Characterization of a cadmium resistance Lactococcus lactis subsp. lactis strain by antioxidant assays and proteome profiles methods.

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Sheng, Yao; Yang, Xuan; Lian, Yuanyuan; Zhang, Boyang; He, Xiaoyun; Xu, Wentao; Huang, Kunlun

2016-09-01

Heavy metal contamination poses a major threat to the environment and human health for their potential toxicity and non-biodegradable properties. At present, some probiotics bacteria are reported to have great potential to eliminate heavy metals from food and water. In this study, resistance properties of a newly isolated Lactococcus lactis subsp. lactis for cadmium were studied by antioxidant assays and proteomics analysis. Antioxidant capacity of this strain was significantly activated under cadmium stress indicated by Fenton reaction, DPPH assay, SOD assay and GSH assay. Intracellular antioxidant enzyme systems, such as superoxide dismutase, glutathione reductase and catalase were suggested to play vital roles in the activated antioxidant capacity. The up-regulated cadA was associated with the activated P-type ATPases that plays an important role in cadmium resistance. Proteomics analysis identified 12 over-expressed proteins under 50mg/L cadmium stress and these proteins are abundant in oxidative stress response and energy metabolism regulation, which were considered as consequences as cadmium resistance of the strain. Thus, the probiotics Lactococcus lactis subsp. lactis may resist cadmium stress through antioxidant approach and enhanced energy metabolism. The food grade lactis strain may be applied in metal decontamination in environment and food/feed. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of novel Clostridium perfringens type E strains that carry an iota toxin plasmid with a functional enterotoxin gene.

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Kazuaki Miyamoto

Full Text Available Clostridium perfringens enterotoxin (CPE is a major virulence factor for human gastrointestinal diseases, such as food poisoning and antibiotic associated diarrhea. The CPE-encoding gene (cpe can be chromosomal or plasmid-borne. Recent development of conventional PCR cpe-genotyping assays makes it possible to identify cpe location (chromosomal or plasmid in type A isolates. Initial studies for developing cpe genotyping assays indicated that all cpe-positive strains isolated from sickened patients were typable by cpe-genotypes, but surveys of C. perfringens environmental strains or strains from feces of healthy people suggested that this assay might not be useful for some cpe-carrying type A isolates. In the current study, a pulsed-field gel electrophoresis Southern blot assay showed that four cpe-genotype untypable isolates carried their cpe gene on a plasmid of ∼65 kb. Complete sequence analysis of the ∼65 kb variant cpe-carrying plasmid revealed no intact IS elements and a disrupted cytosine methyltransferase (dcm gene. More importantly, this plasmid contains a conjugative transfer region, a variant cpe gene and variant iota toxin genes. The toxin genes encoded by this plasmid are expressed based upon the results of RT-PCR assays. The ∼65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates. MLST analyses indicated these isolates belong to a unique cluster of C. perfringens. Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains.

Use of GenoType® MTBDRplus assay to assess drug resistance and mutation patterns of multidrug-resistant tuberculosis isolates in northern India

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A K Maurya

2013-01-01

Full Text Available Purpose: The emergence and spread of multidrug-resistant tuberculosis (MDR-TB is a major public health problem. The diagnosis of MDR-TB is of paramount importance in establishing appropriate clinical management and infection control measures. The aim of this study was to evaluate drug resistance and mutational patterns in clinical isolates MDR-TB by GenoType® MTBDRplus assay. Material and Methods: A total of 350 non-repeated sputum specimens were collected from highly suspected drug-resistant pulmonary tuberculosis (PTB cases; which were processed by microscopy, culture, differentiation and first line drug susceptibility testing (DST using BacT/ALERT 3D system. Results: Among a total of 125 mycobacterium tuberculosis complex (MTBC strains, readable results were obtained from 120 (96% strains by GenoType® MTBDRplus assay. Only 45 MDR-TB isolates were analysed for the performance, frequency and mutational patterns by GenoType® MTBDRplus assay. The sensitivity of the GenoType® MDRTBplus assay for detecting individual resistance to rifampicin (RIF, isoniazid (INH and multidrug resistance was found to be 95.8%, 96.3% and 97.7%, respectively. Mutation in codon S531L of the rpoB gene and codon S315T1 of katG genes were dominated in MDR-TB strains, respectively (P < 0.05. Conclusions: The GenoType® MTBDRplus assay is highly sensitive with short turnaround times and a rapid test for the detection of the most common mutations conferring resistance in MDR-TB strains that can readily be included in a routine laboratory workflow.

Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay

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Denoeud France

2006-02-01

Full Text Available Abstract Background The classification of Brucella into species and biovars relies on phenotypic characteristics and sometimes raises difficulties in the interpretation of the results due to an absence of standardization of the typing reagents. In addition, the resolution of this biotyping is moderate and requires the manipulation of the living agent. More efficient DNA-based methods are needed, and this work explores the suitability of multiple locus variable number tandem repeats analysis (MLVA for both typing and species identification. Results Eighty tandem repeat loci predicted to be polymorphic by genome sequence analysis of three available Brucella genome sequences were tested for polymorphism by genotyping 21 Brucella strains (18 reference strains representing the six 'classical' species and all biovars as well as 3 marine mammal strains currently recognized as members of two new species. The MLVA data efficiently cluster the strains as expected according to their species and biovar. For practical use, a subset of 15 loci preserving this clustering was selected and applied to the typing of 236 isolates. Using this MLVA-15 assay, the clusters generated correspond to the classical biotyping scheme of Brucella spp. The 15 markers have been divided into two groups, one comprising 8 user-friendly minisatellite markers with a good species identification capability (panel 1 and another complementary group of 7 microsatellite markers with higher discriminatory power (panel 2. Conclusion The MLVA-15 assay can be applied to large collections of Brucella strains with automated or manual procedures, and can be proposed as a complement, or even a substitute, of classical biotyping methods. This is facilitated by the fact that MLVA is based on non-infectious material (DNA whereas the biotyping procedure itself requires the manipulation of the living agent. The data produced can be queried on a dedicated MLVA web service site.

A duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains.

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Benacer, Douadi; Zain, Siti Nursheena Mohd; Lewis, John W; Khalid, Mohd Khairul Nizam Mohd; Thong, Kwai Lin

2017-01-01

This study aimed to develop a duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains. Primers were designed to target the rrs (LG1/LG2) and ligB (LP1/LP2) genes to confirm the presence of the Leptospira genus and the pathogenic species, respectively. The assay showed 100% specificity against 17 Leptospira strains with a limit of detection of 23.1pg/µl of leptospiral DNA and sensitivity of 103 leptospires/ml in both spiked urine and water. Our duplex endpoint PCR assay is suitable for rapid early detection of Leptospira with high sensitivity and specificity.

Diphtheria in the Republic of Georgia: Use of Molecular Typing Techniques for Characterization of Corynebacterium diphtheriae Strains

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Sulakvelidze, Alexander; Kekelidze, Merab; Gomelauri, Tsaro; Deng, Yingkang; Khetsuriani, Nino; Kobaidze, Ketino; De Zoysa, Aruni; Efstratiou, Androulla; Morris, J. Glenn; Imnadze, Paata

1999-01-01

Sixty-six Corynebacterium diphtheriae strains (62 of the gravis biotype and 4 of the mitis biotype) isolated during the Georgian diphtheria epidemic of 1993 to 1998 and 13 non-Georgian C. diphtheriae strains (10 Russian and 3 reference isolates) were characterized by (i) biotyping, (ii) toxigenicity testing with the Elek assay and PCR, (iii) the randomly amplified polymorphic DNA (RAPD) technique, and (iv) pulsed-field gel electrophoresis (PFGE). Fifteen selected strains were ribotyped. Six RAPD types and 15 PFGE patterns were identified among all strains examined, and 12 ribotypes were found among the 15 strains that were ribotyped. The Georgian epidemic apparently was caused by one major clonal group of C. diphtheriae (PFGE type A, ribotype R1), which was identical to the predominant epidemic strain(s) isolated during the concurrent diphtheria epidemic in Russia. A dendrogram based on the PFGE patterns revealed profound differences between the minor (nonpredominant) epidemic strains found in Georgia and Russia. The methodologies for RAPD typing, ribotyping, and PFGE typing of C. diphtheriae strains were improved to enable rapid and convenient molecular typing of the strains. The RAPD technique was adequate for biotype differentiation; however, PFGE and ribotyping were better (and equal to each other) at discriminating between epidemiologically related and unrelated isolates. PMID:10488190

Loop-Mediated Isothermal Amplification Assay Targeting the MOMP Gene for Rapid Detection of Chlamydia psittaci Abortus Strain

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Guo-Zhen Lin, Fu-Ying Zheng, Ji-Zhang Zhou, Guang-Hua Wang, Xiao-An Cao, Xiao-Wei Gong and Chang-Qing Qiu*

2012-05-01

Full Text Available For rapid detection of the Chlamydia psittaci abortus strain, a loop-mediated isothermal amplification (LAMP assay was developed and evaluated in this study. The primers for the LAMP assay were designed on the basis of the main outer membrane protein (MOMP gene sequence of C. psittaci. Analysis showed that the assay could detect the abortus strain of C. psittaci with adequate specificity. The sensitivity of the test was the same as that of the nested-conventional PCR and higher than that of chick embryo isolation. Testing of 153 samples indicated that the LAMP assay could detect the genome of the C. psittaci abortus strain effectively in clinical samples. This assay is a useful tool for rapid diagnosis of C. psittaci infection in sheep, swine and cattle.

Rapid detection and typing of pathogenic nonpneumophila Legionella spp. isolates using a multiplex real-time PCR assay.

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Benitez, Alvaro J; Winchell, Jonas M

2016-04-01

We developed a single tube multiplex real-time PCR assay that allows for the rapid detection and typing of 9 nonpneumophila Legionella spp. isolates that are clinically relevant. The multiplex assay is capable of simultaneously detecting and discriminating L. micdadei, L. bozemanii, L. dumoffii, L. longbeachae, L. feeleii, L. anisa, L. parisiensis, L. tucsonensis serogroup (sg) 1 and 3, and L. sainthelensis sg 1 and 2 isolates. Evaluation of the assay with nucleic acid from each of these species derived from both clinical and environmental isolates and typing strains demonstrated 100% sensitivity and 100% specificity when tested against 43 other Legionella spp. Typing of L. anisa, L. parisiensis, and L. tucsonensis sg 1 and 3 isolates was accomplished by developing a real-time PCR assay followed by high-resolution melt (HRM) analysis targeting the ssrA gene. Further typing of L. bozemanii, L. longbeachae, and L. feeleii isolates to the serogroup level was accomplished by developing a real-time PCR assay followed by HRM analysis targeting the mip gene. When used in conjunction with other currently available diagnostic tests, these assays may aid in rapidly identifying specific etiologies associated with Legionella outbreaks, clusters, sporadic cases, and potential environmental sources. Published by Elsevier Inc.

MOLECULAR TYPING OF STREPTOCOCCUS PNEUMONIAE BY THE MULTIPLEX POLYMERASE CHAIN REACTION ASSAY IN ACCORDANCE TO THE PREVALENCE OF SEROTYPES IN THE RUSSIAN FEDERATION

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N. M. Alyab'eva

2013-01-01

Full Text Available Aim: to compare two methods of S. pneumoniae molecular typing: classic serological method and the multiplex polymerase chain reaction (M-PCR assay modified in accordance to the date on the serotypes circulating in Russian Federation. Patients and methods: 420 pneumococcal isolates mainly from non-sterile loci were analyzed. After microbiological identification pneumococci were serologically typed with the means of specific antiserum produced by Staten Serum Institute (Denmark in latex agglutination test and/or capsular swelling method. At the same time we performed series of the M-PCR, which consisted of 7 consecutive reactions at the most. Results: serotype was identified by the means of serological method in all 420 strains of S. pneumoniae; in total we determined 24 different serotypes. By the means of the M-PCR we succeeded in identification of 95% (399/420 examined strains, and 90% of them were typed during the first 3 reactions. All isolates failed to be typed by M-PCR (n =21 have serotypes not included into the composition of the M-PCR. The results of serological and molecular typing were identical in 99,2% (396/399 of the isolates; 3 strains showed contradictory results: serotype 19A was found on serological assay and serotype 19F — on PCR assay. Conclusions: the introduced modification of M-PCR allows correct identification of pneumococcal serotype more than in 90% of strains circulating in the Russian Federation, including all serotypes of pneumococcal polysaccharide conjugate vaccine.

Single assay for simultaneous detection and differential identification of human and avian influenza virus types, subtypes, and emergent variants.

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David Metzgar

Full Text Available For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based or remarkably insensitive (antibody-based. Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A

Single assay for simultaneous detection and differential identification of human and avian influenza virus types, subtypes, and emergent variants.

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Metzgar, David; Myers, Christopher A; Russell, Kevin L; Faix, Dennis; Blair, Patrick J; Brown, Jason; Vo, Scott; Swayne, David E; Thomas, Colleen; Stenger, David A; Lin, Baochuan; Malanoski, Anthony P; Wang, Zheng; Blaney, Kate M; Long, Nina C; Schnur, Joel M; Saad, Magdi D; Borsuk, Lisa A; Lichanska, Agnieszka M; Lorence, Matthew C; Weslowski, Brian; Schafer, Klaus O; Tibbetts, Clark

2010-02-03

For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based) or remarkably insensitive (antibody-based). Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR) have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu) is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A/HN subtype, virulence

A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa

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2010-01-01

Background Chronic lung infection with the bacterium Pseudomonas aeruginosa is one of the hallmarks of cystic fibrosis (CF) and is associated with worsening lung function, increased hospitalisation and reduced life expectancy. A virulent clonal strain of P. aeruginosa (Australian epidemic strain I; AES-I) has been found to be widespread in CF patients in eastern Australia. Methods Suppression subtractive hybridization (SSH) was employed to identify genetic sequences that are present in the AES-I strain but absent from the sequenced reference strain PAO1. We used PCR to evaluate the distribution of several of the AES-I loci amongst a collection of 188 P. aeruginosa isolates which was comprised of 35 AES-I isolates (as determined by PFGE), 78 non-AES-I CF isolates including other epidemic CF strains as well as 69 P. aeruginosa isolates from other clinical and environmental sources. Results We have identified a unique AES-I genetic locus that is present in all 35 AES-I isolates tested and not present in any of the other 153 P. aeruginosa strains examined. We have used this unique AES-I locus to develop a diagnostic PCR and a real-time PCR assay to detect the presence of P. aeruginosa and AES-I in patient sputum samples. Conclusions We have developed diagnostic PCR assays that are 100% sensitive and 100% specific for the P. aeruginosa strain AES-I. We have also shown that Whatman FTA® Elute cards may be used with PCR-based assays to rapidly detect the presence of P. aeruginosa strains in CF sputum. PMID:20637114

A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa

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Murphy Anna

2010-07-01

Full Text Available Abstract Background Chronic lung infection with the bacterium Pseudomonas aeruginosa is one of the hallmarks of cystic fibrosis (CF and is associated with worsening lung function, increased hospitalisation and reduced life expectancy. A virulent clonal strain of P. aeruginosa (Australian epidemic strain I; AES-I has been found to be widespread in CF patients in eastern Australia. Methods Suppression subtractive hybridization (SSH was employed to identify genetic sequences that are present in the AES-I strain but absent from the sequenced reference strain PAO1. We used PCR to evaluate the distribution of several of the AES-I loci amongst a collection of 188 P. aeruginosa isolates which was comprised of 35 AES-I isolates (as determined by PFGE, 78 non-AES-I CF isolates including other epidemic CF strains as well as 69 P. aeruginosa isolates from other clinical and environmental sources. Results We have identified a unique AES-I genetic locus that is present in all 35 AES-I isolates tested and not present in any of the other 153 P. aeruginosa strains examined. We have used this unique AES-I locus to develop a diagnostic PCR and a real-time PCR assay to detect the presence of P. aeruginosa and AES-I in patient sputum samples. Conclusions We have developed diagnostic PCR assays that are 100% sensitive and 100% specific for the P. aeruginosa strain AES-I. We have also shown that Whatman FTA® Elute cards may be used with PCR-based assays to rapidly detect the presence of P. aeruginosa strains in CF sputum.

Development of an indirect enzyme-linked immunosorbent assay (ELISA) to differentiate antibodies against wild-type porcine reproductive and respiratory syndrome from the vaccine strain TJM-F92 based on a recombinant Nsp2 protein.

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Wang, X X; Wang, F X; Li, Z G; Wen, Y J; Wang, X; Song, N; Wu, H

2018-01-01

An accurate ELISA method to differentiate pigs infected with wild-type porcine reproductive and respiratory syndrome (PRRSV) strains from vaccinated ones would help to monitor PRRSV vaccination compliance. The recombinant protein GST-d120aa derived from the continuous deletion of 120 amino acids in the non-structural protein 2 region of the modified-live vaccine strain TJM-F92 was used to develop an indirect enzyme-linked immunosorbent assay (d120-ELISA) for differentiating serum antibodies against TJM-F92 from other PRRSV strains. At the optimized cut-off value which was calculated at an S/P of 0.25, it yielded a sensitivity of 90.7% and a specificity of 95.1%. Cross-reactivity tests suggested that the d120-ELISA was PRRSV-specific. Coefficient of variations of the repeatability tests ranged between 1.41-17.02%. The results suggest that the d120-ELISA is suitable for differentiating animals infected with wild-type strains from those immunized with MLV TJM-F92. Copyright © 2017. Published by Elsevier B.V.

Comparison of multiplex RT-PCR and real-time HybProbe assay for serotyping of dengue virus using reference strains and clinical samples from India

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Anita Chakravarti

2016-01-01

Full Text Available Background: Dengue virus serotyping is crucial from clinical management and epidemiological point of view. Aims: To compare efficacy of two molecular detection and typing methods, namely, multiplex reverse transcription polymerase chain reaction (RT-PCR and real-time Hybprobe assay using a panel of known dilution of four reference Dengue virus strains and a panel of sera collected from clinically suspected dengue patients. Settings: This study was conducted at a tertiary-care teaching hospital in Delhi, India. Materials and Methods: Dengue serotype specific virus strains were used as prototypes for serotyping assays. Viral load was quantified by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR. Acute phase serum samples were collected from 79 patients with clinically suspected Dengue fever on their first day of presentation during September-October 2012. Viral RNA from serum and cell culture supernatant was extracted. Reverse transcription was carried out. Quantitative detection of DENV RNA from reference strain culture supernatants and each of the 79 patient samples by real-time PCR was performed using light cycler Taqman master mix kit. Serotyping was done by multiplex RT-PCR assay and Hybprobe assay. Results: The multiplex RT-PCR assay, though found to be 100% specific, couldn't serotype either patient or reference strains with viral load less than 1000 RNA copies/ml. The Hybprobe assay was found to have 100% specificity and had a lower limit of serotype detection of merely 3.54 RNA copies/ml. Conclusions: HybProbe assay has an important role especially in situations where serotyping is to be performed in clinical samples with low viral load.

Multiple-locus variant-repeat assay (MLVA) is a useful tool for molecular epidemiologic analysis of Streptococcus agalactiae strains causing bovine mastitis.

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Radtke, Andreas; Bruheim, Torkjel; Afset, Jan Egil; Bergh, Kåre

2012-06-15

Group B streptococci (GBS) were considered a major cause of mastitis in cattle until preventive measures succeeded in controlling the disease in the 1970s and 1980s. During the last 5-6 years an increasing number of cases have been observed in some Scandinavian countries. A total of 187 GBS isolates from mastitis cases were collected from 119 animals in 34 Norwegian farms in the period from April 2007 to November 2010. 133 (71%) of the isolates were from farms with automated milking systems. The strains underwent typing of capsular polysaccharides (CPS) and surface proteins, and were analyzed by multi-locus variable repeat assay (MLVA) to investigate the epidemiological relationship of strains within and between farms. The GBS strains were differentiated into 12 types by CPS and surface protein analysis, with CPS types V (54%) and IV (34%) predominating. MLVA was superior to CPS and protein typing for strain differentiation, resolving the 187 strains into 37 types. In 29 of 34 farms all GBS strains had identical MLVA profiles specific for each farm. However, in one farm represented with 48 isolates, four MLVA variants with differences in one repeat locus were observed during the almost 3-year long collection period. Similar variations were observed at four other farms. This might reflect the stability of repeat loci under in vivo conditions. Farms with automated milking systems were overrepresented in this material. In conclusion, the five-loci MLVA allowed rapid high-resolution genotyping of the bovine GBS strains within and between farms. Copyright © 2012 Elsevier B.V. All rights reserved.

Evaluation of GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing of multi-drug resistance tuberculosis in Northern India

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Anand Kumar Maurya

2013-01-01

Full Text Available Background: The problem of multi-drug resistance tuberculosis (MDR-TB is growing in several hotspots throughout the world. Rapid and accurate diagnosis of MDR-TB is crucial to facilitate early treatment and to reduce its spread in the community. The aim of the present study was to evaluate the new, novel GenoType® MTBDRplus assay for rapid detection of drug susceptibility testing (DST of MDR-TB cases in Northern India. Materials and Methods: A total of 550 specimens were collected from highly suspected drug resistant from pulmonary and extra-pulmonary TB cases. All the specimens were processed by Ziehl- Neelsen staining, culture, differentiation by the GenoType® CM assay, first line DST using BacT/ALERT 3D system and GenoType® MTBDRplus assay. The concordance of the GenoType® MTBDRplus assay was calculated in comparison with conventional DST results. Results: Overall the sensitivity for detection of rifampicin, isoniazid and MDR-TB resistance by GenoType® MTBDRplus assay was 98.0%, 98.4% and 98.2% respectively. Out of 55 MDR-TB strains, 45 (81.8%, 52 (94.5% and 17 (30.9% strains showed mutation in rpoB, katG and inhA genes respectively (P < 0.05. The most prominent mutations in rpoB, katG and inhA genes were; 37 (67.3% in S531L, 52 (94.5% in S315T1 and 11 (20% in C15T regions respectively (P < 0.05. Conclusions: Our study demonstrated a high concordance between the GenoType® MTBDRplus assay resistance patterns and those were observed by conventional DST with good sensitivity, specificity with short turnaround times and to control new cases of MDR-TB in countries with a high prevalence of MDR-TB.

Prior Inoculation with Type B Strains of Francisella tularensis Provides Partial Protection against Virulent Type A Strains in Cottontail Rabbits.

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Vienna R Brown

Full Text Available Francisella tularensis is a highly virulent bacterium that is capable of causing severe disease (tularemia in a wide range of species. This organism is characterized into two distinct subspecies: tularensis (type A and holarctica (type B which vary in several crucial ways, with some type A strains having been found to be considerably more virulent in humans and laboratory animals. Cottontail rabbits have been widely implicated as a reservoir species for this subspecies; however, experimental inoculation in our laboratory revealed type A organisms to be highly virulent, resulting in 100% mortality following challenge with 50-100 organisms. Inoculation of cottontail rabbits with the same number of organisms from type B strains of bacteria was found to be rarely lethal and to result in a robust humoral immune response. The objective of this study was to characterize the protection afforded by a prior challenge with type B strains against a later inoculation with a type A strain in North American cottontail rabbits (Sylvilagus spp. Previous infection with a type B strain of organism was found to lengthen survival time and in some cases prevent death following inoculation with a type A2 strain of F. tularensis. In contrast, inoculation of a type A1b strain was uniformly lethal in cottontail rabbits irrespective of a prior type B inoculation. These findings provide important insight about the role cottontail rabbits may play in environmental maintenance and transmission of this organism.

A rapid enzymatic assay for high-throughput screening of adenosine-producing strains

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Dong, Huina; Zu, Xin; Zheng, Ping; Zhang, Dawei

2015-01-01

Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH3, the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples. PMID:25580842

Real-time reverse transcription polymerase chain reaction method for detection of Canine distemper virus modified live vaccine shedding for differentiation from infection with wild-type strains.

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Wilkes, Rebecca P; Sanchez, Elena; Riley, Matthew C; Kennedy, Melissa A

2014-01-01

Canine distemper virus (CDV) remains a common cause of infectious disease in dogs, particularly in high-density housing situations such as shelters. Vaccination of all dogs against CDV is recommended at the time of admission to animal shelters and many use a modified live virus (MLV) vaccine. From a diagnostic standpoint for dogs with suspected CDV infection, this is problematic because highly sensitive diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) tests are able to detect MLV virus in clinical samples. Real-time PCR can be used to quantitate amount of virus shedding and can differentiate vaccine strains from wild-type strains when shedding is high. However, differentiation by quantitation is not possible in vaccinated animals during acute infection, when shedding is low and could be mistaken for low level vaccine virus shedding. While there are gel-based RT-PCR assays for differentiation of vaccine strains from field strains based on sequence differences, the sensitivity of these assays is unable to match that of the real-time RT-PCR assay currently used in the authors' laboratory. Therefore, a real-time RT-PCR assay was developed that detects CDV MLV vaccine strains and distinguishes them from wild-type strains based on nucleotide sequence differences, rather than the amount of viral RNA in the sample. The test is highly sensitive, with detection of as few as 5 virus genomic copies (corresponding to 10(-1) TCID(50)). Sequencing of the DNA real-time products also allows phylogenetic differentiation of the wild-type strains. This test will aid diagnosis during outbreaks of CDV in recently vaccinated animals.

A real-time PCR assay for the detection of atypical strains of Chlamydiaceae from pigeons.

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Aleksandar Zocevic

Full Text Available Recent evidence of the occurrence of atypical Chlamydiaceae strains in pigeons, different from the established Chlamydiaceae, requires the development of a specific and rapid detection tool to investigate their prevalence and significance. Here is described a new real-time PCR assay that allows specific detection of atypical Chlamydiaceae from pigeons. The assay has been used to assess the dissemination of these strains in field samples collected from Parisian pigeon populations in 2009. The results suggest a limited dissemination compared to the usually higher prevalence of Chlamydia psittaci that is the main species associated with avian chlamydiosis.

Microbiological diagnosis and molecular typing of Legionella strains during an outbreak of legionellosis in Southern Germany.

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Essig, Andreas; von Baum, Heike; Gonser, Theodor; Haerter, Georg; Lück, Christian

2016-02-01

An explosive outbreak of Legionnaires' disease with 64 reported cases occurred in Ulm/Neu-Ulm in the South of Germany in December 2009/January 2010 caused by Legionella (L.) pneumophila serogroup 1, monoclonal (mAb) subtype Knoxville, sequence type (ST) 62. Here we present the clinical microbiological results from 51 patients who were diagnosed at the University hospital of Ulm, the results of the environmental investigations and of molecular typing of patients and environmental strains. All 50 patients from whom urine specimens were available were positive for L. pneumophila antigen when an enzyme-linked immunosorbent assay (EIA) was used following concentration of those urine samples that tested initially negative. The sensitivity of the BinaxNow rapid immunographic assay (ICA), after 15 min reading and after 60 min reading were 70% and 84%, respectively. Direct typing confirmed the monoclonal subtype Knoxville in 5 out of 8 concentrated urine samples. Real time PCR testing of respiratory tract specimens for L. pneumophila was positive in 15 out of 25 (60%) patients. Direct nested sequence based typing (nSBT) in some of these samples allowed partial confirmation of ST62. L. pneumophila serogroup 1, monoclonal subtype Knoxville ST62, defined as the epidemic strain was isolated from 8 out of 31 outbreak patients (26%) and from one cooling tower confirming it as the most likely source of the outbreak. While rapid detection of Legionella antigenuria was crucial for the recognition and management of the outbreak, culture and molecular typing of the strains from patients and environmental specimens was the clue for the rapid identification of the source of infection. Copyright © 2016 Elsevier GmbH. All rights reserved.

A simplified multiplex PCR assay for fast and easy discrimination of globally distributed staphylococcal cassette chromosome mec types in meticillin-resistant Staphylococcus aureus.

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Ghaznavi-Rad, Ehsanollah; Nor Shamsudin, Mariana; Sekawi, Zamberi; van Belkum, Alex; Neela, Vasanthakumari

2010-10-01

A multiplex PCR assay was developed for the identification of major types and subtypes of staphylococcal cassette chromosome mec (SCCmec) in meticillin-resistant Staphylococcus aureus (MRSA) strains. The method uses a novel 9 valent multiplex PCR plus two primer pairs for S. aureus identification and detection of meticillin resistance. All 389 clinical MRSA isolates from Malaysia and 18 European isolates from the Harmony collection harbouring different SCCmec types that we tested were correctly characterized by our PCR assay. SCCmec type III and V were by far the most common types among both hospital- and community-acquired Malaysian MRSA isolates, with an apparent emergence of MRSA harbouring the IVh type.

Capsular polysaccharide typing of domestic mastitis-causing Staphylococcus aureus strains and its potential exploration of bovine mastitis vaccine development. I. Capsular polysaccharide typing, isolation and purification of the strains.

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Han, H R; Pak, S I; Kang, S W; Jong, W S; Youn, C J

2000-06-01

One hundred seven isolates of Staphylococcus aureus from bovine mastitis were investigated for colony morphology in serum-soft agar (SSA), autoagglutination in salt, and capsular serotype. Capsular polysaccharide (CP) was purified and quantified from the extracts of clinical isolates. Overall, 89 isolates (83.2%) were diffuse in the SSA, without any difference in the proportion of diffuse colony between type 5 and type 8 strains. Some strains exhibited compact colonies in the SSA and expressed CP as determined by an enzyme-linked immunosorbent assay, indicating that compact morphology does not exclude encapsulation. The majority of the strains (11/12) showed autoagglutination in the salt aggregation test. The serotype 336 accounted for 46.7% of the isolates followed by serotype 5 (12.1%) and serotype 8 (12.1%). Particularly, twenty-six (24.3%) isolates reacted with two serotypes; 7 for type 8/336 and 19 for type 5/336. Five isolates (4.7%) were nontypeable with monoclonal antibodies specific for CP serotype 5, 8, or 336. The CP concentration in culture supernatants varied with the serotypes, and the total amount of CP produced by cells grown in a liquid medium was much less than that produced by cells grown on a solid medium. The Western blotting indicated that the CP bands of S. aureus serotype 5 and 8 were ranged in the molecular mass of 58-84 kilodalton (kDa), with additional bands in the region of approximately >/= 48 or

A TaqMan-based real time PCR assay for specific detection and quantification of Xylella fastidiosa strains causing bacterial leaf scorch in oleander.

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Guan, Wei; Shao, Jonathan; Singh, Raghuwinder; Davis, Robert E; Zhao, Tingchang; Huang, Qi

2013-02-15

A TaqMan-based real-time PCR assay was developed for specific detection of strains of X. fastidiosa causing oleander leaf scorch. The assay uses primers WG-OLS-F1 and WG-OLS-R1 and the fluorescent probe WG-OLS-P1, designed based on unique sequences found only in the genome of oleander strain Ann1. The assay is specific, allowing detection of only oleander-infecting strains, not other strains of X. fastidiosa nor other plant-associated bacteria tested. The assay is also sensitive, with a detection limit of 10.4fg DNA of X. fastidiosa per reaction in vitro and in planta. The assay can also be applied to detect low numbers of X. fastidiosa in insect samples, or further developed into a multiplex real-time PCR assay to simultaneously detect and distinguish diverse strains of X. fastidiosa that may occupy the same hosts or insect vectors. Specific and sensitive detection and quantification of oleander strains of X. fastidiosa should be useful for disease diagnosis, epidemiological studies, management of oleander leaf scorch disease, and resistance screening for oleander shrubs. Published by Elsevier B.V.

Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis.

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Li, Wenbin; Teixeira, Diva C; Hartung, John S; Huang, Qi; Duan, Yongping; Zhou, Lijuan; Chen, Jianchi; Lin, Hong; Lopes, Silvio; Ayres, A Juliano; Levy, Laurene

2013-01-01

The xylem-limited, Gram-negative, fastidious plant bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease affecting approximately half of the citrus plantations in the State of São Paulo, Brazil. The disease was recently found in Central America and is threatening the multi-billion U.S. citrus industry. Many strains of X. fastidiosa are pathogens or endophytes in various plants growing in the U.S., and some strains cross infect several host plants. In this study, a TaqMan-based assay targeting the 16S rDNA signature region was developed for the identification of X. fastidiosa at the species level. Another TaqMan-based assay was developed for the specific identification of the CVC strains. Both new assays have been systematically validated in comparison with the primer/probe sets from four previously published assays on one platform and under similar PCR conditions, and shown to be superior. The species specific assay detected all X. fastidiosa strains and did not amplify any other citrus pathogen or endophyte tested. The CVC-specific assay detected all CVC strains but did not amplify any non-CVC X. fastidiosa nor any other citrus pathogen or endophyte evaluated. Both sets were multiplexed with a reliable internal control assay targeting host plant DNA, and their diagnostic specificity and sensitivity remained unchanged. This internal control provides quality assurance for DNA extraction, performance of PCR reagents, platforms and operators. The limit of detection for both assays was equivalent to 2 to 10 cells of X. fastidiosa per reaction for field citrus samples. Petioles and midribs of symptomatic leaves of sweet orange harbored the highest populations of X. fastidiosa, providing the best materials for detection of the pathogen. These new species specific assay will be invaluable for molecular identification of X. fastidiosa at the species level, and the CVC specific assay will be very powerful for the

A flow cytometry-based assay for quantifying non-plaque forming strains of yellow fever virus.

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Erika Hammarlund

Full Text Available Primary clinical isolates of yellow fever virus can be difficult to quantitate by standard in vitro methods because they may not form discernable plaques or induce a measurable cytopathic effect (CPE on cell monolayers. In our hands, the Dakar strain of yellow fever virus (YFV-Dakar could not be measured by plaque assay (PA, focus-forming assay (FFA, or by measurement of CPE. For these reasons, we developed a YFV-specific monoclonal antibody (3A8.B6 and used it to optimize a highly sensitive flow cytometry-based tissue culture limiting dilution assay (TC-LDA to measure levels of infectious virus. The TC-LDA was performed by incubating serial dilutions of virus in replicate wells of C6/36 cells and stained intracellularly for virus with MAb 3A8.B6. Using this approach, we could reproducibly quantitate YFV-Dakar in tissue culture supernatants as well as from the serum of viremic rhesus macaques experimentally infected with YFV-Dakar. Moreover, the TC-LDA approach was >10-fold more sensitive than standard plaque assay for quantitating typical plaque-forming strains of YFV including YFV-17D and YFV-FNV (French neurotropic vaccine. Together, these results indicate that the TC-LDA technique is effective for quantitating both plaque-forming and non-plaque-forming strains of yellow fever virus, and this methodology may be readily adapted for the study and quantitation of other non-plaque-forming viruses.

Specificity of B-type natriuretic peptide assays

DEFF Research Database (Denmark)

Saenger, Amy K.; Rodriguez-Fraga, Olaia; Ler, Ranka

2017-01-01

BACKGROUND: B-type natriuretic peptides (BNPs) are used clinically to diagnose and monitor heart failure and are present in the circulation as multiple proBNP-derived fragments. We investigated the specificity of BNP immunoassays with glycosylated and nonglycosylated BNP, N-terminal proBNP (NT......-proBNP), and proBNP peptides to probe the cross-reactivity of each assay. METHODS: Nine B-type natriuretic peptides were studied, including synthetic and recombinant BNP (Shionogi, Scios, Mayo), human and synthetic glycosylated and nonglycosylated NT-proBNP (HyTest, Roche Diagnostics), and human glycosylated......-Rad, Goetze] were evaluated. Specificity was assessed by calculating the recovery between baseline and peptide-spiked human plasma pools at target concentrations of 100 ng/L BNP, 300 ng/L proBNP, or 450 ng/L NT-proBNP. All assays were performed in duplicate. RESULTS: BNP and NT-proBNP assays demonstrated...

[Multilocus sequence-typing for characterization of Moscow strains of Haemophilus influenzae type b].

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Platonov, A E; Mironov, K O; Iatsyshina, S B; Koroleva, I S; Platonova, O V; Gushchin, A E; Shipulin, G A

2003-01-01

Haemophilius influenzae, type b (Hib) bacteria, were genotyped by multilocus sequence typing (MLST) using 5 loci (adk, fucK, mdh, pgi, recA). 42 Moscow Hib strains (including 38 isolates form cerebrospinal fluid of children, who had purulent meningitis in 1999-2001, and 4 strains isolated from healthy carriers of Hib), as well as 2 strains from Yekaterinburg were studied. In MLST a strain is characterized, by alleles and their combinations (an allele profile) referred to also as sequence-type (ST). 9 Sts were identified within the Russian Hib bacteria: ST-1 was found in 25 strains (57%), ST-12 was found in 8 strains (18%), ST-11 was found in 4 strains (9%) and ST-15 was found in 2 strains (4.5%); all other STs strains (13, 14, 16, 17, 51) were found in isolated cases (2.3%). A comparison of allelic profiles and of nucleotide sequences showed that 93% of Russian isolates, i.e. strain with ST-1, 11, 12, 13, 15 and 17, belong to one and the same clonal complex. 2 isolates from Norway and Sweden from among 7 foreign Hib strains studied up to now can be described as belonging to the same clonal complex; 5 Hib strains were different from the Russian ones.

A duplex PCR assay for the detection of Ralstonia solanacearum phylotype II strains in Musa spp.

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Gilles Cellier

Full Text Available Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato, no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae.

Real-time RPA assay for rapid detection and differentiation of wild-type pseudorabies and gE-deleted vaccine viruses.

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Wang, Jianchang; Liu, Libing; Wang, Jinfeng; Pang, Xiaoyu; Yuan, Wanzhe

2018-02-15

The objective of this study was to develop a dual real-time recombinase polymerase amplification (RPA) assay using exo probes for the detection and differentiation of pseudorabies virus (PRV). Specific RPA primers and probes were designed for gB and gE genes of PRV within the conserved region of viral genome. The reaction process can be completed in 20 min at 39 °C. The dual real-time RPA assay performed in the single tube was capable of specific detecting and differentiating of the wild-type PRV and gE-deleted vaccine strains, without cross-reactions with other non-targeted pig viruses. The analytical sensitivity of the assay was 10 2 copies for gB and gE genes. The dual real-time RPA demonstrated a 100% diagnostic agreement with the real-time PCR on 4 PRV strains and 37 clinical samples. Through the linear regression analysis, the R 2 value of the real-time RPA and the real-time PCR for gB and gE was 0.983 and 0.992, respectively. The dual real-time RPA assay provides an alternative useful tool for rapid, simple, and reliable detection and differentiation of PRV, especially in remote and rural areas. Copyright © 2017 Elsevier Inc. All rights reserved.

Development of multiple strain competitive index assays for Listeria monocytogenes using pIMC; a new site-specific integrative vector

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Cronin Michael

2008-06-01

Full Text Available Abstract Background The foodborne, gram-positive pathogen, Listeria monocytogenes, is capable of causing lethal infections in compromised individuals. In the post genomic era of L. monocytogenes research, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tag L. monocytogenes strains, with a particular emphasis on the development of multiple strain competitive index assays. Results We have constructed a new site-specific integrative vector, pIMC, based on pPL2, for the selection of L. monocytogenes from complex samples. The pIMC vector was further modified through the incorporation of IPTG inducible markers (antibiotic and phenotypic to produce a suite of four vectors which allowed the discrimination of multiple strains from a single sample. We were able to perform murine infection studies with up to four EGDe isolates within a single mouse and showed that the tags did not impact upon growth rate or virulence. The system also allowed the identification of subtle differences in virulence between strains of L. monocytogenes commonly used in laboratory studies. Conclusion This study has developed a competitive index assay that can be broadly applied to all L. monocytogenes strains. Improved statistical robustness of the data was observed, resulting in fewer mice being required for virulence assays. The competitive index assays provide a powerful method to analyse the virulence or fitness of L. monocytogenes in complex biological samples.

Human papillomavirus detection and typing using a nested-PCR-RFLP assay.

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Coser, Janaina; Boeira, Thaís da Rocha; Fonseca, André Salvador Kazantzi; Ikuta, Nilo; Lunge, Vagner Ricardo

2011-01-01

It is clinically important to detect and type human papillomavirus (HPV) in a sensitive and specific manner. Development of a nested-polymerase chain reaction-restriction fragment length polymorphism (nested-PCR-RFLP) assay to detect and type HPV based on the analysis of L1 gene. Analysis of published DNA sequence of mucosal HPV types to select sequences of new primers. Design of an original nested-PCR assay using the new primers pair selected and classical MY09/11 primers. HPV detection and typing in cervical samples using the nested-PCR-RFLP assay. The nested-PCR-RFLP assay detected and typed HPV in cervical samples. Of the total of 128 clinical samples submitted to simple PCR and nested-PCR for detection of HPV, 37 (28.9%) were positive for the virus by both methods and 25 samples were positive only by nested-PCR (67.5% increase in detection rate compared with single PCR). All HPV positive samples were effectively typed by RFLP assay. The method of nested-PCR proved to be an effective diagnostic tool for HPV detection and typing.

Adhesion activity of glyceraldehyde-3-phosphate dehydrogenase in a Chinese Streptococcus suis type 2 strain.

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Wang, Kaicheng; Lu, Chengping

2007-01-01

A total of 36 streptococcal strains, including seven S. equi ssp.zooepidemicus, two S. suis type 1 (SS1), 24 SS2, two SS9, and one SS7, were tested for glyceraldehyde-3-phosphate dehydrogenase gene (gapdh). Except from non-virulent SS2 strain T1 5, all strains harboured gapdh. The gapdh of Chinese Sichuan SS2 isolate ZY05719 and Jiangsu SS2 isolate HA9801 were sequenced and then compared with published sequences in the GenBank. The comparison revealed a 99.9 % and 99.8 % similarity of ZY05719 and HA9801, respectively, with the published sequence. Adherence assay data demonstrated a significant ((p<0.05)) reduction in adhesion of SS2 in HEp-2 cells pre-incubated with purified GAPDH compared to non pre-incubated controls, suggesting the GAPDH mediates SS2 bacterial adhesion to host cells.

Development of a strain-specific real-time PCR assay for enumeration of a probiotic Lactobacillus reuteri in chicken feed and intestine.

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Verity Ann Sattler

Full Text Available A strain-specific real-time PCR assay was developed for quantification of a probiotic Lactobacillus reuteri (DSM 16350 in poultry feed and intestine. The specific primers were designed based on a genomic sequence of the strain derived from suppression subtractive hybridization with the type strain L. reuteri DSM 20016. Specificity was tested using a set of non-target strains from several sources. Applicability of the real-time PCR assay was evaluated in a controlled broiler feeding trial by using standard curves specific for feed and intestinal matrices. The amount of the probiotic L. reuteri was determined in feed from three feeding phases and in intestinal samples of the jejunum, ileum, and caecum of three, 14, and 39 day old birds. L. reuteri DSM 16350 cells were enumerated in all feeds supplemented with the probiotic close to the inclusion rate of 7.0 × 10(3 cfu/g, however, were not detected in L. reuteri DSM 16350 free feed. In three day old birds L. reuteri DSM 16350 was only detected in intestinal samples from probiotic fed animals ranging from 8.2 ± 7.8 × 10(5 cfu/g in the jejunum, 1.0 ± 1.1×10(7 cfu/g in the ileum, and 2.5 ± 5.7 × 10(5 cfu/g in the caecum. Similar results were obtained for intestinal samples of older birds (14 and 39 days. With increasing age of the animals the amount of L. reuteri signals in the control animals, however, also increased, indicating the appearance of highly similar bacterial genomes in the gut microbiota. The L. reuteri DSM 16350 qPCR assay could be used in future for feeding trials to assure the accurate inclusion of the supplement to the feed and to monitor it's uptake into the GIT of young chicken.

Development of a strain-specific real-time PCR assay for enumeration of a probiotic Lactobacillus reuteri in chicken feed and intestine.

Science.gov (United States)

Sattler, Verity Ann; Mohnl, Michaela; Klose, Viviana

2014-01-01

A strain-specific real-time PCR assay was developed for quantification of a probiotic Lactobacillus reuteri (DSM 16350) in poultry feed and intestine. The specific primers were designed based on a genomic sequence of the strain derived from suppression subtractive hybridization with the type strain L. reuteri DSM 20016. Specificity was tested using a set of non-target strains from several sources. Applicability of the real-time PCR assay was evaluated in a controlled broiler feeding trial by using standard curves specific for feed and intestinal matrices. The amount of the probiotic L. reuteri was determined in feed from three feeding phases and in intestinal samples of the jejunum, ileum, and caecum of three, 14, and 39 day old birds. L. reuteri DSM 16350 cells were enumerated in all feeds supplemented with the probiotic close to the inclusion rate of 7.0 × 10(3) cfu/g, however, were not detected in L. reuteri DSM 16350 free feed. In three day old birds L. reuteri DSM 16350 was only detected in intestinal samples from probiotic fed animals ranging from 8.2 ± 7.8 × 10(5) cfu/g in the jejunum, 1.0 ± 1.1×10(7) cfu/g in the ileum, and 2.5 ± 5.7 × 10(5) cfu/g in the caecum. Similar results were obtained for intestinal samples of older birds (14 and 39 days). With increasing age of the animals the amount of L. reuteri signals in the control animals, however, also increased, indicating the appearance of highly similar bacterial genomes in the gut microbiota. The L. reuteri DSM 16350 qPCR assay could be used in future for feeding trials to assure the accurate inclusion of the supplement to the feed and to monitor it's uptake into the GIT of young chicken.

A simplified multiplex PCR assay for fast and easy discrimination of globally distributed staphylococcal cassette chromosome mec types in meticillin-resistant Staphylococcus aureus

NARCIS (Netherlands)

E. Ghaznavi Rad (Ehsanollah); N.S. Mariana (Nor Shamsudin); Z. Sekawi (Zamberi); A.F. van Belkum (Alex); V. Neela (Vasanthakumari)

2010-01-01

textabstractA multiplex PCR assay was developed for the identification of major types and subtypes of staphylococcal cassette chromosome mec (SCCmec) in meticillin-resistant Staphylococcus aureus (MRSA) strains. The method uses a novel 9 valent multiplex PCR plus two primer pairs for S. aureus

Requirements for Pseudomonas aeruginosa Type I-F CRISPR-Cas Adaptation Determined Using a Biofilm Enrichment Assay.

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Heussler, Gary E; Miller, Jon L; Price, Courtney E; Collins, Alan J; O'Toole, George A

2016-11-15

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated protein) systems are diverse and found in many archaea and bacteria. These systems have mainly been characterized as adaptive immune systems able to protect against invading mobile genetic elements, including viruses. The first step in this protection is acquisition of spacer sequences from the invader DNA and incorporation of those sequences into the CRISPR array, termed CRISPR adaptation. Progress in understanding the mechanisms and requirements of CRISPR adaptation has largely been accomplished using overexpression of cas genes or plasmid loss assays; little work has focused on endogenous CRISPR-acquired immunity from viral predation. Here, we developed a new biofilm-based assay system to enrich for Pseudomonas aeruginosa strains with new spacer acquisition. We used this assay to demonstrate that P. aeruginosa rapidly acquires spacers protective against DMS3vir, an engineered lytic variant of the Mu-like bacteriophage DMS3, through primed CRISPR adaptation from spacers present in the native CRISPR2 array. We found that for the P. aeruginosa type I-F system, the cas1 gene is required for CRISPR adaptation, recG contributes to (but is not required for) primed CRISPR adaptation, recD is dispensable for primed CRISPR adaptation, and finally, the ability of a putative priming spacer to prime can vary considerably depending on the specific sequences of the spacer. Our understanding of CRISPR adaptation has expanded largely through experiments in type I CRISPR systems using plasmid loss assays, mutants of Escherichia coli, or cas1-cas2 overexpression systems, but there has been little focus on studying the adaptation of endogenous systems protecting against a lytic bacteriophage. Here we describe a biofilm system that allows P. aeruginosa to rapidly gain spacers protective against a lytic bacteriophage. This approach has allowed us to probe the requirements for CRISPR adaptation in

Development of a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction assay for the differential diagnosis of Feline leukemia virus vaccine and wild strains.

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Ho, Chia-Fang; Chan, Kun-Wei; Yang, Wei-Cheng; Chiang, Yu-Chung; Chung, Yang-Tsung; Kuo, James; Wang, Chi-Young

2014-07-01

A multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) was developed for the differential diagnosis of Feline leukemia virus (FeLV) vaccine and wild-type strains based on a point mutation between the vaccine strain (S) and the wild-type strain (T) located in the p27 gene. This system was further upgraded to obtain a real-time ARMS RT-PCR (ARMS qRT-PCR) with a high-resolution melt analysis (HRMA) platform. The genotyping of various strains of FeLV was determined by comparing the HRMA curves with the defined wild-type FeLV (strain TW1), and the results were expressed as a percentage confidence. The detection limits of ARMS RT-PCR and ARMS qRT-PCR combined with HRMA were 100 and 1 copies of transcribed FeLV RNA per 0.5 ml of sample, respectively. No false-positive results were obtained with 6 unrelated pathogens and 1 feline cell line. Twelve FeLV Taiwan strains were correctly identified using ARMS qRT-PCR combined with HRMA. The genotypes of the strains matched the defined FeLV wild-type strain genotype with at least 91.17% confidence. A higher degree of sequence polymorphism was found throughout the p27 gene compared with the long terminal repeat region. In conclusion, the current study describes the phylogenetic relationship of the FeLV Taiwan strains and demonstrates that the developed ARMS RT-PCR assay is able to be used to detect the replication of a vaccine strain that has not been properly inactivated, thus acting as a safety check for the quality of FeLV vaccines.

Immunity status of adults and children against poliomyelitis virus type 1 strains CHAT and Sabin (LSc-2ab in Germany

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Diedrich Sabine

2010-12-01

Full Text Available Abstract Background In October 2007, the working group CEN/TC 216 of the European Committee for standardisation suggested that the Sabin oral poliovirus vaccine type 1 strain (LSc-2ab presently used for virucidal tests should be replaced by another attenuated vaccine poliovirus type 1 strain, CHAT. Both strains were historically used as oral vaccines, but the Sabin type 1 strain was acknowledged to be more attenuated. In Germany, vaccination against poliomyelitis was introduced in 1962 using the oral polio vaccine (OPV containing Sabin strain LSc-2ab. The vaccination schedule was changed from OPV to an inactivated polio vaccine (IPV containing wild polio virus type 1 strain Mahoney in 1998. In the present study, we assessed potential differences in neutralising antibody titres to Sabin and CHAT in persons with a history of either OPV, IPV, or OPV with IPV booster. Methods Neutralisation poliovirus antibodies against CHAT and Sabin 1 were measured in sera of 41 adults vaccinated with OPV. Additionally, sera from 28 children less than 10 years of age and immunised with IPV only were analysed. The neutralisation assay against poliovirus was performed according to WHO guidelines. Results The neutralisation activity against CHAT in adults with OPV vaccination history was significantly lower than against Sabin poliovirus type 1 strains (Wilcoxon signed-rank test P Conclusion The lack of neutralising antibodies against the CHAT strain in persons vaccinated with OPV might be associated with an increased risk of reinfection with the CHAT polio virus type 1, and this implies a putative risk of transmission of the virus to polio-free communities. We strongly suggest that laboratory workers who were immunised with OPV receive a booster vaccination with IPV before handling CHAT in the laboratory.

Strain differentiation of polioviruses with monoclonal antibodies.

NARCIS (Netherlands)

A.D.M.E. Osterhaus (Albert); A.L. van Wezel; A.J.H. Stegmann; J.A.A.M. van Asten (Jack)

1984-01-01

textabstractPanels of monoclonal antibodies raised against different poliovirus type 1, 2 and 3 strains, were tested in a micro-neutralization test and in a micro-enzyme linked immunosorbent assay against a large number of poliovirus strains. The results were compared with those obtained with the

Typing of lymphogranuloma venereum Chlamydia trachomatis strains

NARCIS (Netherlands)

Christerson, Linus; de Vries, Henry J. C.; de Barbeyrac, Bertille; Gaydos, Charlotte A.; Henrich, Birgit; Hoffmann, Steen; Schachter, Julius; Thorvaldsen, Johannes; Vall-Mayans, Martí; Klint, Markus; Herrmann, Björn; Morré, Servaas A.

2010-01-01

We analyzed by multilocus sequence typing 77 lymphogranuloma venereum Chlamydia trachomatis strains from men who have sex with men in Europe and the United States. Specimens from an outbreak in 2003 in Europe were monoclonal. In contrast, several strains were in the United States in the 1980s,

Prion strain discrimination based on rapid in vivo amplification and analysis by the cell panel assay.

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Yervand Eduard Karapetyan

Full Text Available Prion strain identification has been hitherto achieved using time-consuming incubation time determinations in one or more mouse lines and elaborate neuropathological assessment. In the present work, we make a detailed study of the properties of PrP-overproducing Tga20 mice. We show that in these mice the four prion strains examined are rapidly and faithfully amplified and can subsequently be discriminated by a cell-based procedure, the Cell Panel Assay.

Typing discrepancy between phenotypic and molecular characterization revealing an emerging biovar 9 variant of smooth phage-resistant B. abortus strain 8416 in China

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YaoXia eKang

2015-12-01

Full Text Available A newly isolated smooth colony morphology phage-resistant (SPR strain 8416 isolated from a 45-year-old cattle farm cleaner with clinical features of brucellosis in China was reported. The most unusual phenotype was its resistance to two Brucella phages Tbilisi and Weybridge, but sensitive to Berkeley 2, a pattern similar to that of B. melitensis biovar 1. VITEK 2 biochemical identification system found that both strain 8416 and B. melitensis strains shared positive ILATk, but negative in other B. abortus strains. However, routine biochemical and phenotypic characteristics of strain 8416 were most similar to that of B. abortus biovar 9 except CO2 requirement. In addition, multiple PCR molecular typing assays including AMOS-PCR, B. abortus special PCR (B-ab PCR and a novel sub-biovar typing PCR, indicated that strain 8416 may belong to either biovar 3b or 9 of B. abortus. Surprisingly, further MLVA typing results showed that strain 8416 was most closely related to B. abortus biovar 3 in the Brucella MLVA database, primarily differing in 4 out of 16 screened loci. Therefore, due to the unusual discrepancy between phenotypic (biochemical reactions and particular phage lysis profile and molecular typing characteristics, strain 8416 couldn’t be exactly classified to any of the existing B. abortus biovars and might be a new variant of B. abortus biovar 9. The present study also indicates that the present phage typing scheme for Brucella spp. is subject to variation and the routine Brucella biovar typing needs further studies.

Typing Discrepancy Between Phenotypic and Molecular Characterization Revealing an Emerging Biovar 9 Variant of Smooth Phage-Resistant B. abortus Strain 8416 in China.

Science.gov (United States)

Kang, Yao-Xia; Li, Xu-Ming; Piao, Dong-Ri; Tian, Guo-Zhong; Jiang, Hai; Jia, En-Hou; Lin, Liang; Cui, Bu-Yun; Chang, Yung-Fu; Guo, Xiao-Kui; Zhu, Yong-Zhang

2015-01-01

A newly isolated smooth colony morphology phage-resistant strain 8416 isolated from a 45-year-old cattle farm cleaner with clinical features of brucellosis in China was reported. The most unusual phenotype was its resistance to two Brucella phages Tbilisi and Weybridge, but sensitive to Berkeley 2, a pattern similar to that of Brucella melitensis biovar 1. VITEK 2 biochemical identification system found that both strain 8416 and B. melitensis strains shared positive ILATk, but negative in other B. abortus strains. However, routine biochemical and phenotypic characteristics of strain 8416 were most similar to that of B. abortus biovar 9 except CO2 requirement. In addition, multiple PCR molecular typing assays including AMOS-PCR, B. abortus special PCR (B-ab PCR) and a novel sub-biovar typing PCR, indicated that strain 8416 may belong to either biovar 3b or 9 of B. abortus. Surprisingly, further MLVA typing results showed that strain 8416 was most closely related to B. abortus biovar 3 in the Brucella MLVA database, primarily differing in 4 out of 16 screened loci. Therefore, due to the unusual discrepancy between phenotypic (biochemical reactions and particular phage lysis profile) and molecular typing characteristics, strain 8416 could not be exactly classified to any of the existing B. abortus biovars and might be a new variant of B. abortus biovar 9. The present study also indicates that the present phage typing scheme for Brucella sp. is subject to variation and the routine Brucella biovar typing needs further studies.

Immunity status of adults and children against poliomyelitis virus type 1 strains CHAT and Sabin (LSc-2ab) in Germany.

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Eggers, Maren; Terletskaia-Ladwig, Elena; Rabenau, Holger F; Doerr, Hans W; Diedrich, Sabine; Enders, Gisela; Enders, Martin

2010-12-09

In October 2007, the working group CEN/TC 216 of the European Committee for standardisation suggested that the Sabin oral poliovirus vaccine type 1 strain (LSc-2ab) presently used for virucidal tests should be replaced by another attenuated vaccine poliovirus type 1 strain, CHAT. Both strains were historically used as oral vaccines, but the Sabin type 1 strain was acknowledged to be more attenuated. In Germany, vaccination against poliomyelitis was introduced in 1962 using the oral polio vaccine (OPV) containing Sabin strain LSc-2ab. The vaccination schedule was changed from OPV to an inactivated polio vaccine (IPV) containing wild polio virus type 1 strain Mahoney in 1998. In the present study, we assessed potential differences in neutralising antibody titres to Sabin and CHAT in persons with a history of either OPV, IPV, or OPV with IPV booster. Neutralisation poliovirus antibodies against CHAT and Sabin 1 were measured in sera of 41 adults vaccinated with OPV. Additionally, sera from 28 children less than 10 years of age and immunised with IPV only were analysed. The neutralisation assay against poliovirus was performed according to WHO guidelines. The neutralisation activity against CHAT in adults with OPV vaccination history was significantly lower than against Sabin poliovirus type 1 strains (Wilcoxon signed-rank test P Sabin 1 varied between 8 and 64. Following IPV booster, anti-CHAT antibodies increased rapidly in sera of CHAT-negative adults with OPV history. Sera from children with IPV history neutralised CHAT and Sabin 1 strains equally. The lack of neutralising antibodies against the CHAT strain in persons vaccinated with OPV might be associated with an increased risk of reinfection with the CHAT polio virus type 1, and this implies a putative risk of transmission of the virus to polio-free communities. We strongly suggest that laboratory workers who were immunised with OPV receive a booster vaccination with IPV before handling CHAT in the laboratory.

Typing of Lymphogranuloma Venereum Chlamydia trachomatis Strains

Science.gov (United States)

Christerson, Linus; de Vries, Henry J.C.; de Barbeyrac, Bertille; Gaydos, Charlotte A.; Henrich, Birgit; Hoffmann, Steen; Schachter, Julius; Thorvaldsen, Johannes; Vall-Mayans, Martí; Klint, Markus; Morré, Servaas A.

2010-01-01

We analyzed by multilocus sequence typing 77 lymphogranuloma venereum Chlamydia trachomatis strains from men who have sex with men in Europe and the United States. Specimens from an outbreak in 2003 in Europe were monoclonal. In contrast, several strains were in the United States in the 1980s, including a variant from Europe. PMID:21029543

Multilocus microsatellite typing (MLMT of strains from Turkey and Cyprus reveals a novel monophyletic L. donovani sensu lato group.

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Evi Gouzelou

Full Text Available BACKGROUND: New foci of human CL caused by strains of the Leishmania donovani (L. donovani complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE using the Montpellier (MON system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic. METHODOLOGY/PRINCIPAL FINDINGS: The Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains and from a VL patient in the south-west (Kuşadasi; EP59 strain. These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains and MON-308 (EP59. A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey. CONCLUSION: The discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding

Effects of canine parvovirus strain variations on diagnostic test results and clinical management of enteritis in dogs.

Science.gov (United States)

Markovich, Jessica E; Stucker, Karla M; Carr, Alaina H; Harbison, Carole E; Scarlett, Janet M; Parrish, Colin R

2012-07-01

To estimate the prevalence of canine parvovirus (CPV) strains among dogs with enteritis admitted to a referral hospital in the southwestern United States during an 11-month period and to compare diagnostic test results, disease severity, and patient outcome among CPV strains. Prospective observational study. 72 dogs with histories and clinical signs of parvoviral enteritis. For each dog, a fecal sample or rectal swab specimen was evaluated for CPV antigen via an ELISA. Subsequently, fecal samples (n = 42 dogs) and pharyngeal swab specimens (16) were obtained and tested for CPV antigen via an ELISA and CPV DNA via a PCR assay. For specimens with CPV-positive results via PCR assay, genetic sequencing was performed to identify the CPV strain. 56 dogs tested positive for CPV via ELISA or PCR assay. For 42 fecal samples tested via both ELISA and PCR assay, 27 had positive results via both assays, whereas 6 had positive PCR assay results only. Ten pharyngeal swab specimens yielded positive PCR assay results. Genetic sequencing was performed on 34 fecal or pharyngeal swab specimens that had CPV-positive PCR assay results; 25 (73.5%) were identified as containing CPV type-2c, and 9 (26.5%) were identified as containing CPV type-2b. No association was found between CPV strain and disease severity or clinical outcome. CPV type-2b and CPV type-2c posed similar health risks for dogs; therefore, genetic sequencing of CPV does not appear necessary for clinical management of infected patients. The diagnostic tests used could detect CPV type-2c.

Characterization of Burkholderia pseudomallei Strains Using a Murine Intraperitoneal Infection Model and In Vitro Macrophage Assays.

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Susan L Welkos

and in the macrophage phagocytosis assay. Strains which were more virulent for mice (e.g., HBPU10304a were often less virulent in the macrophage assays, as determined by several parameters such as intracellular bacterial replication and host cell cytotoxicity.

Characterization of Burkholderia pseudomallei Strains Using a Murine Intraperitoneal Infection Model and In Vitro Macrophage Assays.

Science.gov (United States)

Welkos, Susan L; Klimko, Christopher P; Kern, Steven J; Bearss, Jeremy J; Bozue, Joel A; Bernhards, Robert C; Trevino, Sylvia R; Waag, David M; Amemiya, Kei; Worsham, Patricia L; Cote, Christopher K

2015-01-01

macrophage phagocytosis assay. Strains which were more virulent for mice (e.g., HBPU10304a) were often less virulent in the macrophage assays, as determined by several parameters such as intracellular bacterial replication and host cell cytotoxicity.

[Standard algorithm of molecular typing of Yersinia pestis strains].

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Eroshenko, G A; Odinokov, G N; Kukleva, L M; Pavlova, A I; Krasnov, Ia M; Shavina, N Iu; Guseva, N P; Vinogradova, N A; Kutyrev, V V

2012-01-01

Development of the standard algorithm of molecular typing of Yersinia pestis that ensures establishing of subspecies, biovar and focus membership of the studied isolate. Determination of the characteristic strain genotypes of plague infectious agent of main and nonmain subspecies from various natural foci of plague of the Russian Federation and the near abroad. Genotyping of 192 natural Y. pestis strains of main and nonmain subspecies was performed by using PCR methods, multilocus sequencing and multilocus analysis of variable tandem repeat number. A standard algorithm of molecular typing of plague infectious agent including several stages of Yersinia pestis differentiation by membership: in main and nonmain subspecies, various biovars of the main subspecies, specific subspecies; natural foci and geographic territories was developed. The algorithm is based on 3 typing methods--PCR, multilocus sequence typing and multilocus analysis of variable tandem repeat number using standard DNA targets--life support genes (terC, ilvN, inv, glpD, napA, rhaS and araC) and 7 loci of variable tandem repeats (ms01, ms04, ms06, ms07, ms46, ms62, ms70). The effectiveness of the developed algorithm is shown on the large number of natural Y. pestis strains. Characteristic sequence types of Y. pestis strains of various subspecies and biovars as well as MLVA7 genotypes of strains from natural foci of plague of the Russian Federation and the near abroad were established. The application of the developed algorithm will increase the effectiveness of epidemiologic monitoring of plague infectious agent, and analysis of epidemics and outbreaks of plague with establishing the source of origin of the strain and routes of introduction of the infection.

Mumps Hoshino and Torii vaccine strains were distinguished from circulating wild strains.

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Sawada, Akihito; Yamaji, Yoshiaki; Nakayama, Tetsuo

2013-06-01

Aseptic meningitis and acute parotitis have been observed after mumps vaccination. Mumps outbreaks have been reported in Japan because of low vaccine coverage, and molecular differentiation is required to determine whether these cases are vaccine associated. RT-nested PCR was performed in the small hydrophobic gene region, and viruses were differentiated by restriction fragment length polymorphism assay. A total of 584 nucleotides were amplified. The PCR product of the Hoshino strain was cut into two fragments (313 and 271 nucleotides) by MfeI; that of the Torii strain was digested with EcoT22I, resulting in 332- and 252-nucleotide fragments. Both strains were genotype B and had an XbaI site, resulting in two fragments: 299 and 285 nucleotides. Current circulating wild types were cut only by XbaI or MfeI. However, the MfeI site of the wild types was different from that of the Hoshino strain, resulting in 451- and 133-nucleotide fragments. Using three restriction enzymes, two mumps vaccine strains were distinguished from wild types, and this separation was applied to the identification of vaccine-related adverse events.

A novel rapid direct haemagglutination-inhibition assay for measurements of humoral immune response against non-haemagglutinating Fowlpox virus strains in vaccinated chickens.

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Wambura, Philemon N; Mzula, Alexanda

2017-10-01

Fowlpox (FP) is a serious disease in chickens caused by Fowlpox virus (FPV). One method currently used to control FPV is vaccination followed by confirmation that antibody titres are protective using the indirect haemagglutination assay (IHA). The direct haemagglutination inhibition (HI) assay is not done because most FPV strains do not agglutinate chicken red blood cells (RBCs). A novel FPV strain TPV-1 which agglutinates chicken RBCs was discovered recently and enabled a direct HI assay to be conducted using homologous sera. This study is therefore aimed at assessing the direct HI assay using a recently discovered novel haemagglutinating FPV strain TPV-1 in chickens vaccinated with a commercial vaccine containing a non-haemagglutinating FPV.Chicks vaccinated with FPV at 1 day-old had antibody geometric mean titres (GMT) of log 2 3.7 at 7 days after vaccination and log 2 8.0 at 28 days after vaccination when tested in the direct HI. Chickens vaccinated at 6 weeks-old had antibody geometric mean titres (GMT) of log 2 5.0 at 7 days after vaccination and log 2 8.4 at 28 days after vaccination when tested in the direct HI. The GMT recorded 28 days after vaccination was slightly higher in chickens vaccinated at 6-week-old than in chicks vaccinated at one-day-old. However, this difference was not significant (P > 0.05). All vaccinated chickens showed "takes". No antibody response to FPV and "takes" were detected in unvaccinated chickens (GMT 0.05). These findings indicate that a simple and rapid direct HI assay using the FPV TPV-1 strain as antigen may be used to measure antibody levels in chickens vaccinated with non-haemagglutinating strains of FPV, and that the titres are comparable to those obtained by indirect IHA.

Quality control assessment of human immunodeficiency virus type 2 (HIV-2) viral load quantification assays: results from an international collaboration on HIV-2 infection in 2006

NARCIS (Netherlands)

Damond, Florence; Benard, Antoine; Ruelle, Jean; Alabi, Abraham; Kupfer, Bernd; Gomes, Perpetua; Rodes, Berta; Albert, Jan; Böni, Jürg; Garson, Jeremy; Ferns, Bridget; Matheron, Sophie; Chene, Geneviève; Brun-Vezinet, Françoise; Goubau, Patrick; Campa, Pauline; Descamps, Diane; Simon, François; Taieb, Audrey; Autran, Brigitte; Cotten, Matt; Jaye, Assan; Peterson, Kevin; Rowland-Jones, Sarah; Rockstroh, Jürgen; Schwarze-Zander, Carolynne; de Wolf, Frank; van Sighem, Ard; Reiss, Peter; van der Loeff, Maarten Schim; Schutten, Martin; Camacho, Ricardo; Mansinho, Kamal; Antunes, Francisco; Luis, Franca; Valadas, Emilia; Toro, Carlos; Soriano, Vicente; Gyllensten, Katarina; Sonnerborg, Anders; Yilmaz, Aylin; Gisslén, Magnus; Calmy, Alexandra; Rickenbach, Martin; Pillay, Deenan; Tosswill, Jennifer; Anderson, Jane; Chadwick, David

2008-01-01

Human immunodeficiency virus type 2 (HIV-2) RNA quantification assays used in nine laboratories of the ACHI(E)V(2E) (A Collaboration on HIV-2 Infection) study group were evaluated. In a blinded experimental design, laboratories quantified three series of aliquots of an HIV-2 subtype A strain, each

Mutagenic and antimutagenic activities of Artemisia absinthium volatile oil by the bacterial reverse mutation assay in Salmonella typhimurium strains TA98 and TA100

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Mahboubeh Taherkhani

2014-09-01

Full Text Available Objective: To investigate the mutagenic and antimutagenic activities of Artemisia absinthium L. (A. absinthium essential oil by the bacterial reverse mutation assay in Salmonella typhimurium (S. typhimurium strains. Methods: Water-distilled essential oil of A. absinthium collected from Ardabil, NorthWestern Iran, was investigated for mutagenic and antimutagenic activities. In present study, the mutagenic and antimutagenic activities of A. absinthium oil were investigated by the bacterial revere mutation assay in S. typhimurium TA98 and TA100 strains with and without S9 (microsomal mutagenesis assay. Results: The comparative mutagenicity effect was seen in 1.5 mg/plate by the bacterial reverse mutation assay in S. typhimurium TA98 strains, without S9 and the excellent antimutagenicity effect was seen in 1.5 mg/plate against S. typhimurium TA100, without S9. Conclusions: The mutagenicity and antimutagenicity effects of the volatile oil of A. absinthium were seen without the presence of metabolic activation.

Discriminatory Indices of Typing Methods for Epidemiologic Analysis of Contemporary Staphylococcus aureus Strains.

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Rodriguez, Marcela; Hogan, Patrick G; Satola, Sarah W; Crispell, Emily; Wylie, Todd; Gao, Hongyu; Sodergren, Erica; Weinstock, George M; Burnham, Carey-Ann D; Fritz, Stephanie A

2015-09-01

Historically, a number of typing methods have been evaluated for Staphylococcus aureus strain characterization. The emergence of contemporary strains of community-associated S. aureus, and the ensuing epidemic with a predominant strain type (USA300), necessitates re-evaluation of the discriminatory power of these typing methods for discerning molecular epidemiology and transmission dynamics, essential to investigations of hospital and community outbreaks. We compared the discriminatory index of 5 typing methods for contemporary S. aureus strain characterization. Children presenting to St. Louis Children's Hospital and community pediatric practices in St. Louis, Missouri (MO), with community-associated S. aureus infections were enrolled. Repetitive sequence-based PCR (repPCR), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal protein A (spa), and staphylococcal cassette chromosome (SCC) mec typing were performed on 200 S. aureus isolates. The discriminatory index of each method was calculated using the standard formula for this metric, where a value of 1 is highly discriminatory and a value of 0 is not discriminatory. Overall, we identified 26 distinct strain types by repPCR, 17 strain types by PFGE, 30 strain types by MLST, 68 strain types by spa typing, and 5 strain types by SCCmec typing. RepPCR had the highest discriminatory index (D) of all methods (D = 0.88), followed by spa typing (D = 0.87), MLST (D = 0.84), PFGE (D = 0.76), and SCCmec typing (D = 0.60). The method with the highest D among MRSA isolates was repPCR (D = 0.64) followed by spa typing (D = 0.45) and MLST (D = 0.44). The method with the highest D among MSSA isolates was spa typing (D = 0.98), followed by MLST (D = 0.93), repPCR (D = 0.92), and PFGE (D = 0.89). Among isolates designated USA300 by PFGE, repPCR was most discriminatory, with 10 distinct strain types identified (D = 0.63). We identified 45

A large portion of meningococcal antigen typing system-negative meningococcal strains from spain is killed by sera from adolescents and infants immunized with 4CMenB.

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Abad, R; Biolchi, A; Moschioni, M; Giuliani, M M; Pizza, M; Vázquez, J A

2015-04-01

A new vaccine (the 4CMenB 4-component protein vaccine [Bexsero], which includes PorA, factor H-binding protein [fHbp], neisserial heparin-binding antigen [NHBA], and Neisseria adhesin A [NadA]) against serogroup B meningococci has recently been approved for use in people older than age 2 months in Europe, Australia, and Canada. Preapproval clinical efficacy studies are not feasible for invasive meningococcal disease because its incidence is low/very low, and the serum bactericidal antibody (SBA) titer (or the human SBA [hSBA] titer when human complement is used in the assay) has been used as a surrogate marker of protection. However, the hSBA assay cannot be used on a large scale, and therefore, a meningococcal antigen typing system (MATS) was developed. MATS combines conventional PorA genotyping with an enzyme-linked immunosorbent assay (ELISA) that quantifies both the expression and the cross-reactivity of antigenic variants. The assay has been used to evaluate the potential of the 4CMenB meningococcal group B vaccine to cover group B strains in several countries. Some recent data suggest that MATS is a conservative predictor of strain coverage. We used pooled sera from adolescents and infants to test by the hSBA assay 10 meningococcal group B strains isolated in Spain that were negative for the 3 antigens (n = 9) or that had very low levels of the 3 antigens (n = 1) by MATS. We found that all strains were killed by sera from adolescents and that 5 of the 10 strains were also killed, although at a low titer, by sera from infants. Our data confirm that MATS underestimates vaccine coverage. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DNA type analysis to differentiate strains of Xylophilus ampelinus from Europe and Hokkaido, Japan

OpenAIRE

Komatsu, Tsutomu; Shinmura, Akinori; Kondo, Norio

2016-01-01

Strains of the bacterium Xylophilus ampelinus were collected from Europe and Hokkaido, Japan. Genomic fingerprints generated from 43 strains revealed four DNA types (A-D) using the combined results of Rep-, ERIC-, and Box-PCR. Genetic variation was found among the strains examined; strains collected from Europe belonged to DNA types A or B, and strains collected from Hokkaido belonged to DNA types C or D. However, strains belonging to each DNA type showed the same pathogenicity to grapevines ...

Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains

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K.C. Médici

2010-01-01

Full Text Available Group B rotaviruses (RV-B were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8 were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7% nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8% identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8%. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species.

A duck hepatitis B virus strain with a knockout mutation in the putative X ORF shows similar infectivity and in vivo growth characteristics to wild-type virus

International Nuclear Information System (INIS)

Meier, P.; Scougall, C.A.; Will, H.; Burrell, C.J.; Jilbert, A.R.

2003-01-01

Hepadnaviruses including human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) express X proteins, HBx and DHBx, respectively. Both HBx and DHBx are transcriptional activators and modulate cellular signaling in in vitro assays. To test whether the DHBx protein plays a role in virus infection, we compared the in vivo infectivity and growth characteristics of a DHBV3 strain with a stop codon in the X-like ORF (DHBV3-X-K.O.) to those of the wild-type DHBV3 strain. Here we report that the two strains showed no significant difference in (i) their ability to induce infection that resulted in stable viraemia measured by serum surface antigen (DHBsAg) and DHBV DNA, and detection of viral proteins and replicative DNA intermediates in the liver; (ii) the rate of spread of infection in liver and extrahepatic sites after low-dose virus inoculation; and (iii) the ability to produce transient or persistent infection under balanced age/dose conditions designed to detect small differences between the strains. Thus, none of the infection parameters assayed were detectably affected by the X-ORF knockout mutation, raising the question whether DHBx expression plays a physiological role during in vivo infection with wild-type DHBV

Reverse transcription loop-mediated isothermal amplification assays for rapid identification of eastern and western strains of bluetongue virus in India.

Science.gov (United States)

Maan, S; Maan, N S; Batra, K; Kumar, A; Gupta, A; Rao, Panduranga P; Hemadri, Divakar; Reddy, Yella Narasimha; Guimera, M; Belaganahalli, M N; Mertens, P P C

2016-08-01

Bluetongue virus (BTV) infects all ruminants, including cattle, goats and camelids, causing bluetongue disease (BT) that is often severe in naïve deer and sheep. Reverse-transcription-loop-mediated-isothermal-amplification (RT-LAMP) assays were developed to detect eastern or western topotype of BTV strains circulating in India. Each assay uses four primers recognizing six distinct sequences of BTV genome-segment 1 (Seg-1). The eastern (e)RT-LAMP and western (w)RT-LAMP assay detected BTV RNA in all positive isolates that were tested (n=52, including Indian BTV-1, -2, -3, -5, -9, -10, -16, -21 -23, and -24 strains) with high specificity and efficiency. The analytical sensitivity of the RT-LAMP assays is comparable to real-time RT-PCR, but higher than conventional RT-PCR. The accelerated eRT-LAMP and wRT-LAMP assays generated detectable levels of amplified DNA, down to 0.216 fg of BTV RNA template or 108 fg of BTV RNA template within 60-90min respectively. The assays gave negative results with RNA from foot-and-mouth-disease virus (FMDV), peste des petits ruminants virus (PPRV), or DNA from Capripox viruses and Orf virus (n=10), all of which can cause clinical signs similar to BT. Both RT-LAMP assays did not show any cross-reaction among themselves. The assays are rapid, easy to perform, could be adapted as a 'penside' test making them suitable for 'front-line' diagnosis, helping to identify and contain field outbreaks of BTV. Copyright © 2016 Elsevier B.V. All rights reserved.

Differentiation of highly virulent strains of Streptococcus suis serotype 2 according to glutamate dehydrogenase electrophoretic and sequence type.

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Kutz, Russell; Okwumabua, Ogi

2008-10-01

The glutamate dehydrogenase (GDH) enzymes of 19 Streptococcus suis serotype 2 strains, consisting of 18 swine isolates and 1 human clinical isolate from a geographically varied collection, were analyzed by activity staining on a nondenaturing gel. All seven (100%) of the highly virulent strains tested produced an electrophoretic type (ET) distinct from those of moderately virulent and nonvirulent strains. By PCR and nucleotide sequence determination, the gdh genes of the 19 strains and of 2 highly virulent strains involved in recent Chinese outbreaks yielded a 1,820-bp fragment containing an open reading frame of 1,344 nucleotides, which encodes a protein of 448 amino acid residues with a calculated molecular mass of approximately 49 kDa. The nucleotide sequences contained base pair differences, but most were silent. Cluster analysis of the deduced amino acid sequences separated the isolates into three groups. Group I (ETI) consisted of the seven highly virulent isolates and the two Chinese outbreak strains, containing Ala(299)-to-Ser, Glu(305)-to-Lys, and Glu(330)-to-Lys amino acid substitutions compared with groups II and III (ETII). Groups II and III consisted of moderately virulent and nonvirulent strains, which are separated from each other by Tyr(72)-to-Asp and Thr(296)-to-Ala substitutions. Gene exchange studies resulted in the change of ETI to ETII and vice versa. A spectrophotometric activity assay for GDH did not show significant differences between the groups. These results suggest that the GDH ETs and sequence types may serve as useful markers in predicting the pathogenic behavior of strains of this serotype and that the molecular basis for the observed differences in the ETs was amino acid substitutions and not deletion, insertion, or processing uniqueness.

Extracellular enzyme activities during lignocellulose degradation by Streptomyces spp.: a comparative study of wild-type and genetically manipulated strains

International Nuclear Information System (INIS)

Ramachandra, M.; Crawford, D.L.; Pometto, A.L. III

1987-01-01

The wild-type ligninolytic actinomycete Streptomyces viridosporus T7A and two genetically manipulated strains with enhanced abilities to produce a water-soluble lignin degradation intermediate, an acid-precipitable polymeric lignin (APPL), were grown on lignocellulose in solid-state fermentation cultures. Culture filtrates were periodically collected, analyzed for APPL, and assayed for extracellular lignocellulose-catabolizing enzyme activities. Two APPL-overproducing strains, UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10, had higher and longer persisting peroxidase, esterase, and endoglucanase activities than did the wild-type strain T7A. Results implicated one or more of these enzymes in lignin solubilization. Only mutant T7A-81 had higher xylanase activity than the wild type. The peroxidase was induced by both lignocellulose and APPL. This extracellular enzyme has some similarities to previously described ligninases in fungi. This is the first report of such an enzyme in Streptomyces spp. Four peroxidase isozymes were present, and all catalyzed the oxidation of 3,4-dihydroxyphenylalanine, while one also catalyzed hydrogen peroxide-dependent oxidation of homoprotocatechuic acid and caffeic acid. Three constitutive esterase isozymes were produced which differed in substrate specificity toward α-naphthyl acetate and α-naphthyl butyrate. Three endoglucanase bands, which also exhibited a low level of xylanase activity, were identified on polyacrylamide gels as was one xylanase-specific band. There were no major differences in the isoenzymes produced by the different strains. The probable role of each enzyme in lignocellulose degradation is discussed

A Novel Duplex Real-Time Reverse-Transcription PCR Assay for the Detection of Influenza A and the Novel Influenza A(H1N1 Strain

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Theo P. Sloots

2009-12-01

Full Text Available Timely implementation of antiviral treatment and other public health based responses are dependent on accurate and rapid diagnosis of the novel pandemic influenza A(H1N1 strain. In this study we developed a duplex real-time PCR (RT-PCR (dFLU-TM assay for the simultaneous detection of a broad range of influenza A subtypes and specific detection of the novel H1N1 2009 pandemic strain. The assay was compared to the combined results of two previously described monoplex RT-PCR assays using 183 clinical samples and 10 seasonal influenza A isolates. Overall, the results showed that the dFLU-TM RT-PCR method is suitable for detection of influenza A, including the novel H1N1 pandemic strain, in clinical samples.

Recognition of Highly Diverse Type-1 and -2 Porcine Reproductive and Respiratory Syndrome Viruses (PRRSVs by T-Lymphocytes Induced in Pigs after Experimental Infection with a Type-2 PRRSV Strain.

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Chungwon J Chung

Full Text Available Live attenuated vaccines confer partial protection in pigs before the appearance of neutralizing antibodies, suggesting the contribution of cell-mediated immunity (CMI. However, PRRSV-specific T-lymphocyte responses and protective mechanisms need to be further defined. To this end, the hypothesis was tested that PRRSV-specific T-lymphocytes induced by exposure to type-2 PRRSV can recognize diverse isolates.An IFN-gamma ELISpot assay was used to enumerate PRRSV-specific T-lymphocytes from PRRSVSD23983-infected gilts and piglets born after in utero infection against 12 serologically and genetically distinct type-1 and -2 PRRSV isolates. The IFN-gamma ELISpot assay using synthetic peptides spanning all open reading frames of PRRSVSD23983 was utilized to localize epitopes recognized by T-lymphocytes. Virus neutralization tests were carried out using the challenge strain (type-2 PRRSVSD23983 and another strain (type-2 PRRSVVR2332 with high genetic similarity to evaluate cross-reactivity of neutralizing antibodies in gilts after PRRSVSD23983 infection.At 72 days post infection, T-lymphocytes from one of three PRRSVSD23983-infected gilts recognized all 12 diverse PRRSV isolates, while T-lymphocytes from the other two gilts recognized all but one isolate. Furthermore, five of nine 14-day-old piglets infected in utero with PRRSVSD23983 had broadly reactive T-lymphocytes, including one piglet that recognized all 12 isolates. Overlapping peptides encompassing all open reading frames of PRRSVSD23983 were used to identify ≥28 peptides with T-lymphocyte epitopes from 10 viral proteins. This included one peptide from the M protein that was recognized by T-lymphocytes from all three gilts representing two completely mismatched MHC haplotypes. In contrast to the broadly reactive T-lymphocytes, neutralizing antibody responses were specific to the infecting PRRSVSD23983 isolate.These results demonstrated that T-lymphocytes recognizing antigenically and

Brunenders: a partially attenuated historic poliovirus type I vaccine strain.

Science.gov (United States)

Sanders, Barbara P; Liu, Ying; Brandjes, Alies; van Hoek, Vladimir; de Los Rios Oakes, Isabel; Lewis, John; Wimmer, Eckard; Custers, Jerome H H V; Schuitemaker, Hanneke; Cello, Jeronimo; Edo-Matas, Diana

2015-09-01

Brunenders, a type I poliovirus (PV) strain, was developed in 1952 by J. F. Enders and colleagues through serial in vitro passaging of the parental Brunhilde strain, and was reported to display partial neuroattenuation in monkeys. This phenotype of attenuation encouraged two vaccine manufacturers to adopt Brunenders as the type I component for their inactivated poliovirus vaccines (IPVs) in the 1950s, although today no licensed IPV vaccine contains Brunenders. Here we confirmed, in a transgenic mouse model, the report of Enders on the reduced neurovirulence of Brunenders. Although dramatically neuroattenuated relative to WT PV strains, Brunenders remains more virulent than the attenuated oral vaccine strain, Sabin 1. Importantly, the neuroattenuation of Brunenders does not affect in vitro growth kinetics and in vitro antigenicity, which were similar to those of Mahoney, the conventional type I IPV vaccine strain. We showed, by full nucleotide sequencing, that Brunhilde and Brunenders differ at 31 nucleotides, eight of which lead to amino acid changes, all located in the capsid. Upon exchanging the Brunenders capsid sequence with that of the Mahoney capsid, WT neurovirulence was regained in vivo, suggesting a role for the capsid mutations in Brunenders attenuation. To date, as polio eradication draws closer, the switch to using attenuated strains for IPV is actively being pursued. Brunenders preceded this novel strategy as a partially attenuated IPV strain, accompanied by decades of successful use in the field. Providing data on the attenuation of Brunenders may be of value in the further construction of attenuated PV strains to support the grand pursuit of the global eradication of poliomyelitis.

Evaluation of genotype MTBDRplus assay for rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis clinical isolates from Pakistan

Directory of Open Access Journals (Sweden)

Hasnain Javed

2016-01-01

Conclusions: As evidenced in this study, the major concern with the GenoType MTBDRplus assay were false negative results. In comparison to conventional drug susceptibility testing, the assay was unable to detect 30 (30/100; 30% strains resistant to INH and 23 (23/100; 23% strains resistant to RMP. The GenoType MTBDRplus failed to identify 38 MDR (38/100; 38% strains. Resistance in those strains probably originate from mutations in other codons and/or genes than those covered by the test. For detecting INH and RMP resistance in TB cases, especially in high TB incidence countries, such as Pakistan, molecular approaches should still be a complement rather than areplacement to conventional drug susceptibility testing.

Sensitivity and specificity of four assays to detect human T-lymphotropic virus type I or type I/II antibodies

NARCIS (Netherlands)

Vrielink, H.; Reesink, H. W.; Zaaijer, H. L.; van der Poel, C. L.; Cuypers, H. T.; Lelie, P. N.

1996-01-01

BACKGROUND: Assays that detect human T-lymphotropic virus type I and type II antibody (HTLV-I/II) are widely used in the routine screening of blood donors. STUDY DESIGN AND METHODS: Four commercially available anti-HTLV-I (Fujirebio and Organon Teknika) or -HTLV-I/II assays (Murex and Ortho) were

Use of colony-based bacterial strain typing for tracking the fate of Lactobacillus strains during human consumption

Directory of Open Access Journals (Sweden)

Drevinek Pavel

2009-12-01

Full Text Available Abstract Background The Lactic Acid Bacteria (LAB are important components of the healthy gut flora and have been used extensively as probiotics. Understanding the cultivable diversity of LAB before and after probiotic administration, and being able to track the fate of administered probiotic isolates during feeding are important parameters to consider in the design of clinical trials to assess probiotic efficacy. Several methods may be used to identify bacteria at the strain level, however, PCR-based methods such as Random Amplified Polymorphic DNA (RAPD are particularly suited to rapid analysis. We examined the cultivable diversity of LAB in the human gut before and after feeding with two Lactobacillus strains, and also tracked the fate of these two administered strains using a RAPD technique. Results A RAPD typing scheme was developed to genetically type LAB isolates from a wide range of species, and optimised for direct application to bacterial colony growth. A high-throughput strategy for fingerprinting the cultivable diversity of human faeces was developed and used to determine: (i the initial cultivable LAB strain diversity in the human gut, and (ii the fate of two Lactobacillus strains (Lactobacillus salivarius NCIMB 30211 and Lactobacillus acidophilus NCIMB 30156 contained within a capsule that was administered in a small-scale human feeding study. The L. salivarius strain was not cultivated from the faeces of any of the 12 volunteers prior to capsule administration, but appeared post-feeding in four. Strains matching the L. acidophilus NCIMB 30156 feeding strain were found in the faeces of three volunteers prior to consumption; after taking the Lactobacillus capsule, 10 of the 12 volunteers were culture positive for this strain. The appearance of both Lactobacillus strains during capsule consumption was statistically significant (p Conclusion We have shown that genetic strain typing of the cultivable human gut microbiota can be

High resolution melting curve analysis as a new tool for rapid identification of canine parvovirus type 2 strains.

Science.gov (United States)

Bingga, Gali; Liu, Zhicheng; Zhang, Jianfeng; Zhu, Yujun; Lin, Lifeng; Ding, Shuangyang; Guo, Pengju

2014-01-01

A high resolution melting (HRM) curve method was developed to identify canine parvovirus type 2 (CPV-2) strains by nested PCR. Two sets of primers, CPV-426F/426R and CPV-87R/87F, were designed that amplified a 52 bp and 53 bp product from the viral VP2 capsid gene. The region amplified by CPV-426F/426R included the A4062G and T4064A mutations in CPV-2a, CPV-2b and CPV-2c. The region amplified by CPV-87F/87R included the A3045T mutation in the vaccine strains of CPV-2 and CPV-2a, CPV-2b and CPV-2c. Faecal samples were obtained from 30 dogs that were CPV antigen-positive. The DNA was isolated from the faecal samples and PCR-amplified using the two sets of primers, and genotyped by HRM curve analysis. The PCR-HRM assay was able to distinguish single nucleotide polymorphisms between CPV-2a, CPV-2b and CPV-2c using CPV-426F/426R. CPV-2a was distinguished from CPV-2b and CPV-2c by differences in the melting temperature. CPV-2b and CPV-2c could be distinguished based on the shape of the melting curve after generating heteroduplexes using a CPV-2b reference sample. The vaccine strains of CPV-2 were identified using CPV-87F/87R. Conventional methods for genotyping CPV strains are labor intensive, expensive or time consuming; the present PCR-based HRM assay might be an attractive alternative. Copyright © 2014 Elsevier Ltd. All rights reserved.

Complete genome sequence of Rhodospirillum rubrum type strain (S1).

Science.gov (United States)

Munk, A Christine; Copeland, Alex; Lucas, Susan; Lapidus, Alla; Del Rio, Tijana Glavina; Barry, Kerrie; Detter, John C; Hammon, Nancy; Israni, Sanjay; Pitluck, Sam; Brettin, Thomas; Bruce, David; Han, Cliff; Tapia, Roxanne; Gilna, Paul; Schmutz, Jeremy; Larimer, Frank; Land, Miriam; Kyrpides, Nikos C; Mavromatis, Konstantinos; Richardson, Paul; Rohde, Manfred; Göker, Markus; Klenk, Hans-Peter; Zhang, Yaoping; Roberts, Gary P; Reslewic, Susan; Schwartz, David C

2011-07-01

Rhodospirillum rubrum (Esmarch 1887) Molisch 1907 is the type species of the genus Rhodospirillum, which is the type genus of the family Rhodospirillaceae in the class Alphaproteobacteria. The species is of special interest because it is an anoxygenic phototroph that produces extracellular elemental sulfur (instead of oxygen) while harvesting light. It contains one of the most simple photosynthetic systems currently known, lacking light harvesting complex 2. Strain S1(T) can grow on carbon monoxide as sole energy source. With currently over 1,750 PubMed entries, R. rubrum is one of the most intensively studied microbial species, in particular for physiological and genetic studies. Next to R. centenum strain SW, the genome sequence of strain S1(T) is only the second genome of a member of the genus Rhodospirillum to be published, but the first type strain genome from the genus. The 4,352,825 bp long chromosome and 53,732 bp plasmid with a total of 3,850 protein-coding and 83 RNA genes were sequenced as part of the DOE Joint Genome Institute Program DOEM 2002.

Molecular diversity of neurotoxins from Clostridium botulinum type D strains.

OpenAIRE

Moriishi, K; Syuto, B; Kubo, S; Oguma, K

1989-01-01

The molecular properties of Clostridium botulinum type D South African (D-SA) were compared with those of neurotoxins from type D strain 1873 (D-1873) and type C strains Stockholm and 6813. D-SA toxin, purified 610-fold from the culture supernatant in an overall yield of 30%, consisted of an intact peptide chain with a molecular weight of 140,000. Limited proteolysis of the toxin by trypsin formed a dichain structure consisting of a light chain (Mr, 50,000) and a heavy chain (Mr, 90,000) link...

In vitro profiling of antimethicillin-resistant Staphylococcus aureus activity of thymoquinone against selected type and clinical strains.

Science.gov (United States)

Hariharan, P; Paul-Satyaseela, M; Gnanamani, A

2016-03-01

This study explores antimethicillin-resistant Staphylococcus aureus (MRSA) activity of a bioactive phytochemical constituent, thymoquinone obtained from the medicinal herb, Nigella sativa Linn. Based on initial assessment on crude extract of seeds of Nigella sativa Linn, the pure active constituent was employed in the study. A total of 99 MRSA strains which comprised of 40 types and 59 clinical strains were selected for the study. Minimum inhibitory concentration (MIC), bactericidal activity, postantibiotic effect (PAE) and propensity to select resistant mutants were determined using standard protocols. Results revealed that thymoquinone exhibited MIC in the range of 8-16 μg ml(-1) and MIC90 of 16 μg ml(-1) against MRSA strains. It was bactericidal to MRSA by demonstrating >3 log kill. It showed a longer PAE of 3·2 ± 0·2 h. Upon exposure to high-density inoculum of MRSA, it did not select resistant mutants. Transmission electron microscopy of thymoquinone-treated MRSA showed no lysis but damage to cell wall and cell membrane which corroborated well with the salt tolerance and bacteriolysis assays. In conclusion, MIC90 , bactericidal property, longer PAE, absence of resistant mutant selection and damages in cell membrane and cell wall imply a promising anti-MRSA activity of thymoquinone. This is the first detailed report on anti-MRSA activity of thymoquinone. The assessment was made with both type and clinical strains. Thymoquinone may be a potential lead compound which can be further optimized to discover novel anti-MRSA agents. © 2016 The Society for Applied Microbiology.

High-resolution melt PCR analysis for rapid identification of Chlamydia abortus live vaccine strain 1B among C. abortus strains and field isolates.

Science.gov (United States)

Vorimore, Fabien; Cavanna, Noémie; Vicari, Nadia; Magnino, Simone; Willems, Hermann; Rodolakis, Annie; Siarkou, Victoria I; Laroucau, Karine

2012-09-01

We describe a novel high-resolution melt assay that clearly differentiates Chlamydia abortus live vaccine strain 1B from field C. abortus strains and field wild-type isolates based on previously described single nucleotide polymorphisms. This modern genotyping technique is inexpensive, easy to use, and less time-consuming than PCR-RFLP. Copyright © 2012 Elsevier B.V. All rights reserved.

Types of strain among family members of individuals with autism spectrum disorder across the lifespan.

Science.gov (United States)

Shivers, Carolyn M; Krizova, Katarina; Lee, Gloria K

2017-09-01

Although increased caregiver strain is often found among family caregivers of individuals with autism spectrum disorder, it is still unclear as to how different types of strain relate to amount and types of caregiving across the lifespan. The present study examined different types of strain (i.e. subjective internalized strain, subjective externalized strain, and objective strain) and how such strain relates to the amount of caregiving responsibilities. Data was collected via online survey from a sample of 193 family caregivers of individuals with ASD from the United States, Canada, and the Republic of Ireland. Participants completed measures of strain and caregiving responsibilities, as well as coping, demographics, and services needed and received by the individual with ASD. Caregivers reported higher levels of objective strain than subjective, and caregiving responsibility was related to objective and subjective internalized strain. Coping style was strongly correlated with all types of strain, and unmet service needs were significantly related to objective and subjective internalized strain. Caregiving behaviors were only related to objective strain. The present results indicate that, although caregiving responsibility is related to objective and subjective internalized strain, the relationship is perhaps not as strong as the relationship between coping mechanisms and strain. Future research is needed to understand different types of strain and develop strategies to help caregivers. Copyright © 2017 Elsevier Ltd. All rights reserved.

Differentiation of the virulence potential of Campylobacter jejuni strains by use of gene transcription analysis and a caco-2 assay

DEFF Research Database (Denmark)

Poli, Vanessa Fadanelli Schoenardie; Thorsen, Line; Olesen, Inger

2012-01-01

properties were evaluated by analyzing transcriptions of the virulence genes cdtB, ciaB, cadF and the stress associated genes clpP, htrB using reverse transcription quantitative PCR (RT-qPCR) and by the ability of the C. jejuni strains to adhere to and invade Caco-2 cells. Similar cell survival and no growth...... gene, cipA between DFVF1099 and NCTC11168 resulting in a 14 amino acid deletion and 28 amino acid addition at the N and C terminal ends respectively of the CipA protein of DFVF1099. In contrast to DFVF1099, strains NCTC1168 and TB1048 were able to invade Caco-2 cells. Invasion ability was not affected...... expression of C. jejuni. The clinical strains appeared to be more virulent than the chicken isolate as measured by the Caco-2 invasion assay which could be due to differences in CipA functionality. The RT-qPCR analysis and Caco-2 assay showed to be useful tools for differentiating virulence potentials...

Pyroprinting: a rapid and flexible genotypic fingerprinting method for typing bacterial strains.

Science.gov (United States)

Black, Michael W; VanderKelen, Jennifer; Montana, Aldrin; Dekhtyar, Alexander; Neal, Emily; Goodman, Anya; Kitts, Christopher L

2014-10-01

Bacterial strain typing is commonly employed in studies involving epidemiology, population ecology, and microbial source tracking to identify sources of fecal contamination. Methods for differentiating strains generally use either a collection of phenotypic traits or rely on some interrogation of the bacterial genotype. This report introduces pyroprinting, a novel genotypic strain typing method that is rapid, inexpensive, and discriminating compared to the most sensitive methods already in use. Pyroprinting relies on the simultaneous pyrosequencing of polymorphic multicopy loci, such as the intergenic transcribed spacer regions of rRNA operons in bacterial genomes. Data generated by sequencing combinations of variable templates are reproducible and intrinsically digitized. The theory and development of pyroprinting in Escherichia coli, including the selection of similarity thresholds to define matches between isolates, are presented. The pyroprint-based strain differentiation limits and phylogenetic relevance compared to other typing methods are also explored. Pyroprinting is unique in its simplicity and, paradoxically, in its intrinsic complexity. This new approach serves as an excellent alternative to more cumbersome or less phylogenetically relevant strain typing methods. Copyright © 2014 Elsevier B.V. All rights reserved.

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using Clostridium thermocellum

Energy Technology Data Exchange (ETDEWEB)

Oguntimein, Gbekeloluwa B. [Morgan State Univ., Baltimore, MD (United States); Rodriguez, Jr., Miguel [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); National Lab., Oak Ridge, TN (United States). BioEnergy Science Center; Dumitrache, Alexandru [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); National Lab., Oak Ridge, TN (United States). BioEnergy Science Center; Shollenberger, Todd [National Renewable Energy Lab. (NREL), Golden, CO (United States); Decker, Stephen R. [National Renewable Energy Lab. (NREL), Golden, CO (United States); Davison, Brian H. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); National Lab., Oak Ridge, TN (United States). BioEnergy Science Center; Brown, Steven D. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); National Lab., Oak Ridge, TN (United States). BioEnergy Science Center; LanzaTech, Inc., Skokie, IL (United States)

2017-11-09

Here, to develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential. Clostridium thermocellum parent Δhpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentations when compared to the Δhpt strain. A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.

Discovering and differentiating new and emerging clonal populations of Chlamydia trachomatis with a novel shotgun cell culture harvest assay.

Science.gov (United States)

Somboonna, Naraporn; Mead, Sally; Liu, Jessica; Dean, Deborah

2008-03-01

Chlamydia trachomatis is the leading cause of preventable blindness and bacterial sexually transmitted diseases worldwide. Plaque assays have been used to clonally segregate laboratory-adapted C. trachomatis strains from mixed infections, but no assays have been reported to segregate clones from recent clinical samples. We developed a novel shotgun cell culture harvest assay for this purpose because we found that recent clinical samples do not form plaques. Clones were strain-typed by using outer membrane protein A and 16S rRNA sequences. Surprisingly, ocular trachoma reference strain A/SA-1 contained clones of Chlamydophila abortus. C. abortus primarily infects ruminants and pigs and has never been identified in populations where trachoma is endemic. Three clonal variants of reference strain Ba/Apache-2 were also identified. Our findings reflect the importance of clonal isolation in identifying constituents of mixed infections containing new or emerging strains and of viable clones for research to more fully understand the dynamics of in vivo strain-mixing, evolution, and disease pathogenesis.

[TYPING OF LEPTOSPIRA SPP. STRAINS BASED ON 16S rRNA].

Science.gov (United States)

Ostankova, Yu V; Semenov, A V; Stoyanova, N A; Tokarevich, N K; Lyubimova, N E; Petrova, O A; Ananina, Yu V; Petrov, E M

2016-01-01

Comparative typing of Leptospira spp. strain collection based on analysis of 16S RNA fragment. 2 pairs of primers were used for PCR, that jointly flank 1423b.p. sized fragment. Sequences of Leptospira spp. strain 16S rRNA, presented in the international database, were used for phylogenetic analysis. A high similarity, including interspecies, of the 16S fragment in Leptospira spp. strains was shown independently of the source, serovar and serogroup. Heterogeneity of the primary matrix, spontaneous mutations of hotspots and erroneous nucleotide couplings, characteristic for 16S sequence of pathogenic Leptospira spp. strains, are discussed. Molecular-genetic characteristic of certain reference Leptospira spp. strains by 16S sequence is obtained. Results of the studies give evidence on expedience of introduction into clinical practice of identification of Leptospira spp. by 16S sequence directly from the clinical material, that would allow to significantly reduce identification time, dismiss complex type-specific sera and other labor-intensive methods.

Evaluation of GenoType NTM-DR Assay for Identification of Mycobacterium chimaera.

Science.gov (United States)

Mok, Simone; Rogers, Thomas R; Fitzgibbon, Margaret

2017-06-01

Identification of species within the Mycobacterium avium complex (MAC) is difficult, and most current diagnostic laboratory tests cannot distinguish between species included in the complex. Differentiation of species within the MAC is important, as Mycobacterium chimaera has recently emerged as a major cause of invasive cardiovascular infections following open heart surgery. A new commercial diagnostic assay, GenoType NTM-DR ver. 1.0, is intended to differentiate between three species within the MAC, namely, Mycobacterium avium , Mycobacterium intracellulare , and Mycobacterium chimaera In this study, we investigated an archival collection of 173 MAC isolates using 16S rRNA and 16S-23S internal transcribed spacer (ITS) gene sequencing, and GenoType NTM-DR was evaluated for identifying M. chimaera and other species belonging to the MAC. Species identification of 157/173 (91%) isolates with the GenoType NTM-DR assay was in agreement with 16S rRNA and 16S-23S ITS gene sequencing results. Misidentification occurred with 16 isolates which belonged to four species included in the MAC that are rarely encountered in clinical specimens. Despite some limitations of this assay, GenoType NTM-DR had 100% specificity for identifying M. chimaera This novel assay will enable diagnostic laboratories to differentiate species belonging to the Mycobacterium avium complex and to accurately identify M. chimaera It can produce rapid results and is also more cost efficient than gene sequencing methods. Copyright © 2017 American Society for Microbiology.

Drosophila comet assay: insights, uses, and future perspectives

Science.gov (United States)

Gaivão, Isabel; Sierra, L. María

2014-01-01

The comet assay, a very useful tool in genotoxicity and DNA repair testing, is being applied to Drosophila melanogaster since around 15 years ago, by several research groups. This organism is a valuable model for all kind of processes related to human health, including DNA damage response. The assay has been performed mainly in vivo using different larvae cell types (from brain, midgut, hemolymph, and imaginal disk), but also in vitro with the S2 cell line. Since its first application, it has been used to analyze the genotoxicity and action mechanisms of different chemicals, demonstrating good sensitivity and proving its usefulness. Moreover, it is the only assay that can be used to analyze DNA repair in somatic cells in vivo, comparing the effects of chemicals in different repair strains, and to quantitate repair activities in vitro. Additionally, the comet assay in Drosophila, in vivo and in vitro, has been applied to study the influence of protein overexpression on genome integrity and degradation. Although the assay is well established, it could benefit from some research to determine optimal experimental design to standardize it, and then to allow comparisons among laboratories independently of the chosen cell type. PMID:25221574

Draft Genome Sequence of Mycobacterium chimaera Type Strain Fl-0169

Science.gov (United States)

We report the draft genome sequence of the type strain Mycobacterium chimaera Fl-0169T, a member of the Mycobacterium avium complex (MAC). M. chimaera Fl-0169T was isolated from a patient in Italy and is highly similar to strains of M. chimaera isolated in Ireland, though Fl-016...

[Investigation of OXA type beta-lactamases and PFGE patterns in Acinetobacter baumannii strains resistant to carbapenems].

Science.gov (United States)

Keyik, Serafettin; Arslan, Uğur; Türk Dağı, Hatice; Seyhan, Tuba; Fındık, Duygu

2014-10-01

Acinetobacter baumannii is an important opportunistic and multidrug-resistant pathogen leading to nosocomial infections. Over the last 10 years, a significant and threatening increase in resistance to carbapenems, mainly due to the dissemination of class D beta-lactamases, has been reported in A.baumannii worldwide. The most common types of beta-lactamases causing carbapenem resistance in A.baumannii are the OXA-23, OXA-24, OXA-40, OXA-58 and OXA-143 type serine beta-lactamases. The aim of this study was to investigate the presence of OXA type beta-lactamases in carbapenem-resistant A.baumannii strains and the clonal relationship between the strains. A total of 105 non-duplicate carbapenem-resistant A.baumannii strains isolated from various clinical samples (68 blood, 18 bronchoalveolar lavage, 13 drainage, 3 urine, 2 cerebrospinal fluid and 1 catheter samples) in the Microbiology Laboratories of Selcuk University, Meram (2009-2012) and Selcuklu (2007-2008) Medical School Hospitals, were included in the study. The isolates were identified by conventional methods and Phoenix 100 BD (BD Diagnostic, USA) and Vitek II (bioMerieux, France) automated systems. Carbapenem susceptibility test was performed by Kirby-Bauer disk diffusion method according to the CLSI standards. bla(OXA 23-like), bla(OXA 24-like), bla(OXA 58-like) and bla(OXA 51-like) genes were amplified by multiplex PCR assay and clonal relatedness was investigated by pulsed-field gel electrophoresis (PFGE) using ApaI enzyme. The bla(OXA 51-like) gene was determined in all carbapenem-resistant A.baumannii isolates, while the bla(OXA 23-like) and bla(OXA 58-like) genes were detected in 46.6% and 53.3% of isolates, respectively. However bla(OXA 24-like) gene was not demonstrated in any isolates. bla(OXA 23-like) gene was determined in both Meram and Selcuklu Medical School hospitals, but bla(OXA 58-like) gene was detected only in Meram Medical School hospital. PFGE analysis of the isolates revealed 32 different

Anthelmintic effect of Psidium guajava and Tagetes erecta on wild-type and Levamisole-resistant Caenorhabditis elegans strains.

Science.gov (United States)

Piña-Vázquez, Denia M; Mayoral-Peña, Zyanya; Gómez-Sánchez, Maricela; Salazar-Olivo, Luis A; Arellano-Carbajal, Fausto

2017-04-18

Psidium guajava and Tagetes erecta have been used traditionally to treat gastrointestinal parasites, but their active metabolites and mechanisms of action remain largely unknown. To evaluate the anthelmintic potential of Psidium guajava and Tagetes erecta extracts on Levamisole-sensitive and Levamisole-resistant strains of the model nematode Caenorhabditis elegans. Aqueous extracts of Psidium guajava (PGE) and Tagetes erecta (TEE) were assayed on locomotion and egg-laying behaviors of the wild-type (N2) and Levamisole-resistant (CB193) strains of Caenorhabditis elegans. Both extracts paralyzed wild-type and Levamisole-resistant nematodes in a dose-dependent manner. In wild-type worms, TEE 25mg/mL induced a 75% paralysis after 8h of treatment and PGE 25mg/mL induced a 100% paralysis after 4h of treatment. PGE exerted a similar paralyzing effect on N2 wild-type and CB193 Levamisole-resistant worms, while TEE only partially paralyzed CB193 worms. TEE 25mg/mL decreased N2 egg-laying by 65% with respect to the untreated control, while PGE did it by 40%. Psidium guajava leaves and Tagetes erecta flower-heads possess hydrosoluble compounds that block the motility of Caenorhabditis elegans by a mechanism different to that of the anthelmintic drug Levamisole. Effects are also observable on oviposition, which was diminished in the wild-type worms. The strong anthelmintic effects in crude extracts of these plants warrants future work to identify their active compounds and to elucidate their molecular mechanisms of action. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Genetic homogeneity of Clostridium botulinum type A1 strains with unique toxin gene clusters.

Science.gov (United States)

Raphael, Brian H; Luquez, Carolina; McCroskey, Loretta M; Joseph, Lavin A; Jacobson, Mark J; Johnson, Eric A; Maslanka, Susan E; Andreadis, Joanne D

2008-07-01

A group of five clonally related Clostridium botulinum type A strains isolated from different sources over a period of nearly 40 years harbored several conserved genetic properties. These strains contained a variant bont/A1 with five nucleotide polymorphisms compared to the gene in C. botulinum strain ATCC 3502. The strains also had a common toxin gene cluster composition (ha-/orfX+) similar to that associated with bont/A in type A strains containing an unexpressed bont/B [termed A(B) strains]. However, bont/B was not identified in the strains examined. Comparative genomic hybridization demonstrated identical genomic content among the strains relative to C. botulinum strain ATCC 3502. In addition, microarray data demonstrated the absence of several genes flanking the toxin gene cluster among the ha-/orfX+ A1 strains, suggesting the presence of genomic rearrangements with respect to this region compared to the C. botulinum ATCC 3502 strain. All five strains were shown to have identical flaA variable region nucleotide sequences. The pulsed-field gel electrophoresis patterns of the strains were indistinguishable when digested with SmaI, and a shift in the size of at least one band was observed in a single strain when digested with XhoI. These results demonstrate surprising genomic homogeneity among a cluster of unique C. botulinum type A strains of diverse origin.

Typing clinical and animal environment Aspergillus fumigatus gliotoxin producer strains isolated from Brazil by PCR-RFLP markers.

Science.gov (United States)

Soleiro, C A; Pena, G A; Cavaglieri, L R; Coelho, I; Keller, L M; Dalcero, A M; Rosa, C A R

2013-12-01

Aspergillus fumigatus, a well-known human and animal pathogen causing aspergillosis, has been historically identified by morphological and microscopic features. However, recent studies have shown that species identification on the basis of morphology alone is problematic. The aim of this work was to confirm the taxonomic state at specie level of a set of clinical (human and animal) and animal environment A. fumigatus strains identified by morphological criteria applying a PCR-RFLP assay by an in silico and in situ analysis with three restriction enzymes. The A. fumigatus gliotoxin-producing ability was also determined. Previous to the in situ PCR-RFLP analysis, an in silico assay with BccI, MspI and Sau3AI restriction enzymes was carried out. After that, these enzymes were used for in situ assay. All A. fumigatus strains isolated from corn silage, human aspergillosis and bovine mastitis and high per cent of the strains isolated from cereals, animal feedstuff and sorghum silage were able to produce high gliotoxin levels. Also, all these strains identified by morphological criteria as A. fumigatus, regardless of its isolation source, had band patterns according to A. fumigatus sensu stricto by PCR-RFLP markers. Aspergillus fumigatus is a well-known human and animal pathogen causing aspergillosis. In this study, clinical (human and animal) and animal environment strains were able to produce high gliotoxin levels and had band profiles according to A. fumigatus sensu stricto by PCR-RFLP markers. The results obtained here suggest that strains involved in human and animal aspergillosis could come from the animal environment in which A. fumigatus is frequently found. Its presence in animal environments could affect animal health and productivity; in addition, there are risks of contamination for rural workers during handling and storage of animal feedstuffs. © 2013 The Society for Applied Microbiology.

Analysis by rotavirus gene 6 reverse transcriptase-polymerase chain reaction assay of rotavirus-positive gastroenteritis cases observed during the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST)

Science.gov (United States)

Matson, David O; Vesikari, Timo; Dennehy, Penelope; Dallas, Michael D; Goveia, Michelle G; Itzler, Robbin F; Ciarlet, Max

2014-01-01

During the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST), the period between the administration of dose 1 through 13 days after the administration of dose 3, there were more wild-type rotavirus gastroenteritis (RVGE) cases among vaccine recipients compared with placebo recipients using the protocol-specified microbiological plaque assay in the clinical-efficacy cohort, a subset of subjects where vaccine efficacy against RVGE of any severity was assessed. In this study, a rotavirus genome segment 6-based reverse transcriptase–polymerase chain reaction assay was applied post hoc to clarify the accuracy of type categorization of all these RVGE cases in vaccine recipients during the vaccination phase of REST. The assay characterized 147 (90%) of 163 re-assayed RVGE cases or rotavirus-associated health care contacts as type-determinable: either wild-type or vaccine-type rotavirus strains. In the clinical-efficacy cohort (N = 5673), 19 (18.8%) of 101 samples from RVGE cases contained wild-type rotavirus, 70 (69.3%) vaccine virus, and 12 (11.9%) were indeterminable. In the large-scale cohort (N = 68,038), 10 (34.5%) of 29 samples from RVGE-related health care contacts contained wild-type rotavirus strains, 15 (51.7%) vaccine-type rotavirus strains, and 4 (13.8%) were indeterminable. Of the 33 samples from RVGE cases in placebo recipients, all were confirmed to contain wild-type rotaviruses. Altogether, this post-hoc re-evaluation showed that the majority (75%) of type-determinable RVGE cases or health care contacts that occurred during the vaccination phase of REST in vaccine recipients were associated with vaccine-type rotavirus strains rather than wild-type rotavirus strains. PMID:25424931

Analysis by rotavirus gene 6 reverse transcriptase-polymerase chain reaction assay of rotavirus-positive gastroenteritis cases observed during the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST).

Science.gov (United States)

Matson, David O; Vesikari, Timo; Dennehy, Penelope; Dallas, Michael D; Goveia, Michelle G; Itzler, Robbin F; Ciarlet, Max

2014-01-01

During the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST), the period between the administration of dose 1 through 13 days after the administration of dose 3, there were more wild-type rotavirus gastroenteritis (RVGE) cases among vaccine recipients compared with placebo recipients using the protocol-specified microbiological plaque assay in the clinical-efficacy cohort, a subset of subjects where vaccine efficacy against RVGE of any severity was assessed. In this study, a rotavirus genome segment 6-based reverse transcriptase-polymerase chain reaction assay was applied post hoc to clarify the accuracy of type categorization of all these RVGE cases in vaccine recipients during the vaccination phase of REST. The assay characterized 147 (90%) of 163 re-assayed RVGE cases or rotavirus-associated health care contacts as type-determinable: either wild-type or vaccine-type rotavirus strains. In the clinical-efficacy cohort (N = 5673), 19 (18.8%) of 101 samples from RVGE cases contained wild-type rotavirus, 70 (69.3%) vaccine virus, and 12 (11.9%) were indeterminable. In the large-scale cohort (N = 68,038), 10 (34.5%) of 29 samples from RVGE-related health care contacts contained wild-type rotavirus strains, 15 (51.7%) vaccine-type rotavirus strains, and 4 (13.8%) were indeterminable. Of the 33 samples from RVGE cases in placebo recipients, all were confirmed to contain wild-type rotaviruses. Altogether, this post-hoc re-evaluation showed that the majority (75%) of type-determinable RVGE cases or health care contacts that occurred during the vaccination phase of REST in vaccine recipients were associated with vaccine-type rotavirus strains rather than wild-type rotavirus strains.

Differentiation of herpes simplex virus types 1 and 2 in clinical samples by a real-time taqman PCR assay.

Science.gov (United States)

Corey, Lawrence; Huang, Meei-Li; Selke, Stacy; Wald, Anna

2005-07-01

While the clinical manifestations of HSV-1 and -2 overlap, the site of CNS infection, complications, response to antivirals, frequency of antiviral resistance, and reactivation rate on mucosal surfaces varies between HSV-1 and -2. Detection of HSV DNA by PCR has been shown to be the most sensitive method for detecting HSV in clinical samples. As such, we developed a PCR-based assay to accurately distinguish HSV-1 from HSV-2. Our initial studies indicated the assay using type specific primers was slightly less efficient for detecting HSV-1 and -2 DNA than the high throughput quantitative PCR assay we utilize that employs type common primers to gB. We subsequently evaluated the type specific assay on 3,131 specimens that had HSV DNA detected in the type common PCR assay. The typing results of these specimens were compared with the monoclonal antibody staining results of culture isolates collected from the same patients at the same time, and the HSV serologic status of the patient. The typing assay accurately identified both HSV-1 and -2 with a specificity of >99.5% and was significantly more sensitive than typing by culture and subsequent monoclonal antibody assays. Complete concordance was seen between the typing assay and HSV serologic status of the patient. Dual (HSV-1 and -2) infection in clinical samples was recognized in 2.6% of clinical samples using the new typing assay. This assay, when used in combination with the type common assay, can now accurately type almost all mucosal and visceral HSV isolates by molecular techniques. Copyright (c) 2005 Wiley-Liss, Inc.

Genome Sequences of Three Vaccine Strains and Two Wild-Type Canine Distemper Virus Strains from a Recent Disease Outbreak in South Africa.

Science.gov (United States)

Loots, Angelika K; Du Plessis, Morné; Dalton, Desiré Lee; Mitchell, Emily; Venter, Estelle H

2017-07-06

Canine distemper virus causes global multihost infectious disease. This report details complete genome sequences of three vaccine and two new wild-type strains. The wild-type strains belong to the South African lineage, and all three vaccine strains to the America 1 lineage. This constitutes the first genomic sequences of this virus from South Africa. Copyright © 2017 Loots et al.

Real-time reverse transcription-polymerase chain reaction assays for identification of wild poliovirus 1 & 3.

Science.gov (United States)

Sharma, Deepa K; Nalavade, Uma P; Deshpande, Jagadish M

2015-10-01

The poliovirus serotype identification and intratypic differentiation by real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay is suitable for serotype mixtures but not for intratypic mixtures of wild and vaccine poliovirus strains. This study was undertaken to develop wild poliovirus 1 and 3 (WPV1 and WPV3) specific rRT-PCR assays for use. Specific primers and probes for rRT-PCR were designed based on VP1 sequences of WPV1 and WPV3 isolated in India since 2000. The specificity of the rRT-PCR assays was evaluated using WPV1 and WPV3 of different genetic lineages, non-polio enteroviruses (NPEVs) and mixtures of wild/wild and wild/Sabin vaccine strains. The sensitivity of the assays was determined by testing serial 10-fold dilutions of wild poliovirus 1 and 3 stock suspensions of known titre. No cross-reactivity with Sabin strains, intertypic wild poliovirus isolates or 27 types of NPEVs across all the four Enterovirus species was found for both the wild poliovirus 1 and 3 rRT-PCR assays. All WPV1 and WPV3 strains isolated since 2000 were successfully amplified. The rRT-PCR assays detected 10 4.40 CCID 50 /ml of WPV1 and 10 4.00 CCID 50 /ml of WPV3, respectively either as single isolate or mixture with Sabin vaccine strains or intertypic wild poliovirus. rRT-PCR assays for WPV1 and WPV3 have been validated to detect all the genetic variations of the WPV1 and WPV3 isolated in India for the last decade. When used in combination with the current rRT-PCR assay testing was complete for confirmation of the presence of wild poliovirus in intratypic mixtures.

A novel cell-based assay for measuring neutralizing autoantibodies against type I interferons in patients with autoimmune polyendocrine syndrome type 1.

Science.gov (United States)

Breivik, Lars; Oftedal, Bergithe E V; Bøe Wolff, Anette S; Bratland, Eirik; Orlova, Elizaveta M; Husebye, Eystein S

2014-07-01

An important characteristic of autoimmune polyendocrine syndrome type 1 (APS 1) is the existence of neutralizing autoantibodies (nAbs) against the type I interferons (IFN) -α2 and -ω at frequencies close to 100%. Type 1 IFN autoantibodies are detected by antiviral neutralizing assays (AVA), binding assays with radiolabelled antigens (RLBA), enzyme-linked immunosorbent assay (ELISA), or by reporter-based cell assays. We here present a simple and reliable version of the latter utilizing a commercially available cell line (HEK-Blue IFN-α/β). All 67 APS 1 patients were positive for IFN-ω nAbs, while 90% were positive for IFN-α2 nAbs, a 100% and 96% correlation with RLBA, respectively. All blood donors and non-APS 1 patients were negative. The dilution titer required to reduce the effect of IFN-ω nAbs correlated with the RLBA index. This cell-based autoantibody assay (CBAA) is easy to perform, suitable for high throughput, while providing high specificity and sensitivity. Copyright © 2014 Elsevier Inc. All rights reserved.

SNIT: SNP identification for strain typing

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Reifman Jaques

2011-09-01

Full Text Available Abstract With ever-increasing numbers of microbial genomes being sequenced, efficient tools are needed to perform strain-level identification of any newly sequenced genome. Here, we present the SNP identification for strain typing (SNIT pipeline, a fast and accurate software system that compares a newly sequenced bacterial genome with other genomes of the same species to identify single nucleotide polymorphisms (SNPs and small insertions/deletions (indels. Based on this information, the pipeline analyzes the polymorphic loci present in all input genomes to identify the genome that has the fewest differences with the newly sequenced genome. Similarly, for each of the other genomes, SNIT identifies the input genome with the fewest differences. Results from five bacterial species show that the SNIT pipeline identifies the correct closest neighbor with 75% to 100% accuracy. The SNIT pipeline is available for download at http://www.bhsai.org/snit.html

Draft Genome Sequence of Type Strain Streptococcus gordonii ATCC 10558

DEFF Research Database (Denmark)

Rasmussen, Louise Hesselbjerg; Dargis, Rimtas; Christensen, Jens Jørgen Elmer

2016-01-01

Streptococcus gordonii ATCC 10558T was isolated from a patient with infective endocarditis in 1946 and announced as a type strain in 1989. Here, we report the 2,154,510-bp draft genome sequence of S. gordonii ATCC 10558T. This sequence will contribute to knowledge about the pathogenesis of infect......Streptococcus gordonii ATCC 10558T was isolated from a patient with infective endocarditis in 1946 and announced as a type strain in 1989. Here, we report the 2,154,510-bp draft genome sequence of S. gordonii ATCC 10558T. This sequence will contribute to knowledge about the pathogenesis...

Characterisation of the thermostable protease AprX in strains of Pseudomonas fluorescens and impact on the shelf-life of dairy products: preliminary results

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Nadia Andrea Andreani

2016-12-01

Full Text Available Bacterial proteases are involved in food spoilage and shelf-life reduction. Among the bacterial proteases, a predominant role in spoilage of dairy products seems to be played by the thermostable metallo-protease AprX, which is produced by various strains of Pseudomonas fluorescens. Differences in AprX enzyme activity among different strains were highlighted, but the most proteolytic strains were not identified. In this study, the presence of the aprX gene was evaluated in 69 strains isolated from food matrices and 18 reference strains belonging to the P. fluorescens group, which had been previously typed by the multi locus sequence typing method. Subsequently, a subset of reference strains was inoculated in ultra-high temperature milk, and the expression of the aprX gene was evaluated at 22 and 6°C. On the same milk samples, the proteolytic activity was then evaluated through Azocasein and trinitrobenzenesulfonic acid solution assays. Finally, to assess the applicability of the former assay directly on dairy products the proteolityc activity was tested on industrial ricotta samples using the Azocasein assay. These results demonstrate the spread of aprX gene in most strains tested and the applicability of Azocasein assay to monitor the proteolytic activity in dairy products.

A rapid qualitative assay for detection of Clostridium perfringens in canned food products.

Science.gov (United States)

Dave, Gayatri Ashwinkumar

2017-01-01

Clostridium perfringens (MTCC 1349) is a Gram-positive, anaerobic, endospore forming, and rod-shaped bacterium. This bacterium produces a variety of toxins under strict anaerobic environment. C. perfringens can grow at temperatures ranging between 20°C and 50°C. It is the major causetive agent for gas gangrene, cellulitis, septicemia, necrotic enteritis and food poisoning, which are common toxin induced conditions noted in human and animals. C. perfringens can produce produce four major types of toxins that are used for the classification of strains, classified under type A-E. Across the globe many countries, including the United States, are affected by C. perfringens food poisonings where it is ranked as one of the most common causes of food borne infections. To date, no direct one step assay for the detection of C. perfringens has been developed and only few methods are known for accurate detection of C. perfringens. Long detection and incubation time is the major consideration of these reporter assays. The prensent study proposes a rapid and reliable colorimetric assay for the detection of C. perfringens. In principale, this assay detects the para nitrophenyl (yellow colour end product) liberated due to the hydrolysis of paranitrophenyl phosphetidyl choline (PNPC) through phospholipase C (lecithinase). Constitutive secretion of phospholipase C is a charactristic feature of C. perfringens. This assay detects the presence of the extracellular lecithinse through the PNPC impragnated impregnated probe. The probe is impregnated with peranitrophenyl phosphotidyl choline ester, which is colourless substrate used by lecithinase. The designed assay is specific towards PNPC and detectes very small quantites of lecithinase under conditions used. The reaction is substrate specific, no cross reaction was observed upon incubation with other substrates. In addition, this assay gave negative results with other clostridium strains, no cross reactions were observed with other

High-Resolution Typing Reveals Distinct Chlamydia trachomatis Strains in an At-Risk Population in Nanjing, China

NARCIS (Netherlands)

Bom, Reinier J. M.; van den Hoek, Anneke; Wang, Qianqiu; Long, Fuquan; de Vries, Henry J. C.; Bruisten, Sylvia M.

2013-01-01

We investigated Chlamydia trachomatis strains from Nanjing, China, and whether these strains differed from Amsterdam, the Netherlands. C. trachomatis type was determined with multilocus sequence typing. Most strains were specific to Nanjing, but some clustered with strains from Amsterdam. This

Static strain aging type AISI-304 austenitic stainless steel

International Nuclear Information System (INIS)

Trindade, M.B.

1981-03-01

Static strain aging of type AISI-304 austenitic stainless steel was studied from room temperature up to 623K by conducting tests in which the load was held approximately constant, continuously relaxing and unloaded. The aging times varied between 10s and 100h, using a plastic pre deformation of 9% in most of the cases. The static strain aging of 304 steel furnished an activation energy of 23,800 cal/mol. This implies that vacancies play an important role on the aging process. The curve of the variation of the discontinuous yielding with aging time presented different stages, to which specific mathematical expressions were developed. These facts permited the conclusion that Snoek type mechanisms are responsible for the aging in such conditions. (Author) [pt

Molecular typing of Salmonella enterica serovar typhi isolates from various countries in Asia by a multiplex PCR assay on variable-number tandem repeats.

Science.gov (United States)

Liu, Yichun; Lee, May-Ann; Ooi, Eng-Eong; Mavis, Yeo; Tan, Ai-Ling; Quek, Hung-Hiang

2003-09-01

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.

Strain typing with IS200 fingerprints in Salmonella abortusovis.

Science.gov (United States)

Schiaffino, A; Beuzón, C R; Uzzau, S; Leori, G; Cappuccinelli, P; Casadesús, J; Rubino, S

1996-07-01

A collection of Salmonella abortusovis isolates was examined for the presence of insertion element IS200. All proved to contain three or four copies of the element. One IS200 hybridization band of approximately 9 kb was found in all isolates, indicating that all S. abortusovis strains carry an IS200 element in similar or identical locations; this band can be potentially useful for serovar identification. S. abortusovis collection isolates from distinct geographic areas were highly polymorphic, suggesting that IS200 fingerprints might provide information on the geographic origin of S. abortusovis strains. Isolates obtained from the same geographic area (the island of Sardinia, Italy) were less polymorphic: all shared three constant IS200 hybridization bands, indicating that they derive from a single ancestor. Most strains analyzed contained an additional copy of IS200 in the variable region of the virulence plasmid. Certain Sardinian flocks proved to be infected by only one S. abortusovis strain, while others harbored two strains. Strain typing with IS200 fingerprints proved to be more reliable than plasmid analysis, because the latter yielded a high degree of polymorphism, even among isolates from the same flock.

Antibody induced by immunization with the Jeryl Lynn mumps vaccine strain effectively neutralizes a heterologous wild-type mumps virus associated with a large outbreak.

Science.gov (United States)

Rubin, Steven A; Qi, Li; Audet, Susette A; Sullivan, Bradley; Carbone, Kathryn M; Bellini, William J; Rota, Paul A; Sirota, Lev; Beeler, Judy

2008-08-15

Recent mumps outbreaks in older vaccinated populations were caused primarily by genotype G viruses, which are phylogenetically distinct from the genotype A vaccine strains used in the countries affected by the outbreaks. This finding suggests that genotype A vaccine strains could have reduced efficacy against heterologous mumps viruses. The remote history of vaccination also suggests that waning immunity could have contributed to susceptibility. To examine these issues, we obtained consecutive serum samples from children at different intervals after vaccination and assayed the ability of these samples to neutralize the genotype A Jeryl Lynn mumps virus vaccine strain and a genotype G wild-type virus obtained during the mumps outbreak that occurred in the United States in 2006. Although the geometric mean neutralizing antibody titers against the genotype G virus were approximately one-half the titers measured against the vaccine strain, and although titers to both viruses decreased with time after vaccination, antibody induced by immunization with the Jeryl Lynn mumps vaccine strain effectively neutralized the outbreak-associated virus at all time points tested.

Toxin Gene Analysis of a Variant Strain of Clostridium difficile That Causes Human Clinical Disease

Science.gov (United States)

Sambol, Susan P.; Merrigan, Michelle M.; Lyerly, David; Gerding, Dale N.; Johnson, Stuart

2000-01-01

A toxin variant strain of Clostridium difficile was isolated from two patients with C. difficile-associated disease (CDAD), one of whom died from extensive pseudomembranous colitis. This strain, identified by restriction endonuclease analysis (REA) as type CF2, was not detected by an immunoassay for C. difficile toxin A. Culture supernatants of CF2 failed to elicit significant enterotoxic activity in the rabbit ileal loop assay but did produce atypical cytopathic effects in cell culture assay. Southern hybridization, PCR amplification, and DNA sequence analyses were performed on the toxin A (tcdA) and toxin B (tcdB) genes of type CF2 isolate 5340. Type CF2 5340 tcdA exhibited a 1,821-bp truncation, due to three deletions in the 3′ end of the gene, and a point mutation in the 5′ end of the gene, resulting in a premature stop codon at tcdA position 139. Type CF2 5340 tcdB exhibited multiple nucleotide base substitutions in the 5′ end of the gene compared to tcdB of the standard toxigenic strain VPI 10463. Type CF2 5340 toxin gene nucleotide sequences and deduced amino acid sequences showed a strong resemblance to those of the previously described variant C. difficile strain 1470, a strain reported to have reduced pathogenicity and no association with clinical illness in humans. REA of strain 1470 identified this strain as a distinct type (CF1) within the same REA group as the closely related type CF2. A review of our clinical-isolate collection identified five additional patients infected with type CF2, three of whom had documented CDAD. PCR amplification of the 3′ end of tcdA demonstrated identical 1.8-kb deletions in all seven type CF2 isolates. REA type CF2 is a toxin variant strain of C. difficile that retains the ability to cause disease in humans but is not detected in clinical immunoassays for toxin A. PMID:10992443

Complete genome sequence of Kytococcus sedentarius type strain (strain 541T)

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Sims, David; Brettin, Thomas; Detter, John C.; Han, Cliff; Lapidus, Alla; Copeland, Alex; Glavina Del Rio, Tijana; Nolan, Matt; Chen, Feng; Lucas, Susan; Tice, Hope; Cheng, Jan-Fang; Bruce, David; Goodwin, Lynne; Pitluck, Sam; Ovchinnikova, Galina; Pati, Amrita; Ivanova, Natalia; Mavrommatis, Konstantinos; Chen, Amy; Palaniappan, Krishna; D' haeseleer, Patrick; Chain, Patrick; Bristow, James; Eisen, Jonathan A.; Markowitz, Victor; Hugenholtz, Philip; Schneider, Susanne; Goker, Markus; Pukall, Rudiger; Kyrpides, Nikos C.; Klenk, Hans-Peter

2009-05-20

Kytococcus sedentarius (ZoBell and Upham 1944) Stackebrandt et al. 1995 is the type strain of the species, and is of phylogenetic interest because of its location in the Dermacoccaceae, a poorly studied family within the actinobacterial suborder Micrococcineae. K. sedentarius is known for the production of oligoketide antibiotics as well as for its role as an opportunistic pathogen causing valve endocarditis, hemorrhagic pneumonia, and pitted keratolysis. It is strictly aerobic and can only grow when several amino acids are provided in the medium. The strain described in this report is a free-living, nonmotile, Gram-positive bacterium, originally isolated from a marine environment. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the family Dermacoccaceae and the 2,785,024 bp long single replicon genome with its 2639 protein-coding and 64 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

R5 strains of human immunodeficiency virus type 1 from rapid progressors lacking X4 strains do not possess X4-type pathogenicity in human thymus

NARCIS (Netherlands)

Berkowitz, R. D.; van't Wout, A. B.; Kootstra, N. A.; Moreno, M. E.; Linquist-Stepps, V. D.; Bare, C.; Stoddart, C. A.; Schuitemaker, H.; McCune, J. M.

1999-01-01

Some individuals infected with only R5 strains of human immunodeficiency virus type 1 progress to AIDS as quickly as individuals harboring X4 strains. We determined that three R5 viruses were much less pathogenic than an X4 virus in SCID-hu Thy/Liv mice, suggesting that R5 virus-mediated rapid

Detection by hemi-nested reverse transcription polymerase chain reaction and genetic characterization of wild type strains of Canine distemper virus in suspected infected dogs.

Science.gov (United States)

Di Francesco, Cristina E; Di Francesco, Daniela; Di Martino, Barbara; Speranza, Roberto; Santori, Domenico; Boari, Andrea; Marsilio, Fulvio

2012-01-01

A new highly sensitive and specific hemi-nested reverse transcription polymerase chain reaction (RT-PCR) assay was applied to detect nucleoprotein (NP) gene of Canine distemper virus (CDV) in samples collected from dogs showing respiratory, gastrointestinal, and neurological signs. Thirty-eight out of 86 samples were positive suggesting that despite the vaccination, canine distemper may still represent a high risk to the canine population. The 968 base pair (bp) fragments from the hemagglutinin (H) gene of 10 viral strains detected in positive samples were amplified and analyzed by restriction fragment length polymorphism (RFLP) using AluI and PsiI enzymes in order to differentiate among vaccine and wild-type CDV strains and to characterize the field viral strains. The products of the both enzymatic digestions allowed identification all viruses as wild strains of CDV. In addition, the RFLP analysis with AluI provided additional information about the identity level among the strains analyzed on the basis of the positions of the cleavage site in the nucleotide sequences of the H gene. The method could be a more useful and simpler method for molecular studies of CDV strains.

Galleria mellonella model identifies highly virulent strains among all major molecular types of Cryptococcus gattii.

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Carolina Firacative

Full Text Available Cryptococcosis is mainly caused by Cryptococcus neoformans. However, the number of cases due to C. gattii is increasing, affecting mainly immunocompetent hosts. C. gattii is divided into four major molecular types, VGI to VGIV, which differ in their host range, epidemiology, antifungal susceptibility and geographic distribution. Besides studies on the Vancouver Island outbreak strains, which showed that the subtype VGIIa is highly virulent compared to the subtype VGIIb, little is known about the virulence of the other major molecular types. To elucidate the virulence potential of the major molecular types of C. gattii, Galleria mellonella larvae were inoculated with ten globally selected strains per molecular type. Survival rates were recorded and known virulence factors were studied. One VGII, one VGIII and one VGIV strain were more virulent (p 0.05, 21 (five VGI, five VGII, four VGIII and seven VGIV were less virulent (p <0.05 while one strain of each molecular type were avirulent. Cell and capsule size of all strains increased markedly during larvae infection (p <0.001. No differences in growth rate at 37°C were observed. Melanin synthesis was directly related with the level of virulence: more virulent strains produced more melanin than less virulent strains (p <0.05. The results indicate that all C. gattii major molecular types exhibit a range of virulence, with some strains having the potential to be more virulent. The study highlights the necessity to further investigate the genetic background of more and less virulent strains in order to recognize critical features, other than the known virulence factors (capsule, melanin and growth at mammalian body temperature, that maybe crucial for the development and progression of cryptococcosis.

Acute Heat Stress Changes Protein Expression in the Testes of a Broiler-Type Strain of Taiwan Country Chickens.

Science.gov (United States)

Wang, Shih-Han; Cheng, Chuen-Yu; Chen, Chao-Jung; Chan, Hong-Lin; Chen, Hsin-Hsin; Tang, Pin-Chi; Chen, Chih-Feng; Lee, Yen-Pai; Huang, San-Yuan

2018-03-19

Heat stress leads to decreased fertility in roosters. This study investigated the global protein expression in response to acute heat stress in the testes of a broiler-type strain of Taiwan country chickens (TCCs). Twelve 45-week-old roosters were randomly allocated to the control group maintained at 25°C, and three groups subjected to acute heat stress at 38°C for 4 h, with 0, 2, and 6 h of recovery, respectively. Testis samples were collected for hematoxylin and eosin staining, apoptosis assay, and protein analysis. The results revealed 101 protein spots that differed significantly from the control following exposure to acute heat stress. The proteins that were differentially expressed participated mainly in protein metabolism and other metabolic processes, responses to stimuli, apoptosis, cellular organization, and spermatogenesis. Proteins that negatively regulate apoptosis were downregulated and proteins involved in autophagy and major heat shock proteins (HSP90α, HSPA5, and HSPA8) were upregulated in the testes of heat-stressed chickens. In conclusion, acute heat stress causes a change in protein expression in the testes of broiler-type B strain TCCs and may thus impair cell morphology, spermatogenesis, and apoptosis. The expression of heat shock proteins increased to attenuate the testicular injury induced by acute heat stress.

Identification and antifungal activity of an actinomycete strain against Alternaria spp.

Directory of Open Access Journals (Sweden)

Fen Gao

2014-10-01

Full Text Available Alternaria alternata (Fries Keissler is a phytopathogenic fungus responsible for tobacco brown spot disease. This study aims to evaluate the antifungal activity of strain 163 against A. alternata and clarify its taxonomic status. The evaluation of the antifungal activity of strain 163 and its bacteria-free filtrate of fermentation broth was done through measuring the diameters of inhibition zones, and testing the antimicrobial spectrum and the inhibition effect on mycelial growth in vitro. The biocontrol activity of the bacteria-free filtrate in vivo was evaluated by using detached tobacco leaves method and assaying the inhibition rate to disease incidence in growth chamber. A polyphasic approach was taken in the identification of strain 163. The bacterial strain 163 showed inhibitory effect in vitro against A. alternata. The bacteria-free filtrate of the strain 163 fermentation broth showed a 56.7% inhibition rate in a detached leaf assay. In growth chamber conditions, it showed greater biocontrol activity when applied before plants being inoculated with A. alternata than after, the inhibition rate being 46.05%. Investigations into the morphological, cultural, physiological and biochemical properties of strain 163 found it to be most similar to Streptomyces microflavus. Its classification into cell wall type I and sugar type C further confirmed its Streptomyces characteristics. Construction of a phylogenetic tree based on 16S rDNA verified that strain 163 was most closely related to Streptomyces microflavus. From polyphasic taxonomical analysis, strain 163 was found to be identical to S. microflavus.

Job Strain as a Risk Factor for Type 2 Diabetes

DEFF Research Database (Denmark)

Nyberg, Solja T; Fransson, Eleonor I; Heikkilä, Katriina

2014-01-01

with baseline questionnaires. Incident type 2 diabetes at follow-up was ascertained using national health registers, clinical screening, and self-reports. We analyzed data for each study using Cox regression and pooled the study-specific estimates in fixed-effect meta-analyses. RESULTS: There were 3,703 cases......OBJECTIVE: The status of psychosocial stress at work as a risk factor for type 2 diabetes is unclear because existing evidence is based on small studies and is subject to confounding by lifestyle factors, such as obesity and physical inactivity. This collaborative study examined whether stress...... at work, defined as "job strain," is associated with incident type 2 diabetes independent of lifestyle factors. RESEARCH DESIGN AND METHODS: We extracted individual-level data for 124,808 diabetes-free adults from 13 European cohort studies participating in the IPD-Work Consortium. We measured job strain...

Drug discovery for Chagas disease should consider Trypanosoma cruzi strain diversity

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Bianca Zingales

2014-09-01

Full Text Available This opinion piece presents an approach to standardisation of an important aspect of Chagas disease drug discovery and development: selecting Trypanosoma cruzi strains for in vitro screening. We discuss the rationale for strain selection representing T. cruzi diversity and provide recommendations on the preferred parasite stage for drug discovery, T. cruzi discrete typing units to include in the panel of strains and the number of strains/clones for primary screens and lead compounds. We also consider experimental approaches for in vitro drug assays. The Figure illustrates the current Chagas disease drug-discovery and development landscape.

ELECTROPHORETIC MOBILITIES OF ESCHERICHIA COLI 0157:H7 AND WILD-TYPE ESCHERICHIA COLI STRAINS

Science.gov (United States)

The electrophoretic mobility (EPM) of a number of human-virulent and "wild-type" Escherichia coli strains in phosphate buffered water was measured. The impact of pH, ionic strength, cation type (valence) and concentration, and bacterial strain on the EPM was investigated. Resul...

Bacteriological Typing of C. Diphtheriae Strains Recently Isolated in Teheran

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H. Esterabady

1963-01-01

Full Text Available From a total of 600 nose and throat introduction swabs examined for diphtherie, 200 or 33% were positive. Cultures were carfully classified on the basis of morphological appearance and biochemical characteristics into Gravis, Mitis and Intermedius groups.  A special tellurite serum agar was used for colonial appearance. Neill's broth culture was employed for haemolytic tests. The virulence of each culture was examined in laboratory animals by the agar gel precipitation method of Elek. From 200 cultures tested, 138 or 69% were gravis, 5 or 2.5% were intermedius, and 57 or 28% were mitis. 'I'hre.e strains of gravis type and one strain of mitis type were avirulent

In Vitro and In Vivo Characterization of a Typical and a High Pathogenic Bovine Viral Diarrhea Virus Type II Strains

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Dario Amilcar Malacari

2018-04-01

Full Text Available Non-cytopathic (ncp type 2 bovine viral diarrhea virus (BVDV-2 is widely prevalent in Argentina causing high mortality rates in cattle herds. In this study, we characterized an Argentinean ncp BVDV-2 field isolate (98-124 compared to a high-virulence reference strain (NY-93, using in silico analysis, in vitro assays, and in vivo infections of colostrum-deprived calves (CDC to compare pathogenic characters and virulence. In vitro infection of bovine peripheral blood mononuclear cells (PBMC with BVDV 98-124 induced necrosis shortly after infection while NY-93 strain increased the apoptotic rate in infected cells. Experimental infection of CDC (n = 4 each with these strains caused an enteric syndrome. High pyrexia was detected in both groups. Viremia and shedding were more prolonged in the CDC infected with the NY-93 strain. In addition, NY-93 infection elicited a severe lymphopenia that lasted for 14 days, whereas 98-124 strain reduced the leukocyte counts for 5 days. All infected animals had a diminished lymphoproliferation activity in response to a mitogen. Neutralizing and anti-NS3 antibodies were detected 3 weeks after infection in all infected calves. Virulence was associated with a more severe clinical score, prolonged immune-suppression, and a greater window for transmission. Studies of apoptosis/necrosis performed after in vitro PBMC infection also revealed differences between both strains that might be correlated to the in vivo pathogenesis. Our results identified 98-124 as a low-virulence strain.

Distribution, persistence and interchange of Epstein-Barr virus strains among PBMC, plasma and saliva of primary infection subjects.

Science.gov (United States)

Kwok, Hin; Chan, Koon Wing; Chan, Kwok Hung; Chiang, Alan Kwok Shing

2015-01-01

Our study aimed at investigating the distribution, persistence and interchange of viral strains among peripheral blood mononuclear cells (PBMC), plasma and saliva of primary Epstein-Barr virus (EBV) infection subjects. Twelve infectious mononucleosis (IM) patients and eight asymptomatic individuals (AS) with primary EBV infection were followed longitudinally at several time points for one year from the time of diagnosis, when blood and saliva samples were collected and separated into PBMC, plasma and saliva, representing circulating B cell, plasma and epithelial cell compartments, respectively. To survey the viral strains, genotyping assays for the natural polymorphisms in two latent EBV genes, EBNA2 and LMP1, were performed and consisted of real-time PCR on EBNA2 to distinguish type 1 and 2 viruses, fluorescent-based 30-bp typing assay on LMP1 to distinguish deletion and wild type LMP1, and fluorescent-based heteroduplex tracking assays on both EBNA2 and LMP1 to distinguish defined polymorphic variants. No discernible differences were observed between IM patients and AS. Multiple viral strains were acquired early at the start of infection. Stable persistence of dominant EBV strains in the same tissue compartment was observed throughout the longitudinal samples. LMP1-defined strains, China 1, China 2 and Mediterranean+, were the most common strains observed. EBNA2-defined groups 1 and 3e predominated the PBMC and saliva compartments. Concordance of EBNA2 and LMP1 strains between PBMC and saliva suggested ready interchange of viruses between circulating B cell and epithelial cell pools, whilst discordance of viral strains observed between plasma and PBMC/saliva indicated presence of viral pools in other undetermined tissue compartments. Taken together, the results indicated that the distribution, persistence and interchange of viral strains among the tissue compartments are more complex than those proposed by the current model of EBV life cycle.

Complete genome sequence of Halanaerobium praevalens type strain (GSLT)

Energy Technology Data Exchange (ETDEWEB)

Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Sikorski, Johannes [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Chertkov, Olga [Los Alamos National Laboratory (LANL); Nolan, Matt [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Hammon, Nancy [U.S. Department of Energy, Joint Genome Institute; Deshpande, Shweta [U.S. Department of Energy, Joint Genome Institute; Cheng, Jan-Fang [U.S. Department of Energy, Joint Genome Institute; Tapia, Roxanne [Los Alamos National Laboratory (LANL); Han, Cliff [Los Alamos National Laboratory (LANL); Goodwin, Lynne A. [Los Alamos National Laboratory (LANL); Pitluck, Sam [U.S. Department of Energy, Joint Genome Institute; Huntemann, Marcel [U.S. Department of Energy, Joint Genome Institute; Liolios, Konstantinos [U.S. Department of Energy, Joint Genome Institute; Pagani, Ioanna [U.S. Department of Energy, Joint Genome Institute; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Ovchinnikova, Galina [U.S. Department of Energy, Joint Genome Institute; Pati, Amrita [U.S. Department of Energy, Joint Genome Institute; Chen, Amy [U.S. Department of Energy, Joint Genome Institute; Palaniappan, Krishna [U.S. Department of Energy, Joint Genome Institute; Land, Miriam L [ORNL; Hauser, Loren John [ORNL; Brambilla, Evelyne-Marie [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Kannan, K. Palani [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Rohde, Manfred [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Tindall, Brian [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Goker, Markus [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Detter, J. Chris [U.S. Department of Energy, Joint Genome Institute; Woyke, Tanja [U.S. Department of Energy, Joint Genome Institute; Bristow, James [U.S. Department of Energy, Joint Genome Institute; Eisen, Jonathan [U.S. Department of Energy, Joint Genome Institute; Markowitz, Victor [U.S. Department of Energy, Joint Genome Institute; Hugenholtz, Philip [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute; Klenk, Hans-Peter [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute

2011-01-01

Halanaerobium praevalens Zeikus et al. 1984 is the type species of the genus Halanaero- bium, which in turn is the type genus of the family Halanaerobiaceae. The species is of inter- est because it is able to reduce a variety of nitro-substituted aromatic compounds at a high rate, and because of its ability to degrade organic pollutants. The strain is also of interest be- cause it functions as a hydrolytic bacterium, fermenting complex organic matter and produc- ing intermediary metabolites for other trophic groups such as sulfate-reducing and methano- genic bacteria. It is further reported as being involved in carbon removal in the Great Salt Lake, its source of isolation. This is the first completed genome sequence of a representative of the genus Halanaerobium and the second genome sequence from a type strain of the fami- ly Halanaerobiaceae. The 2,309,262 bp long genome with its 2,110 protein-coding and 70 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Biological assay of attenuated strain NADL-2 and virulent strain NADL-8 of porcine parvovirus.

Science.gov (United States)

Mengeling, W L; Pejsak, Z; Paul, P S

1984-11-01

Attenuated strain NADL-2 and virulent strain NADL-8 of porcine parvovirus (PPV) were titrated in vivo and in vitro under similar conditions to provide a better understanding of some of the factors involved in virulence of PPV in causing maternal reproductive failure of swine. Both strains cause fetal death when they are injected directly into fetal fluids, but only strain NADL-8 does so when administered to pregnant swine. The strains were tested for their hemagglutinating activity (HA), median cell culture infective dose (CCID50), median fetal infective dose (FID50), and median fetal lethal dose (FLD50). The FID50 and FLD50 were determined by injecting virus directly into the amniotic fluid of fetuses in utero at 44 +/- 2 days of gestation and collecting the fetuses 15 +/- 1 days later. Both strains had an HA titer of 64, suggesting that there is a similar number of virions in stock preparations. However, other measurements differed markedly. The CCID50, FID50, and FLD50 were 10(5.5), 10(3.5), and 10(0.5), respectively, for strain NADL-2, and 10(4.5), 10(7.7), and 10(6.3), respectively, for strain NADL-8. Collectively, the values indicate that more than 10,000 times as much strain NADL-2 would need to reach the conceptus transplacentally to establish infection. These observations may help to explain the different consequences of oronasal exposure of pregnant swine to these strains of PPV.

Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus.

Science.gov (United States)

Mari, Viviana; Losurdo, Michele; Lucente, Maria Stella; Lorusso, Eleonora; Elia, Gabriella; Martella, Vito; Patruno, Giovanni; Buonavoglia, Domenico; Decaro, Nicola

2016-03-01

HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)-10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples. Copyright © 2015 Elsevier B.V. All rights reserved.

CodY Promotes Sporulation and Enterotoxin Production by Clostridium perfringens Type A Strain SM101.

Science.gov (United States)

Li, Jihong; Freedman, John C; Evans, Daniel R; McClane, Bruce A

2017-03-01

Clostridium perfringens type D strains cause enterotoxemia and enteritis in livestock via epsilon toxin production. In type D strain CN3718, CodY was previously shown to increase the level of epsilon toxin production and repress sporulation. C. perfringens type A strains producing C. perfringens enterotoxin (CPE) cause human food poisoning and antibiotic-associated diarrhea. Sporulation is critical for C. perfringens type A food poisoning since spores contribute to transmission and resistance in the harsh food environment and sporulation is essential for CPE production. Therefore, the current study asked whether CodY also regulates sporulation and CPE production in SM101, a derivative of C. perfringens type A food-poisoning strain NCTC8798. An isogenic codY -null mutant of SM101 showed decreased levels of spore formation, along with lower levels of CPE production. A complemented strain recovered wild-type levels of both sporulation and CPE production. When this result was coupled with the earlier results obtained with CN3718, it became apparent that CodY regulation of sporulation varies among different C. perfringens strains. Results from quantitative reverse transcriptase PCR analysis clearly demonstrated that, during sporulation, codY transcript levels remained high in SM101 but rapidly declined in CN3718. In addition, abrB gene expression patterns varied significantly between codY -null mutants of SM101 and CN3718. Compared to the levels in their wild-type parents, the level of abrB gene expression decreased in the CN3718 codY -null mutant strain but significantly increased in the SM101 codY -null mutant strain, demonstrating CodY-dependent regulation differences in abrB expression between these two strains. This difference appears to be important since overexpression of the abrB gene in SM101 reduced the levels of sporulation and enterotoxin production, supporting the involvement of AbrB repression in regulating C. perfringens sporulation. Copyright © 2017

A robust SNP barcode for typing Mycobacterium tuberculosis complex strains

KAUST Repository

Coll, Francesc

2014-09-01

Strain-specific genomic diversity in the Mycobacterium tuberculosis complex (MTBC) is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Several systems have been proposed to classify MTBC strains into distinct lineages and families. Here, we investigate single-nucleotide polymorphisms (SNPs) as robust (stable) markers of genetic variation for phylogenetic analysis. We identify ∼92k SNP across a global collection of 1,601 genomes. The SNP-based phylogeny is consistent with the gold-standard regions of difference (RD) classification system. Of the ∼7k strain-specific SNPs identified, 62 markers are proposed to discriminate known circulating strains. This SNP-based barcode is the first to cover all main lineages, and classifies a greater number of sublineages than current alternatives. It may be used to classify clinical isolates to evaluate tools to control the disease, including therapeutics and vaccines whose effectiveness may vary by strain type. © 2014 Macmillan Publishers Limited.

A new real-time PCR assay for rapid identification of the S. aureus/MRSA strains

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Ivan Manga

2013-01-01

Full Text Available The Methicillin-resistant Staphylococcus aureus (MRSA with the livestock-associated MRSA (LA-MRSA are of great interest to scientists and general public. The aim of our study was to present a new more rapid and reliable diagnostic method working on the RT-PCR platform applicable for monitoring of MRSA/S. aureus. The parallel testing of the S. aureus specific nuc gene sequence and the mecA gene sequence was utilised for this purpose. A collection of ten S. aureus/MRSA reference strains, fifteen genetically related non S. aureus reference strains and fifty-six environmental samples was employed for estimation of the assay performance and parameters. The environmental samples acquired in the Czech livestock farms were represented with the livestock and human nasal mucosae or skin swabs, the slaughter meat swabs and were chosen preferentially from individuals with previously confi rmed or suspected positive MRSA/S. aureus cases. The classic selective cultivation approach with the biochemical test and agar disk diffusion test was accepted as reference diagnostic method. As there were no culture positive samples that were negative using RT-PCR, our method featured with 100% sensitivity in comparison to reference method. The limit of detection allowed to identify from tens to hundreds copies of S. aureus/MRSA genome. Further, the RT-PCR assay featured with 100% inclusivity and 95% exclusivity at Cq value below 30. These parameters suggested on powerful and reliable diagnostic method with real potential of practical utilisation. We consider our method as ideal for testing of individual suspected colonies, when the results can be acquired in less than 1.5 hour.

Comparison of two enzyme-linked immunosorbent assays and one rapid immunoblot assay for detection of herpes simplex virus type 2-specific antibodies in serum

NARCIS (Netherlands)

Groen, J; Van Dijk, G; Niesters, H G; Van Der Meijden, W I; Osterhaus, A D

The sensitivities and specificities of three immunoassays for the detection of herpes simplex virus type 2 (HSV-2)-specific immunoglobulin G antibodies in serum, including the one-strip rapid immunoblot assay (RIBA; Chiron Corporation) and two indirect enzyme immunosorbent assays (EIA; Gull

CC8 MRSA strains harboring SCCmec type IVc are predominant in Colombian hospitals.

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J Natalia Jiménez

Full Text Available BACKGROUND: Recent reports highlight the incursion of community-associated MRSA within healthcare settings. However, knowledge of this phenomenon remains limited in Latin America. The aim of this study was to evaluate the molecular epidemiology of MRSA in three tertiary-care hospitals in Medellín, Colombia. METHODS: An observational cross-sectional study was conducted from 2008-2010. MRSA infections were classified as either community-associated (CA-MRSA or healthcare-associated (HA-MRSA, with HA-MRSA further classified as hospital-onset (HAHO-MRSA or community-onset (HACO-MRSA according to standard epidemiological definitions established by the U.S. Centers for Disease Control and Prevention (CDC. Genotypic analysis included SCCmec typing, spa typing, PFGE and MLST. RESULTS: Out of 538 total MRSA isolates, 68 (12.6% were defined as CA-MRSA, 243 (45.2% as HACO-MRSA and 227 (42.2% as HAHO-MRSA. The majority harbored SCCmec type IVc (306, 58.7%, followed by SCCmec type I (174, 33.4%. The prevalence of type IVc among CA-, HACO- and HAHO-MRSA isolates was 92.4%, 65.1% and 43.6%, respectively. From 2008 to 2010, the prevalence of type IVc-bearing strains increased significantly, from 50.0% to 68.2% (p = 0.004. Strains harboring SCCmec IVc were mainly associated with spa types t1610, t008 and t024 (MLST clonal complex 8, while PFGE confirmed that the t008 and t1610 strains were closely related to the USA300-0114 CA-MRSA clone. Notably, strains belonging to these three spa types exhibited high levels of tetracycline resistance (45.9%. CONCLUSION: CC8 MRSA strains harboring SCCmec type IVc are becoming predominant in Medellín hospitals, displacing previously reported CC5 HA-MRSA clones. Based on shared characteristics including SCCmec IVc, absence of the ACME element and tetracycline resistance, the USA300-related isolates in this study are most likely related to USA300-LV, the recently-described 'Latin American variant' of USA300.

Genetic relatedness of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated in south Asia.

Science.gov (United States)

Talukder, Kaisar A; Khajanchi, Bijay K; Islam, M Aminul; Dutta, Dilip K; Islam, Zhahirul; Safa, Ashrafus; Khan, G Y; Alam, Khorshed; Hossain, M A; Malla, Sarala; Niyogi, S K; Rahman, Mustafizur; Watanabe, Haruo; Nair, G Balakrish; Sack, David A

2004-10-01

The aim of the present study was to determine the clonal relationships of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated from south Asia, and S. dysenteriae 1 strains associated with epidemics in 1978, 1984 and 1994. The antimicrobial susceptibilities were examined by NCCLS methods. Molecular epidemiological characterization was performed by plasmid profiling, pulsed-field gel electrophoresis (PFGE) and mutation analysis of the quinolone resistance-determining region (QRDR) of gyrA by sequencing. Plasmid patterns of the current ciprofloxacin-resistant strains from India, Nepal and Bangladesh were very similar to those of the 1978, 1984 and 1994 epidemic isolates of S. dysenteriae 1, except for the presence of a new plasmid of approximately 2.6 MDa, which was found in one recent ciprofloxacin-resistant strain isolated in Bangladesh. PFGE analysis showed that the ciprofloxacin-resistant strains isolated in Bangladesh, India and Nepal belonged to a PFGE type (type A), which was possibly related to that of the 1984 and 1994 clone of S. dysenteriae 1, but different from 1978 epidemic strains. The current ciprofloxacin-resistant strains belong to five subtypes (A3-A7), all of which were found in India, but in Bangladesh and Nepal, only A3 existed. Mutation analysis of the QRDR of gyrA revealed that amino acid substitutions at positions 83 and 87 of ciprofloxacin-resistant strains isolated in Bangladesh were similar to those of the strains isolated in Nepal, but different (at position 87) from ciprofloxacin-resistant strains isolated in India. PFGE and mutation analysis of gyrA showed differences between the current ciprofloxacin-resistant S. dysenteriae 1 strains isolated in south Asia and those associated with epidemics in 1978, 1984 and 1994.

Strain components of nuclear-reactor-type concretes during first heat cycle

International Nuclear Information System (INIS)

Khoury, G.A.

1995-01-01

Strains of three advanced-gas-cooled-reactor-type nuclear reactor concretes were measured during the first heat cycle and their relative thermal stability determined. It was possible to isolate for the first time the shrinkage component for the period during heating. Predictions of the residual strains for the loaded specimens can be made by simple superposition of creep and shrinkage components up to a certain critical temperature, which for basalt concrete is about 500 C and for limestone concrete is about 200-300 C. Above the critical temperature, an expansive ''cracking'' strain component is present. It is shown that the strain behaviour of concrete provides a sensitive indication of its thermal stability during heating and subsequent cooling. (orig.)

Virulence-associated genes, antimicrobial resistance and molecular typing of Salmonella Typhimurium strains isolated from swine from 2000 to 2012 in Brazil.

Science.gov (United States)

Almeida, F; Medeiros, M I C; Kich, J D; Falcão, J P

2016-06-01

The aims of this study were to assess the pathogenic potential, antimicrobial resistance and genotypic diversity of Salmonella Typhimurium strains isolated in Brazil from swine (22) and the surrounding swine environment (5) from 2000 to 2012 and compare them to the profiles of 43 human strains isolated from 1983 to 2010, which had been previously studied. The presence of 12 SPI-1, SPI-2 and plasmid genes was assessed by PCR, the antimicrobial susceptibility to 13 antimicrobials was determined by the disc diffusion assay and genotyping was performed using pulsed-field gel electrophoresis (PFGE), multiple-locus variable-number of tandem repeats analysis (MLVA) and ERIC-PCR. More than 77·8% of the swine strains carried 10 or more of the virulence markers. Ten (37%) strains isolated from swine were multi-drug resistant (MDR). All the molecular typing techniques grouped the strains in two main clusters. Some strains isolated from swine and humans were allocated together in the PFGE-B2, MLVA-A1, MLVA-B and ERIC-A1 clusters. The genotyping results suggest that some strains isolated from swine and humans may descend from a common subtype and may indicate a possible risk of MDR S. Typhimurium with high frequency of virulence genes isolated from swine to contaminate humans in Brazil. This study provided new information about the pathogenic potential, antimicrobial resistance and genotypic diversity of S. Typhimurium isolates from swine origin in Brazil, the fourth largest producer of pigs worldwide. © 2016 The Society for Applied Microbiology.

Molecular typing of uropathogenic E. coli strains by the ERIC-PCR method.

Science.gov (United States)

Ardakani, Maryam Afkhami; Ranjbar, Reza

2016-04-01

Escherichia coli (E. coli) is the most common cause of urinary infections in hospitals. The aim of this study was to evaluate the ERIC-PCR method for molecular typing of uropathogenic E. coli strains isolated from hospitalized patients. In a cross sectional study, 98 E. coli samples were collected from urine samples taken from patients admitted to Baqiyatallah Hospital from June 2014 to January 2015. The disk agar diffusion method was used to determine antibiotic sensitivity. DNA proliferation based on repetitive intergenic consensus was used to classify the E. coli strains. The products of proliferation were electrophoresed on 1.5% agarose gel, and their dendrograms were drawn. The data were analyzed by online Insillico software. The method used in this research proliferated numerous bands (4-17 bands), ranging from 100 to 3000 base pairs. The detected strains were classified into six clusters (E1-E6) with 70% similarity between them. In this study, uropathogenic E. coli strains belonged to different genotypic clusters. It was found that ERIC-PCR had good differentiation power for molecular typing of uropathogenic E. coli strains isolated from the patients in the study.

The amyR-deletion strain of Aspergillus niger CICC2462 is a suitable host strain to express secreted protein with a low background.

Science.gov (United States)

Zhang, Hui; Wang, Shuang; Zhang, Xiang Xiang; Ji, Wei; Song, Fuping; Zhao, Yue; Li, Jie

2016-04-28

The filamentous fungus Aspergillus niger is widely exploited as an important expression host for industrial production. The glucoamylase high-producing strain A. niger CICC2462 has been used as a host strain for the establishment of a secretion expression system. It expresses recombinant xylanase, mannase and asparaginase at a high level, but some high secretory background proteins in these recombinant strains still remain, such as alpha-amylase and alpha-glucosidase; lead to a low-purity of fermentation products. The aim was to construct an A. niger host strain with a low background of protein secretion. The transcription factor amyR was deleted in A. niger CICC2462, and the results from enzyme activity assays and SDS-PAGE analysis showed that the glucoamylase and amylase activities of the ∆amyR strains were significantly lower than those of the wild-type strain. High-throughput RNA-sequencing and shotgun LC-MS/MS proteomic technology analysis demonstrated that the expression of amylolytic enzymes was decreased at both the transcriptional and translational levels in the ∆amyR strain. Interestingly, the ∆amyR strain growth rate better than the wild-type strain. Our findings clearly indicated that the ∆amyR strain of A. niger CICC2462 can be used as a host strain with a low background of protein secretion.

Complete Genome Sequence of Mycobacterium phlei Type Strain RIVM601174

KAUST Repository

Abdallah, A. M.; Rashid, M.; Adroub, S. A.; Arnoux, M.; Ali, Shahjahan; van Soolingen, D.; Bitter, W.; Pain, Arnab

2012-01-01

Mycobacterium phlei is a rapidly growing nontuberculous Mycobacterium species that is typically nonpathogenic, with few reported cases of human disease. Here we report the whole genome sequence of M. phlei type strain RIVM601174.

Complete Genome Sequence of Mycobacterium phlei Type Strain RIVM601174

KAUST Repository

Abdallah, A. M.

2012-05-24

Mycobacterium phlei is a rapidly growing nontuberculous Mycobacterium species that is typically nonpathogenic, with few reported cases of human disease. Here we report the whole genome sequence of M. phlei type strain RIVM601174.

Identification of novel linear megaplasmids carrying a ß-lactamase gene in neurotoxigenic Clostridium butyricum type E strains.

Directory of Open Access Journals (Sweden)

Giovanna Franciosa

Full Text Available Since the first isolation of type E botulinum toxin-producing Clostridium butyricum from two infant botulism cases in Italy in 1984, this peculiar microorganism has been implicated in different forms of botulism worldwide. By applying particular pulsed-field gel electrophoresis run conditions, we were able to show for the first time that ten neurotoxigenic C. butyricum type E strains originated from Italy and China have linear megaplasmids in their genomes. At least four different megaplasmid sizes were identified among the ten neurotoxigenic C. butyricum type E strains. Each isolate displayed a single sized megaplasmid that was shown to possess a linear structure by ATP-dependent exonuclease digestion. Some of the neurotoxigenic C. butyricum type E strains possessed additional smaller circular plasmids. In order to investigate the genetic content of the newly identified megaplasmids, selected gene probes were designed and used in Southern hybridization experiments. Our results revealed that the type E botulinum neurotoxin gene was chromosome-located in all neurotoxigenic C. butyricum type E strains. Similar results were obtained with the 16S rRNA, the tetracycline tet(P and the lincomycin resistance protein lmrB gene probes. A specific mobA gene probe only hybridized to the smaller plasmids of the Italian C. butyricum type E strains. Of note, a ß-lactamase gene probe hybridized to the megaplasmids of eight neurotoxigenic C. butyricum type E strains, of which seven from clinical sources and the remaining one from a food implicated in foodborne botulism, whereas this ß-lactam antibiotic resistance gene was absent form the megaplasmids of the two soil strains examined. The widespread occurrence among C. butyricum type E strains associated to human disease of linear megaplasmids harboring an antibiotic resistance gene strongly suggests that the megaplasmids could have played an important role in the emergence of C. butyricum type E as a human

Evaluation of molecular typing techniques to assign genetic diversity among Saccharomyces cerevisiae strains

NARCIS (Netherlands)

Baleiras Couto, M.M.; Eijsma, B.; Hofstra, H.; Huis in 't Veld, J.H.J.; Vossen, J.M.B.M. van der

1996-01-01

Discrimination of strains within the species Saccharomyces cerevisiae was demonstrated by the use of four different techniques to type 15 strains isolated from spoiled wine and beer. Random amplified polymorphic DNA with specific oligonucleotides and PCR fingerprinting with the microsatellite

Comparison of multilocus sequence typing, RAPD, and MALDI-TOF mass spectrometry for typing of β-lactam-resistant Klebsiella pneumoniae strains.

Science.gov (United States)

Sachse, Svea; Bresan, Stephanie; Erhard, Marcel; Edel, Birgit; Pfister, Wolfgang; Saupe, Angela; Rödel, Jürgen

2014-12-01

Extended spectrum of β-lactam (ESBL) resistance of Klebsiella pneumoniae has become an increasing problem in hospital infections. Typing of isolates is important to establish the intrahospital surveillance of resistant clones. In this study, the discriminatory potential of randomly amplified polymorphic DNA and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses were compared with multilocus sequence typing (MLST) by using 17 β-lactam-resistant K. pneumoniae isolates of different genotypes. MLST alleles were distributed in 8 sequence types (STs). Among ESBL strains of the same ST, the presence of different β-lactamase genes was common. RAPD band patterns also revealed 8 types that corresponded to MLST-defined genotypes in 15 out of 17 cases. MALDI-TOF analysis could differentiate 5 clusters of strains. The results of this work show that RAPD may be usable as a rapid screening method for the intrahospital surveillance of K. pneumoniae, allowing a discrimination of clonally related strains. MALDI-TOF-based typing was not strongly corresponding to genotyping and warrants further investigation. Copyright © 2014 Elsevier Inc. All rights reserved.

Mead production: selection and characterization assays of Saccharomyces cerevisiae strains.

Science.gov (United States)

Pereira, Ana Paula; Dias, Teresa; Andrade, João; Ramalhosa, Elsa; Estevinho, Letícia M

2009-08-01

Mead is a traditional drink, which results from the alcoholic fermentation of diluted honey carried out by yeasts. However, when it is produced in a homemade way, mead producers find several problems, namely, the lack of uniformity in the final product, delayed and arrested fermentations, and the production of "off-flavours" by the yeasts. These problems are usually associated with the inability of yeast strains to respond and adapt to unfavourable and stressful growth conditions. The main objectives of this work were to evaluate the capacity of Saccharomyces cerevisiae strains, isolated from honey of the Trás-os-Montes (Northeast Portugal), to produce mead. Five strains from honey, as well as one laboratory strain and one commercial wine strain, were evaluated in terms of their fermentation performance under ethanol, sulphur dioxide and osmotic stress. All the strains showed similar behaviour in these conditions. Two yeasts strains isolated from honey and the commercial wine strain were further tested for mead production, using two different honey (a dark and a light honey), enriched with two supplements (one commercial and one developed by the research team), as fermentation media. The results obtained in this work show that S. cerevisiae strains isolated from honey, are appropriate for mead production. However it is of extreme importance to take into account the characteristics of the honey, and supplements used in the fermentation medium formulation, in order to achieve the best results in mead production.

Differential growth of Mycobacterium leprae strains (SNP genotypes) in armadillos.

Science.gov (United States)

Sharma, Rahul; Singh, Pushpendra; Pena, Maria; Subramanian, Ramesh; Chouljenko, Vladmir; Kim, Joohyun; Kim, Nayong; Caskey, John; Baudena, Marie A; Adams, Linda B; Truman, Richard W

2018-04-14

Leprosy (Hansen's Disease) has occurred throughout human history, and persists today at a low prevalence in most populations. Caused by Mycobacterium leprae, the infection primarily involves the skin, mucosa and peripheral nerves. The susceptible host range for Mycobacterium leprae is quite narrow. Besides humans, nine banded armadillos (Dasypus novemcinctus) and red squirrels (Sciurus vulgaris) are the only other natural hosts for M. leprae, but only armadillos recapitulate the disease as seen in humans. Armadillos across the Southern United States harbor a single predominant genotypic strain (SNP Type-3I) of M. leprae, which is also implicated in the zoonotic transmission of leprosy. We investigated, whether the zoonotic strain (3I) has any notable growth advantages in armadillos over another genetically distant strain-type (SNP Type-4P) of M. leprae, and if M. leprae strains manifest any notably different pathology among armadillos. We co-infected armadillos (n = 6) with 2 × 10 9 highly viable M. leprae of both strains and assessed the relative growth and dissemination of each strain in the animals. We also analyzed 12 additional armadillos, 6 each individually infected with the same quantity of either strain. The infections were allowed to fulminate and the clinical manifestations of the disease were noted. Animals were humanely sacrificed at the terminal stage of infection and the number of bacilli per gram of liver, spleen and lymph node tissue were enumerated by Q-PCR assay. The growth of M. leprae strain 4P was significantly higher (P leprae strains within armadillos suggest there are notable pathological variations between M. leprae strain-types. Copyright © 2018. Published by Elsevier B.V.

Phage type and sensitivity to antibiotics of Staphylococcus aureus film-forming strains isolated from airway mucosa

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O. S. Voronkova

2014-10-01

Full Text Available Today film-forming strains of bacteria play very important role in clinical pathology. Staphylococci are ones of most dangerous of them. This bacteria can determine different pathological processes, for example, complication of airway mucosa. The ability to form a biofilm is one of the main properties of nosocomial strains. These strains should be monitored and their carriers are to be properly treated. To determine the origin of staphylococci strains we used bacteriophages from the International kit. The aim of research was to determine the phage type of staphylococci film-forming strains, that were isolated from naso-pharingial mucosa. Phage typing has been carried out for 16 film-forming strains of S. aureus. To solve this problem, we used the International phage kit by Fisher’s method. As a result, sensitivity to phages from the International kit showed 53.8% of studied strains of S. aureus. 64.3% of sensitivity strains were lysed by one of the phage, 21.4% – were by two of the phages, 14.3% – by three of the phages. Isolates were sensitive to phages: 81 – 42.9%, 75 – 35.7%, 28.6% were sensitive to phages 47 and 53. All cases of detection of sensitivity to phage 47 coincided with the ability to form biofilm. Among non-film-forming strains there was no sensitive strains for this phage. Film-forming strains resist to erythromycin (62.5%, ciprofloxacin (43.8%, gentamicin (56.3%, tetracycline (87.5%, amoxicillin (93.8%, and cefuroxime (37.5%. All cases of sensitivity to phage 47 coincided with resistance to erythromycin, amoxicillin and tetracycline. For two of these strains, we also defined resistance to gentamicin and for one of them – to ciprofloxacin. Results of research allowed to relate the bacterial cultures for determining the type. This may have implications for studying of film-forming ability, because surface structures of bacterial cell take place in this process. Belonging of an isolate to specific phage type may

Isochronous relaxation curves for type 304 stainless steel after monotonic and cyclic strain

International Nuclear Information System (INIS)

Swindeman, R.W.

1978-01-01

Relaxation tests to 100 hr were performed on type 304 stainless steel in the temperature range 480 to 650 0 C and were used to develop isochronous relaxation curves. Behavior after monotonic and cyclic strain was compared. Relaxation differed only slightly as a consequence of the type of previous strain, provided that plastic flow preceded the relaxation period. We observed that the short-time relaxation behavior did not manifest strong heat-to-heat variation in creep strength

Stressed and strained state for cermetic-rod-type fuel element

International Nuclear Information System (INIS)

Kulikov, I.S.

1987-01-01

Calculation technique for designing the stress-strained state of a cermetic rod-type fuel element has been proposed. The technique is based on the time-dependent step-by-step method and the solution of the deformation equilibrium equation for continuous and thick-wall long cylinders at every temporal step by the finite difference method. Additional strains, caused by thermal expansion and radiation swelling, have been taken into account. The transion from the non-contact model to the stiff-contact model has been provided in the case of cladding-fuel gap dissappearing in one or a number of cross-sections along the fuel element height. The method is supplemented by the formula for fuel cans stability estimation in the case of high coolant external pressure. The example of estimation of the cermetic-rod-type fuel elements are considered as an example

Difference in Degradation Patterns on Inulin-type Fructans among Strains of Lactobacillus delbrueckii and Lactobacillus paracasei.

Science.gov (United States)

Tsujikawa, Yuji; Nomoto, Ryohei; Osawa, Ro

2013-01-01

Lactobacillus delbrueckii strains were assessed for their degradation patterns of various carbohydrates with specific reference to inulin-type fructans in comparison with those of Lactobacillus paracasei strains. Firstly, growth curves on glucose, fructose, sucrose and inulin-type fructans with increasing degrees of fructose polymerization (i.e., 1-kestose, fructo-oligosaccharides and inulin) of the strains were compared. L. paracasei DSM 20020 grew well on all these sugars, while the growth rates of the 4 L. delbrueckii strains were markedly higher on the fructans with a greater degree of polymerization than on fructose and sucrose. Secondly, sugar compositions of spent cultures of the strains of L. delbrueckii and L. paracasei grown in mMRS containing either the fructans or inulin were determined by thin layer chromatography, in which the spent cultures of L. paracasei DSM 20020 showed spots of short fructose and sucrose fractions, whereas those of the L. delbrueckii strains did not show such spots at all. These results suggest that, unlike the L. paracasei strains, the L. delbrueckii strains do not degrade the inulin-type fructans extracellularly, but transport the fructans capable of greater polymerization preferentially into their cells to be degraded intracellularly for their growth.

Characterization of incompletely typed rotavirus strains from Guinea-Bissau: identification of G8 and G9 types and a high frequency of mixed infections

International Nuclear Information System (INIS)

Fischer, T.K.; Page, N.A.; Griffin, D.D.; Eugen-Olsen, J.; Pedersen, A.G.; Valentiner-Branth, P.; Moelbak, K.; Sommerfelt, H.; Nielsen, N. Munk

2003-01-01

Among 167 rotavirus specimens collected from young children in a suburban area of Bissau, Guinea-Bissau, from 1996 to 1998, most identifiable strains belonged to the uncommon P[6], G2 type and approximately 50% remained incompletely typed. In the present study, 76 such strains were further characterized. Due to interprimer interaction during the standard multiplex PCR approach, modifications of this procedure were implemented. The modified analyses revealed a high frequency of G2, G8, and G9 genotypes, often combined with P[4] and/or P[6]. The Guinean G8 and G9 strains were 97 and 98%, respectively, identical to other African G8 and G9 strains. Multiple G and/or P types were identified at a high frequency (59%), including two previously undescribed mixed infections, P[4]P[6], G2G8 and P[4]P[6], G2G9. These mixed infections most likely represent naturally occurring reassortance of rotavirus strains. Detection of such strains among the previously incompletely typed strains indicates a potential underestimation of mixed infections, if only a standard multiplex PCR procedure is followed. Furthermore cross-priming of the G3 primer with the G8 primer binding site and silent mutations at the P[4] and P[6] primer binding sites were detected. These findings highlight the need for regular evaluation of the multiplex primer PCR method and typing primers. The high frequency of uncommon as well as reassortant rotavirus strains in countries where rotavirus is an important cause of child mortality underscores the need for extensive strain surveillance as a basis to develop appropriate rotavirus vaccine candidates

Staphylococcus aureus strains associated with food poisoning outbreaks in France: comparison of different molecular typing methods, including MLVA

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Roussel, Sophie; Felix, Benjamin; Vingadassalon, Noémie; Grout, Joël; Hennekinne, Jacques-Antoine; Guillier, Laurent; Brisabois, Anne; Auvray, Fréderic

2015-01-01

Staphylococcal food poisoning outbreaks (SFPOs) are frequently reported in France. However, most of them remain unconfirmed, highlighting a need for a better characterization of isolated strains. Here we analyzed the genetic diversity of 112 Staphylococcus aureus strains isolated from 76 distinct SFPOs that occurred in France over the last 30 years. We used a recently developed multiple-locus variable-number tandem-repeat analysis (MLVA) protocol and compared this method with pulsed field gel electrophoresis (PFGE), spa-typing and carriage of genes (se genes) coding for 11 staphylococcal enterotoxins (i.e., SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SEJ, SEP, SER). The strains known to have an epidemiological association with one another had identical MLVA types, PFGE profiles, spa-types or se gene carriage. MLVA, PFGE and spa-typing divided 103 epidemiologically unrelated strains into 84, 80, and 50 types respectively demonstrating the high genetic diversity of S. aureus strains involved in SFPOs. Each MLVA type shared by more than one strain corresponded to a single spa-type except for one MLVA type represented by four strains that showed two different-but closely related-spa-types. The 87 enterotoxigenic strains were distributed across 68 distinct MLVA types that correlated all with se gene carriage except for four MLVA types. The most frequent se gene detected was sea, followed by seg and sei and the most frequently associated se genes were sea-seh and sea-sed-sej-ser. The discriminatory ability of MLVA was similar to that of PFGE and higher than that of spa-typing. This MLVA protocol was found to be compatible with high throughput analysis, and was also faster and less labor-intensive than PFGE. MLVA holds promise as a suitable method for investigating SFPOs and tracking the source of contamination in food processing facilities in real time. PMID:26441849

Determination of the molar extinction coefficient for the ferric reducing/antioxidant power assay.

Science.gov (United States)

Hayes, William A; Mills, Daniel S; Neville, Rachel F; Kiddie, Jenna; Collins, Lisa M

2011-09-15

The FRAP reagent contains 2,4,6-tris(2-pyridyl)-s-triazine, which forms a blue-violet complex ion in the presence of ferrous ions. Although the FRAP (ferric reducing/antioxidant power) assay is popular and has been in use for many years, the correct molar extinction coefficient of this complex ion under FRAP assay conditions has never been published, casting doubt on the validity of previous calibrations. A previously reported value of 19,800 is an underestimate. We determined that the molar extinction coefficient was 21,140. The value of the molar extinction coefficient was also shown to depend on the type of assay and was found to be 22,230 under iron assay conditions, in good agreement with published data. Redox titration indicated that the ferrous sulfate heptahydrate calibrator recommended by Benzie and Strain, the FRAP assay inventors, is prone to efflorescence and, therefore, is unreliable. Ferrous ammonium sulfate hexahydrate in dilute sulfuric acid was a more stable alternative. Few authors publish their calibration data, and this makes comparative analyses impossible. A critical examination of the limited number of examples of calibration data in the published literature reveals only that Benzie and Strain obtained a satisfactory calibration using their method. Copyright © 2011 Elsevier Inc. All rights reserved.

CRISPR is an optimal target for the design of specific PCR assays for salmonella enterica serotypes Typhi and Paratyphi A.

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Laetitia Fabre

Full Text Available BACKGROUND: Serotype-specific PCR assays targeting Salmonella enterica serotypes Typhi and Paratyphi A, the causal agents of typhoid and paratyphoid fevers, are required to accelerate formal diagnosis and to overcome the lack of typing sera and, in some situations, the need for culture. However, the sensitivity and specificity of such assays must be demonstrated on large collections of strains representative of the targeted serotypes and all other bacterial populations producing similar clinical symptoms. METHODOLOGY: Using a new family of repeated DNA sequences, CRISPR (clustered regularly interspaced short palindromic repeats, as a serotype-specific target, we developed a conventional multiplex PCR assay for the detection and differentiation of serotypes Typhi and Paratyphi A from cultured isolates. We also developed EvaGreen-based real-time singleplex PCR assays with the same two sets of primers. PRINCIPAL FINDINGS: We achieved 100% sensitivity and specificity for each protocol after validation of the assays on 188 serotype Typhi and 74 serotype Paratyphi A strains from diverse genetic groups, geographic origins and time periods and on 70 strains of bacteria frequently encountered in bloodstream infections, including 29 other Salmonella serotypes and 42 strains from 38 other bacterial species. CONCLUSIONS: The performance and convenience of our serotype-specific PCR assays should facilitate the rapid and accurate identification of these two major serotypes in a large range of clinical and public health laboratories with access to PCR technology. These assays were developed for use with DNA from cultured isolates, but with modifications to the assay, the CRISPR targets could be used in the development of assays for use with clinical and other samples.

Primary isolation strain determines both phage type and receptors recognised by Campylobacter jejuni bacteriophages.

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Martine C Holst Sørensen

Full Text Available In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN of CPS as a phage receptor. We therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb, host range and morphological appearance correlated with the isolation strain. Thus, according to C. jejuni phage grouping, NCTC12662 and NCTC12658 selected for CP81-type phages, while RM1221 selected for CP220-type phages. Furthermore, using acapsular ∆kpsM mutants we demonstrated that phages isolated on NCTC12658 and NCTC12662 were dependent on the capsule for infection. In contrast, CP220-type phages isolated on RM1221 were unable to infect non-motile ∆motA mutants, hence requiring motility for successful infection. Hence, the primary phage isolation strain determines both phage type (CP81 or CP220 as well as receptors (CPS or flagella recognised by the isolated phages.

Probe-based real-time PCR method for multilocus melt typing of Xylella fastidiosa strains.

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Brady, Jeff A; Faske, Jennifer B; Ator, Rebecca A; Castañeda-Gill, Jessica M; Mitchell, Forrest L

2012-04-01

Epidemiological studies of Pierce's disease (PD) can be confounded by a lack of taxonomic detail on the bacterial causative agent, Xylella fastidiosa (Xf). PD in grape is caused by strains of Xylella fastidiosa subsp. fastidiosa, but is not caused by other subspecies of Xf that typically colonize plants other than grape. Detection assays using ELISA and qPCR are effective at detecting and quantifying Xf presence or absence, but offer no information on Xf subspecies or strain identity. Surveying insects or host plants for Xf by current ELISA or qPCR methods provides only presence/absence and quantity information for any and all Xf subspecies, potentially leading to false assessments of disease threat. This study uses a series of adjacent-hybridizing DNA melt analysis probes that are capable of efficiently discriminating Xf subspecies and strain relationships in rapid real-time PCR reactions. Copyright © 2012 Elsevier B.V. All rights reserved.

Influence of dynamic strain ageing on tensile strain energy of type 316L(N) austenitic stainless steel

International Nuclear Information System (INIS)

Isaac Samuel, B.; Choudhary, B.K.; Bhanu Sankara Rao, K.

2010-01-01

Tensile tests were conducted on type 316 L(N) stainless steel over a wide temperature range of 300-1123 K employing strain rates ranging from 3.16 X 10 -5 to 3.16 X 10 -3/s . The variation of strain energy in terms of modulus of resilience and modulus of toughness over the wide range of temperatures and strain rates were examined. The variation in modulus of resilience with temperature and strain rate did not show the signatures of dynamic strain ageing (DSA). However, the modulus of toughness exhibited a plateau at the intermediate temperatures of 523-1023 K. Further, the distribution of energy absorbed till necking and energy absorbed from necking till fracture were found to characterise the deformation and damage processes, respectively, and exhibited anomalous variations in the temperature range 523-823 K and 823-1023 K, respectively. In addition to the observed manifestations of DSA such as serrated load-elongation curve, peaks/plateaus in flow stress, ultimate tensile strength and work hardening rate, negative strain rate sensitivity and ductility minima, the observed anomalous variations in modulus of toughness at intermediate temperatures (523-1023 K) can be regarded as yet another key manifestation of DSA. At temperatures above 1023 K, a sharp decrease in the modulus of toughness and also in the strain energies up to necking and from necking to fracture observed, with increasing temperature and decreasing strain rate, reveal the onset of dynamic recovery leading to early cross slip and climb processes. (author)

Strain typing of acetic acid bacteria responsible for vinegar production by the submerged elaboration method.

Science.gov (United States)

Fernández-Pérez, Rocío; Torres, Carmen; Sanz, Susana; Ruiz-Larrea, Fernanda

2010-12-01

Strain typing of 103 acetic acid bacteria isolates from vinegars elaborated by the submerged method from ciders, wines and spirit ethanol, was carried on in this study. Two different molecular methods were utilised: pulsed field gel electrophoresis (PFGE) of total DNA digests with a number of restriction enzymes, and enterobacterial repetitive intergenic consensus (ERIC) - PCR analysis. The comparative study of both methods showed that restriction fragment PFGE of SpeI digests of total DNA was a suitable method for strain typing and for determining which strains were present in vinegar fermentations. Results showed that strains of the species Gluconacetobacter europaeus were the most frequent leader strains of fermentations by the submerged method in the studied vinegars, and among them strain R1 was the predominant one. Results showed as well that mixed populations (at least two different strains) occurred in vinegars from cider and wine, whereas unique strains were found in spirit vinegars, which offered the most stressing conditions for bacterial growth. Copyright © 2010 Elsevier Ltd. All rights reserved.

Genome sequence of the thermophile Bacillus coagulans Hammer, the type strain of the species.

Science.gov (United States)

Su, Fei; Tao, Fei; Tang, Hongzhi; Xu, Ping

2012-11-01

Here we announce a 3.0-Mb assembly of the Bacillus coagulans Hammer strain, which is the type strain of the species within the genus Bacillus. Genomic analyses based on the sequence may provide insights into the phylogeny of the species and help to elucidate characteristics of the poorly studied strains of Bacillus coagulans.

Genome Sequence of the Thermophile Bacillus coagulans Hammer, the Type Strain of the Species

OpenAIRE

Su, Fei; Tao, Fei; Tang, Hongzhi; Xu, Ping

2012-01-01

Here we announce a 3.0-Mb assembly of the Bacillus coagulans Hammer strain, which is the type strain of the species within the genus Bacillus. Genomic analyses based on the sequence may provide insights into the phylogeny of the species and help to elucidate characteristics of the poorly studied strains of Bacillus coagulans.

Effects of strain differences and vehicles on results of local lymph node assays.

Science.gov (United States)

Anzai, Takayuki; Ullmann, Ludwig G; Hayashi, Daisuke; Satoh, Tetsuo; Kumazawa, Takeshi; Sato, Keizo

2010-01-01

The Local Lymph Node Assay (LLNA) is now regarded as the worldwide standard. The analysis of accumulated LLNA data reveals that the animal strains and vehicles employed are likely to affect LLNA results. Here we show that an obvious strain difference in the local lymph node response was observed between DMSO-treated CBA/CaOlaHsd and CBA/CaHsdRcc mice. We also show that a vehicle difference in the response was observed when CBA/CaHsdRcc mice were exposed to 6 vehicles; 4:1 v/v acetone/olive oil (AOO), ethanol/water (70% EtOH), N,N-dimethylformamide (DMF), 2-butanone (BN), propylene glycol (PG), and dimethylsulfoxide (DMSO). The dpm/LN level was lowest in the 70% EtOH group and highest in the DMSO group. When alpha-hexylcinnamaldehyde (HCA) was used as a sensitizer for the LLNA, HCA was a weak sensitizer when AOO or DMSO was used as a vehicle, but a moderate sensitizer when the other 4 vehicles were used. This study showed that there are vehicle differences in the local lymph node response (dpm/LN level) in the LLNA and that the sensitization potency of HCA may be classified in different categories when using different vehicles. This suggests that careful consideration should be exercised in selecting a vehicle for the LLNA. A further comprehensive study will be needed to investigate why vehicle differences are observed in the LLNA.

Experimental infection of chickens and turkeys with Mycoplasma gallisepticum reference strain S6 and North Carolina field isolate RAPD type B.

Science.gov (United States)

Sanei, B; Barnes, H J; Vaillancourt, J P; Leyc, D H

2007-03-01

During an epidemic of mycoplasmosis in chicken and turkey flocks in North Carolina between 1999 and 2001, isolates of Mycoplasma gallisepticum (MG) from affected flocks were characterized by random amplification of polymorphic DNA (RAPD), and eight distinct RAPD types were identified. MG RAPD type B accounted for more than 90% of the isolates and was associated with moderate-to-severe clinical signs and mortality. The virulence of MG RAPD type B for chickens and turkeys was compared with sham-inoculated negative controls and MG S6 (a virulent strain)-inoculated positive controls. Clinical signs occurred in chickens and turkeys inoculated with either MG RAPD type B or MG S6. However, they were not as frequent or severe as those seen in naturally affected flocks, and there was no mortality in the experimental groups. Based on gross and microscopic findings, MG RAPD type B was equal to or more virulent than MG S6. All MG-inoculated birds were culture and PCR positive at 7 and 14 days postinoculation (PI). Among serological tests, the serum plate agglutination test was positive for the majority of chickens and turkeys (58%-100%) infected with either strain of MG at both 7 and 14 days PI. The hemagglutination inhibition test was negative for all birds at 7 days PI and positive for a few chickens (8%-17%) and several turkey sera (40%-60%) at 14 days PI. Only a single serum was positive by enzyme-linked immunosorbent assay (an MG S6-infected turkey) at 14 days PI.

Enhanced Molecular Typing of Treponema pallidum subspecies pallidum Strains From 4 Italian Hospitals Shows Geographical Differences in Strain Type Heterogeneity, Widespread Resistance to Macrolides, and Lack of Mutations Associated With Doxycycline Resistance.

Science.gov (United States)

Giacani, Lorenzo; Ciccarese, Giulia; Puga-Salazar, Christian; Dal Conte, Ivano; Colli, Laura; Cusini, Marco; Ramoni, Stefano; Delmonte, Sergio; DʼAntuono, Antonietta; Gaspari, Valeria; Drago, Francesco

2018-04-01

Although syphilis rates have been relatively high in Italy for more than 15 years, no data on the molecular types of Treponema pallidum subspecies pallidum circulating in this country are yet available. Likewise, no data on how widespread is resistance to macrolide or tetracycline antibiotics in these strains exist. Such data would, however, promote comprehensive studies on the molecular epidemiology of syphilis infections in Italy and inform future interventions aiming at syphilis control in this and other European countries. Swabs from oral, genital, cutaneous, or anal lesions were obtained from 60 syphilis patients attending dermatology clinics in Milan, Turin, Genoa, and Bologna. Molecular typing of T. pallidum DNA was performed to provide a snapshot of the genetic diversity of strains circulating in Northern Italy. Samples were also screened for mutations conferring resistance to macrolides and tetracyclines. T. pallidum DNA was detected in 88.3% (53/60) of the specimens analyzed. Complete and partial T. pallidum typing data were obtained for 77.3% (41/53) and 15.0% (8/53) of samples, respectively, whereas 4 samples could not be typed despite T. pallidum DNA being detected. The highest strain type heterogeneity was seen in samples from Bologna and Milan, followed by Genoa. Minimal diversity was detected in samples from Turin, despite the highest number of typeable samples collected there. Resistance to macrolides was detected in 94.3% (50/53) of the strains, but no known mutations associated with tetracycline resistance were found. Genetic diversity among T. pallidum strains circulating in Northern Italy varies significantly among geographical areas regardless of physical distance. Resistance to macrolides is widespread.

Discriminating the hemolytic risk of blood type A plasmas using the complement hemolysis using human erythrocytes (CHUHE) assay.

Science.gov (United States)

Cunnion, Kenji M; Hair, Pamela S; Krishna, Neel K; Sass, Megan A; Enos, Clinton W; Whitley, Pamela H; Maes, Lanne Y; Goldberg, Corinne L

2017-03-01

The agglutination-based cross-matching method is sensitive for antibody binding to red blood cells but is only partially predictive of complement-mediated hemolysis, which is important in many acute hemolytic transfusion reactions. Here, we describe complement hemolysis using human erythrocytes (CHUHE) assays that directly evaluate complement-mediated hemolysis between individual serum-plasma and red blood cell combinations. The CHUHE assay is used to evaluate correlations between agglutination titers and complement-mediated hemolysis as well as the hemolytic potential of plasma from type A blood donors. Plasma or serum from each type A blood donor was incubated with AB or B red blood cells in the CHUHE assay and measured for free hemoglobin release. CHUHE assays for serum or plasma demonstrate a wide, dynamic range and high sensitivity for complement-mediated hemolysis for individual serum/plasma and red blood cell combinations. CHUHE results suggest that agglutination assays alone are only moderately predictive of complement-mediated hemolysis. CHUHE results also suggest that plasma from particular type A blood donors produce minimal complement-mediated hemolysis, whereas plasma from other type A blood donors produce moderate to high-level complement-mediated hemolysis, depending on the red blood cell donor. The current results indicate that the CHUHE assay can be used to assess complement-mediated hemolysis for plasma or serum from a type A blood donor, providing additional risk discrimination over agglutination titers alone. © 2016 AABB.

Complete genome sequence of Hippea maritima type strain (MH2T)

Energy Technology Data Exchange (ETDEWEB)

Huntemann, Marcel [U.S. Department of Energy, Joint Genome Institute; Lu, Megan [Los Alamos National Laboratory (LANL); Nolan, Matt [U.S. Department of Energy, Joint Genome Institute; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Hammon, Nancy [U.S. Department of Energy, Joint Genome Institute; Deshpande, Shweta [U.S. Department of Energy, Joint Genome Institute; Cheng, Jan-Fang [U.S. Department of Energy, Joint Genome Institute; Tapia, Roxanne [Los Alamos National Laboratory (LANL); Han, Cliff [Los Alamos National Laboratory (LANL); Goodwin, Lynne A. [Los Alamos National Laboratory (LANL); Pitluck, Sam [U.S. Department of Energy, Joint Genome Institute; Liolios, Konstantinos [U.S. Department of Energy, Joint Genome Institute; Pagani, Ioanna [U.S. Department of Energy, Joint Genome Institute; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Ovchinnikova, Galina [U.S. Department of Energy, Joint Genome Institute; Pati, Amrita [U.S. Department of Energy, Joint Genome Institute; Chen, Amy [U.S. Department of Energy, Joint Genome Institute; Palaniappan, Krishna [U.S. Department of Energy, Joint Genome Institute; Land, Miriam L [ORNL; Hauser, Loren John [ORNL; Jeffries, Cynthia [Oak Ridge National Laboratory (ORNL); Detter, J. Chris [U.S. Department of Energy, Joint Genome Institute; Brambilla, Evelyne-Marie [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Rohde, Manfred [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Spring, Stefan [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Goker, Markus [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Woyke, Tanja [U.S. Department of Energy, Joint Genome Institute; Bristow, James [U.S. Department of Energy, Joint Genome Institute; Eisen, Jonathan [U.S. Department of Energy, Joint Genome Institute; Markowitz, Victor [U.S. Department of Energy, Joint Genome Institute; Hugenholtz, Philip [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute; Klenk, Hans-Peter [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute

2011-01-01

Hippea maritima (Miroshnichenko et al. 1999) is the type species of the genus Hippea, which belongs to the family Desulfurellaceae within the class Deltaproteobacteria. The anaerobic, moderately thermophilic marine sulfur-reducer was first isolated from shallow-water hot vents in Matipur Harbor, Papua New Guinea. H. maritima was of interest for genome se- quencing because of its isolated phylogenetic location, as a distant next neighbor of the ge- nus Desulfurella. Strain MH2T is the first type strain from the order Desulfurellales with a com- pletely sequenced genome. The 1,694,430 bp long linear genome with its 1,723 protein- coding and 57 RNA genes consists of one circular chromosome and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Succinate Dehydrogenase Activity Assay in situ with Blue Tetrazolium Salt in Crabtree-Positive Saccharomyces cerevisiae Strain

Directory of Open Access Journals (Sweden)

Joanna Berlowska

2008-01-01

Full Text Available A spectrophotometric method for determining succinate dehydrogenase (SDH activity assay in azide-sensitive yeast Saccharomyces cerevisiae has been developed. The permeabilization of yeast cells by 0.05 % digitonin permitted to study yeast enzymatic activity in situ. The reduction of blue tetrazolium salt (BT to blue tetrazolium formazan (BTf was conducted in the presence of phenazine methosulphate (PMS as an exogenous electron carrier, and sodium azide (SA as an inhibitor of cytochrome oxidase (Cyt pathway. Various factors such as type of substrate, BT concentration, cell number, temperature and time of incubation, and different Cyt pathway blockers were optimized. In earlier studies, dimethyl sulfoxide (DMSO had been selected as the best solvent for extraction of BTf from yeast cells. The linear correlation between permeabilized yeast cell density and amount of formed formazan was evidenced in the range from 9·10^7 to 5·10^8 cells per sample solution. Below the yeast cell concentration of 10^7 the absorbance values were too low to detect formazans with good precision. This standarized procedure allows the estimation of SDH activity in whole cells, depending on vitality level of yeast populations. Significant increases of succinate dehydrogenase activities were observed in sequential passages as the result of the increase of activity of the strain and adaptation to cultivation conditions.

Complete Genome Sequence of Mycobacterium vaccae Type Strain ATCC 25954

KAUST Repository

Ho, Y. S.

2012-10-26

Mycobacterium vaccae is a rapidly growing, nontuberculous Mycobacterium species that is generally not considered a human pathogen and is of major pharmaceutical interest as an immunotherapeutic agent. We report here the annotated genome sequence of the M. vaccae type strain, ATCC 25954.

Complete Genome Sequence of Mycobacterium vaccae Type Strain ATCC 25954

KAUST Repository

Ho, Y. S.; Adroub, S. A.; Abadi, Maram; Al Alwan, B.; Alkhateeb, R.; Gao, G.; Ragab, A.; Ali, Shahjahan; van Soolingen, D.; Bitter, W.; Pain, Arnab; Abdallah, A. M.

2012-01-01

Mycobacterium vaccae is a rapidly growing, nontuberculous Mycobacterium species that is generally not considered a human pathogen and is of major pharmaceutical interest as an immunotherapeutic agent. We report here the annotated genome sequence of the M. vaccae type strain, ATCC 25954.

Different distribution patterns of ten virulence genes in Legionella reference strains and strains isolated from environmental water and patients.

Science.gov (United States)

Zhan, Xiao-Yong; Hu, Chao-Hui; Zhu, Qing-Yi

2016-04-01

Virulence genes are distinct regions of DNA which are present in the genome of pathogenic bacteria and absent in nonpathogenic strains of the same or related species. Virulence genes are frequently associated with bacterial pathogenicity in genus Legionella. In the present study, an assay was performed to detect ten virulence genes, including iraA, iraB, lvrA, lvrB, lvhD, cpxR, cpxA, dotA, icmC and icmD in different pathogenicity islands of 47 Legionella reference strains, 235 environmental strains isolated from water, and 4 clinical strains isolated from the lung tissue of pneumonia patients. The distribution frequencies of these genes in reference or/and environmental L. pneumophila strains were much higher than those in reference non-L. pneumophila or/and environmental non-L. pneumophila strains, respectively. L. pneumophila clinical strains also maintained higher frequencies of these genes compared to four other types of Legionella strains. Distribution frequencies of these genes in reference L. pneumophila strains were similar to those in environmental L. pneumophila strains. In contrast, environmental non-L. pneumophila maintained higher frequencies of these genes compared to those found in reference non-L. pneumophila strains. This study illustrates the association of virulence genes with Legionella pathogenicity and reveals the possible virulence evolution of non-L. pneumophia strains isolated from environmental water.

Automated 5 ' nuclease assay for detection of virulence factors in porcine Escherichia coli

DEFF Research Database (Denmark)

Frydendahl, K.; Imberechts, H.; Lehmann, S.

2001-01-01

(STa, STb, EAST1) and heat labile LT) enterotoxins and the verocytotoxin variant 2e (VT2e). To correctly identify false negative results, an endogenous internal control targeting the E. coil 16S rRNA gene was incorporated in each test tube. The assay was evaluated using a collection of E. coil...... reference strains which have previously been examined with phenotypical assays or DNA hybridization. Furthermore, the assay was evaluated by testing porcine E. coil field strains, previously characterized. The 5' nuclease assay correctly detected the presence of virulence genes in all reference strains....... When testing field strains there was generally excellent agreement with results obtained by laboratories in Belgium and Germany. In conclusion, the 5' nuclease assay developed is a fast and specific tool for detection of E. coli virulence genes in the veterinary diagnostic laboratory....

Population Genetic Structure of Listeria monocytogenes Strains as Determined by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing

DEFF Research Database (Denmark)

Henri, Clémentine; Félix, Benjamin; Guillier, Laurent

2016-01-01

on the basis of different pulsed-field gel electrophoresis (PFGE) clusters, serotypes, and strain origins and typed by multilocus sequence typing (MLST), and the MLST results were supplemented with MLST data available from Institut Pasteur, representing human and additional food strains from France....... The distribution of sequence types (STs) was compared between food and clinical strains on a panel of 675 strains. High congruence between PFGE and MLST was found. Out of 73 PFGE clusters, the two most prevalent corresponded to ST9 and ST121. Using original statistical analysis, we demonstrated that (i...

The mechanism of critical strain and serration type of the serrated flow in Mg–Nd–Zn alloy

Energy Technology Data Exchange (ETDEWEB)

Wang, W.H. [The Group of Magnesium Alloys and Their Applications, Institute of Metal Research Chinese Academy of Sciences, 62 Wencui Road, Shenyang 110016 (China); School of Materials Science and Engineering, Shenyang Ligong University, 6 Nanpingzhong Road, Shenyang 110159 (China); Wu, D., E-mail: dwu@imr.ac.cn [The Group of Magnesium Alloys and Their Applications, Institute of Metal Research Chinese Academy of Sciences, 62 Wencui Road, Shenyang 110016 (China); Shah, S.S.A. [The Group of Magnesium Alloys and Their Applications, Institute of Metal Research Chinese Academy of Sciences, 62 Wencui Road, Shenyang 110016 (China); Chen, R.S., E-mail: rschen@imr.ac.cn [The Group of Magnesium Alloys and Their Applications, Institute of Metal Research Chinese Academy of Sciences, 62 Wencui Road, Shenyang 110016 (China); Lou, C.S. [School of Materials Science and Engineering, Shenyang Ligong University, 6 Nanpingzhong Road, Shenyang 110159 (China)

2016-01-01

In present research the serrated flow has been observed successfully after a critical amount of strain. Two relationships between the critical strain and temperature i.e. normal and inverse, corresponding to each serration type were studied. In order to investigate systematically the onset of serrated flow and serration type in NZ31 alloy, samples in solutionized condition were tensile tested at the temperature ranging from 100 °C to 300 °C with the strain rate ranging from 1×10{sup −4} s{sup −1} to 1×10{sup −2} s{sup −1}. Results showed that normal critical strain appeared with type A and B serrated flow at temperature from 150°C to 250 °C, and inverse critical strain appeared with type C at temperature from 275 °C to 300 °C. Through analyzing the mechanism of three serration types, we found that the production of serration required improvement in diffusion for solute atoms for pinning process at low temperature, and enhance the moving ability of dislocations for unpinning process at high temperature, which need the assistance of the strain and stress respectively. So, in this work, the critical strain for pinning and the critical stress for unpinning processes were defined, which give a better explanation to the variation tendency of two definitions in accordance with temperature. Furthermore, this relationship results in the critical strain for onset of serrated flow changing from normal to inverse and corresponding different serrations.

Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples.

Science.gov (United States)

Pulford, D J; Gias, E; Bueno, I M; McFadden, Amj

2016-01-01

To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis. Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay. The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R(2) = 0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with T. orientalis, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with T. orientalis ranged between 5.6 × 10(4) and 3.3 × 10(6) genomes per

POTENTIAL PRODUCTION OF CYCLOPIAZONIC ACID BY PENICILLIUM CAMEMBERTI STRAINS ISOLATED FROM CAMEMBERT TYPE CHEESE

Directory of Open Access Journals (Sweden)

Miroslava Císarová

2012-10-01

Full Text Available The aim of this study was to isolate the strains of fungi from Camembert type cheese, identify them and to test isolated strains of Penicillium camemberti for their ability to produce cyclopiazonic acid. The description of micro- and macromorphological features was used for identification of Penicillium camemberti strains. Strains were subsequently in vitro tested on their potential ability to produce mycotoxin cyclopiazonic acid (CPA. All of the 14 strains of Penicillium camemberti, which were obtained from 20 samples of Camembert type cheese, were cultivated 7, 14, 21, 27 and 30 days on CYA medium at 10±1°C, 15±1°C and 25±1°C in the dark. For determination of CPA production ability by P. camemberti isolates in vitro was TLC used. After 7 days of cultivation cyclopiazonic acid was produced only by 5 from 14 strains cultivated at all cultivation temperatures. After 14 and 21 days of cultivation was CPA produced by 6 strains at all of cultivation temperatures. After 27 and 30 days of cultivation was CPA identified in 7 strains cultivated at all temperatures of cultivation. The other strains also produced mycotoxin, however, not at each temperature. The most productive at all temperatures and after all days were 5 out of 14 tested strains (S9, S10, S13, S18 and S19. Strains S6 and S16 did not produce CPA at any temperature. The lowest production after all days of cultivation was found at 10±1 °C (44% and the highest at 25±1 °C (85%.

A novel multiplex poliovirus binding inhibition assay applicable for large serosurveillance and vaccine studies, without the use of live poliovirus.

Science.gov (United States)

Schepp, Rutger M; Berbers, Guy A M; Ferreira, José A; Reimerink, Johan H; van der Klis, Fiona R

2017-03-01

Large-scale serosurveillance or vaccine studies for poliovirus using the "gold standard" WHO neutralisation test (NT) are very laborious and time consuming. With the polio eradication at hand and with the removal of live attenuated Sabin strains from the oral poliovirus vaccine (OPV), starting with type 2 (as of April 2016), laboratories will need to conform to much more stringent laboratory biosafety regulations when handling live poliovirus strains. In this study, a poliovirus binding inhibition multiplex immunoassay (polio MIA) using inactivated poliovirus vaccine (IPV-Salk) was developed for simultaneous quantification of serum antibodies directed to all three poliovirus types. Our assay shows a good correlation with the NT and an excellent correlation with the ELISA-based binding inhibition assay (POBI). The assay is highly type-specific and reproducible. Additionally, serum sample throughput increases about fivefold relative to NT and POBI and the amount of serum needed is reduced by more than 90%. In conclusion, the polio MIA can be used as a safe and high throughput application, especially for large-scale surveillance and vaccine studies, reducing laboratory time and serum amounts needed. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Effect of Attachment Type on Denture Strain in Maxillary Implant Overdentures: Part 1. Overdenture with Palate.

Science.gov (United States)

Takahashi, Toshihito; Gonda, Tomoya; Maeda, Yoshinobu

This study examined the effects of attachments on strain in maxillary implant overdentures supported by two or four implants. A maxillary edentulous model with implants inserted into anterior, premolar, and molar areas was fabricated, and three types of unsplinted attachments-ball, locator, and magnet-were set on the implants distributed under various conditions. Maxillary experimental dentures were fabricated, and two strain gauges were attached at the anterior midline on the labial and palatal sides. A vertical occlusal load of 98 N was applied and shear strain of the dentures was measured. On both sides, magnet attachments resulted in the lowest shear strain, while ball attachments resulted in the highest shear strain under most conditions. However, differences in shear strain among the three attachment types were not significant when supported by four implants, especially molar implants. Shear strain of the maxillary implant overdenture was lowest when using magnet attachments. Magnet attachments mounted on four implants are recommended to prevent denture complications when using maxillary implant overdentures.

Characterization of incompletely typed rotavirus strains from Guinea-Bissau: identification of G8 and G9 types and a high frequency of mixed infections

DEFF Research Database (Denmark)

Fischer, TK; Page, NA; Griffin, DD

2003-01-01

Among 167 rotavirus specimens collected from young children in a suburban area of Bissau, Guinea-Bissau, from 1996 to 1998, most identifiable strains belonged to the uncommon P[6], G2 type and approximately 50% remained incompletely typed. In the present study, 76 such strains were further......%, respectively, identical to other African G8 and G9 strains. Multiple G and/or P types were identified at a high frequency (59%), including two previously undescribed mixed infections, P[4]P[6], G2G8 and P[4]P[6], G2G9. These mixed infections most likely represent naturally occurring reassortance of rotavirus......] and P[6] primer binding sites were detected. These findings highlight the need for regular evaluation of the multiplex primer PCR method and typing primers. The high frequency of uncommon as well as reassortant rotavirus strains in countries where rotavirus is an important cause of child mortality...

Species attribution and strain typing of Oenococcus oeni (formerly Leuconostoc oenos) with restriction endonuclease fingerprints.

Science.gov (United States)

Viti, C; Giovannetti, L; Granchi, L; Ventura, S

1996-10-01

In several wines, malolactic fermentation is required to improve the organoleptic characters and to stabilize the final product. In order to establish a controlled malolactic fermentation in wine, easy identification and sensitive typing of strains of Oenococcus oeni (new name of the malolactic bacterium Leuconostoc oenos) used as starter cultures are necessary. To accomplish these tasks, several strains of Oenococcus oeni isolated from wines of the Chianti region (Italy), along with reference strains and strains of L. mesenteroides subsp. mesenteroides, L. carnosum, L. fallax, L. pseudomesenteroides, L. lactis and Weisella paramesenteroides, were studied with RFLP of ribosomal genes and ultrasensitive total DNA restriction pattern analysis performed on polyacrylamide gel. With each of four restriction endonucleases used, identical restriction profiles of ribosomal genes were obtained for all strains of O. oeni. These ribopatterns, being strongly dissimilar to profiles of the other lactic acid bacteria tested, appear to be well suited for the attribution of wine lactic acid bacteria to the species O. oeni. Cluster analysis performed on two total DNA restriction profile data sets showed that the species O. oeni possesses a good degree of genomic homogeneity. Very sensitive typing of strains of O. oeni was obtained with total DNA restriction profiles. The potential of an integrated approach using restriction profiles for species assignment and typing of selected malolactic bacteria is demonstrated.

[Identification and typing of hospital strains of Acinetobacter calcoaceticus-Acinetobacter baumanni complex].

Science.gov (United States)

Nemec, A; Urbásková, P; Grimont, F; Vránková, J; Melter, O; Schindler, J

1996-05-01

A collection of 95 strains of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex, isolated between 1991 and 1993 in the Prague Burn Center (BC), was studied. Ninety-one strains were isolated from 43 patients: 50 of them from burnt sites, 22 from endotracheal tube, 13 from urine, 3 from blood and 3 from venous catheter, and 4 strains were isolated from the hospital environment and the nursing staff. The strains were classified by restriction endonuclease fingerprinting of total DNA, plasmid profile analysis, ribotyping, comparison of antibiograms, biotyping and according to epidemiological data, into 31 relatedness groups each of them including 1 to 29 strains, likely to be isolates of the same strain. None of the methods used enabled to distinguish all groups. The importance of the polyphasic approach is emphasized since three multiresistant strains, isolated almost simultaneously in the BC, needed at least two methods to be distinguished (e.g. ribotyping and biotyping). Twenty-eight representative strains of different groups were identified by ribotyping: 18 of them were allocated to genomospecies 2 (A. baumannii), 5 to genomospecies 3 and 5 to genomospecies 13 sensu Tjernberg and Ursing. Only A. baumannii was found to spread among patients. Strains of two multiresistant groups persisted in the BC throughout the period studied and strains of one of these groups were responsible for an outbreak in the autumn of 1993. The methods mentioned above were used to describe 12 multiresistant strains isolated in three hospital wards in other localities. When ribotyped these strains were identified as A. baumannii. The strains of the same origin were identical in their typing profiles while the strains of different origins were easy to differentiate using any of the above methods; nevertheless, 2 of these groups were almost identical to 2 groups of multiresistant strains isolated in the BC.

Specificity and Strain-Typing Capabilities of Nanorod Array-Surface Enhanced Raman Spectroscopy for Mycoplasma pneumoniae Detection.

Directory of Open Access Journals (Sweden)

Kelley C Henderson

Full Text Available Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the human respiratory tract that accounts for > 20% of all community-acquired pneumonia (CAP. At present the most effective means for detection and strain-typing is quantitative polymerase chain reaction (qPCR, which can exhibit excellent sensitivity and specificity but requires separate tests for detection and genotyping, lacks standardization between available tests and between labs, and has limited practicality for widespread, point-of-care use. We have developed and previously described a silver nanorod array-surface enhanced Raman Spectroscopy (NA-SERS biosensing platform capable of detecting M. pneumoniae with statistically significant specificity and sensitivity in simulated and true clinical throat swab samples, and the ability to distinguish between reference strains of the two main genotypes of M. pneumoniae. Furthermore, we have established a qualitative lower endpoint of detection for NA-SERS of < 1 genome equivalent (cell/μl and a quantitative multivariate detection limit of 5.3 ± 1 cells/μl. Here we demonstrate using partial least squares- discriminatory analysis (PLS-DA of sample spectra that NA-SERS correctly identified M. pneumoniae clinical isolates from globally diverse origins and distinguished these from a panel of 12 other human commensal and pathogenic mycoplasma species with 100% cross-validated statistical accuracy. Furthermore, PLS-DA correctly classified by strain type all 30 clinical isolates with 96% cross-validated accuracy for type 1 strains, 98% cross-validated accuracy for type 2 strains, and 90% cross-validated accuracy for type 2V strains.

Analysis of the diversity of murine antibodies to dextran B1355. II. Demonstration of multiple idiotypes with variable expression in several strains

International Nuclear Information System (INIS)

Hansburg, D.; Briles, D.E.; Davie, J.M.

1977-01-01

We have developed radioimmunoassays that detect idiotypic (variable region) differences among the α(1 → 3) dextran-binding myeloma proteins U102, J558, and M104 as well as an assay that detects variable region determinants common to all three proteins. Using these assays, we have examined 7S and 19S anti-α(1 → 3) dextran antibodies induced in five murine strains of the a 1 I/sub g/C/sub H/ linkage group and the recombinant strain BAB/14. All idiotypes were expressed in both 19S and 7S antibodies from all strains, but with considerable strain-specific variability in penetrance. In two strains, one additional type of antibody, which lacked all four idiotypic determinants, generally constituted the bulk of total anti-α(1 → 3) dextran antibodies

Complete genome sequence of Tolumonas auensis type strain (TA 4T)

Energy Technology Data Exchange (ETDEWEB)

Chertkov, Olga; Copeland, Alex; Lucas1, Susa; Lapidus, Alla; Berry, KerrieW.; Detter, JohnC.; Glavina Del Rio, Tijana; Hammon, Nancy; Dalin, Eileen; Tice, Hope; Pitluck, Sam; Richardson, Paul; Bruce, David; Goodwin, Lynne; Han, Cliff; Tapia, Roxanne; Saunders, Elizabeth; Schmutz, Jeremy; Brettin, Thomas; Larimer, Frank; Land, Miriam; Hauser, Loren; Spring, Stefan; Rohde, Manfred; Kyrpides, NikosC.; Ivanova, Natalia; G& #246; ker, Markus; Beller, HarryR.; Klenk, Hans-Peter; Woyke, Tanja

2011-10-04

Tolumonas auensis (Fischer-Romero et al. 1996) is currently the only validly named species of the genus Tolumonas in the family Aeromonadaceae. The strain is of interest because of its ability to produce toluene from phenylalanine and other phenyl precursors, as well as phenol from tyrosine. This is of interest because toluene is normally considered to be a tracer of anthropogenic pollution in lakes, but T. auensis represents a biogenic source of toluene. Other than Aeromonas hydrophila subsp. hydrophila, T. auensis strain TA 4T is the only other member in the family Aeromonadaceae with a completely sequenced type-strain genome. The 3,471,292-bp chromosome with a total of 3,288 protein-coding and 116 RNA genes was sequenced as part of the DOE Joint Genome Institute Program JBEI 2008.

Complete genome sequence of Tolumonas auensis type strain (TA 4T)

Energy Technology Data Exchange (ETDEWEB)

Chertkov, Olga [Los Alamos National Laboratory (LANL); Copeland, A [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Berry, Alison M [California Institute of Technology, University of California, Davis; Detter, J. Chris [U.S. Department of Energy, Joint Genome Institute; Glavina Del Rio, Tijana [U.S. Department of Energy, Joint Genome Institute; Hammon, Nancy [U.S. Department of Energy, Joint Genome Institute; Dalin, Eileen [U.S. Department of Energy, Joint Genome Institute; Tice, Hope [U.S. Department of Energy, Joint Genome Institute; Pitluck, Sam [U.S. Department of Energy, Joint Genome Institute; Richardson, P M [U.S. Department of Energy, Joint Genome Institute; Bruce, David [Los Alamos National Laboratory (LANL); Goodwin, Lynne A. [Los Alamos National Laboratory (LANL); Han, Cliff [Los Alamos National Laboratory (LANL); Tapia, Roxanne [Los Alamos National Laboratory (LANL); Saunders, Elizabeth H [Los Alamos National Laboratory (LANL); Schmutz, Jeremy [Stanford University; Brettin, Thomas S [ORNL; Larimer, Frank W [ORNL; Land, Miriam L [ORNL; Hauser, Loren John [ORNL; Spring, Stefan [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Rohde, Manfred [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Goker, Markus [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Beller, Harry R. [Lawrence Berkeley National Laboratory (LBNL); Klenk, Hans-Peter [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Woyke, Tanja [U.S. Department of Energy, Joint Genome Institute

2011-01-01

Tolumonas auensis Fischer-Romero et al. 1996 is currently the only validly named species of the genus Tolumonas in the family Aeromonadaceae. The strain is of interest because of its ability to produce toluene from phenylalanine and other phenyl precursors, as well as phenol from tyrosine. This is of interest because toluene is normally considered to be a tracer of anthropogenic pollution in lakes, but T. auensis represents a biogenic source of toluene. Oth- er than Aeromonas hydrophila subsp. hydrophila, T. auensis strain TA 4T is the only other member in the family Aeromonadaceae with a completely sequenced type-strain genome. The 3,471,292 bp chromosome with a total of 3,288 protein-coding and 116 RNA genes was sequenced as part of the DOE Joint Genome Institute Program JBEI 2008.

The Evolution of Strain Typing in the Mycobacterium tuberculosis Complex.

Science.gov (United States)

Merker, Matthias; Kohl, Thomas A; Niemann, Stefan; Supply, Philip

2017-01-01

Tuberculosis (TB) is a contagious disease with a complex epidemiology. Therefore, molecular typing (genotyping) of Mycobacterium tuberculosis complex (MTBC) strains is of primary importance to effectively guide outbreak investigations, define transmission dynamics and assist global epidemiological surveillance of the disease. Large-scale genotyping is also needed to get better insights into the biological diversity and the evolution of the pathogen. Thanks to its shorter turnaround and simple numerical nomenclature system, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) typing, based on 24 standardized plus 4 hypervariable loci, optionally combined with spoligotyping, has replaced IS6110 DNA fingerprinting over the last decade as a gold standard among classical strain typing methods for many applications. With the continuous progress and decreasing costs of next-generation sequencing (NGS) technologies, typing based on whole genome sequencing (WGS) is now increasingly performed for near complete exploitation of the available genetic information. However, some important challenges remain such as the lack of standardization of WGS analysis pipelines, the need of databases for sharing WGS data at a global level, and a better understanding of the relevant genomic distances for defining clusters of recent TB transmission in different epidemiological contexts. This chapter provides an overview of the evolution of genotyping methods over the last three decades, which culminated with the development of WGS-based methods. It addresses the relative advantages and limitations of these techniques, indicates current challenges and potential directions for facilitating standardization of WGS-based typing, and provides suggestions on what method to use depending on the specific research question.

Characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking

NARCIS (Netherlands)

van der Schaar, Hilde M.; Rust, Michael J.; Waarts, Barry-Lee; van der Ende-Metselaarl, Heidi; Kuhn, Richard J.; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.

In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain SI on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by

Characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking

NARCIS (Netherlands)

van der Schaar, Hilde M.; Rust, Michael J.; Waarts, Barry-Lee; van der Ende-Metselaarl, Heidi; Kuhn, Richard J.; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.

2007-01-01

In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain SI on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by

Antigenic characterization of a formalin-inactivated poliovirus vaccine derived from live-attenuated Sabin strains.

Science.gov (United States)

Tano, Yoshio; Shimizu, Hiroyuki; Martin, Javier; Nishimura, Yorihiro; Simizu, Bunsiti; Miyamura, Tatsuo

2007-10-10

A candidate inactivated poliovirus vaccine derived from live-attenuated Sabin strains (sIPV), which are used in the oral poliovirus vaccine (OPV), was prepared in a large-production scale. The modification of viral antigenic epitopes during the formalin inactivation process was investigated by capture ELISA assays using type-specific and antigenic site-specific monoclonal antibodies (MoAbs). The major antigenic site 1 was modified during the formalin inactivation of Sabin 1. Antigenic sites 1-3 were slightly modified during the formalin inactivation of Sabin 2 strain. Sites 1 and 3 were altered on inactivated Sabin 3 virus. These alterations were different to those shown by wild-type Saukett strain, used in conventional IPV (cIPV). It has been previously reported that type 1 sIPV showed higher immunogenicity to type 1 cIPV whereas types 2 and 3 sIPV induced lower level of immunogenicity than their cIPV counterparts. Our results suggest that the differences in epitope structure after formalin inactivation may account, at least in part, for the observed differences in immunogenicity between Sabin and wild-type inactivated poliovaccines.

Antibodies with higher bactericidal activity induced by a Neisseria gonorrhoeae Rmp deletion mutant strain.

Directory of Open Access Journals (Sweden)

Guocai Li

Full Text Available Neisseria gonorrhoeae (N. gonorrhoeae outer membrane protein reduction modifiable protein (Rmp has strong immunogenicity. However, anti-Rmp antibodies block rather than preserve the antibacterial effects of protective antibodies, which hampers the development of vaccines for gonococcal infections. We herein constructed an Rmp deletion mutant strain of N. gonorrhoeae by gene homologous recombination. The 261-460 nucleotide residues of Rmp gene amplified from N. gonorrhoeae WHO-A strain were replaced with a kanamycin-resistant Kan gene amplified from pET-28a. The resultant hybridized DNA was transformed into N. gonorrhoeae WHO-A strain. PCR was used to screen the colonies in which wild-type Rmp gene was replaced with a mutant gene fragment. Western blotting revealed that the Rmp deletion mutant strain did not express Rmp protein. Rmp deletion did not alter the morphological and Gram staining properties of the mutant strain that grew slightly more slowly than the wild-type one. Rmp gene mutated stably throughout 25 generations of passage. Antibody-mediated complement-dependent cytotoxicity assay indicated that the antibodies induced by the mutant strain had evidently higher bactericidal activities than those induced by the wild-type strain. Further modification of the Rmp deletion mutant strain is still required in the development of novel live attenuated vaccines for gonorrhea by Opa genes deletion or screening of phenotypic variant strains that do not express Opa proteins.

Improvement of heavy metal stress and toxicity assays by coupling a transgenic reporter in a mutant nematode strain

Energy Technology Data Exchange (ETDEWEB)

Chu, K.-W. [Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China); Chan, Shirley K.W. [Atmospheric, Marine and Coastal Environment Program, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China); Chow, King L. [Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China) and Atmospheric, Marine and Coastal Environment Program, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China)]. E-mail: bokchow@ust.hk

2005-09-30

Previous studies have demonstrated that wild type Caenorhabditis elegans displays high sensitivity to heavy metals in a lethality test at a level comparable to that of other bioindicator organisms. Taking advantage of the genetics of this model organism, we have tested a number of mutant strains for enhanced sensitivity in heavy metal induced lethality and stress response. These mutants are defective in genes controlling dauer formation, longevity or response to reactive oxygen species (ROS). Among the tested mutants, a double mutant daf-16 unc-75 strain was identified to have superior sensitivity. It has a 6-, 3- and 2-fold increase in sensitivity to cadmium, copper and zinc, respectively, as compared with that of wild type animals. When a fluorescent reporter transgene was coupled with this double mutant for stress detection, a 10-fold enhancement of sensitivity to cadmium over the wild type strain was observed. These transgenic animals, superior to most of the model organisms currently used in bioassays for environmental pollutants, offer a fast and economic approach to reveal the bioavailability of toxic substance in field samples. This study also demonstrates that combination of genetic mutations and transgenesis is a viable approach to identify sensitive indicator animals for environmental monitoring.

Improvement of heavy metal stress and toxicity assays by coupling a transgenic reporter in a mutant nematode strain

International Nuclear Information System (INIS)

Chu, K.-W.; Chan, Shirley K.W.; Chow, King L.

2005-01-01

Previous studies have demonstrated that wild type Caenorhabditis elegans displays high sensitivity to heavy metals in a lethality test at a level comparable to that of other bioindicator organisms. Taking advantage of the genetics of this model organism, we have tested a number of mutant strains for enhanced sensitivity in heavy metal induced lethality and stress response. These mutants are defective in genes controlling dauer formation, longevity or response to reactive oxygen species (ROS). Among the tested mutants, a double mutant daf-16 unc-75 strain was identified to have superior sensitivity. It has a 6-, 3- and 2-fold increase in sensitivity to cadmium, copper and zinc, respectively, as compared with that of wild type animals. When a fluorescent reporter transgene was coupled with this double mutant for stress detection, a 10-fold enhancement of sensitivity to cadmium over the wild type strain was observed. These transgenic animals, superior to most of the model organisms currently used in bioassays for environmental pollutants, offer a fast and economic approach to reveal the bioavailability of toxic substance in field samples. This study also demonstrates that combination of genetic mutations and transgenesis is a viable approach to identify sensitive indicator animals for environmental monitoring

Complete Genome Sequence of Plesiomonas shigelloides Type Strain NCTC10360

Science.gov (United States)

Fazal, Mohammed-Abbas; Burnett, Edward; Deheer-Graham, Ana; Oliver, Karen; Holroyd, Nancy; Russell, Julie E.

2016-01-01

Plesiomonas shigelloides is a Gram-negative rod within the Enterobacteriaceae family. It is a gastrointestinal pathogen of increasing notoriety, often associated with diarrheal disease. P. shigelloides is waterborne, and infection is often linked to the consumption of seafood. Here, we describe the first complete genome for P. shigelloides type strain NCTC10360. PMID:27660796

A nested PCR approach for unambiguous typing of pestiviruses infecting cattle.

Science.gov (United States)

Decaro, Nicola; Sciarretta, Rossana; Lucente, Maria Stella; Mari, Viviana; Amorisco, Francesca; Colaianni, Maria Loredana; Cordioli, Paolo; Parisi, Antonio; Lelli, Rossella; Buonavoglia, Canio

2012-02-01

An atypical pestivirus ('Hobi'-like pestivirus, putative bovine viral diarrhoea 3, BVDV-3) was identified firstly in contaminated foetal calf serum batches and isolated subsequently from an outbreak of respiratory disease in a cattle herd in Italy. The isolation of the novel pestivirus from animals affected clinically posed concerns about the validity of BVDV eradication programs, considering that 'Hobi'-like pestivirus (BVDV-3) is undetected or mistyped by the molecular diagnostic tools currently employed. In this paper, the development of a nested PCR (nPCR) assay for unambiguous typing of all bovine pestiviruses is reported. The assay consisted of a first-round amplification using an oligonucleotide pair which binds to conserved sequences located in the 5' untranslated region and capsid gene, followed by a heminested PCR using virus-specific forward primers. The assay performances were evaluated analytically, showing good sensitivity and specificity. By analysis of 100 BVDV-positive samples typed using a nPCR assay discriminating ruminant pestiviruses, five samples recognised previously as BVDV-2 were not typed when submitted to the new assay (n=2) or reacted as 'Hobi'-like pestivirus BVDV-3 (n=3). Sequence analysis of the first-round amplification products showed that the untyped strains were border disease viruses, whereas the other three strains were true 'Hobi'-like viruses. The development of a molecular assay able to identify simultaneously all bovine pestiviruses known currently will help warrant biosafety of live vaccines and other biological products and assess the molecular epidemiology of 'Hobi'-like pestivirus, thus leading to the improvement of the eradication programs through unambiguous typing of pestiviruses infecting cattle. Copyright © 2011 Elsevier Ltd. All rights reserved.

Detection and typing of highly pathogenic porcine reproductive and respiratory syndrome virus by multiplex real-time rt-PCR.

Directory of Open Access Journals (Sweden)

Kerstin Wernike

Full Text Available Porcine reproductive and respiratory syndrome (PRRS causes economic losses in the pig industry worldwide, and PRRS viruses (PRRSV are classified into the two distinct genotypes "North American (NA, type 2" and "European (EU, type 1". In 2006, a highly pathogenic NA strain of PRRSV (HP-PRRSV, characterized by high fever as well as high morbidity and mortality, emerged in swine farms in China. Therefore, a real-time reverse transcription polymerase chain reaction (RT-qPCR assay specific for HP-PRRSV was developed and combined with type 1- and type 2-specific RT-qPCR systems. Furthermore, an internal control, based on a heterologous RNA, was successfully introduced. This final multiplex PRRSV RT-qPCR, detecting and typing PRRSV, had an analytical sensitivity of less than 200 copies per µl for the type 1-assay and 20 copies per µl for the type 2- and HP assays and a high diagnostic sensitivity. A panel of reference strains and field isolates was reliably detected and samples from an animal trial with a Chinese HP-PRRS strain were used for test validation. The new multiplex PRRSV RT-qPCR system allows for the first time the highly sensitive detection and rapid differentiation of PRRSV of both genotypes as well as the direct detection of HP-PRRSV.

Beyond PrPres Type 1/Type 2 Dichotomy in Creutzfeldt-Jakob Disease

Science.gov (United States)

Simon, Stéphanie; Lugan, Séverine; Bilheude, Jean-Marc; Perret-Liaudet, Armand; Ironside, James W.; Haik, Stéphane; Basset-Leobon, Christelle; Lacroux, Caroline; Peoch', Katell; Streichenberger, Nathalie; Langeveld, Jan; Head, Mark W.; Grassi, Jacques; Hauw, Jean-Jacques; Schelcher, Francois; Delisle, Marie Bernadette; Andréoletti, Olivier

2008-01-01

Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently subclassified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K (PK) digested abnormal prion protein (PrPres) identified on Western blotting (type 1 or type 2). These biochemically distinct PrPres types have been considered to represent potential distinct prion strains. However, since cases of CJD show co-occurrence of type 1 and type 2 PrPres in the brain, the basis of this classification system and its relationship to agent strain are under discussion. Different brain areas from 41 sCJD and 12 iatrogenic CJD (iCJD) cases were investigated, using Western blotting for PrPres and two other biochemical assays reflecting the behaviour of the disease-associated form of the prion protein (PrPSc) under variable PK digestion conditions. In 30% of cases, both type 1 and type 2 PrPres were identified. Despite this, the other two biochemical assays found that PrPSc from an individual patient demonstrated uniform biochemical properties. Moreover, in sCJD, four distinct biochemical PrPSc subgroups were identified that correlated with the current sCJD clinico-pathological classification. In iCJD, four similar biochemical clusters were observed, but these did not correlate to any particular PRNP 129 polymorphism or western blot PrPres pattern. The identification of four different PrPSc biochemical subgroups in sCJD and iCJD, irrespective of the PRNP polymorphism at codon 129 and the PrPres isoform provides an alternative biochemical definition of PrPSc diversity and new insight in the perception of Human TSE agents variability. PMID:18389084

Casting of organic glass by radiation-induced polymerization of glass-forming monomers at low temperature. II. Optical strain of remaining stress type

International Nuclear Information System (INIS)

Okubo, H.; Yoshii, F.; Kaetsu, I.; Honda, S.

1978-01-01

Previously it was found that casting could be carried out efficiently without strain formation by radiation-induced polymerization of glass-forming monomers. Two types of strain were observed in casting: thermal stream type, which was studied previously, and remained stress type. In this report, the effect of various factors on the formation of remaining stress-type strain in radiation-induced casting polymerization was studied. It was found that the molecular weight of prepolymer did not affect strain formation, while prepolymer concentration and viscosity of the system had a serious influence on strain formation. It could be deduced that this type of strain formed as a result of remaining inner stress due to poor relaxation of the shrinking stress. It was realized that less volume shrinkage of glass-forming monomers accompanying casting polymerization reduced the strain formation of this type in radiation-induced casting polymerization at low temperatures

A rapid minor groove binder PCR method for distinguishing the vaccine strain Brucella abortus 104M.

Science.gov (United States)

Nan, Wenlong; Qin, Lide; Wang, Yong; Zhang, Yueyong; Tan, Pengfei; Chen, Yuqi; Mao, Kairong; Chen, Yiping

2018-01-24

Brucellosis is a widespread zoonotic disease caused by Gram-negative Brucella bacteria. Immunisation with attenuated vaccine is an effective method of prevention, but it can interfere with diagnosis. Live, attenuated Brucella abortus strain 104M has been used for the prevention of human brucellosis in China since 1965. However, at present, no fast and reliable method exists that can distinguish this strain from field strains. Single nucleotide polymorphism (SNP)-based assays offer a new approach for such discrimination. SNP-based minor groove binder (MGB) and Cycleave assays have been used for rapid identification of four Brucella vaccine strains (B. abortus strains S19, A19 and RB51, and B. melitensis Rev1). The main objective of this study was to develop a PCR assay for rapid and specific detection of strain 104M. We developed a SNP-based MGB PCR assay that could successfully distinguish strain 104M from 18 representative strains of Brucella (B. abortus biovars 1, 2, 3, 4, 5, 6, 7 and 9, B. melitensis biovars 1, 2 and 3, B. suis biovars 1, 2, 3 and 4, B. canis, B. neotomae, and B. ovis), four Brucella vaccine strains (A19, S19, S2, M5), and 55 Brucella clinical field strains. The assay gave a negative reaction with four non-Brucella species (Escherichia coli, Pasteurella multocida, Streptococcus suis and Pseudomonas aeruginosa). The minimum sensitivity of the assay, evaluated using 10-fold dilutions of chromosomal DNA, was 220 fg for the 104M strain and 76 fg for the single non-104M Brucella strain tested (B. abortus A19). The assay was also reproducible (intra- and inter-assay coefficients of variation = 0.006-0.022 and 0.012-0.044, respectively). A SNP-based MGB PCR assay was developed that could straightforwardly and unambiguously distinguish B. abortus vaccine strain 104M from non-104M Brucella strains. Compared to the classical isolation and identification approaches of bacteriology, this real-time PCR assay has substantial advantages in terms of

Identification of Variable-Number Tandem-Repeat (VNTR) Sequences in Legionella pneumophila and Development of an Optimized Multiple-Locus VNTR Analysis Typing Scheme▿

Science.gov (United States)

Pourcel, Christine; Visca, Paolo; Afshar, Baharak; D'Arezzo, Silvia; Vergnaud, Gilles; Fry, Norman K.

2007-01-01

The utility of a genotypic typing assay for Legionella pneumophila was investigated. A multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) scheme using PCR and agarose gel electrophoresis is proposed based on eight minisatellite markers. Panels of well-characterized strains were examined in a multicenter analysis to validate the assay and to compare its performance to that of other genotyping assays. Excellent typeability, reproducibility, stability, and epidemiological concordance were observed. The MLVA type or profile is composed of a string of allele numbers, corresponding to the number of repeats at each VNTR locus, separated by commas, in a predetermined order. A database containing information from 99 L. pneumophila serogroup 1 strains and four strains of other serogroups and their MLVA profiles, which can be queried online, is available from http://bacterial-genotyping.igmors.u-psud.fr/. PMID:17251393

The use of subcutaneous fat tissue for amyloid typing by enzyme-linked immunosorbent assay

DEFF Research Database (Denmark)

Olsen, K E; Sletten, K; Westermark, Per

1999-01-01

for typing the most common systemic amyloidoses of AL, AA, and transthyretin types by enzyme-linked immunosorbent assay (ELISA), using abdominal wall subcutaneous fat biopsy specimens. The method was tested on 21 abdominal fat biopsy specimens that were sent to the laboratory. Of these, 15 contained amyloid......The amyloidoses are biochemically heterogeneous diseases with pathophysiologic deposits of various proteins. The clinical course, prognosis, and therapy are different for each type of amyloidosis and, therefore, a type-specific diagnosis is demanded as early as possible. We describe a method...

Complete Genome Sequence of Mycobacterium xenopi Type Strain RIVM700367

KAUST Repository

Abdallah, A. M.; Rashid, M.; Adroub, S. A.; Elabdalaoui, H.; Ali, Shahjahan; van Soolingen, D.; Bitter, W.; Pain, Arnab

2012-01-01

Mycobacterium xenopi is a slow-growing, thermophilic, water-related Mycobacterium species. Like other nontuberculous mycobacteria, M. xenopi more commonly infects humans with altered immune function, such as chronic obstructive pulmonary disease patients. It is considered clinically relevant in a significant proportion of the patients from whom it is isolated. We report here the whole genome sequence of M. xenopi type strain RIVM700367.

Complete Genome Sequence of Mycobacterium xenopi Type Strain RIVM700367

KAUST Repository

Abdallah, A. M.

2012-05-24

Mycobacterium xenopi is a slow-growing, thermophilic, water-related Mycobacterium species. Like other nontuberculous mycobacteria, M. xenopi more commonly infects humans with altered immune function, such as chronic obstructive pulmonary disease patients. It is considered clinically relevant in a significant proportion of the patients from whom it is isolated. We report here the whole genome sequence of M. xenopi type strain RIVM700367.

Topological characterization of static strain aging of type AISI 304 austenitic stainless steel

International Nuclear Information System (INIS)

Monteiro, S.N.; Miranda, P.E.V. de

1981-01-01

Static strain aging of type AISI 304 austenitic stainless steel was studied from room temperature up to 623K by conducting tests in which the load was held approximately constant. The aging times varied between 10s and 100h, using a plastic pre-deformation of 9%. The static strain aging of 304 steel furnished an activation energy of 23.800 cal/mol. This implies that vacancies play an important role on the aging process. The curve of the variation of the discontinuous yielding with aging time presented different stages, to which specific mathematical expressions were developed. These facts permited the conclusion that Snock type mechanisms are responsible for the aging in such conditions. (Author) [pt

Development of a hydrolysis probe-based real-time assay for the detection of tropical strains of Fusarium oxysporum f. sp. cubense race 4

Science.gov (United States)

Cerf-Wendling, Isabelle; Hostachy, Bruno; Viljoen, Altus; Ioos, Renaud

2017-01-01

Fusarium oxysporum f. sp. cubense (Foc) is one of the most important threats to global banana production. Strategies to control the pathogen are lacking, with plant resistance offering the only long-term solution, if sources of resistance are available. Prevention of introduction of Foc into disease-free areas thus remains a key strategy to continue sustainable banana production. In recent years, strains of Foc affecting Cavendish bananas have destroyed plantations in a number of countries in Asia and in the Middle East, and one African country. One vegetative compatibility group (VCG), 01213/16, is considered the major threat to bananas in tropical and subtropical climatic conditions. However, other genetically related VCGs, such as 0121, may potentially jeopardize banana cultures if they were introduced into disease-free areas. To prevent the introduction of these VCGs into disease-free Cavendish banana-growing countries, a real-time PCR test was developed to accurately detect both VCGs. A previously described putative virulence gene was used to develop a specific combination of hydrolysis probe/primers for the detection of tropical Foc race 4 strains. The real-time PCR parameters were optimized by following a statistical approach relying on orthogonal arrays and the Taguchi method in an attempt to enhance sensitivity and ensure high specificity of the assay. This study also assessed critical performance criteria, such as repeatability, reproducibility, robustness, and specificity, with a large including set of 136 F. oxysporum isolates, including 73 Foc pathogenic strains representing 24 VCGs. The validation data demonstrated that the new assay could be used for regulatory testing applications on banana plant material and can contribute to preventing the introduction and spread of Foc strains affecting Cavendish bananas in the tropics. PMID:28178348

Assay of the β-glucosidase activity with natural labelled and artificial substrates in leukocytes from homozygotes and heterozygotes with the Norrbottnian type (Type 3) of Gaucher disease

International Nuclear Information System (INIS)

Svennerholm, L.; Haakansson, G.; Dreborg, S.

1980-01-01

Leukocytes were isolated from 14 patients (7 males and 7 females) with Gaucher disease of the Norrbottnian type (Type 3), 32 obligate heterozygotes (16 males and 16 females) for this disease and 20 controls (10 males and 10 females). After collection, the cells were transported in dry ice to the laboratory, where they were assayed. The assays were repeated after the cells had been stored for 12 months. β-Glucosidase activity was assayed with D-[glucose-U- 14 C]glucosylceramide at pH 5.8 with Cutscum-Na-cholate as a detergent and 4-methylumbelliferyl-β-glucoside at pH 4.1 with Triton-Na-taurocholate as a detergent. The activities of two marker enzymes, 4-methylumbelliferyl-β-galactosidase and N-acetyl-β-glucosaminidase, were assayed in aliquots of the same leukocyte samples. (Auth.)

Bio-Engineering High Performance Microbial Strains for MEOR

Energy Technology Data Exchange (ETDEWEB)

Xiangdong Fang; Qinghong Wang; Patrick Shuler

2007-12-30

The main objectives of this three-year research project are: (1) to employ the latest advances in genetics and bioengineering, especially Directed Protein Evolution technology, to improve the effectiveness of the microbial enhanced oil recovery (MEOR) process. (2) to improve the surfactant activity and the thermal stability of bio-surfactant systems for MEOR; and (3) to develop improved laboratory methods and tools that screen quickly candidate bio-systems for EOR. Biosurfactants have been receiving increasing attention as Enhanced Oil Recovery (EOR) agents because of their unique properties (i.e., mild production conditions, lower toxicity, and higher biodegradability) compared to their synthetic chemical counterparts. Rhamnolipid as a potent natural biosurfactant has a wide range of potential applications, including EOR and bioremediation. During the three-year of the project period, we have successfully cloned the genes involved in the rhamnolipid bio-synthesis. And by using the Transposon containing Rhamnosyltransferase gene rhlAB, we engineered the new mutant strains P. aeruginosa PEER02 and E. coli TnERAB so they can produce rhamnolipid biosurfactans. We were able to produce rhamnolipds in both P. aeroginosa PAO1-RhlA- strain and P. fluorescens ATCC15453 strain, with the increase of 55 to 175 fold in rhamnolipid production comparing with wild type bacteria strain. We have also completed the first round direct evolution studies using Error-prone PCR technique and have constructed the library of RhlAB-containing Transposon to express mutant gene in heterologous hosts. Several methods, such as colorimetric agar plate assay, colorimetric spectrophotometer assay, bioactive assay and oil spreading assay have been established to detect and screen rhamnolipid production. Our engineered P. aeruginosa PEER02 strain can produce rhamnolipids with different carbon sources as substrate. Interfacial tension analysis (IFT) showed that different rhamnolipids from different

Comparison of ribotyping, randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis in typing of Lactobacillus rhamnosus and L. casei strains.

Science.gov (United States)

Tynkkynen, S; Satokari, R; Saarela, M; Mattila-Sandholm, T; Saxelin, M

1999-09-01

A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 334, respectively, but did not give any signal with the recently revived species L. zeae, which contains the type strain ATCC 15820 and the strain ATCC 393, which was previously classified as L. casei. Our results are in accordance with the suggested new classification of the L. casei group. Altogether, 21 of the 24 strains studied were identified with the species-specific primers. In strain typing, PFGE was the most discriminatory method, revealing 17 genotypes for the 24 strains studied. Ribotyping and RAPD analysis yielded 15 and 12 genotypes, respectively.

Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay.

Science.gov (United States)

Binet, Rachel; Deer, Deanne M; Uhlfelder, Samantha J

2014-06-01

Faster detection of contaminated foods can prevent adulterated foods from being consumed and minimize the risk of an outbreak of foodborne illness. A sensitive molecular detection method is especially important for Shigella because ingestion of as few as 10 of these bacterial pathogens can cause disease. The objectives of this study were to compare the ability of four DNA extraction methods to detect Shigella in six types of produce, post-enrichment, and to evaluate a new and rapid conventional multiplex assay that targets the Shigella ipaH, virB and mxiC virulence genes. This assay can detect less than two Shigella cells in pure culture, even when the pathogen is mixed with background microflora, and it can also differentiate natural Shigella strains from a control strain and eliminate false positive results due to accidental laboratory contamination. The four DNA extraction methods (boiling, PrepMan Ultra [Applied Biosystems], InstaGene Matrix [Bio-Rad], DNeasy Tissue kit [Qiagen]) detected 1.6 × 10(3)Shigella CFU/ml post-enrichment, requiring ∼18 doublings to one cell in 25 g of produce pre-enrichment. Lower sensitivity was obtained, depending on produce type and extraction method. The InstaGene Matrix was the most consistent and sensitive and the multiplex assay accurately detected Shigella in less than 90 min, outperforming, to the best of our knowledge, molecular assays currently in place for this pathogen. Published by Elsevier Ltd.

Complete genome sequence of Hydrogenobacter thermophilus type strain (TK-6T)

Energy Technology Data Exchange (ETDEWEB)

Zeytun, Ahmet [Los Alamos National Laboratory (LANL); Sikorski, Johannes [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Nolan, Matt [Joint Genome Institute, Walnut Creek, California; Lapidus, Alla L. [Joint Genome Institute, Walnut Creek, California; Lucas, Susan [Joint Genome Institute, Walnut Creek, California; Han, James [Joint Genome Institute; Tice, Hope [Joint Genome Institute, Walnut Creek, California; Cheng, Jan-Fang [Joint Genome Institute, Walnut Creek, California; Tapia, Roxanne [Los Alamos National Laboratory (LANL); Goodwin, Lynne A. [Los Alamos National Laboratory (LANL); Pitluck, Sam [Joint Genome Institute, Walnut Creek, California; Liolios, Konstantinos [Joint Genome Institute, Walnut Creek, California; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Mikhailova, Natalia [U.S. Department of Energy, Joint Genome Institute; Ovchinnikova, Galina [U.S. Department of Energy, Joint Genome Institute; Pati, Amrita [U.S. Department of Energy, Joint Genome Institute; Chen, Amy [Joint Genome Institute, Walnut Creek, California; Palaniappan, Krishna [Joint Genome Institute, Walnut Creek, California; Ngatchou, Olivier Duplex [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Land, Miriam L [ORNL; Hauser, Loren John [ORNL; Chang, Yun-Juan [ORNL; Jeffries, Cynthia [Oak Ridge National Laboratory (ORNL); Han, Cliff [Los Alamos National Laboratory (LANL); Detter, J. Chris [Joint Genome Institute, Walnut Creek, California; Ubler, Susanne [Universitat Regensburg, Regensburg, Germany; Rohde, Manfred [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Tindall, Brian [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Wirth, Reinhard [Universitat Regensburg, Regensburg, Germany; Woyke, Tanja [Joint Genome Institute, Walnut Creek, California; Bristow, James [Joint Genome Institute, Walnut Creek, California; Eisen, Jonathan [Joint Genome Institute, Walnut Creek, California; Markowitz, Victor [Joint Genome Institute, Walnut Creek, California; Hugenholtz, Philip [U.S. Department of Energy, Joint Genome Institute; Klenk, Hans-Peter [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Kyrpides, Nikos C [Joint Genome Institute, Walnut Creek, California

2011-01-01

Hydrogenobacter thermophilus Kawasumi et al. 1984 is the type species of the genus Hydrogenobacter. H. thermophilus was the first obligate autotrophic organism reported among aerobic hydrogen-oxidizing bacteria. Strain TK-6T is of interest because of the unusually efficient hydrogen-oxidizing ability of this strain, which results in a faster generation time compared to other autotrophs. It is also able to grow anaerobically using nitrate as an electron acceptor when molecular hydrogen is used as the energy source, and able to aerobically fix CO2 via the reductive tricarboxylic acid cycle. This is the fifth completed genome sequence in the family Aquificaceae, and the second genome sequence determined from a strain derived from the original isolate. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,742,932 bp long genome with its 1,899 protein-coding and 49 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Induction of pure and sectored mutant clones in excision-proficient and deficient strains of yeast.

Science.gov (United States)

Eckardt, F; Haynes, R H

1977-06-01

We have found that UV-induced mutation frequency in a forward non-selective assay system (scoring white adex ade2 double auxotroph mutants among the red pigmented ade2 clones) increases linearly with dose up to a maximum frequency of about 3 X 10(-3) mutants per survivor and then declines in both RAD wild-type and rad2 excision deficient strains of Saccharomyces cerevisiae. Mutation frequencies of the RAD and the rad2 strains plotted against survival are nearly identical over the entire survival range. On this basis we conclude that unexcised pyrimidine dimers are the predominant type of pre-mutational lesions in both strains. In the RAD wild-type strain pure mutant clones outnumber sectors in a 10:1 ratio at all doses used; in rad2 this ratio varies from 1:1 at low doses up to 10:1 at high doses. As others have concluded for wild-type strains we find also in the rad2 strain that pure clone formation cannot be accounted for quantitatively by lethal sectoring events alone. We conclude that heteroduplex repair is a crucial step in pure mutant clone formation and we examine the plausibility of certain macromolecular mechanisms according to which heteroduplex repair may be coupled with replication, repair and sister strand exchange in yeast mutagenesis.

Induction of pure and sectored mutant clones in excision-proficient and deficient strains of yeast

International Nuclear Information System (INIS)

Eckardt, F.; Haynes, R.H.

1977-01-01

It was found that UV-induced mutation frequency in a forward non-selective assay system (scoring white adex ade2 double auxotroph mutants among the red pigmented ade2 clones) increases linearly with dose up to a maximum frequency of about 3 x 10 -3 mutants per survivor and then declines in both RAD wild-type and rad2 excision deficient strains of Saccharomyces cerevisiae. Mutation frequencies of the RAD and the rad2 strains plotted against survival are nearly identical over the entire survival range. On this basis it is concluded that unexcised pyrimidine dimers are the predominant type of pre-mutational lesions in both strains. In the RAD wild-type strain pure mutant clones outnumber sectors in a 10:1 ratio at all doses used; in rad2 this ratio varies from 1:1 at low doses up to 10:1 at high doses. In agreement with conclusions of others, it was also found that for wild-type strains in the rad2 strain pure clone formation cannot be accounted for quantitatively by lethal sectoring events alone. It is concluded that heteroduplex repair is a crucial step in pure mutant clone formation and the plausibility of certain macromolecular mechanisms according to which heteroduplex repair may be coupled with replication, repair and sister strand exchange in yeast mutagenesis is examined

Spatial encoding in spinal sensorimotor circuits differs in different wild type mice strains

Directory of Open Access Journals (Sweden)

Schouenborg Jens

2008-05-01

Full Text Available Abstract Background Previous studies in the rat have shown that the spatial organisation of the receptive fields of nociceptive withdrawal reflex (NWR system are functionally adapted through experience dependent mechanisms, termed somatosensory imprinting, during postnatal development. Here we wanted to clarify 1 if mice exhibit a similar spatial encoding of sensory input to NWR as previously found in the rat and 2 if mice strains with a poor learning capacity in various behavioural tests, associated with deficient long term potention, also exhibit poor adaptation of NWR. The organisation of the NWR system in two adult wild type mouse strains with normal long term potentiation (LTP in hippocampus and two adult wild type mouse strains exhibiting deficiencies in corresponding LTP were used and compared to previous results in the rat. Receptive fields of reflexes in single hindlimb muscles were mapped with CO2 laser heat pulses. Results While the spatial organisation of the nociceptive receptive fields in mice with normal LTP were very similar to those in rats, the LTP impaired strains exhibited receptive fields of NWRs with aberrant sensitivity distributions. However, no difference was found in NWR thresholds or onset C-fibre latencies suggesting that the mechanisms determining general reflex sensitivity and somatosensory imprinting are different. Conclusion Our results thus confirm that sensory encoding in mice and rat NWR is similar, provided that mice strains with a good learning capability are studied and raise the possibility that LTP like mechanisms are involved in somatosensory imprinting.

Comparison of two commercial carbapenemase gene confirmatory assays in multiresistant Enterobacteriaceae and Acinetobacter baumannii-complex.

Science.gov (United States)

Rösner, Stephan; Gehlweiler, Kevin; Küsters, Uta; Kolbert, Mathias; Hübner, Kirsten; Pfennigwerth, Niels; Mack, Dietrich

2018-01-01

Multidrug-resistant Gram-negative bacilli (MDR-GNB) producing carbapenemases are increasing at an alarming speed. Rapid confirmation of carbapenemase type will be an important diagnostic step in clinical microbiology laboratories not only to reduce the risk of transmissions but also for optimising antibiotic therapy in the future. We compared diagnostic reliability of two commercially available molecular assays (Check-Direct CPE vs. AID line probe assay) for detection and typing of carbapenemase genes in 80 well-characterized isolates of MDR-GNB. Respective strains were isolated in various clinical specimens at our clinical microbiology laboratory. The reference standard included confirmation of carbapenemase-production at the molecular level at the German National Reference Laboratory for Multidrug-resistant Gram-negative bacteria (Ruhr-University Bochum, Germany). 53 Enterobacteriaceae and 27 members of the A. baumannii-complex were used in this study. The tested assays appeared highly reliable to confirm carbapenemase-producing Enterobacteriaceae (CPE) with respective sensitivities of 97.7%, but are currently unsuitable for analysis of members of the A. baumannii-complex. Both assays are easy to perform and rapid tools for confirmation and typing of the most common carbapenemase genes in Enterobacteriaceae. Implementation should be possible for any clinical microbiology laboratory with Check-Direct CPE being easier to handle and having less technological requirements.

Comparison of two commercial carbapenemase gene confirmatory assays in multiresistant Enterobacteriaceae and Acinetobacter baumannii-complex.

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Stephan Rösner

Full Text Available Multidrug-resistant Gram-negative bacilli (MDR-GNB producing carbapenemases are increasing at an alarming speed. Rapid confirmation of carbapenemase type will be an important diagnostic step in clinical microbiology laboratories not only to reduce the risk of transmissions but also for optimising antibiotic therapy in the future. We compared diagnostic reliability of two commercially available molecular assays (Check-Direct CPE vs. AID line probe assay for detection and typing of carbapenemase genes in 80 well-characterized isolates of MDR-GNB. Respective strains were isolated in various clinical specimens at our clinical microbiology laboratory. The reference standard included confirmation of carbapenemase-production at the molecular level at the German National Reference Laboratory for Multidrug-resistant Gram-negative bacteria (Ruhr-University Bochum, Germany. 53 Enterobacteriaceae and 27 members of the A. baumannii-complex were used in this study. The tested assays appeared highly reliable to confirm carbapenemase-producing Enterobacteriaceae (CPE with respective sensitivities of 97.7%, but are currently unsuitable for analysis of members of the A. baumannii-complex. Both assays are easy to perform and rapid tools for confirmation and typing of the most common carbapenemase genes in Enterobacteriaceae. Implementation should be possible for any clinical microbiology laboratory with Check-Direct CPE being easier to handle and having less technological requirements.

A rapid cycleave PCR method for distinguishing the vaccine strain Brucella abortus A19 in China.

Science.gov (United States)

Nan, Wenlong; Zhang, Yueyong; Tan, Pengfei; Xu, Zouliang; Chen, Yuqi; Mao, Kairong; Chen, Yiping

2016-05-01

Brucellosis is a widespread zoonotic disease caused by Brucella spp. Immunization with attenuated vaccines has proved to be an effective method of prevention; however, it may also interfere with diagnosis. Brucella abortus strain A19, which is homologous to B. abortus strain S19, is widely used for the prevention of bovine brucellosis in China. For effective monitoring of the control of brucellosis, it is essential to distinguish A19 from field strains. Single-nucleotide polymorphism-based assays offer a new approach to such discrimination studies. In the current study, we developed a cycleave PCR assay that successfully distinguished attenuated vaccine strains A19 and S19 from 22 strains of B. abortus and 57 strains of 5 other Brucella species. The assay gave a negative reaction with 4 non-Brucella species. The minimum sensitivity of the assay, evaluated using 10-fold dilutions of chromosomal DNA, was 7.6 fg for the A19 strain and 220 fg for the single non-A19/non-S19 Brucella strain tested (B. abortus 104M). The assay was also reproducible (intra- and interassay coefficients of variation: 0.003-0.01 and 0.004-0.025, respectively). The cycleave assay gave an A19/S19-specific reaction in 3 out of 125 field serum samples, with the same 3 samples being positive in an alternative A19/S19-specific molecular assay. The cycleave assay gave a total of 102 Brucella-specific reactions (3 being the A19/S19-specific reactions), whereas an alternative Brucella-specific assay gave 92 positive reactions (all also positive in the cycleave assay). Therefore, this assay represents a simple, rapid, sensitive, and specific tool for use in brucellosis control. © 2016 The Author(s).

Use of the VNTR typing technique to determine the origin of Mycobacterium tuberculosis strains isolated from Filipino patients in Korea.

Science.gov (United States)

Lee, Jihye; Tupasi, Thelma E; Park, Young Kil

2014-05-01

With increasing international interchange of personnel, international monitoring is necessary to decrease tuberculosis incidence in the world. This study aims to develop a new tool to determine origin of Mycobacterium tuberculosis strains isolated from Filipino patients living in Korea. Thirty-two variable number tandem repeat (VNTR) loci were used for discrimination of 50 Filipino M. tuberculosis strains isolated in the Philippines, 317 Korean strains isolated in Korea, and 8 Filipino strains isolated in Korea. We found that the VNTR loci 0580, 0960, 2531, 2687, 2996, 0802, 2461, 2163a, 4052, 0424, 1955, 2074, 2347, 2401, 3171, 3690, 2372, 3232, and 4156 had different mode among copy numbers or exclusively distinct copy number in VNTR typing between Filipino and Korean M. tuberculosis strains. When these differences of the VNTR loci were applied to 8 Filipino M. tuberculosis strains isolated in Korea, 6 of them revealed Filipino type while 2 of them had Korean type. Using the differences of mode or repeated number of VNTR loci were very useful in distinguishing the Filipino strain from Korean strain.

Complete Genome Sequences of emm111 Type Streptococcus pyogenes Strain GUR, with Antitumor Activity, and Its Derivative Strain GURSA1 with an Inactivated emm Gene

DEFF Research Database (Denmark)

Suvorova, Maria A; Tsapieva, Anna N; Bak, Emilie Glad

2017-01-01

We present here the complete genome sequence of Streptococcus pyogenes type emm111 strain GUR, a throat isolate from a scarlet fever patient, which has been used to treat cancer patients in the former Soviet Union. We also present the complete genome sequence of its derivative strain GURSA1...

Complete genome sequence of Capnocytophaga ochracea type strain (VPI 2845T)

Energy Technology Data Exchange (ETDEWEB)

Mavromatis, Konstantinos; Gronow, Sabine; Saunders, Elizabeth; Land, Miriam; Lapidus, Alla; Copeland, Alex; Glavina Del Rio, Tijana; Nolan, Matt; Lucas, Susan; Chen, Feng; Tice1, Hope; Cheng, Jan-Fang; Bruce, David; Goodwin, Lynne; Pitluck, Sam; Pati, Amrita; Ivanova, Natalia; Chen, Amy; Palaniappan, Krishna; Chain, Patrick; Hauser, Loren; Chang, Yun-Juan; Jefferies, Cynthia C.; Brettin, Thomas; Detter, John C.; Han, Cliff; Bristow, James; Goker, Markus; Rohde, Manfred; Eisen, Jonathan A.; Markowitz, Victor; Kyrpides, Nikos C.; Klenk, Hans-Peter; Hugenholtz, Philip

2009-05-20

Capnocytophaga ochracea (Prevot et al. 1956) Leadbetter et al. 1982 is the type species of the genus Capnocytophaga. It is of interest because of its location in the Flavobacteriaceae, a genomically yet uncharted family within the order Flavobacteriales. The species grows as fusiform to rod shaped cells which tend to form clumps and are able to move by gliding. C. ochracea is known as a capnophilic organism with the ability to grow under anaerobic as well as under aerobic conditions (oxygen concentration larger than 15percent), here only in the presence of 5percent CO2. Strain VPI 2845T, the type strain of the species, is portrayed in this report as a gliding, Gram-negative bacterium, originally isolated from a human oral cavity. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence from the flavobacterial genus Capnocytophaga, and the 2,612,925 bp long single replicon genome with its 2193 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Development of a Novel Protein Microarray Method for Serotyping Salmonella enterica Strains

OpenAIRE

Cai, H. Y.; Lu, L.; Muckle, C. A.; Prescott, J. F.; Chen, S.

2005-01-01

An antibody microarray assay was developed for Salmonella serotyping based on the Kauffmann-White scheme. A model (8 by 15) array was constructed using 35 antibodies for identification of 20 common Salmonella serovars and evaluated using 117 target and 73 nontarget Salmonella strains. The assay allowed complete serovar identification of 86 target strains and partial identification of 30 target strains and allowed exclusion of the 73 nontarget strains from the target serovars.

Toxoplasma gondii: II. Tachyzoite antigenic characterization of eigth strains

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Regina Mitsuka

1998-01-01

Full Text Available Eight Toxoplasma gondii strains were analyzed using ELISA and Western blot techniques, in order to demonstrate possible immunological differences. The analyzed strains were: LIV IV, LIV V and S 11 isolated from swine, RH and VPS from a human being, AS 28 from a wild mouse, HV III from a dog and CN from a cat. With the ELISA assay the eight strains showed similar reactivity with homologous and heterologous sera. The antigenic suspension, consisting of total cellular extract of tachyzoites, was effective in the indirect ELISA assay, with the positive sera reacting strongly and the negative not reacting with the antigens. The Western blot analysis showed that the T. gondii strains have similar antigenic profiles with a few variations. Three bands were observed in all strains: one of about 33 kDa (p33, another of 54 kDa (p54 and a third one of 66 kDa (p66. The HV III strain, isolated from a dog, did not show three antigens (50, 70 and 75 kDa that were present in the others. However, this difference was not detected by the ELISA assay. Only two antigens (62 kDa of the CN and 67 kDa of the LIV IV were strain-specific antigens.

Whole genome sequence of Enterobacter ludwigii type strain EN-119T, isolated from clinical specimens.

Science.gov (United States)

Li, Gengmi; Hu, Zonghai; Zeng, Ping; Zhu, Bing; Wu, Lijuan

2015-04-01

Enterobacter ludwigii strain EN-119(T) is the type strain of E. ludwigii, which belongs to the E. cloacae complex (Ecc). This strain was first reported and nominated in 2005 and later been found in many hospitals. In this paper, the whole genome sequencing of this strain was carried out. The total genome size of EN-119(T) is 4952,770 bp with 4578 coding sequences, 88 tRNAs and 10 rRNAs. The genome sequence of EN-119(T) is the first whole genome sequence of E. ludwigii, which will further our understanding of Ecc. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Rapid and specific identification of Brucella abortus using the loop-mediated isothermal amplification (LAMP) assay.

Science.gov (United States)

Kang, Sung-Il; Her, Moon; Kim, Ji-Yeon; Lee, Jin Ju; Lee, Kichan; Sung, So-Ra; Jung, Suk Chan

2015-06-01

A rapid and accurate diagnosis of brucellosis is required to reduce and prevent the spread of disease among animals and the risk of transfer to humans. In this study, a Brucella abortus-specific (Ba) LAMP assay was developed, that had six primers designed from the BruAb2_0168 region of chromosome I. The specificity of this LAMP assay was confirmed with Brucella reference strains, B. abortus vaccine strains, B. abortus isolates and phylogenetically or serologically related strains. The detection limit of target DNA was up to 20 fg/μl within 60 min. The sensitivity of the new LAMP assay was equal to or slightly higher than other PCR based assays. Moreover, this Ba-LAMP assay could specifically amplify all B. abortus biovars compared to previous PCR assays. To our knowledge, this is the first report of specific detection of B. abortus using a LAMP assay. The Ba-LAMP assay can offer a rapid, sensitive and accurate diagnosis of bovine brucellosis in the field. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Molecular typing of 12 Brucella strains isolated in Guizhou province in 2010-2013].

Science.gov (United States)

Wang, Yue; Chen, Hong; Liu, Ying; Zhou, Jingzhu; Li, Shijun; Hang, Yan; Tang, Guangpeng; Wang, Dingming; Chen, Guichun

2015-09-01

To identify and characterize the Brucella strains from Guizhou province in 2010-2013. A total of 12 strains of Brucella suspicious bacteria were isolated in Guizhou province from 2010 to 2013. Four strains (GZLL3, GZLL4, GZLL11 and SH2) were isolated from goat blood samples and eight strains (SH4, GZZY, GZSQ, GZZA, BR13001, BR13004, BR13005 and BR13006) were isolated from blood samples of patient 12 Brucella suspicious strains were identified and characterized using conventional methods. Brucella genus specific gene BCSP31-based PCR (BCSP31-PCR) was used to identify the genus of Brucella and IS711 insert sequence-based PCR (AMOS-PCR) was applied to identify the species of Brucella strains. Goats and patients originated Brucella strains were comparatively analysed using Pulse-field Gel Electrophoresis (PFGE). Both of conventional methods and PCR identified the 12 Brucella suspicious strains as B. melitensis biotype 3. BCSP31-PCR identification results showed that a specific DNA bands (223 bp) were detected in all the 12 strains and positive control samples with no DNA band in negative samples. AMOS-PCR amplified a 731 bp-DNA bands in all the 12 strains, with 731 bp, 498 bp and 275 bp in M5, S2 and A19 strains, respectively, and no DNA band was detected in the negative control samples. PFGE analysis showed that 12 Brucella isolates from patients and goats showed consistent PFGE patterns with the digestion of restriction enzyme Xba I. The epidemic species/type of Brucella in both human and animal in Guizhou province was B. melitensis biotype 3 and goat was the main animal source of infection of brucellosis in Guizhou province.

Complete genome sequence of Marivirga tractuosa type strain (H-43).

OpenAIRE

Pagani, Ioanna; Chertkov, Olga; Lapidus, Alla; Lucas, Susan; Del Rio, Tijana Glavina; Tice, Hope; Copeland, Alex; Cheng, Jan-Fang; Nolan, Matt; Saunders, Elizabeth; Pitluck, Sam; Held, Brittany; Goodwin, Lynne; Liolios, Konstantinos; Ovchinikova, Galina

2011-01-01

Marivirga tractuosa (Lewin 1969) Nedashkovskaya et al. 2010 is the type species of the genus Marivirga, which belongs to the family Flammeovirgaceae. Members of this genus are of interest because of their gliding motility. The species is of interest because representative strains show resistance to several antibiotics, including gentamicin, kanamycin, neomycin, polymixin and streptomycin. This is the first complete genome sequence of a member of the family Flammeovirgaceae. Here we describe t...

Correlation of mutations and recombination with growth kinetics of poliovirus vaccine strains.

Science.gov (United States)

Pliaka, V; Kyriakopoulou, Z; Tsakogiannis, D; Ruether, I G A; Gartzonika, C; Levidiotou-Stefanou, S; Krikelis, A; Markoulatos, P

2010-12-01

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and, in rare cases, may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains, and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight oral poliovirus vaccine (OPV) isolates (two non-recombinants and six recombinants), as well as of Sabin vaccine strains, was evaluated using two different assays, the reproductive capacity at different temperatures (Rct) test and the one-step growth curve test in Hep-2 cells at two different temperatures (37°C and 40°C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All of the recombinant OPV isolates showed a thermoresistant phenotype in the Rct test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37°C and 40°C in the one-step growth curve test. All of the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature-sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light on the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.

Strains of avian paramyxovirus type 1 of low pathogenicity for chickens isolated from poultry and wild birds in Denmark

DEFF Research Database (Denmark)

Jørgensen, Poul Henrik; Handberg, Kurt; Ahrens, Peter

2004-01-01

Twenty-one strains of avian paramyxovirus type 1 of low virulence for chickens were isolated in Denmark between 1996 and the beginning of 2003. The low virulence of the strains was demonstrated by sequencing the fusion (F) gene at the cleavage site motif and in some cases by determining the intra......Twenty-one strains of avian paramyxovirus type 1 of low virulence for chickens were isolated in Denmark between 1996 and the beginning of 2003. The low virulence of the strains was demonstrated by sequencing the fusion (F) gene at the cleavage site motif and in some cases by determining...

Experience of social role strain in Korean women with type 2 diabetes.

Science.gov (United States)

Park, Hyunjeong; Wenzel, Jennifer A

2013-06-01

To expand our understanding of the experience of social role strain in the context of diabetes care among middle-aged married Korean women with type 2 diabetes. Diabetes remains an international concern. There are special challenges experienced by middle-aged married women who may not prioritize self-care and disease management. These challenges may be heightened in certain cultures due to traditional female and family roles along with other social norms and values. Descriptive qualitative study. This qualitative descriptive study involves in-depth interviews conducted between January-February 2007 with ten middle-aged married Korean women purposively selected to represent both higher and lower levels of role strain as measured by the measure of role gratification and strain instrument from the companion study, which was conducted simultaneously. Korean women in this study reported 'resentment regarding previous role strain'. This psychosocial burden was heightened by a noted pattern of 'sacrificing self in favour of others', which complicated both their personal lives and their ability to take care of themselves physically. Added to this were feelings of guilt related to their diabetes and the requirements of day-to-day management expressed as, 'my diabetes makes me a liability'. The women's role-strain experience related to their diabetes was intertwined with their past and current daily life. Further explication and interventions to address and manage role strain could potentially improve women's disease management and overall quality of life. © 2012 Blackwell Publishing Ltd.

Comparison of the Abbott RealTime CT new formulation assay with two other commercial assays for detection of wild-type and new variant strains of Chlamydia trachomatis

DEFF Research Database (Denmark)

Møller, Jens Kjølseth; Pedersen, Lisbeth Nørum; Persson, Kenneth

2010-01-01

In an analytical methods comparison study on clinical samples, the Abbott RealTime CT new formulation assay (m2000 real-time PCR) consisting of a duplex PCR targeting different parts of the cryptic plasmid in Chlamydia trachomatis was compared with version 2 of the Roche COBAS(R) TaqMan(R) CT ass...

Complete genome sequence of 'Thermobaculum terrenum' type strain (YNP1).

Science.gov (United States)

Kiss, Hajnalka; Cleland, David; Lapidus, Alla; Lucas, Susan; Del Rio, Tijana Glavina; Nolan, Matt; Tice, Hope; Han, Cliff; Goodwin, Lynne; Pitluck, Sam; Liolios, Konstantinos; Ivanova, Natalia; Mavromatis, Konstantinos; Ovchinnikova, Galina; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam; Hauser, Loren; Chang, Yun-Juan; Jeffries, Cynthia D; Lu, Megan; Brettin, Thomas; Detter, John C; Göker, Markus; Tindall, Brian J; Beck, Brian; McDermott, Timothy R; Woyke, Tanja; Bristow, James; Eisen, Jonathan A; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C; Klenk, Hans-Peter; Cheng, Jan-Fang

2010-10-27

'Thermobaculum terrenum' Botero et al. 2004 is the sole species within the proposed genus 'Thermobaculum'. Strain YNP1(T) is the only cultivated member of an acid tolerant, extremely thermophilic species belonging to a phylogenetically isolated environmental clone group within the phylum Chloroflexi. At present, the name 'Thermobaculum terrenum' is not yet validly published as it contravenes Rule 30 (3a) of the Bacteriological Code. The bacterium was isolated from a slightly acidic extreme thermal soil in Yellowstone National Park, Wyoming (USA). Depending on its final taxonomic allocation, this is likely to be the third completed genome sequence of a member of the class Thermomicrobia and the seventh type strain genome from the phylum Chloroflexi. The 3,101,581 bp long genome with its 2,872 protein-coding and 58 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Effect of wheelchair mass, tire type and tire pressure on physical strain and wheelchair propulsion technique.

Science.gov (United States)

de Groot, Sonja; Vegter, Riemer J K; van der Woude, Lucas H V

2013-10-01

The purpose of this study was to evaluate the effect of wheelchair mass, solid vs. pneumatic tires and tire pressure on physical strain and wheelchair propulsion technique. 11 Able-bodied participants performed 14 submaximal exercise blocks on a treadmill with a fixed speed (1.11 m/s) within 3 weeks to determine the effect of tire pressure (100%, 75%, 50%, 25% of the recommended value), wheelchair mass (0 kg, 5 kg, or 10 kg extra) and tire type (pneumatic vs. solid). All test conditions (except pneumatic vs. solid) were performed with and without instrumented measurement wheels. Outcome measures were power output (PO), physical strain (heart rate (HR), oxygen uptake (VO2), gross mechanical efficiency (ME)) and propulsion technique (timing, force application). At 25% tire pressure PO and subsequently VO2 were higher compared to 100% tire pressure. Furthermore, a higher tire pressure led to a longer cycle time and contact angle and subsequently lower push frequency. Extra mass did not lead to an increase in PO, physical strain or propulsion technique. Solid tires led to a higher PO and physical strain. The solid tire effect was amplified by increased mass (tire × mass interaction). In contrast to extra mass, tire pressure and tire type have an effect on PO, physical strain or propulsion technique of steady-state wheelchair propulsion. As expected, it is important to optimize tire pressure and tire type. Copyright © 2013 IPEM. Published by Elsevier Ltd. All rights reserved.

Characterization of incompletely typed rotavirus strains from Guinea-Bissau: identification of G8 and G9 types and a high frequency of mixed infections

DEFF Research Database (Denmark)

Fischer, T.K.; Page, N.A.; Griffin, D.D.

2003-01-01

%, respectively, identical to other African G8 and G9 strains. Multiple G and/or P types were identified at a high frequency (59%), including two previously undescribed mixed infections, P[4]P[6], G2G8 and P[4]P[6], G2G9. These mixed infections most likely represent naturally occurring reassortance of rotavirus......] and P[6] primer binding sites were detected. These findings highlight the need for regular evaluation of the multiplex primer PCR method and typing primers. The high frequency of uncommon as well as reassortant rotavirus strains in countries where rotavirus is an important cause of child mortality...... underscores the need for extensive strain surveillance as a basis to develop appropriate rotavirus vaccine candidates....

A piezoelectric-based infinite stiffness generation method for strain-type load sensors

International Nuclear Information System (INIS)

Zhang, Shuwen; Shao, Shubao; Xu, Minglong; Chen, Jie

2015-01-01

Under certain application conditions like nanoindentation technology and the mechanical property measurement of soft materials, the elastic deformation of strain-type load sensors affects their displacement measurement accuracy. In this work, a piezoelectric-based infinite stiffness generation method for strain-type load sensors that compensates for this elastic deformation is presented. The piezoelectric material-based deformation compensation method is proposed. An Hottinger Baldwin Messtechnik GmbH (HBM) Z30A/50N load sensor acts as the foundation of the method presented in this work. The piezoelectric stack is selected based on its size, maximum deformation value, blocking force and stiffness. Then, a clamping and fixing structure is designed to integrate the HBM sensor with the piezoelectric stack. The clamping and fixing structure, piezoelectric stack and HBM load sensor comprise the sensing part of the enhanced load sensor. The load-deformation curve and the voltage-deformation curve of the enhanced load sensor are then investigated experimentally. Because a hysteresis effect exists in the piezoelectric structure, the relationship between the control signal and the deformation value of the piezoelectric material is nonlinear. The hysteresis characteristic in a quasi-static condition is studied and fitted using a quadratic polynomial, and its coefficients are analyzed to enable control signal prediction. Applied arithmetic based on current theory and the fitted data is developed to predict the control signal. Finally, the experimental effects of the proposed method are presented. It is shown that when a quasi-static load is exerted on this enhanced strain-type load sensor, the deformation is reduced and the equivalent stiffness appears to be almost infinite. (paper)

The Mechanism for Type I Interferon Induction by Mycobacterium tuberculosis is Bacterial Strain-Dependent.

Directory of Open Access Journals (Sweden)

Kirsten E Wiens

2016-08-01

Full Text Available Type I interferons (including IFNαβ are innate cytokines that may contribute to pathogenesis during Mycobacterium tuberculosis (Mtb infection. To induce IFNβ, Mtb must gain access to the host cytosol and trigger stimulator of interferon genes (STING signaling. A recently proposed model suggests that Mtb triggers STING signaling through bacterial DNA binding cyclic GMP-AMP synthase (cGAS in the cytosol. The aim of this study was to test the generalizability of this model using phylogenetically distinct strains of the Mtb complex (MTBC. We infected bone marrow derived macrophages with strains from MTBC Lineages 2, 4 and 6. We found that the Lineage 6 strain induced less IFNβ, and that the Lineage 2 strain induced more IFNβ, than the Lineage 4 strain. The strains did not differ in their access to the host cytosol and IFNβ induction by each strain required both STING and cGAS. We also found that the three strains shed similar amounts of bacterial DNA. Interestingly, we found that the Lineage 6 strain was associated with less mitochondrial stress and less mitochondrial DNA (mtDNA in the cytosol compared with the Lineage 4 strain. Treating macrophages with a mitochondria-specific antioxidant reduced cytosolic mtDNA and inhibited IFNβ induction by the Lineage 2 and 4 strains. We also found that the Lineage 2 strain did not induce more mitochondrial stress than the Lineage 4 strain, suggesting that additional pathways contribute to higher IFNβ induction. These results indicate that the mechanism for IFNβ by Mtb is more complex than the established model suggests. We show that mitochondrial dynamics and mtDNA contribute to IFNβ induction by Mtb. Moreover, we show that the contribution of mtDNA to the IFNβ response varies by MTBC strain and that additional mechanisms exist for Mtb to induce IFNβ.

Preliminary characterization of Rhizobium strains isolated from ...

African Journals Online (AJOL)

SERVER

2008-03-18

Mar 18, 2008 ... residues for animal feed and helps to maintain soil fertility through .... A loopful of agar slopes of the strains assayed for vitamin production was ... B12 Assay Medium, amended with 2 mmol l-1 Ca-pantothenate, biotin and ...

Clostridium difficile infection: Early history, diagnosis and molecular strain typing methods.

Science.gov (United States)

Rodriguez, C; Van Broeck, J; Taminiau, B; Delmée, M; Daube, G

2016-08-01

Recognised as the leading cause of nosocomial antibiotic-associated diarrhoea, the incidence of Clostridium difficile infection (CDI) remains high despite efforts to improve prevention and reduce the spread of the bacterium in healthcare settings. In the last decade, many studies have focused on the epidemiology and rapid diagnosis of CDI. In addition, different typing methods have been developed for epidemiological studies. This review explores the history of C. difficile and the current scope of the infection. The variety of available laboratory tests for CDI diagnosis and strain typing methods are also examined. Copyright © 2016 Elsevier Ltd. All rights reserved.

Typing Chlamydia trachomatis: from egg yolk to nanotechnology

DEFF Research Database (Denmark)

Pedersen, Lisbeth Nørum; Herrmann, Bjørn; Møller, Jens Kjølseth

2009-01-01

A historical review is provided of the various methods used for half a century to differentiate and type Chlamydia trachomatis strains. Typing of C. trachomatis is an important tool for revealing transmission patterns in sexual networks, and enabling association with clinical manifestations......, insufficient epidemiological resolution is achieved by characterization of both MOMP and omp1. This calls for new high-resolution genotyping methods applying for example a multilocus variable number tandem repeat assay (MLVA) or multilocus sequence typing (MLST). The futuristic nanotechnology already seems...

Trypanosoma cruzi strains isolated from human, vector, and animal reservoir in the same endemic region in Mexico and typed as T. cruzi I, discrete typing unit 1 exhibit considerable biological diversity

Directory of Open Access Journals (Sweden)

María del Carmen Sánchez-Guillén

2006-09-01

Full Text Available In this study, three strains of Trypanosoma cruzi were isolated at the same time and in the same endemic region in Mexico from a human patient with chronic chagasic cardiomyopathy (RyC-H; vector (Triatoma barberi (RyC-V; and rodent reservoir (Peromyscus peromyscus (RyC-R. The three strains were characterized by multilocus enzyme electrophoresis, random amplified polymorphic DNA, and by pathological profiles in experimental animals (biodemes. Based on the analysis of genetic markers the three parasite strains were typed as belonging to T. cruzi I major group, discrete typing unit 1. The pathological profile of RyC-H and RyC-V strains indicated medium virulence and low mortality and, accordingly, the strains should be considered as belonging to biodeme Type III. On the other hand, the parasites from RyC-R strain induced more severe inflammatory processes and high mortality (> 40% and were considered as belonging to biodeme Type II. The relationship between genotypes and biological characteristics in T. cruzi strains is still debated and not clearly understood. An expert committee recommended in 1999 that Biodeme Type III would correspond to T. cruzi I group, whereas Biodeme Type II, to T. cruzi II group. Our findings suggest that, at least for Mexican isolates, this correlation does not stand and that biological characteristics such as pathogenicity and virulence could be determined by factors different from those identified in the genotypic characterization

Characterization of Staphylococcus aureus strains involved in human and bovine mastitis.

Science.gov (United States)

Delgado, Susana; García, Pilar; Fernández, Leonides; Jiménez, Esther; Rodríguez-Baños, Mercedes; del Campo, Rosa; Rodríguez, Juan M

2011-07-01

Staphylococcus aureus is one of the main etiological agents of mastitis in different mammalian species. At present, it is unknown whether strains isolated from human mastitis cases share phenotypic properties and genetic background with those obtained from animal mastitis cases. Therefore, the objective of this study was to characterize S. aureus strains isolated from women with lactational mastitis and to compare them with the strains responsible for bovine mastitis and noninfectious strains. All the strains were genotyped by both pulsed field gel electrophoresis and multilocus sequence typing and submitted to a characterization scheme that included diverse assays related to pathogenic potential and antibiotic resistance. Apart from siderophore production, no significant association was observed between the strains from bovine and human mastitis. Statistical differences between human- and bovine-mastitis-associated strains were detected for some traits and virulence determinants, such as the presence of prophages and cna and hlb genes, which were more frequently found within the bovine group. On the contrary, resistance to penicillin was significantly higher among strains isolated from human lactational mastitis, probably related to the common presence of the blaZ gene. A high genetic diversity was found among the strains involved in mastitis in breastfeeding women. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Complete Genome Sequence of Mycobacterium fortuitum subsp. fortuitum Type Strain DSM46621

KAUST Repository

Ho, Y. S

2012-10-26

Mycobacterium fortuitum is a member of the rapidly growing nontuberculous mycobacteria (NTM). It is ubiquitous in water and soil habitats, including hospital environments. M. fortuitum is increasingly recognized as an opportunistic nosocomial pathogen causing disseminated infection. Here we report the genome sequence of M. fortuitum subsp. fortuitum type strain DSM46621.

Complete Genome Sequence of Mycobacterium fortuitum subsp. fortuitum Type Strain DSM46621

KAUST Repository

Ho, Y. S; Adroub, S. A.; Aleisa, F.; Mahmood, H.; Othoum, G.; Rashid, F.; Zaher, M.; Ali, Shahjahan; Bitter, W.; Pain, Arnab; Abdallah, A. M.

2012-01-01

Mycobacterium fortuitum is a member of the rapidly growing nontuberculous mycobacteria (NTM). It is ubiquitous in water and soil habitats, including hospital environments. M. fortuitum is increasingly recognized as an opportunistic nosocomial pathogen causing disseminated infection. Here we report the genome sequence of M. fortuitum subsp. fortuitum type strain DSM46621.

Complete genome sequence of Capnocytophaga ochracea type strain (VPI 2845T)

Energy Technology Data Exchange (ETDEWEB)

Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Gronow, Sabine [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Saunders, Elizabeth H [Los Alamos National Laboratory (LANL); Land, Miriam L [ORNL; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Copeland, A [U.S. Department of Energy, Joint Genome Institute; Glavina Del Rio, Tijana [U.S. Department of Energy, Joint Genome Institute; Nolan, Matt [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Chen, Feng [U.S. Department of Energy, Joint Genome Institute; Bruce, David [Los Alamos National Laboratory (LANL); Tice, Hope [U.S. Department of Energy, Joint Genome Institute; Cheng, Jan-Fang [U.S. Department of Energy, Joint Genome Institute; Goodwin, Lynne A. [Los Alamos National Laboratory (LANL); Pitluck, Sam [U.S. Department of Energy, Joint Genome Institute; Pati, Amrita [U.S. Department of Energy, Joint Genome Institute; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Chen, Amy [U.S. Department of Energy, Joint Genome Institute; Palaniappan, Krishna [U.S. Department of Energy, Joint Genome Institute; Chain, Patrick S. G. [Lawrence Livermore National Laboratory (LLNL); Hauser, Loren John [ORNL; Chang, Yun-Juan [ORNL; Jeffries, Cynthia [Oak Ridge National Laboratory (ORNL); Brettin, Thomas S [ORNL; Detter, J. Chris [U.S. Department of Energy, Joint Genome Institute; Han, Cliff [Los Alamos National Laboratory (LANL); Bristow, James [U.S. Department of Energy, Joint Genome Institute; Goker, Markus [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Eisen, Jonathan [U.S. Department of Energy, Joint Genome Institute; Markowitz, Victor [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute; Klenk, Hans-Peter [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Hugenholtz, Philip [U.S. Department of Energy, Joint Genome Institute

2009-01-01

Capnocytophaga ochracea (Pr vot et al. 1956) Leadbetter et al. 1982 is the type species of the genus Capnocytophaga. It is of interest because of its location in the Flavobacteriaceae, a genomically not yet charted family within the order Flavobacteriales. The species grows as fusiform to rod shaped cells which tend to form clumps and are able to move by gliding. C. ochracea is known as a capnophilic (CO2-requiring) organism with the ability to grow under anaerobic as well as aerobic conditions (oxygen concentration larger than 15%), here only in the presence of 5% CO2. Strain VPI 2845T, the type strain of the species, is portrayed in this report as a gliding, Gram-negative bacterium, originally isolated from a human oral cavity. Here we describe the features of this organism, together with the complete genome se-quence, and annotation. This is the first completed genome sequence from the flavobacterial genus Capnocytophaga, and the 2,612,925 bp long single replicon genome with its 2193 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

The biological properties of different Epstein-Barr virus strains explain their association with various types of cancers.

Science.gov (United States)

Tsai, Ming-Han; Lin, Xiaochen; Shumilov, Anatoliy; Bernhardt, Katharina; Feederle, Regina; Poirey, Remy; Kopp-Schneider, Annette; Pereira, Bruno; Almeida, Raquel; Delecluse, Henri-Jacques

2017-02-07

The Epstein-Barr virus (EBV) is etiologically associated with the development of multiple types of tumors, but it is unclear whether this diversity is due to infection with different EBV strains. We report a comparative characterization of SNU719, GP202, and YCCEL1, three EBV strains that were isolated from gastric carcinomas, M81, a virus isolated in a nasopharyngeal carcinoma and several well-characterized laboratory type A strains. We found that B95-8, Akata and GP202 induced cell growth more efficiently than YCCEL1, SNU719 and M81 and this correlated positively with the expression levels of the viral BHRF1 miRNAs. In infected B cells, all strains except Akata and B95-8 induced lytic replication, a risk factor for carcinoma development, although less efficiently than M81. The panel of viruses induced tumors in immunocompromised mice with variable speed and efficacy that did not strictly mirror their in vitro characteristics, suggesting that additional parameters play an important role. We found that YCCEL1 and M81 infected primary epithelial cells, gastric carcinoma cells and gastric spheroids more efficiently than Akata or B95-8. Reciprocally, Akata and B95-8 had a stronger tropism for B cells than YCCEL1 or M81. These data suggest that different EBV strains will induce the development of lymphoid tumors with variable efficacy in immunocompromised patients and that there is a parallel between the cell tropism of the viral strains and the lineage of the tumors they induce. Thus, EBV strains can be endowed with properties that will influence their transforming abilities and the type of tumor they induce.

High-throughput typing method to identify a non-outbreak-involved Legionella pneumophila strain colonizing the entire water supply system in the town of Rennes, France.

Science.gov (United States)

Sobral, D; Le Cann, P; Gerard, A; Jarraud, S; Lebeau, B; Loisy-Hamon, F; Vergnaud, G; Pourcel, C

2011-10-01

Two legionellosis outbreaks occurred in the city of Rennes, France, during the past decade, requiring in-depth monitoring of Legionella pneumophila in the water network and the cooling towers in the city. In order to characterize the resulting large collection of isolates, an automated low-cost typing method was developed. The multiplex capillary-based variable-number tandem repeat (VNTR) (multiple-locus VNTR analysis [MLVA]) assay requiring only one PCR amplification per isolate ensures a high level of discrimination and reduces hands-on and time requirements. In less than 2 days and using one 4-capillary apparatus, 217 environmental isolates collected between 2000 and 2009 and 5 clinical isolates obtained during outbreaks in 2000 and 2006 in Rennes were analyzed, and 15 different genotypes were identified. A large cluster of isolates with closely related genotypes and representing 77% of the population was composed exclusively of environmental isolates extracted from hot water supply systems. It was not responsible for the known Rennes epidemic cases, although strains showing a similar MLVA profile have regularly been involved in European outbreaks. The clinical isolates in Rennes had the same genotype as isolates contaminating a mall's cooling tower. This study further demonstrates that unknown environmental or genetic factors contribute to the pathogenicity of some strains. This work illustrates the potential of the high-throughput MLVA typing method to investigate the origin of legionellosis cases by allowing the systematic typing of any new isolate and inclusion of data in shared databases.

Anticoccidial efficacy testing: In vitro Eimeria tenella assays as replacement for animal experiments.

Science.gov (United States)

Thabet, Ahmed; Zhang, Runhui; Alnassan, Alaa-Aldin; Daugschies, Arwid; Bangoura, Berit

2017-01-15

Availability of an accurate in vitro assay is a crucial demand to determine sensitivity of Eimeria spp. field strains toward anticoccidials routinely. In this study we tested in vitro models of Eimeria tenella using various polyether ionophores (monensin, salinomycin, maduramicin, and lasalocid) and toltrazuril. Minimum inhibitory concentrations (MIC 95 , MIC 50/95 ) for the tested anticoccidials were defined based on a susceptible reference (Houghton strain), Ref-1. In vitro sporozoite invasion inhibition assay (SIA) and reproduction inhibition assay (RIA) were applied on sensitive laboratory (Ref-1 and Ref-2) and field (FS-1, FS-2, and FS-3) strains to calculate percent of inhibition under exposure of these strains to the various anticoccidials (%I SIA and%I RIA, respectively). The in vitro data were related to oocyst excretion, lesion scores, performance, and global resistance indices (GI) assessed in experimentally infected chickens. Polyether ionophores applied in the RIA were highly effective at MIC 95 against Ref-1 and Ref-2 (%I RIA ≥95%). In contrast, all tested field strains displayed reduced to low efficacy (%I RIA animal model (p89%) against all strains used in this study. However, adjusted GI (GI adj ) for toltrazuril-treated groups exhibited differences between reference and field strains which might indicate varying sensitivity. RIA is a suitable in vitro tool to detect sensitivity of E. tenella towards polyether ionophores, and may thus help to reduce, replace, or refine use of animal experimentation for in vivo sensitivity assays. Copyright © 2016 Elsevier B.V. All rights reserved.

Cadmium tolerance, cysteine and thiol peptide levels in wild type and chromium-tolerant strains of Scenedesmus acutus (Chlorophyceae)

Energy Technology Data Exchange (ETDEWEB)

Torricelli, Elena; Gorbi, Gessica; Pawlik-Skowronska, Barbara; Di Toppi, Luigi Sanita; Corradi, Maria Grazia

2004-07-14

Two strains of the unicellular green alga Scenedesmus acutus with different sensitivity to hexavalent chromium were compared for their tolerance of cadmium, by means of growth and recovery tests, and determination of cysteine, reduced glutathione and phytochelatin content, after short-term exposure to various cadmium concentrations (from 1.125 to 27 {mu}M). Growth experiments showed that, after 7-day treatments with cadmium, the chromium-tolerant strain reached a significantly higher cell density and, after 24-h exposure to Cd, was able to resume growth significantly better than the wild type. Constitutive level of cysteine was higher in the chromium-tolerant strain, while glutathione levels were similar in the two strains. The higher content of cysteine and the maintenance of both reduced glutathione and phytochelatin high levels in the presence of cadmium, support the higher cadmium co-tolerance of the chromium-tolerant strain in comparison with the wild type one.

Cadmium tolerance, cysteine and thiol peptide levels in wild type and chromium-tolerant strains of Scenedesmus acutus (Chlorophyceae)

International Nuclear Information System (INIS)

Torricelli, Elena; Gorbi, Gessica; Pawlik-Skowronska, Barbara; Di Toppi, Luigi Sanita; Corradi, Maria Grazia

2004-01-01

Two strains of the unicellular green alga Scenedesmus acutus with different sensitivity to hexavalent chromium were compared for their tolerance of cadmium, by means of growth and recovery tests, and determination of cysteine, reduced glutathione and phytochelatin content, after short-term exposure to various cadmium concentrations (from 1.125 to 27 μM). Growth experiments showed that, after 7-day treatments with cadmium, the chromium-tolerant strain reached a significantly higher cell density and, after 24-h exposure to Cd, was able to resume growth significantly better than the wild type. Constitutive level of cysteine was higher in the chromium-tolerant strain, while glutathione levels were similar in the two strains. The higher content of cysteine and the maintenance of both reduced glutathione and phytochelatin high levels in the presence of cadmium, support the higher cadmium co-tolerance of the chromium-tolerant strain in comparison with the wild type one

STRESS - STRAIN CURVE ANALYSIS OF WOVEN FABRICS MAD E FROM COMBED YARNS TYPE WOOL

Directory of Open Access Journals (Sweden)

VÎLCU Adrian

2014-05-01

Full Text Available The paper analyses the tensile behavior of woven fabrics made from 45%Wool + 55% PES used for garments. Analysis of fabric behavior during wearing has shown that these are submitted to simple and repeated uni-axial or bi-axial tensile strains. The level of these strains is often within the elastic limit, rarely going over yielding. Therefore the designer must be able to evaluate the mechanical behavior of such fabrics in order to control the fabric behavior in the garment. This evaluation is carried out based on the tensile testing, using certain indexes specific to the stress-strain curve. The paper considers an experimental matrix based on woven fabrics of different yarn counts, different or equal yarn count for warp and weft systems and different structures. The fabrics were tested using a testing machine and the results were then compared in order to determine the fabrics’ tensile behavior and the factors of influence that affect it.From the point of view of tensile testing, the woven materials having twill weave are preferable because this type of structure is characterized by higher durability and better yarn stability in the fabric. In practice, the woven material must exhibit an optimum behavior to repeated strains, flexions and abrasions during wearing process. The analysis of fabrics tensile properties studied by investigation of stress-strain diagrams reveals that the main factors influencing the tensile strength are: yarns fineness, technological density of those two systems of yarns and the weaving type.

Colony Dimorphism in Bradyrhizobium Strains

Science.gov (United States)

Sylvester-Bradley, Rosemary; Thornton, Philip; Jones, Peter

1988-01-01

Ten isolates of Bradyrhizobium spp. which form two colony types were studied; the isolates originated from a range of legume species. The two colony types differed in the amount of gum formed or size or both, depending on the strain. Whole 7-day-old colonies of each type were subcultured to determine the proportion of cells which had changed to the other type. An iterative computerized procedure was used to determine the rate of switching per generation between the two types and to predict proportions reached at equilibrium for each strain. The predicted proportions of the wetter (more gummy) or larger colony type at equilibrium differed significantly between strains, ranging from 0.9999 (strain CIAT 2383) to 0.0216 (strain CIAT 2469), because some strains switched faster from dry to wet (or small to large) and others switched faster from wet to dry (or large to small). Predicted equilibrium was reached after about 140 generations in strain USDA 76. In all but one strain (CIAT 3030) the growth rate of the wetter colony type was greater than or similar to that of the drier type. The mean difference in generation time between the two colony types was 0.37 h. Doubling times calculated for either colony type after 7 days of growth on the agar surface ranged from 6.0 to 7.3 h. The formation of two persistent colony types by one strain (clonal or colony dimorphism) may be a common phenomenon among Bradyrhizobium strains. Images PMID:16347599

Complete genome sequence of thermophilic Bacillus smithii type strain DSM 4216T

DEFF Research Database (Denmark)

Bosma, Elleke Fenna; Koehorst, Jasper J.; van Hijum, Sacha A. F. T.

2016-01-01

determined the complete genomic sequence of the B. smithii type strain DSM 4216T, which consists of a 3,368,778 bp chromosome (GenBank accession number CP012024.1) and a 12,514 bp plasmid (GenBank accession number CP012025.1), together encoding 3880 genes. Genome annotation via RAST was complemented...

Comparative genomics of human and non-human Listeria monocytogenes sequence type 121 strains.

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Kathrin Rychli

Full Text Available The food-borne pathogen Listeria (L. monocytogenes is able to survive for months and even years in food production environments. Strains belonging to sequence type (ST121 are particularly found to be abundant and to persist in food and food production environments. To elucidate genetic determinants characteristic for L. monocytogenes ST121, we sequenced the genomes of 14 ST121 strains and compared them with currently available L. monocytogenes ST121 genomes. In total, we analyzed 70 ST121 genomes deriving from 16 different countries, different years of isolation, and different origins-including food, animal and human ST121 isolates. All ST121 genomes show a high degree of conservation sharing at least 99.7% average nucleotide identity. The main differences between the strains were found in prophage content and prophage conservation. We also detected distinct highly conserved subtypes of prophages inserted at the same genomic locus. While some of the prophages showed more than 99.9% similarity between strains from different sources and years, other prophages showed a higher level of diversity. 81.4% of the strains harbored virtually identical plasmids. 97.1% of the ST121 strains contain a truncated internalin A (inlA gene. Only one of the seven human ST121 isolates encodes a full-length inlA gene, illustrating the need of better understanding their survival and virulence mechanisms.

Molecular Mechanisms of Attenuation of the Sabin Strain of Poliovirus Type 3

OpenAIRE

Guest, Stephen; Pilipenko, Evgeny; Sharma, Kamal; Chumakov, Konstantin; Roos, Raymond P.

2004-01-01

Mutations critical for the central nervous system (CNS) attenuation of the Sabin vaccine strains of poliovirus (PV) are located within the viral internal ribosome entry site (IRES). We examined the interaction of the IRESs of PV type 3 (PV3) and Sabin type 3 (Sabin3) with polypyrimidine tract-binding protein (PTB) and a neural cell-specific homologue, nPTB. PTB and nPTB were found to bind to a site directly adjacent to the attenuating mutation, and binding at this site was less efficient on t...

Detection and Molecular Characterization of Escherichia coli Strains Producers of Extended-Spectrum and CMY-2 Type Beta-Lactamases, Isolated from Turtles in Mexico.

Science.gov (United States)

Cortés-Cortés, Gerardo; Lozano-Zarain, Patricia; Torres, Carmen; Castañeda, Miguel; Sánchez, Gabriela Moreno; Alonso, Carla A; López-Pliego, Liliana; Mayen, María G Gutiérrez; Martínez-Laguna, Ygnacio; Rocha-Gracia, Rosa Del Carmen

2016-09-01

Multidrug-resistant bacteria are a growing problem in different environments and hosts, but scarce information exists about their prevalence in reptiles. The aim of this study was to analyze the resistance mechanisms, molecular typing, and plasmid content of cefotaxime-resistant (CTX(R)) Escherichia coli isolates recovered from cloacal samples of 71 turtles sheltered in a herpetarium in Mexico. CTX(R)-E. coli were recovered in 11 of 71 samples (15.5%), and one isolate/sample was characterized. Extended-spectrum β-lactamase (ESBL)-producing E. coli isolates were detected in four samples (5.6%): two strains carried the blaCTX-M-2 gene (phylogroup D and ST2732) and two contained the blaCTX-M-15 gene (phylogroup B1 and lineages ST58 and ST156). The blaCMY-2 gene was detected by PCR in E. coli isolates of eight samples (9.8%) (one of them also carried blaCTX-M-2); these isolates were distributed into phylogroups A (n = 1), B1 (n = 6), and D (n = 1) and typed as ST155, ST156, ST2329, and ST2732. Plasmid-mediated quinolone resistance (PMQR) genes were detected in five isolates [aac(6')Ib-cr, qnrA, qnrB19, and oqxB]. From three to five replicon plasmids were detected among the strains, being IncFIB, IncI1, IncFrep, and IncK the most prevalent. ESBL or pAmpC genes were transferred by conjugation in four strains, and the blaCTX-M-15 and blaCMY-2 genes were localized in IncFIB or IncI1 plasmids by Southern blot hybridization assays. Class 1 and/or class 2 integrons were detected in eight strains with six different structures of gene cassette arrays. Nine pulsed-field gel electrophoresis patterns were found among the 11 studied strains. To our knowledge, this is the first detection of ESBL, CMY-2, PMQR, and mobile determinants of antimicrobial resistance in E. coli of turtle origin, highlighting the potential dissemination of multidrug-resistant bacteria from these animals to other environments and hosts, including humans.

Genotypic and Phenotypic Characterization of Enterotoxigenic Escherichia coli Strains Isolated from Peruvian Children ▿

Science.gov (United States)

Rivera, F. P.; Ochoa, T. J.; Maves, R. C.; Bernal, M.; Medina, A. M.; Meza, R.; Barletta, F.; Mercado, E.; Ecker, L.; Gil, A. I.; Hall, E. R.; Huicho, L.; Lanata, C. F.

2010-01-01

Enterotoxigenic Escherichia coli (ETEC) is a major cause of childhood diarrhea. The present study sought to determine the prevalence and distribution of toxin types, colonization factors (CFs), and antimicrobial susceptibility of ETEC strains isolated from Peruvian children. We analyzed ETEC strains isolated from Peruvian children between 2 and 24 months of age in a passive surveillance study. Five E. coli colonies per patient were studied by multiplex real-time PCR to identify ETEC virulence factors. ETEC-associated toxins were confirmed using a GM1-based enzyme-linked immunosorbent assay. Confirmed strains were tested for CFs by dot blot assay using 21 monoclonal antibodies. We analyzed 1,129 samples from children with diarrhea and 744 control children and found ETEC in 5.3% and 4.3%, respectively. ETEC was more frequently isolated from children >12 months of age than from children <12 months of age (P < 0.001). Fifty-two percent of ETEC isolates from children with diarrhea and 72% of isolates from controls were heat-labile enterotoxin (LT) positive and heat-stable enterotoxin (ST) negative; 25% and 19%, respectively, were LT negative and ST positive; and 23% and 9%, respectively, were LT positive and ST positive. CFs were identified in 64% of diarrheal samples and 37% of control samples (P < 0.05). The most common CFs were CS6 (14% and 7%, respectively), CS12 (12% and 4%, respectively), and CS1 (9% and 4%, respectively). ST-producing ETEC strains caused more severe diarrhea than non-ST-producing ETEC strains. The strains were most frequently resistant to ampicillin (71%) and co-trimoxazole (61%). ETEC was thus found to be more prevalent in older infants. LT was the most common toxin type; 64% of strains had an identified CF. These data are relevant in estimating the burden of disease due to ETEC and the potential coverage of children in Peru by investigational vaccines. PMID:20631096

Detection and strain typing of ancient Mycobacterium leprae from a medieval leprosy hospital.

Science.gov (United States)

Taylor, G Michael; Tucker, Katie; Butler, Rachel; Pike, Alistair W G; Lewis, Jamie; Roffey, Simon; Marter, Philip; Lee, Oona Y-C; Wu, Houdini H T; Minnikin, David E; Besra, Gurdyal S; Singh, Pushpendra; Cole, Stewart T; Stewart, Graham R

2013-01-01

Nine burials excavated from the Magdalen Hill Archaeological Research Project (MHARP) in Winchester, UK, showing skeletal signs of lepromatous leprosy (LL) have been studied using a multidisciplinary approach including osteological, geochemical and biomolecular techniques. DNA from Mycobacterium leprae was amplified from all nine skeletons but not from control skeletons devoid of indicative pathology. In several specimens we corroborated the identification of M. leprae with detection of mycolic acids specific to the cell wall of M. leprae and persistent in the skeletal samples. In five cases, the preservation of the material allowed detailed genotyping using single-nucleotide polymorphism (SNP) and multiple locus variable number tandem repeat analysis (MLVA). Three of the five cases proved to be infected with SNP type 3I-1, ancestral to contemporary M. leprae isolates found in southern states of America and likely carried by European migrants. From the remaining two burials we identified, for the first time in the British Isles, the occurrence of SNP type 2F. Stable isotope analysis conducted on tooth enamel taken from two of the type 3I-1 and one of the type 2F remains revealed that all three individuals had probably spent their formative years in the Winchester area. Previously, type 2F has been implicated as the precursor strain that migrated from the Middle East to India and South-East Asia, subsequently evolving to type 1 strains. Thus we show that type 2F had also spread westwards to Britain by the early medieval period.

Detection and strain typing of ancient Mycobacterium leprae from a medieval leprosy hospital.

Directory of Open Access Journals (Sweden)

G Michael Taylor

Full Text Available Nine burials excavated from the Magdalen Hill Archaeological Research Project (MHARP in Winchester, UK, showing skeletal signs of lepromatous leprosy (LL have been studied using a multidisciplinary approach including osteological, geochemical and biomolecular techniques. DNA from Mycobacterium leprae was amplified from all nine skeletons but not from control skeletons devoid of indicative pathology. In several specimens we corroborated the identification of M. leprae with detection of mycolic acids specific to the cell wall of M. leprae and persistent in the skeletal samples. In five cases, the preservation of the material allowed detailed genotyping using single-nucleotide polymorphism (SNP and multiple locus variable number tandem repeat analysis (MLVA. Three of the five cases proved to be infected with SNP type 3I-1, ancestral to contemporary M. leprae isolates found in southern states of America and likely carried by European migrants. From the remaining two burials we identified, for the first time in the British Isles, the occurrence of SNP type 2F. Stable isotope analysis conducted on tooth enamel taken from two of the type 3I-1 and one of the type 2F remains revealed that all three individuals had probably spent their formative years in the Winchester area. Previously, type 2F has been implicated as the precursor strain that migrated from the Middle East to India and South-East Asia, subsequently evolving to type 1 strains. Thus we show that type 2F had also spread westwards to Britain by the early medieval period.

Lectin typing of Campylobacter jejuni using a novel quartz crystal microbalance technique

Energy Technology Data Exchange (ETDEWEB)

Yakovleva, Maria E., E-mail: maria.yakovleva@gmail.com [Department of Infectious Diseases and Medical Microbiology, Lund University, 223 62 Lund (Sweden); Moran, Anthony P. [Department of Microbiology, School of Natural Sciences, National University of Ireland, Galway (Ireland); Safina, Gulnara R. [Department of Analytical and Marine Chemistry, University of Gothenburg, 412 96 Gothenburg (Sweden); Wadstroem, Torkel [Department of Infectious Diseases and Medical Microbiology, Lund University, 223 62 Lund (Sweden); Danielsson, Bengt [Acromed Invest AB, Magistratsvaegen 10, 226 43 Lund (Sweden)

2011-05-23

Seven Campylobacter jejuni strains were characterised by a lectin typing assay. The typing system was based on a quartz crystal microbalance technique (QCM) with four commercially available lectins (wheat germ agglutinin, Maackia amurensis lectin, Lens culinaris agglutinin, and Concanavalin A), which were chosen for their differing carbohydrate specificities. Initially, the gold surfaces of the quartz crystals were modified with 11-mercaptoundecanoic acid followed by lectin immobilisation using a conventional amine-coupling technique. Bacterial cells were applied for lectin typing without preliminary treatment, and resonant frequency and dissipation responses were recorded. The adhesion of microorganisms on lectin surfaces was confirmed by atomic force microscopy. Scanning was performed in the tapping mode and the presence of bacteria on lectin-coated surfaces was successfully demonstrated. A significant difference in the dissipation response was observed for different C. jejuni strains which made it possible to use this parameter for discriminating between bacterial strains. In summary, the QCM technique proved a powerful tool for the recognition and discrimination of C. jejuni strains. The approach may also prove applicable to strain discrimination of other bacterial species, particularly pathogens.

Lectin typing of Campylobacter jejuni using a novel quartz crystal microbalance technique

International Nuclear Information System (INIS)

Yakovleva, Maria E.; Moran, Anthony P.; Safina, Gulnara R.; Wadstroem, Torkel; Danielsson, Bengt

2011-01-01

Seven Campylobacter jejuni strains were characterised by a lectin typing assay. The typing system was based on a quartz crystal microbalance technique (QCM) with four commercially available lectins (wheat germ agglutinin, Maackia amurensis lectin, Lens culinaris agglutinin, and Concanavalin A), which were chosen for their differing carbohydrate specificities. Initially, the gold surfaces of the quartz crystals were modified with 11-mercaptoundecanoic acid followed by lectin immobilisation using a conventional amine-coupling technique. Bacterial cells were applied for lectin typing without preliminary treatment, and resonant frequency and dissipation responses were recorded. The adhesion of microorganisms on lectin surfaces was confirmed by atomic force microscopy. Scanning was performed in the tapping mode and the presence of bacteria on lectin-coated surfaces was successfully demonstrated. A significant difference in the dissipation response was observed for different C. jejuni strains which made it possible to use this parameter for discriminating between bacterial strains. In summary, the QCM technique proved a powerful tool for the recognition and discrimination of C. jejuni strains. The approach may also prove applicable to strain discrimination of other bacterial species, particularly pathogens.

Genetic Variation of pln Loci Among Probiotic Lactobacillus plantarum Group Strains with Antioxidant and Cholesterol-Lowering Ability.

Science.gov (United States)

Devi, Sundru Manjulata; Halami, Prakash M

2017-10-13

In the present study, 14 different plantaricin-encoding genes of pln loci were studied and compared to available sequences from public domain database of probiotic Lactobacillus plantarum strains. Based upon the presence and absence of selected genes, pln locus was grouped into eight clusters. Further, quantitative real-time PCR (qRT-PCR) analysis for seven genes has discriminated the complex pln locus into five types which includes WCFS1 (in Lactobacillus plantarum subsp. plantarum MCC 2976 and MCC 2974 and Lactobacillus paraplantarum MCC 2978), closely related to J51 (in Lb. paraplantarum MCC 2973 and MCC 2977), J23 (in Lb. plantarum MTCC 5422), NC8 (in Lb. paraplantarum MTCC 9483), and a new E1 type (in Lb. plantarum subsp. plantarum E1). It was observed that the plnA, EF, NC8βα, NC81F, NC8HK, and G were expressed in E1 strain. Further, southern hybridization confirmed the chromosome-encoded plantaricin in Lb. plantarum group (LPG) strains. Several PCR assays and DNA sequence analysis of the regions amplified in pln loci of E1 isolate suggested a hybrid variant of NC8 and J51 plantaritypes. This indicates the wide distribution of plantaricin with remarkable variation, diversity, and plasticity among the LPG strains of vegetable origin. Further, the selected strains were able to reduce the growth of Kocuria rhizophila ATCC 9341 by 40-54% within 6 h of co-incubation under in vitro pathogen exclusion assay. These isolates also possessed cholesterol-lowering and antioxidant activity suggesting their application in the development of functional foods.

Analytical and clinical performance of the CDC real time RT-PCR assay for detection and typing of dengue virus.

Science.gov (United States)

Santiago, Gilberto A; Vergne, Edgardo; Quiles, Yashira; Cosme, Joan; Vazquez, Jesus; Medina, Juan F; Medina, Freddy; Colón, Candimar; Margolis, Harold; Muñoz-Jordán, Jorge L

2013-01-01

Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV). There are four genetically distinct DENVs (DENV-1-4) that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1-4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA) for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1-4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1-4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases.

Genome Analysis of Listeria monocytogenes Sequence Type 8 Strains Persisting in Salmon and Poultry Processing Environments and Comparison with Related Strains

Science.gov (United States)

Fagerlund, Annette; Langsrud, Solveig; Schirmer, Bjørn C. T.; Møretrø, Trond; Heir, Even

2016-01-01

Listeria monocytogenes is an important foodborne pathogen responsible for the disease listeriosis, and can be found throughout the environment, in many foods and in food processing facilities. The main cause of listeriosis is consumption of food contaminated from sources in food processing environments. Persistence in food processing facilities has previously been shown for the L. monocytogenes sequence type (ST) 8 subtype. In the current study, five ST8 strains were subjected to whole-genome sequencing and compared with five additionally available ST8 genomes, allowing comparison of strains from salmon, poultry and cheese industry, in addition to a human clinical isolate. Genome-wide analysis of single-nucleotide polymorphisms (SNPs) confirmed that almost identical strains were detected in a Danish salmon processing plant in 1996 and in a Norwegian salmon processing plant in 2001 and 2011. Furthermore, we show that L. monocytogenes ST8 was likely to have been transferred between two poultry processing plants as a result of relocation of processing equipment. The SNP data were used to infer the phylogeny of the ST8 strains, separating them into two main genetic groups. Within each group, the plasmid and prophage content was almost entirely conserved, but between groups, these sequences showed strong divergence. The accessory genome of the ST8 strains harbored genetic elements which could be involved in rendering the ST8 strains resilient to incoming mobile genetic elements. These included two restriction-modification loci, one of which was predicted to show phase variable recognition sequence specificity through site-specific domain shuffling. Analysis indicated that the ST8 strains harbor all important known L. monocytogenes virulence factors, and ST8 strains are commonly identified as the causative agents of invasive listeriosis. Therefore, the persistence of this L. monocytogenes subtype in food processing facilities poses a significant concern for food safety

Rapid Screening of MDR-TB in Cases of Extra Pulmonary Tuberculosis Using Geno Type MTBDRplus.

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Richa Kumari

Full Text Available Drug resistance in tuberculosis is a major public health challenge in developing countries. The limited data available on drug resistance in extra pulmonary tuberculosis stimulated us to design our study on anti-tuberculosis drug resistance pattern in cases of extra pulmonary tuberculosis in a tertiary referral hospital of North India. We performed Geno Type MTBDRplus assay in comparison with conventional drug susceptibility testing by proportion method to study the mutation patterns in rpoB, katG and inhA genes.A total of 510 extra pulmonary samples were included in this study. After the smear microscopy, all the specimens were subjected for culture on Lowenstein Jensen (LJ media. Phenotypic drug susceptibility testing (DST was performed on LJ media for all the MTB isolates and compared with the results of Geno Type MTBDRplus assay which was performed with the DNA isolated from the culture by conventional method.Of 510 specimens cultured, the total culture positivity obtained was 11.8% (60 encompassing 54 (10.6% Mycobacterium tuberculosis and 6 (1.2% non-tubercular mycobacteria (NTM. DST results by Geno Type MTBDRplus assay and solid culture methods were compared in 51 MTB isolates excluding the two Rif indeterminate and one invalid test. Geno Type MTBDRplus accurately identified 13 of 14 rifampicin-resistant strains, 14 of 15 isoniazid-resistant strains and 13 of 14 as multi drug resistant tuberculosis (MDR-TB in comparison with conventional method. Sensitivity and specificity were 92.86% and 97.30% respectively for detection of RIF resistance, 93.33% and 94.44% respectively for detection of INH resistance, 92.86% and 97.30% respectively for detection of MDR-TB, while the overall concordance of Geno Type MTBDRplus assay with conventional DST was 94.11%. The turn-around time for performing Geno Type MTBDRplus assay test was 48 hours.The problem of MDR in extra pulmonary tuberculosis (EPTB cannot be overlooked and due attention on patients

Klebsiella pneumoniae lipopolysaccharide O typing: revision of prototype strains and O-group distribution among clinical isolates from different sources and countries

DEFF Research Database (Denmark)

Hansen, D S; Mestre, F; Alberti, S

1999-01-01

of the currently recognized O types. The method was validated by studying the prototype strains that have defined the O groups by the classical tube agglutinatination O-typing method. Based on these results, we confirmed the O types of 60 of 64 typeable strains, and we propose a revised O-antigenic scheme...

Molecular typing of Brucella melitensis endemic strains and differentiation from the vaccine strain Rev-1.

Science.gov (United States)

Noutsios, Georgios T; Papi, Rigini M; Ekateriniadou, Loukia V; Minas, Anastasios; Kyriakidis, Dimitrios A

2012-03-01

In the present study forty-four Greek endemic strains of Br. melitensis and three reference strains were genotyped by Multi locus Variable Number Tandem Repeat (ML-VNTR) analysis based on an eight-base pair tandem repeat sequence that was revealed in eight loci of Br. melitensis genome. The forty-four strains were discriminated from the vaccine strain Rev-1 by Restriction Fragment Length Polymorphism (RFLP) and Denaturant Gradient Gel Electrophoresis (DGGE). The ML-VNTR analysis revealed that endemic, reference and vaccine strains are genetically closely related, while most of the loci tested (1, 2, 4, 5 and 7) are highly polymorphic with Hunter-Gaston Genetic Diversity Index (HGDI) values in the range of 0.939 to 0.775. Analysis of ML-VNTRs loci stability through in vitro passages proved that loci 1 and 5 are non stable. Therefore, vaccine strain can be discriminated from endemic strains by allele's clusters of loci 2, 4, 6 and 7. RFLP and DGGE were also employed to analyse omp2 gene and reveled different patterns among Rev-1 and endemic strains. In RFLP, Rev-1 revealed three fragments (282, 238 and 44 bp), while endemic strains two fragments (238 and 44 bp). As for DGGE, the electrophoretic mobility of Rev-1 is different from the endemic strains due to heterologous binding of DNA chains of omp2a and omp2b gene. Overall, our data show clearly that it is feasible to genotype endemic strains of Br. melitensis and differentiate them from vaccine strain Rev-1 with ML-VNTR, RFLP and DGGE techniques. These tools can be used for conventional investigations in brucellosis outbreaks.

A multiplex ligation detection assay for the characterization of Salmonella enterica strains

NARCIS (Netherlands)

Aarts, H.J.M.; Vos, P.; Larsson, J.T.; Hoek, van A.H.A.M.; Huehn, S.; Weijers, T.; Gronlund, H.A.; Malorny, B.

2011-01-01

A proof of principle of a multi-target assay for genotyping Salmonella has been developed targeting 62 genomic marker sequences of Salmonella related to pathogenicity. The assay is based on multiplex ligation detection reaction (LDR) followed by customized ArrayTube (R) microarray detection. The

A Biology Laboratory Exercise Using Macromolecule Assays to Distinguish Four Types of Milk

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Charlotte W. Pratt

2011-03-01

Full Text Available One of the drawbacks of cookbook-style laboratory exercises for General Biology courses is that students are not challenged to develop skills in scientific reasoning, such as formulating hypotheses and designing and carrying out experiments. Several traditional laboratory curricula include exercises involving semi-quantitative colorimetric assays to detect proteins (biuret test, reducing sugars (Benedict’s test, starch (Lugol’s test, and lipids (Sudan red test in a variety of easily prepared solutions (glucose, albumin, glycine, etc. and familiar food items (lemon juice, cornstarch, egg white, etc.. An extension of this lab exercise was developed to allow students to use their knowledge of the macromolecule assays to design an experiment to distinguish four types of “milk”: whole milk, skim milk, cream, and soy milk (rice milk or almond milk could also be included.

Using the overlay assay to qualitatively measure bacterial production of and sensitivity to pneumococcal bacteriocins.

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Maricic, Natalie; Dawid, Suzanne

2014-09-30

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

A Real-Time PCR with Melting Curve Analysis for Molecular Typing of Vibrio parahaemolyticus.

Science.gov (United States)

He, Peiyan; Wang, Henghui; Luo, Jianyong; Yan, Yong; Chen, Zhongwen

2018-05-23

Foodborne disease caused by Vibrio parahaemolyticus is a serious public health problem in many countries. Molecular typing has a great scientific significance and application value for epidemiological research of V. parahaemolyticus. In this study, a real-time PCR with melting curve analysis was established for molecular typing of V. parahaemolyticus. Eighteen large variably presented gene clusters (LVPCs) of V. parahaemolyticus which have different distributions in the genome of different strains were selected as targets. Primer pairs of 18 LVPCs were distributed into three tubes. To validate this newly developed assay, we tested 53 Vibrio parahaemolyticus strains, which were classified in 13 different types. Furthermore, cluster analysis using NTSYS PC 2.02 software could divide 53 V. parahaemolyticus strains into six clusters at a relative similarity coefficient of 0.85. This method is fast, simple, and conveniently for molecular typing of V. parahaemolyticus.

A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus

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Cui Shang-jin

2010-05-01

Full Text Available Abstract A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV. A pair of primers (P1 and P4 specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV, canine parvovirus (CPV, canine coronavirus (CCV, rabies virus (RV, or canine adenovirus (CAV. The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance.

A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus

Science.gov (United States)

2010-01-01

A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV). A pair of primers (P1 and P4) specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV), canine parvovirus (CPV), canine coronavirus (CCV), rabies virus (RV), or canine adenovirus (CAV). The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance. PMID:20433759

Accessible switching of electronic defect type in SrTi O3 via biaxial strain

Science.gov (United States)

Chi, Yen-Ting; Youssef, Mostafa; Sun, Lixin; Van Vliet, Krystyn J.; Yildiz, Bilge

2018-05-01

Elastic strain is used widely to alter the mobility of free electronic carriers in semiconductors, but a predictive relationship between elastic lattice strain and the extent of charge localization of electronic defects is still underdeveloped. Here we considered SrTi O3 , a prototypical perovskite as a model functional oxide for thin film electronic devices and nonvolatile memories. We assessed the effects of biaxial strain on the stability of electronic defects at finite temperature by combining density functional theory (DFT) and quasiharmonic approximation (QHA) calculations. We constructed a predominance diagram for free electrons and small electron polarons in this material, as a function of biaxial strain and temperature. We found that biaxial tensile strain in SrTi O3 can stabilize the small polaron, leading to a thermally activated and slower electronic transport, consistent with prior experimental observations on SrTi O3 and distinct from our prior theoretical assessment of the response of SrTi O3 to hydrostatic stress. These findings also resolved apparent conflicts between prior atomistic simulations and conductivity experiments for biaxially strained SrTi O3 thin films. Our computational approach can be extended to other functional oxides, and for the case of SrTi O3 our findings provide concrete guidance for conditions under which strain engineering can shift the electronic defect type and concentration to modulate electronic transport in thin films.

Multiplexed Microsphere Suspension-Array Assay for Urine Mitochondrial DNA Typing by C-Stretch Length in Hypervariable Regions.

Science.gov (United States)

Aoki, Kimiko; Tanaka, Hiroyuki; Kawahara, Takashi

2018-07-01

The standard method for personal identification and verification of urine samples in doping control is short tandem repeat (STR) analysis using nuclear DNA (nDNA). The DNA concentration of urine is very low and decreases under most conditions used for sample storage; therefore, the amount of DNA from cryopreserved urine samples may be insufficient for STR analysis. We aimed to establish a multiplexed assay for urine mitochondrial DNA typing containing only trace amounts of DNA, particularly for Japanese populations. A multiplexed suspension-array assay using oligo-tagged microspheres (Luminex MagPlex-TAG) was developed to measure C-stretch length in hypervariable region 1 (HV1) and 2 (HV2), five single nucleotide polymorphisms (SNPs), and one polymorphic indel. Based on these SNPs and the indel, the Japanese population can be classified into five major haplogroups (D4, B, M7a, A, D5). The assay was applied to DNA samples from urine cryopreserved for 1 - 1.5 years (n = 63) and fresh blood (n = 150). The assay with blood DNA enabled Japanese subjects to be categorized into 62 types, exhibiting a discriminatory power of 0.960. The detection limit for cryopreserved urine was 0.005 ng of nDNA. Profiling of blood and urine pairs revealed that 5 of 63 pairs showed different C-stretch patterns in HV1 or HV2. The assay described here yields valuable information in terms of the verification of urine sample sources employing only trace amounts of recovered DNA. However, blood cannot be used as a reference sample.

Genetic relationships between clinical and non-clinical strains of Yersinia enterocolitica biovar 1A as revealed by multilocus enzyme electrophoresis and multilocus restriction typing

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Virdi Jugsharan S

2010-05-01

Full Text Available Abstract Background Genetic relationships among 81 strains of Y. enterocolitica biovar 1A isolated from clinical and non-clinical sources were discerned by multilocus enzyme electrophoresis (MLEE and multilocus restriction typing (MLRT using six loci each. Such studies may reveal associations between the genotypes of the strains and their sources of isolation. Results All loci were polymorphic and generated 62 electrophoretic types (ETs and 12 restriction types (RTs. The mean genetic diversity (H of the strains by MLEE and MLRT was 0.566 and 0.441 respectively. MLEE (DI = 0.98 was more discriminatory and clustered Y. enterocolitica biovar 1A strains into four groups, while MLRT (DI = 0.77 identified two distinct groups. BURST (Based Upon Related Sequence Types analysis of the MLRT data suggested aquatic serotype O:6,30-6,31 isolates to be the ancestral strains from which, clinical O:6,30-6,31 strains might have originated by host adaptation and genetic change. Conclusion MLEE revealed greater genetic diversity among strains of Y. enterocolitica biovar 1A and clustered strains in four groups, while MLRT grouped the strains into two groups. BURST analysis of MLRT data nevertheless provided newer insights into the probable evolution of clinical strains from aquatic strains.

Electrochemiluminescence assays for insulin and glutamic acid decarboxylase autoantibodies improve prediction of type 1 diabetes risk.

Science.gov (United States)

Miao, Dongmei; Steck, Andrea K; Zhang, Li; Guyer, K Michelle; Jiang, Ling; Armstrong, Taylor; Muller, Sarah M; Krischer, Jeffrey; Rewers, Marian; Yu, Liping

2015-02-01

We recently developed new electrochemiluminescence (ECL) insulin autoantibody (IAA) and glutamic acid decarboxylase 65 autoantibody (GADA) assays that discriminate high-affinity, high-risk diabetes-specific autoantibodies from low-affinity, low-risk islet autoantibodies (iAbs) detected by radioassay (RAD). Here, we report a further validation of the ECL-IAA and -GADA assays in 3,484 TrialNet study participants. The ECL assay and RAD were congruent in those with prediabetes and in subjects with multiple autoantibodies, but only 24% (P<0.0001) of single RAD-IAA-positive and 46% (P<0.0001) of single RAD-GADA-positive were confirmed by the ECL-IAA and -GADA assays, respectively. During a follow-up (mean, 2.4 years), 51% of RAD-IAA-positive and 63% of RAD-GADA-positive subjects not confirmed by ECL became iAb negative, compared with only 17% of RAD-IAA-positive (P<0.0001) and 15% of RAD-GADA-positive (P<0.0001) subjects confirmed by ECL assays. Among subjects with multiple iAbs, diabetes-free survival was significantly shorter if IAA or GADA was positive by ECL and negative by RAD than if IAA or GADA was negative by ECL and positive by RAD (P<0.019 and P<0.0001, respectively). Both positive and negative predictive values in terms of progression to type 1 diabetes mellitus were superior for ECL-IAA and ECL-GADA, compared with RADs. The prevalence of the high-risk human leukocyte antigen-DR3/4, DQB1*0302 genotype was significantly higher in subjects with RAD-IAA or RAD-GADA confirmed by ECL. In conclusion, both ECL-IAA and -GADA are more disease-specific and better able to predict the risk of progression to type 1 diabetes mellitus than the current standard RADs.

Comparison of Ribotyping, Randomly Amplified Polymorphic DNA Analysis, and Pulsed-Field Gel Electrophoresis in Typing of Lactobacillus rhamnosus and L. casei Strains

OpenAIRE

Tynkkynen, Soile; Satokari, Reetta; Saarela, Maria; Mattila-Sandholm, Tiina; Saxelin, Maija

1999-01-01

A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 33...

A multiplex ligation detection assay for the characterization of Salmonella enterica strains

DEFF Research Database (Denmark)

Aarts, Henk J.M.; Vos, Pieter; Larsson, Jonas T.

2011-01-01

A proof of principle of a multi-target assay for genotyping Salmonella has been developed targeting 62 genomic marker sequences of Salmonella related to pathogenicity. The assay is based on multiplex ligation detection reaction (LDR) followed by customized ArrayTube® microarray detection. The fea...... assessors that support bio-traceability models....

Complete genome sequence of Desulfomicrobium baculatum type strain (XT)

Energy Technology Data Exchange (ETDEWEB)

Copeland, Alex; Spring, Stefan; Goker, Markus; Schneider, Susanne; Lapidus, Alla; Glavina Del Rio, Tijana; Tice, Hope; Cheng, Jan-Fang; Lucas, Susan; Chen, Feng; Nolan, Matt; Bruce, David; Goodwin, Lynne; Pitluck, Sam; Ivanova, Natalia; Mavrommatis, Konstantinos; Ovchinnikova, Galina; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam; Hauser, Loren; Chang, Yun-Juan; Jefferies, Cynthia C; Meincke, Linda; Sims, David; Brettin, Thomas; Detter, John C; Han, Cliff; Chain, Patrick; Bristow, James; Eisen, Jonathan; Markowitz, Victor; Hugenholtz, Philip; Klenk, Hans-Peter; Kyrpides, Nikos C; Lucas, Susan

2009-05-20

Desulfomicrobium baculatum is the type species of the genus Desulfomicrobium, which is the type genus of the family Desulfomicrobiaceae. It is of phylogenetic interest because of the isolated location of the family Desulfomicrobiaceae within the order Desulfovibrionales. D. baculatum strain XT is a Gram-negative, motile, sulfate-reducing bacterium isolated from water-saturated manganese carbonate ore. It is strictly anaerobic and does not require NaCl for growth, although NaCl concentrations up to 6percent (w/v) are tolerated. The metabolism is respiratory or fermentative. In the presence of sulfate, pyruvate and lactate are incompletely oxidized to acetate and CO2. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of a member of the deltaproteobacterial family Desulfomicrobiaceae, and this 3,942,657 bp long single replicon genome with its 3494 protein-coding and 72 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

Enzyme-linked immunosorbent serum assay specific for the 7S domain of Collagen Type IV (P4NP 7S)

DEFF Research Database (Denmark)

Leeming, Diana J; Nielsen, Mette J; Dai, Yueqin

2012-01-01

Aim:  The present study describes the ability of a newly developed N-terminal pro-peptides of type IV collagen 7S domain (P4NP 7S) competitive enzyme-linked immunosorbent assay (ELISA) for describing liver fibrosis. The assay applies a monoclonal antibody specific for a PIVNP 7S epitope 100...... were significantly elevated in rat with liver fibrosis as seen by histology (CCL4: 283% elevated in the highest quartile of total hepatic collagen compared with controls, P = 0.001; BDL: 183% elevated at week 4 compared with sham, P type IV collagen...... expression in BDL rats (r = 0.49, P serum assay specific for P4NP 7S was highly related to liver fibrosis...

Presence and Functionality of Mating Type Genes in the Supposedly Asexual Filamentous Fungus Aspergillus oryzae

Science.gov (United States)

Wada, Ryuta; Maruyama, Jun-ichi; Yamaguchi, Haruka; Yamamoto, Nanase; Wagu, Yutaka; Paoletti, Mathieu; Archer, David B.; Dyer, Paul S.

2012-01-01

The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the reference strain RIB40. We now report the existence of a complementary MAT1-2 gene and the sequencing of an idiomorphic region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of the MAT1-1 and MAT1-2 genotypes among 180 strains assayed, including industrial tane-koji isolates. Strains used for sake and miso production showed a near-1:1 ratio of the MAT1-1 and MAT1-2 mating types, whereas strains used for soy sauce production showed a significant bias toward the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and gene expression was compared by DNA microarray and quantitative real-time PCR (qRT-PCR) methodologies under conditions in which MAT genes were expressed. Thirty-three genes were found to be upregulated more than 10-fold in either the MAT1-1 host strain or the MAT1-2 gene replacement strain relative to each other, showing that both the MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating type-dependent manner, the first such report for a supposedly asexual fungus. MAT1-1 expression specifically upregulated an α-pheromone precursor gene, but the functions of most of the genes affected were unknown. The results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed. PMID:22327593

Radiometric macrophage culture assay for rapid evaluation of antileprosy activity of rifampin

Energy Technology Data Exchange (ETDEWEB)

Mittal, A.; Seshadri, P.S.; Prasad, H.K.; Sathish, M.; Nath, I.

1983-10-01

The antileprosy effect of rifampin was evaluated by a newly developed rapid in vitro assay wherein 31 human-derived strains and 1 armadillo-derived strain of Mycobacterium leprae were maintained for 2 and 3 weeks, respectively, in murine and human macrophages in the presence of (3H)thymidine. Of these strains, 27 showed significant incorporation of the radiolabel in cultures of live bacilli as compared with control cultures of heat-killed bacilli of the same strain. Consistent and significant inhibition of (3H)thymidine uptake was observed in M. leprae resident cultures with 3 to 200 ng of rifampin per ml as compared with similar cultures without the drug. In general, an increase in percent inhibition was seen from 3 to 20 ng/ml, with marginal increases at 40, 50, and 100 ng/ml. M. leprae strains appear to be remarkably susceptible to this drug in the in vitro assay.

Radiometric macrophage culture assay for rapid evaluation of antileprosy activity of rifampin

International Nuclear Information System (INIS)

Mittal, A.; Seshadri, P.S.; Prasad, H.K.; Sathish, M.; Nath, I.

1983-01-01

The antileprosy effect of rifampin was evaluated by a newly developed rapid in vitro assay wherein 31 human-derived strains and 1 armadillo-derived strain of Mycobacterium leprae were maintained for 2 and 3 weeks, respectively, in murine and human macrophages in the presence of [3H]thymidine. Of these strains, 27 showed significant incorporation of the radiolabel in cultures of live bacilli as compared with control cultures of heat-killed bacilli of the same strain. Consistent and significant inhibition of [3H]thymidine uptake was observed in M. leprae resident cultures with 3 to 200 ng of rifampin per ml as compared with similar cultures without the drug. In general, an increase in percent inhibition was seen from 3 to 20 ng/ml, with marginal increases at 40, 50, and 100 ng/ml. M. leprae strains appear to be remarkably susceptible to this drug in the in vitro assay

Extended biofilm susceptibility assay for Staphylococcus aureus bovine mastitis isolates: evidence for association between genetic makeup and biofilm susceptibility.

Science.gov (United States)

Melchior, M B; van Osch, M H J; Lam, T J G M; Vernooij, J C M; Gaastra, W; Fink-Gremmels, J

2011-12-01

Staphylococcus aureus is one of the most prevalent causes of bovine mastitis. The antimicrobial treatment of this disease is currently based on antimicrobial susceptibility tests according to Clinical and Laboratory Standards Institute standards. However, various authors have shown a discrepancy between the results of this standard susceptibility test and the actual cure rate of the applied antimicrobial treatment. Increasing evidence suggests that in vivo biofilm formation by Staph. aureus, which is not assessed in the antimicrobial susceptibility tests, is associated with this problem, resulting in disappointing cure rates, especially for infections of longer duration. Previous data obtained with a limited number of strains showed that the extended biofilm antimicrobial susceptibility (EBS) assay reveals differences between strains, which cannot be derived from a standard susceptibility test or from a 24-h biofilm susceptibility test. The objective of this study was to test a collection of Staph. aureus bovine mastitis strains in the EBS assay and to model the effect of antimicrobial exposure, duration of antimicrobial exposure, and genotype profile of the strains on antimicrobial susceptibility. With the results from a previous study with the same collection of strains, the effect of genotype represented by accessory gene regulator gene (agr-type), the presence of insertional sequence 257 (IS257), intercellular adhesion (ica), and the β-lactamase (blaZ) gene were entered as explanatory factors in a logistic regression model. The agr locus of Staph. aureus controls the expression of most of the virulence factors, represses the transcription of several cell wall-associated proteins, and activates several exoproteins during the post-exponential phase. The IS257 gene has been related to biofilm formation in vitro and was found earlier in 50% of the agr-type 2 strains. The ica gene cluster encodes for the production of an extracellular polysaccharide adhesin, termed

Multi-Locus Next-Generation Sequence Typing of DNA Extracted From Pooled Colonies Detects Multiple Unrelated Candida albicans Strains in a Significant Proportion of Patient Samples

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Ningxin Zhang

2018-06-01

Full Text Available The yeast Candida albicans is an important opportunistic human pathogen. For C. albicans strain typing or drug susceptibility testing, a single colony recovered from a patient sample is normally used. This is insufficient when multiple strains are present at the site sampled. How often this is the case is unclear. Previous studies, confined to oral, vaginal and vulvar samples, have yielded conflicting results and have assessed too small a number of colonies per sample to reliably detect the presence of multiple strains. We developed a next-generation sequencing (NGS modification of the highly discriminatory C. albicans MLST (multilocus sequence typing method, 100+1 NGS-MLST, for detection and typing of multiple strains in clinical samples. In 100+1 NGS-MLST, DNA is extracted from a pool of colonies from a patient sample and also from one of the colonies. MLST amplicons from both DNA preparations are analyzed by high-throughput sequencing. Using base call frequencies, our bespoke DALMATIONS software determines the MLST type of the single colony. If base call frequency differences between pool and single colony indicate the presence of an additional strain, the differences are used to computationally infer the second MLST type without the need for MLST of additional individual colonies. In mixes of previously typed pairs of strains, 100+1 NGS-MLST reliably detected a second strain. Inferred MLST types of second strains were always more similar to their real MLST types than to those of any of 59 other isolates (22 of 31 inferred types were identical to the real type. Using 100+1 NGS-MLST we found that 7/60 human samples, including three superficial candidiasis samples, contained two unrelated strains. In addition, at least one sample contained two highly similar variants of the same strain. The probability of samples containing unrelated strains appears to differ considerably between body sites. Our findings indicate the need for wider surveys to

An improved Armstrong-Frederick-Type Plasticity Model for Stable Cyclic Stress-Strain Responses Considering Nonproportional Hardening

Science.gov (United States)

Li, Jing; Zhang, Zhong-ping; Li, Chun-wang

2018-03-01

This paper modified an Armstrong-Frederick-type plasticity model for investigating the stable cyclic deformation behavior of metallic materials with different sensitivity to nonproportional loadings. In the modified model, the nonproportionality factor and nonproportional cyclic hardening coefficient coupled with the Jiang-Sehitoglu incremental plasticity model were used to estimate the stable stress-strain responses of the two materials (1045HR steel and 304 stainless steel) under various tension-torsion strain paths. A new equation was proposed to calculate the nonproportionality factor on the basis of the minimum normal strain range. Procedures to determine the minimum normal strain range were presented for general multiaxial loadings. Then, the modified model requires only the cyclic strain hardening exponent and cyclic strength coefficient to determine the material constants. It is convenient for predicting the stable stress-strain responses of materials in engineering application. Comparisons showed that the modified model can reflect the effect of nonproportional cyclic hardening well.

Multilocus sequence typing and virulence analysis of Haemophilus parasuis strains isolated in five provinces of China.

Science.gov (United States)

Wang, Liyan; Ma, Lina; Liu, Yongan; Gao, Pengcheng; Li, Youquan; Li, Xuerui; Liu, Yongsheng

2016-10-01

Haemophilus parasuis is the etiological agent of Glässers disease, which causes high morbidity and mortality in swine herds. Although H. parasuis strains can be classified into 15 serovars with the Kielstein-Rapp-Gabrielson serotyping scheme, a large number of isolates cannot be classified and have been designated 'nontypeable' strains. In this study, multilocus sequence typing (MLST) of H. parasuis was used to analyze 48 H. parasuis field strains isolated in China and two strains from Australia. Twenty-six new alleles and 29 new sequence types (STs) were detected, enriching the H. parasuis MLST databases. A BURST analysis indicated that H. parasuis lacks stable population structure and is highly heterogeneous, and that there is no association between STs and geographic area. When an UPGMA dendrogram was constructed, two major clades, clade A and clade B, were defined. Animal experiments, in which guinea pigs were challenged intraperitoneally with the bacterial isolates, supported the hypothesis that the H. parasuis STs in clade A are generally avirulent or weakly virulent, whereas the STs in clade B tend to be virulent. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of Clostridium difficile Strains in British Columbia, Canada: A Shift from NAP1 Majority (2008 to Novel Strain Types (2013 in One Region

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Agatha N. Jassem

2016-01-01

Full Text Available Background. Clostridium difficile is a major cause of gastrointestinal illness. Epidemic NAP1 strains contain toxins A and B, a deletion in repressor tcdC, and a binary toxin. Objectives. To determine the molecular epidemiology of C. difficile in British Columbia and compare between two time points in one region. Methods. C. difficile isolates from hospital and community laboratories (2008 and one Island Health hospital laboratory (2013 were characterized by pulsed-field gel electrophoresis, PCR-ribotyping, toxin possession, tcdC genotype, and antimicrobial susceptibility. Results. In 2008, 42.7% of isolates had NAP1 designation. Hospital-collected isolates were associated with older patients and more NAP1 types. Unlike other isolates, most NAP1 isolates possessed binary toxin and a 19 bp loss in tcdC. All isolates were susceptible to metronidazole and vancomycin. A 2013 follow-up revealed a 28.9% decrease in NAP1 isolates and 20.0% increase in isolates without NAP designation in one region. Then, community-associated cases were seen in younger patients, while NAP types were evenly distributed. Isolates without NAP designation did not cluster with a PFGE pattern or ribotype. Conclusions. Evaluation of C. difficile infections within British Columbia revealed demographic associations, epidemiological shifts, and characteristics of strain types. Continuous surveillance of C. difficile will enable detection of emerging strains.

Comparison of the proteomes of three yeast wild type strains: CEN.PK2, FY1679 and W303

DEFF Research Database (Denmark)

Rogowska-Wrzesinska, A.; Mose Larsen, P.; Blomberg, A.

2001-01-01

Yeast deletion strains created during gene function analysis projects very often show drastic phenotypic differences depending on the genetic background used. These results indicate the existence of important molecular differences between the CEN.PK2, FY1679 and W303 wild type strains...

lambda. -prophage induction in repair-deficient and wild type E. coli strains by. gamma. -rays and heavy ions

Energy Technology Data Exchange (ETDEWEB)

Bonev, M.N.; Kozubek, S.; Krasavin, E.A.; Amirtajev, K.G. (Joint Inst. for Nuclear Research, Dubna (USSR))

1990-05-01

{lambda}-prophage induction in repair-deficient and wild-type E. coli strains by heavy ions and {gamma}-rays was investigated. The dose dependence of the fraction of induced cells has been measured and its initial slope ({lambda}-induction potency) determined. Induction by {gamma}-rays was found to be more efficient in a polA-repair-deficient strain; the value of {lambda}-induction potency is zero in lexA{sup -} and recA{sup -} strains. The {lambda}-induction potency potency increased with LET for wild-type cells but remained constant in polA{sup -} mutant cells. It is suggested that DNA damage triggering the {lambda}-prophage induction in the case of ionizing radiation could be a type of DNA single-strand break with complex structures which cannot be repaired by fast repair processes, and requires a substantial level of energy deposition for induction in a DNA molecule. (author).

Subtyping of Chilean Methicillin-Resistant Staphylococcus aureus strains carrying the staphylococcal cassette chromosome mec type I

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Gustavo Medina

2012-09-01

Full Text Available The cassette chromosome mec (SCCmec present in methicillin-resistant Staphylococcus aureus (MRSA has two essential components, the ccr gene complex and the mec gene complex. Additionally, SCCmec has non-essential components called J regions which are used for MRSA subtyping. This study was performed to determine subtypes MRSA strains carrying SCCmec type I based on polymorphism of regions located downstream of the mecA gene. A total of 98 MRSA strains carrying SCCmec type I isolated from patients hospitalized at the County Hospital of Valdivia (Chile between May 2007 and May 2008, were analyzed by multiplex PCR designed to amplify the mecA gene and 7 DNA hypervariable regions located around the mecA gene. MRSA strains were classified into seventeen genotypes accordingly to amplification patterns of DNA hypervariable regions. Five genotypes showed amplification patterns previously described. The remaining twelve genotypes showed new amplification patterns. Genotypes 18 and Genotype 19 were the most frequently detected. Regions HVR, Ins117 and pI258 stand out as being present in more than 60% of tested isolates. The acquisition of hypervariable regions by MRSA is a continuous horizontal transfer process through which the SCCmec have been preserved intact, or even may give rise to new types and subtypes of SCCmec. Therefore it is possible to infer that most MRSA strains isolated at the County Hospital of Valdivia (Chile were originated from two local clones which correspond to Genotype 18 and Genotype 19.

Analytical and clinical performance of the CDC real time RT-PCR assay for detection and typing of dengue virus.

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Gilberto A Santiago

Full Text Available Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV. There are four genetically distinct DENVs (DENV-1-4 that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1-4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1-4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1-4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases.

Identification of a novel prophage-like gene cluster actively expressed in both virulent and avirulent strains of Leptospira interrogans serovar Lai.

Science.gov (United States)

Qin, Jin-Hong; Zhang, Qing; Zhang, Zhi-Ming; Zhong, Yi; Yang, Yang; Hu, Bao-Yu; Zhao, Guo-Ping; Guo, Xiao-Kui

2008-06-01

DNA microarray analysis was used to compare the differential gene expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22-kb genomic island covering a cluster of 34 genes (i.e., genes LA0186 to LA0219) was actively expressed in both strains but concomitantly upregulated in strain 56601 in contrast to that of IPAV. Reverse transcription-PCR assays proved that the gene cluster comprised five transcripts. Gene annotation of this cluster revealed characteristics of a putative prophage-like remnant with at least 8 of 34 sequences encoding prophage-like proteins, of which the LA0195 protein is probably a putative prophage CI-like regulator. The transcription initiation activities of putative promoter-regulatory sequences of transcripts I, II, and III, all proximal to the LA0195 gene, were further analyzed in the Escherichia coli promoter probe vector pKK232-8 by assaying the reporter chloramphenicol acetyltransferase (CAT) activities. The strong promoter activities of both transcripts I and II indicated by the E. coli CAT assay were well correlated with the in vitro sequence-specific binding of the recombinant LA0195 protein to the corresponding promoter probes detected by the electrophoresis mobility shift assay. On the other hand, the promoter activity of transcript III was very low in E. coli and failed to show active binding to the LA0195 protein in vitro. These results suggested that the LA0195 protein is likely involved in the transcription of transcripts I and II. However, the identical complete DNA sequences of this prophage remnant from these two strains strongly suggests that possible regulatory factors or signal transduction systems residing outside of this region within the genome may be responsible for the differential expression profiling in these two strains.

A high-throughput cellular assay to quantify the p53-degradation activity of E6 from different human papillomavirus types.

Science.gov (United States)

Gagnon, David; Archambault, Jacques

2015-01-01

A subset of human papillomaviruses (HPVs), known as the high-risk types, are the causative agents of cervical cancer and other malignancies of the anogenital region and oral mucosa. The capacity of these viruses to induce cancer and to immortalize cells in culture relies in part on a critical function of their E6 oncoprotein, that of promoting the poly-ubiquitination of the cellular tumor suppressor protein p53 and its subsequent degradation by the proteasome. Here, we describe a cellular assay to measure the p53-degradation activity of E6 from different HPV types. This assay is based on a translational fusion of p53 to Renilla luciferase (Rluc-p53) that remains sensitive to degradation by high-risk E6 and whose steady-state levels can be accurately measured in standard luciferase assays. The p53-degradation activity of any E6 protein can be tested and quantified in transiently transfected cells by determining the amount of E6-expression vector required to reduce by half the levels of RLuc-p53 luciferase activity (50 % effective concentration [EC50]). The high-throughput and quantitative nature of this assay makes it particularly useful to compare the p53-degradation activities of E6 from several HPV types in parallel.

Genetic Relationships among Reptilian and Mammalian Campylobacter fetus Strains Determined by Multilocus Sequence Typing

NARCIS (Netherlands)

Dingle, K.E.; Blaser, M.J.; Tu, Z.C.; Pruckler, J.; Fitzgerald, C.; Bergen, van M.A.P.; Lawson, A.J.; Owen, R.J.; Wagenaar, J.A.

2010-01-01

Reptile Campylobacter fetus isolates and closely related strains causing human disease were characterized by multilocus sequence typing. They shared similar to 90% nucleotide sequence identity with classical mammalian C. fetus, and there was evidence of recombination among members of these two

Radiation-sensitive mutant of hypertoxinogenic strain 569B of Vibro cholerae

International Nuclear Information System (INIS)

Das, G.; Das, J.

1983-01-01

A radiation-sensitive mutant of the hypertoxinogenic strain 569B of Vibrio cholerae was isolated and characterized. The mutant, designated V. cholerae 569Bsub(s), lacks both excision- and medium-dependent dark-repair mechanisms of UV-induced DNA damage while retaining the wild-type photoreactivating capability. Analysis of the UV-irradiated cell DNA by velocity sedimentation in alkaline sucrose gradient suggests that UV-induced pyrimidine dimers may not be incised in these cells. In contrast to the wild-type cells, the mutant cell DNA was degraded after treatment with nalidixic acid. The mutant cells failed to produce any detectable amount of cholera toxin as measured by ileal-loop assay. (orig.)

Multilocus Sequence Typing Reveals Relevant Genetic Variation and Different Evolutionary Dynamics among Strains of Xanthomonas arboricola pv. juglandis

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Marco Scortichini

2010-11-01

Full Text Available Forty-five Xanthomonas arboricola pv. juglandis (Xaj strains originating from Juglans regia cultivation in different countries were molecularly typed by means of MultiLocus Sequence Typing (MLST, using acnB, gapA, gyrB and rpoD gene fragments. A total of 2.5 kilobases was used to infer the phylogenetic relationship among the strains and possible recombination events. Haplotype diversity, linkage disequilibrium analysis, selection tests, gene flow estimates and codon adaptation index were also assessed. The dendrograms built by maximum likelihood with concatenated nucleotide and amino acid sequences revealed two major and two minor phylotypes. The same haplotype was found in strains originating from different continents, and different haplotypes were found in strains isolated in the same year from the same location. A recombination breakpoint was detected within the rpoD gene fragment. At the pathovar level, the Xaj populations studied here are clonal and under neutral selection. However, four Xaj strains isolated from walnut fruits with apical necrosis are under diversifying selection, suggesting a possible new adaptation. Gene flow estimates do not support the hypothesis of geographic isolation of the strains, even though the genetic diversity between the strains increases as the geographic distance between them increases. A triplet deletion, causing the absence of valine, was found in the rpoD fragment of all 45 Xaj strains when compared with X. axonopodis pv. citri strain 306. The codon adaptation index was high in all four genes studied, indicating a relevant metabolic activity.

Rapid MALDI-TOF Mass Spectrometry Strain Typing during a Large Outbreak of Shiga-Toxigenic Escherichia coli

Science.gov (United States)

Christner, Martin; Trusch, Maria; Rohde, Holger; Kwiatkowski, Marcel; Schlüter, Hartmut; Wolters, Manuel; Aepfelbacher, Martin; Hentschke, Moritz

2014-01-01

Background In 2011 northern Germany experienced a large outbreak of Shiga-Toxigenic Escherichia coli O104:H4. The large amount of samples sent to microbiology laboratories for epidemiological assessment highlighted the importance of fast and inexpensive typing procedures. We have therefore evaluated the applicability of a MALDI-TOF mass spectrometry based strategy for outbreak strain identification. Methods Specific peaks in the outbreak strain’s spectrum were identified by comparative analysis of archived pre-outbreak spectra that had been acquired for routine species-level identification. Proteins underlying these discriminatory peaks were identified by liquid chromatography tandem mass spectrometry and validated against publicly available databases. The resulting typing scheme was evaluated against PCR genotyping with 294 E. coli isolates from clinical samples collected during the outbreak. Results Comparative spectrum analysis revealed two characteristic peaks at m/z 6711 and m/z 10883. The underlying proteins were found to be of low prevalence among genome sequenced E. coli strains. Marker peak detection correctly classified 292 of 293 study isolates, including all 104 outbreak isolates. Conclusions MALDI-TOF mass spectrometry allowed for reliable outbreak strain identification during a large outbreak of Shiga-Toxigenic E. coli. The applied typing strategy could probably be adapted to other typing tasks and might facilitate epidemiological surveys as part of the routine pathogen identification workflow. PMID:25003758

Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum

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Al-Khedery Basima

2012-11-01

Full Text Available Abstract Background Anaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain. Results Although in all strains the T4SS conforms to the previously described split loci for vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S. Conclusions Our study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in

High-Resolution Amplified Fragment Length Polymorphism Typing of Lactococcus lactis Strains Enables Identification of Genetic Markers for Subspecies-Related Phenotypes▿

Science.gov (United States)

Kütahya, Oylum Erkus; Starrenburg, Marjo J. C.; Rademaker, Jan L. W.; Klaassen, Corné H. W.; van Hylckama Vlieg, Johan E. T.; Smid, Eddy J.; Kleerebezem, Michiel

2011-01-01

A high-resolution amplified fragment length polymorphism (AFLP) methodology was developed to achieve the delineation of closely related Lactococcus lactis strains. The differentiation depth of 24 enzyme-primer-nucleotide combinations was experimentally evaluated to maximize the number of polymorphisms. The resolution depth was confirmed by performing diversity analysis on 82 L. lactis strains, including both closely and distantly related strains with dairy and nondairy origins. Strains clustered into two main genomic lineages of L. lactis subsp. lactis and L. lactis subsp. cremoris type-strain-like genotypes and a third novel genomic lineage rooted from the L. lactis subsp. lactis genomic lineage. Cluster differentiation was highly correlated with small-subunit rRNA homology and multilocus sequence analysis (MLSA) studies. Additionally, the selected enzyme-primer combination generated L. lactis subsp. cremoris phenotype-specific fragments irrespective of the genotype. These phenotype-specific markers allowed the differentiation of L. lactis subsp. lactis phenotype from L. lactis subsp. cremoris phenotype strains within the same L. lactis subsp. cremoris type-strain-like genomic lineage, illustrating the potential of AFLP for the generation of phenotype-linked genetic markers. PMID:21666014

Preclinical detection of porcine circovirus type 2 infection using an ultrasensitive nanoparticle DNA probe-based PCR assay.

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Yong Huang

Full Text Available Porcine circovirus type 2 (PCV2 has emerged as one of the most important pathogens affecting swine production globally. Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR for PCV2 detection. Magnetic microparticles coated with PCV2 specific DNA probes were used to enrich PCV2 DNA from samples, then gold nanoparticles coated with PCV2 specific oligonucleotides were added to form a sandwich nucleic acid-complex. After the complex was formed, the oligonucleotides were released and characterized by PCR. This assay exhibited about 500-fold more sensitive than conventional PCR, with a detection limit of 2 copies of purified PCV2 genomic DNA and 10 viral copies of PCV2 in serum. The assay has a wide detection range for all of PCV2 genotypes with reliable reproducibility. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1, porcine parvovirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus and classical swine fever virus. The positive detection rate of PCV2 specific UNDP-PCR in 40 preclinical field samples was 27.5%, which appeared greater than that by conventional and real-time PCR and appeared application potency in evaluation of the viral loads levels of preclinical infection samples. The UNDP-PCR assay reported here can reliably rule out false negative results from antibody-based assays, provide a nucleic acid extraction free, specific, ultrasensitive, economic and rapid diagnosis method for preclinical PCV2 infection in field, which may help prevent large-scale outbreaks.

Rep-PCR typing of Staphylococcus spp. strains in meat paste production line and identification of their origin

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Ivan Manga

2015-05-01

Full Text Available A meat paste production line and its microbial parameters have been evaluated in single Czech company. The raw meat paste samples before heat treatment were tested positively for the presence of three staphylococci species: Staphylococcus aureus, Staphylococcus haemolyticus and Staphylococcus epidermidis. Subsequent microbial analysis of meat paste components and ingredients (fresh meat, water, spices, equipment identified only the spices used as positive for S. aureus (coriander, cinnamon, badian, mustard – (10 - 40 cfu/g and S. haemolyticus strains (juniper, ginger. The collection of sixteen collected strains (S. aureus (n = 4, S. haemolyticus (n = 4, S. epidermidis (n = 8 has been typed with the rep-PCR method utilising (GTG5 primer. Analysis of the fingerprints using the unweighted pair-group method using arithmetic averages (UPGMA clustering method revealed presence of eleven strain clusters with similarity lower than 90%: two fingerprint clusters of S. aureus, three individual clusters characteristic for S. haemolyticus and six different S. epidermidis specific clusters. The S. aureus strains from different types of spice were identical, resp. very similar. Molecular tracking composed from the rep-PCR analysis of acquired isolates and comparison among all collected fingerprints confirmed the spices to be the source of both S. aureus and S. haemolyticus strains identified in raw meat paste. The additional rep-PCR analysis of the S. epidermidis collection confirmed usability and performance of this method. The antibiotic susceptibility to fourteen individual antibiotics has been examined among the collected staphylococci strains. The predominant erythromycin resistance (68.8% was followed with the resistance to amoxicillin/clavulanic acid (56.2%. Other resistances observed were less frequent (clindamycin – 12.5%, oxacillin – 6.3%, tetracycline – 6.3%, sulphamethoxazole-trimethoprim – 6.3%, chloramphenicol – 6.3%, novobiocin – 6

Detection and Typing of Human Papilloma Viruses by Nested Multiplex Polymerase Chain Reaction Assay in Cervical Cancer

Science.gov (United States)

Jalal Kiani, Seyed; Shatizadeh Malekshahi, Somayeh; Yousefi Ghalejoogh, Zohreh; Ghavvami, Nastaran; Shafiei Jandaghi, Nazanin Zahra; Shahsiah, Reza; Jahanzad, Isa; Yavarian, Jila

2015-01-01

Background: Cervical cancer is the leading cause of death from cancer in under-developed countries. Human papilloma virus (HPV) 16 and 18 are the most prevalent types associated with carcinogenesis in the cervix. Conventional Polymerase Chain Reaction (PCR), type-specific and consensus primer-based PCR followed by sequencing, Restriction Fragment Length Polymorphism (RFLP) or hybridization by specific probes are common methods for HPV detection and typing. In addition, some researchers have developed a multiplex PCR for simultaneous detection and typing of different HPVs. Objectives: The aim of the present study was to investigate the prevalence of HPV infection and its types in cervical Squamous Cell Carcinoma (SCC) using the Nested Multiplex PCR (NMPCR) assay. Patients and Methods: Sixty-six samples with histologically confirmed SCC were evaluated. Total DNA was isolated by phenol–chloroform extraction and ethanol precipitation. Nested multiplex PCR was performed with first-round PCR by GP-E6/E7 consensus primers for amplification of the genomic DNA of all known mucosal HPV genotypes and second-round PCR by type-specific multiplex PCR primer cocktails. Results: Human papilloma virus infection was detected in 78.8% of samples, with the highest prevalence of HPV 16 (60.6%) while concurrent infections with two types was detected in 10.6%. Conclusions: The NMPCR assay is more convenient and easy for analysis of results, which is important for fast diagnosis and patient management, in a type-specific manner. PMID:26865940

75 FR 59611 - Microbiology Devices; Reclassification of Herpes Simplex Virus Types 1 and 2 Serological Assays...

Science.gov (United States)

2010-09-28

... DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration 21 CFR Part 866 [Docket No. FDA-2009-N-0344] Microbiology Devices; Reclassification of Herpes Simplex Virus Types 1 and 2 Serological Assays; Confirmation of Effective Date AGENCY: Food and Drug Administration, HHS. ACTION: Direct...

Translation efficiency determines differences in cellular infection among dengue virus type 2 strains

International Nuclear Information System (INIS)

Edgil, Dianna; Diamond, Michael S.; Holden, Katherine L.; Paranjape, Suman M.; Harris, Eva

2003-01-01

We have investigated the molecular basis for differences in the ability of natural variants of dengue virus type 2 (DEN2) to replicate in primary human cells. The rates of virus binding, virus entry, input strand translation, and RNA stability of low-passage Thai and Nicaraguan and prototype DEN2 strains were compared. All strains exhibited equivalent binding, entry, and uncoating, and displayed comparable stability of positive strand viral RNA over time in primary cells. However, the low-passage Nicaraguan isolates were much less efficient in their ability to translate viral proteins. Sequence analysis of the full-length low-passage Nicaraguan and Thai viral genomes identified specific differences in the 3' untranslated region (3'UTR). Substitution of the different sequences into chimeric RNA reporter constructs demonstrated that the changes in the 3'UTR directly affected the efficiency of viral translation. Thus, differences in infectivity among closely related DEN2 strains correlate with efficiency of translation of input viral RNA

Feline coronavirus type II strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type I and canine coronavirus

NARCIS (Netherlands)

Horzinek, M.C.; Herrewegh, A.A.; Rottier, P.J.M.; Groot, R.J. de

1998-01-01

Recent evidence suggests that the type II feline coronavirus (FCoV) strains 79-1146 and 79-1683 have arisen from a homologous RNA recombination event between FCoV type I and canine coronavirus (CCV). In both cases, the template switch apparently took place between the S and M genes, giving rise to

Effects that different types of sports have on the hearts of children and adolescents and the value of two-dimensional strain-strain-rate echocardiography.

Science.gov (United States)

Binnetoğlu, Fatih Köksal; Babaoğlu, Kadir; Altun, Gürkan; Kayabey, Özlem

2014-01-01

Whether the hypertrophy found in the hearts of athletes is physiologic or a risk factor for the progression of pathologic hypertrophy remains controversial. The diastolic and systolic functions of athletes with left ventricular (LV) hypertrophy usually are normal when measured by conventional methods. More precise assessment of global and regional myocardial function may be possible using a newly developed two-dimensional (2D) strain echocardiographic method. This study evaluated the effects that different types of sports have on the hearts of children and adolescents and compared the results of 2D strain and strain-rate echocardiographic techniques with conventional methods. Athletes from clubs for five different sports (basketball, swimming, football, wrestling, and tennis) who had practiced regularly at least 3 h per week during at least the previous 2 years were included in the study. The control group consisted of sedentary children and adolescents with no known cardiac or systemic diseases (n = 25). The athletes were grouped according to the type of exercise: dynamic (football, tennis), static (wrestling), or static and dynamic (basketball, swimming). Shortening fraction and ejection fraction values were within normal limits for the athletes in all the sports disciplines. Across all 140 athletes, LV geometry was normal in 58 athletes (41.4 %), whereas 22 athletes (15.7 %) had concentric remodeling, 20 (14.3 %) had concentric hypertrophy, and 40 (28.6 %) had eccentric hypertrophy. Global LV longitudinal strain values obtained from the average of apical four-, two-, and three-chamber global strain values were significantly lower for the basketball players than for all the other groups (p < 0.001).

[The development of reagents set in the format of DNA-chip for genetic typing of strains of Vibrio cholerae].

Science.gov (United States)

Pudova, E A; Markelov, M L; Dedkov, V G; Tchekanova, T A; Sadjin, A I; Kirdiyashkina, N P; Bekova, M V; Deviyatkin, A A

2014-05-01

The necessity of development of methods of genic diagnostic of cholera is conditioned by continuation of the Seventh pandemic of cholera, taxonomic variability of strains of Vibrio cholerae involved into pandemic and also permanent danger of delivery of disease to the territory of the Russian Federation. The methods of genic diagnostic of cholera make it possible in a comparatively short time to maximally minutely characterize strains isolated from patients or their environment. The article presents information about working out reagents set for genetic typing of agents of cholera using DNA-chip. The makeup of DNA-chip included oligonucleotide probes making possible to differentiate strains of V. cholerae on serogroups and biovars and to determine their pathogenicity. The single DNA-chip makes it possible to genetically type up to 12 samples concurrently. At that, duration of analysis without accounting stage of DNA separation makes up to 5 hours. In the progress of work, 23 cholera and non-cholera strains were analyzed. The full compliance of DNA-chip typing results to previously known characteristics of strains. Hence, there is a reason to consider availability of further development of reagents set and possibility of its further application in laboratories of regional level and reference centers.

Development of an Assay for the Detection of PrPres in Blood and Urine Based on PMCA Assay an ELISA Methods

National Research Council Canada - National Science Library

Rohwer, Robert G; Gregori, Luisa L

2005-01-01

.... The assay is been developed with test material from two animal models: the hamster infected with the 263K strain of scrapie and the sheep either naturally or experimentally infected with scrapie...

Fructose 6-phosphate phosphoketolase activity in wild-type strains of Lactobacillus, isolated from the intestinal tract of pigs.

Science.gov (United States)

Bolado-Martínez, E; Acedo-Félix, E; Peregrino-Uriarte, A B; Yepiz-Plascencia, G

2012-01-01

Phosphoketolases are key enzymes of the phosphoketolase pathway of heterofermentative lactic acid bacteria, which include lactobacilli. In heterofermentative lactobacilli xylulose 5-phosphate phosphoketolase (X5PPK) is the main enzyme of the phosphoketolase pathway. However, activity of fructose 6-phosphate phosphoketolase (F6PPK) has always been considered absent in lactic acid bacteria. In this study, the F6PPK activity was detected in 24 porcine wild-type strains of Lactobacillus reuteri and Lactobacillus mucosae, but not in the Lactobacillus salivarius or in L. reuteri ATCC strains. The activity of F6PPK increased after treatment of the culture at low-pH and diminished after porcine bile-salts stress conditions in wild-type strains of L. reuteri. Colorimetric quantification at 505 nm allowed to differentiate between microbial strains with low activity and without the activity of F6PPK. Additionally, activity of F6PPK and the X5PPK gene expression levels were evaluated by real time PCR, under stress and nonstress conditions, in 3 L. reuteri strains. Although an exact correlation, between enzyme activity and gene expression was not obtained, it remains possible that the xpk gene codes for a phosphoketolase with dual substrate, at least in the analyzed strains of L. reuteri.

Complete genome sequence of the gliding, heparinolytic Pedobacter saltans type strain (113).

Science.gov (United States)

Liolios, Konstantinos; Sikorski, Johannes; Lu, Meagan; Nolan, Matt; Lapidus, Alla; Lucas, Susan; Hammon, Nancy; Deshpande, Shweta; Cheng, Jan-Fang; Tapia, Roxanne; Han, Cliff; Goodwin, Lynne; Pitluck, Sam; Huntemann, Marcel; Ivanova, Natalia; Pagani, Ioanna; Mavromatis, Konstantinos; Ovchinikova, Galina; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam; Hauser, Loren; Brambilla, Evelyne-Marie; Kotsyurbenko, Oleg; Rohde, Manfred; Tindall, Brian J; Abt, Birte; Göker, Markus; Detter, John C; Woyke, Tanja; Bristow, James; Eisen, Jonathan A; Markowitz, Victor; Hugenholtz, Philip; Klenk, Hans-Peter; Kyrpides, Nikos C

2011-10-15

Pedobacter saltans Steyn et al. 1998 is one of currently 32 species in the genus Pedobacter within the family Sphingobacteriaceae. The species is of interest for its isolated location in the tree of life. Like other members of the genus P. saltans is heparinolytic. Cells of P. saltans show a peculiar gliding, dancing motility and can be distinguished from other Pedobacter strains by their ability to utilize glycerol and the inability to assimilate D-cellobiose. The genome presented here is only the second completed genome sequence of a type strain from a member of the family Sphingobacteriaceae to be published. The 4,635,236 bp long genome with its 3,854 protein-coding and 67 RNA genes consists of one chromosome, and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Characterization of rotavirus strains detected among children and adults with acute gastroenteritis in Ganozan, Saudi Arabia

International Nuclear Information System (INIS)

Kheyami, Ali M.; Dove, W.; Cunliffe, Nigel A.; Hart, C.A.; Areeshi, Mohammed Y.; Nakagomi, O.

2008-01-01

Objective was to assess the circulating rotavirus strains among hospitalized children and adults in Gizan City. This cross-sectional study was based in 5 hospitals in the Gizan area. Stool samples were collected between November 2004 and March 2005 from sequential patients with acute dehydrating diarrhea. Rotavirus antigen was detected in stool by enzyme-linked immunosorbent assay. The diversity of rotavirus strains was investigated using electropherotyping and reverse transcription-polymerase chain reaction amplification of the VP7 and VP4 genes (G and P genotyping). Rotavirus was detected in 54 of 454 (12%) subjects. The ages of those infected with rotavirus ranged from 15 days to 20 years, with a median age of 36 months. The highest rotavirus detection rate (24%) occurred in children in aged 48-59 months. Overall, 50 (93%) of strains could be assigned both a G- and P- type; G1P (8) was the most frequently detected strain type (n=48, 89%) with one rotavirus each of G2P (4) and G9P (8). Rotavirus strains circulating in Gizan would be well covered by current rotavirus vaccines. Rotavirus serotype G9 has been detected in Saudi Arabia for the first time. Continued surveillance of rotavirus strains is required. (author)

Full-length genome sequence analysis of four subgroup J avian leukosis virus strains isolated from chickens with clinical hemangioma.

Science.gov (United States)

Lin, Lulu; Wang, Peikun; Yang, Yongli; Li, Haijuan; Huang, Teng; Wei, Ping

2017-12-01

Since 2014, cases of hemangioma associated with avian leukosis virus subgroup J (ALV-J) have been emerging in commercial chickens in Guangxi. In this study, four strains of the subgroup J avian leukosis virus (ALV-J), named GX14HG01, GX14HG04, GX14LT07, and GX14ZS14, were isolated from chickens with clinical hemangioma in 2014 by DF-1 cell culture and then identified with ELISA detection of ALV group specific antigen p27, the detection of subtype specific PCR and indirect immunofluorescence assay (IFA) with ALV-J specific monoclonal antibody. The complete genomes of the isolates were sequenced and it was found that the gag and pol were relatively conservative, while env was variable especially the gp85 gene. Homology analysis of the env gene sequences showed that the env gene of all the four isolates had higher similarities with the hemangioma (HE)-type reference strains than that of the myeloid leukosis (ML)-type strains, and moreover, the HE-type strains' specific deletion of 205-bp sequence covering the rTM and DR1 in 3'UTR fragment was also found in the four isolates. Further analysis on the sequences of subunits of env gene revealed an interesting finding: the gp85 of isolates GX14ZS14 and GX14HG04 had a higher similarity with HPRS-103 and much lower similarity with the HE-type reference strains resulting in GX14ZS14, GX14HG04, and HPRS-103 being clustered in the same branch, while gp37 had higher similarities with the HE-type reference strains when compared to that of HPRS-103, resulted in GX14ZS14, GX14HG04, and HE-type reference strains being clustered in the same branch. The results suggested that isolates GX14ZS14 and GX14HG04 may be the recombinant strains of the foreign strain HPRS-103 with the local epidemic HE-type strains of ALV-J.

Bacillus 'next generation' diagnostics: Moving from detection towards sub-typing and risk related strain profiling

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Monika eEhling-Schulz

2013-02-01

Full Text Available The highly heterogeneous genus Bacillus comprises the largest species group of endospore forming bacteria. Because of their ubiquitous nature, Bacillus spores can enter food production at several stages resulting in significant economic losses and posing a potential risk to consumers due the capacity of certain Bacillus strains for toxin production. In the past, food microbiological diagnostics was focused on the determination of species using conventional culture based methods, which are still widely used. However, due to the extreme intraspecies diversity found in the genus Bacillus, DNA based identification and typing methods are gaining increasing importance in routine diagnostics. Several studies showed that certain characteristics are rather strain dependent than species specific. Therefore, the challenge for current and future Bacillus diagnostics is not only the efficient and accurate identification on species level but also the development of rapid methods to identify strains with specific characteristics (such as stress resistance or spoilage potential, trace contamination sources, and last but not least discriminate potential hazardous strains from non-toxic strains.

Selection of bacteriocin producer strains of lactic acid bacteria from a dairy environment.

Science.gov (United States)

Lasagno, M; Beoleito, V; Sesma, F; Raya, R; Font de Valdez, G; Eraso, A

2002-01-01

Two strains showing bacteriocin production were selected from a total of 206 lactic acid bacteria isolated from samples of milk, milk serum, whey and homemade cheeses in Southern Cordoba, Argentina. This property was detected by means of well diffusion assays. The strains were identified as Enterococcus hirae and Enterococcus durans. The protein nature of those substances was proved by showing their sensitivity to type IV and XXV proteases, papaine, trypsin, pepsin and K proteinase. The bacteriocins inhibited the growth of Listeria monocytogenes, Bacillus cereus, Clostridium perfringes and two strains of Staphylococcus aureus, an A-enterotoxin and a B-enterotoxin producers. All of these bacteria are common pathogens usually associated with food borne diseases (ETA). These lactic acid bacteria or their bacteriocins could be suitable candidates for food preservation and specially useful in the our regional dairy industry.

Bactericidal antibody against a representative epidemiological meningococcal serogroup B panel confirms that MATS underestimates 4CMenB vaccine strain coverage.

Science.gov (United States)

Frosi, Giacomo; Biolchi, Alessia; Lo Sapio, Morena; Rigat, Fabio; Gilchrist, Stefanie; Lucidarme, Jay; Findlow, Jamie; Borrow, Ray; Pizza, Mariagrazia; Giuliani, Marzia Monica; Medini, Duccio

2013-10-09

4CMenB (Bexsero), a vaccine developed against invasive meningococcal disease caused by capsular group B strains (MenB), was recently licensed for use by the European Medicines Agency. Assessment of 4CMenB strain coverage in specific epidemiologic settings is of primary importance to predict vaccination impact on the burden of disease. The Meningococcal Antigen Typing System (MATS) was developed to predict 4CMenB strain coverage, using serum bactericidal antibody assay with human complement (hSBA) data from a diverse panel of strains not representative of any specific epidemiology. To experimentally validate the accuracy of MATS-based predictions against strains representative of a specific epidemiologic setting. We used a stratified sampling method to identify a representative sample from all MenB disease isolates collected from England and Wales in 2007-2008, tested the strains in the hSBA assay with pooled sera from infant and adolescent vaccinees, and compared these results with MATS. MATS predictions and hSBA results were significantly associated (P=0.022). MATS predicted coverage of 70% (95% CI, 55-85%) was largely confirmed by 88% killing in the hSBA (95% CI, 72-95%). MATS had 78% accuracy and 96% positive predictive value against hSBA. MATS is a conservative predictor of strain coverage by the 4CMenB vaccine in infants and adolescents. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Comparison of different serological assays for the differential diagnosis of brucellosis

International Nuclear Information System (INIS)

Moreno, E.; Rojas, N.; Nielsen, K.; Gall, D.

1998-01-01

Two indirect and two competitive Enzyme-linked immunosorbent assays (I-ELISA102, I-ELISA103, C-ELISA1 and C-ELISA2 respectively) have been evaluated in comparison with traditional test such as Radial Immunodiffusion (RID), Complement Fixation (CF), Rose Bengal Agglutination (RB) and Rivanol agglutination (RV). The sera analysed included 1018 sera obtained from non-vaccinated bovines, 848 sera from brucellosis free herds calf vaccinated with Strain-19, 295 sera obtained from brucellosis free herds adult vaccinated with Strain-19 and 665 sera from Brucella abortus biotype 1 (field strain) infected herds. Cut-off of values calculated by ROC analysis were established for each ELISA. Although all ELISAs fulfilled the requirements for sensitivity and specificity, in our hands C-ELISA2 performed slightly better than the other assays for differentiating infected from vaccinated bovines. The specificity of this test was similar to that of RID assay which is known to have high specificity for differentiating adult vaccinated from infected bovines. The kappa value among the different tests was good and within the limits of reproducibility and performance expected for the different assays. From the different immunoenzymatic assays, the C-ELISA2, which uses LPS as antigen and a monoclonal antibody against the C/Y epitope as competing reagent, seems to be the most promising of the ELISAs and therefore can be recommended for screening a large number of serum samples on a laboratory basis. (author)

Identification of concomitant infection with Chlamydia trachomatis IncA-negative mutant and wild-type strains by genomic, transcriptional, and biological characterizations.

Science.gov (United States)

Suchland, Robert J; Jeffrey, Brendan M; Xia, Minsheng; Bhatia, Ajay; Chu, Hencelyn G; Rockey, Daniel D; Stamm, Walter E

2008-12-01

Clinical isolates of Chlamydia trachomatis that lack IncA on their inclusion membrane form nonfusogenic inclusions and have been associated with milder, subclinical infections in patients. The molecular events associated with the generation of IncA-negative strains and their roles in chlamydial sexually transmitted infections are not clear. We explored the biology of the IncA-negative strains by analyzing their genomic structure, transcription, and growth characteristics in vitro and in vivo in comparison with IncA-positive C. trachomatis strains. Three clinical samples were identified that contained a mixture of IncA-positive and -negative same-serovar C. trachomatis populations, and two more such pairs were found in serial isolates from persistently infected individuals. Genomic sequence analysis of individual strains from each of two serovar-matched pairs showed that these pairs were very similar genetically. In contrast, the genome sequence of an unmatched IncA-negative strain contained over 5,000 nucleotide polymorphisms relative to the genome sequence of a serovar-matched but otherwise unlinked strain. Transcriptional analysis, in vitro culture kinetics, and animal modeling demonstrated that IncA-negative strains isolated in the presence of a serovar-matched wild-type strain are phenotypically more similar to the wild-type strain than are IncA-negative strains isolated in the absence of a serovar-matched wild-type strain. These studies support a model suggesting that a change from an IncA-positive strain to the previously described IncA-negative phenotype may involve multiple steps, the first of which involves a translational inactivation of incA, associated with subsequent unidentified steps that lead to the observed decrease in transcript level, differences in growth rate, and differences in mouse infectivity.

Phenotypic and genetic characterization of Paecilomyces lilacinus strains with biocontrol activity against root-knot nematodes.

Science.gov (United States)

Gunasekera, T S; Holland, R J; Gillings, M R; Briscoe, D A; Neethling, D C; Williams, K L; Nevalainen, K M

2000-09-01

Efficient selection of fungi for biological control of nematodes requires a series of screening assays. Assessment of genetic diversity in the candidate species maximizes the variety of the isolates tested and permits the assignment of a particular genotype with high nematophagous potential using a rapid novel assay. Molecular analyses also facilitate separation between isolates, allowing the identification of proprietary strains and trace biocontrol strains in the environment. The resistance of propagules to UV radiation is an important factor in the survival of a biocontrol agent. We have analyzed 15 strains of the nematophagous fungus Paecilomyces lilacinus using these principles. Arbitrarily primed DNA and allozyme assays were applied to place the isolates into genetic clusters, and demonstrated that some genetically related P. lilacinus strains exhibit widespread geographic distributions. When exposed to UV radiation, some weakly nematophagous strains were generally more susceptible than effective isolates. A microtitre tray-based assay used to screen the pathogenic activity of each isolate to Meloidogyne javanica egg masses revealed that the nematophagous ability varied between 37%-100%. However, there was no clear relationship between nematophagous ability and genetic clusters. Molecular characterizations revealed sufficient diversity to allow tracking of strains released into the environment.

Multilocus sequence typing, biochemical and antibiotic resistance characterizations reveal diversity of North American strains of the honey bee pathogen Paenibacillus larvae.

Science.gov (United States)

Krongdang, Sasiprapa; Evans, Jay D; Pettis, Jeffery S; Chantawannakul, Panuwan

2017-01-01

Paenibacillus larvae is a Gram positive bacterium and the causative agent of the most widespread fatal brood disease of honey bees, American foulbrood (AFB). A total of thirty-three independent Paenibacillus larvae isolates from various geographical origins in North America and five reference strains were investigated for genetic diversity using multilocus sequence typing (MLST). This technique is regarded to be a powerful tool for epidemiological studies of pathogenic bacteria and is widely used in genotyping assays. For MLST, seven housekeeping gene loci, ilvD (dihydroxy-acid dyhydrogenase), tri (triosephosphate isomerase), purH (phospharibosyl-aminoimidazolecarboxamide), recF (DNA replication and repair protein), pyrE (orotate phosphoribosyltransferase), sucC (succinyl coenzyme A synthetase β subunit) and glpF (glycerol uptake facilitator protein) were studied and applied for primer designs. Previously, ERIC type DNA fingerprinting was applied to these same isolates and the data showed that almost all represented the ERIC I type, whereas using BOX-PCR gave an indication of more diversity. All isolates were screened for resistance to four antibiotics used by U.S. beekeepers, showing extensive resistance to tetracycline and the first records of resistance to tylosin and lincomycin. Our data highlight the intraspecies relationships of P. larvae and the potential application of MLST methods in enhancing our understanding of epidemiological relationships among bacterial isolates of different origins.

Multilocus sequence typing, biochemical and antibiotic resistance characterizations reveal diversity of North American strains of the honey bee pathogen Paenibacillus larvae.

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Sasiprapa Krongdang

Full Text Available Paenibacillus larvae is a Gram positive bacterium and the causative agent of the most widespread fatal brood disease of honey bees, American foulbrood (AFB. A total of thirty-three independent Paenibacillus larvae isolates from various geographical origins in North America and five reference strains were investigated for genetic diversity using multilocus sequence typing (MLST. This technique is regarded to be a powerful tool for epidemiological studies of pathogenic bacteria and is widely used in genotyping assays. For MLST, seven housekeeping gene loci, ilvD (dihydroxy-acid dyhydrogenase, tri (triosephosphate isomerase, purH (phospharibosyl-aminoimidazolecarboxamide, recF (DNA replication and repair protein, pyrE (orotate phosphoribosyltransferase, sucC (succinyl coenzyme A synthetase β subunit and glpF (glycerol uptake facilitator protein were studied and applied for primer designs. Previously, ERIC type DNA fingerprinting was applied to these same isolates and the data showed that almost all represented the ERIC I type, whereas using BOX-PCR gave an indication of more diversity. All isolates were screened for resistance to four antibiotics used by U.S. beekeepers, showing extensive resistance to tetracycline and the first records of resistance to tylosin and lincomycin. Our data highlight the intraspecies relationships of P. larvae and the potential application of MLST methods in enhancing our understanding of epidemiological relationships among bacterial isolates of different origins.

Complete genome sequence of Saccharomonospora viridis type strain (P101T)

Energy Technology Data Exchange (ETDEWEB)

Pati, Amrita; Sikorski, Johannes; Nolan, Matt; Lapidus, Alla; Copeland, Alex; Glavina Del Rio, Tijana; Lucas, Susan; Chen, Feng; Tice, Hope; Pitluck, Sam; Cheng, Jan-Fang; Chertkov, Olga; Brettin, Thomas; Han, Cliff; Detter, John C.; Kuske, Cheryl; Bruce, David; Goodwin, Lynne; Chain, Patrick; D' haeseleer, Patrik; Chen, Amy; Palaniappan, Krishna; Ivanova, Natalia; Mavromatis, Konstantinos; Mikhailova, Natalia; Rohde, Manfred; Tindall, Brian J.; Goker, Markus; Bristow, Jim; Eisen, Jonathan A.; Markowitz, Victor; Hugenholtz, Philip; Kyrpides1, Nikos C.; Klenk, Hans-Peter

2009-05-20

Saccharomonospora viridis (Schuurmans et al. 1956) Nonomurea and Ohara 1971 is the type species of the genus Saccharomonospora which belongs to the family Pseudonocardiaceae. S. viridis is of interest because it is a Gram-negative organism classified amongst the usually Gram-positive actinomycetes. Members of the species are frequently found in hot compost and hay, and its spores can cause farmer?s lung disease, bagassosis, and humidifier fever. Strains of the species S. viridis have been found to metabolize the xenobiotic pentachlorophenol (PCP). The strain described in this study has been isolated from peat-bog in Ireland. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the family Pseudonocardiaceae, and the 4,308,349 bp long single replicon genome with its 3906 protein-coding and 64 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

Histophilus somni IbpA Fic cytotoxin is conserved in disease strains and most carrier strains from cattle, sheep and bison.

Science.gov (United States)

Zekarias, B; O'Toole, D; Lehmann, J; Corbeil, L B

2011-04-21

Histophilus somni causes bovine pneumonia, septicemia, myocarditis, thrombotic meningoencephalitis and arthritis, as well as a genital or upper respiratory carrier state in normal animals. However, differences in virulence factors among strains are not well studied. The surface and secreted immunoglobulin binding protein A (IbpA) Fic motif of H. somni causes bovine alveolar type 2 (BAT2) cells to retract, allowing virulent bacteria to cross the alveolar monolayer. Because H. somni IbpA is an important virulence factor, its presence was evaluated in different strains from cattle, sheep and bison to define whether there are syndrome specific markers and whether antigenic/molecular/functional conservation occurs. A few preputial carrier strains lacked IbpA by Western blotting but all other tested disease or carrier strains were IbpA positive. These positive strains had either both IbpA DR1/Fic and IbpA DR2/Fic or only IbpA DR2/Fic by PCR. IbpA Fic mediated cytotoxicity for BAT2 cells and sequence analysis of IbpA DR2/Fic from selected strains revealed conservation of sequence and function in disease and IbpA positive carrier strains. Passive protection of mice against H. somni septicemia with antibody to IbpA DR2/Fic, along with previous data, indicates that the IbpA DR1/Fic and/or DR2/Fic domains are candidate vaccine antigens for protection against many strains of H. somni. Since IbpA DR2/Fic is conserved in most carrier strains, they may be virulent if introduced to susceptible animals at susceptible sites. Conservation of the protective IbpA antigen in all disease isolates tested is encouraging for development of protective vaccines and diagnostic assays. Copyright © 2010 Elsevier B.V. All rights reserved.

Real-time PCR assays for detection of Brucella spp. and the identification of genotype ST27 in bottlenose dolphins (Tursiops truncatus).

Science.gov (United States)

Wu, Qingzhong; McFee, Wayne E; Goldstein, Tracey; Tiller, Rebekah V; Schwacke, Lori

2014-05-01

Rapid detection of Brucella spp. in marine mammals is challenging. Microbiologic culture is used for definitive diagnosis of brucellosis, but is time consuming, has low sensitivity and can be hazardous to laboratory personnel. Serological methods can aid in diagnosis, but may not differentiate prior exposure versus current active infection and may cross-react with unrelated Gram-negative bacteria. This study reports a real-time PCR assay for the detection of Brucella spp. and application to screen clinical samples from bottlenose dolphins stranded along the coast of South Carolina, USA. The assay was found to be 100% sensitive for the Brucella strains tested, and the limit of detection was 0.27fg of genomic DNA from Brucella ceti B1/94 per PCR volume. No amplification was detected for the non-Brucella pathogens tested. Brucella DNA was detected in 31% (55/178) of clinical samples tested. These studies indicate that the real-time PCR assay is highly sensitive and specific for the detection of Brucella spp. in bottlenose dolphins. We also developed a second real-time PCR assay for rapid identification of Brucella ST27, a genotype that is associated with human zoonotic infection. Positive results were obtained for Brucella strains which had been identified as ST27 by multilocus sequence typing. No amplification was found for other Brucella strains included in this study. ST27 was identified in 33% (18/54) of Brucella spp. DNA-positive clinical samples. To our knowledge, this is the first report on the use of a real-time PCR assay for identification of Brucella genotype ST27 in marine mammals. Copyright © 2014 Elsevier B.V. All rights reserved.

CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections.

Directory of Open Access Journals (Sweden)

Laëtitia Fabre

Full Text Available Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool.

Evaluation of antifungal activity from Bacillus strains against ...

African Journals Online (AJOL)

In this study, 30 bacterial strains isolated from marine biofilms were screened for their antifungal activity against Rhizoctonia solani by dual culture assay. Two bacterial strains, Bacillus subtilis and Bacillus cereus, showed a clear antagonism against R. solani on potato dextrose agar (PDA) medium. The antagonistic activity ...

A Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action

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Paul Priyesh Vijayakumar

2015-06-01

Full Text Available Lactic acid bacteria (LAB have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS by the FDA for use as food ingredients. In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives. In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92. Activity differences between non-neutralized and neutralized Bac+ preps in agar spot assays could not readily be attributed to acid because a bacteriocin-negative control strain was not inhibitory to Listeria in these assays. When neutralized and non-neutralized Bac+ preps were used in microplate growth inhibition assays against L. monocytogenes 39-2 we observed some differences attributed to acid inhibition. A microplate growth inhibition assay was used to compare inhibitory reactions of wild-type and bacteriocin-resistant variants of L. monocytogenes to differentiate bacteriocins with different modes-of-action (MOA whereby curvaticins FS47 and Beef3, and pediocin Bac3 were categorized to be in MOA1; enterocins FS92 and FS56-1 in MOA2; and lacticin FLS1 in MOA3. The microplate bacteriocin MOA assay establishes a platform to evaluate the best combination of bacteriocin preparations for use in food applications as biopreservatives against L. monocytogenes.

A Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action.

Science.gov (United States)

Vijayakumar, Paul Priyesh; Muriana, Peter M

2015-06-11

Lactic acid bacteria (LAB) have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS) by the FDA for use as food ingredients. In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives. In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps) produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92. Activity differences between non-neutralized and neutralized Bac+ preps in agar spot assays could not readily be attributed to acid because a bacteriocin-negative control strain was not inhibitory to Listeria in these assays. When neutralized and non-neutralized Bac+ preps were used in microplate growth inhibition assays against L. monocytogenes 39-2 we observed some differences attributed to acid inhibition. A microplate growth inhibition assay was used to compare inhibitory reactions of wild-type and bacteriocin-resistant variants of L. monocytogenes to differentiate bacteriocins with different modes-of-action (MOA) whereby curvaticins FS47 and Beef3, and pediocin Bac3 were categorized to be in MOA1; enterocins FS92 and FS56-1 in MOA2; and lacticin FLS1 in MOA3. The microplate bacteriocin MOA assay establishes a platform to evaluate the best combination of bacteriocin preparations for use in food applications as biopreservatives against L. monocytogenes.

Evidence for immunisation failure in vaccinated adult dogs infected with canine parvovirus type 2c.

Science.gov (United States)

Decaro, Nicola; Desario, Costantina; Elia, Gabriella; Martella, Vito; Mari, Viviana; Lavazza, Antonio; Nardi, Manuela; Buonavoglia, Canio

2008-01-01

An outbreak of canine parvovirus type 2c (CPV-2c) infection in vaccinated adult dogs is reported. The disease occurred in a breeding kennel in Italy and affected 11 dogs aged between 6 months and 2.5 years, that had been repeatedly administered vaccines containing a type 2 (old type) CPV strain. CPV infection was demonstrated in all diseased dogs by an immunochromatographic test. A CPV strain was isolated from the intestinal content of a 20-month-old pregnant Bernese mountain bitch that underwent a fatal outcome. The strain was characterised as CPV-2c by means of real-time PCR assays using minor groove binder probes. The present report provides further concerns about the real efficacy of type 2-based vaccines against the antigenic variants of CPV and stresses the need for developing new vaccines prepared with the variants currently circulating in the dog population.

Identification of enzymes and quantification of metabolic fluxes in the wild type and in a recombinant Aspergillus oryzae strain

DEFF Research Database (Denmark)

Pedersen, Henrik; Carlsen, Morten; Nielsen, Jens Bredal

1999-01-01

Two alpha-amylase-producing strains of Aspergillus oryzae, a wild-type strain and a recombinant containing additional copies of the alpha-amylase gene, were characterized,vith respect to enzyme activities, localization of enzymes to the mitochondria or cytosol, macromolecular composition...

Genetical and functional investigation of fliC genes encoding flagellar serotype H4 in wildtype strains of Escherichia coli and in a laboratory E. coli K-12 strain expressing flagellar antigen type H48

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Schaudinn Christoph

2005-01-01

Full Text Available Abstract Background Serotyping of O-(lipopolysaccharide and H-(flagellar antigens is a wideley used method for identification of pathogenic strains and clones of Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies. Results The complete nucleotide sequence of fliC genes present in E. coli reference strains U9-41 (O2:K1:H4 and P12b (O15:H17 was determined and both were found 99.3% (1043 of 1050 nucleotides identical in their coding sequence. A PCR/RFLP protocol was developed for typing of fliC-H4 strains and 88 E. coli strains reacting with H4 antiserum were investigated. Nucleotide sequencing of complete fliC genes of six E. coli strains which were selected based on serum agglutination titers, fliC-PCR genotyping and reference data revealed 96.6 to 100% identity on the amino acid level. The functional expression of flagellin encoded by fliC-H4 from strain U9-41 and from our strain P12b which is an H4 expressing variant type was investigated in the E. coli K-12 strain JM109 which encodes flagellar type H48. The fliC recombinant plasmid carrying JM109 strains reacted with both H4 and H48 specific antisera whereas JM109 reacted only with the H48 antiserum. By immunoelectron microscopy, we could show that the flagella made by the fliC-H4 recombinant plasmid carrying strain are constituted of H48 and H4 flagellins which are co-assembled into functional flagella. Conclusion The flagellar serotype H4 is encoded by closely related fliC genes present in serologically different types of E. coli strainswhich were isolated at different time periods and geographical locations. Our expression studies show for the first time, that flagellins of

Complete genome sequence of Truepera radiovictrix type strain (RQ-24).

Science.gov (United States)

Ivanova, Natalia; Rohde, Christine; Munk, Christine; Nolan, Matt; Lucas, Susan; Del Rio, Tijana Glavina; Tice, Hope; Deshpande, Shweta; Cheng, Jan-Fang; Tapia, Roxane; Han, Cliff; Goodwin, Lynne; Pitluck, Sam; Liolios, Konstantinos; Mavromatis, Konstantinos; Mikhailova, Natalia; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam; Hauser, Loren; Chang, Yun-Juan; Jeffries, Cynthia D; Brambilla, Evelyne; Rohde, Manfred; Göker, Markus; Tindall, Brian J; Woyke, Tanja; Bristow, James; Eisen, Jonathan A; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C; Klenk, Hans-Peter; Lapidus, Alla

2011-02-22

Truepera radiovictrix Albuquerque et al. 2005 is the type species of the genus Truepera within the phylum "Deinococcus/Thermus". T. radiovictrix is of special interest not only because of its isolated phylogenetic location in the order Deinococcales, but also because of its ability to grow under multiple extreme conditions in alkaline, moderately saline, and high temperature habitats. Of particular interest is the fact that, T. radiovictrix is also remarkably resistant to ionizing radiation, a feature it shares with members of the genus Deinococcus. This is the first completed genome sequence of a member of the family Trueperaceae and the fourth type strain genome sequence from a member of the order Deinococcales. The 3,260,398 bp long genome with its 2,994 protein-coding and 52 RNA genes consists of one circular chromosome and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Distinction between infections with European and American/vaccine type PRRS virus after vaccination with a modified-live PRRS virus vaccine

DEFF Research Database (Denmark)

Bøtner, Anette; Strandbygaard, Bertel; Sørensen, K. J.

2000-01-01

types of PRRSV was made on a serological basis. The immunoperoxidase monolayer assay (IPMA), carried out using a Danish strain (IPMA/DK) and the vaccine strain (IPMA/vac) in parallel, allows the distinction of infections with EU and US strains of PRRSV. In herds infected with the EU type, the titer...... in individual samples is higher in the IPMA/DK compared to the titer in the IPMA/vac, while in herds infected with the vaccine/US type, the titers are highest in the IPMA/vac. Furthermore, a double blocking ELISA has been developed, which enables large scale screening for and simultaneous distinction between...... ELISA-Vac), which enables us to serologically distinguish between EU and US strains of PRRSV infections. In herds infected with the Danish strain of PRRSV, most animals have a ratio below 1, while in herds infected with the vaccine/US strain most animals have a ratio above 2. The distinction between...

Conjugated Linoleic Acid Stimulates Apoptosis in RH and Tehran Strains of Toxoplasma gondii, in Vitro.

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Jebreil Shamseddin

2015-06-01

Full Text Available The aim of this study was to evaluate the effects of conjugated linoleic acid (CLA on apoptosis of tachyzoites of T. gondii, RH strain (type I and the cyst-forming Tehran strain (type II in vitro.Toxoplasma strains were injected into the peritoneal cavity of BALB/c mice. The Tehran strain forms cysts in the brain of mice. Bradyzoites within the cysts are reactivated to proliferative tachyzoites, by dexamethasone. Tachyzoites were aspirated from the peritoneum of infected mice, and the percentage of viable parasites was estimated with trypan blue staining. Tachyzoites were inoculated into HeLa cells cultivated in DMEM medium. Different concentrations of CLA were evaluated on T. gondii in HeLa cells by the tetrazolium (MTT colorimetric assay. Differentiation between apoptosis and cell death was determined by flow cytometry using Annexin V and propidium iodide (PI double staining. The statistical analysis performed by GraphPad Prism version 6.00.CLA induces apoptosis in virulent (RH and avirulent (Tehran strains of T. gondii. The results of MTT indicated that CLA could decrease the proliferation of tachyzoites of both strains in HeLa cells.Conjugated linoleic acid has anti-toxoplasmacidal activity on tachyzoites of T. gondii. Therefore, we recommended further studies on this component in order to achieve a new drug against the parasite.

First Worldwide Proficiency Study on Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis Complex Strains

Science.gov (United States)

de Beer, Jessica L.; Kremer, Kristin; Ködmön, Csaba; Supply, Philip

2012-01-01

Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future. PMID:22170917

Development of a loop-mediated Isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus

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Kawahara Ryuji

2008-09-01

Full Text Available Abstract Background Vibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH and TDH-related hemolysin (TRH are known as major virulence determinants of V. parahaemolyticus. Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH. Total V. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total V. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP assay for the sensitive and rapid detection of Vibrio parahaemolyticus. Results The assay provided markedly more sensitive and rapid detection of V. parahaemolyticus strains than conventional biochemical and PCR assays. The assay correctly identified 143 V. parahaemolyticus strains, but did not detect 33 non-parahaemolyticus Vibrio and 56 non-Vibrio strains. Sensitivity of the LAMP assay for direct detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples was 5.3 × 102 CFU per ml/g (2.0 CFU per reaction. The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13–22 min in a single colony on TCBS agar from each of 143 V. parahaemolyticus strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of V. parahaemolyticus required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination. Conclusion The LAMP assay is a sensitive, rapid and simple tool for the detection of V. parahaemolyticus and will facilitate the surveillance for control of contamination of V

Pathogenicity Assay of Vibrio harveyi in Tiger Shrimp Larvae Employing Rifampicin-Resistant as A Molecular Marker

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. Widanarni

2007-12-01

Full Text Available Rifampicin-resistant marker was employed as a reporter to assay pathogenicity of Vibrio harveyi  in shrimp larvae.  V. harveyi M. G3 and G7 that difference not schizotyping as shown by Pulsed-Filed Gel Electrophoresis (PFGE used in this study. Spontaneous mutation was conducted to generate V. harveyi resistant to rifampicin. Two groups of shrimp post-larvae (PL5 were immersed for 30 min in 106 CFU/ml of mutants and wild type of V. harveyi, respectively; and then placed in a 2 liter shrimp rearing tank for five days. A control group was immersed in sterile seawater. Growth curve analysis and pathogenicity assay of V. harveyi  showed that each of the V. harveyi mutant exhibited almost identical profiles to that of the wild type parental strain and did not show alteration in their pathogenicity. Sample from dead shrimp larvae showed that the dead shrimp larvae were infected by V. harveyi RfR, indicated that rifampicin-resistant marker effective as a reporter to assay pathogenicity of Vibrio harveyi in shrimp larvae. Key words: shrimp larvae, Vibrio harveyi, rifampicin-resistant, molecular marker

[Diagnostic values of type III Procollagen N-terminal peptide and combination assay of type III procollagen N-terminal peptide with CEA and CA 19-9 in gastric cancer].

Science.gov (United States)

Akazawa, S; Harada, A; Futatsuki, K

1984-07-01

It is known that interstitial collagens are initially synthesized as precursors (procollagen), which possess extra peptide segments at both ends of the molecules. The authors attempted to detect the aminoterminal peptide of type III procollagen (type III-N-peptide) and also to measure the carcinoembryonic antigen (CEA) and carbohydrate antigen (CA 19-9) together in sera of patients with gastric cancer. The results showed that: (1) mean serum levels and positive ratios of the type III-N-peptide increased as the clinical stage of the patients with gastric cancer advanced; (2) serum levels of the type III-N-peptide were not correlated either with those of CEA or CA 19-9; (3) positive ratios of type III-N-peptide, CEA and CA 19-9 were 51.7%, 44.8% and 48.3%, respectively: (4) positive ratio in combination of the type III-N-peptide with CEA was 69.3% and that in combination of the type III-N-peptide with CEA and CA 19-9 was 72.4%. These results suggest that type III-N-peptide is available for diagnosis of gastric cancer and, that the combination assay of type III-N-peptide with CEA and CA 19-9 is more effective than a single assay for diagnosis.

Strains of Lactococcus lactis with a partial pyrimidine requirement show sensitivity toward aspartic acid

DEFF Research Database (Denmark)

Wadskov-Hansen, Steen Lyders Lerche; Martinussen, Jan

2009-01-01

The growth rate of the widely used laboratory strain Lactococcus lactis subsp. cremoris LM0230 was reduced if aspartic acid were present in the growth medium. The strain LM0230 is a plasmid- and phage-cured derivative of L. lactis subsp. cremoris C2, the ancestor of the original dairy isolate L...... with the wild-type strain, and this varied with the concentration of aspartic acid. The observed effect of aspartate could be explained by the accumulation of the toxic pyrimidine de novo pathway intermediate, carbamoyl aspartate. Assays of the pyrimidine biosynthetic enzymes of L. lactis LM0230 showed...... that the partial pyrimidine requirement can be explained by a low specific activity of the pyrimidine biosynthetic enzymes. In conclusion, L. lactis LM0230 during the process of plasmid- and prophage-curing has acquired a partial pyrimidine requirement resulting in sensitivity toward aspartic acid....

Serotype, mating type and ploidy of Cryptococcus neoformans strains isolated from patients in Brazil Sorotipos, "mating type" e ploidia de amostras de C. neoformans isoladas de pacientes no Brasil

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Misako OHKUSU

2002-12-01

Full Text Available Serotype, mating type and ploidy of 84 strains of Cryptococcus neoformans isolated from 61 AIDS and 23 non-AIDS patients admitted in a tertiary teaching hospital in São Paulo, Brazil were examined. Among 61 strains isolated from AIDS patients, 60 strains were var. grubii (serotype A. Only one strain was var. gattii (serotype B. No var. neoformans (serotype D was found. Among 23 strains isolated from non-AIDS patients, 15 were var. grubii (serotype A and the remaining 8 were var. gattii, all of which were serotype B. Seventy-three of the 75 serotype A strains were the heterothallic alpha type (MATalpha and the remaining 2 were untypable (asexual. Most of the MATalpha strains (69/73 were haploid and the remaining 4 strains were diploid. Similarly, both of the 2 asexual strains among the 75 serotype A strains were haploid. There were no alpha-mating type (MATalpha strains among the 84 isolates. All of the 8 var. gattii strains were serotype B and haploid. Among a total of 84 strains tested, neither serotype AD nor serotype D were found. Neither triploid nor tetraploid were found. These results suggest that the serological, sexual and ploidy characteristics in C. neoformans strains isolated from AIDS patients in São Paulo were rather simple, whereas strains isolated from non-AIDS patients presented serotype A and B with predominance of serotype A.Foram estudados os sorotipos, "mating type" e ploidia de 84 amostras de C. neoformans isoladas de 61 pacientes com AIDS e 23 não-AIDS em São Paulo. Das amostras isoladas de pacientes com AIDS, 60 foram identificadas como var. grubii (sorotipo A e 1 como var. gattii (sorotipo B. Não houve isolamento do sorotipo D. Entre as amostras isoladas, de pacientes não-AIDS, 15 foram de var. grubii (sorotipo A e as 8 restantes de var. gattii, todos do sorotipo B. Setenta e três dos 75 sorotipos A foram identificadas como cepas heterotálicas do fenótipo alfa (MATalfa e as 2 remanescentes n

Using PCR-based detection and genotyping to trace Streptococcus salivarius meningitis outbreak strain to oral flora of radiology physician assistant.

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Velusamy Srinivasan

Full Text Available We recently investigated three cases of bacterial meningitis that were reported from a midwestern radiology clinic where facemasks were not worn during spinal injection of contrast agent during myelography procedures. Using pulsed field gel electrophoresis we linked a case strain of S. salivarius to an oral specimen of a radiology physician assistant (RPA. We also used a real-time PCR assay to detect S. salivarius DNA within a culture-negative cerebrospinal fluid (CSF specimen. Here we extend this investigation through using a nested PCR/sequencing strategy to link the culture-negative CSF specimen to the case strain. We also provide validation of the real-time PCR assay used, demonstrating that it is not solely specific for Streptococcus salivarius, but is also highly sensitive for detection of the closely related oral species Streptococcus vestibularis. Through using multilocus sequence typing and 16S rDNA sequencing we further strengthen the link between the CSF case isolate and the RPA carriage isolate. We also demonstrate that the newly characterized strains from this study are distinct from previously characterized S. salivarius strains associated with carriage and meningitis.

Quantitative and multiplexed detection for blood typing based on quantum dot-magnetic bead assay.

Science.gov (United States)

Xu, Ting; Zhang, Qiang; Fan, Ya-Han; Li, Ru-Qing; Lu, Hua; Zhao, Shu-Ming; Jiang, Tian-Lun

2017-01-01

Accurate and reliable blood grouping is essential for safe blood transfusion. However, conventional methods are qualitative and use only single-antigen detection. We overcame these limitations by developing a simple, quantitative, and multiplexed detection method for blood grouping using quantum dots (QDs) and magnetic beads. In the QD fluorescence assay (QFA), blood group A and B antigens were quantified using QD labeling and magnetic beads, and the blood groups were identified according to the R value (the value was calculated with the fluorescence intensity from dual QD labeling) of A and B antigens. The optimized performance of QFA was established by blood typing 791 clinical samples. Quantitative and multiplexed detection for blood group antigens can be completed within 35 min with more than 10 5 red blood cells. When conditions are optimized, the assay performance is satisfactory for weak samples. The coefficients of variation between and within days were less than 10% and the reproducibility was good. The ABO blood groups of 791 clinical samples were identified by QFA, and the accuracy obtained was 100% compared with the tube test. Receiver-operating characteristic curves revealed that the QFA has high sensitivity and specificity toward clinical samples, and the cutoff points of the R value of A and B antigens were 1.483 and 1.576, respectively. In this study, we reported a novel quantitative and multiplexed method for the identification of ABO blood groups and presented an effective alternative for quantitative blood typing. This method can be used as an effective tool to improve blood typing and further guarantee clinical transfusion safety.

Complete genome sequence of Mahella australiensis type strain (50-1 BONT)

Energy Technology Data Exchange (ETDEWEB)

Sikorski, Johannes [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Teshima, Hazuki [Los Alamos National Laboratory (LANL); Nolan, Matt [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Hammon, Nancy [U.S. Department of Energy, Joint Genome Institute; Deshpande, Shweta [U.S. Department of Energy, Joint Genome Institute; Cheng, Jan-Fang [U.S. Department of Energy, Joint Genome Institute; Pitluck, Sam [U.S. Department of Energy, Joint Genome Institute; Liolios, Konstantinos [U.S. Department of Energy, Joint Genome Institute; Pagani, Ioanna [U.S. Department of Energy, Joint Genome Institute; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Huntemann, Marcel [U.S. Department of Energy, Joint Genome Institute; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Ovchinnikova, Galina [U.S. Department of Energy, Joint Genome Institute; Pati, Amrita [U.S. Department of Energy, Joint Genome Institute; Tapia, Roxanne [Los Alamos National Laboratory (LANL); Han, Cliff [Los Alamos National Laboratory (LANL); Goodwin, Lynne A. [Los Alamos National Laboratory (LANL); Chen, Amy [U.S. Department of Energy, Joint Genome Institute; Palaniappan, Krishna [U.S. Department of Energy, Joint Genome Institute; Land, Miriam L [ORNL; Hauser, Loren John [ORNL; Ngatchou, Olivier Duplex [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Rohde, Manfred [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Pukall, Rudiger [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Spring, Stefan [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Abt, Birte [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Goker, Markus [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Detter, J. Chris [U.S. Department of Energy, Joint Genome Institute; Woyke, Tanja [U.S. Department of Energy, Joint Genome Institute; Bristow, James [U.S. Department of Energy, Joint Genome Institute; Markowitz, Victor [U.S. Department of Energy, Joint Genome Institute; Hugenholtz, Philip [U.S. Department of Energy, Joint Genome Institute; Eisen, Jonathan [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute; Klenk, Hans-Peter [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute

2011-01-01

Mahella australiensis Bonilla Salinas et al. 2004 is the type species of the genus Mahella, which belongs to the family Thermoanaerobacteraceae. The species is of interest because it differs from other known anaerobic spore-forming bacteria in its G+C content, and in certain phenotypic traits, such as carbon source utilization and relationship to temperature. Moreo- ver, it has been discussed that this species might be an indigenous member of petroleum and oil reservoirs. This is the first completed genome sequence of a member of the genus Mahella and the ninth completed type strain genome sequence from the family Thermoanaerobacte- raceae. The 3,135,972 bp long genome with its 2,974 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Complete genome sequence of Tsukamurella paurometabola type strain (no. 33T)

Energy Technology Data Exchange (ETDEWEB)

Munk, Christine [U.S. Department of Energy, Joint Genome Institute; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Nolan, Matt [U.S. Department of Energy, Joint Genome Institute; Tice, Hope [U.S. Department of Energy, Joint Genome Institute; Cheng, Jan-Fang [U.S. Department of Energy, Joint Genome Institute; Glavina Del Rio, Tijana [U.S. Department of Energy, Joint Genome Institute; Goodwin, Lynne A. [Los Alamos National Laboratory (LANL); Pitluck, Sam [U.S. Department of Energy, Joint Genome Institute; Liolios, Konstantinos [U.S. Department of Energy, Joint Genome Institute; Huntemann, Marcel [U.S. Department of Energy, Joint Genome Institute; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Mikhailova, Natalia [U.S. Department of Energy, Joint Genome Institute; Pati, Amrita [U.S. Department of Energy, Joint Genome Institute; Chen, Amy [U.S. Department of Energy, Joint Genome Institute; Palaniappan, Krishna [U.S. Department of Energy, Joint Genome Institute; Tapia, Roxanne [Los Alamos National Laboratory (LANL); Han, Cliff [Los Alamos National Laboratory (LANL); Land, Miriam L [ORNL; Hauser, Loren John [ORNL; Chang, Yun-Juan [ORNL; Jeffries, Cynthia [Oak Ridge National Laboratory (ORNL); Brettin, Thomas S [ORNL; Yasawong, Montri [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Brambilla, Evelyne-Marie [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Rohde, Manfred [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Sikorski, Johannes [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Goker, Markus [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Woyke, Tanja [U.S. Department of Energy, Joint Genome Institute; Bristow, James [U.S. Department of Energy, Joint Genome Institute; Eisen, Jonathan [U.S. Department of Energy, Joint Genome Institute; Markowitz, Victor [U.S. Department of Energy, Joint Genome Institute; Hugenholtz, Philip [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute; Klenk, Hans-Peter [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany

2011-01-01

Tsukamurella paurometabola corrig. (Steinhaus 1941) Collins et al. 1988 is the type species of the genus Tsukamurella, which is the type genus to the family Tsukamurellaceae. The spe- cies is not only of interest because of its isolated phylogenetic location, but also because it is a human opportunistic pathogen with some strains of the species reported to cause lung in- fection, lethal meningitis, and necrotizing tenosynovitis. This is the first completed genome sequence of a member of the genus Tsukamurella and the first genome sequence of a member of the family Tsukamurellaceae. The 4,479,724 bp long genome contains a 99,806 bp long plasmid and a total of 4,335 protein-coding and 56 RNA genes, and is a part of the Ge- nomic Encyclopedia of Bacteria and Archaea project.

Wholesomeness studies on gamma-irradiated smoked fish using short-term mutagenicity assays

International Nuclear Information System (INIS)

De la Rosa, A.M.; Banzon, R.B.

1985-12-01

The effect of gamma irradiation on the mutagenicity potential of wood-smoked mackerel (Rastrelliger sp.) was investigated. Smoked fish were irradiated with dose of 2.0, 4.0, 6.0 and 8.0 KGy, and tested for mutagenic activity using the Salmonella plate incorporation assay, host-mediated assay, and micronucleus test. The DMSO extract of unirradiated smoked fish was found to be mutagenic, without metabolic activation in Salmonella strains TA 100 and TA 104, both sensitive to base-pair substitution mutations. Strains TA 98 and TA 97 which are sensitive to frameshift mutations showed no mutagenic activity towards the same DMSO extract. The observed response towards the Salmonella strains was not affected by irradiation in the range of radiation doses studied. The presence of protamutagens in the DMSO extract of unirradiated smoked fish was not detected using the host-mediated assay. In another in-vivo test however, the same DMSO extract induced the formation of micronuclei in the bonemarrow cells of mice. Gamma irradiation up to a dose of 8.0 KGy did not affect the observed mutagenicity of wood-smoked fish. (author)

Yellow fever virus isolated from a fatal post vaccination event: an experimental comparative study with the 17DD vaccine strain in the Syrian hamster (Mesocricetus auratus

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Sueli Guerreiro Rodrigues

2004-01-01

Full Text Available In order to investigate the pathogenicity of the virus strain GOI 4191 that was isolated from a fatal adverse event after yellow fever virus (YFV vaccination, an experimental assay using hamsters (Mesocricetus auratus as animal model and YFV 17DD vaccine strain as virus reference was accomplished. The two virus strains were inoculated by intracerebral, intrahepatic and subcutaneous routes. The levels of viremia, antibody response, and aminotransferases were determined in sera; while virus, antigen and histopathological changes were determined in the viscera. No viremia was detected for either strain following infection; the immune response was demonstrated to be more effective to strain GOI 4191; and no significant aminotransferase levels alterations were detected. Strain GOI 4191 was recovered only from the brain of animals inoculated by the IC route. Viral antigens were detected in liver and brain by immunohistochemical assay. Histothological changes in the viscera were characterized by inflammatory infiltrate, hepatocellular necrosis, and viral encephalitis. Histological alterations and detection of viral antigen were observed in the liver of animals inoculated by the intrahepatic route. These findings were similar for both strains used in the experiment; however, significant differences were observed from those results previously reported for wild type YFV strains.

Colistin-Resistant Acinetobacter baumannii Clinical Strains with Deficient Biofilm Formation

Science.gov (United States)

Dafopoulou, Konstantina; Xavier, Basil Britto; Hotterbeekx, An; Janssens, Lore; Lammens, Christine; Dé, Emmanuelle; Goossens, Herman; Tsakris, Athanasios; Malhotra-Kumar, Surbhi

2015-01-01

In two pairs of clinical colistin-susceptible/colistin-resistant (Csts/Cstr) Acinetobacter baumannii strains, the Cstr strains showed significantly decreased biofilm formation in static and dynamic assays (P Cstr strain and a frameshift mutation in CarO and the loss of a 47,969-bp element containing multiple genes associated with biofilm production in the other. PMID:26666921

Complete genome sequence of Nakamurella multipartita type strain (Y-104).

Science.gov (United States)

Tice, Hope; Mayilraj, Shanmugam; Sims, David; Lapidus, Alla; Nolan, Matt; Lucas, Susan; Glavina Del Rio, Tijana; Copeland, Alex; Cheng, Jan-Fang; Meincke, Linda; Bruce, David; Goodwin, Lynne; Pitluck, Sam; Ivanova, Natalia; Mavromatis, Konstantinos; Ovchinnikova, Galina; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam; Hauser, Loren; Chang, Yun-Juan; Jeffries, Cynthia D; Detter, John C; Brettin, Thomas; Rohde, Manfred; Göker, Markus; Bristow, Jim; Eisen, Jonathan A; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C; Klenk, Hans-Peter; Chen, Feng

2010-03-30

Nakamurella multipartita (Yoshimi et al. 1996) Tao et al. 2004 is the type species of the monospecific genus Nakamurella in the actinobacterial suborder Frankineae. The nonmotile, coccus-shaped strain was isolated from activated sludge acclimated with sugar-containing synthetic wastewater, and is capable of accumulating large amounts of polysaccharides in its cells. Here we describe the features of the organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of a member of the family Nakamurellaceae. The 6,060,298 bp long single replicon genome with its 5415 protein-coding and 56 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

Genotypic and Phenotypic Assessment of Hyaluronidase among Type Strains of a Select Group of Staphylococcal Species

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Mark E. Hart

2009-01-01

Full Text Available Hyaluronidases degrade hyaluronic acid, a major polysaccharide of the extracellular matrix of tissues, and are considered important for virulence in a number of Gram-positive and -negative bacteria. The purpose of the present study was to determine the prevalence of hyaluronidase among clinical strains of Staphylococcus aureus and among other Staphylococcus species. Spent media and chromosomal DNA were assessed for hyaluronidase activity and the absence or presence of a hyaluronidase gene (hysA by Southern analysis, respectively. All S. aureus strains examined exhibited at least one hybridizing band (half of the strains exhibited two or more hybridizing bands when probed for hysA and all but three of these strains produced hyaluronidase. In contrast, none of the type strains of 19 other species exhibited either hyaluronidase activity or hybridizing bands when probed for hysA. These data support the hypothesis that among members of the Staphylococcus genus only strains of S. aureus possess the enzyme hyaluronidase. This would suggest that hyaluronidase represents yet another potential virulence factor employed by S. aureus to cause disease and may represent a diagnostically important characteristic for distinguishing S. aureus from other members of this genus.

Molecular Typing of Acinetobacter Baumannii Clinical Strains in Tehran by Pulsed-Field Gel Electrophoresis

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Neda Farahani

2013-03-01

Full Text Available Background & Objective : Currently, Acinetobacter baumannii is an important nosocomial pathogen insofar as its hospital outbreaks have been described from various geographical areas. Since the discrimination of strains within a species is important for delineating nosocomial outbreaks, this study was conducted with the aim of genotyping the A. baumannii clinical strains in Tehran via the pulsed-field gel electrophoresis (PFGE method, which is the most accurate method used for the typing of bacterial species.   Materials & methods: This study was performed on 70 isolates of acinetobacter baumannii isolated from patients from Baqiyatallah, Rasoole Akram, and Milad hospitals in Tehran. Cultural and biochemical methods were used for the identification of the isolates in species level, and then susceptibility tests were carried out on 50 isolates of A. baumannii using the disk diffusion method. The PFGE method was performed on the isolates by Apa I restriction enzyme. Finally, the results of the PFGE were analyzed. Result: Acinetobacter baumannii strains isolated from hospitals in Tehran showed seven different genetic patterns, two of which were sporadic . Also, genotypic profiles were different in each hospital, and different patterns of genetic resistance to common antibiotics were observed. Conclusion: A lthough diversity was observed among the strains of A. baumannii by the PFGE method in Tehran, no epidemic strains were found among them.  

Genotyping assay for differentiation of wild-type and vaccine viruses in subjects immunized with live attenuated influenza vaccine.

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Victoria Matyushenko

Full Text Available Live attenuated influenza vaccines (LAIVs are considered as safe and effective tool to control influenza in different age groups, especially in young children. An important part of the LAIV safety evaluation is the detection of vaccine virus replication in the nasopharynx of the vaccinees, with special attention to a potential virus transmission to the unvaccinated close contacts. Conducting LAIV clinical trials in some geographical regions with year-round circulation of influenza viruses warrants the development of robust and reliable tools for differentiating vaccine viruses from wild-type influenza viruses in nasal pharyngeal wash (NPW specimens of vaccinated subjects. Here we report the development of genotyping assay for the detection of wild-type and vaccine-type influenza virus genes in NPW specimens of young children immunized with Russian-backbone seasonal trivalent LAIV using Sanger sequencing from newly designed universal primers. The new primer set allowed amplification and sequencing of short fragments of viral genes in NPW specimens and appeared to be more sensitive than conventional real-time RT-PCR protocols routinely used for the detection and typing/subtyping of influenza virus in humans. Furthermore, the new assay is capable of defining the origin of wild-type influenza virus through BLAST search with the generated sequences of viral genes fragments.

Preplastic strain effect on chromium carbides precipitation of type 316 stainless steel during high-temperature ageing

International Nuclear Information System (INIS)

Mao, X.; Zhao, W.

1992-01-01

Long exposure of Type 316 stainless steel to elevated temperature (400-900 o C) is known to cause high-temperature embrittlement due to chromium carbides and σ-phase precipitating in grain boundaries. Numerous investigations have been published on the mechanical properties and microstructure changes occurring during such exposure. However, no investigations exist on the preplastic deformation effect on chromium carbide precipitation in the grain matrix and grain boundary during high-temperature ageing of Type 316 stainless steel and then its effects on the room-temperature tensile properties. Since the stainless steel sometimes is deformed before use at high temperatures, it is necessary to study the preplastic strain effect of the stainless steel on the microstructure change and mechanical property change during high-temperature exposure. The purpose of the present investigation was to carry out such a study. The conclusions reached are as follows. First, chromium carbides are precipitated in deformation lines (slip lines) and then the amount of chromium carbides precipitation in the grain boundary is relatively reduced in predeformed stainless steel after ageing. Secondly, plastic strain pretreatments of and subsequent ageing treatments of Type 316 stainless steel can improve its tensile ductility. Finally, secondary cracking of aged stainless steel occurs in a normal tensile test. The secondary cracking can be reduced by adding preplastic strain into the material. (Author)

Complete genome sequence of Pedobacter heparinus type strain (HIM 762-3T)

Energy Technology Data Exchange (ETDEWEB)

Han, Cliff; Spring, Stefan; Lapidus, Alla; Glavina Del Rio, Tijana; Tice, Hope; Copeland, Alex; Cheng, Jan-Fang; Lucas, Susan; Chen, Feng; Nolan, Matt; Bruce, David; Goodwin, Lynne; Pitluck, Sam; Ivanova, Natalia; Mavrommatis, Konstantinos; Mikhailova, Natalia; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam; Hauser, Loren; Chang, Yun-Juan; Jefferies, Cynthia C.; Saunders, Elizabeth; Chertkov, Olga; Brettin, Thomas; Goker, Markus; Rohde, Manfred; Bristow, James; Eisen, Jonathan A.; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C.; Klenk, Hans-Peter; Detter, John C.

2009-05-20

Pedobacter heparinus (Payza and Korn 1956) Steyn et al. 1998 comb. nov. is the type species of the rapidly growing genus Pedobacter within the family Sphingobacteriaceae of the phylum 'Bacteroidetes'. P. heparinus is of interest, because it was the first isolated strain shown to grow with heparin as sole carbon and nitrogen source and because it produces several enzymes involved in the degradation of mucopolysaccharides. All available data about this species are based on a sole strain that was isolated from dry soil. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first report on a complete genome sequence of a member of the genus Pedobacter, and the 5,167,383 bp long single replicon genome with its 4287 protein-coding and 54 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

Genotyping of Bacillus anthracis strains based on automated capillary 25-loci Multiple Locus Variable-Number Tandem Repeats Analysis

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Ciervo Alessandra

2006-04-01

Full Text Available Abstract Background The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR Analysis (MLVA assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary. Results Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats. Conclusion In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the

Evaluation of the Precision ID Ancestry Panel for crime case work: A SNP typing assay developed for typing of 165 ancestral informative markers.

Science.gov (United States)

Pereira, Vania; Mogensen, Helle S; Børsting, Claus; Morling, Niels

2017-05-01

The application of massive parallel sequencing (MPS) methodologies in forensic genetics is promising and it is gradually being implemented in forensic genetic case work. One of the major advantages of these technologies is that several traditional electrophoresis assays can be combined into one single MPS assay. This reduces both the amount of sample used and the time of the investigations. This study assessed the utility of the Precision ID Ancestry Panel (Thermo Fisher Scientific, Waltham, USA) in forensic genetics. This assay was developed for the Ion Torrent PGM™ System and genotypes 165 ancestry informative SNPs. The performance of the assay and the accompanying software solution for ancestry inference was assessed by typing 142 Danes and 98 Somalis. Locus balance, heterozygote balance, and noise levels were calculated and future analysis criteria for crime case work were estimated. Overall, the Precision ID Ancestry Panel performed well, and only minor changes to the recommended protocol were implemented. Three out of the 165 loci (rs459920, rs7251928, and rs7722456) had consistently poor performance, mainly due to misalignment of homopolymeric stretches. We suggest that these loci should be excluded from the analyses. The different statistical methods for reporting ancestry in forensic genetic case work are discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of Attachment Type on Implant Strain in Maxillary Implant Overdentures: Comparison of Ball, Locator, and Magnet Attachments. Part 1. Overdenture with Palate.

Science.gov (United States)

Takahashi, Toshihito; Gonda, Tomoya; Maeda, Yoshinobu

Implant overdentures with attachments have been used in clinical practice and the effect of attachments on implant strain has been frequently reported. However, most studies have focused on mandibular overdentures; there are few reports on maxillary overdentures. The purpose of this study was to examine the influence of attachment type on implant strain in maxillary overdentures under various implant configurations. A maxillary edentulous model with implants and experimental overdentures were fabricated. Four strain gauges were attached to each implant, positioned in anterior, premolar, and molar areas. Three types of unsplinted attachments-ball, locator, and magnet-were set on the implants under various implant configurations. A vertical occlusal load of 98 N was applied through the mandibular complete denture, and implant strain was compared using the Kruskal-Wallis test. Ball attachments caused the greatest amount of strain, while magnet attachments caused the least amount under all conditions. For all attachments, two anterior implants caused significantly more strain than four implants (P magnet attachments are recommended to reduce implant stress. Using only two implants, especially two anterior implants, is not recommended regardless of attachment type.

Composition and strain effects in Type I and Type II heterostructure ZnSe/Cd(Zn)S and ZnSe/Cd1-xZnxS core/shell quantum dots

Science.gov (United States)

Gheshlaghi, Negar; Pisheh, Hadi Sedaghat; Ünlü, Hilmi

2017-11-01

We investigated the effect of ternary shell alloy composition on the bandgap and diameter of core of ZnSe / Cd1 - x Znx S heterostructure core/shell quantum dots, which were synthesized by using a simple colloidal technique. Characterization by using the x-ray diffraction (XRD), transmission electron microscopy (TEM), UV-Vis absorption and fluorescence emission spectroscopic techniques indicate that (i) there is a transition of ZnSe / Cd0.6 Zn0.4 S Type-I heterostructure (electrons and holes tend to localize in core) to ZnSe / Cd0.75 Zn0.25 S quasi-Type-II heterostructures (holes tend to localized in the core and electrons are delocalized) and (ii) then after large red shift and Stock-shift in PL emission spectra but not a distinct absorption peak in UV spectra become noticeable in ZnSe/Cd0.75Zn0.25 S quasi-Type II and ZnSe/CdS Type II heterostructures (electrons are localized in core and holes are localized in shell). Furthermore, the increase of Cd:S ratio in shell alloy composition shifts the XRD peaks to lower 2θ degrees which corresponds to tensile strain in the ZnSe core. Finally, the hydrostatic interfacial strain has effect on the squeezing or stretching the capped core: A decrease of compressive force on core from ZnSe/ZnS to tensile force in ZnSe/CdS with increase in Cd:S ratio indicates that transition of compressive strain to tensile strain takes place with the transition from Type-I to II heterostructure.

Multicenter evaluation of the new Abbott Realtime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA

NARCIS (Netherlands)

M. Schutten (Martin); D. Peters (D.); N. Back (Nicole); A.W. van den Beld (Annewieke); B. Beuselinck (B.); V. Foulongne (V.); A.M. Geretti (Anna Maria); L. Pandiani (L.); M. Tiemann; H.G.M. Niesters (Bert)

2007-01-01

textabstractThe analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche

Different levels of immunogenicity of two strains of Fowlpox virus as recombinant vaccine vectors eliciting T-cell responses in heterologous prime-boost vaccination strategies.

Science.gov (United States)

Cottingham, Matthew G; van Maurik, Andre; Zago, Manola; Newton, Angela T; Anderson, Richard J; Howard, M Keith; Schneider, Jörg; Skinner, Michael A

2006-07-01

The FP9 strain of F has been described as a more immunogenic recombinant vaccine vector than the Webster FPV-M (FPW) strain (R. J. Anderson et al., J. Immunol. 172:3094-3100, 2004). This study expands the comparison to include two separate recombinant antigens and multiple, rather than single, independent viral clones derived from the two strains. Dual-poxvirus heterologous prime-boost vaccination regimens using individual clones of recombinant FP9 or FPW in combination with recombinant modified V Ankara expressing the same antigen were evaluated for their ability to elicit T-cell responses against recombinant antigens from Plasmodium berghei (circumsporozoite protein) or human immunodeficiency virus type 1 (a Gag-Pol-Nef fusion protein). Gamma interferon enzyme-linked immunospot assay and fluorescence-activated cell sorting assays of the responses to specific epitopes confirmed the approximately twofold-greater cellular immunogenicity of FP9 compared to FPW, when given as the priming or boosting immunization. Equality of transgene expression in mouse cells infected with the two strains in vitro was verified by Western blotting. Directed partial sequence analysis and PCR analysis of FPW and comparison to available whole-genome sequences revealed that many loci that are mutated in the highly attenuated and culture-adapted FP9 strain are wild type in FPW, including the seven multikilobase deletions. These "passage-specific" alterations are hypothesized to be involved in determining the immunogenicity of fowlpox virus as a recombinant vaccine vector.

A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification

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Luiz Tadeu M Figueiredo

1997-05-01

Full Text Available We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37oC for RT followed by a variable amount of cycles of two-step PCR amplification (92oC for 60 sec, 53oC for 60 sec with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 102.8 TCID50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique

Bioethanol strains of Saccharomyces cerevisiae characterised by microsatellite and stress resistance

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Vanda Renata Reis

Full Text Available Abstract Strains of Saccharomyces cerevisiae may display characteristics that are typical of rough-type colonies, made up of cells clustered in pseudohyphal structures and comprised of daughter buds that do not separate from the mother cell post-mitosis. These strains are known to occur frequently in fermentation tanks with significant lower ethanol yield when compared to fermentations carried out by smooth strains of S. cerevisiae that are composed of dispersed cells. In an attempt to delineate genetic and phenotypic differences underlying the two phenotypes, this study analysed 10 microsatellite loci of 22 S. cerevisiae strains as well as stress resistance towards high concentrations of ethanol and glucose, low pH and cell sedimentation rates. The results obtained from the phenotypic tests by Principal-Component Analysis revealed that unlike the smooth colonies, the rough colonies of S. cerevisiae exhibit an enhanced resistance to stressful conditions resulting from the presence of excessive glucose and ethanol and high sedimentation rate. The microsatellite analysis was not successful to distinguish between the colony phenotypes as phenotypic assays. The relevant industrial strain PE-2 was observed in close genetic proximity to rough-colony although it does not display this colony morphology. A unique genetic pattern specific to a particular phenotype remains elusive.

Clonal structure of Trypanosoma cruzi Colombian strain (biodeme Type III: biological, isoenzymic and histopathological analysis of seven isolated clones

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Camandaroba Edson Luiz Paes

2001-01-01

Full Text Available The clonal structure of the Colombian strain of Trypanosoma cruzi, biodeme Type III and zymodeme 1, was analyzed in order to characterize its populations and to establish its homogeneity or heterogeneity. Seven isolated clones presented the basic characteristics of Biodeme Type III, with the same patterns of parasitemic curves, tissue tropism to skeletal muscle and myocardium, high pathogenicity with extensive necrotic-inflammatory lesions from the 20th to 30th day of infection. The parental strain and its clones C1, C3, C4 and C6, determined the higher levels of parasitemia, 20 to 30 days of infection, with high mortality rate up to 30 days (79 to 100%; clones C2, C5 and C7 presented lower levels of parasitemia, with low mortality rates (7.6 to 23%. Isoenzymic patterns, characteristic of zymodeme 1, (Z1 were similar for the parental strain and its seven clones. Results point to a phenotypic homogeneity of the clones isolated from the Colombian strain and suggest the predominance of a principal clone, responsible for the biological behavior of the parental strain and clones.

Strain and high-temperature discrimination using a Type II fiber Bragg grating and a miniature fiber Fabry-Perot interferometer.

Science.gov (United States)

Jiang, Yajun; Yang, Dexing; Yuan, Yuan; Xu, Jian; Li, Dong; Zhao, Jianlin

2016-08-10

A novel method for simultaneous measurement of strain and high temperature using a Type II fiber Bragg grating (FBG) and a miniature fiber Fabry-Perot interferometer (MFFPI) is proposed. The MFFPI is produced by fusion splicing a short section of quartz capillary tube with two single-mode fibers, and then it is exposed by a focused femtosecond laser and a phase mask to inscribe a Type II FBG nearby. The reflection spectrum of this sensor is the superposition of the reflection spectrum of the FBG and the interference fringe of the MFFPI. This sensor shows perfect high-temperature and strain responses. Because of the different responses to the uniform variations of strain and temperature, by measuring the reflection peak of FBG and one of the interference dips of the MFFPI, strain and temperature can be simultaneously determined. The resolutions of this particular sensor in measuring strain and temperature are estimated to be ±8.4  μϵ and ±3.3°C, respectively, in the range from 0 to 1122 μϵ and from 23°C to 600°C.

Isolation of coagulase-positive staphylococci from bitches' colostrum and milk and genetic typing of methicillin-resistant Staphylococcus pseudintermedius strains.

Science.gov (United States)

Rota, Ada; Corrò, Michela; Drigo, Ilenia; Bortolami, Alessio; Börjesson, Stefan

2015-07-23

Among the coagulase-positive, potentially pathogenic staphylococci, Staphylococcus pseudintermedius has been frequently isolated from bitches' milk. This organism colonizes the mammary gland or causes infection, while S. aureus has been only occasionally reported. The objective of this study was to investigate the occurrence and persistence of coagulase-positive staphylococci in the colostrum and milk of postpartum bitches, either treated or untreated with antimicrobials, and to assess the incidence, antibiotic resistance profile and genetic type of the methicillin-resistant strains. On postpartum D1, D7 and D15, drops of secretion were collected from the mammary glands of 27 postpartum bitches, nine of which were treated with antimicrobials. Coagulase-positive staphylococci were identified, antimicrobial susceptibility and the presence of mecA were tested and the genetic profile of methicillin-resistant strains was assessed. Staphylococcus pseudintermedius was the only coagulase-positive staphylococcus isolated, and its presence was detected in 21 out of 27 bitches and in 66 out of 145 swabs. In a single bitch, it caused puerperal mastitis. In untreated bitches, the frequency of isolation was lower in colostrum than in milk. All of the isolates except one were resistant to at least three antimicrobial classes, while 14 out of 66 S. pseudintermedius strains were methicillin-resistant mecA positive (MRSP) and were isolated from eight bitches housed in the same breeding kennel. A significant association was found between antimicrobial treatment and the presence of MRSP. Six of the 12 typed isolates belonged to spa-type t02 carrying SCCmec II/III, and another six were non-typeable with spa carrying SCCmec IV. The t02-SCCmec II/III isolates were sequence type (ST) 71; four NT-SCCmec IV isolates were ST258 and two were ST369. PFGE showed that isolates from the same dog had identical band patterns, while isolates from different dogs had unique band patterns. MRSP strains

Standardization of micro-enzyme-linked immunosorbent assay (ELISA) and Western blot for detection of Trypanosoma cruzi antibodies using extracts from Mexican strains as antigens.

Science.gov (United States)

Sánchez, B; Monteón, V; Reyes, P A; Espinoza, B

2001-01-01

This report describes two assays for the detection of anti-Trypanosoma cruzi antibodies using Mexican strains of the parasite and the concordance with two assays previously evaluated at the Instituto Nacional de Cardiología Ignacio Chávez in Mexico City. Micro-enzyme-linked immunosorbent assay (ELISA) and Western blot were used for the detection of T. cruzi antibodies with a total extract of epimastigote from Ninoa and Queretaro, which are Mexican strains of T. cruzi. To standardize these methods, a total of 246 serum samples was used. In addition, sera from six confirmed Mexican chronic individuals in the asymptomatic phase were also used for comparison with the Argentinean antigen. ELISA was 100% specific in that no false positive results were found with sera of both healthy individuals and non-Chagasic cardiopaths. Sera from individuals infected with Leishmania sp. showed approximately 16% of cross-reaction with ELISA. The test showed a positive predictive value of 90% and a negative predictive value of 100%. Western blot was also a highly sensitive test for detecting chronic Chagasic symptomatic patients from Mexico because no false negative results were obtained. Furthermore, it was possible to use Western blot to detect seven immunodominant antigens of approximately 30, 32, 40, 42, 65, 70, and 83 kDa. Concordance with two previous standardized tests at the Instituto Nacional de Cardiología showed a Kappa index of 0.96, indicating high concordance between the results obtained at these two laboratories. Finally, ELISA using Ninoa antigen extract was more sensitive than ELISA with an Argentinean extract, which failed to detect individuals in the chronic asymptomatic phase (undetermined phase) of infection. This study indicates that ELISA and Western blot using Ninoa and/or Queretaro extracts of T. cruzi as antigens are useful tools in the detection of individuals who have been exposed to T. cruzi both in the undetermined/asymptomatic and symptomatic phases

The use of quantitative PCR for identification and quantification of Brachyspira pilosicoli, Lawsonia intracellularis and Escherichia coli fimbrial types F4 and F18 in pig feces

DEFF Research Database (Denmark)

Ståhl, Marie; Kokotovic, Branko; Hjulsager, Charlotte Kristiane

2011-01-01

Four quantitative PCR (qPCR) assays were evaluated for quantitative detection of Brachyspira pilosicoli, Lawsonia intracellularis, and E. coli fimbrial types F4 and F18 in pig feces. Standard curves were based on feces spiked with the respective reference strains. The detection limits from...... the spiking experiments were 102 bacteria/g feces for BpiloqPCR and Laws-qPCR, 103 CFU/g feces for F4-qPCR and F18-qPCR. The PCR efficiency for all four qPCR assays was between 0.91 and 1.01 with R2 above 0.993. Standard curves, slopes and elevation, varied between assays and between measurements from pure...... DNA from reference strains and feces spiked with the respective strains. The linear ranges found for spiked fecal samples differed both from the linear ranges from pure culture of the reference strains and between the qPCR tests. The linear ranges were five log units for F4- qPCR, and Laws-qPCR, six...

Optimization and validation of a neutralizing antibody assay for HIV-1 in A3R5 cells.

Science.gov (United States)

Sarzotti-Kelsoe, Marcella; Daniell, Xiaoju; Todd, Christopher A; Bilska, Miroslawa; Martelli, Amanda; LaBranche, Celia; Perez, Lautaro G; Ochsenbauer, Christina; Kappes, John C; Rountree, Wes; Denny, Thomas N; Montefiori, David C

2014-07-01

A3R5 is a human CD4(+) lymphoblastoid cell line that was engineered to express CCR5 and is useful for the detection of weak neutralizing antibody responses against tier 2 strains of HIV-1. Here we describe the optimization and validation of the HIV-1 neutralizing antibody assay that utilizes A3R5 cells, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay utilizes Renilla luciferase-expressing replication competent infectious molecular clones (IMC) encoding heterologous env genes from different HIV-1 clades. Key assay validation parameters tested included specificity, accuracy, precision, limit of detection and quantitation, specificity, linearity and range, and robustness. Plasma samples demonstrated higher non-specific activity than serum samples in the A3R5 assay. This assay can tolerate a wide range of virus input but is more sensitive to cell concentration. The higher sensitivity of the A3R5 assay in neutralization responses to tier 2 strains of HIV-1 makes it complementary to, but not a substitute for the TZM-bl assay. The validated A3R5 assay is employed as an endpoint immunogenicity test for vaccine-elicited neutralizing antibodies against tier 2 strains of HIV-1, and to identify correlates of protection in HIV-1 vaccine trials conducted globally. Copyright © 2014 Elsevier B.V. All rights reserved.