Complement sensitive strain, Escherichia coli 0111, originally used to characterise Complement activity in human milk was successfully applied to freshly drawn bovine milk samples, thus, providing an opportunity to compare Complement activities in both human and bovine milks. Although not identical in response, the levels of Complement activity in bovine milk were found to be closely comparable with that of human milk. Differential counts of Esch. coli 0111 after 2 h incubation were 6.20 and 6.06 log CFU/ml, for raw bovine and human milks, respectively - the lower value representing a stronger Complement response. Exposing bovine milk to a range of thermal treatments e.g. 42, 45, 65, 72, 85 or 95 °C for 10 min, progressively inhibited Complement activity by increasing temperature, thus confirming the heat labile nature of this immune protein system. Low level Complement activity was found, however, in 65 and 72 °C heat treated samples and in retailed pasteurised milk which highlights the outer limit to which high temperature, short time (HTST) industrial thermal processes should be applied if retention of activity is a priority. Concentration of Complement in the fat phase was evident following cream separation, and this was also reflected in the further loss of activity recorded in low fat variants of retailed pasteurised milk. Laboratory-based churning of the cream during simulated buttermaking generated an aqueous (buttermilk) phase with higher levels of Complement activity than the fat phase, thus pointing to a likely association with the milk fat globule membrane (MFGM) layer.

  10. Capillary electrophoresis-single strand conformation polymorphism for the detection of multiple mutations leading to tuberculosis drug resistance.

    Science.gov (United States)

    Krothapalli, Sowmya; May, Michael K; Hestekin, Christa N

    2012-10-01

    Drug resistant tuberculosis (TB) is a major health problem in both developed and developing countries. Mutations in the Mycobacterium (M.) tuberculosis bacterial genome, such as those to the rpoB gene and mabA-inhA promoter region, have been linked to TB drug resistance in against rifampicin and isoniazid, respectively. The rapid, accurate, and inexpensive identification of these and other mutations leading to TB drug resistance is an essential tool for improving human health. Capillary electrophoresis (CE) single strand conformation polymorphism (SSCP) can be a highly sensitive technique for the detection of genetic mutation that has not been previously explored for drug resistance mutations in M. tuberculosis. This work explores the potential of CE-SSCP through the optimization of variables such as polymer separation matrix concentration, capillary wall coating, electric field strength, and temperature on resolution of mutation detection. The successful detection of an rpoB gene mutation and two mabA-inhA promoter region mutations while simultaneously differentiating a TB-causing mycobacteria from a non-TB bacteria was accomplished using the optimum conditions of 4.5% (w/v) PDMA in a PDMA coated capillary at 20°C using a separation voltage of 278 V/cm. This multiplexed analysis that can be completed in a few hours demonstrates the potential of CE-SSCP to be an inexpensive and rapid analysis method.

  11. Direct screening of tetracyclines in water and bovine milk using room temperature phosphorescence detection

    Energy Technology Data Exchange (ETDEWEB)

    Traviesa-Alvarez, J.M. [Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, c/Julian Claveria 8, 33006 Oviedo (Spain); Costa-Fernandez, J.M. [Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, c/Julian Claveria 8, 33006 Oviedo (Spain); Pereiro, R. [Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, c/Julian Claveria 8, 33006 Oviedo (Spain); Sanz-Medel, A. [Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, c/Julian Claveria 8, 33006 Oviedo (Spain)]. E-mail: asm@uniovi.es

    2007-04-18

    A fast and simple flow-through optosensor was designed and characterized for the direct screening of four tetracycline (TCC) antibiotics (tetracycline, oxytetracycline, chlortetracycline and doxycycline) in water and bovine milk samples. The proposed optosensor provides rapid binary yes/no overall responses, being appropriate for the screening of this family of antibiotics above or below a pre-set concentration threshold. The experimental set-up is based on a flow-injection manifold coupled on-line to a phosphorescence detector. Aliquots of the samples are pretreated with Eu(III) to form room temperature phosphorescent metal chelates and injected in the flow manifold. Those chelates are then on-line retained on a conventional flow-cell (packed with polymeric Amberlite XAD-4 particles) which is placed inside the cell holder of the phosphorimeter. After the emission is registered, the antibiotic-metal complexes are eluted from the packed resin with 1 M HCl (for milk samples a second regeneration step, using methanol, should be performed). A sample throughput of about 20 samples per hour was obtained. Optimum experimental conditions include a pH 9, a Eu(III) concentration of 2 x 10{sup -4} M and 8 mM sodium sulphite as chemical deoxygenant. The phosphorescence emitted by the europium-TCC complexes was measured at 394 and 617 nm for excitation and emission wavelengths, respectively. The unreliability region, given by the probability of false positives and false negatives, respectively (set at 5% in both cases) was in the range between 0.2 and 11.6 nM for detection of tetracyclines in water samples (at a cut-off level of 4 nM) and in the range between 165 and 238 nM for detection of tetracyclines in milk (cut-off level fixed at the normative EU level of 200 nM). Finally, the applicability of the proposed screening optosensor was tested for the reliable control of tetracyclines in contaminated and uncontaminated water and milk samples.

  12. Development of a sandwich Dot-ELISA for detecting bovine viral diarrhea virus antigen with E2 recombinant protein

    Institute of Scientific and Technical Information of China (English)

    Yuelan ZHAO; Yuzhu ZUO; Lei ZHANG; Jinghui FAN; Hanchun YANG; Jianhua QIN

    2009-01-01

    The IgG antibodies of rabbit anti-E2 protein of the bovine viral diarrhea virus were prepared by a general method from high efficiency serum immunized by E2 recombinant protein antigen expressed in E. coli prokaryotic expression system and were labeled to make enzymelabeled antibody with the method of NaIO4. A sandwich Dot enzyme-linked immunosorbent assay (Dot-ELISA) for the detection of BVDV was developed. The optimal reaction conditions of Dot-ELISAwere determined. The results show that optimal coating antibody was 300 μg·mL-1, the working concentration of HRP-labeled antibody was 1:50. The optimal blocking reagent and time were 5% bovine serum and 45 rain. The minimum detection of the content of antigen reached 1.35μg·mL-1. Compared with the routine IDEXX ELISA test kit with the whole virus, its specificity, sensitivity and coincidence rate were 90.48%, 96.55% and 95.24%, respectively. Compared with the sandwich Dot-ELISA with the negative staining electron microscope and RT-PCR, the coincidence rates were 90.9% and 93.1%, respectively. In addition, Bovine viral diarrhea virus (BVDV) antigen of 178 samples collected from cow farms in the Hebei Province, China, were detected by the developed Dot-ELISA and the IDEXX BVDV antigen Test Kit simultaneously, BVDV antigen positive rate was 39.89%-41.01%. The result of detecting clinical samples demonstrated that the established method showed its specificity, sensitivity and repeatability, whereas the results were easily interpreted without an ELISA reader.

  13. A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots

    Science.gov (United States)

    Gliddon, H. D.; Howes, P. D.; Kaforou, M.; Levin, M.; Stevens, M. M.

    2016-05-01

    The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis.The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar

  14. An easy and sensitive sandwich assay for detection of Mycobacterium tuberculosis Ag85B antigen using quantum dots and gold nanorods.

    Science.gov (United States)

    Kim, Eun Ju; Kim, Eun Bee; Lee, Seung Woo; Cheon, Seon Ah; Kim, Hwa-Jung; Lee, Jaebeom; Lee, Mi-Kyung; Ko, Sungho; Park, Tae Jung

    2017-01-15

    Mycobacterium tuberculosis is a serious global infectious pathogen causing tuberculosis (TB). The development of an easy and sensitive method for the detection of M. tuberculosis is in urgent need due to complex and low specificity of the current assays. Herein, we present a novel method for M. tuberculosis detection based on a sandwich assay via antigen-antibody interaction using silica-coated quantum dots (SiQDs) and gold nanorods (AuNRs). A genetically engineered recombinant antibody (GBP-50B14 and SiBP-8B3) was bound to surfaces of AuNRs and SiQDs respectively, without any surface modification. The antigen-antibody interaction was revealed using M. tuberculosis-specific secretory antigen, Ag85B. Two biocomplexes showed a quenching effect in the presence of the target antigen through a sandwich assay. The assay response was in the range of 1×10(-3)-1×10(-10)μgmL(-1) (R=0.969) and the limit of detection for Ag85B was 13.0pgmL(-1). The Ag85B was selectively detected using three different proteins (CFP10, and BSA), and further specifically confirmed by the use of spiked samples. Compared with existing methods, a highly sensitive and selective method for Ag85B-expressing M. tuberculosis detection has been developed for better diagnosis of TB.

  15. Comparison of histopathology and real-time polymerase chain reaction (RT-PCR) for detection of Mycobacterium tuberculosis in fistula-in-ano.

    Science.gov (United States)

    Garg, Pankaj

    2017-07-01

    Histopathology is commonly used to diagnose tuberculosis in fistula-in-ano. The aim was to compare the sensitivity of polymerase chain reaction and histopathology in detecting tuberculosis in fistula-in-ano. The histopathology and polymerase chain-reaction of tissue (fistula tract) was done in all the consecutive operated cases. When pus sample was also available, polymerase chain reaction-pus was also done RESULTS: Three hundred forty seven samples (179 patients) were tested over 2 years (median 6.5 months). The mean age was 38.8 ± 10.7 years, and male/female was 170/9. Histopathology and polymerase chain reaction of tissue (fistula tract) was done in 152 and 165 patients, respectively. Polymerase chain reaction (pus) could be done in 30 patients. Overall, tuberculosis was detected in 20/179 (11.2%) patients. Of these, tuberculosis was detected by histopathology (tissue) in 1/152 (0.7%) and by polymerase chain reaction (tissue) in 14/165 (8.5%) patients. In pus, polymerase chain reaction detected tuberculosis in 6/30 (20%) patients. Both polymerase chain reaction of tissue and pus were positive in one patient. Polymerase chain reaction (tissue) and polymerase chain reaction (pus) were significantly more sensitive than histopathology (tissue) for detecting tuberculosis [histopathology 1/152 vs. polymerase chain reaction (tissue) 14/165, p = 0.0009] [histopathology 1/152 vs. polymerase chain reaction (pus) 6/30, p Polymerase chain reaction was significantly more sensitive than histopathology in detecting tuberculosis in fistula-in-ano. Histopathology might be missing out tuberculosis in many patients leading to recurrence of the fistula.

  16. Tuberculosis in wild and domestic animals in South Africa

    NARCIS (Netherlands)

    Michel, A.L.

    2008-01-01

    Bovine tuberculosis is an endemic disease with a low prevalence in South African cattle. This is mostly the result of a national bovine tuberculosis control scheme which has been in place for nearly 40 years and has prevented outbreaks from spreading and causing large-scale losses, thereby also mini

  17. Detection of Mycobacterium tuberculosis based on H37R(v) binding peptides using surface functionalized magnetic microspheres coupled with quantum dots – a nano detection method for Mycobacterium tuberculosis.

    Science.gov (United States)

    Yang, Hua; Qin, Lianhua; Wang, Yilong; Zhang, Bingbo; Liu, Zhonghua; Ma, Hui; Lu, Junmei; Huang, Xiaochen; Shi, Donglu; Hu, Zhongyi

    2015-01-01

    Despite suffering from the major disadvantage of low sensitivity, microscopy of direct smear with the Ziehl-Neelsen stain is still broadly used for detection of acid-fast bacilli and diagnosis of tuberculosis. Here, we present a unique detection method of Mycobacterium tuberculosis (MTB) using surface functionalized magnetic microspheres (MMSs) coupled with quantum dots (QDs), conjugated with various antibodies and phage display-derived peptides. The principle is based upon the conformation of the sandwich complex composed of bacterial cells, MMSs, and QDs. The complex system is tagged with QDs for providing the fluorescent signal as part of the detection while magnetic separation is achieved by MMSs. The peptide ligand H8 derived from the phage display library Ph.D.-7 is developed for MTB cells. Using the combinations of MMS-polyclonal antibody+QD-H8 and MMS-H8+QD-H8, a strong signal of 10(3) colony forming units (CFU)/mL H37R(v) was obtained with improved specificity. MS-H8+QD-H8 combination was further optimized by adjusting the concentrations of MMSs, QDs, and incubation time for the maximum detection signal. The limit of detection for MTB was found to reach 10(3) CFU/mL even for the sputum matrices. Positive sputum samples could be distinguished from control. Thus, this novel method is shown to improve the detection limit and specificity of MTB from the sputum samples, and to reduce the testing time for accurate diagnosis of tuberculosis, which needs further confirmation of more clinical samples.

  18. A Nonsynonymous SNP Catalog of Mycobacterium tuberculosis Virulence Genes and Its Use for Detecting New Potentially Virulent Sublineages

    Science.gov (United States)

    Mikheecheva, Natalya E.; Zaychikova, Marina V.; Melerzanov, Alexander V.

    2017-01-01

    Mycobacterium tuberculosis is divided into several distinct lineages, and various genetic markers such as IS-elements, VNTR, and SNPs are used for lineage identification. We propose an M. tuberculosis classification approach based on functional polymorphisms in virulence genes. An M. tuberculosis virulence genes catalog has been established, including 319 genes from various protein groups, such as proteases, cell wall proteins, fatty acid and lipid metabolism proteins, sigma factors, toxin–antitoxin systems. Another catalog of 1,573 M. tuberculosis isolates of different lineages has been developed. The developed SNP-calling program has identified 3,563 nonsynonymous SNPs. The constructed SNP-based phylogeny reflected the evolutionary relationship between lineages and detected new sublineages. SNP analysis of sublineage F15/LAM4/KZN revealed four lineage-specific mutations in cyp125, mce3B, vapC25, and vapB34. The Ural lineage has been divided into two geographical clusters based on different SNPs in virulence genes. A new sublineage, B0/N-90, was detected inside the Beijing-B0/W-148 by SNPs in irtB, mce3F and vapC46. We have found 27 members of B0/N-90 among the 227 available genomes of the Beijing-B0/W-148 sublineage. Whole-genome sequencing of strain B9741, isolated from an HIV-positive patient, was demonstrated to belong to the new B0/N-90 group. A primer set for PCR detection of B0/N-90 lineage-specific mutations has been developed. The prospective use of mce3 mutant genes as genetically engineered vaccine is discussed. PMID:28338924

  19. A Nonsynonymous SNP Catalog of Mycobacterium tuberculosis Virulence Genes and Its Use for Detecting New Potentially Virulent Sublineages.

    Science.gov (United States)

    Mikheecheva, Natalya E; Zaychikova, Marina V; Melerzanov, Alexander V; Danilenko, Valery N

    2017-04-01

    Mycobacterium tuberculosis is divided into several distinct lineages, and various genetic markers such as IS-elements, VNTR, and SNPs are used for lineage identification. We propose an M. tuberculosis classification approach based on functional polymorphisms in virulence genes. An M. tuberculosis virulence genes catalog has been established, including 319 genes from various protein groups, such as proteases, cell wall proteins, fatty acid and lipid metabolism proteins, sigma factors, toxin-antitoxin systems. Another catalog of 1,573 M. tuberculosis isolates of different lineages has been developed. The developed SNP-calling program has identified 3,563 nonsynonymous SNPs. The constructed SNP-based phylogeny reflected the evolutionary relationship between lineages and detected new sublineages. SNP analysis of sublineage F15/LAM4/KZN revealed four lineage-specific mutations in cyp125, mce3B, vapC25, and vapB34. The Ural lineage has been divided into two geographical clusters based on different SNPs in virulence genes. A new sublineage, B0/N-90, was detected inside the Beijing-B0/W-148 by SNPs in irtB, mce3F and vapC46. We have found 27 members of B0/N-90 among the 227 available genomes of the Beijing-B0/W-148 sublineage. Whole-genome sequencing of strain B9741, isolated from an HIV-positive patient, was demonstrated to belong to the new B0/N-90 group. A primer set for PCR detection of B0/N-90 lineage-specific mutations has been developed. The prospective use of mce3 mutant genes as genetically engineered vaccine is discussed. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  20. Role of digital tomosynthesis and dual energy subtraction digital radiography in detection of parenchymal lesions in active pulmonary tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Madhurima, E-mail: madhurimashrm88@gmail.com [Department of Radiodiagnosis and Imaging, PGIMER, Chandigarh 160012 (India); Sandhu, Manavjit Singh, E-mail: manavjitsandhu@yahoo.com [Department of Radiodiagnosis and Imaging, PGIMER, Chandigarh 160012 (India); Gorsi, Ujjwal, E-mail: ujjwalgorsi@gmail.com [Department of Radiodiagnosis and Imaging, PGIMER, Chandigarh 160012 (India); Gupta, Dheeraj, E-mail: dheeraj1910@gmail.com [Department of Pulmonary Medicine, PGIMER, Chandigarh 160012 (India); Khandelwal, Niranjan, E-mail: khandelwaln@hotmail.com [Department of Radiodiagnosis and Imaging, PGIMER, Chandigarh 160012 (India)

    2015-09-15

    Highlights: • Digital tomosynthesis and dual energy subtraction digital radiography are modifications of digital radiography. • These modalities perform better than digital radiography in detection of parenchymal lesions in active pulmonary tuberculosis. • Digital tomosynthesis has a sensitivity of 100% in detection of cavities. • Centrilobular nodules seen on CT in active pulmonary tuberculosis, were also demonstrated on digital tomosynthesis in our study. • Digital tomosynthesis can be used for diagnosis and follow up of patients in pulmonary tuberculosis, thereby reducing the number of CT examinations. - Abstract: Objective: To assess the role of digital tomosynthesis (DTS) and dual energy subtraction digital radiography (DES-DR) in detection of parenchymal lesions in active pulmonary tuberculosis (TB) and to compare them with digital radiography (DR). Materials and methods: This prospective study was approved by our institutional review committee. DTS and DES-DR were performed in 62 patients with active pulmonary TB within one week of multidetector computed tomography (MDCT) study. Findings of active pulmonary TB, that is consolidation, cavitation and nodules were noted on digital radiography (DR), DTS and DES-DR in all patients. Sensitivity, specificity, positive and negative predictive values of all 3 modalities was calculated with MDCT as reference standard. In addition presence of centrilobular nodules was also noted on DTS. Results: Our study comprised of 62 patients (33 males, 29 females with age range 18–82 years). Sensitivity and specificity of DTS for detection of nodules and cavitation was better than DR and DES-DR. Sensitivity and specificity of DTS for detection of consolidation was comparable to DR and DES-DR. DES-DR performed better than DR in detection of nodules and cavitation. DTS was also able to detect centrilobular nodules with sensitivity and specificity of 57.4% and 86.5% respectively. Conclusion: DTS and DES-DR perform better

  1. Multiplex real-time PCR melting curve assay to detect drug-resistant mutations of Mycobacterium tuberculosis.

    Science.gov (United States)

    Luo, Tao; Jiang, Lili; Sun, Weiming; Fu, G; Mei, Jian; Gao, Qian

    2011-09-01

    Early diagnosis of drug-resistant Mycobacterium tuberculosis is urgently needed to optimize treatment regimens and to prevent the transmission of resistant strains. Real-time PCR assays have been developed to detect drug resistance rapidly, but none of them have been widely applied due to their complexity, high cost, or requirement for advanced instruments. In this study, we developed a real-time PCR method based on melting curve analysis of dually labeled probes. Six probes targeting the rpoB 81-bp core region, katG315, the inhA promoter, the ahpC promoter, and embB306 were designed and validated with clinical isolates. First, 10 multidrug-resistant (MDR) strains with a wide mutation spectrum were used to analyze the melting temperature (T(m)) deviations of different mutations by single real-time PCR. All mutations can be detected by significant T(m) reductions compared to the wild type. Then, three duplex real-time PCRs, with two probes in each, were developed to detect mutations in 158 MDR isolates. Comparison of the results with the sequencing data showed that all mutations covered by the six probes were detected with 100% sensitivity and 100% specificity. Our method provided a new way to rapidly detect drug-resistant mutations in M. tuberculosis. Compared to other real-time PCR methods, we use fewer probes, which are labeled with the same fluorophore, guaranteeing that this assay can be used for detection in a single fluorescent channel or can be run on single-channel instruments. In conclusion, we have developed a widely applicable real-time PCR assay to detect drug-resistant mutations in M. tuberculosis.

  2. Evaluation of the MeltPro TB/STR assay for rapid detection of streptomycin resistance in Mycobacterium tuberculosis.

    Science.gov (United States)

    Zhang, Ting; Hu, Siyu; Li, Guoli; Li, Hui; Liu, Xiaoli; Niu, Jianjun; Wang, Feng; Wen, Huixin; Xu, Ye; Li, Qingge

    2015-03-01

    Rapid and comprehensive detection of drug-resistance is essential for the control of tuberculosis, which has facilitated the development of molecular assays for the detection of drug-resistant mutations in Mycobacterium tuberculosis. We hereby assessed the analytical and clinical performance of an assay for streptomycin-resistant mutations. MeltPro TB/STR is a closed-tube, dual-color, melting curve analysis-based, real-time PCR test designed to detect 15 streptomycin-resistant mutations in rpsL 43, rpsL 88, rrs 513, rrs 514, rrs 517, and rrs 905-908 of M. tuberculosis. Analytical studies showed that the accuracy was 100%, the limit of detection was 50-500 bacilli per reaction, the reproducibility in the form of Tm variation was within 1.0 °C, and we could detect 20% STR resistance in mixed bacterial samples. The cross-platform study demonstrated that the assay could be performed on six models of real-time PCR instruments. A multicenter clinical study was conducted using 1056 clinical isolates, which were collected from three geographically different healthcare units, including 709 STR-susceptible and 347 STR-resistant isolates characterized on Löwenstein-Jensen solid medium by traditional drug susceptibility testing. The results showed that the clinical sensitivity and specificity of the MeltPro TB/STR was 88.8% and 95.8%, respectively. Sequencing analysis confirmed the accuracy of the mutation types. Among all the 8 mutation types detected, rpsL K43R (AAG → AGG), rpsL K88R (AAG → AGG) and rrs 514 A → C accounted for more than 90%. We concluded that MeltPro TB/STR represents a rapid and reliable assay for the detection of STR resistance in clinical isolates.

  3. A Fluorescent Probe for Detecting Mycobacterium tuberculosis and Identifying Genes Critical for Cell Entry

    Directory of Open Access Journals (Sweden)

    Dong Yang

    2016-12-01

    Full Text Available ABSTRACTThe conventional method for quantitating Mycobacterium tuberculosis (Mtb in vitro and in vivo relies on bacterial colony forming unit (CFU enumeration on agar plates. Due to the slow growth rate of Mtb, it takes 3-6 weeks to observe visible colonies on agar plates. Imaging technologies that are capable of quickly quantitating both active and dormant tubercle bacilli in vitro and in vivo would accelerate research towards the development of anti-TB chemotherapies and vaccines. We have developed a fluorescent probe that can directly label the Mtb cell wall components. The fluorescent probe, designated as DLF-1, has a strong affinity to the D-Ala-D-Ala unit of the late peptidoglycan intermediates in the bacterial cell wall. We demonstrate that DLF-1 is capable of detecting Mtb in both the active replicating and dormant states in vitro at 100 nM without inhibiting bacterial growth. The DLF-1 fluorescence signal correlated well with CFU of the labeled bacteria (R2=1 and 0.99 for active replicating and dormant Mtb, respectively. DLF-1 can also quantitate labeled Mtb inside of cells. The utility of DLF-1 probe to quantitate Mtb was also successfully applied to identify genes critical for cell invasion. In conclusion, this novel near infrared imaging probe provides a powerful new tool for enumerating Mtb with potential future use in bacterial virulence study.

  4. Detection of tuberculosis using digital chest radiography: automated reading vs. interpretation by clinical officers.

    Science.gov (United States)

    Maduskar, P; Muyoyeta, M; Ayles, H; Hogeweg, L; Peters-Bax, L; van Ginneken, B

    2013-12-01

    A busy urban health centre in Lusaka, Zambia. To compare the accuracy of automated reading (CAD4TB) with the interpretation of digital chest radiograph (CXR) by clinical officers for the detection of tuberculosis (TB). A retrospective analysis was performed on 161 subjects enrolled in a TB specimen bank study. CXRs were analysed using CAD4TB, which computed an image abnormality score (0-100). Four clinical officers scored the CXRs for abnormalities consistent with TB. We compared the automated readings and the readings by clinical officers against the bacteriological and radiological results used as reference. We report here the area under the receiver operating characteristic curve (AUC) and kappa (κ) statistics. Of 161 enrolled subjects, 97 had bacteriologically confirmed TB and 120 had abnormal CXR. The AUCs for CAD4TB and the clinical officers were respectively 0.73 and 0.65-0.75 in comparison with the bacteriological reference, and 0.91 and 0.89-0.94 in comparison with the radiological reference. P values indicated no significant differences, except for one clinical officer who performed significantly worse than CAD4TB (P < 0.05) using the bacteriological reference. κ values for CAD4TB and clinical officers with radiological reference were respectively 0.61 and 0.49-0.67. CXR assessment using CAD4TB and by clinical officers is comparable. CAD4TB has potential as a point-of-care test and for the automated identification of subjects who require further examinations.

  5. Lymphocyte proliferation to mycobacterial antigens is detectable across a spectrum of HIV-associated tuberculosis

    Directory of Open Access Journals (Sweden)

    Bakari Muhammad

    2009-02-01

    Full Text Available Abstract Background Identifying novel TB diagnostics is a major public health priority. We explored the diagnostic characteristics of antimycobacterial lymphocyte proliferation assays (LPA in HIV-infected subjects with latent or active TB. Methods HIV-infected subjects with bacille Calmette Guérin (BCG scars and CD4 counts ≥ 200 cells/mm3 entering a TB booster vaccine trial in Tanzania had baseline in vivo and in vitro immune tests performed: tuberculin skin tests (TST, LPA and five day assays of interferon gamma (IFN-γ release. Assay antigens were early secreted antigenic target 6 (ESAT-6, antigen 85 (Ag85, and Mycobacterium tuberculosis whole cell lysate (WCL. Subjects were screened for active TB at enrollment by history, exam, sputum smear and culture. We compared antimycobacterial immune responses between subjects with and without latent or active TB at enrollment. Results Among 1885 subjects screened, 635 had latent TB and 13 had active TB. Subjects with latent TB were more likely than subjects without TB to have LPA responses to ESAT-6 (13.2% vs. 5.5%, P Conclusion Lymphoproliferative responses to mycobacteria are detectable during HIV-associated active TB, and are less sensitive but more specific than TST. Trial registration ClinicalTrials.gov Identifier NCT00052195.

  6. [Comparison of methods of detection of bovine adenovirus serotype 3 in infected culture of calf kidney cells (MDBK)].

    Science.gov (United States)

    Kaliuzhnaia, A N; Trifonov, V D; Zil'berman, M I; Zalmanzon, E S; Kaledin, A S

    1988-10-01

    The comparative study of the dynamics of the main antigen (hexon) and viral DNA of the bovine adenovirus type 3 accumulation in the established cell line MDBK under the conditions of single- or multistep cycle of infection has been undertaken. The quantitative immunoelectrophoresis and immunoenzyme assay detected the viral antigens on the late stages of infection in the period of cellular monolayer degradation. The immunofluorescence reaction and histochemical immunoenzyme method detected the antigen in the infected cells concurrently with the primary expression of the viral cytopathic effect. The reaction of the spot molecular hybridization with the [32P]-DNA probes detected the viral DNA considerably earlier than the antigen was detected by the immunological methods, before the appearance of degenerative changes in the infected cells. Preference of the immunoenzyme assay and DNA-probes in diagnosis of the virus are discussed.

  7. Subgrouping of bovine respiratory syncytial virus strains detected in lung tissue

    NARCIS (Netherlands)

    Schrijver, R.S.; Daus, E.; Kramps, J.A.; Langedijk, J.P.M.; Buijs, R.; Middel, W.G.J.; Taylor, G.; Furze, J.; Huyben, M.W.C.; Oirschot, van J.T.

    1996-01-01

    Bovine respiratory syncytial virus is an important respiratory pathogen in cattle. Recently, subgroups of BRSV have been identified, based on antigenic differences. However, little is known about subgroups of BRSV that circulate in the cattle population. Therefore, we determined the reactivity of

  8. Protein biomarker-based screening for detection of recombinant bovine somatotropin abuse in dairy cows

    NARCIS (Netherlands)

    Ludwig, S.K.J.

    2014-01-01

    Recombinant bovine somatotropin (rbST) is a 22 kDa proteohormone, which can be used to increase milk production in dairy cows. It has been marketed since 1994 and while its use in food production is approved in several countries, such as the US, it is banned in the EU since 2000. To enforce the ban

  9. Genetic diversity of Brazilian bovine pestiviruses detected between 1995 and 2014

    Science.gov (United States)

    Pestivirus infections in ruminants result in significant economic losses worldwide. The etiological agents are three species from the genus Pestivirus, family Flaviviridae, including Bovine Viral Diarrhea Virus type 1 (BVDV-1), BVDV-2, Border Disease Virus (BDV), and an atypical pestivirus named HoB...

  10. Schmallenberg virus detection in bovine semen after experimental infection of bulls.

    NARCIS (Netherlands)

    Poel, van der W.H.M.; Parlevliet, J.M.; Verstraten, E.R.A.M.; Kooi, E.A.; Hakze-van der Honing, van der R.W.; Stockhofe-Zurwieden, N.

    2014-01-01

    To study Schmallenberg virus (SBV) excretion in bovine semen after experimental infection, two bulls were inoculated subcutaneously with a SBV isolate (1 ml Vero cell culture 106 TCID50). After inoculation (at day 0), semen was collected daily from both animals for 21 days and samples were tested fo

  11. Protein biomarker-based screening for detection of recombinant bovine somatotropin abuse in dairy cows

    NARCIS (Netherlands)

    Ludwig, S.K.J.

    2014-01-01

    Recombinant bovine somatotropin (rbST) is a 22 kDa proteohormone, which can be used to increase milk production in dairy cows. It has been marketed since 1994 and while its use in food production is approved in several countries, such as the US, it is banned in the EU since 2000. To enforce the ban

  12. Novel real-time simultaneous amplification and testing method to accurately and rapidly detect Mycobacterium tuberculosis complex.

    Science.gov (United States)

    Cui, Zhenling; Wang, Yongzhong; Fang, Liang; Zheng, Ruijuan; Huang, Xiaochen; Liu, Xiaoqin; Zhang, Gang; Rui, Dongmei; Ju, Jinliang; Hu, Zhongyi

    2012-03-01

    The aim of this study was to establish and evaluate a simultaneous amplification and testing method for detection of the Mycobacterium tuberculosis complex (SAT-TB assay) in clinical specimens by using isothermal RNA amplification and real-time fluorescence detection. In the SAT-TB assay, a 170-bp M. tuberculosis 16S rRNA fragment is reverse transcribed to DNA by use of Moloney murine leukemia virus (M-MLV) reverse transcriptase, using specific primers incorporating the T7 promoter sequence, and undergoes successive cycles of amplification using T7 RNA polymerase. Using a real-time PCR instrument, hybridization of an internal 6-carboxyfluorescein-4-[4-(dimethylamino)phenylazo] benzoic acid N-succinimidyl ester (FAM-DABCYL)-labeled fluorescent probe can be used to detect RNA amplification. The SAT-TB assay takes less than 1.5 h to perform, and the sensitivity of the assay for detection of M. tuberculosis H37Rv is 100 CFU/ml. The TB probe has no cross-reactivity with nontuberculous mycobacteria or other common respiratory tract pathogens. For 253 pulmonary tuberculosis (PTB) specimens and 134 non-TB specimens, the SAT-TB results correlated with 95.6% (370/387 specimens) of the Bactec MGIT 960 culture assay results. The sensitivity, specificity, and positive and negative predictive values of the SAT-TB test for the diagnosis of PTB were 67.6%, 100%, 100%, and 62.0%, respectively, compared to 61.7%, 100%, 100%, and 58.0% for Bactec MGIT 960 culture. For PTB diagnosis, the sensitivities of the SAT-TB and Bactec MGIT 960 culture methods were 97.6% and 95.9%, respectively, for smear-positive specimens and 39.2% and 30.2%, respectively, for smear-negative specimens. In conclusion, the SAT-TB assay is a novel, simple test with a high specificity which may enhance the detection rate of TB. It is therefore a promising tool for rapid diagnosis of M. tuberculosis infection in clinical microbiology laboratories.

  13. Value of the polymerase chain reaction method for detecting tuberculosis in the bronchial tissue involved by anthracosis.

    Science.gov (United States)

    Mirsadraee, Majid; Shafahie, Ahmad; Reza Khakzad, Mohammad; Sankian, Mojtaba

    2014-04-01

    Anthracofibrosis is the black discoloration of the bronchial mucosa with deformity and obstruction. Association of this disease with tuberculosis (TB) was approved. The objective of this study was to find the additional benefit of assessment of TB by the polymerase chain reaction (PCR) method. Bronchoscopy was performed on 103 subjects (54 anthracofibrosis and 49 control subjects) who required bronchoscopy for their pulmonary problems. According to bronchoscopic findings, participants were classified to anthracofibrosis and nonanthracotic groups. They were examined for TB with traditional methods such as direct smear (Ziehl-Neelsen staining), Löwenstein-Jensen culture, and histopathology and the new method "PCR" for Mycobacterium tuberculosis genome (IS6110). Age, sex, smoking, and clinical findings were not significantly different in the TB and the non-TB groups. Acid-fast bacilli could be detected by a direct smear in 12 (25%) of the anthracofibrosis subjects, and adding the results of culture and histopathology traditional tests indicated TB in 27 (31%) of the cases. Mycobacterium tuberculosis was diagnosed by PCR in 18 (33%) patients, but the difference was not significant. Detection of acid-fast bacilli in control nonanthracosis subjects was significantly lower (3, 6%), but PCR (20, 40%) and accumulation of results from all traditional methods (22, 44%) showed a nonsignificant difference. The PCR method showed a result equal to traditional methods including accumulation of smear, culture, and histopathology.

  14. Evaluation of a commercial enzyme-linked immunosorbent assay for the diagnosis of bovine tuberculosis from milk samples from dairy cows.

    Science.gov (United States)

    Buddle, Bryce M; Wilson, Tania; Luo, Dongwen; Voges, Hinrich; Linscott, Richard; Martel, Edmond; Lawrence, John C; Neill, Mark A

    2013-12-01

    Milk samples from dairy cows provide a ready source of material for measuring antibody responses to Mycobacterium bovis antigens. In this study, we evaluated the IDEXX enzyme-linked immunosorbent assay (ELISA) for the measurement of antibody responses to M. bovis antigens MPB70 and MPB83 in milk samples from New Zealand cattle. Test sensitivities for individual milk and serum samples were assessed in samples collected from 44 M. bovis-infected cows, and test specificities were assessed in milk samples collected from 356 cows from tuberculosis (TB)-free herds. Milk vat samples were collected from 505 herds from regions with relatively high or low prevalences of infection. The ELISA had a sensitivity of 50% and a specificity of 97.5% for milk samples, and the test sensitivities for milk and serum samples were the same. Dilution of the positive test milk samples in milk from noninfected cows at 1/10, 1/20, and 1/50 dilutions reduced the proportions of positive responses to 13/21, 9/21, and 4/21, respectively. Small differences were observed in the ELISA responses of milk samples from individual TB-free cows collected at different times during lactation. No significant differences were detected in the ELISA responses of milk vat samples collected from infected and noninfected herds. This study shows that milk samples can be substituted for serum samples for screening individual cows for M. bovis infection, and pooling of milk samples from 10 to 20 animals can result in a reduction in the sensitivity by approximately 50%. However, screening of milk vat samples is unlikely to be useful in countries with low prevalences of M. bovis in cattle and large herd sizes.

  15. Improved detection of Mycobacterium bovis infection in bovine lymph node tissue using immunomagnetic separation (IMS-based methods.

    Directory of Open Access Journals (Sweden)

    Linda D Stewart

    Full Text Available Immunomagnetic separation (IMS can selectively isolate and concentrate Mycobacterium bovis cells from lymph node tissue to facilitate subsequent detection by PCR (IMS-PCR or culture (IMS-MGIT. This study describes application of these novel IMS-based methods to test for M. bovis in a survey of 280 bovine lymph nodes (206 visibly lesioned (VL, 74 non-visibly lesioned (NVL collected at slaughter as part of the Northern Ireland bovine TB eradication programme. Their performance was evaluated relative to culture. Overall, 174 (62.1% lymph node samples tested positive by culture, 162 (57.8% by IMS-PCR (targeting IS6110, and 191 (68.2% by IMS-MGIT culture. Twelve (6.9% of the 174 culture positive lymph node samples were not detected by either of the IMS-based methods. However, an additional 79 M. bovis positive lymph node samples (27 (13.1% VL and 52 (70.3% NVL were detected by the IMS-based methods and not by culture. When low numbers of viable M. bovis are present in lymph nodes (e.g. in NVLs of skin test reactor cattle decontamination prior to culture may adversely affect viability, leading to false negative culture results. In contrast, IMS specifically captures whole M. bovis cells (live, dead or potentially dormant which are not subject to any deleterious treatment before detection by PCR or MGIT culture. During this study only 2.7% of NVL lymph nodes tested culture positive, whereas 70.3% of the same samples tested M. bovis positive by the IMS-based tests. Results clearly demonstrate that not only are the IMS-based methods more rapid but they have greater detection sensitivity than the culture approach currently used for the detection of M. bovis infection in cattle. Adoption of the IMS-based methods for lymph node testing would have the potential to improve M. bovis detection in clinical samples.

  16. Development of real-time immuno-PCR for the quantitative detection of mycobacterial PstS1 in tuberculosis patients.

    Science.gov (United States)

    Sharma, Suman; Raj, Ankush; Singh, Netrapal; Dahiya, Bhawna; Sheoran, Abhishek; Gupta, Krishna B; Mehta, Promod K

    2017-01-01

    A novel indirect real-time immuno-polymerase chain reaction (RT-I-PCR) assay, an evolution of I-PCR, was developed for the quantitative detection of Mycobacterium tuberculosis PstS1 (Rv0934) with a wide dynamic range of 10ng/mL to 1pg/mL in body fluids of tuberculosis (TB) patients, which may monitor the dynamics of disease. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Use of a molecular diagnostic test in AFB smear positive tuberculosis suspects greatly reduces time to detection of multidrug resistant tuberculosis.

    Directory of Open Access Journals (Sweden)

    Nestani Tukvadze

    Full Text Available BACKGROUND: The WHO has recommended the implementation of rapid diagnostic tests to detect and help combat M/XDR tuberculosis (TB. There are limited data on the performance and impact of these tests in field settings. METHODS: The performance of the commercially available Genotype MTBDRplus molecular assay was compared to conventional methods including AFB smear, culture and drug susceptibility testing (DST using both an absolute concentration method on Löwenstein-Jensen media and broth-based method using the MGIT 960 system. Sputum specimens were obtained from TB suspects in the country of Georgia who received care through the National TB Program. RESULTS: Among 500 AFB smear-positive sputum specimens, 458 (91.6% had both a positive sputum culture for Mycobacterium tuberculosis and a valid MTBDRplus assay result. The MTBDRplus assay detected isoniazid (INH resistance directly from the sputum specimen in 159 (89.8% of 177 specimens and MDR-TB in 109 (95.6% of 114 specimens compared to conventional methods. There was high agreement between the MTBDRplus assay and conventional DST results in detecting MDR-TB (kappa = 0.95, p<0.01. The most prevalent INH resistance mutation was S315T (78% in the katG codon and the most common rifampicin resistance mutation was S531L (68% in the rpoB codon. Among 13 specimens from TB suspects with negative sputum cultures, 7 had a positive MTBDRplus assay (3 with MDR-TB. The time to detection of MDR-TB was significantly less using the MTBDRplus assay (4.2 days compared to the use of standard phenotypic tests (67.3 days with solid media and 21.6 days with broth-based media. CONCLUSIONS: Compared to conventional methods, the MTBDRplus assay had high accuracy and significantly reduced time to detection of MDR-TB in an area with high MDR-TB prevalence. The use of rapid molecular diagnostic tests for TB and drug resistance should increase the proportion of patients promptly placed on appropriate therapy.

  18. Development of competitive ELISA for the detection of bovine serum albumin using single-chain variable fragments.

    Science.gov (United States)

    Liu, Yuan; Lin, Manman; Zhang, Xifeng; Hu, Xiaodan; Lin, Jieru; Hao, Jia; He, Dan; Zhang, Xiao; Xu, Chongxin; Zhong, Jianfeng; Xie, Yajing; Zhang, Cunzheng; Liu, Xianjin

    2017-05-15

    Soluble anti-bovine serum albumin (BSA) single-chain variable fragments (scFvs) were expressed in E. Coli. HB2151. The antigen-binding equilibrium dissociation constant of the scFvs was determined to be 2.9 × 10(-8) M by surface plasmon resonance analysis. A competitive ELISA for the detection of BSA was developed using the antibody fragment above. The limits of detection (I10) and I50 were 0.002 and 0.74 μg/ml respectively, with a recovery between 87.8 and 119.2% in spiked milk samples. The assay has the potential to be used to detect concentration of BSA in milk or other matrix instead of the ELISA based on traditional antibodies.

  19. Evaluation of DNA extraction techniques for detecting Mycobacterium tuberculosis complex organisms in Asian elephant trunk wash samples.

    Science.gov (United States)

    Kay, Meagan K; Linke, Lyndsey; Triantis, Joni; Salman, M D; Larsen, R Scott

    2011-02-01

    Rapid and sensitive diagnostic assays for the detection of tuberculous mycobacteria in elephants are lacking. DNA extraction with PCR analysis is useful for tuberculosis screening in many species but has not been validated on elephant trunk wash samples. We estimated the analytical sensitivity and specificity of three DNA extraction methods to detect Mycobacterium tuberculosis complex organisms in trunk wash specimens. A ZR soil microbe DNA kit (ZR) and a traditional salt and ethanol precipitation (TSEP) approach were evaluated under three different treatment conditions: heat treatment, phenol treatment, and contamination with Mycobacterium avium. A third approach, using a column filtration method, was evaluated for samples contaminated with soil. Trunk wash samples from uninfected elephants were spiked with various concentrations of M. bovis cells and subjected to the described treatment conditions prior to DNA extraction. Extracted DNA was amplified using IS6110-targeted PCR analysis. The ZR and TSEP methods detected as low as 1 to 5 M. bovis cells and 10 M. bovis cells, respectively, per 1.5 ml of trunk wash under all three conditions. Depending on the amount of soil present, the column filtration method detected as low as 5 to 50 M. bovis cells per 1.5 ml of trunk wash. Analytical specificity was assessed by DNA extraction from species of nontuberculous mycobacteria and amplification using the same PCR technique. Only M. bovis DNA was amplified, indicating 100% analytical specificity of this PCR technique. Our results indicate that these DNA extraction techniques offer promise as useful tests for detection of M. tuberculosis complex organisms in elephant trunk wash specimens.

  20. Multi-Fluorescence Real-Time PCR Assay for Detection of RIF & INH Resistance of M. tuberculosis

    Directory of Open Access Journals (Sweden)

    Jingfu ePeng

    2016-04-01

    Full Text Available Background: Failure to early detect multidrug-resistant tuberculosis (MDR-TB results in treatment failure and poor clinical outcomes, and highlights the need to rapidly detect resistance to rifampicin (RIF and isoniazid (INHMethods: In Multi-Fluorescence quantitative Real-Time PCR (MF-qRT-PCR assay, 10 probes labeled with 4 kinds of fluorophores were designed to detect the mutations in regions of rpoB, katG, mabA-inhA, oxyR-ahpC and rrs. The efficiency of MF-qRT-PCR assay was tested using 261 bacterial isolates and 33 clinical sputum specimens. Among these samples, 227 Mycobacterium tuberculosis isolates were analyzed using drug susceptibility testing (DST, DNA sequencing and MF-qRT-PCR assay.Results: Compared with DST, MF-qRT-PCR sensitivity and specificity for RIF-resistance were 94.6% and 100%, respectively. And the detection sensitivity and specificity for INH-resistance were 85.9% and 95.3%, respectively. Compared with DNA sequencing, the sensitivity and specificity of our assay were 97.2% and 100% for RIF-resistance and 97.9% and 96.4% for INH-resistance. Compared with Phenotypic strain identification, MF-qRT-PCR can distinguish 227 Mycobacterium tuberculosis complexes (MTC from 34 Non-tuberculous mycobacteria (NTM isolates with 100% accuracy rate.Conclusions: MF-qRT-PCR assay was an efficient, accurate, reliable and easy-operated method for detection of RIF and INH-resistance, and distinction of MTC and NTM of clinical isolates.

  1. Detection of mycobacteria, Mycobacterium avium subspecies, and Mycobacterium tuberculosis complex by a novel tetraplex real-time PCR assay.

    Science.gov (United States)

    Sevilla, Iker A; Molina, Elena; Elguezabal, Natalia; Pérez, Valentín; Garrido, Joseba M; Juste, Ramón A

    2015-03-01

    Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses.

  2. FIND Tuberculosis Strain Bank: a Resource for Researchers and Developers Working on Tests To Detect Mycobacterium tuberculosis and Related Drug Resistance.

    Science.gov (United States)

    Tessema, Belay; Nabeta, Pamela; Valli, Eloise; Albertini, Audrey; Collantes, Jimena; Lan, Nguyen Huu; Romancenco, Elena; Tukavdze, Nestani; Denkinger, Claudia M; Dolinger, David L

    2017-04-01

    The spread of multidrug-resistant (MDR) tuberculosis (TB) and extensively drug-resistant (XDR) TB hampers global efforts in the fight against tuberculosis. To enhance the development and evaluation of diagnostic tests quickly and efficiently, well-characterized strains and samples from drug-resistant tuberculosis patients are necessary. In this project, the Foundation for Innovative New Diagnostics (FIND) has focused on the collection, characterization, and storage of such well-characterized reference materials and making them available to researchers and developers. The collection is being conducted at multiple centers in Southeast Asia, South America, Eastern Europe, and soon the sub-Saharan Africa regions. Strains are characterized for their phenotypic resistances and MICs to first-line drugs (FLDs) and second-line drugs (SLDs) using the automated MGIT 960 system following validated procedures and WHO criteria. Analysis of resistance-associated mutations is done by whole-genome sequencing (WGS) using the Illumina NextSeq system. Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis and WGS are used to determine strain lineages. All strains are maintained frozen at -80°C ± 10°C as distinct mother and daughter lots. All strains are extensively quality assured. The data presented here represent an analysis of the initial part of the collection. Currently, the bank contains 118 unique strains with extracted genomic DNA and matched sputum, serum, and plasma samples and will be expanded to a minimum of 1,000 unique strains over the next 3 years. Analysis of the current strains by phenotypic resistance testing shows 102 (86.4%), 10 (8.5%), and 6 (5.1%) MDR, XDR, and mono/poly resistant strains, respectively. Two of the strains are resistant to all 11 drugs that were phenotypically tested. WGS mutation analysis revealed FLD resistance-associated mutations in the rpoB, katG, inhA, embB, embA, and pncA genes; SLD resistance in the gyrA, gyr

  3. Determining resistance to mastitis in a bovine subject comprises detecting the presence or absence of a genetic marker that is linked to a trait indicative of mastitis resistance

    DEFF Research Database (Denmark)

    2007-01-01

    The invention relates to a method for determining mastitis resistance in bovine subjects, wherein mastitis resistance comprise resistance to both sub-clinical and clinical mastitis. In particular, the method of the invention involves identification of genetic markers and/or Quantitative Trait Locus...... (QTL) for the determination of mastitis resistance in a bovine subject. The determination of mastitis resistance involves resolution of the specific microsatellite status. Furthermore, the invention relates to a diagnostic kit for detection of genetic marker(s) associated with mastitis resistance....... The method and kit of the present invention can be applied for selection of bovine subjects for breeding purposes. Thus, the invention provides a method of genetically selecting bovine subjects with mastitis resistance, thereby yielding cows less prone to mastitis...

  4. Magnetic bead and gold nanoparticle probes based immunoassay for β-casein detection in bovine milk samples.

    Science.gov (United States)

    Li, Y S; Meng, X Y; Zhou, Y; Zhang, Y Y; Meng, X M; Yang, L; Hu, P; Lu, S Y; Ren, H L; Liu, Z S; Wang, X R

    2015-04-15

    In this work, a double-probe based immunoassay was developed for rapid and sensitive determination of β-casein in bovine milk samples. In the method, magnetic beads (MBs), employed as supports for the immobilization of anti-β-casein polyclonal antibody (PAb), were used as the capture probe. Colloidal gold nanoparticles (AuNPs), employed as a bridge for loading anti-β-casein monoclonal antibody (McAb) and horseradish peroxidase (HRP), were used as the amplification probe. The presence of β-casein causes the sandwich structures of MBs-PAb-β-casein-McAb-AuNPs through the interaction between β-casein and the anti-β-casein antibodies. The HRP, used as an enzymatic-amplified tracer, can catalytically oxidize the substrate 3,3',5,5'-tetramethylbenzidine (TMB), generating optical signals that are proportional to the quantity of β-casein. The linear range of the immunoassay was from 6.5 to 1520ngmL(-1). The limit of detection (LOD) was 4.8ngmL(-1) which was 700 times lower than that of MBs-antibody-HRP based immunoassay and 6-7 times lower than that from the microplate-antibody-HRP based assay. The recoveries of β-casein from bovine milk samples were from 95.0% to 104.3% that had a good correlation coefficient (R(2)=0.9956) with those obtained by an official standard Kjeldahl method. For higher sensitivity, simple sample pretreatment and shorter time requirement of the antigen-antibody reaction, the developed immunoassay demonstrated the viability for detection of β-casein in bovine milk samples.

  5. Molecular identification of coagulase-negative staphylococci isolated from bovine mastitis and detection of β-lactam resistance.

    Science.gov (United States)

    Srednik, Mariela Elizabeth; Grieben, Mario Andres; Bentancor, Adriana; Gentilini, Elida Raquel

    2015-09-27

    Bovine mastitis is a frequent cause of economic loss in dairy herds. Coagulase-negative staphylococci (CoNS) are increasing in importance as causeof bovine intramammary infection (IMI) throughout the world in recent years. CoNShave been isolated from milk samples collected from cows with clinical andsubclinical mastitis in several countries. Identification of mastitis pathogensis important when selection appropriate antimicrobial therapy. A total of 93 strains of Staphylococcusspp isolated from bovine clinical andsubclinical mastitis in Argentina during 2010-2013 were identified by Restriction Fragment Length Polymorphism (RFLP)-PCR using the gap gene. The isolates were tested by PCR for the presence of blaZ gene and mecA gene and were tested by disk diffusion for the susceptibilityto penicillin and cefoxitin. The most common CoNS species was S.chromogenes 46.2% (43/93), followed by S. devriesei 11.8% (11/93) and S. haemolyticus 9.7% (9/93). The blaZ gene was detected in 19 (20.4%) but only 16 (17.2%) isolates were resistant to penicillin; the mecA was detected in6 (6.5%) isolates but only 4 (4.3) were resistant to cefoxitin. The 6 mecA-positive isolates showed oxaxillinMICs ≤ 0.5 μg/ml. CoNSare important minor mastitis pathogens and can be the cause of substantial economic losses. Despite the low resistance to PEN in Argentina, the presenceof MR isolates found in this study emphasize the importance of identificationof CoNS when an IMI is present because of the potentially risk of lateraltransfer of resistance genes between staphylococcal species.

  6. Detection of PrP(Sc) in peripheral tissues of clinically affected cattle after oral challenge with bovine spongiform encephalopathy.

    Science.gov (United States)

    Franz, Martin; Eiden, Martin; Balkema-Buschmann, Anne; Greenlee, Justin; Schatzl, Hermann; Fast, Christine; Richt, Jürgen; Hildebrandt, Jan-Peter; Groschup, Martin H

    2012-12-01

    Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease that mainly affects cattle. Transmission of BSE to humans caused a variant form of Creutzfeldt-Jakob disease. Following infection, the protease-resistant, disease-associated isoform of prion protein (PrP(Sc)) accumulates in the central nervous system and in other tissues. Many countries have defined bovine tissues that may contain prions as specified risk materials, which must not enter the human or animal food chains and therefore must be discarded. Ultrasensitive techniques such as protein misfolding cyclic amplification (PMCA) have been developed to detect PrP(Sc) when present in minuscule amounts that are not readily detected by other diagnostic methods such as immunohistochemistry or Western blotting. This study was conducted to determine when and where PrP(Sc) can be found by PMCA in cattle orally challenged with BSE. A total of 48 different tissue samples from four cattle infected orally with BSE at various clinical stages of disease were examined using a standardized PMCA protocol. The protocol used brain homogenate from bovine PrP transgenic mice (Tgbov XV) as substrate and three consecutive rounds of PMCA. Using this protocol, PrP(Sc) was found in the brain, spinal cord, nerve ganglia, optic nerve and Peyer's patches. The presence of PrP(Sc) was confirmed in adrenal glands, as well as in mesenteric lymph nodes - a finding that was reported recently by another group. Interestingly, additional positive results were obtained for the first time in the oesophagus, abomasum, rumen and rectum of clinically affected cattle.

  7. Interferon Gamma-Based Detection of Latent Tuberculosis Infection in the Border States of Nuevo Leon and Tamaulipas, Mexico.

    Science.gov (United States)

    Oren, Eyal; Alatorre-Izaguirre, Gabriela; Vargas-Villarreal, Javier; Moreno-Treviño, Maria Guadalupe; Garcialuna-Martinez, Javier; Gonzalez-Salazar, Francisco

    2015-01-01

    Nearly one-third of the world's population is infected with latent tuberculosis (LTBI). Tuberculosis (TB) rates in the border states are higher than national rates in both the US and Mexico, with the border accounting for 30% of total registered TB cases in both countries. However, LTBI rates in the general population in Mexican border states are unknown. In this region, LTBI is diagnosed using the tuberculin skin test (TST). New methods of detection more specific than TST have been developed, although there is currently no gold standard for LTBI detection. Our objective is to demonstrate utility of the Quantiferon TB gold In-Tube (QFT-GIT) test compared with the TST to detect LTBI among border populations. This is an observational, cross-sectional study carried out in border areas of the states of Nuevo Leon and Tamaulipas, Mexico. Participants (n = 210) provided a TST and blood sample for the QFT-GIT. Kappa coefficients assessed the agreement between TST and QFT-GIT. Participant characteristics were compared using Fisher exact tests. Thirty-eight percent of participants were diagnosed with LTBI by QFT-GIT. The proportion of LTBI detected using QFT-GIT was almost double [38% (79/210)] that found by TST [19% (39/210)] (P < 0.001). Concordance between TST and QFT-GIT was low (kappa = 0.37). We recommend further studies utilizing the QFT-GIT test to detect LTBI among border populations.

  8. Performance of a molecular assay to detect Mycobacterium tuberculosis complex DNA in clinical specimens: multicenter study in Brazil

    Science.gov (United States)

    Verza, Mirela; Schmid, Karen Barros; Barcellos, Regina Bones; Linck, Natali; Bello, Graziele Lima; Scapin, Daniel; Sperhacke, Rosa Dea; Silva, Márcia Susana Nunes; Wollheim, Claudia; Rivero, Martha Gabriela Celle; Kritski, Afrânio Lineu; Rezende, Leonides; Oliveira, Martha Maria; Costa, Elis Regina Dalla; Rossetti, Maria Lucia Rosa

    2017-01-01

    BACKGROUND In high tuberculosis (TB) burden countries, there are few data on the performance of new molecular commercialised assays developed locally. OBJECTIVE To evaluate the performance of a new molecular commercialised assay for TB diagnosis (Detect-TB) in three laboratories. METHODS A total of 302 sputum samples from an equal number of patients with presumptive diagnosis of pulmonary tuberculosis (PTB) were submitted for routine smear microscopy, culture, and Detect-TB assay at three different sites in Brazil (the cities of Caxias do Sul, São Paulo and Canoas). FINDINGS Seventy four (24.7%) TB cases were diagnosed (65 bacteriologically confirmed). When compared to smear microscopy/culture results, the overall sensitivity and specificity of Detect-TB assay was 84.6% (CI 95%; 73.7-91.6) and 93.1% (CI 95%; 89.1-95.8), respectively. When compared to bacteriological and clinical diagnostic criteria, the sensitivity and specificity of Detect-TB assay was 74.3% (CI 95%; 63.3-82.9) and 92.9% (CI 95%; 88.7-95.6), respectively. Among the three sites - Caxias do Sul, São Paulo and Canoas - the sensitivity and specificity were respectively 94.7% and 97.8%; 71.4% and 93.9%, 82.1% and 88.9%. MAIN CONCLUSIONS These findings suggest that the Detect-TB assay could be applied routinely in reference laboratories across different regions in Brazil. PMID:28177043

  9. Recombinant Jembrana disease virus proteins as antigens for the detection of antibody to bovine lentiviruses.

    Science.gov (United States)

    Burkala, E J; Narayani, I; Hartaningsih, N; Kertayadnya, G; Berryman, D I; Wilcox, G E

    1998-09-01

    Jembrana disease virus (JDV) is a recently identified bovine lentivirus causing an acute severe disease syndrome in banteng cattle (Bos javanicus) and a milder disease syndrome in Bos taurus cattle in Indonesia. The virus is closely related genetically to the previously identified bovine lentivirus, bovine immunodeficiency virus (BIV). Recombinant clones were produced which contained the capsid (CA) and transmembrane (TM) subunits of the respective gag and env open reading frames of JDV. The proteins were expressed as fusions to the glutathione-s-transferase (GST) enzyme in Escherichia coli and purification was achieved using affinity chromatography via immobilized reduced glutathione. The soluble recombinant CA and TM antigens of JDV were reacted in western immunoblots with both serum antibodies from JDV-infected Bos javanicus cattle and Bos taurus cattle immunized with BIV. The recombinant CA protein of JDV reacted equally well with both the JDV and BIV antisera. The recombinant TM protein of JDV also reacted with antibody from the JDV infected cattle and with the BIV antisera. The results indicated conservation of immunogenic epitopes of the CA and TM proteins of the two viruses. The production of the recombinant proteins should enable the development of rapid and sensitive serological tests for JDV and BIV, and tools for further study of the immune response to JDV and the differential epidemiology of JDV infections in cattle.

  10. Efficient diagnosis of tuberculous meningitis by detection of Mycobacterium tuberculosis DNA in cerebrospinal fluid filtrates using PCR.

    Science.gov (United States)

    Haldar, Sagarika; Sharma, Neera; Gupta, V K; Tyagi, Jaya Sivaswami

    2009-05-01

    Tuberculous meningitis (TBM) is the most devastating form of meningitis and prompt diagnosis holds the key to its management. Conventional microbiology has limited utility and nucleic acid-based methods have not been widely accepted for various reasons. In view of the paucibacillary nature of cerebrospinal fluid (CSF) and the recent demonstration of free Mycobacterium tuberculosis DNA in clinical specimens, the present study was designed to evaluate the utility of CSF 'filtrates' for the diagnosis of TBM using PCR. One hundred and sixty-seven CSF samples were analysed from patients with 'suspected' TBM (n=81) and a control group including other cases of meningitis or neurological disorders (n=86). CSF 'sediments' and 'filtrates' were analysed individually for M. tuberculosis DNA by quantitative real-time PCR (qRT-PCR) and conventional PCR. Receiver-operating characteristic curves were generated from qRT-PCR data and cut-off values of 84 and 30 were selected for calling a 'filtrate' or 'sediment' sample positive, respectively. Based on these, TBM was diagnosed with 87.6% and 53.1% sensitivity (Ptuberculosis DNA was detected using devR PCR assays in 'sediment' and 'filtrate' fractions of all samples. From this study, we conclude that (i) CSF 'filtrates' contain a substantial amount of M. tuberculosis DNA and (ii) 'filtrates' and not 'sediments' are likely to reliably provide a PCR-based diagnosis in 'suspected' TBM patients.