Lactoferricin Peptides Increase Macrophages' Capacity To Kill Mycobacterium avium.

Science.gov (United States)

Silva, Tânia; Moreira, Ana C; Nazmi, Kamran; Moniz, Tânia; Vale, Nuno; Rangel, Maria; Gomes, Paula; Bolscher, Jan G M; Rodrigues, Pedro N; Bastos, Margarida; Gomes, Maria Salomé

2017-01-01

Mycobacterial infections cause a significant burden of disease and death worldwide. Their treatment is long, toxic, costly, and increasingly prone to failure due to bacterial resistance to currently available antibiotics. New therapeutic options are thus clearly needed. Antimicrobial peptides represent an important source of new antimicrobial molecules, both for their direct activity and for their immunomodulatory potential. We have previously reported that a short version of the bovine antimicrobial peptide lactoferricin with amino acids 17 to 30 (LFcin17-30), along with its variants obtained by specific amino acid substitutions, killed Mycobacterium avium in broth culture. In the present work, those peptides were tested against M. avium living inside its natural host cell, the macrophage. We found that the peptides increased the antimicrobial action of the conventional antibiotic ethambutol inside macrophages. Moreover, the d-enantiomer of the lactoferricin peptide (d-LFcin17-30) was more stable and induced significant killing of intracellular mycobacteria by itself. Interestingly, d-LFcin17-30 did not localize to M. avium -harboring phagosomes but induced the production of proinflammatory cytokines and increased the formation of lysosomes and autophagosome-like vesicles. These results lead us to conclude that d-LFcin17-30 primes macrophages for intracellular microbial digestion through phagosomal maturation and/or autophagy, culminating in mycobacterial killing. IMPORTANCE The genus Mycobacterium comprises several pathogenic species, including M. tuberculosis , M. leprae , M. avium , etc. Infections caused by these bacteria are particularly difficult to treat due to their intrinsic impermeability, low growth rate, and intracellular localization. Antimicrobial peptides are increasingly acknowledged as potential treatment tools, as they have a high spectrum of activity, low tendency to induce bacterial resistance, and immunomodulatory properties. In this study, we

Mycobacterium avium intracellulare complex causing olecranon bursitis and prosthetic joint infection in an immunocompromised host

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Eugene M. Tan

2016-01-01

Full Text Available Case: A 73-year-old immunocompromised male presented with recurrent left elbow swelling due to Mycobacterium avium intracellulare complex (MAC olecranon bursitis. 3 years after completing MAC treatment, he underwent right total knee arthroplasty (TKA. 1 year later, he developed TKA pain and swelling and was diagnosed with MAC prosthetic joint infection (PJI. He underwent TKA resection, reimplantation, and 12 months of anti-MAC therapy. This patient is the seventh case report of MAC olecranon bursitis and the third case report of MAC PJI. He is the only report of both MAC olecranon bursitis and PJI occurring in the same patient. Informed consent: This patient was informed and agreed to the publication of this material.

In vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae to ticarcillin in combination with clavulanic acid.

OpenAIRE

Casal, M J; Rodriguez, F C; Luna, M D; Benavente, M C

1987-01-01

The in vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae (M. chelonei) to ticarcillin in combination with calvulanic acid (CA) was studied by the agar dilution method. All the M. tuberculosis, M. bovis, and M. africanum strains were inhibited at a ticarcillin concentration of 32 micrograms/ml or lower in combination with 5 micrograms of CA. M. chelonae and M. avium strains ...

THE ISOLATION AND IDENTIFICATION OF MYCOBACTERIUM AVIUM COMPLEX (MAC) RECOVERED FROM LOS ANGELES POTABLE WATER, A POSSIBLE SOURCE OF INFECTION IN AIDS PATIENTS

Science.gov (United States)

Los Angeles water was investigated as a possible source of Mycobacterium avium complex (MAC) infection in patients with AIDS. MAC consists of M.avium (MA), M. intracellulare (MI) and Mycobacterium X (MX)(positive for MAC by DNA probe but not MA or MI). The study included 13 reser...

Experimental Inoculation of BFDV-Positive Budgerigars (Melopsittacus undulatus with Two Mycobacterium avium subsp. avium Isolates

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Aleksandra Ledwoń

2014-01-01

Full Text Available Beak and feather disease virus- (BFDV- positive (naturally infected but clinically healthy budgerigars (Melopsittacus undulatus were inoculated with two isolates of Mycobacterium avium subsp. avium isolated from naturally infected golden pheasant (Chrysolophus pictus and peafowl (Pavo cristatus. During a period of more than two months after inoculation, samples of cloacal and crop swabs, faeces, and blood were obtained for BFDV and Mycobacterium avium testing with PCR. Birds were euthanized nine weeks after inoculation. All infected budgerigars developed signs typical of mycobacteriosis, but more advanced clinical and pathological changes were visible in the group infected with the pheasant isolate. Only a few cloacal and crop swab samples were positive for Mycobacterium avium subsp. avium despite advanced pathological changes in the internal organs. In the groups infected with mycobacterium isolates the frequency of BFDV-positive samples was higher than in the control group. In the infected groups the frequency of BFDV was substantially higher in the cloacal swabs of birds inoculated with the pheasant isolate than in the peafowl-isolate-infected group.

Experimental inoculation of BFDV-positive budgerigars (Melopsittacus undulatus) with two Mycobacterium avium subsp. avium isolates.

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Ledwoń, Aleksandra; Sapierzyński, Rafał; Augustynowicz-Kopeć, Ewa; Szeleszczuk, Piotr; Kozak, Marcin

2014-01-01

Beak and feather disease virus- (BFDV-) positive (naturally infected) but clinically healthy budgerigars (Melopsittacus undulatus) were inoculated with two isolates of Mycobacterium avium subsp. avium isolated from naturally infected golden pheasant (Chrysolophus pictus) and peafowl (Pavo cristatus). During a period of more than two months after inoculation, samples of cloacal and crop swabs, faeces, and blood were obtained for BFDV and Mycobacterium avium testing with PCR. Birds were euthanized nine weeks after inoculation. All infected budgerigars developed signs typical of mycobacteriosis, but more advanced clinical and pathological changes were visible in the group infected with the pheasant isolate. Only a few cloacal and crop swab samples were positive for Mycobacterium avium subsp. avium despite advanced pathological changes in the internal organs. In the groups infected with mycobacterium isolates the frequency of BFDV-positive samples was higher than in the control group. In the infected groups the frequency of BFDV was substantially higher in the cloacal swabs of birds inoculated with the pheasant isolate than in the peafowl-isolate-infected group.

Transcriptional profiling of ileocecal valve of Holstein dairy cows infected with mycobacterium avium subsp. paratuberculosis

Science.gov (United States)

Johne’s disease is a chronic infection of the small intestine caused by Mycobacterium avium subspecies paratuberculosis (MAP), an intracellular bacterium. The events of pathogen survival within the host cell(s), chronic inflammation and the progression from asymptomatic subclinical stage to an advan...

A Mycobacterium avium subsp. paratuberculosis predicted serine protease is associated with acid stress and intraphagosomal survival

Science.gov (United States)

Mycobacterium avium subsp. paratuberculosis (MAP) is an intracellular pathogen that persists inside host macrophages despite severe oxidative stress and nutrient deprivation. Intrabacterial pH homeostasis is vital to pathogenic mycobacteria to preserve cellular biological processes and stability of ...

Mycobacterium avium subsp. avium found in raptors exposed to infected domestic fowl.

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Kriz, Petr; Kaevska, Marija; Bartejsova, Iva; Pavlik, Ivo

2013-09-01

We report a case of a falcon breeding facility, where raptors (both diurnal and nocturnal) were raised in contact with domestic fowl (Gallus gallus f. domesticus) infected by Mycobacterium avium subsp. avium. Fecal and environmental samples from 20 raptors and four common ravens (Corvus corax) were collected. Mycobacterium a. avium DNA was detected in feces of four raptors (bald eagle [Haliaeetus leucocephalus], eagle owl [Bubo bubo], barn owl [Tyto alba], and little owl [Athene noctua]) using triplex quantitative real-time PCR. As both the flock of domestic fowl and one of the infected raptors had the same origin (zoological collection), they might have had a common source of colonization/infection. However, the detection of M. a. avium in feces of three other raptors may point at transmission of the agent between the birds in the facility. Contact of raptors with domestic fowl infected by M. a. avium may pose a risk for transmission of the infection for them; however, raptors from the falcon breeding facility seemed to be relatively resistant to the infection.

Environmental Mycobacterium avium subsp. paratuberculosis hosted by free-living amoebae

Science.gov (United States)

Mycobacterium avium subsp. paratuberculosis is responsible for paratuberculosis in animals. This disease, leading to an inflammation of the gastrointestinal tract, has a high impact on animal health and an important economic burden. The environmental life cycle of Mycobacterium avium subsp. paratube...

Molecular analysis of clinical isolates previously diagnosed as Mycobacterium intracellulare reveals incidental findings of "Mycobacterium indicus pranii" genotypes in human lung infection.

Science.gov (United States)

Kim, Su-Young; Park, Hye Yun; Jeong, Byeong-Ho; Jeon, Kyeongman; Huh, Hee Jae; Ki, Chang-Seok; Lee, Nam Yong; Han, Seung-Jung; Shin, Sung Jae; Koh, Won-Jung

2015-09-30

Mycobacterium intracellulare is a major cause of Mycobacterium avium complex lung disease in many countries. Molecular studies have revealed several new Mycobacteria species that are closely related to M. intracellulare. The aim of this study was to re-identify and characterize clinical isolates from patients previously diagnosed with M. intracellulare lung disease at the molecular level. Mycobacterial isolates from 77 patients, initially diagnosed with M. intracellulare lung disease were re-analyzed by multi-locus sequencing and pattern of insertion sequences. Among the 77 isolates, 74 (96 %) isolates were designated as M. intracellulare based on multigene sequence-based analysis. Interestingly, the three remaining strains (4 %) were re-identified as "Mycobacterium indicus pranii" according to distinct molecular phylogenetic positions in rpoB and hsp65 sequence-based typing. In hsp65 sequevar analysis, code 13 was found in the majority of cases and three unreported codes were identified. In 16S-23S rRNA internal transcribed spacer (ITS) sequevar analysis, all isolates of both species were classified within the Min-A ITS sequevar. Interestingly, four of the M. intracellulare isolates harbored IS1311, a M. avium-specific element. Two of three patients infected with "M. indicus pranii" had persistent positive sputum cultures after antibiotic therapy, indicating the clinical relevance of this study. This analysis highlights the importance of precise identification of clinical isolates genetically close to Mycobacterium species, and suggests that greater attention should be paid to nontuberculous mycobacteria lung disease caused by "M. indicus pranii".

MYCOBACTERIUM AVIUM AND DRINKING WATER WHAT ARE THE CONNECTIONS?

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Background: Human Mycobacterium avium infections are only known to be acquired from environmental sources such as water and soil. We compared M. avium isolates from clinical and drinking water sources using molecular tools. Methods: M. avium was isolated from water samples colle...

A single or multistage mycobacterium avium subsp. paratuberculosis subunit vaccine

DEFF Research Database (Denmark)

2014-01-01

The present invention provides one or more immunogenic polypeptides for use in a preventive or therapeutic vaccine against latent or active infection in a human or animal caused by a Mycobacterium species, e.g. Mycobacterium avium subsp. paratuberculosis. Furthermore a single or multi-phase vaccine...... comprising the one or more immunogenic polypeptides is provided for administration for the prevention or treatment of infection with a Mycobacterium species, e.g. Mycobacterium avium subsp. paratuberculosis. Additionally, nucleic acid vaccines, capable of in vivo expression of the multi-phase vaccine...

Detection of Mycobacterium avium subspecies in the gut associated lymphoid tissue of slaughtered rabbits.

Science.gov (United States)

Arrazuria, Rakel; Sevilla, Iker A; Molina, Elena; Pérez, Valentín; Garrido, Joseba M; Juste, Ramón A; Elguezabal, Natalia

2015-06-11

Rabbits are susceptible to infection by different species of the genus Mycobacterium. Particularly, development of specific lesions and isolation of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis, both subspecies of the M. avium complex, has been reported in wildlife conditions. Although, rabbit meat production worldwide is 200 million tons per year, microbiological data on this source of meat is lacking and more specifically reports of mycobacterial presence in industrially reared rabbit for human consumption have not been published. To this end, we sought mycobacteria by microbiological and histopathological methods paying special attention to Mycobacterium avium subsp. paratuberculosis in rabbits from commercial rabbitries from the North East of Spain. M. avium subsp. paratuberculosis was not detected either by culture or PCR. However, Mycobacterium avium subsp. avium was detected in 15.15% (10/66) and Mycobacterium avium subsp. hominissuis was detected in 1.51% (1/66) of gut associated lymphoid tissue of sampled animals by PCR, whereas caecal contents were negative. 9% (6/66) of the animals presented gross lesions suggestive of lymphoid activation, 6% (4/66) presented granulomatous lesions and 3% (2/66) contained acid fast bacilli. Mycobacterial isolation from samples was not achieved, although colonies of Thermoactinomycetes sp. were identified by 16s rRNA sequencing in 6% (4/66) of sampled animals. Apparently healthy farmed rabbits that go to slaughter may carry M. avium subspecies in gut associated lymphoid tissue.

A novel multi-antigen virally vectored vaccine against Mycobacterium avium subspecies paratuberculosis.

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Tim J Bull

Full Text Available BACKGROUND: Mycobacterium avium subspecies paratuberculosis causes systemic infection and chronic intestinal inflammation in many species including primates. Humans are exposed through milk and from sources of environmental contamination. Hitherto, the only vaccines available against Mycobacterium avium subspecies paratuberculosis have been limited to veterinary use and comprised attenuated or killed organisms. METHODS: We developed a vaccine comprising a fusion construct designated HAV, containing components of two secreted and two cell surface Mycobacterium avium subspecies paratuberculosis proteins. HAV was transformed into DNA, human Adenovirus 5 (Ad5 and Modified Vaccinia Ankara (MVA delivery vectors. Full length expression of the predicted 95 kDa fusion protein was confirmed. PRINCIPAL FINDINGS: Vaccination of naïve and Mycobacterium avium subspecies paratuberculosis infected C57BL/6 mice using DNA-prime/MVA-boost or Ad5-prime/MVA-boost protocols was highly immunogenic resulting in significant IFN-gamma ELISPOT responses by splenocytes against recombinant vaccine antigens and a range of HAV specific peptides. This included strong recognition of a T-cell epitope GFAEINPIA located near the C-terminus of the fusion protein. Antibody responses to recombinant vaccine antigens and HAV specific peptides but not GFAEINPIA, also occurred. No immune recognition of vaccine antigens occurred in any sham vaccinated Mycobacterium avium subspecies paratuberculosis infected mice. Vaccination using either protocol significantly attenuated pre-existing Mycobacterium avium subspecies paratuberculosis infection measured by qPCR in spleen and liver and the Ad5-prime/MVA-boost protocol also conferred some protection against subsequent challenge. No adverse effects of vaccination occurred in any of the mice. CONCLUSIONS/SIGNIFICANCE: A range of modern veterinary and clinical vaccines for the treatment and prevention of disease caused by Mycobacterium avium

A novel multi-antigen virally vectored vaccine against Mycobacterium avium subspecies paratuberculosis.

Science.gov (United States)

Bull, Tim J; Gilbert, Sarah C; Sridhar, Saranya; Linedale, Richard; Dierkes, Nicola; Sidi-Boumedine, Karim; Hermon-Taylor, John

2007-11-28

Mycobacterium avium subspecies paratuberculosis causes systemic infection and chronic intestinal inflammation in many species including primates. Humans are exposed through milk and from sources of environmental contamination. Hitherto, the only vaccines available against Mycobacterium avium subspecies paratuberculosis have been limited to veterinary use and comprised attenuated or killed organisms. We developed a vaccine comprising a fusion construct designated HAV, containing components of two secreted and two cell surface Mycobacterium avium subspecies paratuberculosis proteins. HAV was transformed into DNA, human Adenovirus 5 (Ad5) and Modified Vaccinia Ankara (MVA) delivery vectors. Full length expression of the predicted 95 kDa fusion protein was confirmed. Vaccination of naïve and Mycobacterium avium subspecies paratuberculosis infected C57BL/6 mice using DNA-prime/MVA-boost or Ad5-prime/MVA-boost protocols was highly immunogenic resulting in significant IFN-gamma ELISPOT responses by splenocytes against recombinant vaccine antigens and a range of HAV specific peptides. This included strong recognition of a T-cell epitope GFAEINPIA located near the C-terminus of the fusion protein. Antibody responses to recombinant vaccine antigens and HAV specific peptides but not GFAEINPIA, also occurred. No immune recognition of vaccine antigens occurred in any sham vaccinated Mycobacterium avium subspecies paratuberculosis infected mice. Vaccination using either protocol significantly attenuated pre-existing Mycobacterium avium subspecies paratuberculosis infection measured by qPCR in spleen and liver and the Ad5-prime/MVA-boost protocol also conferred some protection against subsequent challenge. No adverse effects of vaccination occurred in any of the mice. A range of modern veterinary and clinical vaccines for the treatment and prevention of disease caused by Mycobacterium avium subspecies paratuberculosis are needed. The present vaccine proved to be highly

Mycobacterium avium complex disseminated infection in a kidney transplant recipient.

Science.gov (United States)

Fadlallah, J; Rammaert, B; Laurent, S; Lanternier, F; Pol, S; Franck, N; Mamzer, M F; Dupin, N; Lortholary, O

2016-02-01

Mycobacterium avium-intracellulare complex (MAC) infections are well known in immunocompromised patients, notably in human immunodeficiency virus infection, but remain scarcely described in kidney transplantation. Moreover, cutaneous involvement in this infection is very unusual. We describe here a disseminated infection caused by MAC in a kidney transplant recipient revealed by cutaneous lesions. This case highlights the need for an exhaustive, iterative microbiologic workup in the context of an atypical disease presentation in a renal transplant patient, regardless of the degree of immunosuppression. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Putative in vitro expressed gene fragments unique to Mycobacterium avium subspecies para tuberculosis

DEFF Research Database (Denmark)

Nielsen, Kirstine Klitgaard; Ahrens, Peter

2002-01-01

By a suppression subtractive hybridization based method, nine novel Mycobacterium avium subsp. para tuberculosis (M. para tuberculosis) fragments of between 318 and 596 bp have been identified and characterized. Database search revealed little or no similarity with other mycobacteria. The uniquen......By a suppression subtractive hybridization based method, nine novel Mycobacterium avium subsp. para tuberculosis (M. para tuberculosis) fragments of between 318 and 596 bp have been identified and characterized. Database search revealed little or no similarity with other mycobacteria....... The uniqueness and diagnostic potential of seven of these fragments in relation to M. paratuberculosis closest relative Mycobacterium avium subsp. avium (M. avium) was confirmed by species-specific PCR and Southern blot. Furthermore, RT-PCR indicated that eight of the nine fragments originate from areas...

Characterization of a Mycobacterium avium subsp. avium Operon Associated with Virulence and Drug Detoxification

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Mariana Noelia Viale

2014-01-01

Full Text Available The lprG-p55 operon of Mycobacterium tuberculosis and Mycobacterium bovis is involved in the transport of toxic compounds. P55 is an efflux pump that provides resistance to several drugs, while LprG is a lipoprotein that modulates the host's immune response against mycobacteria. The knockout mutation of this operon severely reduces the replication of both mycobacterial species during infection in mice and increases susceptibility to toxic compounds. In order to gain insight into the function of LprG in the Mycobacterium avium complex, in this study, we assayed the effect of the deletion of lprG gene in the D4ER strain of Mycobacterium avium subsp. avium. The replacement of lprG gene with a hygromycin cassette caused a polar effect on the expression of p55. Also, a twofold decrease in ethidium bromide susceptibility was observed and the resistance to the antibiotics rifampicin, amikacin, linezolid, and rifabutin was impaired in the mutant strain. In addition, the mutation decreased the virulence of the bacteria in macrophages in vitro and in a mice model in vivo. These findings clearly indicate that functional LprG and P55 are necessary for the correct transport of toxic compounds and for the survival of MAA in vitro and in vivo.

Mycobacterium avium Infection after Acupoint Embedding Therapy

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Jiao Zhang, MD

2017-09-01

Full Text Available Summary:. Nontuberculous mycobacterium is a ubiquitous environmental organism that is unusual to cause a true infection, but it can cause severe cutaneous infections. In this case report, we present a successful treatment for a Chinese patient with Mycobacterium avium cutaneous infection after acupoint embedding therapy. We managed to conduct pathogenic detection, drug sensitive test, and multidisciplinary consultation. Finally, a systematic treatment strategy of nontuberculous mycobacterium was performed. Twenty-two-month follow-up revealed excellent outcome without any recurrence.

Exploring MALDI-TOF MS approach for a rapid identification of Mycobacterium avium ssp. paratuberculosis field isolates.

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Ricchi, M; Mazzarelli, A; Piscini, A; Di Caro, A; Cannas, A; Leo, S; Russo, S; Arrigoni, N

2017-03-01

The aim of the study was to explore the suitability of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid and correct identification of Mycobacterium avium ssp. paratuberculosis (MAP) field isolates. MALDI-TOF MS approach is becoming one of the most popular tests for the identification of intact bacterial cells which has been shown to be fast and reliable. For this purpose, 36 MAP field isolates were analysed through MALDI-TOF MS and the spectra compared with two different databases: one provided by the vendor of the system employed (Biotyper ver. 3·0; Bruker Daltonics) and a homemade database containing spectra from both tuberculous and nontuberculous Mycobacteria. Moreover, principal component analysis procedure was employed to confirm the ability of MALDI-TOF MS to discriminate between very closely related subspecies. Our results suggest MAP can be differentiated from other Mycobacterium species, both when the species are very close (M. intracellulare) and when belonging to different subspecies (M. avium ssp. avium and M. avium ssp. silvaticum). The procedure applied is fast, easy to perform, and achieves an earlier accurate species identification of MAP and nontuberculous Mycobacteria in comparison to other procedures. The gold standard test for the diagnosis of paratuberculosis is still isolation of MAP by cultural methods, but additional assays, such as qPCR and subculturing for determination of mycobactin dependency are required to confirm its identification. We have provided here evidence pertaining to the usefulness of MALDI-TOF MS approach for a rapid identification of this mycobacterium among other members of M. avium complex. © 2016 The Society for Applied Microbiology.

Identification of Mycobacterium avium subsp. hominissuis Isolated From Drinking Water

Science.gov (United States)

Mycobacterium avium (MA) is divided into four subspecies based primarily on host-range and consists of MA subsp. avium (birds), MA subsp. silvaticum (wood pigeons), MA subsp. paratuberculosis (broad, poorly-defined host range), and the recently described MA subsp. hominissuis (hu...

Seroprevalence of Mycobacterium avium SSP paratuberculosis ...

African Journals Online (AJOL)

This study aimed to determine the seroprevalence of antibodies for Mycobacterium avium subspecies paratuberculosis (MAP) in dairy cattle in the Jimma zone of Ethiopia in 2011. A random sample of 29 herds was selected, and all mature cattle within these herds had a blood sample taken. Serum was tested in duplicate, ...

ELECTROPHORETIC MOBILITY OF MYCOBACTERIUM AVIUM COMPLEX ORGANISMS

Science.gov (United States)

The electrophoretic mobilities (EPMs) of thirty Mycobacterium avium Complex (MAC) organisms were measured. The EPMs of fifteen clinical isolates ranged from -1.9 to -5.0 µm cm V-1s-1, and the EPMs of fifteen environmental isolates ranged from -1...

Isolation of mycobacteria other than Mycobacterium avium from porcine lymph nodes

NARCIS (Netherlands)

Ingen, van J.; Wisselink, H.J.; Solt-Smits, van C.B.; Boeree, M.J.; Soolingen, D.

2010-01-01

Mycobacterium avium causes lymphadenitis in pigs. This presents an economical burden, as these pigs meat is considered inappropriate for consumption. In humans, lymphadenitis due to nontuberculous mycobacteria (NTM) primarily affects children and is caused by a variety of NTM, though M. avium

Isolation of mycobacteria other than Mycobacterium avium from porcine lymph nodes.

NARCIS (Netherlands)

Ingen, J. van; Wisselink, H.J.; Solt-Smits, C.B. van; Boeree, M.J.; Soolingen, D. van

2010-01-01

Mycobacterium avium causes lymphadenitis in pigs. This presents an economical burden, as these pigs meat is considered inappropriate for consumption. In humans, lymphadenitis due to nontuberculous mycobacteria (NTM) primarily affects children and is caused by a variety of NTM, though M. avium

Inhibition of Adherence of Mycobacterium avium to Plumbing Surface Biofilms of Methylobacterium spp.

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Mari Carmen Muñoz Egea

2017-09-01

Full Text Available Both Mycobacterium spp. and Methylobacterium spp. are opportunistic premise plumbing pathogens that are found on pipe surfaces in households. However, examination of data published in prior microbiological surveys indicates that Methylobacterium spp. and Mycobacterium spp. tend not to coexist in the same household plumbing biofilms. That evidence led us to test the hypothesis that Methylobacterium spp. in biofilms could inhibit the adherence of Mycobacterium avium. Measurements of adherence of M. avium cells to stainless steel coupons using both culture and PCR-based methods showed that the presence of Methylobacterium spp. biofilms substantially reduced M. avium adherence and vice versa. That inhibition of M. avium adherence was not reduced by UV-irradiation, cyanide/azide exposure, or autoclaving of the Methylobacterium spp. biofilms. Further, there was no evidence of the production of anti-mycobacterial compounds by biofilm-grown Methylobacterium spp. cells. The results add to understanding of the role of microbial interactions in biofilms as a driving force in the proliferation or inhibition of opportunistic pathogens in premise plumbing, and provide a potential new avenue by which M. avium exposures may be reduced for at-risk individuals.

Mean effective sensitivity for Mycobacterium avium subsp paratuberculosis infection in cattle herds

DEFF Research Database (Denmark)

Kirkeby, Carsten; Græsbøll, Kaare; Hisham Beshara Halasa, Tariq

2015-01-01

Background: Mycobacterium avium subsp. paratuberculosis (MAP) infections in cattle are generally challenging to detect and cost-effective test strategies are consequently difficult to identify. MAP-specific antibody ELISAs for milk and serum are relatively inexpensive, but their utility is influe......Background: Mycobacterium avium subsp. paratuberculosis (MAP) infections in cattle are generally challenging to detect and cost-effective test strategies are consequently difficult to identify. MAP-specific antibody ELISAs for milk and serum are relatively inexpensive, but their utility...

Full genome sequence of a Danish isolate of Mycobacterium avium subspecies paratuberculosis, strain Ejlskov2007

DEFF Research Database (Denmark)

Afzal, Mamuna; Abidi, Soad; Mikkelsen, Heidi

We have sequenced a Danish isolate of Mycobacterium avium subspecies paratuberculosis, strain Ejlskov2007. The strain was isolated from faecal material of a 48 month old second parity Danish Holstein cow, with clinical symptoms of chronic diarrhoea and emaciation. The cultures were grown on Löwen......We have sequenced a Danish isolate of Mycobacterium avium subspecies paratuberculosis, strain Ejlskov2007. The strain was isolated from faecal material of a 48 month old second parity Danish Holstein cow, with clinical symptoms of chronic diarrhoea and emaciation. The cultures were grown......, consisting of 4317 unique gene families. Comparison with M. avium paratuberculosis strain K10 revealed only 3436 genes in common (~70%). We have used GenomeAtlases to show conserved (and unique) regions along the Ejlskov2007 chromosome, compared to 2 other Mycobacterium avium sequenced genomes. Pan......-genome analyses of the sequenced Mycobacterium genomes reveal a surprisingly open and diverse set of genes for this bacterial genera....

Improved detection of Mycobacterium avium complex with the Bactec radiometric system

International Nuclear Information System (INIS)

Hoffner, S.E.

1988-01-01

A reconsideration of the laboratory methods used for primary isolation of mycobacteria other than Mycobacterium tuberculosis is needed due to the increasingly recognized importance of such mycobacterial infections in immunocompromised patients. One example of this is the severe opportunistic infections caused by Mycobacterium avium complex among AIDS patients. In this study, the Bactec radiometric system was compared to conventional culture on solid medium for the detection of M. avium complex in 3,612 selected clinical specimens, mainly of extrapulmonary origin. Of a total number of 63 M. avium complex isolates, the Bactec system detected 58 (92%), compared to 37 (59%) for conventional culture. A much more rapid detection was attained with radiometric technique than with conventional culture. The mean detection time for the cultures positive with both methods was 7.1 and 28.3 days, respectively. The Bactec radiometric system achieves a rapid and significantly more sensitive detection and seems to be an excellent complement to conventional culture in the laboratory diagnosis of infections with the M. avium complex

Effectiveness of bronchoscopy in the diagnosis of bronchial-type mycobacterium avium-intracellulare complex pulmonary disease

International Nuclear Information System (INIS)

Sato, Kazuhiro; Kourakata, Hiroyo

2004-01-01

Mycobacterium avium-intracellulare complex (MAC) pulmonary disease with associated nodules and bronchiectasis is an increasingly prevalent condition. This condition is often difficult to diagnose in the early stages of the disease, because of the limited effectiveness of sputum culture cytology. The effectiveness of bronchoscopy in the isolation and diagnosis of MAC in respiratory secretions is still unclear. Over a three-year period, we examined the effectiveness of bronchoscopy in 45 non-HIV-infected patients who had clusters of small peripheral lung nodules. These nodules were associated with changes of the draining bronchi detected by high-resolution CT (HRCT). A total of 22 of 45 patients (48.9%) had cultures positive for MAC. In the MAC-positive group, 10 patients tested positive for disease in sputum and 22 tested positive for disease in bronchial washings. A total of 13 of 45 patients (28.9%) fulfilled the American Thoracic Society criteria for pulmonary MAC disease, and 9 (20.0%) others with cultures positive for MAC did not fulfill the criteria. Radiographic measures and sputum cultures of 13 of 16 patients (81.3%) with negative cultures revealed no further disease progression. We found that HRCT was a useful technique in the diagnosis of MAC-pulmonary disease. We also found that bronchoscopy was a more sensitive diagnostic technique than sputum culture, analysis in the differential diagnosis of MAC pulmonary diseases. (author)

Broncho-pleural fistula with hydropneumothorax at CT: Diagnostic implications in mycobacterium avium complex lung disease with pleural involvement

International Nuclear Information System (INIS)

Yoon, Hyun Jung; Chung, Myung Jin; Lee, Kyung Soo; Park, Hye Yun; Koh, Won Jung; Kim, Jung Soo

2016-01-01

To determine the patho-mechanism of pleural effusion or hydropneumothorax in Mycobacterium avium complex (MAC) lung disease through the computed tomographic (CT) findings. We retrospectively collected data from 5 patients who had pleural fluid samples that were culture-positive for MAC between January 2001 and December 2013. The clinical findings were investigated and the radiological findings on chest CT were reviewed by 2 radiologists. The 5 patients were all male with a median age of 77 and all had underlying comorbid conditions. Pleural fluid analysis revealed a wide range of white blood cell counts (410-100690/µL). The causative microorganisms were determined as Mycobacterium avium and Mycobacterium intracellulare in 1 and 4 patients, respectively. Radiologically, the peripheral portion of the involved lung demonstrated fibro-bullous changes or cavitary lesions causing lung destruction, reflecting the chronic, insidious nature of MAC lung disease. All patients had broncho-pleural fistulas (BPFs) and pneumothorax was accompanied with pleural effusion. In patients with underlying MAC lung disease who present with pleural effusion, the presence of BPFs and pleural air on CT imaging are indicative that spread of MAC infection is the cause of the effusion

Broncho-pleural fistula with hydropneumothorax at CT: Diagnostic implications in mycobacterium avium complex lung disease with pleural involvement

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Yoon, Hyun Jung; Chung, Myung Jin; Lee, Kyung Soo; Park, Hye Yun; Koh, Won Jung [Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Kim, Jung Soo [Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Inha University Hospital, Inha University School of Medicine, Incheon (Korea, Republic of)

2016-04-15

To determine the patho-mechanism of pleural effusion or hydropneumothorax in Mycobacterium avium complex (MAC) lung disease through the computed tomographic (CT) findings. We retrospectively collected data from 5 patients who had pleural fluid samples that were culture-positive for MAC between January 2001 and December 2013. The clinical findings were investigated and the radiological findings on chest CT were reviewed by 2 radiologists. The 5 patients were all male with a median age of 77 and all had underlying comorbid conditions. Pleural fluid analysis revealed a wide range of white blood cell counts (410-100690/µL). The causative microorganisms were determined as Mycobacterium avium and Mycobacterium intracellulare in 1 and 4 patients, respectively. Radiologically, the peripheral portion of the involved lung demonstrated fibro-bullous changes or cavitary lesions causing lung destruction, reflecting the chronic, insidious nature of MAC lung disease. All patients had broncho-pleural fistulas (BPFs) and pneumothorax was accompanied with pleural effusion. In patients with underlying MAC lung disease who present with pleural effusion, the presence of BPFs and pleural air on CT imaging are indicative that spread of MAC infection is the cause of the effusion.

Hemolysin as a Virulence Factor for Systemic Infection with Isolates of Mycobacterium avium Complex

Science.gov (United States)

Maslow, Joel N.; Dawson, David; Carlin, Elizabeth A.; Holland, Steven M.

1999-01-01

Isolates of the Mycobacterium avium complex were examined for hemolysin expression. Only invasive isolates of M. avium were observed to be hemolytic (P < 0.001), with activity the greatest for isolates of serovars 4 and 8. Thus, M. avium hemolysin appears to represent a virulence factor necessary for invasive disease. PMID:9889239

Intracellular pH of Mycobacterium avium subsp. paratuberculosis following exposure to antimicrobial compounds monitored at the single cell level

DEFF Research Database (Denmark)

Gaggìa, Francesca; Nielsen, Dennis Sandris; Biavati, Bruno

2010-01-01

for 24h revealed the presence of a subpopulation of cells probably resistant to the antimicrobial compounds tested. Use of nisin and bacteriocin-producing LAB strains could lead to new intervention strategies for the control of MAP based on in vivo application of probiotic cultures as feed additives......Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease; moreover, it seems to be implicated in the development of Crohn's disease in humans. In the present study, fluorescence ratio imaging microscopy (FRIM) was used to assess changes in intracellular pH (p......H(i)) of one strain of MAP after exposure to nisin and neutralized cell-free supernatants (NCSs) from five bacteriocin-producing lactic acid bacteria (LAB) with known probiotic properties. The evaluation of pH(i) by FRIM provides information about the physiological state of bacterial cells, bypassing the long...

Roles for Cell Wall Glycopeptidolipid in Surface Adherence and Planktonic Dispersal of Mycobacterium avium

Science.gov (United States)

The opportunistic pathogen Mycobacterium avium is a significant inhabitant of biofilms in drinking water distribution systems. M. avium expresses on its cell surface serovar-specific glycopeptidolipids (ssGPLs). Studies have implicated the core GPL in biofilm formation by M. aviu...

Ag85-focused T-cell immune response controls Mycobacterium avium chronic infection.

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Bruno Cerqueira-Rodrigues

Full Text Available CD4+ T cells are essential players for the control of mycobacterial infections. Several mycobacterial antigens have been identified for eliciting a relevant CD4+ T cell mediated-immune response, and numerous studies explored this issue in the context of Mycobacterium tuberculosis infection. Antigen 85 (Ag85, a highly conserved protein across Mycobacterium species, is secreted at the early phase of M. tuberculosis infection leading to the proliferation of Ag85-specific CD4+ T cells. However, in the context of Mycobacterium avium infection, little is known about the expression of this antigen and the elicited immune response. In the current work, we investigated if a T cell receptor (TCR repertoire mostly, but not exclusively, directed at Ag85 is sufficient to mount a protective immune response against M. avium. We show that P25 mice, whose majority of T cells express a transgenic TCR specific for Ag85, control M. avium infection at the same level as wild type (WT mice up to 20 weeks post-infection (wpi. During M. avium infection, Ag85 antigen is easily detected in the liver of 20 wpi mice by immunohistochemistry. In spite of the propensity of P25 CD4+ T cells to produce higher amounts of interferon-gamma (IFNγ upon ex vivo stimulation, no differences in serum IFNγ levels are detected in P25 compared to WT mice, nor enhanced immunopathology is detected in P25 mice. These results indicate that a T cell response dominated by Ag85-specific T cells is appropriate to control M. avium infection with no signs of immunopathology.

Mycobacterium avium-intracellulare infection during HIV disease. Persisting problems

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Roberto Manfredi

2008-12-01

Full Text Available Still in the era of combined antiretroviral therapy, late recognition of HIV disease or lack of sufficient immune recovery pose HIV-infected patients at risk to develop opportunistic infections by nontuberculous mycobacteria (NTM, which are environmental organisms commonly retrieved in soil and superficial waters.Among these microorganisms, the most frequent is represented by Mycobacterium avium complex (MAC. Health care professionals who face HIV-infected patients should suspect disseminated mycobacterial disease when a deep immunodeficiency is present, (a CD4+ lymphocyte count below 50 cells/μL often associated with constitutional signs and symptoms, and non-specific laboratory abnormalities. Mycobacterial culture of peripheral blood is a reliable technique for diagnosing disseminated disease. Among drugs active against NTM, as well as some anti-tubercular compounds, the rifampin derivative rifabutin, and some novel fluoroquinolones, the availability of macrolides, has greatly contributed to improve both prophylaxis and treatment outcome of disseminated MAC infections. Although multiple questions remain about which regimens may be regarded as optimal, general recommendations can be expressed on the ground of existing evidences.Treatment should begin with associated clarithromycin (or azithromycin, plus ethambutol and rifabutin (with the rifabutin dose depending on other concomitant medications that might result in drug-drug interactions.A combined three-drug regimen is preferred for patients who cannot be prescribed an effective antiretroviral regimen immediately. Patients with a CD4+ lymphocyte count below 50 cells/μL, who do not have clinical evidence of active mycobacterial disease, should receive a primary prophylaxis with either clarithromycin or azithromycin, with or without rifabutin.

Genome-wide sequence variations among Mycobacterium avium subspecies paratuberculosis.

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Chung-Yi eHsu

2011-12-01

Full Text Available Mycobacterium avium subspecies paratuberculosis (M. ap, the causative agent of Johne’s disease (JD, infects many farmed ruminants, wildlife animals and humans. To better understand the molecular pathogenesis of these infections, we analyzed the whole genome sequences of several M. ap and M. avium subspecies avium (M. avium strains isolated from various hosts and environments. Using Next-generation sequencing technology, all 6 M. ap isolates showed a high percentage of homology (98% to the reference genome sequence of M. ap K-10 isolated from cattle. However, 2 M. avium isolates (DT 78 and Env 77 showed significant sequence diversity from the reference strain M. avium 104. The genomes of M. avium isolates DT 78 and Env 77 exhibited only 87% and 40% homology, respectively, to the M. avium 104 reference genome. Within the M. ap isolates, genomic rearrangements (insertions/deletions, Indels were not detected, and only unique single nucleotide polymorphisms (SNPs were observed among the 6 M. ap strains. While most of the SNPs (~100 in M. ap genomes were non-synonymous, a total of ~ 6000 SNPs were detected among M. avium genomes, most of them were synonymous suggesting a differential selective pressure between M. ap and M. avium isolates. In addition, SNPs-based phylo-genomic analysis showed that isolates from goat and Oryx are closely related to the cattle (K-10 strain while the human isolate (M. ap 4B is closely related to the environmental strains, indicating environmental source to human infections. Overall, SNPs were the most common variations among M. ap isolates while SNPs in addition to Indels were prevalent among M. avium isolates. Genomic variations will be useful in designing host-specific markers for the analysis of mycobacterial evolution and for developing novel diagnostics directed against Johne’s disease in animals.

Assessing the inactivation of Mycobacterium avium subsp. paratuberculosis during composting of livestock carcasses.

Science.gov (United States)

Tkachuk, Victoria L; Krause, Denis O; McAllister, Tim A; Buckley, Katherine E; Reuter, Tim; Hendrick, Steve; Ominski, Kim H

2013-05-01

Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants, with substantial economic impacts on the cattle industry. Johne's disease is known for its long latency period, and difficulties in diagnosis are due to insensitivities of current detection methods. Eradication is challenging as M. avium subsp. paratuberculosis can survive for extended periods within the environment, resulting in new infections in naïve animals (W. Xu et al., J. Environ. Qual. 38:437-450, 2009). This study explored the use of a biosecure, static composting structure to inactivate M. avium subsp. paratuberculosis. Mycobacterium smegmatis was also assessed as a surrogate for M. avium subsp. paratuberculosis. Two structures were constructed to hold three cattle carcasses each. Naturally infected tissues and ground beef inoculated with laboratory-cultured M. avium subsp. paratuberculosis and M. smegmatis were placed in nylon and plastic bags to determine effects of temperature and compost environment on viability over 250 days. After removal, samples were cultured and growth of both organisms was assessed after 12 weeks. After 250 days, M. avium subsp. paratuberculosis was still detectable by PCR, while M. smegmatis was not detected after 67 days of composting. Furthermore, M. avium subsp. paratuberculosis remained viable in both implanted nylon and plastic bags over the composting period. As the compost never reached a homogenous thermophilic (55 to 65°C) state throughout each structure, an in vitro experiment was conducted to examine viability of M. avium subsp. paratuberculosis after exposure to 80°C for 90 days. Naturally infected lymph tissues were mixed with and without compost. After 90 days, M. avium subsp. paratuberculosis remained viable despite exposure to temperatures typically higher than that achieved in compost. In conclusion, it is unlikely composting can be used as a means of inactivating M. avium subsp. paratuberculosis associated with cattle

Assessing the Inactivation of Mycobacterium avium subsp. paratuberculosis during Composting of Livestock Carcasses

Science.gov (United States)

Tkachuk, Victoria L.; Krause, Denis O.; McAllister, Tim A.; Buckley, Katherine E.; Reuter, Tim; Hendrick, Steve

2013-01-01

Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants, with substantial economic impacts on the cattle industry. Johne's disease is known for its long latency period, and difficulties in diagnosis are due to insensitivities of current detection methods. Eradication is challenging as M. avium subsp. paratuberculosis can survive for extended periods within the environment, resulting in new infections in naïve animals (W. Xu et al., J. Environ. Qual. 38:437-450, 2009). This study explored the use of a biosecure, static composting structure to inactivate M. avium subsp. paratuberculosis. Mycobacterium smegmatis was also assessed as a surrogate for M. avium subsp. paratuberculosis. Two structures were constructed to hold three cattle carcasses each. Naturally infected tissues and ground beef inoculated with laboratory-cultured M. avium subsp. paratuberculosis and M. smegmatis were placed in nylon and plastic bags to determine effects of temperature and compost environment on viability over 250 days. After removal, samples were cultured and growth of both organisms was assessed after 12 weeks. After 250 days, M. avium subsp. paratuberculosis was still detectable by PCR, while M. smegmatis was not detected after 67 days of composting. Furthermore, M. avium subsp. paratuberculosis remained viable in both implanted nylon and plastic bags over the composting period. As the compost never reached a homogenous thermophilic (55 to 65°C) state throughout each structure, an in vitro experiment was conducted to examine viability of M. avium subsp. paratuberculosis after exposure to 80°C for 90 days. Naturally infected lymph tissues were mixed with and without compost. After 90 days, M. avium subsp. paratuberculosis remained viable despite exposure to temperatures typically higher than that achieved in compost. In conclusion, it is unlikely composting can be used as a means of inactivating M. avium subsp. paratuberculosis associated with cattle

Disseminated Mycobacterium avium complex in an immunocompetent host

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Joseph M Yabes

2017-01-01

Full Text Available Disseminated Mycobacterium avium complex (DMAC has historically been described in the immunocompromised. The current epidemiologic research suggests that the incidence of nontuberculous mycobacterial infections is increasing. We present a case of DMAC infection manifesting as hepatic granulomas in a 35-year-old immunocompetent female. This case suggests DMAC infection in a patient without traditional epidemiological risk factors.

Characterization of clinical and environmental Mycobacterium avium spp. isolates and their interaction with human macrophages

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Members of the Mycobacterium avium complex (MAC) are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking water and clinical isolates from infected individuals. There is a need to develop new screening methodologies tha...

Mycobacterium avium subsp. paratuberculosis infection, immunology and pathology of livestock

Science.gov (United States)

Mycobacterium avium subsp. paratuberculosis (MAP) infection in ruminants leads to a chronic and progressive enteric disease (Johne’s disease) that results in loss of intestinal function, poor body condition, and eventual death. Transmission is primarily through a fecal-oral route in neonates but con...

Development of a novel oral vaccine against Mycobacterium avium paratuberculosis and Johne disease

Science.gov (United States)

Johnston, C; Coffey, A; Sleator, RD

2010-01-01

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne disease, a granulomatous enteritis of cattle and other domesticated and wild ruminant species. Johne disease is prevalent worldwide and has a significant impact on the global agricultural economy. Current vaccines against Johne are insufficient in stemming its spread, and associated side-effects prevent their widespread use in control programs. Effective and safe vaccine strategies are needed. The main purpose of this paper is to propose and evaluate the development of a novel oral subunit-vaccine using a patho-biotechnological approach. This novel strategy, which harnesses patho-genetic elements from the intracellular pathogen Listeria monocytogenes, may provide a realistic route towards developing an effective next generation subunit vaccine against Johne disease and paratuberculosis. PMID:21326921

Mycobacterium avium subsp. avium in lymph nodes and diaphragms of pigs from one infected herd in the Czech Republic.

Science.gov (United States)

Kriz, Petr; Kaevska, Marija; Slana, Iva; Bartejsova, Iva; Pavlik, Ivo

2014-01-01

This study was performed on 40 finished pigs from one herd naturally infected with Mycobacterium avium subsp. avium. The aim was to investigate the presence and amount of M. a. avium in samples of lymph nodes and diaphragm tissues collected during routine postmortem inspection using the triplex quantitative real time PCR (qPCR) method. We collected, in total, 107 samples: various lymph nodes affected by gross tuberculosis (TB)-like lesions from 17 pig carcasses, as well as samples of head and mesenteric lymph nodes from 23 carcasses without TB-like lesions. Samples of diaphragm tissues were collected from all carcasses. M. a. avium was detected in one or more tissue samples collected from half of the slaughtered pigs tested. Samples of diaphragm tissues of three pigs with detected TB-like lesions contained M. a. avium (10(2) to 10(3) cells per g of sample); the organism was not detected in diaphragm tissues from pigs without TB-like lesions. The qPCR method may be useful for quantification of M. a. avium in pigs for the purposes of foodborne risk assessment.

Shared Mycobacterium avium genotypes observed among unlinked clinical and environmental isolates

Science.gov (United States)

Our understanding of the sources of Mycobacterium avium infection is partially based on genotypic matching of pathogen isolates from cases and environmental sources. These approaches assume that genotypic identity is rare in isolates from unlinked cases or sources. To test this, ...

Shared Mycobacterium avium genotypes observed among unlinked clinical and environmental isolates*

Science.gov (United States)

Our understanding of the sources of Mycobacterium avium infection is partially based on genotypic matching of pathogen isolates from cases and environmental sources. These approaches assume that genotypic identity is rare in isolates from unlinked cases or sources. To test this a...

Serovars of Mycobacterium avium Complex isolated from patients in Denmark

DEFF Research Database (Denmark)

Askgaard, D. S.; Giese, Steen Bjørck; Thybo, S.

1994-01-01

Danish isolates of Mycobacterium avium complex were serotyped by the use of seroagglutination. The most prevalent serovars among patients with AIDS (n = 89) were 4 and 6, while among non-AIDS patients the most prevalent serovars were 1, 6, and 4, with no major differences between those in patients...... with pulmonary disease (n = 65) and those in patients with lymph node infection (n = 58). The results suggest a Scandinavian distribution of serovars with a predominance of serovar 6 and fail to demonstrate any selective protection against different serovars by Mycobacterium bovis ECG vaccination....

Association between milk antibody and interferon-gamma responses in cattle from Mycobacterium avium subsp. paratuberculosis infected herds

DEFF Research Database (Denmark)

Mikkelsen, Heidi; Jungersen, Gregers; Nielsen, Søren Saxmose

2009-01-01

Paratuberculosis is a chronic infection of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). It is possible to detect infection with paratuberculosis at different stages of disease by means of various diagnostic test strategies. The objective of the present study was to evalu......Paratuberculosis is a chronic infection of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). It is possible to detect infection with paratuberculosis at different stages of disease by means of various diagnostic test strategies. The objective of the present study...

Composition and potency characterization of Mycobacterium avium subsp. paratuberculosis purified protein derivatives

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Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives (PPDs) are immunologic reagents prepared from cultured filtrates of the type strain ATCC 19698. Traditional production consists of floating culture incubation at 37oC, organism inactivation by autoclaving, coarse filtrat...

ISOLATION OF THE GENOME SEQUENCE STRAIN MYCOBACTERIUM AVIUM 104 FROM MULTIPLE PATIENTS OVER A 17-YEAR PERIOD

Science.gov (United States)

The genome sequence strain 104 of the opportunistic pathogen Mycobacterium avium was isolated form an adult AIDS patient in Southern California in 1983. Isolates of non-paratuberculosis M. avium from 207 other patients in Southern California and elsewhere were examined for genoty...

Draft Genome Sequence of Mycobacterium chimaera Type ...

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-0169T possesses unique virulence genes. Evidence suggests that M. avium, M. intracellulare, and M. chimaera are differently virulent and a comparative genomic analysis is critically needed to identify diagnostic targets that reliably differentiate species of MAC. With treatment costs for Mycobacterium infections estimated to be >$1.8 B annually in the U.S., correct species identification will result in improved treatment selection, lower costs, and improved patient outcomes.

Mycobacterium avium genotype is associated with the therapeutic response to lung infection

Science.gov (United States)

Kikuchi, T; Kobashi, Y; Hirano, T; Tode, N; Santoso, A; Tamada, T; Fujimura, S; Mitsuhashi, Y; Honda, Y; Nukiwa, T; Kaku, M; Watanabe, A; Ichinose, M; Drancourt, M

2014-01-01

Factors that can interfere with the successful treatment of Mycobacterium avium lung infection have been inadequately studied. To identify a potent predictor of therapeutic responses of M. avium lung infection, we analyzed variable number tandem repeats (VNTR) at 16 minisatellite loci of M. avium clinical isolates. Associations between the VNTR profiling data and a therapeutic response were evaluated in 59 subjects with M. avium lung infection. M. avium lung infection of 30 subjects in whom clarithromycin-containing regimens produced microbiological and radiographic improvement was defined as responsive disease, while that of the remaining 29 subjects was defined as refractory disease. In phylogenetic analysis using the genotypic distance aggregated from 16-dimensional VNTR data, 59 M. avium isolates were divided into three clusters, which showed a nearly significant association with therapeutic responses (p 0.06). We then subjected the raw 16-dimensional VNTR data directly to principal component analysis, and identified the genetic features that were significantly associated with the therapeutic response (p VNTR data from only four minisatellite loci. In conclusion, we identified four mycobacterial minisatellite loci that together were associated with the therapeutic response of M. avium lung infections. PMID:23829301

Inactivation of Mycobacterium avium with free chlorine.

Science.gov (United States)

Luh, Jeanne; Mariñas, Benito J

2007-07-15

The inactivation kinetics of Mycobacterium avium with free chlorine was characterized by two stages: an initial phase at a relatively fast rate followed by a slower second stage of pseudo first-order kinetics. The inactivation rate of each stage was approximately the same for all experiments performed at a certain condition of pH and temperature; however, variability was observed for the disinfectant exposure at which the transition between the two stages occurred. This variability was not a function of the initial disinfectant concentration, the initial bacterial density, or the bacterial stock. However, the transition to the second stage varied more significantly at high temperatures (30 degrees C), while lower variability was observed at lower temperatures (5 and 20 degrees C). Experiments conducted at pH values in the range of 6-9 revealed that the inactivation of M. avium was primarily due to hypochlorous acid, with little contribution from hypochlorite ion within this pH range. The inactivation kinetics was represented with a two-population model. The activation energies for the resulting pseudo first-order rate constants for the populations with fast and slow kinetics were 100.3 and 96.5 kJ/mol, respectively. The magnitude of these values suggested that for waters of relatively high pH and low temperatures, little inactivation of M. avium would be achieved within treatment plants, providing a seeding source for distribution systems.

Mycobacterium avium subspecies paratuberculosis recombinant proteins modulate antimycobacterial functions of bovine macrophages

Science.gov (United States)

It has been shown that Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) activates the Mitogen Activated Protein Kinase (MAPK) p38 pathway, yet it is unclear which components of M. paratuberculosis are involved in the process. Therefore, a set of 42 M. paratuberculosis recombinan...

Systems Analysis of Early Host Gene Expression Provides Clues for Transient Mycobacterium avium ssp avium vs. Persistent Mycobacterium avium ssp paratuberculosis Intestinal Infections.

Science.gov (United States)

Khare, Sangeeta; Drake, Kenneth L; Lawhon, Sara D; Nunes, Jairo E S; Figueiredo, Josely F; Rossetti, Carlos A; Gull, Tamara; Everts, Robin E; Lewin, Harris A; Adams, Leslie Garry

It has long been a quest in ruminants to understand how two very similar mycobacterial species, Mycobacterium avium ssp. paratuberculosis (MAP) and Mycobacterium avium ssp. avium (MAA) lead to either a chronic persistent infection or a rapid-transient infection, respectively. Here, we hypothesized that when the host immune response is activated by MAP or MAA, the outcome of the infection depends on the early activation of signaling molecules and host temporal gene expression. To test our hypothesis, ligated jejuno-ileal loops including Peyer's patches in neonatal calves were inoculated with PBS, MAP, or MAA. A temporal analysis of the host transcriptome profile was conducted at several times post-infection (0.5, 1, 2, 4, 8 and 12 hours). When comparing the transcriptional responses of calves infected with the MAA versus MAP, discordant patterns of mucosal expression were clearly evident, and the numbers of unique transcripts altered were moderately less for MAA-infected tissue than were mucosal tissues infected with the MAP. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis. Bayesian network modeling identified mechanistic genes, gene-to-gene relationships, pathways and Gene Ontologies (GO) biological processes that are involved in specific cell activation during infection. MAP and MAA had significant different pathway perturbation at 0.5 and 12 hours post inoculation. Inverse processes were observed between MAP and MAA response for epithelial cell proliferation, negative regulation of chemotaxis, cell-cell adhesion mediated by integrin and regulation of cytokine-mediated signaling. MAP inoculated tissue had significantly lower expression of phagocytosis receptors such as mannose receptor and complement receptors. This study reveals that perturbation of genes and cellular pathways during MAP infection resulted in host evasion by mucosal membrane barrier weakening to access entry in the ileum

The persistence of Mycobacterium avium in a drinking water system, what is the risk to human health?

Science.gov (United States)

Drinking water is believed to be a major source of human exposure to nontuberculous mycobacteria (NTM) such as Mycobacterium avium. We monitored the prevalence of M. avium in a drinking water system during the addition of filtration treatment. Our goal was to determine if the pre...

Thermal Inactivation of Mycobacterium avium subsp. paratuberculosis in Artificially Contaminated Milk by Direct Steam Injection

Science.gov (United States)

Butot, Sophie; Jagadeesan, Balamurugan; Bakker, Douwe; Donaghy, John

2016-01-01

ABSTRACT The efficiency of direct steam injection (DSI) at 105°C for 3 s to inactivate Mycobacterium avium subsp. paratuberculosis in milk at a pilot-plant scale was investigated. Milk samples were artificially contaminated with M. avium subsp. paratuberculosis and also with cow fecal material naturally infected with M. avium subsp. paratuberculosis. We also tested milk artificially contaminated with Mycobacterium smegmatis as a candidate surrogate to compare thermal inactivation between M. smegmatis and M. avium subsp. paratuberculosis. Following the DSI process, no viable M. avium subsp. paratuberculosis or M. smegmatis was recovered using culture methods for both strains. For pure M. avium subsp. paratuberculosis cultures, a minimum reduction of 5.6 log10 was achieved with DSI, and a minimum reduction of 5.7 log10 was found with M. smegmatis. The minimum log10 reduction for wild-type M. avium subsp. paratuberculosis naturally present in feces was 3.3. In addition, 44 dairy and nondairy powdered infant formula (PIF) ingredients used during the manufacturing process of PIF were tested for an alternate source for M. avium subsp. paratuberculosis and were found to be negative by quantitative PCR (qPCR). In conclusion, the results obtained from this study indicate that a >7-fold-log10 reduction of M. avium subsp. paratuberculosis in milk can be achieved with the applied DSI process. IMPORTANCE M. avium subsp. paratuberculosis is widespread in dairy herds in many countries. M. avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle, and infected animals can directly or indirectly (i.e., fecal contamination) contaminate milk. Despite much research and debate, there is no conclusive evidence that M. avium subsp. paratuberculosis is a zoonotic bacterium, i.e., one that causes disease in humans. The presence of M. avium subsp. paratuberculosis or its DNA has been reported in dairy products, including pasteurized milk, cheese, and infant formula

Different Mycobacterium avium subsp. paratuberculosis MIRU-VNTR patterns coexist within cattle herds

NARCIS (Netherlands)

Hulzen, van K.J.E.; Heuven, H.C.M.; Nielen, M.; Hoeboer, J.; Santema, W.J.; Koets, A.P.

2011-01-01

A better understanding of the biodiversity of Mycobacterium avium subsp. paratuberculosis (MAP) offers more insight in the epidemiology of paratuberculosis and therefore may contribute to the control of the disease. The aim of this study was to investigate the genetic diversity in bovine MAP

Concomitant Mycobacterium avium infection and Hodgkin's disease in a lymph node from an HIV-negative child.

Science.gov (United States)

de Armas, Yaxsier; Capó, Virginia; González, Ida; Mederos, Lilian; Díaz, Raúl; de Waard, Jacobus H; Rodríguez, Alberto; García, Yarmila; Cabanas, Ricardo

2011-03-01

We report a case of an immunocompetent child with simultaneously an infection with Mycobacterium avium and Hodgkin's disease in a cervical lymph node. A positive PCR result for M. avium on a biopsy of the lymph node directed the definitive diagnosis for both etiologies and avoided a possible dissemination of this infection after chemotherapy was started.

Infections with Mycobacterium tuberculosis and Mycobacterium avium among HIV-infected patients after the introduction of highly active antiretroviral therapy. EuroSIDA Study Group JD

DEFF Research Database (Denmark)

Kirk, O; Gatell, J M; Mocroft, A

2000-01-01

the introduction of HAART, using data from the EuroSIDA study, a European, multicenter observational cohort of more than 7,000 patients. Overall incidences of Mycobacterium tuberculosis (TB) and Mycobacterium avium complex (MAC) were 0.8 and 1.4 cases/100 person-years of follow-up (PYF), decreasing from 1.8 (TB...

AMPLIFIED FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF MYCOBACTERIUM AVIUM COMPLEX ISOLATES RECOVERED FROM SOUTHERN CALIFORNIA

Science.gov (United States)

Fine-scale genotyping methods are necessary in order to identify possible sources of human exposure to opportunistic pathogens belonging to the Mycobacterium avium complex (MAC). In this study, amplified fragment length polymorphism (AFLP) analysis was evaluated for fingerprintin...

Rapid identification of Mycobacterium avium ssp paratuberculosis laboratory strains by IS900-Nested polymerase chain reaction.

Science.gov (United States)

Taheri, Mohammad Mohammad; Mosavari, Nader; Feizabadi, Mohammad Mehdi; Tadayon, Keyvan; Keshavarz, Rouholah; Pajoohi, Reza Aref; Soleimani, Kioomars; Pour, Shojaat Dashti

2016-12-01

Mycobacterium avium ssp paratuberculosis (MAP) causes paratuberculosis (Johne's disease) in ruminants. As a species, M. avium comprises M. avium subsp. hominissuis and a number of clones that are known to have evolved from this subspecies, namely M. avium subsp. avium (MAA), M. avium subsp. silvaticum, and MAP. Despite the very high genomic similarity of MAP and MAA, the insertion sequence IS900, which is 1,451-bp long, is now understood to be exclusively present in 10-20 copies in the genome of MAP. In the present study, a multidiscipline polymerase chain reaction (PCR)-based algorithm targeting16SrRNA, IS6110, IS901, IS1245, and IS900 markers has been employed to differentiate between six laboratory strains of M. avium complex (including MAP 316F, III&V, and 2e plus MAA D4), Mycobacterium tuberculosis DT, and Mycobacterium bovis AN5 strains used at the Razi Institute (Tehran, Iran) for the preparation of paratuberculin, avian, human, and bovine tuberculin, respectively. Three laboratory strains of III&V, 2e, and 316F were subcultured on Herrold's egg yolk medium, whereas the MAA strain of D4 along with M. bovis AN5 and M. tuberculosis DT were subcultured on Lowenstein-Jensen slopes. All the inoculated culture tubes were incubated for 8weeks at 37°C. Eventually, their genomic DNA was extracted according to the method of van Soolingen. Five individual PCRs were conducted on these templates to amplify 16SrRNA (genus-specific marker shared by all mycobacteria), IS900 (MAP-specific marker), IS901 (MAA-specific marker), IS1245 (M. avium complex (MAC)-specific marker), and IS6110 (M. tuberculosis complex (MTC)-specific marker) loci. Consequently, a 543-bp amplicon was amplified by all the six strains in PCR against 16SrRNA, an indication of their identity as members of Mycobacterium genus. A 245-bp fragment was detected in only IS6110-PCR with M. bovis AN5 as well as M. tuberculosis DT. In the IS1245 assessment, the MAA strain of D4 produced a 427-bp amplicon, whereas

Mycobacterium avium subsp. paratuberculosis: presencia en los alimentos y su relación con la enfermedad de Crohn Mycobacterium avium subsp. paratuberculosis in food and its relationship with Crohn's disease

Directory of Open Access Journals (Sweden)

K. Cirone

2007-03-01

Full Text Available La paratuberculosis o enfermedad de Johne es una enteritis crónica producida por Mycobacterium avium subsp. paratuberculosis, que afecta a bovinos y a otras especies. En la Argentina se ha caracterizado en rodeos bovinos y de ciervos, con aislamientos tipificados en distintos patrones genéticos. M. avium subsp. paratuberculosis ha sido vinculado en humanos con una inflamación crónica del intestino, denominada enfermedad de Crohn. Existen evidencias clínicas y experimentales que relacionan a M. avium subsp. paratuberculosis con la enfermedad en el humano, mediante su detección por PCR y por cultivo a partir de biopsias de órganos, de leche materna y de sangre de pacientes afectados. La leche y sus subproductos serían posibles fuentes de infección y se ha sugerido que M. avium subsp. paratuberculosis resistiría las condiciones de pasteurización. Diversos trabajos de investigación demostraron que esta micobacteria podría estar presente en leches comercializadas en diversos países, como Reino Unido, Estados Unidos, República Checa, y también en la Argentina. La presencia de M. avium subsp. paratuberculosis en productos lácteos y agua de consumo ha sido relacionada con la resistencia del microorganismo tanto a los procesos de elaboración como a los factores climáticos adversos, lo que enfatiza el rol de los alimentos y del agua como vías de transmisión al humano. Las investigaciones en curso podrían ratificar el riesgo y las implicancias de la exposición del humano a M. avium subsp. paratuberculosis a través de los alimentos y del agua contaminados, para determinar la importancia de la paratuberculosis como enfermedad zoonótica.Paratuberculosis or Johne's disease is a chronic enteritis of the cattle and other small ruminant animals caused by Mycobacterium avium subsp. paratuberculosis. In Argentina, the strains were characterized in beef and dairy cattle and deer in different genetic patterns by molecular tools. M. avium

Utilization of Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) to Identify environmental Strains of Mycobacterium Complex

Science.gov (United States)

Species within the Mycobacterium avium Complex (MAC) group are found to be both prevalent and persistent in drinking water distribution systems. The MAC is composed of two predominant species: M. avium and M. intracellulare. These species have the ability to survive drinking ...

Epidemiology and Ecology of Opportunistic Premise Plumbing Pathogens: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa

Science.gov (United States)

BACKGROUND: Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa are opportunistic premise plumbing pathogens (OPPPs) that persist and grow in household plumbing, habitats they share with humans. Infections caused by these OPPPs involve individuals with preexis...

Detection of Mycobacterium avium subspecies paratuberculosis in Drinking Water and Biofilms Using Quantitative PCR

Science.gov (United States)

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease in domestic animals and has been implicated in Crohn’s disease in humans. This bacterium is a slow growing, gram-positive, acid-fast organism which can be difficult to culture from the environment. For ...

Inferring biomarkers for Mycobacterium avium subsp. paratuberculosis infection and disease progression using experimental data

Science.gov (United States)

Available diagnostic assays for Mycobacterium avium subsp paratuberculosis (MAP) have poor sensitivities and cannot detect early stages of the infection, therefore, there is need to find new diagnostic markers for early infection detection and disease stages. We analyzed longitudinal IFN- gamma, ELI...

Molecular analysis and MIRU-VNTR typing of Mycobacterium avium subsp. avium, 'hominissuis' and silvaticum strains of veterinary origin.

Science.gov (United States)

Rónai, Zsuzsanna; Csivincsik, Ágnes; Dán, Ádám; Gyuranecz, Miklós

2016-06-01

Besides Mycobacterium avium subsp. paratuberculosis (MAP), M. avium subsp. avium (MAA), M. avium subsp. silvaticum (MAS), and 'M. avium subsp. hominissuis' (MAH) are equally important members of M. avium complex, with worldwide distribution and zoonotic potential. Genotypic discrimination is a prerequisite to epidemiological studies which can facilitate disease prevention through revealing infection sources and transmission routes. The primary aim of this study was to identify the genetic diversity within 135 MAA, 62 MAS, and 84 MAH strains isolated from wild and domestic mammals, reptiles and birds. Strains were tested for the presence of large sequence polymorphism LSP(A)17 and were submitted to Mycobacterial interspersed repetitive units-variable-number tandem repeat (MIRU-VNTR) analysis at 8 loci, including MIRU1, 2, 3, and 4, VNTR25, 32, and 259, and MATR9. In 12 strains hsp65 sequence code type was also determined. LSP(A)17 was present only in 19.9% of the strains. All LSP(A)17 positive strains belonged to subspecies MAH. The discriminatory power of the MIRU-VNTR loci set used reached 0.9228. Altogether 54 different genotypes were detected. Within MAH, MAA, and MAS strains 33, 16, and 5 different genotypes were observed. The described genotypes were not restricted to geographic regions or host species, but proved to be subspecies specific. Our knowledge about MAS is limited due to isolation and identification difficulties. This is the first study including a large number of MAS field strains. Our results demonstrate the high diversity of MAH and MAA strains and the relative uniformity of MAS strains. Copyright © 2016 Elsevier B.V. All rights reserved.

Detection of Mycobacterium avium subsp. paratuberculosis in Drinking Water and Biofilms Using Quantitative PCR

Science.gov (United States)

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease in domestic animals and has been implicated in Crohn’s disease in humans. Cows infected with Johne’s disease shed large quantities of MAP into soil. Further, MAP has been isolated from surface water, is resi...

Mycobacterium avium subspecies paratuberculosis: A possible causative agent in human morbidity and risk to public health safety

Directory of Open Access Journals (Sweden)

Mary Garvey

2018-05-01

Full Text Available Mycobacterium avium subspecies paratuberculosis is a bacterial parasite and the causative agent of paratuberculosis, a disease predominately found in cattle and sheep. Infection with this microorganism results in substantial farming economic losses and animal morbidity. The link between infection with this pathogen and human disease has been theorised for many years with Crohn’s disease being one of many suspected resultant conditions. Mycobacterium avium may be spread from animal to human hosts by water and foodborne transmission routes, where the foodborne route of exposure represents a significant risk for susceptible populations, namely children and the immune-compromised. Following colonisation of the host, the parasitic organism evades the host immune system by use of molecular mimicry, displaying peptide sequences similar to that of the host cells causing a disruption of self-verses non self-recognition. Theoretically, this failure to recognise the invading organism as distinct from host cells may result in numerous autoimmune conditions. Here, the author presents current information assessing the link between numerous diseases states in humans such inflammatory bowel disease, Type 1 diabetes, rheumatoid arthritis, Hashimoto\\'s thyroiditis, multiple sclerosis and autism following infection with Mycobacterium avium paratuberculosis. The possibility of zoonotic transmission of the organism and its significant risk to public health safety as a consequence is also discussed.

Lymphoproliferative and gamma interferon responses to stress-regulated Mycobacterium avium subsp. paratuberculosis recombinant proteins

Science.gov (United States)

Johne’s disease in ruminants is a chronic infection of the intestines caused by Mycobacterium avium subsp. paratuberculosis. Economic losses associated with Johne’s disease arise due to premature culling, reduced production of milk and wool and mortalities. The disease is characterised by a long inc...

A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

Science.gov (United States)

Pooley, Hannah B.; de Silva, Kumudika; Purdie, Auriol C.; Begg, Douglas J.; Whittington, Richard J.

2016-01-01

ABSTRACT Determining the viability of bacteria is a key outcome of in vitro cellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viable M. avium subsp. paratuberculosis cells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and dead M. avium subsp. paratuberculosis organisms and their accuracy at low bacterial concentrations. Using the culture-based method, M. avium subsp. paratuberculosis growth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viable M. avium subsp. paratuberculosis cells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples from in vitro cellular infection assays. IMPORTANCE Rapid quantification of the viability of Mycobacterium avium subsp. paratuberculosis in samples from in vitro cellular infection assays is important, as it allows these assays to be carried out on a large scale. In vitro cellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regarding M. avium subsp. paratuberculosis viability after an in vitro infection assay; however, with the quantitative PCR and liquid culture method

Mycobacterium intracellulare Infection Mimicking Progression of Scleroderma

DEFF Research Database (Denmark)

Krabbe, Simon; Engelhart, Merete; Thybo, Sören

2017-01-01

This case report describes a patient with scleroderma who developed Mycobacterium intracellulare infection, which for more than a year mimicked worsening of her connective tissue disorder. The patient was diagnosed with scleroderma based on puffy fingers that developed into sclerodactyly, abnormal......, unfortunately with significant scarring. Immunodeficiency testing was unremarkable. In summary, an infection with Mycobacterium intracellulare was mistaken for an unusually severe progression of scleroderma....

Cellular immune responses to ESAT-6 discriminate between patients with pulmonary disease due to Mycobacterium avium complex and those with pulmonary disease due to Mycobacterium tuberculosis

DEFF Research Database (Denmark)

Lein, A D; von Reyn, C F; Ravn, P

1999-01-01

ESAT-6 (for 6-kDa early secreted antigenic target) is a secreted antigen found almost exclusively in organisms of the Mycobacterium tuberculosis complex. We compared in vitro gamma interferon (IFN-gamma) responses by peripheral blood mononuclear cells to this antigen in patients with pulmonary...... disease due to either Mycobacterium avium complex (MAC) or Mycobacterium tuberculosis with those in healthy, skin test-negative, control subjects. Significant IFN-gamma responses to ESAT-6 were detected in 16 (59%) of 27 M. tuberculosis pulmonary disease patients, 0 (0%) of 8 MAC disease patients, and 0...... (0%) of 8 controls. Significant IFN-gamma responses to M. tuberculosis purified protein derivative were detected in 23 (85%) of 27 M. tuberculosis disease patients, 2 (25%) of 8 MAC disease patients, and 5 (63%) of 8 healthy controls. M. avium sensitin was recognized in 24 (89%) of 27 M. tuberculosis...

Comparison of a Variable-Number Tandem-Repeat (VNTR) Method for Typing Mycobacterium avium with Mycobacterial Interspersed Repetitive-Unit-VNTR and IS1245 Restriction Fragment Length Polymorphism Typing▿ †

OpenAIRE

Inagaki, Takayuki; Nishimori, Kei; Yagi, Tetsuya; Ichikawa, Kazuya; Moriyama, Makoto; Nakagawa, Taku; Shibayama, Takami; Uchiya, Kei-ichi; Nikai, Toshiaki; Ogawa, Kenji

2009-01-01

Mycobacterium avium complex (MAC) infections are increasing annually in various countries, including Japan, but the route of transmission and pathophysiology of the infection remain unclear. Currently, a variable-number tandem-repeat (VNTR) typing method using the Mycobacterium avium tandem repeat (MATR) loci (MATR-VNTR) is employed in Japan for epidemiological studies using clinical isolates of M. avium. In this study, the usefulness of this MATR-VNTR typing method was compared with that of ...

Mycobacterium avium subsp. Paratuberculosis (MAP) as a modifying factor in Crohn's disease.

LENUS (Irish Health Repository)

Sibartie, Shomik

2010-02-01

Crohn\\'s disease (CD) is a multifactorial syndrome with genetic and environmental contributions. Mycobacterium avium subspecies paratuberculosis (MAP) has been frequently isolated from mucosal tissues of patients with CD but the cellular immune response to this bacterium has been poorly described. Our aim was to examine the influence of MAP on T-cell proliferation and cytokine responses in patients with inflammatory bowel disease (IBD).

Mycobacterium spp. in wild game in Slovenia.

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Pate, Mateja; Zajc, Urška; Kušar, Darja; Žele, Diana; Vengušt, Gorazd; Pirš, Tina; Ocepek, Matjaž

2016-02-01

Wildlife species are an important reservoir of mycobacterial infections that may jeopardise efforts to control and eradicate bovine tuberculosis (bTB), caused by Mycobacterium bovis. Slovenia is officially free of bTB, but no data on the presence of mycobacteria in wild animals has been reported. In this study, samples of liver and lymph nodes were examined from 306 apparently healthy free-range wild animals of 13 species in Slovenia belonging to the families Cervidae, Suidae, Canidae, Mustelidae and Bovidae. Mycobacteria were isolated from 36/306 (11.8%) animals (red deer, roe deer, fallow deer, wild boar and jackal) and identified by PCR, commercial diagnostic kits and sequencing. Non-tuberculous mycobacteria identified in five species were Mycobacterium peregrinum, M. avium subsp. hominissuis, M. intracellulare, M. confluentis, M. fortuitum, M. terrae, M. avium subsp. avium, M. celatum, M. engbaekii, M. neoaurum, M. nonchromogenicum and M. vaccae. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evaluation of a bovine antibody test for diagnosing Mycobacterium avium complex in patients with cystic fibrosis

DEFF Research Database (Denmark)

Qvist, Tavs; Pressler, Tacjana; Katzenstein, Terese L.

2017-01-01

Introduction: The aim of this study was to test a commercial bovine enzyme-linked immunosorbent assay for investigating antibody activity against Mycobacterium avium complex. Methods: All patients at the Copenhagen Cystic Fibrosis (CF) Center who had culture for nontuberculous mycobacteria...... before and after culture conversion was performed in case patients. Results: Out of 286 included subjects, six had clinical M. avium complex pulmonary disease at the time of sera sampling. These patients presented with higher antibody test values (P-value ... at ruling out pulmonary disease. Screening sera from patients with CF could guide clinicians to focus attention on patients at higher risk of M. avium complex pulmonary disease. Pediatr Pulmonol. 2017;52:34–40....

Characterisation of an ELISA detecting immunoglobulin G to Mycobacterium avium subsp. paratuberculosis in bovine colostrum

DEFF Research Database (Denmark)

Zervens, Lisa Marie-Louise; Nielsen, Søren Saxmose; Jungersen, Gregers

2013-01-01

Although colostrum has been used to detect specific immunoglobulin (Ig) G to Mycobacterium avium subsp. paratuberculosis (MAP) in cattle, confounding, non-specific reactions can be a problem. The objectives of this study were to determine the proportion of non-specific ELISA reactions in samples...

Efficacy of novel lipid-formulated whole bacterial cell vaccines against Mycobacterium avium subsp paratuberculosis in sheep

NARCIS (Netherlands)

Griffin, J.F.T.; Hughes, A.D.; Liggett, S.; Farquhar, P.A.; Mackintosh, C.G.; Bakker, D.

2009-01-01

Mycobacterium avium subsp. paratuberculosis [MAP], the Causative agent of enteric Johne's disease, incurs significant economic losses to the livestock industry. Prophylactic vaccination can be employed as a control means, however mineral oil-based vaccines Currently in practice have limited

COMPARISON OF MYCOBACTERIUM AVIUM ISOLATES FROM DRINKING WATER AND FROM THE POPULATION SERVED BY THE SYSTEM

Science.gov (United States)

Background: Current evidence suggests that drinking water, soil, and produce are potential sources of Mycobacterium avium infections, a pathogen not known to be transmitted person-to-person. Methods: We sampled water during 2000 - 2002 from a large municipal drinking wate...

Faecal bacterial composition in dairy cows shedding Mycobacterium avium subsp. paratuberculosis in faeces in comparison with nonshedding cows.

Science.gov (United States)

Kaevska, Marija; Videnska, Petra; Sedlar, Karel; Bartejsova, Iva; Kralova, Alena; Slana, Iva

2016-06-01

The aim of this study was to determine possible differences in the faecal microbiota of dairy cows infected with Mycobacterium avium subsp. paratuberculosis (Johne's disease) in comparison with noninfected cows from the same herds. Faecal samples from cows in 4 herds were tested for M. avium subsp. paratuberculosis by real-time PCR, and faecal bacterial populations were analysed by 454 pyrosequencing of the 16S rRNA gene. The most notable differences between shedding and nonshedding cows were an increase in the genus Psychrobacter and a decrease in the genera Oscillospira, Ruminococcus, and Bifidobacterium in cows infected with M. avium subsp. paratuberculosis. The present study is the first to report the faecal microbial composition in dairy cows infected with M. avium subsp. paratuberculosis.

Direct detection of Mycobacterium avium in environmental water and scale samples by loop-mediated isothermal amplification.

Science.gov (United States)

Nishiuchi, Yukiko; Tamaru, Aki; Suzuki, Yasuhiko; Kitada, Seigo; Maekura, Ryoji; Tateishi, Yoshitaka; Niki, Mamiko; Ogura, Hisashi; Matsumoto, Sohkichi

2014-06-01

We previously demonstrated the colonization of Mycobacterium avium complex in bathrooms by the conventional culture method. In the present study, we aimed to directly detect M. avium organisms in the environment using loop-mediated isothermal amplification (LAMP), and to demonstrate the efficacy of LAMP by comparing the results with those obtained by culture. Our data showed that LAMP analysis has detection limits of 100 fg DNA/reaction for M. avium. Using an FTA(®) elute card, DNA templates were extracted from environmental samples from bathrooms in the residences of 29 patients with pulmonary M. avium disease. Of the 162 environmental samples examined, 143 (88%) showed identical results by both methods; 20 (12%) and 123 (76%) samples were positive and negative, respectively, for M. avium. Of the remaining 19 samples (12%), seven (5%) and 12 (7%) samples were positive by the LAMP and culture methods, respectively. All samples that contained over 20 colony forming units/primary isolation plate, as measured by the culture method, were also positive by the LAMP method. Our data demonstrate that the combination of the FTA elute card and LAMP can facilitate prompt detection of M. avium in the environment.

Typing of clinical Mycobacterium avium complex strains cultured during a 2-year period in Denmark by using IS1245

DEFF Research Database (Denmark)

Bauer, Jeanett; Andersen, Åse B.; Askgaard, Dorthe

1999-01-01

In the present study restriction fragment length polymorphism analyses with the recently described insertion sequence IS1245 as a probe was performed with clinical Mycobacterium avium complex strains cultured in Denmark during a 2-year period. The overall aim of the study was to disclose potentia...... as potting soil) and veterinary samples were found to contain viable M avium isolates belonging to genotypes also found in humans....

Isolation of Mycobacterium avium subspecies paratuberculosis Reactive T-cells from Intestinal Biopsies of Crohn's Disease Patients

Science.gov (United States)

Crohn’s disease (CD) is a chronic granulomatous inflammation of the intestine. The etiology is still unknown. One hypothesis is that CD is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP) in genetically predisposed individuals. MAP causes a similar disease in ruminants,...

IL-32 expression in the airway epithelial cells of patients with Mycobacterium avium complex lung disease.

NARCIS (Netherlands)

Bai, X.; Ovrutsky, A.R.; Kartalija, M.; Chmura, K.; Kamali, A.; Honda, J.R.; Oberley-Deegan, R.E.; Dinarello, C.A.; Crapo, J.D.; Chang, L.Y.; Chan, E.D.

2011-01-01

Lung disease due to Mycobacterium avium complex (MAC) organisms is increasing. A greater understanding of the host immune response to MAC organisms will provide a foundation to develop novel therapies for these recalcitrant infections. IL-32 is a newly described pro-inflammatory cytokine that

MYCOBACTERIUM AVIUM SUSP. PARATUBERCULOSIS IN DAIRY PRODUCTION

Directory of Open Access Journals (Sweden)

G. Marchetti

2012-08-01

Full Text Available Mycobacterium avium subsp. paratuberculosis (MAP is the etiologic agent of paratuberculosis. The disease affects cows and other ruminants and causes high economic losses, mainly for dairy production. MAP may also have a role in the development of Crohn’s disease in humans. Infected animals shed viable MAP with milk and faeces and humans may assume MAP via the consumption of contaminated milk and dairy products. Current methods of milk pasteurization are not sufficient to kill all MAP cells present in milk and MAP has been found in raw or pasteurized milk and isolated from cheese. The aim of this paper is to review the current knowledge about MAP in dairy production. We analyzed studies on milk contamination, effect of pasteurization and methods for identification of MAP that can be applied to dairy products.

Molecular Analysis of Mycobacterium avium Isolates by Using Pulsed-Field Gel Electrophoresis and PCR

Science.gov (United States)

Pestel-Caron, Martine; Graff, Gabriel; Berthelot, Gilles; Pons, Jean-Louis; Lemeland, Jean-François

1999-01-01

Genetic relationships among 46 isolates of Mycobacterium avium recovered from 37 patients in a 2,500-bed hospital from 1993 to 1998 were assessed by pulsed-field gel electrophoresis (PFGE) and PCR amplification of genomic sequences located between the repetitive elements IS1245 and IS1311. Each technique enabled the identification of 27 to 32 different patterns among the 46 isolates, confirming that the genetic heterogeneity of M. avium strains is high in a given community. Furthermore, this retrospective analysis of sporadic isolates allowed us (i) to suggest the existence of two remanent strains in our region, (ii) to raise the question of the possibility of nosocomial acquisition of M. avium strains, and (iii) to document laboratory contamination. The methods applied in the present study were found to be useful for the typing of M. avium isolates. In general, both methods yielded similar results for both related and unrelated isolates. However, the isolates in five of the six PCR clusters were distributed among two to three PFGE patterns, suggesting that this PCR-based method may have limitations for the analysis of strains with low insertion sequence copy numbers or for resolution of extended epidemiologic relationships. PMID:10405383

Killing of mycobacterium avium by lactoferricin peptides: improved activity of arginine- and D-amino-acid-containing molecules

NARCIS (Netherlands)

Silva, T.; Magalhães, B.; Maia, S.; Gomes, P.; Nazmi, K.; Bolscher, J.G.M.; Rodriques, P.N.; Bastos, M.; Gomes, M.S.

2014-01-01

Mycobacterium avium causes respiratory disease in susceptible individuals, as well as disseminated infections in immunocompromised hosts, being an important cause of morbidity and mortality among these populations. Current therapies consist of a combination of antibiotics taken for at least 6

Gamma-delta T cell responses in subclinical and clinical stages of Bovine Mycobacterium Avium Paratuberculosis infection

Science.gov (United States)

The early immune response to Mycobacterium avium subsp. paratuberculosis (MAP) in cattle is characterized by a Th1-like immune response effective in controlling bacterial proliferation during the subclinical stage of infection. In young calves nearly 60% of circulating lymphocytes are gamma delta T ...

Immunoreactivity of protein tyrosine phosphatase A (PtpA) in sera from sheep infected with Mycobacterium avium subspecies paratuberculosis.

Science.gov (United States)

Gurung, Ratna B; Begg, Douglas J; Purdie, Auriol C; Bach, Horacio; Whittington, Richard J

2014-07-15

Evasion of host defense mechanisms and survival inside infected host macrophages are features of pathogenic mycobacteria including Mycobacterium avium subspecies paratuberculosis, the causative agent of Johne's disease in ruminants. Protein tyrosine phosphatase A (PtpA) has been identified as a secreted protein critical for survival of mycobacteria within infected macrophages. The host may mount an immune response to such secreted proteins. In this study, the humoral immune response to purified recombinant M. avium subsp. paratuberculosis PtpA was investigated using sera from a cohort of sheep infected with M. avium subsp. paratuberculosis and compared with uninfected healthy controls. A significantly higher level of reactivity to PtpA was observed in sera collected from M. avium subspecies paratuberculosis infected sheep when compared to those from uninfected healthy controls. PtpA could be a potential candidate antigen for detection of humoral immune responses in sheep infected with M. avium subspecies paratuberculosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of new antigen candidates for the early diagnosis of Mycobacterium avium subsp. paratuberculosis infection in goats

NARCIS (Netherlands)

Souriau, Armel; Freret, Sandrine; Foret, Benjamin; Willemsen, Peter T.J.; Bakker, Douwe; Guilloteau, Laurence A.

2017-01-01

Currently Mycobacterium avium subsp. paratuberculosis (MAP) infection is diagnosed through indirect tests based on the immune response induced by the infection. The antigens commonly used in IFN-γ release assays (IGRA) are purified protein derivative tuberculins (PPD). However, PPDs, lack both

Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model

Science.gov (United States)

Understanding the pathogenic mechanisms and host responses to Johne’s disease, a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP), is complicated by the multifaceted disease progression, late-onset host reaction, and the lack of ex vivo infection models ...

Mycobacterium avium subsp. hominissuis infection in swine associated with peat used for bedding.

Science.gov (United States)

Johansen, Tone Bjordal; Agdestein, Angelika; Lium, Bjørn; Jørgensen, Anne; Djønne, Berit

2014-01-01

Mycobacterium avium subsp. hominissuis is an environmental bacterium causing opportunistic infections in swine, resulting in economic losses. Additionally, the zoonotic aspect of such infections is of concern. In the southeastern region of Norway in 2009 and 2010, an increase in condemnation of pig carcasses with tuberculous lesions was seen at the meat inspection. The use of peat as bedding in the herds was suspected to be a common factor, and a project examining pigs and environmental samples from the herds was initiated. Lesions detected at meat inspection in pigs originating from 15 herds were sampled. Environmental samples including peat from six of the herds and from three peat production facilities were additionally collected. Samples were analysed by culture and isolates genotyped by MLVA analysis. Mycobacterium avium subsp. hominissuis was detected in 35 out of 46 pigs, in 16 out of 20 samples of peat, and in one sample of sawdust. MLVA analysis demonstrated identical isolates from peat and pigs within the same farms. Polyclonal infection was demonstrated by analysis of multiple isolates from the same pig. To conclude, the increase in condemnation of porcine carcasses at slaughter due to mycobacteriosis seemed to be related to untreated peat used as bedding.

CD4 T Cells From Intestinal Biopsies of Crohn's Disease Patients React to Mycobacterium avium subspecies paratuberculosis

Science.gov (United States)

The role of Mycobacterium avium subspecies paratuberculosis (MAP) in Crohn’s disease (CD) remains controversial. One issue that has been raised is the lack of data showing a cellular immune response to MAP. Earlier studies have mostly focused on responses in peripheral blood which have several limit...

Effect of lipoarabinomannan from Mycobacterium avium subsp avium in Freund's incomplete adjuvant on the immune response of cattle.

Science.gov (United States)

Colavecchia, S B; Jolly, A; Fernández, B; Fontanals, A M; Fernández, E; Mundo, S L

2012-02-01

The aim of the present study was to determine whether lipoarabinomannan (LAM), in combination with Freund's incomplete adjuvant (FIA), was able to improve cell-mediated and antibody-mediated immune responses against ovalbumin (OVA) in cattle. Twenty-three calves were assigned to four treatment groups, which were subcutaneously immunized with either OVA plus FIA, OVA plus FIA and LAM from Mycobacterium avium subsp avium, FIA plus LAM, or FIA alone. Lymphoproliferation, IFN-γ production and cell subpopulations on peripheral blood mononuclear cells before and 15 days after treatment were evaluated. Delayed hypersensitivity was evaluated on day 57. Specific humoral immune response was measured by ELISA. Inoculation with LAM induced higher levels of lymphoproliferation and IFN-γ production in response to ConA and OVA (P < 0.05). Specific antibody titers were similar in both OVA-immunized groups. Interestingly, our results showed that the use of LAM in vaccine preparations improved specific cell immune response evaluated by lymphoproliferation and IFN-γ production by at least 50 and 25%, respectively, in cattle without interfering with tuberculosis and paratuberculosis diagnosis.

Obtención y evaluación de un derivado proteico purificado de una cepa argentina de Mycobacterium avium subsp. paratuberculosis Production and evaluation of a purified protein derivative from an Argentine strain of Mycobacterium avium subsp. paratuberculosis (MAP

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Andrea Gioffré

2012-09-01

Full Text Available Los derivados proteicos purificados (PPD son mezclas antigénicas no definidas obtenidas de distintas micobacterias. Los PPD bovino (PPDb y PPD aviar (PPDa son los antígenos que se emplean para evaluar la respuesta inmunitaria celular en infecciones como tuberculosis y paratuberculosis en el bovino. El PPDa comercial se produce a partir de Mycobacterium avium subsp. avium, y no a partir de la subespecie paratuberculosis. En este trabajo se seleccionó una cepa local de Mycobacterium avium subsp. paratuberculosis cuyo patrón molecular por RFLP es el más frecuente entre los aislamientos de nuestro país que han sido estudiados, y a partir de esta, se obtuvo un derivado proteico purificado: PPDj-IB. Se emplearon tanto el PPDa comercial como el PPDj-IB como antígenos en la prueba de liberación de gamma-interferón en animales de un tambo con paratuberculosis y en animales control. Aun cuando ambos PPD fueron capaces de estimular diferencialmente la liberación de la citoquina en el tambo infectado (respecto de los tambos control, no hubo diferencias significativas en los niveles de estimulación producidos y solo dos animales fueron positivos mediante el empleo de PPDj-IB. A partir del análisis por Western blot se demostró que el contenido de lipoarabinomano y del antígeno Apa/ModD era distinto en los PDD evaluados. Estas diferencias podrían explicar, en parte, las diferencias en los niveles de estimulación en términos individuales. Si bien el empleo de PPDj-IB no mejoró significativamente los resultados de la prueba de liberación de ?IFN, es importante destacar que se logró producir en el laboratorio un PPD apto para su empleo en ensayos in vitro.Purified Protein Derivatives (PPDs are non-defined antigens prepared from mycobacteria cultures. They are usually employed to evaluate the specific cellular immune response both in animals and humans. Bovine and avian PPDs are usually employed as antigens in mycobacterial infections such as

Infection with the Mycobacterium avium complex in patients without predisposing conditons: a case report and literature review

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Andrea Barral Martins

Full Text Available Nontuberculous Mycobacteria (NTM, especially Mycobacterium avium-intracellulare complex (MAC, has been considered responsible for human disease, especially in HIV patients. Nevertheless, it has been diagnosed in immunocompetent elderly men, frequently with previous pulmonary disease: chronic obstructive lung disease (COPD, complications of tuberculosis, pulmonary fibrosis and bronchiectasis. We relate the case of a female patient, 51 years old, with continuously acid fast bacilli (AFB smears and with three previous treatments, which were conducted at the multiresistant tuberculosis (MRTB service. MAC was identified in the sputum culture, and she received treatment for one year. The posterior sputum exams were negative. The cavity lesions observed in the high-resolution computed tomography (HRCT were reduced, and some of the nodule lesions became bronchiectasis, even after the end of treatment. We agree with the literature reports that indicate that MAC is the cause of bronchiectasis. It is necessary to identify the type of mycobacteria in immunocompetent individuals with positive AFB smears that do not become negative with tuberculosis treatment.

THE EFFECT OF TEMPERATURE ON THE GROWTH OF MYCOBACTERIUM AVIUM COMPLEX (MAC) ORGANISMS

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MAC organisms are able to grow, persist, and colonize in water distribution systems and may amplify in hospital hot water systems. This study examined the response of MAC organisms (M. avium, M. intracellulare, and MX) to a range of temperatures commonly associated with drinking...

Mycobacterium avium complex--the role of potable water in disease transmission.

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Whiley, H; Keegan, A; Giglio, S; Bentham, R

2012-08-01

Mycobacterium avium complex (MAC) is a group of opportunistic pathogens of major public health concern. It is responsible for a wide spectrum of disease dependent on subspecies, route of infection and patients pre-existing conditions. Presently, there is limited research on the incidence of MAC infection that considers both pulmonary and other clinical manifestations. MAC has been isolated from various terrestrial and aquatic environments including natural waters, engineered water systems and soils. Identifying the specific environmental sources responsible for human infection is essential in minimizing disease prevalence. This paper reviews current literature and case studies regarding the wide spectrum of disease caused by MAC and the role of potable water in disease transmission. Potable water was recognized as a putative pathway for MAC infection. Contaminated potable water sources associated with human infection included warm water distribution systems, showers, faucets, household drinking water, swimming pools and hot tub spas. MAC can maintain long-term contamination of potable water sources through its high resistance to disinfectants, association with biofilms and intracellular parasitism of free-living protozoa. Further research is required to investigate the efficiency of water treatment processes against MAC and into construction and maintenance of warm water distribution systems and the role they play in MAC proliferation. No claim to Australian Government works Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Dose response models and a quantitative microbial risk assessment framework for the Mycobacterium avium complex that account for recent developments in molecular biology, taxonomy, and epidemiology.

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Hamilton, Kerry A; Weir, Mark H; Haas, Charles N

2017-02-01

Mycobacterium avium complex (MAC) is a group of environmentally-transmitted pathogens of great public health importance. This group is known to be harbored, amplified, and selected for more human-virulent characteristics by amoeba species in aquatic biofilms. However, a quantitative microbial risk assessment (QMRA) has not been performed due to the lack of dose response models resulting from significant heterogeneity within even a single species or subspecies of MAC, as well as the range of human susceptibilities to mycobacterial disease. The primary human-relevant species and subspecies responsible for the majority of the human disease burden and present in drinking water, biofilms, and soil are M. avium subsp. hominissuis, M. intracellulare, and M. chimaera. A critical review of the published literature identified important health endpoints, exposure routes, and susceptible populations for MAC risk assessment. In addition, data sets for quantitative dose-response functions were extracted from published in vivo animal dosing experiments. As a result, seven new exponential dose response models for human-relevant species of MAC with endpoints of lung lesions, death, disseminated infection, liver infection, and lymph node lesions are proposed. Although current physical and biochemical tests used in clinical settings do not differentiate between M. avium and M. intracellulare, differentiating between environmental species and subspecies of the MAC can aid in the assessment of health risks and control of MAC sources. A framework is proposed for incorporating the proposed dose response models into susceptible population- and exposure route-specific QMRA models. Copyright © 2016 Elsevier Ltd. All rights reserved.

Isolation, Identification, and Characterization of a New Highly Pathogenic Field Isolate of Mycobacterium avium spp. avium

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Liangquan Zhu

2018-01-01

Full Text Available Avian tuberculosis is a chronic, contagious zoonotic disease affecting birds, mammals, and humans. The disease is most often caused by Mycobacterium avium spp. avium (MAA. Strain resources are important for research on avian tuberculosis and vaccine development. However, there has been little reported about the newly identified MAA strain in recent years in China. In this study, a new strain was isolated from a fowl with symptoms of avian tuberculosis by bacterial culture. The isolated strain was identified to be MAA by culture, staining, and biochemical and genetic analysis, except for different colony morphology. The isolated strain was Ziehl-Zeelsen staining positive, resistant to p-nitrobenzoic acid, and negative for niacin production, Tween-80 hydrolysis, heat stable catalase and nitrate production. The strain had the DnaJ gene, IS1245, and IS901, as well. Serum agglutination indicated that the MAA strain was of serotype 1. The MAA strain showed strong virulence via mortality in rabbits and chickens. The prepared tuberculin of the MAA strain had similar potency compared to the MAA reference strain and standard tuberculin via a tuberculin skin test. Our studies suggested that this MAA strain tends to be a novel subtype, which might enrich the strain resource of avian tuberculosis.

Molecular characterization of Mycobacterium avium subspecies hominissuis isolated from humans, cattle and pigs in the Uganda cattle corridor using VNTR analysis.

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Muwonge, Adrian; Oloya, James; Kankya, Clovice; Nielsen, Sigrun; Godfroid, Jacques; Skjerve, Eystein; Djønne, Berit; Johansen, Tone B

2014-01-01

Members of the Mycobacterium avium complex (MAC) cause disease in both human and animals. Their ubiquitous nature makes them both successful microbes and difficult to source track. The precise characterization of MAC species is a fundamental step in epidemiological studies and evaluating of possible reservoirs. This study aimed at identifying and characterizing Mycobacterium avium subsp. hominissuis isolated from human, slaughter cattle and pigs in various parts of the Uganda cattle corridor (UCC) at two temporal points using variable number of tandem repeat (VNTR) analysis. A total of 46 M. avium isolates; 31 from 997 pigs, 12 from 43 humans biopsies and three from 61 cattle lesions were identified to subspecies level using IS1245 and IS901 PCR, thereafter characterized using VNTR. Twelve loci from two previously described VNTR methods were used and molecular results were analyzed and interpreted using Bionumerics 6.1. 37 of the isolates were identified as M. avium subsp. hominissuis and four as M. avium subsp. avium, while five could not be differentiated, possibly due to mixed infection. There was distinct clustering that coincides with the temporal and spatial differences of the isolates. The isolates from humans and cattle in the North Eastern parts of the UCC shared identical VNTR genotypes. The panel of loci gave an overall discriminatory power of 0.88. Some loci were absent in several isolates, probably reflecting differences in isolates from Uganda/Africa compared to isolates previously analyzed by these methods in Europe and Asia. The findings indicate a molecular difference between M. avium subsp. hominissuis isolates from pigs in Mubende and cattle and human in the rest of the UCC. Although human and cattle shared VNTR genotypes in the North Eastern parts of the UCC, it is most likely a reflection of a shared environmental source. Copyright © 2013 Elsevier B.V. All rights reserved.

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

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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.

COMPARISON OF MYCOBACTERIUM AVIUM ISOLATES FROM A DRINKING WATER DISTRIBUTION SYSTEM AND FROM THE POPULATION SERVED BY THE SYSTEM

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Background: Current evidence suggests that drinking water, soil, and produce are potential sources of Mycobacterium avium infections, a pathogen not known to be transmitted person-to-person. Methods: We sampled water during 2000-2002 from a large municipal drinking water ...

Soft tissue abscess and lymphadenitis due to Mycobacterium avium Complex as an expression of immune reconstitution inflammatory syndrome after a second scheme of highly active antiretroviral therapy Linfadenitis y absceso subcutáneo por Complejo Mycobacterium avium como manifestación de síndrome inflamatorio de reconstitución inmune luego de un segundo esquema de terapia antirretroviral de gran actividad

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Marcelo Corti

2007-08-01

Full Text Available Immune reconstitution inflammatory syndrome (IRIS is an atypical and unexpected reaction related to highly active antiretroviral therapy (HAART in human immunodeficiency virus (HIV infected patients. IRIS includes an atypical response to an opportunistic pathogen (generally Mycobacterium tuberculosis, Mycobacterium avium complex, cytomegalovirus and herpes varicella-zoster, in patients responding to HAART with a reduction of plasma viral load and evidence of immune restoration based on increase of CD4+ T-cell count. We reported a case of a patient with AIDS which, after a first failure of HAART, developed a subcutaneous abscess and supraclavicular lymphadenitis as an expression of IRIS due to Mycobacterium avium complex after starting a second scheme of HAART.El síndrome inflamatorio de reconstitución inmune (SIRI es una reacción atípica e inesperada relacionada con el tratamiento antirretroviral de gran actividad (TARGA en pacientes infectados por el virus de la inmunodeficiencia humana (VIH. El SIRI representa una respuesta inflamatoria frente a un patógeno oportunista (generalmente Mycobacterium tuberculosis, Complejo Mycobacterium avium, citomegalovirus y herpes varicela-zóster en pacientes que responden a la TARGA con una marcada reducción de la carga viral en plasma y evidencia de una recuperación inmunológica expresada por el incremento de los niveles de linfocitos T CD4+. Presentamos el caso de un paciente con síndrome de inmunodeficiencia adquirida que desarrolló un absceso subcutáneo en muslo derecho y una adenitis supraclavicular izquierda como manifestación de SIRI por Complejo Mycobacterium avium luego del inicio de un segundo esquema de TARGA.

Rapid and sensitive method to identify Mycobacterium avium subsp. paratuberculosis in cow's milk by DNA methylase genotyping.

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Mundo, Silvia Leonor; Gilardoni, Liliana Rosa; Hoffman, Federico José; Lopez, Osvaldo Jorge

2013-03-01

Paratuberculosis is an infectious, chronic, and incurable disease that affects ruminants, caused by Mycobacterium avium subsp. paratuberculosis. This bacterium is shed primarily through feces of infected cows but can be also excreted in colostrum and milk and might survive pasteurization. Since an association of genomic sequences of M. avium subsp. paratuberculosis in patients with Crohn's disease has been described; it is of interest to rapidly detect M. avium subsp. paratuberculosis in milk for human consumption. IS900 insertion is used as a target for PCR amplification to identify the presence of M. avium subsp. paratuberculosis in biological samples. Two target sequences were selected: IS1 (155 bp) and IS2 (94 bp). These fragments have a 100% identity among all M. avium subsp. paratuberculosis strains sequenced. M. avium subsp. paratuberculosis was specifically concentrated from milk samples by immunomagnetic separation prior to performing PCR. The amplicons were characterized using DNA methylase Genotyping, i.e., the amplicons were methylated with 6-methyl-adenine and digested with restriction enzymes to confirm their identity. The methylated amplicons from 100 CFU of M. avium subsp. paratuberculosis can be visualized in a Western blot format using an anti-6-methyl-adenine monoclonal antibody. The use of DNA methyltransferase genotyping coupled to a scintillation proximity assay allows for the detection of up to 10 CFU of M. avium subsp. paratuberculosis per ml of milk. This test is rapid and sensitive and allows for automation and thus multiple samples can be tested at the same time.

The role of Mycobacterium avium complex fibronectin attachment protein in adherence to the human respiratory mucosa.

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Middleton, A M; Chadwick, M V; Nicholson, A G; Dewar, A; Groger, R K; Brown, E J; Wilson, R

2000-10-01

Mycobacterium avium complex (MAC) are opportunistic respiratory pathogens that infect non-immunocompromised patients with established lung disease, although they can also cause primary infections. The ability to bind fibronectin is conserved among many mycobacterial species. We have investigated the adherence of a sputum isolate of MAC to the mucosa of organ cultures constructed with human tissue and the contribution of M. avium fibronectin attachment protein (FAP) to the process. MAC adhered to fibrous, but not globular mucus, and to extracellular matrix (ECM) in areas of epithelial damage, but not to intact extruded cells and collagen fibres. Bacteria occasionally adhered to healthy unciliated epithelium and to cells that had degenerated exposing their contents, but never to ciliated cells. The results obtained with different respiratory tissues were similar. Two ATCC strains of MAC gave similar results. There was a significant reduction (P fibrous mucus was unchanged. Immunogold labelling demonstrated fibronectin in ECM as well as in other areas of epithelial damage, but only ECM bound FAP. A Mycobacterium smegmatis strain had the same pattern of adherence to the mucosa as MAC. When the FAP gene was deleted, the strain demonstrated reduced adherence to ECM, and adherence was restored when the strain was transfected with an M. avium FAP expression construct. We conclude that MAC adheres to ECM in areas of epithelial damage via FAP and to mucus with a fibrous appearance via another adhesin. Epithelial damage exposing ECM and poor mucus clearance will predispose to MAC airway infection.

Virulence of Mycobacterium avium Subsp. hominissuis Human Isolates in an in vitro Macrophage Infection Model

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Laura Rindi

2018-01-01

Full Text Available Background: Mycobacterium avium subsp. hominissuis (MAH is an environmental opportunistic pathogen for humans and swine worldwide; in humans, the vast majority of MAH infections is due to strains belonging to specific genotypes, such as the internal transcribed spacer (ITS-sequevars Mav-A and Mav-B that mostly cause pulmonary infections in elderly patients and severe disseminated infections in acquired immunodeficiency syndrome patients, respectively. To test whether the different types of infections in distinct patients' populations might reflect a different virulence of the infecting genotypes, MAH human isolates, genotyped by ITS sequencing and MIRU-VNTR minisatellite analysis, were studied for the capacity to infect and replicate in human macrophages in vitro. Methods: Cultures of human peripheral blood mononuclear cells and phagocytic human leukemic cell line THP-1 cells were infected with each MAH isolate and intracellular colony-forming units (CFU were determined. Results: At 2 h after infection, i.e., immediately after cell entry, the numbers of intracellular bacteria did not differ between Mav-A and Mav-B organisms in both phagocytic cell types. At 5 days, Mav-A organisms, sharing highly related VNTR-MIRU genotypes, yielded numbers of intracellular CFUs significantly higher than Mav-B organisms in both phagocytic cell types. MIRU-VNTR-based minimum spanning tree analysis of the MAH isolates showed a divergent phylogenetic pathway of Mav-A and Mav-B organisms. Conclusion: Mav-A and Mav-B sequevars might have evolved different pathogenetic properties that might account for their association with different human infections.

ZAP-70, CTLA-4, and proximal T cell receptor signaling in cows infected with Mycobacterium avium subsp. paratuberculosis

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Paratuberculosis is a chronic intestinal disease of ruminant animals caused by Mycobacterium avium subsp. paratuberculosis (MAP). A hallmark of paratuberculosis is a transition from a cell-mediated Th1 type response to a humoral Th2 response with the progression of disease from a subclinical to clin...

Disseminated Mycobacterium avium complex infection in an immunocompetent pregnant woman

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Kim Woo

2006-10-01

Full Text Available Abstract Background Disseminated mycobacterium avium complex (MAC occurs mainly in immunocompromised hosts, which is associated with abnormal cellular immunity. Case presentation A 26-year-old pregnant woman presented with fever and general weakness. Miliary lung nodules were noted on chest X-ray. Under the impression of miliary tuberculosis, anti-tuberculosis medication was administered. However, the patient was not improved. Further work-up demonstrated MAC in the sputum and placenta. The patient was treated successfully with clarithromycin-based combination regimen. Conclusion This appears to be the first case of disseminated MAC in an otherwise healthy pregnant woman. Clinicians should be alert for the diagnosis of MAC infection in diverse clinical conditions.

The characteristics of patients with pulmonary Mycobacterium avium-intracellulare complex disease diagnosed by bronchial lavage culture compared to those diagnosed by sputum culture.

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Maekawa, Koichi; Naka, Megumi; Shuto, Saki; Harada, Yuka; Ikegami, Yumiko

2017-09-01

The utility of bronchoscopy for the diagnosis of pulmonary Mycobacterium avium-intracellulare complex (MAC) disease has been reported; however, which patients require bronchoscopy remains unclear. Our objective was to identify the characteristics of the patients in whom bronchoscopy is needed for the diagnosis of MAC disease. Fifty-four patients with pulmonary MAC disease were divided into two groups according to established diagnostic criteria: 39 patients were diagnosed by sputum culture and 15 patients were diagnosed by bronchial lavage culture. We analysed the differences in demographic and clinical characteristics as well as microbiological and radiological data between the two groups. There were no significant differences in age, sex, smoking status, MAC species, underlying diseases, or steroid use. Significantly more patients diagnosed by sputum culture than bronchial lavage culture had a positive sputum smear for acid-fast bacilli (79.5% vs. 0.0%, respectively; p disease, bronchiectasis, and cavities. However, more patients diagnosed by sputum culture than bronchial lavage culture had abnormalities in the left upper division (48.7% vs. 13.3%, respectively; p = 0.017) and higher numbers of affected lobes (4.3 ± 1.4 vs. 3.3 ± 1.6, respectively; p = 0.034). If patients suspected of having pulmonary MAC disease have a negative sputum smear, no symptoms, no abnormal findings in the left upper division, or fewer affected lobes on computed tomography, bronchoscopy might be needed for the diagnosis. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Analysis of Mycobacterium avium subspecies paratuberculosis mutant libraries reveals loci-dependent transposition biases and strategies to novel mutant discovery

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Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of Johne’s disease, is one of the most important bacterial pathogens in ruminants. The lack of efficacious control measures demands a thorough understanding of MAP pathogenesis to develop new vaccines and diagnostic tests. The ge...

Antibodies Induced by Lipoarabinomannan in Bovines: Characterization and Effects on the Interaction between Mycobacterium Avium Subsp. Paratuberculosis and Macrophages In Vitro.

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Jolly, Ana; Colavecchia, Silvia Beatriz; Fernández, Bárbara; Fernández, Eloy; Mundo, Silvia Leonor

2011-01-01

Lipoarabinomannan (LAM) is a major glycolipidic antigen on the mycobacterial envelope. The aim of this study was to characterize the humoral immune response induced by immunization with a LAM extract in bovines and to evaluate the role of the generated antibodies in the in vitro infection of macrophages with Mycobacterium avium subsp. paratuberculosis (MAP). Sera from fourteen calves immunized with LAM extract or PBS emulsified in Freund's Incomplete Adjuvant and from five paratuberculosis-infected bovines were studied. LAM-immunized calves developed specific antibodies with IgG1 as the predominant isotype. Serum immunoglobulins were isolated and their effect was examined in MAP ingestion and viability assays using a bovine macrophage cell line. Our results show that the antibodies generated by LAM immunization significantly increase MAP ingestion and reduce its intracellular viability, suggesting an active role in this model.

Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transcription biases and strategies to novel mutant discovery

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Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in ruminants and it has been implicated as a cause of Crohn’s disease in humans. The generation of comprehensive random mutant banks by transposon mutagenesis is a fundamental wide genomic technology utilized...

Survival of Mycobacterium avium in drinking water biofilms as affected by water flow velocity, availability of phosphorus, and temperature.

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Torvinen, Eila; Lehtola, Markku J; Martikainen, Pertti J; Miettinen, Ilkka T

2007-10-01

Mycobacterium avium is a potential pathogen occurring in drinking water systems. It is a slowly growing bacterium producing a thick cell wall containing mycolic acids, and it is known to resist chlorine better than many other microbes. Several studies have shown that pathogenic bacteria survive better in biofilms than in water. By using Propella biofilm reactors, we studied how factors generally influencing the growth of biofilms (flow rate, phosphorus concentration, and temperature) influence the survival of M. avium in drinking water biofilms. The growth of biofilms was followed by culture and DAPI (4',6'-diamidino-2-phenylindole) staining, and concentrations of M. avium were determined by culture and fluorescence in situ hybridization methods. The spiked M. avium survived in biofilms for the 4-week study period without a dramatic decline in concentration. The addition of phosphorus (10 microg/liter) increased the number of heterotrophic bacteria in biofilms but decreased the culturability of M. avium. The reason for this result is probably that phosphorus increased competition with other microbes. An increase in flow velocity had no effect on the survival of M. avium, although it increased the growth of biofilms. A higher temperature (20 degrees C versus 7 degrees C) increased both the number of heterotrophic bacteria and the survival of M. avium in biofilms. In conclusion, the results show that in terms of affecting the survival of slowly growing M. avium in biofilms, temperature is a more important factor than the availability of nutrients like phosphorus.

Divergent cellular responses during asymptomatic subclinical and clinical states of disease in cows naturally infected with Mycobacterium avium subsp. paratuberculosis

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Infection of the host with Mycobacterium avium subsp. paratuberculosis (MAP) results in a chronic and progressive enteritis that traverses both subclinical and clinical stages. The mechanism(s) for the shift from asymptomatic subclinical disease state to advanced clinical disease are not fully under...

Epidemiological and economic consequences of purchasing livestock infected with Mycobacterium avium subsp. paratuberculosis

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Kirkeby, Carsten Thure; Græsbøll, Kaare; Nielsen, Søren Saxmose

2017-01-01

Paratuberculosis (PTB) is a chronic disease which may lead to reduced milk yield, lower animal welfare and death in cattle. The causative agent is Mycobacterium avium subsp. paratuberculosis (MAP). The economic consequences are particularly important incentives in the control and eradication...... of the infection. One strategy to control PTB in a herd is to purchase animals from farms with a low risk of MAP infection. We wanted to investigate the epidemiological and economic consequences of buying livestock from different supplier farms of low, medium or high risk, as well as farms with unknown status. We...

CD4 T Cell Dependent Colitis Exacerbation Following Re-Exposure of Mycobacterium avium ssp. paratuberculosis.

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Suwandi, Abdulhadi; Bargen, Imke; Pils, Marina C; Krey, Martina; Zur Lage, Susanne; Singh, Anurag K; Basler, Tina; Falk, Christine S; Seidler, Ursula; Hornef, Mathias W; Goethe, Ralph; Weiss, Siegfried

2017-01-01

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic inflammatory bowel disease of cattle characterized by intermittent to chronic diarrhea. In addition, MAP has been isolated from Crohn's disease (CD) patients. The impact of MAP on severity of clinical symptoms in JD as well as its role in CD are yet unknown. We have previously shown that MAP is able to colonize inflamed enteric tissue and to exacerbate the inflammatory tissue response (Suwandi et al., 2014). In the present study, we analyzed how repeated MAP administration influences the course of dextran sulfate sodium (DSS)-induced colitis. In comparison to mice exposed to DSS or MAP only, repeated exposure of DSS-treated mice to MAP (DSS/MAP) revealed a significantly enhanced clinical score, reduction of colon length as well as severe CD4 + T cell infiltration into the colonic lamina propria . Functional analysis identified a critical role of CD4 + T cells in the MAP-induced disease exacerbation. Additionally, altered immune responses were observed when closely related mycobacteria species such as M. avium ssp. avium and M. avium ssp. hominissuis were administered. These data reveal the specific ability of MAP to aggravate intestinal inflammation and clinical symptoms. Overall, this phenotype is compatible with similar disease promoting capabilites of MAP in JD and CD.

Culture Phenotypes of Genomically and Geographically Diverse Mycobacterium avium subsp. paratuberculosis Isolates from Different Hosts▿

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Whittington, Richard J.; Marsh, Ian B.; Saunders, Vanessa; Grant, Irene R.; Juste, Ramon; Sevilla, Iker A.; Manning, Elizabeth J. B.; Whitlock, Robert H.

2011-01-01

Mycobacterium avium subsp. paratuberculosis causes paratuberculosis (Johne's disease) in ruminants in most countries. Historical data suggest substantial differences in culturability of M. avium subsp. paratuberculosis isolates from small ruminants and cattle; however, a systematic comparison of culture media and isolates from different countries and hosts has not been undertaken. Here, 35 field isolates from the United States, Spain, Northern Ireland, and Australia were propagated in Bactec 12B medium and Middlebrook 7H10 agar, genomically characterized, and subcultured to Lowenstein-Jensen (LJ), Herrold's egg yolk (HEY), modified Middlebrook 7H10, Middlebrook 7H11, and Watson-Reid (WR) agars, all with and without mycobactin J and some with sodium pyruvate. Fourteen genotypes of M. avium subsp. paratuberculosis were represented as determined by BstEII IS900 and IS1311 restriction fragment length polymorphism analysis. There was no correlation between genotype and overall culturability, although most S strains tended to grow poorly on HEY agar. Pyruvate was inhibitory to some isolates. All strains grew on modified Middlebrook 7H10 agar but more slowly and less prolifically on LJ agar. Mycobactin J was required for growth on all media except 7H11 agar, but growth was improved by the addition of mycobactin J to 7H11 agar. WR agar supported the growth of few isolates. The differences in growth of M. avium subsp. paratuberculosis that have historically been reported in diverse settings have been strongly influenced by the type of culture medium used. When an optimal culture medium, such as modified Middlebrook 7H10 agar, is used, very little difference between the growth phenotypes of diverse strains of M. avium subsp. paratuberculosis was observed. This optimal medium is recommended to remove bias in the isolation and cultivation of M. avium subsp. paratuberculosis. PMID:21430104

MOLECULAR COMPARISON OF MYCOBACTERIUM AVIUM ISOLATED FROM A FRINKING WATER DISTRIBUTION SYSTEM AND FROM THE POPULATION SERVED BY THE SYSTEM

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There is evidence that drinking water, soil, and produce may be sources of Mycobacterium avium infections, a pathogen not known to be transmitted person-to-person. We sampled water from a large municipal drinking water distribution system in which surface source water is used. M...

Molecular characterization and drug susceptibility profile of a Mycobacterium avium subspecies avium isolate from a dog with disseminated infection.

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Armas, Federica; Furlanello, Tommaso; Camperio, Cristina; Trotta, Michele; Novari, Gianluca; Marianelli, Cinzia

2016-01-12

Mycobacterium avium complex (MAC) infections have been described in many mammalian species including humans and pets. We isolated and molecularly typed the causative agent of a rare case of disseminated mycobacteriosis in a dog. We identified the pathogen as a M. avium subspecies avium by sequencing the partial genes gyrB and rpsA. Considering the zoonotic potential of this infection, and in an attempt to ensure the most effective treatment for the animal, we also determined the drug susceptibility profile of the isolate to the most common drugs used to treat MAC disease in humans. The pathogen was tested in vitro against the macrolide clarithromycin, as well as against amikacin, ciprofloxacin, rifampicin, ethambutol and linezolid by the resazurin microdilution assay. It was found to be sensitive to all tested drugs save ethambutol. Despite the fact that the pathogen was sensitive to the therapies administered, the dog's overall clinical status worsened, and the animal died shortly after antimicrobial susceptibility results became available. Nucleotide sequencing of the embB gene, the target gene most commonly associated with ethambutol resistance, showed new missense mutations when compared to sequences available in public databases. In conclusion, we molecularly identified the MAC pathogen and determined its drug susceptibility profile in a relatively short period of time (seven days). We also characterized new genetic mutations likely to have been involved in the observed ethambutol resistance. Our results confirm the usefulness of both the gyrB and the rpsA genes as biomarkers for an accurate identification and differentiation of MAC pathogens.

Immunization with a DNA Vaccine Cocktail Induces a Th1 Response and Protects Mice Against Mycobacterium avium subsp. paratuberculosis Challenge

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Several novel antigens of Mycobacterium avium subsp. paratuberculosis have been studied as vaccine components and their immunogenicity has been evaluated. Previously, we reported that 85 antigen complex (85A, 85B, and 85C), superoxide dismutase (SOD), and 35kDa protein could induce significant lymph...

Short communication: Recovery of viable Mycobacterium avium subspecies paratuberculosis from retail pasteurized whole milk in Brazil.

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Carvalho, I A; Pietralonga, P A G; Schwarz, D G G; Faria, A C S; Moreira, M A S

2012-12-01

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic granulomatous enteritis that affects all ruminants worldwide. Some researchers have indicated a possible role of MAP in Crohn's disease. Despite extensive research and large and important advances in the past few decades, the etiology of Crohn's disease remains indefinite. The most probable transmission route of MAP from animals to humans is milk and dairy products. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide, and some studies have reported that MAP is resistant to pasteurization. In Brazil, MAP has been reported in raw milk samples; however, Brazilian retail pasteurized milk has not yet been tested for viable MAP. The aim of this study was to investigate MAP in pasteurized milk in the region of Viçosa (Minas Gerais, Brazil). Thirty-seven samples were collected and processed for culture of MAP. One colony similar to MAP was observed and confirmed by IS900-nested PCR and sequencing. Analysis revealed 97 to 99% identity with the MAP K-10 strain. This study is the first report of the presence of MAP in retail pasteurized whole milk in Brazil. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Relationship between presence of cows with milk positive for Mycobacterium avium subsp. paratuberculosis-specific antibody by enzyme-linked immunosorbent assay and viable M. avium subsp. paratuberculosis in dust in cattle barns.

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Eisenberg, Susanne W F; Chuchaisangrat, Ruj; Nielen, Mirjam; Koets, Ad P

2013-09-01

Paratuberculosis, or Johne's disease, in cattle is caused by Mycobacterium avium subsp. paratuberculosis, which has recently been suspected to be transmitted through dust. This longitudinal study on eight commercial M. avium subsp. paratuberculosis-positive dairy farms studied the relationship between the number of cows with M. avium subsp. paratuberculosis antibody-positive milk and the presence of viable M. avium subsp. paratuberculosis in settled-dust samples, including their temporal relationship. Milk and dust samples were collected in parallel monthly for 2 years. M. avium subsp. paratuberculosis antibodies in milk were measured by enzyme-linked immunosorbent assay (ELISA) and used as a proxy for M. avium subsp. paratuberculosis shedding. Settled-dust samples were collected by using electrostatic dust collectors (EDCs) at six locations in housing for dairy cattle and young stock. The presence of viable M. avium subsp. paratuberculosis was identified by liquid culture and PCR. The results showed a positive relationship (odds ratio [OR], 1.2) between the number of cows with ELISA-positive milk and the odds of having positive EDCs in the same airspace as the adult dairy cattle. Moreover, the total number of lactating cows also showed an OR slightly above 1. This relationship remained the same for settled-dust samples collected up to 2 months before or after the time of milk sampling. The results suggest that removal of adult cows with milk positive for M. avium subsp. paratuberculosis-specific antibody by ELISA might result in a decrease in the presence of viable M. avium subsp. paratuberculosis in dust and therefore in the environment. However, this decrease is likely delayed by several weeks at least. In addition, the data support the notion that M. avium subsp. paratuberculosis exposure of young stock is reduced by separate housing.

Systemic infection of Mycobacterium avium subspecies hominissuis and fungus in a pet dog.

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Kim, Myung-Chul; Kim, JaeMyung; Kang, WoonKi; Jang, Yunho; Kim, Yongbaek

2016-01-01

A 3-year-old neutered female poodle with a long history of dermatophytic skin disease was presented with lethargy, anorexia and progressive weight loss. Abdominal ultrasonography revealed markedly enlarged mesenteric lymph nodes and multiple hypoechoic foci in the spleen. Cytology of the mesenteric lymph nodes and spleen showed granulomatous inflammation with fungal organisms and negatively stained intracytoplasmic bacterial rods consistent with Mycobacteria spp. Based on culture, multiplex polymerase chain reaction and sequence analysis, the bacterium was identified as Mycobacterium avium subspecies hominissuis. Despite treatment with antibiotics, the dog's condition deteriorated, and it died approximately 3 weeks after first presentation.

Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius

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Christopher D Johnston

2014-09-01

Full Text Available It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of two MAP genes (MAP2121c and MAP3733c can enhance the heterologous expression of two antigens (MMP and MptD respectively, analogous to the form to which they are produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, codon optimised MptD displayed the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adhered with the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne’s disease.

Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius.

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Johnston, Christopher D; Bannantine, John P; Govender, Rodney; Endersen, Lorraine; Pletzer, Daniel; Weingart, Helge; Coffey, Aidan; O'Mahony, Jim; Sleator, Roy D

2014-01-01

It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, we previously engineered MAP3733c (encoding MptD) and show herein that MptD displays the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne's disease.

The Consensus from the Mycobacterium avium ssp. paratuberculosis (MAP Conference 2017

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J. Todd Kuenstner

2017-09-01

Full Text Available On March 24 and 25, 2017 researchers and clinicians from around the world met at Temple University in Philadelphia to discuss the current knowledge of Mycobacterium avium ssp. paratuberculosis (MAP and its relationship to human disease. The conference was held because of shared concern that MAP is a zoonotic bacterium that poses a threat not only to animal health but also human health. In order to further study this problem, the conferees discussed ways to improve MAP diagnostic tests and discussed potential future anti-MAP clinical trials. The conference proceedings may be viewed on the www.Humanpara.org website. A summary of the salient work in this field is followed by recommendations from a majority of the conferees.

Efficacy of various pasteurization time-temperature conditions in combination with homogenization on inactivation of Mycobacterium avium subsp. paratuberculosis in milk.

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Grant, Irene R; Williams, Alan G; Rowe, Michael T; Muir, D Donald

2005-06-01

The effect of various pasteurization time-temperature conditions with and without homogenization on the viability of Mycobacterium avium subsp. paratuberculosis was investigated using a pilot-scale commercial high-temperature, short-time (HTST) pasteurizer and raw milk spiked with 10(1) to 10(5) M. avium subsp. paratuberculosis cells/ml. Viable M. avium subsp. paratuberculosis was cultured from 27 (3.3%) of 816 pasteurized milk samples overall, 5 on Herrold's egg yolk medium and 22 by BACTEC culture. Therefore, in 96.7% of samples, M. avium subsp. paratuberculosis had been completely inactivated by HTST pasteurization, alone or in combination with homogenization. Heat treatments incorporating homogenization at 2,500 lb/in2, applied upstream (as a separate process) or in hold (at the start of a holding section), resulted in significantly fewer culture-positive samples than pasteurization treatments without homogenization (P HTST pasteurization with or without homogenization was estimated to be 4.0 to 5.2 log10. The impact of homogenization on clump size distribution in M. avium subsp. paratuberculosis broth suspensions was subsequently assessed using a Mastersizer X spectrometer. These experiments demonstrated that large clumps of M. avium subsp. paratuberculosis cells were reduced to single-cell or "miniclump" status by homogenization at 2,500 lb/in2. Consequently, when HTST pasteurization was being applied to homogenized milk, the M. avium subsp. paratuberculosis cells would have been present as predominantly declumped cells, which may possibly explain the greater inactivation achieved by the combination of pasteurization and homogenization.

Persistence of Mycobacterium avium subsp. paratuberculosis at a Farm-Scale Biogas Plant Supplied with Manure from Paratuberculosis-Affected Dairy Cattle▿

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Slana, I.; Pribylova, R.; Kralova, A.; Pavlik, I.

2011-01-01

In this study, products from all steps of anaerobic digestion at a farm-scale biogas plant supplied with manure from paratuberculosis-affected dairy cattle were examined and quantified for the presence of the causal agent of paratuberculosis, Mycobacterium avium subsp. paratuberculosis, using culture and quantitative real-time PCR (qPCR). Viable M. avium subsp. paratuberculosis cells were detected using culture in fermentors for up to 2 months; the presence of M. avium subsp. paratuberculosis DNA (101 cells/g) was demonstrated in all anaerobic fermentors and digestate 16 months after initiation of work at a biogas plant, using IS900 qPCR. F57 qPCR was able to detect M. avium subsp. paratuberculosis DNA (102 cells/g) at up to 12 months. According to these results, a fermentation process that extended beyond 2 months removed all viable M. avium subsp. paratuberculosis cells and therefore rendered its product M. avium subsp. paratuberculosis free. However, M. avium subsp. paratuberculosis DNA was found during all the examined periods (more than 1 year), which could be explained by either residual DNA being released from dead cells or by the presence of viable cells whose amount was under the limit of cultivability. As the latter hypothesis cannot be excluded, the safety of the final products of digestion used for fertilization or animal bedding cannot be defined, and further investigation is necessary to confirm or refute this risk. PMID:21398476

Efficacy of Various Pasteurization Time-Temperature Conditions in Combination with Homogenization on Inactivation of Mycobacterium avium subsp. paratuberculosis in Milk

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Grant, Irene R.; Williams, Alan G.; Rowe, Michael T.; Muir, D. Donald

2005-01-01

The effect of various pasteurization time-temperature conditions with and without homogenization on the viability of Mycobacterium avium subsp. paratuberculosis was investigated using a pilot-scale commercial high-temperature, short-time (HTST) pasteurizer and raw milk spiked with 101 to 105 M. avium subsp. paratuberculosis cells/ml. Viable M. avium subsp. paratuberculosis was cultured from 27 (3.3%) of 816 pasteurized milk samples overall, 5 on Herrold's egg yolk medium and 22 by BACTEC culture. Therefore, in 96.7% of samples, M. avium subsp. paratuberculosis had been completely inactivated by HTST pasteurization, alone or in combination with homogenization. Heat treatments incorporating homogenization at 2,500 lb/in2, applied upstream (as a separate process) or in hold (at the start of a holding section), resulted in significantly fewer culture-positive samples than pasteurization treatments without homogenization (P pasteurization with or without homogenization was estimated to be 4.0 to 5.2 log10. The impact of homogenization on clump size distribution in M. avium subsp. paratuberculosis broth suspensions was subsequently assessed using a Mastersizer X spectrometer. These experiments demonstrated that large clumps of M. avium subsp. paratuberculosis cells were reduced to single-cell or “miniclump” status by homogenization at 2,500 lb/in2. Consequently, when HTST pasteurization was being applied to homogenized milk, the M. avium subsp. paratuberculosis cells would have been present as predominantly declumped cells, which may possibly explain the greater inactivation achieved by the combination of pasteurization and homogenization. PMID:15932977

Mycobacterium avium MAV2052 protein induces apoptosis in murine macrophage cells through Toll-like receptor 4.

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Lee, Kang-In; Choi, Han-Gyu; Son, Yeo-Jin; Whang, Jake; Kim, Kwangwook; Jeon, Heat Sal; Park, Hye-Soo; Back, Yong Woo; Choi, Seunga; Kim, Seong-Woo; Choi, Chul Hee; Kim, Hwa-Jung

2016-04-01

Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨm), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨm loss, and apoptosis were all significantly reduced in TLR4(-/-) macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.

Hepatite granulomatosa em bovino causada por Mycobacterium avium subsp. paratuberculosis

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A.B.F Rodrigues

2010-12-01

Full Text Available Samples from intestines, liver, and lymph nodes were collected from a dairy steer with clinical suspicion of paratuberculosis. The samples were processed for histologic examination with hematoxylin-eosin and Zihel-Neelsen (ZN staining for the detection of acid-fast bacilli (AFB, and submitted to immunohistochemistry (IHC. Macroscopic changes were observed in the small intestines, with thickening and corrugation of the mucosa. The main microscopic changes were found in small intestines, lymph vessels in the mesentery, and mesenteric lymph nodes characterized by enteritis, lymphangiectasia, and lymphadenitis. Liver presented with granulomatous hepatitis, an uncommon histopathological feature for paratuberculosis. The clinical features associated with positive culture of Mycobacterium avium subsp. paratuberculosis and detection of AFB by ZN and IHC in the cytoplasm of macrophages (epithelioid in the intestinal mucosa and submucosa, lymph nodes, and liver were important to confirm the diagnosis of paratuberculosis.

A longitudinal study of factors influencing the result of a Mycobacterium avium ssp. paratuberculosis antibody ELISA in milk or dairy cows

NARCIS (Netherlands)

Eisenberg, S.W.F.; Veldman, E.; Rutten, V.P.M.G.; Koets, A.P.

2015-01-01

The influence of milk yield and milk composition on the diagnosis of Mycobacterium avium ssp. paratuberculosis (MAP) by milk ELISA in the context of the total IgG secretion patterns in milk throughout lactation and serum concentrations were investigated. A 2-yr trial was performed in which 1,410

Dam Mycobacterium avium subspecies parartuberculosis (MAP) infection status does not predetermine calves for future shedding when raised in a contaminated environment

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Eisenberg, S.W.F.; Rutten, Victor P.M.G.; Koets, A.P.

2015-01-01

Uptake of Mycobacterium avium subsp. paratuberculosis (MAP) by calves in the first days of life from colostrum, milk and faeces is regarded an important moment of transmission. The objective of this study was to quantify the
association between the MAP status of dams as determined by the

Facts, myths and hypotheses on the zoonotic nature of Mycobacterium avium subspecies paratuberculosis.

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Atreya, Raja; Bülte, Michael; Gerlach, Gerald-F; Goethe, Ralph; Hornef, Mathias W; Köhler, Heike; Meens, Jochen; Möbius, Petra; Roeb, Elke; Weiss, Siegfried

2014-10-01

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis (Johne's disease [JD]), a chronic granulomatous enteritis in ruminants. JD is one of the most widespread bacterial diseases of domestic animals with significant economic impact. The histopathological picture of JD resembles that of Crohn's disease (CD), a human chronic inflammatory bowel disease of still unresolved aetiology. An aetiological relevance of MAP for CD has been proposed. This and the ambiguity of other published epidemiological findings raise the question whether MAP represents a zoonotic agent. In this review, we will discuss evidence that MAP has zoonotic capacity. Copyright © 2014 Elsevier GmbH. All rights reserved.

Effective heat inactivation of Mycobacterium avium subsp. paratuberculosis in raw milk contaminated with naturally infected feces.

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Rademaker, Jan L W; Vissers, Marc M M; Te Giffel, Meike C

2007-07-01

The effectiveness of high-temperature, short holding time (HTST) pasteurization and homogenization with respect to inactivation of Mycobacterium avium subsp. paratuberculosis was evaluated quantitatively. This allowed a detailed determination of inactivation kinetics. High concentrations of feces from cows with clinical symptoms of Johne's disease were used to contaminate raw milk in order to realistically mimic possible incidents most closely. Final M. avium subsp. paratuberculosis concentrations varying from 10(2) to 3.5 x 10(5) cells per ml raw milk were used. Heat treatments including industrial HTST were simulated on a pilot scale with 22 different time-temperature combinations, including 60 to 90 degrees C at holding (mean residence) times of 6 to 15 s. Following 72 degrees C and a holding time of 6 s, 70 degrees C for 10 and 15 s, or under more stringent conditions, no viable M. avium subsp. paratuberculosis cells were recovered, resulting in >4.2- to >7.1-fold reductions, depending on the original inoculum concentrations. Inactivation kinetic modeling of 69 quantitative data points yielded an E(a) of 305,635 J/mol and an lnk(0) of 107.2, corresponding to a D value of 1.2 s at 72 degrees C and a Z value of 7.7 degrees C. Homogenization did not significantly affect the inactivation. The conclusion can be drawn that HTST pasteurization conditions equal to 15 s at > or =72 degrees C result in a more-than-sevenfold reduction of M. avium subsp. paratuberculosis.

Effective Heat Inactivation of Mycobacterium avium subsp. paratuberculosis in Raw Milk Contaminated with Naturally Infected Feces▿

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Rademaker, Jan L. W.; Vissers, Marc M. M.; te Giffel, Meike C.

2007-01-01

The effectiveness of high-temperature, short holding time (HTST) pasteurization and homogenization with respect to inactivation of Mycobacterium avium subsp. paratuberculosis was evaluated quantitatively. This allowed a detailed determination of inactivation kinetics. High concentrations of feces from cows with clinical symptoms of Johne's disease were used to contaminate raw milk in order to realistically mimic possible incidents most closely. Final M. avium subsp. paratuberculosis concentrations varying from 102 to 3.5 × 105 cells per ml raw milk were used. Heat treatments including industrial HTST were simulated on a pilot scale with 22 different time-temperature combinations, including 60 to 90°C at holding (mean residence) times of 6 to 15 s. Following 72°C and a holding time of 6 s, 70°C for 10 and 15 s, or under more stringent conditions, no viable M. avium subsp. paratuberculosis cells were recovered, resulting in >4.2- to >7.1-fold reductions, depending on the original inoculum concentrations. Inactivation kinetic modeling of 69 quantitative data points yielded an Ea of 305,635 J/mol and an lnk0 of 107.2, corresponding to a D value of 1.2 s at 72°C and a Z value of 7.7°C. Homogenization did not significantly affect the inactivation. The conclusion can be drawn that HTST pasteurization conditions equal to 15 s at ≥72°C result in a more-than-sevenfold reduction of M. avium subsp. paratuberculosis. PMID:17496131

WC1+ gamma delta T cells from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis respond differentially to stimulation with PPD-J.

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A role for gamma delta T cells in protection against mycobacterial infections including Johne’s disease (JD) has been suggested. In neonatal calves where the risk to infection with Mycobacterium avium subsp. paratuberculosis (MAP) is high, the majority of circulating CD3+ lymphocytes are gamma delta...

Detection of Mycobacterium avium subspecies paratuberculosis of dairy cows in Bogor

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Widagdo Sri Nugroho

2009-12-01

Full Text Available Johne’s disease (JD or partuberculosis is a chronic granulomatous enteritis in ruminants caused by infection of Mycobacterium avium paratuberculosis subspecies (MAP. The disease has been detected serologically in Indonesia. It’s potential to spread to other herds and could create great economic losses. The objectives of current study were to detect MAP in milk and faeces of dairy cows as well as to evaluate the association between farm management factors and presence of the bacteria in dairy cows in Bogor. The sample size was calculated using the formula to detect disease with the prevalence assumed to be 5% using 95% significant level. Milk and faeces samples were taken from 62 dairy cows which were suspected as suffering from MAP infection. Detection of MAP was done by isolation in Herrold’ egg yolk medium with mycobactin J (HEYMj, acid-fast bacilli Ziehl-Neelsen staining, PCR IS900 and F57. Biochemical test to confirm M. tuberculosis presence was also conducted. Fifteen isolates of Mycobacterium sp. were found from the faeces samples but not from the corresponding milk samples. However, conventional PCR conducted on the isolate as well as the milk samples, gave negative results. Biochemical test proved that all Mycobacterium sp. isolates were not M. tuberculosis. This study indicated the prevalence of MAP in Bogor was less than 5%. These findings should be continued by observational study to achieve the comprehensive information at the cattle and herd level. Bovine Tuberculosis monitoring should be done also to protect dairy herd and food safety for the community.

Typing of Human Mycobacterium avium Isolates in Italy by IS1245-Based Restriction Fragment Length Polymorphism Analysis

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Lari, Nicoletta; Cavallini, Michela; Rindi, Laura; Iona, Elisabetta; Fattorini, Lanfranco; Garzelli, Carlo

1998-01-01

All but 2 of 63 Mycobacterium avium isolates from distinct geographic areas of Italy exhibited markedly polymorphic, multibanded IS1245 restriction fragment length polymorphism (RFLP) patterns; 2 isolates showed the low-number banding pattern typical of bird isolates. By computer analysis, 41 distinct IS1245 patterns and 10 clusters of essentially identical strains were detected; 40% of the 63 isolates showed genetic relatedness, suggesting the existence of a predominant AIDS-associated IS1245 RFLP pattern. PMID:9817900

Isolation of Mycobacterium avium subsp paratuberculosis (Map) from feral cats on a dairy farm with Map-infected cattle.

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Palmer, Mitchell V; Stoffregen, William C; Carpenter, Jeremy G; Stabel, Judith R

2005-07-01

Paratuberculosis is an economically important disease of dairy cattle caused by Mycobacterium avium subsp. paratuberculosis (Map). The role of nonruminant, nondomestic animals in the epidemiology of paratuberculosis in cattle is unclear. To examine nonruminant, nondomestic animals for the presence of Map, 25 feral cats, nine mice (species unknown), eight rabbits (Sylvilagus floridanus), six raccoons (Procyon lotor), and three opossums (Didelphis virginiana) were collected from a mid-western dairy with known Map-infected cattle. Mycobacterium avium subsp. paratuberculosis was isolated from the mesenteric lymph node from seven of 25 (28%) feral cats. Ileum was culture-positive for three of these seven cats, and an isolation of Map was also made from the ileum of one of nine (11%) mice. Tissue samples from other species were negative as determined by Map culture; microscopic lesions consistent with paratuberculosis were not seen in any animal. Restriction fragment polymorphism analysis of isolates from cats and dairy cattle suggest interspecies transmission. The means by which interspecies transmission occurred may be through ingestion of Map-contaminated feces or waste milk or through ingestion of Map-infected prey. Shedding of Map from infected cats was not evaluated. The epidemiologic role of Map-infected feral cats on dairy farms requires further investigation.

Genome sequencing of ovine isolates of Mycobacterium avium subspecies paratuberculosis offers insights into host association

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Bannantine John P

2012-03-01

Full Text Available Abstract Background The genome of Mycobacterium avium subspecies paratuberculosis (MAP is remarkably homogeneous among the genomes of bovine, human and wildlife isolates. However, previous work in our laboratories with the bovine K-10 strain has revealed substantial differences compared to sheep isolates. To systematically characterize all genomic differences that may be associated with the specific hosts, we sequenced the genomes of three U.S. sheep isolates and also obtained an optical map. Results Our analysis of one of the isolates, MAP S397, revealed a genome 4.8 Mb in size with 4,700 open reading frames (ORFs. Comparative analysis of the MAP S397 isolate showed it acquired approximately 10 large sequence regions that are shared with the human M. avium subsp. hominissuis strain 104 and lost 2 large regions that are present in the bovine strain. In addition, optical mapping defined the presence of 7 large inversions between the bovine and ovine genomes (~ 2.36 Mb. Whole-genome sequencing of 2 additional sheep strains of MAP (JTC1074 and JTC7565 further confirmed genomic homogeneity of the sheep isolates despite the presence of polymorphisms on the nucleotide level. Conclusions Comparative sequence analysis employed here provided a better understanding of the host association, evolution of members of the M. avium complex and could help in deciphering the phenotypic differences observed among sheep and cattle strains of MAP. A similar approach based on whole-genome sequencing combined with optical mapping could be employed to examine closely related pathogens. We propose an evolutionary scenario for M. avium complex strains based on these genome sequences.

Differences in antimicrobial susceptibility of pigmented and unpigmented colonial variants of Mycobacterium avium.

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Stormer, R S; Falkinham, J O

1989-01-01

Unpigmented colonial variants were isolated from pigmented Mycobacterium avium isolates recovered from patients with acquired immunodeficiency syndrome and the environment. The variants were interconvertible: the rate of transition from unpigmented to pigmented type was 4.0 x 10(-5) variants per cell per generation. The unpigmented variants were more tolerant to antibiotics, especially beta-lactams, and Cd2+ and Cu2+ salts than were their pigmented parents. Both pigmented and unpigmented variants of the strains produced beta-lactamase, although beta-lactamase did not appear to be a determinant of beta-lactam susceptibility. Pigmented variants grew more rapidly in a number of commonly used mycobacterial media, were more hydrophobic, and had higher carotenoid contents than their unpigmented segregants. PMID:2808669

Drug susceptibility testing of Mycobacterium Avium subsp. Avium isolates from naturally infected domestic pigeons to avian tuberculosis

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Kaveh Parvandar

2016-01-01

Conclusion: We suggest drug susceptibility testing for more nontuberculous mycobateria, particularly M. avium complex isolated from infected birds and humans, as well as molecular basics of drug sensitivity in order to detect resistance genes of pathogenic M. avium subsp. avium.

Effect of Soil Slope on the Appearance of Mycobacterium avium subsp. paratuberculosis in Water Running off Grassland Soil after Application of Contaminated Slurry

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Alfaro, M.; Salazar, F.; Troncoso, E.; Mitchell, R. M.; Ramirez, L.; Naguil, A.; Zamorano, P.; Collins, M. T.

2013-01-01

The study assessed the effect of soil slope on Mycobacterium avium subsp. paratuberculosis transport into rainwater runoff from agricultural soil after application of M. avium subsp. paratuberculosis-contaminated slurry. Under field conditions, 24 plots of undisturbed loamy soil 1 by 2 m2 were placed on platforms. Twelve plots were used for water runoff: 6 plots at a 3% slope and 6 plots at a 15% slope. Half of the plots of each slope were treated with M. avium subsp. paratuberculosis-contaminated slurry, and half were not treated. Using the same experimental design, 12 plots were established for soil sampling on a monthly basis using the same spiked slurry application and soil slopes. Runoff following natural rainfall was collected and analyzed for M. avium subsp. paratuberculosis, coliforms, and turbidity. M. avium subsp. paratuberculosis was detected in runoff from all plots treated with contaminated slurry and one control plot. A higher slope (15%) increased the likelihood of M. avium subsp. paratuberculosis detection but did not affect the likelihood of finding coliforms. Daily rainfall increased the likelihood that runoff would have coliforms and the coliform concentration, but it decreased the M. avium subsp. paratuberculosis concentration in the runoff. When there was no runoff, rain was associated with increased M. avium subsp. paratuberculosis concentrations. Coliform counts in runoff were related to runoff turbidity. M. avium subsp. paratuberculosis presence/absence, however, was related to turbidity. Study duration decreased bacterial detection and concentration. These findings demonstrate the high likelihood that M. avium subsp. paratuberculosis in slurry spread on pastures will contaminate water runoff, particularly during seasons with high rainfall. M. avium subsp. paratuberculosis contamination of water has potential consequences for both animal and human health. PMID:23542616

Comparative evaluation of positive tests to Mycobacterium avium subsp. paratuberculosis in clinically healthy sheep and goats in south-west Greece using molecular techniques, serology, and culture.

Science.gov (United States)

Ikonomopoulos, John; Balaskas, Christos; Kantzoura, Bagia; Fragiadaki, Eirini; Pavlik, Ivo; Bartos, Milan; Lukas, John C; Gazouli, Maria

2007-09-01

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of paratuberculosis, which affects mainly ruminants although there is a growing concern about its possible implication in Crohn's disease in humans especially in connection with environmental spread and risks to the food chain. Retail cheese may represent a significant source of human exposure to MAP and the aim of this study was to assess MAP status in clinically healthy sheep and goats in Greece, comparing techniques routinely used in the positive diagnosis of the disease. From a total of 30 flocks, 632 sheep and goats had faecal, serum, and whole-blood samples examined by culture, complement fixation test (CFT), and polymerase chain reaction (PCR) targeted at IS900, IS1245, and IS6110. PCR produced positive results in 21% of the animals tested, with 5.6%, 3.9%, and 11.5% being identified as MAP, Mycobacterium avium subsp. avium, and Mycobacterium tuberculosis complex, respectively. CFT produced positive and suspicious results in 4.4% and 14.4% of the cases. Faecal cultures were negative in all but a single case that was identified as restriction fragment length polymorphism (RFLP)-type BC1. Agreement between results obtained by PCR and CFT was poor with isolated cases although an assessment of the MAP positive tests produced similar results for both methods. The findings indicate the need for additional measures of control, although the costs may be substantial if public health protection justifies elimination of MAP from livestock.

Mycobacterium avium complex pulmonary disease: characteristics and treatment in an Irish patient cohort.

LENUS (Irish Health Repository)

Judge, EP

2016-04-01

The prevalence of Mycobacterium avium complex (MAC) pulmonary disease is increasing globally. However, reliable national and international data relating to its epidemiology and management is lacking. During the period 2003-2014, MAC was isolated from the pulmonary samples of 75 patients at the Irish Mycobacteria Reference Laboratory (IMRL). Most patients (42, 56%) had underlying pulmonary disease, and 37 (49%) had clinical\\/radiographic characteristics consistent with MAC pulmonary disease. However, only 18 patients (24%) fulfilled internationally accepted criteria for diagnosis\\/treatment of this disease. Treatment was started in 13 (72%) of these cases, which is similar to internationally published treatment rates. The diagnosis of significant MAC pulmonary disease can be difficult, and treatment is not always warranted even when diagnostic criteria are met.

IS1245 restriction fragment length polymorphism typing of Mycobacterium avium from patients admitted to a reference hospital in Campinas, Brazil

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A.C. Panunto

2003-10-01

Full Text Available Mycobacterium avium is an important pathogen among immunodeficient patients, especially patients with AIDS. The natural history of this disease is unclear. Several environmental sources have been implicated as the origin of this infection. Polyclonal infection with this species is observed, challenging the understanding of its pathogenesis and treatment. In the present study 45 M. avium strains were recovered from 39 patients admitted to a reference hospital between 1996 and 1998. Species identification was performed using a species-specific nucleic acid hybridization test (AccuProbe® from Gen-Probe®. Strains were genotyped using IS1245 restriction fragment length polymorphism typing. Blood was the main source of the organism. In one patient with disseminated disease, M. avium could be recovered more than once from potentially sterile sites. Strains isolated from this patient had different genotypes, indicating that the infection was polyclonal. Four patient clones were characterized in this population, the largest clone being detected in eight patients. This finding points to a common-source transmission of the organism.

Variable-Number Tandem-Repeat Analysis of Respiratory and Household Water Biofilm Isolates of “Mycobacterium avium subsp. hominissuis” with Establishment of a PCR Database

Science.gov (United States)

Iakhiaeva, Elena; Howard, Susan T.; Brown Elliott, Barbara A.; McNulty, Steven; Newman, Kristopher L.; Falkinham, Joseph O.; Williams, Myra; Kwait, Rebecca; Lande, Leah; Vasireddy, Ravikiran; Turenne, Christine

2016-01-01

“Mycobacterium avium subsp. hominissuis” is an important cause of pulmonary disease. It is acquired from environmental sources, but there is no methodology for large population studies. We evaluated the potential of variable-number tandem-repeat (VNTR) analysis. Clinical and household biofilm M. avium isolates underwent molecular identification. Testing for IS901 was done to separate M. avium subsp. avium from M. avium subsp. hominissuis. VNTR types were defined using VNTR loci, and subtyping was performed using 3′ hsp65 and internal transcribed spacer (ITS) sequencing. Forty-nine VNTR types and eight subtypes of M. avium subsp. hominissuis (IS901 negative) were identified among 416 isolates of M. avium from 121 patients and 80 biofilm sites. Of those types, 67% were found only among patient isolates, 11% only among household water isolates, and 23% among both. Of 13 VNTR types that included ≥4 patients, the majority (61.5%) represented geographic clustering (same city). Most VNTR types with multiple patients belonged to the same 3′ hsp65 sequence code (sequevar). A total of 44 isolates belonging to four M. avium subsp. hominissuis VNTR types (8%), including three with the rare Mav-F ITS sequence and 0/8 subspecies, produced amplicons with IS901 PCR primers. By sequencing, all 44 amplicons were not IS901 but ISMav6, which was recently observed in Japan but had not been previously described among U.S. isolates. VNTR analysis of M. avium subsp. hominissuis isolates is easier and faster than pulsed-field gel electrophoresis. Seven VNTR loci separated 417 isolates into 49 types. No isolates of M. avium subsp. avium were identified. The distributions of the VNTR copy numbers, the allelic diversity, and the low prevalence of ISMav6 differed from the findings for respiratory isolates reported from Japan. PMID:26739155

Semiquantitative Culture Analysis during Therapy for Mycobacterium avium Complex Lung Disease.

Science.gov (United States)

Griffith, David E; Adjemian, Jennifer; Brown-Elliott, Barbara A; Philley, Julie V; Prevots, D Rebecca; Gaston, Christopher; Olivier, Kenneth N; Wallace, Richard J

2015-09-15

Microbiologically based criteria such as sputum culture conversion to negative have traditionally been used to define treatment success for mycobacterial diseases. There are, however, limited data regarding whether nontuberculous mycobacterial sputum culture conversion or semiquantitative culture analysis correlates with subjective or nonmicrobiologic objective indices of treatment response. To determine whether a semiquantitative mycobacterial culture scale correlated with clinical disease status and was predictive of long-term sputum mycobacterial culture conversion to negative in a cohort of patients with nodular/bronchiectatic Mycobacterium avium complex lung disease undergoing therapy. One hundred and eighty patients undergoing standard macrolide-based therapy for M. avium complex lung disease were monitored at standard frequent intervals with symptomatic, radiographic, and microbiologic data collected, including semiquantitative mycobacterial culture analysis. Analyses were used to evaluate clinical and microbiologic predictors of long-term sputum conversion to culture negative. After 12 months of therapy, 148 (82%) patients had sputum conversion to culture negative. Baseline semiquantitative sputum culture scores did not differ between patients with sputum conversion and those without. The change in sputum culture semiquantitative score from baseline to Month 3 was highly predictive of subsequent sputum long-term conversion status indicative of treatment success, as was improvement in cough, and especially early radiographic improvement. Early semiquantitative sputum agar plate culture results can be used to predict symptomatic and radiographic improvement as well as long-term sputum culture conversion to negative in this population. We suggest that semiquantitative sputum culture scores can be a useful tool for evaluating new nontuberculous mycobacterial lung disease therapies.

Occurrence of Mycobacterium avium subsp. paratuberculosis in milk at dairy cattle farms

DEFF Research Database (Denmark)

Okura, Hisako; Toft, Nils; Nielsen, Søren Saxmose

2012-01-01

Presence of Mycobacterium avium subsp. paratuberculosis (MAP) in milk for human consumption is a concern due to its possible relationship with Crohn’s disease in humans. Pasteurization effectively reduces the MAP load by four to five logs, but the efficacy depends on the MAP concentration, which...... depends on the prevalence among contributing herds and individuals. Considerable variation of MAP in bulk tank milk (BTM) and individual cow’s milk (IM) is reported, but factors associated with MAP occurrence in milk at farm level have not been described. This study systematically reviewed published...... studies aiming at estimating the occurrence of MAP in on-farm BTM and IM by meta-analysis. A total of 692 articles were identified through electronic databases and initially screened using title and abstract. The quality of the 61 potentially relevant articles was assessed using full text and 31 articles...

Utilization of a ts-sacB selection system for the generation of a Mycobacterium avium serovar-8 specific glycopeptidolipid allelic exchange mutant

Science.gov (United States)

Irani, Vida R; Lee, Sun-Hwa; Eckstein, Torsten M; Inamine, Julia M; Belisle, John T; Maslow, Joel N

2004-01-01

Background Mycobacterium avium are ubiquitous environmental organisms and a cause of disseminated infection in patients with end-stage AIDS. The glycopeptidolipids (GPL) of M. avium are proposed to participate in the pathogenesis of this organism, however, establishment of a clear role for GPL in disease production has been limited by the inability to genetically manipulate M. avium. Methods To be able to study the role of the GPL in M. avium pathogenesis, a ts-sacB selection system, not previously used in M. avium, was employed as a means to achieve homologous recombination for the rhamnosyltransferase (rtfA) gene of a pathogenic serovar 8 strain of M. avium to prevent addition of serovar-specific sugars to rhamnose of the fatty acyl-peptide backbone of GPL. The genotype of the resultant rtfA mutant was confirmed by polymerase chain reaction and southern hybridization. Disruption in the proximal sugar of the haptenic oligosaccharide resulted in the loss of serovar specific GPL with no change in the pattern of non-serovar specific GPL moieties as shown by thin layer chromatography and gas chromatography/mass spectrometry. Complementation of wild type (wt) rtfA in trans through an integrative plasmid restored serovar-8 specific GPL expression identical to wt serovar 8 parent strain. Results In this study, we affirm our results that rtfA encodes an enzyme responsible for the transfer of Rha to 6d-Tal and provide evidence of a second allelic exchange mutagenesis system suitable for M. avium. Conclusion We report the second allelic exchange system for M. avium utilizing ts-sacB as double-negative and xylE as positive counter-selection markers, respectively. This system of allelic exchange would be especially useful for M. avium strains that demonstrate significant isoniazid (INH) resistance despite transformation with katG. Through the construction of mutants in GPL or other mycobacterial components, their roles in M. avium pathogenesis, biosynthesis, or drug

Utilization of a ts-sacB selection system for the generation of a Mycobacterium avium serovar-8 specific glycopeptidolipid allelic exchange mutant

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Belisle John T

2004-09-01

Full Text Available Abstract Background Mycobacterium avium are ubiquitous environmental organisms and a cause of disseminated infection in patients with end-stage AIDS. The glycopeptidolipids (GPL of M. avium are proposed to participate in the pathogenesis of this organism, however, establishment of a clear role for GPL in disease production has been limited by the inability to genetically manipulate M. avium. Methods To be able to study the role of the GPL in M. avium pathogenesis, a ts-sacB selection system, not previously used in M. avium, was employed as a means to achieve homologous recombination for the rhamnosyltransferase (rtfA gene of a pathogenic serovar 8 strain of M. avium to prevent addition of serovar-specific sugars to rhamnose of the fatty acyl-peptide backbone of GPL. The genotype of the resultant rtfA mutant was confirmed by polymerase chain reaction and southern hybridization. Disruption in the proximal sugar of the haptenic oligosaccharide resulted in the loss of serovar specific GPL with no change in the pattern of non-serovar specific GPL moieties as shown by thin layer chromatography and gas chromatography/mass spectrometry. Complementation of wild type (wt rtfA in trans through an integrative plasmid restored serovar-8 specific GPL expression identical to wt serovar 8 parent strain. Results In this study, we affirm our results that rtfA encodes an enzyme responsible for the transfer of Rha to 6d-Tal and provide evidence of a second allelic exchange mutagenesis system suitable for M. avium. Conclusion We report the second allelic exchange system for M. avium utilizing ts-sacB as double-negative and xylE as positive counter-selection markers, respectively. This system of allelic exchange would be especially useful for M. avium strains that demonstrate significant isoniazid (INH resistance despite transformation with katG. Through the construction of mutants in GPL or other mycobacterial components, their roles in M. avium pathogenesis

Environmental Survival of Mycobacterium avium subsp. paratuberculosis in Different Climatic Zones of Eastern Australia

Science.gov (United States)

Begg, Douglas J.; Dhand, Navneet K.; Watt, Bruce; Whittington, Richard J.

2014-01-01

The duration of survival of both the S and C strains of Mycobacterium avium subsp. paratuberculosis in feces was quantified in contrasting climatic zones of New South Wales, Australia, and detailed environmental temperature data were collected. Known concentrations of S and C strains in feces placed on soil in polystyrene boxes were exposed to the environment with or without the provision of shade (70%) at Bathurst, Armidale, Condobolin, and Broken Hill, and subsamples taken every 2 weeks were cultured for the presence of M. avium subsp. paratuberculosis. The duration of survival ranged from a minimum of 1 week to a maximum of 16 weeks, and the provision of 70% shade was the most important factor in extending the survival time. The hazard of death for exposed compared to shaded samples was 20 and 9 times higher for the S and C strains, respectively. Site did not affect the survival of the C strain, but for the S strain, the hazard of death was 2.3 times higher at the two arid zone sites (Broken Hill and Condobolin) than at the two temperate zone sites (Bathurst and Armidale). Temperature measurements revealed maximum temperatures exceeding 60°C and large daily temperature ranges at the soil surface, particularly in exposed boxes. PMID:24463974

Exposure of young dairy cattle to Mycobacterium avium subsp. paratuberculosis (MAP) through intensive grazing of contaminated pastures in a herd positive for Johne's disease.

Science.gov (United States)

Fecteau, Marie-Eve; Whitlock, Robert H; Buergelt, Claus D; Sweeney, Raymond W

2010-02-01

This study investigated the susceptibility of 1- to 2-year-old cattle to Mycobacterium avium subsp. paratuberculosis (MAP) on pasture previously grazed by infected cattle. The exposure of yearling cattle to pastures contaminated with MAP resulted in infection with MAP, showing that age resistance to infection can be overcome by pressure of infection.

Causation of Crohn’s Disease by Mycobacterium avium Subspecies Paratuberculosis

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John Hermon-Taylor

2000-01-01

Full Text Available Mycobacterium avium subspecies paratuberculosis (MAP is a member of the M avium complex (MAC. It differs genetically from other MAC in having 14 to 18 copies of IS900 and a single cassette of DNA involved in the biosynthesis of surface carbohydrate. Unlike other MAC, MAP is a specific cause of chronic inflammation of the intestine in many animal species, including primates. The disease ranges from pluribacillary to paucimicrobial, with chronic granulomatous inflammation like leprosy in humans. MAP infection can persist for years without causing clinical disease. The herd prevalence of MAP infection in Western Europe and North America is reported in the range 21% to 54%. These subclinically infected animals shed MAP in their milk and onto pastures. MAP is more robust than tuberculosis, and the risk that is conveyed to human populations in retail milk and in domestic water supplies is high. MAP is harboured in the ileocolonic mucosa of a proportion of normal people and can be detected in a high proportion of full thickness samples of inflamed Crohn’s disease gut by improved culture systems and IS900 polymerase chain reaction if the correct methods are used. MAP in Crohn’s disease is present in a protease-resistant nonbacillary form, can evade immune recognition and probably causes an immune dysregulation. As with other MAC, MAP is resistant to most standard antituberculous drugs. Treatment of Crohn’s disease with combinations of drugs more active against MAC such as rifabutin and clarithromycin can bring about a profound improvement and, in a few cases, apparent disease eradication. New drugs as well as effective MAP vaccines for animals and humans are needed. The problems caused by MAP constitute a public health issue of tragic proportions for which a range of remedial measures are urgently needed.

Characterization of Mycobacterium paratuberculosis by gas-liquid and thin-layer chromatography and rapid demonstration of mycobactin dependence using radiometric methods

International Nuclear Information System (INIS)

Damato, J.J.; Knisley, C.; Collins, M.T.

1987-01-01

Thirty-six Mycobacterium paratuberculosis isolates of bovine, caprine, and ovine origins were evaluated by using gas-liquid chromatography (GLC), thin-layer chromatography (TLC), and BACTEC 7H12 Middlebrook TB medium in an effort to more rapidly differentiate this group of organisms from other mycobacteria. Bacterial suspensions (0.1 ml) were inoculated by syringe into 7H12 broth containing 2 micrograms of mycobactin P per ml and control broth without mycobactin P. Cultures were incubated at 37 0 C and read daily with a BACTEC Model 301. After 8 days of incubation, the growth index readings for the test broths containing mycobactin P were twice those of the control broths without mycobactin P. Sixty-five isolates of mycobacteria other than M. paratuberculosis were also examined. No difference was noted between the growth index readings of control and mycobactin-containing broths. Except for Mycobacterium avium-Mycobacterium intracellulare, TLC studies differentiated M. paratuberculosis from the other mycobacterial species tested. The GLC data reveal that all M. paratuberculosis isolates had a distinctive peak (14A) which was not found among M. avium-M. intracellulare complex organisms. These data indicate that 7H12 radiometric broth was able to rapidly demonstrate the mycobactin dependence of M. paratuberculosis and GLC and TLC procedures were capable of rapidly differentiating this organism from the other mycobacteria studied

Methylobacterium spp. as an indicator for the presence or absence of Mycobacterium spp.

OpenAIRE

Falkinham III, Joseph O.; Williams, Myra D.; Kwait, Rebecca; Lande, Leah

2016-01-01

Objective/Background: A published survey of bacteria in showerhead biofilm samples revealed that Methylobacterium spp. and Mycobacterium spp. seldom coexisted in biofilms. Method: To confirm that information, biofilm samples were collected from household plumbing of Mycobacterium avium patients and Methylobacterium spp. and M. avium numbers were measured by direct colony counts. Results: The results demonstrated that if Methylobacterium spp. were present, Mycobacterium spp. were absent,...

Short communication: Correlation between within-herd antibody-prevalence and bulk tank milk antibody levels to Mycobacterium avium ssp. paratuberculosis using 2 commercial immunoassays.

Science.gov (United States)

Pesqueira, M N; Yus, E; Factor, C; Mato, I; Sanjuán, M L; Eiras, C; Arnaiz, I; Diéguez, F J

2017-09-01

The objective of this study was to determine the correlation between the results obtained with the ELISA technique for antibodies to Mycobacterium avium ssp. paratuberculosis in serum and bulk tank milk at the herd level. For this purpose, 203 samples of bulk tank milk were analyzed with 2 commercial ELISA from dairy herds with a prevalence of seropositive animals that was also determined. In regard to the reference test (results in blood serum), the sensitivity of the bulk tank milk test to detect high-positive herds (≥10% seroprevalence) ranged from 85.7 to 71.4%. The specificity to detect herds with no seropositive animals ranged from 70.5 to 53%. In a quantitative approach, Pearson correlation coefficients, reported as a measure of the linear association between herd seroprevalences and transformed optical density values recorded in bulk tank milk, were 0.39 and 0.54 for the studied ELISA. Although the test results were relatively fairly correlated with the within-herd prevalence, the practical utility of bulk tank milk testing for Mycobacterium avium ssp. paratuberculosis seems limited, especially regarding specificity. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Hypersensitivity pneumonitis with Mycobacterium avium complex among spa workers.

Science.gov (United States)

Moraga-McHaley, Stephanie Ann; Landen, Michael; Krapfl, Heidi; Sewell, C Mack

2013-01-01

The New Mexico Department of Health (NMDOH) investigated the cause of two cases of hypersensitivity pneumonitis (HP) in spa maintenance workers with laboratory confirmed Mycobacterium avium complex (MAC). The investigation occurred in tandem with worker protection and swimming pool regulatory investigations by the New Mexico Environment Department at the spa where the workers were employed. The investigation was conducted in order to identify unreported cases, exposure source(s), and to prevent further worker exposure. NMDOH surveyed 57 spa employees about symptoms and exposures, categorized jobs according to self-reported exposure to water, and computed odds ratios for symptom reporting by exposure category. Environmental isolates from spa water and filter swabs were cultured and compared to patient isolates by the Environmental and Applied Microbiology Team, Centers for Disease Control and Prevention (CDC). Workers with the highest exposure reported more HP-like symptoms (OR = 9.6), as did intermediate exposure workers (OR = 6.5), compared to workers with no aerosolized water exposure. Two of 13 environmental isolates were closely related to one of the patient isolates. Workers were likely exposed during spray cleaning of cartridge filters in a poorly ventilated work space. Recommendations include inhibiting organism growth in spa systems, assuring the use of respiratory protection, and adequately ventilating work spaces where filters and equipment are cleaned.

Killing of Mycobacterium avium by lactoferricin peptides: improved activity of arginine- and D-amino-acid-containing molecules.

Science.gov (United States)

Silva, Tânia; Magalhães, Bárbara; Maia, Sílvia; Gomes, Paula; Nazmi, Kamran; Bolscher, Jan G M; Rodrigues, Pedro N; Bastos, Margarida; Gomes, Maria Salomé

2014-06-01

Mycobacterium avium causes respiratory disease in susceptible individuals, as well as disseminated infections in immunocompromised hosts, being an important cause of morbidity and mortality among these populations. Current therapies consist of a combination of antibiotics taken for at least 6 months, with no more than 60% overall clinical success. Furthermore, mycobacterial antibiotic resistance is increasing worldwide, urging the need to develop novel classes of antimicrobial drugs. One potential and interesting alternative strategy is the use of antimicrobial peptides (AMP). These are present in almost all living organisms as part of their immune system, acting as a first barrier against invading pathogens. In this context, we investigated the effect of several lactoferrin-derived AMP against M. avium. Short peptide sequences from both human and bovine lactoferricins, namely, hLFcin1-11 and LFcin17-30, as well as variants obtained by specific amino acid substitutions, were evaluated. All tested peptides significantly inhibited the axenic growth of M. avium, the bovine peptides being more active than the human. Arginine residues were found to be crucial for the display of antimycobacterial activity, whereas the all-d-amino-acid analogue of the bovine sequence displayed the highest mycobactericidal activity. These findings reveal the promising potential of lactoferricins against mycobacteria, thus opening the way for further research on their development and use as a new weapon against mycobacterial infections. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The clinical efficacy of a clarithromycin-based regimen for Mycobacterium avium complex disease: A nationwide post-marketing study.

Science.gov (United States)

Kadota, Jun-Ichi; Kurashima, Atsuyuki; Suzuki, Katsuhiro

2017-05-01

The revised 2007 American Thoracic Society/Infectious Diseases Society of America statement recommend clarithromycin-based combination therapy for treatment of Mycobacterium avium complex lung disease and stipulates approximately 1 year of continuous treatment after bacilli negative conversion. However, supporting data are insufficient. Our objective was to obtain data on the clinical outcome of clarithromycin-based daily regimens by conducting a nationwide retrospective post-marketing study of M. avium complex lung disease. In accordance with the Japanese guidelines, patients were enrolled in this survey according to their chest radiographic findings and microbiologic test results. They were treated with a multidrug regimen including clarithromycin, rifampicin, and ethambutol (clarithromycin-based regimen) until bacilli negative conversion, and the treatment was continued for approximately 1 year after the initial conversion. Data were collected before administration, at the time of bacilli negative conversion, at the end of treatment, and at 6 months after the end of treatment. Of the 466 subjects enrolled in the study, 271 patients who received clarithromycin at 800 mg/day underwent evaluation for M. avium complex disease. The final bacilli negative conversion rate in those patients was 94.7%. The bacteriological relapse rate was 5.0% (5/100 patients). Bacteriological relapse was noted in patients treated for less than 15 months after conversion. No life-threatening or serious adverse drug reactions were observed. This study demonstrated that a clarithromycin-based daily regimen can yield a high bacteriological conversion rate in M. avium complex disease. After conversion, treatment for less than 15 months might be insufficient to prevent bacteriological relapse. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Infección pulmonar por Mycobacterium avium en paciente VIH/SIDA: Primer reporte en Perú

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Juan Carrasco

Full Text Available El complejo Mycobacterium avium (MAC es un patógeno que se encuentra en el medioambiente y causa infecciones tanto en pacientes inmunocompetentes como inmunocomprometidos. Se presenta el caso de un paciente VIH positivo varón de 38 años infectado por P. jirovecii y aparentemente infectado por Mycobacterium tuberculosis desde el año 2009, el cual fue tratado con antibioticoterapia para pneumocistosis y terapia antituberculosis (TB logrando mejoría parcial. En el año 2012 se le realizó nuevamente examen de cultivo y un nuevo tratamiento anti TB, frente a la sospecha de estar en presencia de una cepa de TB multidrogorresistente se recomienda realizar la identificación micobacteriana. El examen de cultivo fue positivo y el resultado genotípico resultó positivo para MAC. Se reporta el primer caso de un paciente VIH/SIDA con infección pulmonar por MAC en el Perú, así como una breve revisión de los aspectos epidemiológicos, clínicos y de tratamiento

Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation, Structural Integrity, and Tolerance to Antibiotics.

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Sasha J Rose

Full Text Available Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH. In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain and MAH 104 (reference strain were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections.

Mycobacterium avium restriction fragment lenght polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains

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Patrícia Carvalho de Sequeira

2005-11-01

Full Text Available Simple double repetitive element polymerase chain reaction (MaDRE-PCR and Pvu II-IS1245 restriction fragment length polymorphism (RFLP typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 Aids inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

Mycobacterium avium Subspecies paratuberculosis Infection in Cases of Irritable Bowel Syndrome and Comparison with Crohn's Disease and Johne's Disease: Common Neural and Immune Pathogenicities▿

OpenAIRE

Scanu, Antonio M.; Bull, Tim J.; Cannas, Sara; Sanderson, Jeremy D.; Sechi, Leonardo A.; Dettori, Giuseppe; Zanetti, Stefania; Hermon-Taylor, John

2007-01-01

Mycobacterium avium subsp. paratuberculosis causes Johne's disease, a systemic infection and chronic inflammation of the intestine that affects many species, including primates. Infection is widespread in livestock, and human populations are exposed. Johne's disease is associated with immune dysregulation, with involvement of the enteric nervous system overlapping with features of irritable bowel syndrome in humans. The present study was designed to look for an association between Mycobacteri...

Epidemiological characterization and risk factors associated with Mycobacterium avium subsp. paratuberculosis infection in dairy goats in the Brazilian semiarid region

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Theonys Diógenes Freitas

2015-02-01

Full Text Available The aim of this investigation was to conduct an epidemiological study and identify risk factors associated with the occurrence of paratuberculosis (Johne’s disease in dairy goats within the semiarid region of Paraíba State. The study was done during the period of March 2009 to July 2011, during which 727 female goats from 86 flocks from the city of Monteiro, Paraíba were investigated. For the serological diagnosis of Mycobacterium avium subsp. paratuberculosis (Map infection indirect ELISA tests (screening and confirmatory were performed. Of the 727 animals used six (0.82% were seropositive at the confirmatory test after screening, and of the 86 flocks six (6.97% presented at least one seropositive animal. In positive flocks the frequency of reactive animals ranged from 5.26% to 16.60%. Risk factors identified were production system (weaning and reproduction (odds ratio = 36.0; 95% CI = 2.6 –486.1; p < 0,001 and absence of technical infrastructure (odds ratio = 54.0; 95% CI = 4.5 –642.9; p < 0,001. It was concluded that Mycobacterium avium subsp. paratuberculosis is present in dairy goat flocks in the region; however, its influence on decrease productivity as well as the risk of transmission to humans through animal products must totally evaluated. Based on the analysis of risk factors, improvements are recommended for the technical infrastructure and the management of breeding goats.

Comparative Analyses of Nonpathogenic, Opportunistic, and Totally Pathogenic Mycobacteria Reveal Genomic and Biochemical Variabilities and Highlight the Survival Attributes of Mycobacterium tuberculosis

Science.gov (United States)

Singh, Yadvir; Kohli, Sakshi; Ahmad, Javeed; Ehtesham, Nasreen Z.; Tyagi, Anil K.

2014-01-01

ABSTRACT Mycobacterial evolution involves various processes, such as genome reduction, gene cooption, and critical gene acquisition. Our comparative genome size analysis of 44 mycobacterial genomes revealed that the nonpathogenic (NP) genomes were bigger than those of opportunistic (OP) or totally pathogenic (TP) mycobacteria, with the TP genomes being smaller yet variable in size—their genomic plasticity reflected their ability to evolve and survive under various environmental conditions. From the 44 mycobacterial species, 13 species, representing TP, OP, and NP, were selected for genomic-relatedness analyses. Analysis of homologous protein-coding genes shared between Mycobacterium indicus pranii (NP), Mycobacterium intracellulare ATCC 13950 (OP), and Mycobacterium tuberculosis H37Rv (TP) revealed that 4,995 (i.e., ~95%) M. indicaus pranii proteins have homology with M. intracellulare, whereas the homologies among M. indicus pranii, M. intracellulare ATCC 13950, and M. tuberculosis H37Rv were significantly lower. A total of 4,153 (~79%) M. indicus pranii proteins and 4,093 (~79%) M. intracellulare ATCC 13950 proteins exhibited homology with the M. tuberculosis H37Rv proteome, while 3,301 (~82%) and 3,295 (~82%) M. tuberculosis H37Rv proteins showed homology with M. indicus pranii and M. intracellulare ATCC 13950 proteomes, respectively. Comparative metabolic pathway analyses of TP/OP/NP mycobacteria showed enzymatic plasticity between M. indicus pranii (NP) and M. intracellulare ATCC 13950 (OP), Mycobacterium avium 104 (OP), and M. tuberculosis H37Rv (TP). Mycobacterium tuberculosis seems to have acquired novel alternate pathways with possible roles in metabolism, host-pathogen interactions, virulence, and intracellular survival, and by implication some of these could be potential drug targets. PMID:25370496

[Comparison of usefulness between variable numbers of tandem repeats (VNTR) analysis and restriction fragment length polymorphism (RFLP) in the genotyping of Mycobacterium avium].

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Kazumi, Yuko; Udagawa, Tadashi; Maeda, Shinji; Murase, Yoshirou; Sugawara, Isamu; Okumura, Masao; Azuma, Yuka; Goto, Mieko; Tsunematsu, Noriko

2007-10-01

Comparison of usefulness of IS1245 RFLP and VNTR in M. avium genotyping. Thirty-six cases (55 strains) from sputum and BALF and twelve cases (29 strains) isolated from blood of HIV-infected patients were used. VNTR and RFLP using IS1245 were performed. Multiple samples were taken from 16 patients and 52 clinical isolates were used for VNTR and RFLP for comparison. (1) VNTR and RFLP results were identical in 12 out of 16 cases whose samples were collected several times. (2) Eight isolates were obtained from one patient. In this eight isolates, there were the cases of M. avium polyclonal infection and of mixed infection with M. intracellulare. VNTR patterns were two types and RFLP were 5 kinds of different in this case. (3) VNTR patterns of six isolates from one HIV-infected patient were identical, but there were three variations in RFLP patterns. There were three cases of mixed infections with M. tuberculosis or M. intracellulare, and six strains polyclonal infection of M. avium (7.1 %) in 84 isolates. These 6 clinical isolates were derived from sputum or BALF (5 strains) and HIV-infected blood (one strain). VNTR patterns were similar in four pairs (9 strains) who did not contact closely, but they were distinguished clearly by RFLP. Seventeen strains had three or less IS1245-related bands in RFLP analyses of 89 strains. As there is a possibility of polyclonal infection with M. avium and mixed infection with other species, the single clonal infection should be confirmed first by VNTR. When single colony was obtained, VNTR and RFLP were performed for genotyping of M. avium. Furthermore, strains with less bands by RFLP should be carefully judged in terms of both VNTR and RFLP. It is recommended that the specimens should be collected from each patient several times.

Clarithromycin therapy for bacteremic Mycobacterium avium complex disease. A randomized, double-blind, dose-ranging study in patients with AIDS. AIDS Clinical Trials Group Protocol 157 Study Team.

Science.gov (United States)

Chaisson, R E; Benson, C A; Dube, M P; Heifets, L B; Korvick, J A; Elkin, S; Smith, T; Craft, J C; Sattler, F R

1994-12-15

To determine the antimicrobial activity and tolerability of clarithromycin for treating bacteremic Mycobacterium avium complex disease in patients with the acquired immunodeficiency syndrome (AIDS). A randomized, double-blind, dose-ranging study. Outpatient clinics. 154 patients with human immunodeficiency virus (HIV) infection and blood cultures positive for M. avium complex who had symptomatic disease. Random assignment to clarithromycin at dosages of 500 mg, 1000 mg, or 2000 mg twice daily for 12 weeks. Median number of colony-forming units of M. avium complex per milliliter of blood. Clarithromycin decreased mycobacterial CFUs from 2.7 to 2.8 log 10/mL of blood at baseline to less than 0 log 10/mL during follow-up (P groups. Clarithromycin-resistant isolates of M. avium complex developed in 46% of patients at a median of 16 weeks. Median survival was longer in patients assigned to 500 mg twice daily (median, 249 days) than in patients assigned to 1000 mg or 2000 mg. Death in the first 12 weeks was lowest in the 500-mg group (P = 0.007). Clarithromycin therapy acutely decreased M. avium complex bacteremia in patients with HIV infection by more than 99%. Clarithromycin, 500 mg twice daily, was well tolerated and associated with better survival. Emergence of clarithromycin-resistant organisms was an important problem.

Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat (MIRU-VNTR) Genotyping of Mycobacterium intracellulare for Strain Comparison with Establishment of a PCR-Based Database

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Iakhiaeva, Elena; McNulty, Steven; Brown Elliott, Barbara A.; Falkinham, Joseph O.; Williams, Myra D.; Vasireddy, Ravikiran; Wilson, Rebecca W.; Turenne, Christine

2013-01-01

Strain comparison is important to population genetics and to evaluate relapses in patients with Mycobacterium avium complex (MAC) lung disease, but the “gold standard” of pulsed-field gel electrophoresis (PFGE) is time-consuming and complex. We used variable-number tandem repeats (VNTR) for fingerprinting of respiratory isolates of M. intracellulare from patients with underlying bronchiectasis, to establish a nonsequence-based database for population analysis. Different genotypes identified by PFGE underwent species identification using a 16S rRNA gene multiplex PCR. Genotypes of M. intracellulare were confirmed by internal transcribed spacer 1 (ITS1) sequencing and characterized using seven VNTR primers. The pattern of VNTR amplicon sizes and repeat number defined each specific VNTR type. Forty-two VNTR types were identified among 84 genotypes. PFGE revealed most isolates with the same VNTR type to be clonal or exhibit similar grouping of bands. Repetitive sequence-based PCR (rep-PCR) showed minimal pattern diversity between VNTR types compared to PFGE. Fingerprinting of relapse isolates from 31 treated patients using VNTR combined with 16S multiplex PCR unambiguously and reliably distinguished different genotypes from the same patient, with results comparable to those of PFGE. VNTR for strain comparison is easier and faster than PFGE, is as accurate as PFGE, and does not require sequencing. Starting with a collection of 167 M. intracellulare isolates, VNTR distinguished M. intracellulare into 42 clonal groups. Comparison of isolates from different geographic areas, habitats, and clinical settings is now possible. PMID:23175249

Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping of mycobacterium intracellulare for strain comparison with establishment of a PCR-based database.

Science.gov (United States)

Iakhiaeva, Elena; McNulty, Steven; Brown Elliott, Barbara A; Falkinham, Joseph O; Williams, Myra D; Vasireddy, Ravikiran; Wilson, Rebecca W; Turenne, Christine; Wallace, Richard J

2013-02-01

Strain comparison is important to population genetics and to evaluate relapses in patients with Mycobacterium avium complex (MAC) lung disease, but the "gold standard" of pulsed-field gel electrophoresis (PFGE) is time-consuming and complex. We used variable-number tandem repeats (VNTR) for fingerprinting of respiratory isolates of M. intracellulare from patients with underlying bronchiectasis, to establish a nonsequence-based database for population analysis. Different genotypes identified by PFGE underwent species identification using a 16S rRNA gene multiplex PCR. Genotypes of M. intracellulare were confirmed by internal transcribed spacer 1 (ITS1) sequencing and characterized using seven VNTR primers. The pattern of VNTR amplicon sizes and repeat number defined each specific VNTR type. Forty-two VNTR types were identified among 84 genotypes. PFGE revealed most isolates with the same VNTR type to be clonal or exhibit similar grouping of bands. Repetitive sequence-based PCR (rep-PCR) showed minimal pattern diversity between VNTR types compared to PFGE. Fingerprinting of relapse isolates from 31 treated patients using VNTR combined with 16S multiplex PCR unambiguously and reliably distinguished different genotypes from the same patient, with results comparable to those of PFGE. VNTR for strain comparison is easier and faster than PFGE, is as accurate as PFGE, and does not require sequencing. Starting with a collection of 167 M. intracellulare isolates, VNTR distinguished M. intracellulare into 42 clonal groups. Comparison of isolates from different geographic areas, habitats, and clinical settings is now possible.

A Mycobacterium avium subsp. paratuberculosis predicted serine protease is associated with acid stress and intraphagosomal survival

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Abirami Kugadas

2016-08-01

Full Text Available AbstractThe ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP, the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophage and MAC-T cells and coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc2 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increase bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5 conditions. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted.

Immunogenicity of Mycobacterium avium subsp. paratuberculosis specific peptides for inclusion in a subunit vaccine against paratuberculosis

DEFF Research Database (Denmark)

Mikkelsen, Heidi; Tollefsen, S.; Olsen, I.

Paratuberculosis in ruminants is caused by an infection with Mycobacterium avium subspecies paratuberculosis (MAP) and is a chronic disease characterized by granulomatous enteritis. Available vaccines against paratuberculosis consist of variations of whole bacteria with adjuvant showing various...... efficacies. The main problem with available vaccines is their interference with surveillance and diagnosis of bovine tuberculosis and paratuberculosis. Our ultimate aim is to develop a subunit vaccine consisting of selected MAP peptides, which allow differentiation of infected from vaccinated animals. Here......, 118 peptides were identified by in silico analysis and synthesized chemically. Peptides were tested for reactivity and immunogenicity with T-cell lines generated from PBMCs isolated from MAP infected goats and with blood samples from MAP infected calves. Immunogenicity of peptides was evaluated using...

Description of a Novel Adhesin of Mycobacterium avium Subsp. paratuberculosis

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Mariana Noelia Viale

2014-01-01

Full Text Available The binding and ingestion of Mycobacterium avium subsp. paratuberculosis (MAP by host cells are fibronectin (FN dependent. In several species of mycobacteria, a specific family of proteins allows the attachment and internalization of these bacteria by epithelial cells through interaction with FN. Thus, the identification of adhesion molecules is essential to understand the pathogenesis of MAP. The aim of this study was to identify and characterize FN binding cell wall proteins of MAP. We searched for conserved adhesins within a large panel of surface immunogenic proteins of MAP and investigated a possible interaction with FN. For this purpose, a cell wall protein fraction was obtained and resolved by 2D electrophoresis. The immunoreactive spots were identified by MALDI-TOF MS and a homology search was performed. We selected elongation factor Tu (EF-Tu as candidate for further studies. We demonstrated the FN-binding capability of EF-Tu using a ligand blot assay and also confirmed the interaction with FN in a dose-dependent manner by ELISA. The dissociation constant of EF-Tu was determined by surface plasmon resonance and displayed values within the μM range. These data support the hypothesis that this protein could be involved in the interaction of MAP with epithelial cells through FN binding.

Polyphasic taxonomic analysis establishes Mycobacterium indicus pranii as a distinct species.

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Vikram Saini

Full Text Available BACKGROUND: Mycobacterium indicus pranii (MIP, popularly known as Mw, is a cultivable, non-pathogenic organism, which, based on its growth and metabolic properties, is classified in Runyon Group IV along with M. fortuitum, M. smegmatis and M. vaccae. The novelty of this bacterium was accredited to its immunological ability to undergo antigen driven blast transformation of leukocytes and delayed hypersensitivity skin test in leprosy patients, a disease endemic in the Indian sub-continent. Consequently, MIP has been extensively evaluated for its biochemical and immunological properties leading to its usage as an immunomodulator in leprosy and tuberculosis patients. However, owing to advances in sequencing and culture techniques, the citing of new strains with almost 100% similarity in the sequences of marker genes like 16S rRNA, has compromised the identity of MIP as a novel species. Hence, to define its precise taxonomic position, we have carried out polyphasic taxonomic studies on MIP that integrate its phenotypic, chemotaxonomic and molecular phylogenetic attributes. METHODOLOGY/PRINCIPAL FINDINGS: The comparative analysis of 16S rRNA sequence of MIP by using BLAST algorithm at NCBI (nr database revealed a similarity of > or =99% with M. intracellulare, M. arosiense, M. chimaera, M. seoulense, M. avium subsp. hominissuis, M. avium subsp. paratuberculosis and M. bohemicum. Further analysis with other widely used markers like rpoB and hsp65 could resolve the phylogenetic relationship between MIP and other closely related mycobacteria apart from M. intracellulare and M. chimaera, which shares > or =99% similarity with corresponding MIP orthologues. Molecular phylogenetic analysis, based on the concatenation of candidate orthologues of 16S rRNA, hsp65 and rpoB, also substantiated its distinctiveness from all the related organisms used in the analysis excluding M. intracellulare and M. chimaera with which it exhibited a close proximity. This

Typing of Mycobacterium avium subspecies paratuberculosis isolates from Newfoundland using fragment analysis.

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Milka P Podder

Full Text Available Short Sequence Repeat (SSR typing of Mycobacterium avium subspecies paratuberculosis (Map isolates is one of the most commonly used method for genotyping this pathogen. Currently used techniques have challenges in analyzing mononucleotide repeats >15 bp, which include some of the Map SSRs. Fragment analysis is a relatively simple technique, which can accurately measure the size of DNA fragments and can be used to calculate the repeat length of the target SSR loci. In the present study, fragment analysis was used to analyze 4 Map SSR loci known to provide sufficient discriminatory power to determine the relationship between Map isolates. Eighty-five Map isolates from 18 animals from the island of Newfoundland were successfully genotyped using fragment analysis. To the best of our knowledge, this is the first report on Map SSR diversity from Newfoundland dairy farms. Previously unreported Map SSR-types or combinations were also identified during the course of the described work. In addition, multiple Map SSR-types were isolated from a single animal in many cases, which is not a common finding.

Rapid detection of Mycobacterium avium subsp. paratuberculosis ...

African Journals Online (AJOL)

Therefore, alternative diagnostic tests such as PCR, are needed for quick detection of infected animals. In this study, the conventional enrichment and isolation procedure and two IS900-based PCR methods for detection of Mycobactrium avium subsp. paratuberculosis in clinical samples from zoo animals and cattle were ...

Protein Kinase G Induces an Immune Response in Cows Exposed to Mycobacterium avium Subsp. paratuberculosis

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Horacio Bach

2018-01-01

Full Text Available To establish infection, pathogens secrete virulence factors, such as protein kinases and phosphatases, to modulate the signal transduction pathways used by host cells to initiate immune response. The protein MAP3893c is annotated in the genome sequence of Mycobacterium avium subspecies paratuberculosis (MAP, the causative agent of Johne’s disease, as the serine/threonine protein kinase G (PknG. In this work, we report that PknG is a functional kinase that is secreted within macrophages at early stages of infection. The antigen is able to induce an immune response from cattle exposed to MAP in the form of interferon gamma production after stimulation of whole blood with PknG. These findings suggest that PknG may contribute to the pathogenesis of MAP by phosphorylating macrophage signalling and/or adaptor molecules as observed with other pathogenic mycobacterial species.

New polymorphisms within the variable number tandem repeat (VNTR) 7 locus of Mycobacterium avium subsp. paratuberculosis.

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Fawzy, Ahmad; Zschöck, Michael; Ewers, Christa; Eisenberg, Tobias

2016-06-01

Variable number tandem repeat (VNTR) is a frequently employed typing method of Mycobacterium avium paratuberculosis (MAP) isolates. Based on whole genome sequencing in a previous study, allelic diversity at some VNTR loci seems to over- or under-estimate the actual phylogenetic variance among isolates. Interestingly, two closely related isolates on one farm showed polymorphism at the VNTR 7 locus, raising concerns about the misleading role that it might play in genotyping. We aimed to investigate the underlying basis of VNTR 7-polymorphism by analyzing sequence data for published genomes and field isolates of MAP and other M. avium complex (MAC) members. In contrast to MAP strains from cattle, strains from sheep displayed an "imperfect" repeat within VNTR 7, which was identical to respective allele types in other MAC genomes. Subspecies- and strain-specific single nucleotide polymorphisms (SNPs) and two novel (16 and 56 bp) repeats were detected. Given the combination of the three existing repeats, there are at least five different patterns for VNTR 7. The present findings highlight a higher polymorphism and probable instability of VNTR 7 locus that needs to be considered and challenged in future studies. Until then, sequencing of this locus in future studies is important to correctly assign the underlying allele types.(1). Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection of Mycobacterium avium subsp. paratuberculosis in milk from clinically affected cows by PCR and culture

DEFF Research Database (Denmark)

Giese, Steen Bjørck; Ahrens, Peter

2000-01-01

Milk and faeces samples from cows with clinical symptoms of paratuberculosis were examined for the presence of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) by culture and PCR. M. paratuberculosis was cultivated in variable numbers from faeces or intestinal mucosa in eight of 11...... animals. In milk from five cows (all faeces culture positive), we cultivated a few colonies of M. paratuberculosis (culture positive, and one cow was milk culture positive). One cow was culture negative on intestinal...... mucosa, but culture positive in milk, and two cows were negative in culture and PCR from both faeces and milk. In conclusion, the presence of M. paratuberculosis could be detected in raw milk by PCR, but cultivation of milk was more sensitive. (C) 2000 Elsevier Science B.V. All rights reserved....

Chylous Ascites in a Patient with HIV/AIDS: A Late Complication of Mycobacterium avium Complex-Immune Reconstitution Inflammatory Syndrome

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Imam H. Shaik

2014-01-01

Full Text Available Chylous ascites is very rare in HIV/AIDS and its association with Mycobacterium avium complex-immune reconstitution inflammatory syndrome (MAC-IRIS has been rarely reported. Here, we report a case of a young African-American male who developed chylous ascites as a late sequela to immune reconstitution inflammatory syndrome while on treatment for MAC. Antiretroviral drug-naive patients who start HAART in close proximity to the diagnosis of an opportunistic infection and have a rapid decline in HIV RNA level should be monitored for development of IRIS. Although the long term prognosis is poor, early diagnosis and treatment help to improve quality of life.

Patchy uptake of gallium in the lungs of AIDS patients with atypical mycobacterial infection

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Skarzynski, J.J.; Sherman, W.; Lee, H.K.; Berger, H.

1987-01-01

The gallium scans of seven AIDS patients who cultured positive for atypical mycobacterium were reviewed. Six cultured positive for Mycobacterium avium intracellulare, while one for Mycobacterium xenopi. A patchy uptake pattern of gallium in the lungs of these patients was identified

Mycobacterium avium subspecies paratuberculosis is not associated with Type-2 Diabetes Mellitus

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Zanetti Stefania

2008-04-01

Full Text Available Abstract Background The role of pathogenic mycobacteria in diabetes has been a focus of speculation since a decade without any meaningful insights into the mechanism of diabetes causation vis a vis mycobacterial factors. Two of our studies based on PCR identification of mycobacterial DNA and detection of antibodies specific to the recombinant antigens and whole cell lysates of the Mycobacterium avium subsp. paratuberculosis (MAP shown a clear association of MAP with the presence of type 1 diabetes mellitus (T1DM. Methods In this study, we sought to investigate if or not type 2 diabetes (T2DM patients harbour humoral responses to MAP. Using three different MAP antigen preparations, humoral antibody profiles were estimated for 57 T2DM patients and 57 healthy controls. Statistical analysis was performed with the Chi-square test with Yates' corrections. Results We observed insignificant levels of humoral antibodies against recombinant heparin binding haemagglutinin (HbHA, glycosyl transferase (Gsd and MAP whole cell lysate in the blood of subjects with T2DM as compared to healthy controls. Conclusion We found no obvious association of MAP with the incidence of T2DM in Sardinian patients.

Mycobacterium intracellulare Pleurisy Identified on Liquid Cultures of the Pleural Fluid and Pleural Biopsy.

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Lim, Jong Gu; O, Sei Won; Lee, Ki Dong; Suk, Dong Keun; Jung, Tae Young; Shim, Tae Sun; Chon, Gyu Rak

2013-03-01

Pleural effusion is a rare complication in non-tuberculous mycobacterial infection. We report a case of Mycobacterium intracellulare pleuritis with idiopathic pulmonary fibrosis in a 69-year-old man presenting with dyspnea. Pleural effusion revealed lymphocyte dominant exudate. M. intracellulare was identified using a polymerase chain reaction-restriction fragment length polymorphism method and liquid cultures of pleural effusion and pleural biopsy. After combination therapy for M. intracellulare pulmonary disease, the patient was clinically well at a 1-month follow-up.

The discovery, function and development of the variable number tandem repeats in different Mycobacterium species.

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Sun, Zhaogang; Li, Weimin; Xu, Shaofa; Huang, Hairong

2016-09-01

The method of genotyping by variable number tandem repeats (VNTRs) facilitates the epidemiological studies of different Mycobacterium species worldwide. Until now, the VNTR method is not fully understood, for example, its discovery, function and classification. The inconsistent nomenclature and terminology of VNTR is especially confusing. In this review, we first describe in detail the VNTRs in Mycobacterium tuberculosis (M. tuberculosis), as this pathogen resulted in more deaths than any other microbial pathogen as well as for which extensive studies of VNTRs were carried out, and then we outline the recent progress of the VNTR-related epidemiological research in several other Mycobacterium species, such as M. abscessus, M. africanum, M. avium, M. bovis, M. canettii, M. caprae, M. intracellulare, M. leprae, M. marinum, M. microti, M. pinnipedii and M. ulcerans from different countries and regions. This article is aimed mainly at the practical notes of VNTR to help the scientists in better understanding and performing this method.

Apparent prevalence of beef carcasses contaminated with Mycobacterium avium subsp. paratuberculosis sampled from Danish slaughter cattle

DEFF Research Database (Denmark)

Okura, Hisako; Toft, Nils; Pozzato, Nicola

2011-01-01

Presence of Mycobacterium avium subsp. paratuberculosis (MAP) in beef has been reported as a public health concern because asymptomatically infected cattle may contain MAP in tissues that are used for human consumption. Associations between MAP carcasses contamination and animal characteristics...... of two dairy cows were positive by culture whereas 4% of the animals were estimated with =10¿CFU/gram muscle based on realtime PCR. Age was found to be associated with carcass contamination with MAP. The observed viable MAP prevalence in beef carcasses was low. However, detection of MAP and MAP DNA...... such as age, breed, production type, and carcass classification were assessed. Cheek muscles from 501 carcasses were sampled cross-sectionally at a Danish abattoir and tested for presence of viable MAP and MAP DNA by bacterial culture and IS900 realtime PCR, respectively. Cheek muscle tissues from carcasses...

Mycobacterium intracellulare Pleurisy Identified on Liquid Cultures of the Pleural Fluid and Pleural Biopsy

OpenAIRE

Lim, Jong Gu; O, Sei Won; Lee, Ki Dong; Suk, Dong Keun; Jung, Tae Young; Shim, Tae Sun; Chon, Gyu Rak

2013-01-01

Pleural effusion is a rare complication in non-tuberculous mycobacterial infection. We report a case of Mycobacterium intracellulare pleuritis with idiopathic pulmonary fibrosis in a 69-year-old man presenting with dyspnea. Pleural effusion revealed lymphocyte dominant exudate. M. intracellulare was identified using a polymerase chain reaction-restriction fragment length polymorphism method and liquid cultures of pleural effusion and pleural biopsy. After combination therapy for M. intracellu...

Comparison of Variable-Number Tandem-Repeat Markers typing and IS1245 Restriction Fragment Length Polymorphism fingerprinting of Mycobacterium avium subsp. hominissuis from human and porcine origins

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Marttila Harri

2010-03-01

Full Text Available Abstract Background Animal mycobacterioses are regarded as a potential zoonotic risk and cause economical losses world wide. M. avium subsp. hominissuis is a slow-growing subspecies found in mycobacterial infected humans and pigs and therefore rapid and discriminatory typing methods are needed for epidemiological studies. The genetic similarity of M. avium subsp. hominissuis from human and porcine origins using two different typing methods have not been studied earlier. The objective of this study was to compare the IS1245 RFLP pattern and MIRU-VNTR typing to study the genetic relatedness of M. avium strains isolated from slaughter pigs and humans in Finland with regard to public health aspects. Methods A novel PCR-based genotyping method, variable number tandem repeat (VNTR typing of eight mycobacterial interspersed repetitive units (MIRUs, was evaluated for its ability to characterize Finnish Mycobacterium avium subsp. hominissuis strains isolated from pigs (n = 16 and humans (n = 13 and the results were compared with those obtained by the conventional IS1245 RFLP method. Results The MIRU-VNTR results showed a discriminatory index (DI of 0,92 and the IS1245 RFLP resulted in DI 0,98. The combined DI for both methods was 0,98. The MIRU-VNTR test has the advantages of being simple, reproducible, non-subjective, which makes it suitable for large-scale screening of M. avium strains. Conclusions Both typing methods demonstrated a high degree of similarity between the strains of human and porcine origin. The parallel application of the methods adds epidemiological value to the comparison of the strains and their origins. The present approach and results support the hypothesis that there is a common source of M. avium subsp. hominissuis infection for pigs and humans or alternatively one species may be the infective source to the other.

Vaccination with peptides of Mycobacterium avium subsp. paratuberculosis (MAP) reduces MAP burden of infected goats

DEFF Research Database (Denmark)

Melvang, Heidi Mikkelsen; Hassan, Sufia Butt; Thakur, Aneesh

Mycobacterium avium subsp. paratuberculosis (Map) is the cause of paratuberculosis, a chronic enteritis of ruminants that is widespread worldwide. We investigated the effect of post-exposure vaccination with Map specific peptides in a goat model aiming at developing a Map vaccine that will neither...... unique to Map from selected proteins (n =68). For vaccination, 23 MAP peptides (20 µg each) were selected and formulated with Montanide ISA 61 VG adjuvant. At age three weeks 10 goats were orally inoculated with 4x10E9 live Map and assigned to two groups of 5 goats each: 5 vaccinated (V) at 14 and 18...... weeks post inoculation (PI) and 5 unvaccinated (C). At termination 32 weeks PI, Map burdens in 15 intestinal tissues and lymph nodes were determined by IS900 qPCR. Of the 75 tissue samples from the 5 C goats only 5 samples were IS900 qPCR negative. In contrast, only 9 samples in total from 5 V goats...

Mycobacterium talmoniae sp. nov., a slowly growing mycobacterium isolated from human respiratory samples.

Science.gov (United States)

Davidson, Rebecca M; DeGroote, Mary Ann; Marola, Jamie L; Buss, Sarah; Jones, Victoria; McNeil, Michael R; Freifeld, Alison G; Elaine Epperson, L; Hasan, Nabeeh A; Jackson, Mary; Iwen, Peter C; Salfinger, Max; Strong, Michael

2017-08-01

A novel slowly growing, non-chromogenic species of the class Actinobacteria was isolated from a human respiratory sample in Nebraska, USA, in 2012. Analysis of the internal transcribed spacer sequence supported placement into the genus Mycobacterium with high sequence similarity to a previously undescribed strain isolated from a patient respiratory sample from Oregon, USA, held in a collection in Colorado, USA, in 2000. The two isolates were subjected to phenotypic testing and whole genome sequencing and found to be indistinguishable. The bacteria were acid-fast stain-positive, rod-shaped and exhibited growth after 7-10 days on solid media at temperatures ranging from 25 to 42°C. Colonies were non-pigmented, rough and slightly raised. Analyses of matrix-assisted laser desorption ionization time-of-flight profiles showed no matches against a reference library of 130 mycobacterial species. Full-length 16S rRNA gene sequences were identical for the two isolates, the average nucleotide identity (ANI) between their genomes was 99.7 % and phylogenetic comparisons classified the novel mycobacteria as the basal most species in the slowly growing Mycobacterium clade. Mycobacterium avium is the most closely related species based on rpoB gene sequence similarity (92 %), but the ANI between the genomes was 81.5 %, below the suggested cut-off for differentiating two species (95 %). Mycolic acid profiles were more similar to M. avium than to Mycobacterium simiae or Mycobacterium abscessus. The phenotypic and genomic data support the conclusion that the two related isolates represent a novel Mycobacterium species for which the name Mycobacterium talmoniae sp. nov. is proposed. The type strain is NE-TNMC-100812T (=ATCC BAA-2683T=DSM 46873T).

Detection of Mycobacterium avium subsp. paratuberculosis in Milk from Clinically Affected Cows by PCR and culture

DEFF Research Database (Denmark)

Giese, Steen Bjørck; Ahrens, Peter

1999-01-01

Milk and faecal samples from cows with clinical symptoms of paratuberculosis were examined for the presence of Mycobacterium avium subsp.paratuberculosis (M. a. paratuberculosis) by culture and PCR. M. a. paratuberculosis was isolated in varied numbers from faeces or intestinal mucosa in 8 of 11...... animals. In milk from 5 cows (all faecal culture-positive) we cultivated a few colonies of M. a. paratuberculosis (less than 100 CFU per mi). Milk samples from 2 cows were PCR-positive (both animals were faecal culture-positive, and 1 cow was milk culture positive). One cow was culture......-negative on intestinal mucosa, but culture-positive in milk, and both faeces and milk were negative in culture and PCR from 2 cows. In conclusion the presence of M. a. paratuberculosis could be detected in raw milk by PCR but cultivation of milk was more sensitive in detecting the organism....

Antimycobacterial potential of the juniper berry essential oil in tap water.

Science.gov (United States)

Peruč, Dolores; Gobin, Ivana; Abram, Maja; Broznić, Dalibor; Svalina, Tomislav; Štifter, Sanja; Staver, Mladenka Malenica; Tićac, Brigita

2018-03-01

Mycobacterium avium complex-related diseases are often associated with poorly maintained hot water systems. This calls for the development of new control strategies. The aim of this study was to investigate the activity of essential oils (EOs) from the Mediterranean plants, common juniper, immortelle, sage, lavandin, laurel, and white cedar against Mycobacterium avium ssp. avium, Mycobacterium intracellulare, and Mycobacterium gordonae in culturing broth and freshwater as their most common habitat. To do that, we developed a new method of water microdilution to determine their minimal effective concentrations (MEC). The most active EO was the one from the common juniper with the MEC of 1.6 mg mL-1. Gas chromatography / mass spectrometry the juniper EO identified monoterpenes (70.54 %) and sesquiterpenes (25.9 %) as dominant component groups. The main monoterpene hydrocarbons were α-pinene, sabinene, and β-pinene. The juniper EO significantly reduced the cell viability of M. intracellulare and M. gordonae at MEC, and of M. avium at 2xMEC. Microscopic analysis confirmed its inhibitory effect by revealing significant morphological changes in the cell membrane and cytoplasm of all three bacteria. The mode of action of the juniper EO on the cell membrane was confirmed by a marked leakage of intracellular material. Juniper EO has a great practical potential as a complementary or alternative water disinfectant in hot water systems such as baths, swimming pools, spa pools, hot tubs, or even foot baths/whirlpools.

A Closed-tube Loop-Mediated Isothermal Amplification Assay for the Visual Endpoint Detection of Brucella spp. and Mycobacterium avium subsp. paratuberculosis.

Science.gov (United States)

Trangoni, Marcos D; Gioffré, Andrea K; Cravero, Silvio L

2017-01-01

LAMP (loop-mediated isothermal amplification) is an isothermal nucleic acid amplification technique that is characterized by its efficiency, rapidity, high yield of final product, robustness, sensitivity, and specificity, with the blueprint that it can be implemented in laboratories of low technological complexity. Despite the conceptual complexity underlying the mechanistic basis for the nucleic acid amplification, the technique is simple to use and the amplification and detection can be carried out in just one step. In this chapter, we present a protocol based on LAMP for the rapid identification of isolates of Brucella spp. and Mycobacterium avium subsp. paratuberculosis, two major bacterial pathogens in veterinary medicine.

Immunogenicity of PtpA secreted during Mycobacterium avium ssp. paratuberculosis infection in cattle.

Science.gov (United States)

Bach, Eviatar; Raizman, Eran A; Vanderwal, Rich; Soto, Paolete; Chaffer, Marcelo; Keefe, Greg; Pogranichniy, Roman; Bach, Horacio

2018-04-01

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease. To survive within host macrophages, the pathogen secretes a battery of proteins to interfere with the immunological response of the host. One of these proteins is tyrosine phosphate A (PtpA), which has been identified as a secreted protein critical for survival of its close relative M. tuberculosis within infected macrophages. In this study, the immune response to recombinant PtpA used as an antigen was investigated in a cohort of ∼1000 cows infected with MAP compared to negative control animals using ELISA. The sera from MAP-infected cows had significantly higher levels of antibodies against PtpA when compared to uninfected cows. The data presented here indicate that the antibodies produced against PtpA are sensitive enough to detect infected animals before the appearance of the disease symptoms. The use of PtpA as an antigen can be developed as an early diagnostic test. Moreover, PtpA is a candidate antigen for detection of humoral immune responses in cows infected with MAP. Copyright © 2018 Elsevier B.V. All rights reserved.

Short communication: Investigation into Mycobacterium avium ssp. paratuberculosis in pasteurized milk in Italy.

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Serraino, A; Bonilauri, P; Giacometti, F; Ricchi, M; Cammi, G; Piva, S; Zambrini, V; Canever, A; Arrigoni, N

2017-01-01

This study investigated the presence of viable Mycobacterium avium ssp. paratuberculosis (MAP) in pasteurized milk produced by Italian industrial dairy plants to verify the prediction of a previously performed risk assessment. The study analyzed 160 one-liter bottles of pasteurized milk from 2 dairy plants located in 2 different regions. Traditional cultural protocols were applied to 500mL of pasteurized milk for each sample. The investigation focused also on the pasteurization parameters and data on the microbiological characteristics of raw milk (total bacterial count) and pasteurized milk (Enterobacteriaceae and Listeria monocytogenes). No sample was positive for MAP, the pasteurization parameters complied with European Union legislation, and the microbiological analysis of raw and pasteurized milk showed good microbiological quality. The results show that a 7-log (or >7) reduction could be a plausible value for commercial pasteurization. The combination of hygiene practices at farm level and commercial pasteurization yield very low or absent levels of MAP contamination in pasteurized milk, suggesting that pasteurized milk is not a significant source of human exposure to MAP in the dairies investigated. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Activity of Medicinal Plant Extracts on Multiplication of Mycobacterium tuberculosis under Reduced Oxygen Conditions Using Intracellular and Axenic Assays

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Purva D. Bhatter

2016-01-01

Full Text Available Aim. Test the activity of selected medicinal plant extracts on multiplication of Mycobacterium tuberculosis under reduced oxygen concentration which represents nonreplicating conditions. Material and Methods. Acetone, ethanol and aqueous extracts of the plants Acorus calamus L. (rhizome, Ocimum sanctum L. (leaf, Piper nigrum L. (seed, and Pueraria tuberosa DC. (tuber were tested on Mycobacterium tuberculosis H37Rv intracellularly using an epithelial cell (A549 infection model. The extracts found to be active intracellularly were further studied axenically under reducing oxygen concentrations. Results and Conclusions. Intracellular multiplication was inhibited ≥60% by five of the twelve extracts. Amongst these 5 extracts, in axenic culture, P. nigrum (acetone was active under aerobic, microaerophilic, and anaerobic conditions indicating presence of multiple components acting at different levels and P. tuberosa (aqueous showed bactericidal activity under microaerophilic and anaerobic conditions implying the influence of anaerobiosis on its efficacy. P. nigrum (aqueous and A. calamus (aqueous and ethanol extracts were not active under axenic conditions but only inhibited intracellular growth of Mycobacterium tuberculosis, suggesting activation of host defense mechanisms to mediate bacterial killing rather than direct bactericidal activity.

Draft Genome Sequence of Mycobacterium chimaera Type Strain Fl-0169

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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...

Analysis of the bovine monocyte-derived macrophage response to Mycobacterium avium subspecies paratuberculosis infection using RNA-seq

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Maura E Casey

2015-02-01

Full Text Available Johne’s disease, caused by infection with Mycobacterium avium subsp. paratuberculosis, (MAP, is a chronic intestinal disease of ruminants with serious economic consequences for cattle production in the United States and elsewhere. During infection, MAP bacilli are phagocytosed and subvert host macrophage processes, resulting in subclinical infections that can lead to immunopathology and dissemination of disease. Analysis of the host macrophage transcriptome during infection can therefore shed light on the molecular mechanisms and host-pathogen interplay associated with Johne’s disease. Here we describe results of an in vitro study of the bovine monocyte-derived macrophage (MDM transcriptome response during MAP infection using RNA-seq. MDM were obtained from seven age- and sex-matched Holstein-Friesian cattle and were infected with MAP across a six-hour infection time course with non-infected controls. We observed 245 and 574 differentially expressed genes in MAP-infected versus non-infected control samples (adjusted P value ≤ 0.05 at 2 and 6 hours post-infection, respectively. Functional analyses of these differentially expressed genes, including biological pathway enrichment, highlighted potential functional roles for genes that have not been previously described in the host response to infection with MAP bacilli. In addition, differential expression of pro- and anti-inflammatory cytokine genes, such as those associated with the IL-10 signaling pathway, and other immune-related genes that encode proteins involved in the bovine macrophage response to MAP infection emphasize the balance between protective host immunity and bacilli survival and proliferation. Systematic comparisons of RNA-seq gene expression results with Affymetrix® microarray data generated from the same experimental samples also demonstrated that RNA-seq represents a superior technology for studying host transcriptional responses to intracellular infection.

Interaction between Mycobacterium avium subsp. paratuberculosis and environmental protozoa

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Rowe Michael T

2006-07-01

Full Text Available Abstract Background Interactions between Mycobacterium avium subsp. paratuberculosis (Map and free-living protozoa in water are likely to occur in nature. The potential impact of ingestion of Map by two naturally occurring Acanthamoeba spp. on this pathogen's survival and chlorine resistance was investigated. Results Between 4.6 and 9.1% of spiked populations of three Map strains (NCTC 8578, B2 and ATCC 19698, which had been added at a multiplicity of infection of 10:1, were ingested by Acanthamoeba castellanii CCAP 1501/1B and A. polyphaga CCAP 1501/3B during co-culture for 3 h at 25°C. Map cells were observed to be present within the vacuoles of the amoebae by acid-fast staining. During extended co-culture of Map NCTC 8578 at 25°C for 24 d with both A. castellanii and A. polyphaga Map numbers did not change significantly during the first 7 days of incubation, however a 1–1.5 log10 increase in Map numbers was observed between days 7 and 24 within both Acanthamoeba spp. Ingested Map cells were shown to be more resistant to chlorine inactivation than free Map. Exposure to 2 μg/ml chlorine for 30 min resulted in a log10 reduction of 0.94 in ingested Map but a log10 reduction of 1.73 in free Map (p Conclusion This study demonstrated that ingestion of Map by and survival and multiplication of Map within Acanthamoeba spp. is possible, and that Map cells ingested by amoebae are more resistant to inactivation by chlorine than free Map cells. These findings have implications with respect to the efficacy of chlorination applied to Map infected surface waters.

Linezolid as treatment for pulmonary Mycobacterium avium disease.

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Deshpande, Devyani; Srivastava, Shashikant; Pasipanodya, Jotam G; Gumbo, Tawanda

2017-09-01

To identify the pharmacokinetic/pharmacodynamic parameters and exposures of linezolid in the treatment of pulmonary Mycobacterium avium complex (MAC) disease. Human-derived monocytes infected with MAC were inoculated into hollow-fibre systems for dose-effect and dose-scheduling studies. We mimicked linezolid concentration-time profiles achieved in adult human lungs treated for 28 days. Sampling to confirm that the intended linezolid pharmacokinetics had been achieved, and for enumeration of MAC colony-forming units, was performed based on repetitive sampling from each system over the 28 days. We then performed 10 000 patient Monte Carlo simulations to identify doses associated with optimal effect in the clinic. Linezolid achieved a hitherto unprecedented feat of at least 1.0 log10 cfu/mL reduction. Efficacy was most closely linked to the AUC0-24/MIC ratio. The AUC0-24/MIC ratio associated with no change in bacterial burden or bacteriostasis was 7.82, while that associated with 1.0 log10 cfu/mL kill was 42.06. The clinical dose of 600 mg/day achieved or exceeded the bacteriostasis exposure in 98.73% of patients. The proportion of 10 000 patients treated with the standard 1200 mg/day who achieved the exposure for 1.0 log10 cfu/mL kill was 70.64%, but was 90% for 1800 mg/day. The proposed MIC breakpoint for linezolid is 16 mg/L, with which 49%-80% of clinical isolates would be considered resistant. Linezolid is associated with a bactericidal effect in pulmonary MAC that is greater than that seen with other recommended drugs. However, because of the MIC distribution, doses that would optimize the bactericidal effect would be associated with a high adverse event rate. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The effect of Mycobacterium avium ssp. paratuberculosis infection on clinical mastitis occurrence in dairy cows.

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Rossi, G; Grohn, Y T; Schukken, Y H; Smith, R L

2017-09-01

Endemic diseases can be counted among the most serious sources of losses for livestock production. In dairy farms in particular, one of the most common diseases is Johne's disease, caused by Mycobacterium avium ssp. paratuberculosis (MAP). Infection with MAP causes direct costs because it affects milk production, but it has also been suspected to increase the risk of clinical mastitis (CM) among infected animals. This might contribute to further costs for farmers. We asked whether MAP infection represents a risk factor for CM and, in particular, whether CM occurrences were more common in MAP-infected animals. Our results, obtained by survival analysis, suggest that MAP-infected cows had an increased probability of experiencing CM during lactation. These results highlight the need to account for the interplay of infectious diseases and other health conditions in economic and epidemiological modeling. In this case, accounting for MAP-infected cows having an increased CM occurrence might have nonnegligible effects on the estimated benefit of MAP control. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Decolorization of Malachite Green and Crystal Violet by Waterborne Pathogenic Mycobacteria

OpenAIRE

Jones, Jefferson J.; Falkinham III, Joseph O.

2003-01-01

Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium marinum, and Mycobacterium chelonae tolerate high concentrations of the dyes malachite green and crystal violet. Cells of strains of those species decolorized (reduced) both malachite green and crystal violet. Because decolorized malachite green lacked antimicrobial activity, the resistance of these mycobacteria could be due, in part, to their ability to decolorize the dyes. Small amounts of malachite...

Use of Novel Recombinant Antigens in the Interferon Gamma Assay for Detection of Mycobacterium Avium Subsp. Paratuberculosis Infection in Cattle

DEFF Research Database (Denmark)

Mikkelsen, Heidi; Aagaard, Claus; Nielsen, Søren Saxmose

2012-01-01

of the study were to evaluate immunogenicity and specificity of 14 novel recombinant antigens for use in the IFN-γ assay and to assess the consistency of IFN-γ responses. The antigens used were 4 ESAT-6 family members, 4 latency proteins, 4 secreted proteins including Ag85B, 3 other antigens and PPDj......Early stage Mycobacterium avium subsp. paratuberculosis (MAP) infection can be detected by measuring antigen specific cell mediated immune responses by the interferon gamma (IFN-γ) assay. Available IFN-γ assay use purified protein derivate of Johnin (PPDj) leading to low specificity. The objectives...... of the infected and non-infected herds were significantly (Passay using PPDj did not correlate with the results using the novel antigens since 5 of the 17 animals that were positive to PPDj were...

Decolorization of Malachite Green and Crystal Violet by Waterborne Pathogenic Mycobacteria

Science.gov (United States)

Jones, Jefferson J.; Falkinham III, Joseph O.

2003-01-01

Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium marinum, and Mycobacterium chelonae tolerate high concentrations of the dyes malachite green and crystal violet. Cells of strains of those species decolorized (reduced) both malachite green and crystal violet. Because decolorized malachite green lacked antimicrobial activity, the resistance of these mycobacteria could be due, in part, to their ability to decolorize the dyes. Small amounts of malachite green and its reduced, decolorized product were detected in the lipid fraction of M. avium strain A5 cells grown in the presence of malachite green, suggesting that a minor component of resistance could be due to sequestering the dyes in the extensive mycobacterial cell surface lipid. The membrane fraction of M. avium strain A5 had at least a fivefold-higher specific decolorization rate than did the crude extract, suggesting that the decolorization activity is membrane associated. The malachite green-decolorizing activity of the membrane fraction of M. avium strain A5 was abolished by either boiling or proteinase exposure, suggesting that the decolorizing activity was due to a protein. Decolorization activity of membrane fractions was stimulated by ferrous ion and inhibited by dinitrophenol and metyrapone. PMID:12821489

Rapid detection and identification of pathogenic mycobacteria by combining radiometric and nucleic acid probe methods

International Nuclear Information System (INIS)

Ellner, P.D.; Kiehn, T.E.; Cammarata, R.; Hosmer, M.

1988-01-01

The combination of radiometric methodology (BACTEC 12B) and probe technology for recovery and identification of mycobacteria was studied in two large hospital laboratories. The sediment from vials with positive growth indices was tested with DNA probes specific for Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare. The sensitivity of the radiometric method and the specificity of the probes resulted in a marked reduction in the time to the final report. Biochemical testing could be eliminated on isolates giving a positive reaction with one of the probes. Some 176 isolates of M. tuberculosis, 110 of M. avium, and 5 of M. intracellulare were recovered. Two-thirds of these isolates were detected and identified within 2 weeks of inoculation and the remainder was detected by 4 weeks, a reduction of 5 to 7 weeks to the final report

Killing of intracellular Mycobacterium tuberculosis by receptor-mediated drug delivery

International Nuclear Information System (INIS)

Majumdar, S.; Basu, S.K.

1991-01-01

p-Aminosalicylic acid (PAS) conjugated to maleylated bovine serum albumin (MBSA) was taken up efficiently through high-affinity MBSA-binding sites on macrophages. Binding of the radiolabeled conjugate to cultured mouse peritoneal macrophages at 4 degrees C was competed for by MBSA but not by PAS. At 37 degrees C, the radiolabeled conjugate was rapidly degraded by the macrophages, leading to release of acid-soluble degradation products in the medium. The drug conjugate was nearly 100 times as effective as free PAS in killing the intracellular mycobacteria in mouse peritoneal macrophages infected in culture with Mycobacterium tuberculosis. The killing of intracellular mycobacteria mediated by the drug conjugate was effectively prevented by simultaneous addition of excess MBSA (100 micrograms/ml) or chloroquine (3 microM) to the medium, whereas these agents did not affect the microbicidal action of free PAS. These results suggest that (i) uptake of the PAS-MBSA conjugate was mediated by cell surface receptors on macrophages which recognize MBSA and (ii) lysosomal hydrolysis of the internalized conjugate resulted in intracellular release of a pharmacologically active form of the drug, which led to selective killing of the M. tuberculosis harbored by mouse macrophages infected in culture. This receptor-mediated modality of delivering drugs to macrophages could contribute to greater therapeutic efficacy and minimization of toxic side effects in the management of tuberculosis and other intracellular mycobacterial infections

Concurrent resolution of chronic diarrhea likely due to Crohn's disease and infection with Mycobacterium avium paratuberculosis

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Shoor Vir Singh

2016-10-01

Full Text Available Examination of samples of stool from a 61 year old male patient, presenting with the clinical symptoms of Crohn’s disease (CD, revealed massive shedding of acid fast bacilli with the morphology of Mycobacterium avium paratuberculosis (MAP, the causative agent of Johne’s disease in cattle. MAP was cultured from the stool. Biotyping of the bacterium isolated from cultures of stool demonstrated it was the Indian Bison biotype of MAP, the dominant biotype infecting livestock and humans in India. Based on this finding and because the patient was unresponsive to standard therapy used in India to treat patients with gastrointestinal inflammatory disorders, the patient was placed on a regimen of multi-antibiotic therapy, currently used to treat tuberculosis and CD. After one year of treatment, the patient’s health was restored, concurrent with cessation of shedding of MAP in his stool. This patient is the first case shown to shed MAP from the stool who was cured of infection with antibiotics and who was concurrently cured of clinical signs of CD.

Mycobacterium avium ssp. paratuberculosis (MAP): Identificação água e fatores de risco para a presença em amostras de biópsias intestinais

OpenAIRE

Braga, Isis de Freitas Espeschit

2015-01-01

Mycobacterium avium ssp. paratuberculosis (MAP) é o agente etiológico da doença de Johne ou paratuberculose, enterite granulomatosa crônica caracterizada por diarreia persistente e perda de peso progressiva que acomete ruminantes. Pode também ser isolado a partir de amostras intestinais de pacientes humanos, com doenças intestinais, principalmente portadores da doença de Crohn. Essa é uma doença de etiologia desconhecida, que se caracteriza por inflamação crônica, focal, assimétrica transmura...

Subversion of Schwann Cell Glucose Metabolism by Mycobacterium leprae*

Science.gov (United States)

Medeiros, Rychelle Clayde Affonso; Girardi, Karina do Carmo de Vasconcelos; Cardoso, Fernanda Karlla Luz; Mietto, Bruno de Siqueira; Pinto, Thiago Gomes de Toledo; Gomez, Lilian Sales; Rodrigues, Luciana Silva; Gandini, Mariana; Amaral, Julio Jablonski; Antunes, Sérgio Luiz Gomes; Corte-Real, Suzana; Rosa, Patricia Sammarco; Pessolani, Maria Cristina Vidal; Nery, José Augusto da Costa; Sarno, Euzenir Nunes; Batista-Silva, Leonardo Ribeiro; Sola-Penna, Mauro; Oliveira, Marcus Fernandes; Moraes, Milton Ozório; Lara, Flavio Alves

2016-01-01

Mycobacterium leprae, the intracellular etiological agent of leprosy, infects Schwann promoting irreversible physical disabilities and deformities. These cells are responsible for myelination and maintenance of axonal energy metabolism through export of metabolites, such as lactate and pyruvate. In the present work, we observed that infected Schwann cells increase glucose uptake with a concomitant increase in glucose-6-phosphate dehydrogenase (G6PDH) activity, the key enzyme of the oxidative pentose pathway. We also observed a mitochondria shutdown in infected cells and mitochondrial swelling in pure neural leprosy nerves. The classic Warburg effect described in macrophages infected by Mycobacterium avium was not observed in our model, which presented a drastic reduction in lactate generation and release by infected Schwann cells. This effect was followed by a decrease in lactate dehydrogenase isoform M (LDH-M) activity and an increase in cellular protection against hydrogen peroxide insult in a pentose phosphate pathway and GSH-dependent manner. M. leprae infection success was also dependent of the glutathione antioxidant system and its main reducing power source, the pentose pathway, as demonstrated by a 50 and 70% drop in intracellular viability after treatment with the GSH synthesis inhibitor buthionine sulfoximine, and aminonicotinamide (6-ANAM), an inhibitor of G6PDH 6-ANAM, respectively. We concluded that M. leprae could modulate host cell glucose metabolism to increase the cellular reducing power generation, facilitating glutathione regeneration and consequently free-radical control. The impact of this regulation in leprosy neuropathy is discussed. PMID:27555322

Genotyping of Mycobacterium avium subsp. avium isolates from naturally infected lofts of domestic pigeons by IS901 RFLP

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K Parvandar Asadollahi

2015-01-01

In conclusion: It is suggested that more DNA fingerprinting tests for non-tuberculous Mycobacteria, particularly M. avium complex isolated from infected birds and humans, be conducted to find the source of their infections.

Composition and Potency Characterization of Mycobacterium avium subsp. paratuberculosis Purified Protein Derivatives.

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Randal T Capsel

Full Text Available Mycobacterium avium subsp. paratuberculosis (MAP purified protein derivatives (PPDs are immunologic reagents prepared from cultured filtrates of the type strain. Traditional production consists of floating culture incubation at 37°C, organism inactivation by autoclaving, coarse filtration, and protein precipitation. Three traditional production PPDs were used in this study including lot 9801, which served as a reference and has been used in the field for decades. Alternative production PPDs (0902A and 0902B, in which the autoclaving step was removed, were also analyzed in this study. SDS-PAGE analysis revealed protein smearing in traditional PPDs, but distinct bands were observed in the alternative PPD preparations. Antibody bound distinct protein bands in the alternative PPDs by immunoblot analysis, whereas an immunoreactive smear was observed with the traditional PPDs. Mass spectrometry identified 194 proteins among three PPD lots representing the two different production methods, ten of which were present in all PPDs examined. Selected proteins identified by mass spectrometry were recombinantly expressed and purified from E. coli and evaluated by the guinea pig potency test. Seven recombinant proteins showed greater erythema as compared to the reference PPD lot 9801 in paired guinea pigs and were able to stimulate interferon-gamma production in blood from Johne's positive animals. These results suggest that autoclaving culture suspensions is not a necessary step in PPD production and specific proteins could supplant the PPD antigen for intradermal skin testing procedures and for use as in-vitro assay reagents.

Short communication: effect of homogenization on heat inactivation of Mycobacterium avium subspecies paratuberculosis in milk.

Science.gov (United States)

Hammer, P; Kiesner, C; Walte, H-G C

2014-01-01

Mycobacterium avium ssp. paratuberculosis (MAP) can be present in cow milk and low numbers may survive high-temperature, short-time (HTST) pasteurization. Although HTST treatment leads to inactivation of at least 5 log10 cycles, it might become necessary to enhance the efficacy of HTST by additional treatments such as homogenization if the debate about the role of MAP in Crohn's disease of humans concludes that MAP is a zoonotic agent. This study aimed to determine whether disrupting the clumps of MAP in milk by homogenization during the heat treatment process would enhance the inactivation of MAP. We used HTST pasteurization in a continuous-flow pilot-plant pasteurizer and evaluated the effect of upstream, downstream, and in-hold homogenization on inactivation of MAP. Reduction of MAP at 72°C with a holding time of 28s was between 3.7 and 6.9 log10 cycles, with an overall mean of 5.5 log10 cycles. None of the 3 homogenization modes applied showed a statistically significant additional effect on the inactivation of MAP during HTST treatment. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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

NARCIS (Netherlands)

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

2006-01-01

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

Decreased serum protein associated with Mycobacterium avium subspecies paratuberculosis shedding in German Holstein cows.

Science.gov (United States)

Donat, K; Erhardt, G; Soschinka, A; Brandt, H R

2014-04-19

Using well established metabolic parameters, this study aimed to substantiate differences in protein and energy metabolism between Mycobacterium avium subspecies paratuberculosis (MAP) positive and negative dairy cows tested by faecal culture. A total of 227 MAP-positive and 239 MAP-negative German Holstein cows kept in 13 MAP-positive dairy herds were selected for metabolic testing. The serum concentrations of total protein (TP), bilirubin, cholesterol and betahydroxybutyrate were measured as well as the activities of Glutamate-Dehydrogenase (GLDH) and Aspartate-Aminotransferase. MAP-positive cows were characterised by a decreased mean TP (66.5 g/l) compared to the MAP-negative controls (73.2 g/l). Mean log10 GLDH activities tended to be higher in MAP-positive than MAP-negative cows. Concerning TP, there was a significant interaction between MAP status and farm. Within four farms, the difference between MAP-positive and MAP-negative animals differed significantly, while in the other farms this difference was not significant. It is concluded that a decreased TP and an increased GLDH indicate alterations in protein metabolism. These findings suggest an enhanced liver cell turnover in MAP-positive cows. The results contribute to an understanding of the metabolic alterations in MAP-positive dairy cows.

Potentiating day-old blood samples for detection of interferon-gamma responses following infection with Mycobacterium avium subsp. paratuberculosis

DEFF Research Database (Denmark)

Mikkelsen, Heidi; Nielsen, Søren Saxmose; Jungersen, Gregers

time interval from blood sampling to culture. The objective of the study was to assess options for use of day-old blood samples for early-stage diagnosis of MAP infections. Bovine interleukin 12 (IL-12) can induce, and IL-10 reduce, IFN-γ production. Therefore, addition of IL-12 and anti-IL-10 could...... result in production of IFN-γ in samples previously exposed to MAP antigens. Whole blood samples were collected from heifers in a Danish dairy herd known to be infected with MAP. The samples were collected on three sample dates, and on each date the blood samples were stimulated with PPDj and recombinant......The interferon gamma (IFN-γ) test measuring specific cell-mediated immune responses in whole blood can be used for diagnosis at an early stage of Mycobacterium avium subsp. paratuberculosis (MAP) infection. A major obstacle for the practical use of IFN-γ testing is the recommended maximum 8 hour...

Development of a method to measure intracellular growth rate of parasitic acid-fast bacteria using radio-isotope and its improvement

International Nuclear Information System (INIS)

Nakata, Noboru; Fukutomi, Yasuo

1999-01-01

Development of measurement method for intracellular growth rate was attempted using gene-transfected acid-fast bacteria and Mycobacterium leprae. M. leprae was inoculated into a well, which was filled with fetus bovine serum containing a cover slip pasted with mouse monocyte-derived malignant cell lines, J774 and P388D1 and cultured for 3-4 hours. Then, the cells on the cover slip were mobilized with 0.1 N NaOH. The metabolic activity of M. leprae was assessed based on the β-oxidation activity of 14 C-palmitic acid. Then, it was investigated whether TNF is produced by the cell culture added with M. leprae or LPS. J774 cells abundantly produced TNF after sensitization with LPS and its production was depending on the amount of added bacteria, whereas TNF production after sensitization with LPS or M. leprae was little in P388D1 cells. Staining for acid-fast bacteria revealed that either of these cell lines has phagocytic activity for M. leprae. To identify the bacterial factor involved to the intracellular proliferation of acid-fast bacteria, transposon insertion mutagenesis was attempted to M. avium complex (MAC) and the degrees of drug-resistance in M. avium mino, M. intracellulare JATA-52 and 8 clinically isolated M. intracellulare strains were determined. M. intracellulare JATA-52 was resistant to kanamycin and plasmid pAL8 and pYT937 were both able to transform the strain with dose-dependency. Since M. intracellulare is pathogenic to human and the strain proliferates with a generation time shorter than that of M. tuberculosis, the former strain is thought suitable for the analysis of a mutated gene. Thus, it became possible to study transposition insertion mutagenesis in M. intracellulare. (M.N.)

Development of a method to measure intracellular growth rate of parasitic acid-fast bacteria using radio-isotope and its improvement

Energy Technology Data Exchange (ETDEWEB)

Nakata, Noboru; Fukutomi, Yasuo [National Inst. of Infectious Diseases, Tokyo (Japan)

1999-02-01

Development of measurement method for intracellular growth rate was attempted using gene-transfected acid-fast bacteria and Mycobacterium leprae. M. leprae was inoculated into a well, which was filled with fetus bovine serum containing a cover slip pasted with mouse monocyte-derived malignant cell lines, J774 and P388D1 and cultured for 3-4 hours. Then, the cells on the cover slip were mobilized with 0.1 N NaOH. The metabolic activity of M. leprae was assessed based on the {beta}-oxidation activity of {sup 14}C-palmitic acid. Then, it was investigated whether TNF is produced by the cell culture added with M. leprae or LPS. J774 cells abundantly produced TNF after sensitization with LPS and its production was depending on the amount of added bacteria, whereas TNF production after sensitization with LPS or M. leprae was little in P388D1 cells. Staining for acid-fast bacteria revealed that either of these cell lines has phagocytic activity for M. leprae. To identify the bacterial factor involved to the intracellular proliferation of acid-fast bacteria, transposon insertion mutagenesis was attempted to M. avium complex (MAC) and the degrees of drug-resistance in M. avium mino, M. intracellulare JATA-52 and 8 clinically isolated M. intracellulare strains were determined. M. intracellulare JATA-52 was resistant to kanamycin and plasmid pAL8 and pYT937 were both able to transform the strain with dose-dependency. Since M. intracellulare is pathogenic to human and the strain proliferates with a generation time shorter than that of M. tuberculosis, the former strain is thought suitable for the analysis of a mutated gene. Thus, it became possible to study transposition insertion mutagenesis in M. intracellulare. (M.N.)

Development and Validation of a Liquid Medium (M7H9C) for Routine Culture of Mycobacterium avium subsp. paratuberculosis To Replace Modified Bactec 12B Medium

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Whittington, Ann-Michele; Waldron, Anna; Begg, Douglas J.; de Silva, Kumi; Purdie, Auriol C.; Plain, Karren M.

2013-01-01

Liquid culture of Mycobacterium avium subsp. paratuberculosis from clinical samples, such as feces, is the most sensitive antemortem test for the diagnosis of Johne's disease in ruminants. In Australia, New Zealand, the United States, and some other countries, the Bactec 460 system with modified Bactec 12B medium (Becton, Dickinson) has been the most commonly used liquid culture system, but it was discontinued in 2012. In this study, a new liquid culture medium, M7H9C, was developed. It consists of a Middlebrook 7H9 medium base with added Casitone, albumin, dextrose, catalase, egg yolk, mycobactin J, and a cocktail of antibiotics. We found that polyoxyethylene stearate (POES) was not essential for the cultivation of M. avium subsp. paratuberculosis in either the Bactec 12B or the M7H9C medium. The limit of detection determined using pure cultures of the C and S strains of M. avium subsp. paratuberculosis was 7 bacilli per 50 μl inoculum in the two media. The new medium was validated using 784 fecal and tissue samples from sheep and cattle, >25% of which contained viable M. avium subsp. paratuberculosis. Discrepant results for the clinical samples between the two media were mostly associated with samples that contained <10 viable bacilli per gram, but these results were relatively uncommon, and the performances of the two media were not significantly different. M7H9C medium was less than half the cost of the Bactec 12B medium and did not require regular examination during incubation, but a confirmatory IS900 PCR test had to be performed on every culture after the predetermined incubation period. PMID:24048541

Interleukin-1 or tumor necrosis factor-alpha augmented the cytotoxic effect of mycobacteria on human fibroblasts: application to evaluation of pathogenesis of clinical isolates of Mycobacterium tuberculosis and M. avium complex.

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Takii, T; Abe, C; Tamura, A; Ramayah, S; Belisle, J T; Brennan, P J; Onozaki, K

2001-03-01

Mycobacteria-induced in vitro events reflecting human tuberculosis can contribute to the evaluation of the pathogenesis of Mycobacterium tuberculosis (MTB). In this study, we propose such an in vitro method based on live mycobacteria-induced cytotoxicity to human cell lines. When human lung-derived normal fibroblast cell line MRC-5 was infected with various strains of mycobacteria (M. tuberculosis H(37)Rv and H(37) Ra, Mycobacterium avium 427S and 2151SmO, and Mycobacterium bovis BCG Pasteur and Tokyo), the fibroblasts were killed by mycobacteria according to the degree of virulence. Other human originated macrophage (U-937, THP-1), myeloid (HL-60), and epithelial carcinoma (A549) cell lines exhibited a similar cytotoxic response to virulent mycobacteria. MRC-5 was most susceptible to virulent mycobacteria among various human cell lines examined. The cytotoxicity was enhanced by the proinflammatory cytokines, interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-alpha), which in the absence of mycobacteria stimulate the growth of normal human fibroblasts. This in vitro evaluation system was applied to clinical isolates of drug-sensitive MTB (DS-MTB), drug-resistant MTB (DR-MTB) including multidrug-resistant (MDR-MTB), and M. avium complex (MAC). MTB strains (n = 24) exhibited strong cytotoxic activity, but MAC strains (n = 5) had only weak activity. Furthermore, there was no significant difference in cytotoxicity between DS-MTB (n = 11) and DR-MTB (n = 13). Collectively, these results suggest that this new in vitro system is useful for evaluating the pathogenesis of mycobacteria and that there was no difference in the pathogenesis between drug-susceptible and drug-resistant clinical isolates.

Mechanisms of Mycobacterium avium-induced resistance against insulin-dependent diabetes mellitus (IDDM) in non-obese diabetic (NOD) mice: role of Fas and Th1 cells.

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Martins, T C; Aguas, A P

1999-02-01

NOD mice spontaneously develop autoimmune diabetes. One of the manipulations that prevent diabetes in NOD mice is infection with mycobacteria or immunization of mice with mycobacteria-containing adjuvant. Infection of NOD mice with Mycobacterium avium, done before the mice show overt diabetes, results in permanent protection of the animals from diabetes and this protective effect is associated with increased numbers of CD4+ T cells and B220+ B cells. Here, we investigate whether the M. avium-induced protection of NOD mice from diabetes was associated with changes in the expression of Fas (CD95) and FasL by immune cells, as well as alterations in cytotoxic activity, interferon-gamma (IFN-gamma) and IL-4 production and activation of T cells of infected animals. Our data indicate that protection of NOD mice from diabetes is a Th1-type response that is mediated by up-regulation of the Fas-FasL pathway and involves an increase in the cytotoxicity of T cells. These changes are consistent with induction by the infection of regulatory T cells with the ability of triggering deletion or anergy of peripheral self-reactive lymphocytes that cause the autoimmune disease of NOD mice.

Intestinal infection following aerosol challenge of calves with Mycobacterium avium subspecies paratuberculosis

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Eisenberg Susanne WF

2011-12-01

Full Text Available Abstract A challenge experiment was performed to investigate whether administration of Mycobacterium avium subsp. paratuberculosis (MAP via the respiratory route leads to MAP infection in calves. Eighteen calves from test negative dams were randomly allocated to four groups. Six calves were challenged with MAP nasally and six calves were challenged by transtracheal injection; three orally challenged calves served as positive controls, and three non challenged calves as negative controls. The challenge was performed as a nine-fold trickle dose, 107 CFU in total. Blood and faecal samples were collected frequently. Calves were euthanized three months post-challenge and extensively sampled. Blood samples were tested for the presence of antibodies and interferon gamma producing cells by ELISA. Faecal and tissue samples were cultured in a liquid culture system and the presence of MAP was confirmed by IS900 realtime PCR. Fourteen out of fifteen calves had no MAP antibody response. The negative controls remained negative; all positive controls became infected. Two nasally challenged calves showed a Purified Protein Derivative Avian (PPDA specific interferon gamma response. In all nasally challenged calves, MAP positive intestinal samples were detected. In three calves of the nasal group MAP positive retropharyngeal lymph nodes or tonsils were detected. In all calves of the transtracheal group MAP positive intestinal tissues were detected as well and three had a MAP positive tracheobronchial lymph node. These findings indicate that inhalation of MAP aerosols can result in infection. These experimental results may be relevant for transmission under field conditions since viable MAP has been detected in dust on commercial dairy farms.

Cell wall peptidolipids of Mycobacterium avium: from genetic prediction to exact structure of a nonribosomal peptide

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Total lipids from an M. avium subsp. paratuberculosis (Map) ovine strain (S-type) contained no identifiable glycopeptidolipids or lipopentapeptide, yet both lipids are present in other M. avium subspecies. We determined the genetic and phenotypic basis for this difference using sequence analysis and...

Consequence of Mycobacterium avium complex pulmonary disease judging from the change of the chest CT image

International Nuclear Information System (INIS)

Fujiwara, Kiyohiro

2008-01-01

The long term consequence of the disease in Mycobacterium avium complex pulmonary disease (MACPD) is scarcely reported. This paper describes consequences of CT images and clinical symptoms in MACPD patients with rather poorer prognosis than usual during chemotherapy for one or more years in authors' hospital until May 2007. Subjects are 17 patients (average age 65.3 y, M 6/F 11) diagnosed as MACPD by the criteria by Jap. Soc. Tuberculosis (2003), whose follow up period is 14-105 (av. 58.1) months, and are classified in tuberculoid type (tt, 2 cases), bronchiectasis post surgery (2) and bronchia type (bt, 13, mostly primary MACPD). Chemotherapy is done with clarithromycin (CAM)+ethambutol (EB)+rifampicin (RHP) (+streptomycin (SM) for progression). Consequences of typical chest CT images are presented for each classification in this paper. Cavitation is seen even in bt as well as in tt and, if observed, the disease tends to deteriorate. In the secondary MACPD post surgery, the exacerbation of clinical symptom is often more severe despite slow changes in CT finding than in bt. Thus, careful follow up is necessary for the two cases above. (R.T.)

Bulk tank milk ELISA for detection of antibodies to Mycobacterium avium subsp paratuberculosis: Correlation between repeated tests and within-herd antibody-prevalence

DEFF Research Database (Denmark)

Nielsen, Søren Saxmose; Toft, Nils

2014-01-01

Detection of bulk tank milk (BTM) antibodies using ELISA (BTM-ELISA) may constitute an inexpensive test for surveillance of Mycobacterium avium subsp. paratuberculosis (MAP) infection in dairy cattle herds provided that the test is accurate and consistent. The objectives of this study were...... Danish Holstein herds over a period of one year. All samples were tested using a commercial indirect ELISA for detection of MAP specific antibodies. The individual cow's results were dichotomised and used to estimate the within-herd antibody prevalence at each test-date. These prevalences were...... to 0.60 when corrected for the within-herd antibody prevalence. Although the test-results were relatively consistent and correlated with the within-herd prevalence, the magnitude of the test-values makes it difficult to use the BTM-ELISA for surveillance of MAP infections in practice....

Methylobacterium spp. as an indicator for the presence or absence of Mycobacterium spp.

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Falkinham, Joseph O; Williams, Myra D; Kwait, Rebecca; Lande, Leah

2016-06-01

A published survey of bacteria in showerhead biofilm samples revealed that Methylobacterium spp. and Mycobacterium spp. seldom coexisted in biofilms. To confirm that information, biofilm samples were collected from household plumbing of Mycobacterium avium patients and Methylobacterium spp. and M. avium numbers were measured by direct colony counts. The results demonstrated that if Methylobacterium spp. were present, Mycobacterium spp. were absent, and the opposite. The data demonstrate that microbial populations in biofilms can influence the presence or absence of opportunistic premise plumbing pathogens and, thereby, increase the range of strategies to reduce exposure to waterborne pathogens. Finally, by assessing for the visual presence of methylobacteria as pink pigmentation on showers and shower curtains, homeowners and managers of hospitals and other buildings can quickly determine whether a premise plumbing biofilm sample has mycobacteria with a high degree of assurance. Copyright © 2016 Asian African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

The presence of opportunistic pathogens, Legionella spp., L. pneumophila and Mycobacterium avium complex, in South Australian reuse water distribution pipelines.

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Whiley, H; Keegan, A; Fallowfield, H; Bentham, R

2015-06-01

Water reuse has become increasingly important for sustainable water management. Currently, its application is primarily constrained by the potential health risks. Presently there is limited knowledge regarding the presence and fate of opportunistic pathogens along reuse water distribution pipelines. In this study opportunistic human pathogens Legionella spp., L. pneumophila and Mycobacterium avium complex were detected using real-time polymerase chain reaction along two South Australian reuse water distribution pipelines at maximum concentrations of 10⁵, 10³ and 10⁵ copies/mL, respectively. During the summer period of sampling the concentration of all three organisms significantly increased (P < 0.05) along the pipeline, suggesting multiplication and hence viability. No seasonality in the decrease in chlorine residual along the pipelines was observed. This suggests that the combination of reduced chlorine residual and increased water temperature promoted the presence of these opportunistic pathogens.

Prevalence of Mycobacterium avium subspecies paratuberculosis and hepatitis E in New World camelids in Austria.

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Stanitznig, A; Khol, J L; Lambacher, B; Franz, S; Wittek, T; Kralik, P; Slana, I; Vasickova, P

2017-07-07

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis in domestic ruminants and New World Camelids (NWC). Hepatitis E virus (HEV) is an important public health concern worldwide. The virus has been identified in several species, some of them serving as a reservoir for zoonotic HEV strains. Husbandry and breeding of llamas and alpacas have increased in Austria in recent years. Therefore, the aim of the present study was to evaluate the prevalence of MAP and HEV in NWC in Austria. Altogether 445 animals, originating from 78 farms were enrolled in the study. Of the animals sampled, 184 (41.35%) were llamas and 261 (58.65%) were alpacas. 443 blood samples for MAP-ELISA and 399 faecal samples for quantitative PCR (qPCR) and culture for MAP as well as for HEV detection by RT-qPCR have been collected. All of the 399 animals tested for shedding of MAP were negative by faecal solid culture. Using qPCR, 15 (3.8%) of the animals were MAP positive and 384 (96.2%) negative. Out of the 443 serum samples examined for specific antibodies against MAP by ELISA, 6 (1.4%) were positive, 1 (0.2%) was questionable and 436 (98.4%) samples were negative. All faecal samples were tested negative for HEV.

Gold nanoparticle-based probes for the colorimetric detection of Mycobacterium avium subspecies paratuberculosis DNA.

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Ganareal, Thenor Aristotile Charles S; Balbin, Michelle M; Monserate, Juvy J; Salazar, Joel R; Mingala, Claro N

2018-02-12

Gold nanoparticle (AuNP) is considered to be the most stable metal nanoparticle having the ability to be functionalized with biomolecules. Recently, AuNP-based DNA detection methods captured the interest of researchers worldwide. Paratuberculosis or Johne's disease, a chronic gastroenteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), was found to have negative effect in the livestock industry. In this study, AuNP-based probes were evaluated for the specific and sensitive detection of MAP DNA. AuNP-based probe was produced by functionalization of AuNPs with thiol-modified oligonucleotide and was confirmed by Fourier-Transform Infrared (FTIR) spectroscopy. UV-Vis spectroscopy and Scanning Electron Microscopy (SEM) were used to characterize AuNPs. DNA detection was done by hybridization of 10 μL of DNA with 5 μL of probe at 63 °C for 10 min and addition of 3 μL salt solution. The method was specific to MAP with detection limit of 103 ng. UV-Vis and SEM showed dispersion and aggregation of the AuNPs for the positive and negative results, respectively, with no observed particle growth. This study therefore reports an AuNP-based probes which can be used for the specific and sensitive detection of MAP DNA. Copyright © 2018 Elsevier Inc. All rights reserved.

[Identification of novel variable number tandem repeat (VNTR) loci in Mycobacterium avium and development of an effective means of VNTR typing].

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Kurokawa, Kazuhiro; Uchiya, Kei-Ichi; Yagi, Tetsuya; Takahashi, Hiroyasu; Niimi, Masaki; Ichikawa, Kazuya; Inagaki, Takayuki; Moriyama, Makoto; Nikai, Toshiaki; Hayashi, Yuta; Nakagawa, Taku; Ogawa, Kenji

2012-07-01

To make more effective use of variable number tandem repeat (VNTR) typing, we identified novel VNTR loci in Mycobacterium avium and used them for modified M. avium tandem repeat-VNTR (MATR-VNTR) typing. Analysis of a DNA sample extracted from a clinical isolate (strain HN135) with the FLX system genome sequencer (Roche Diagnostic System) led to discovery of several novel VNTR loci. The allelic diversity of the novel VNTR loci was evaluated for 71 clinical isolates and compared with the diversity of the MATR-VNTR loci. To improve efficacy of MATR-VNTR typing, we tested typing using 2 sets of loci selected from the newly identified loci and the MATR loci, i.e., one set containing 7 and another 16 loci. Hunter Gaston's discriminatory index (HGDI) was calculated for these sets. Six VNTR loci were newly identified, of which 5 showed a high diversity. The HGDI was 0.980 for the improved new typing using a set of 7 loci, and 0.995 for another set of 16 loci, while it was 0.992 for the conventional MATR-VNTR typing. VNTR typing with the set of the 7 loci enabled a rapid analysis, and another set of 16 loci enabled a precise analysis, as compared with conventional MATR-VNTR typing. A method that uses only VNTR loci with relatively high allelic diversity is considered to be a useful tool for VNTR typing of MAC isolates.

Comparison of prevalence estimation of Mycobacterium avium subsp. paratuberculosis infection by sampling slaughtered cattle with macroscopic lesions vs. systematic sampling.

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Elze, J; Liebler-Tenorio, E; Ziller, M; Köhler, H

2013-07-01

The objective of this study was to identify the most reliable approach for prevalence estimation of Mycobacterium avium ssp. paratuberculosis (MAP) infection in clinically healthy slaughtered cattle. Sampling of macroscopically suspect tissue was compared to systematic sampling. Specimens of ileum, jejunum, mesenteric and caecal lymph nodes were examined for MAP infection using bacterial microscopy, culture, histopathology and immunohistochemistry. MAP was found most frequently in caecal lymph nodes, but sampling more tissues optimized the detection rate. Examination by culture was most efficient while combination with histopathology increased the detection rate slightly. MAP was detected in 49/50 animals with macroscopic lesions representing 1.35% of the slaughtered cattle examined. Of 150 systematically sampled macroscopically non-suspect cows, 28.7% were infected with MAP. This indicates that the majority of MAP-positive cattle are slaughtered without evidence of macroscopic lesions and before clinical signs occur. For reliable prevalence estimation of MAP infection in slaughtered cattle, systematic random sampling is essential.

Different Mycobacterium avium subsp. paratuberculosis MIRU-VNTR patterns coexist within cattle herds.

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van Hulzen, K J E; Heuven, H C M; Nielen, M; Hoeboer, J; Santema, W J; Koets, A P

2011-03-24

A better understanding of the biodiversity of Mycobacterium avium subsp. paratuberculosis (MAP) offers more insight in the epidemiology of paratuberculosis and therefore may contribute to the control of the disease. The aim of this study was to investigate the genetic diversity in bovine MAP isolates using PCR-based methods detecting genetic elements called Variable-Number Tandem Repeats (VNTRs) and Mycobacterial Interspersed Repetitive Units (MIRUs) to determine if multiple MAP strains can coexist on farms with endemic MAP infection. For 52 temporal isolates originating from infected cattle from 32 commercial dairy herds with known trading history, MIRU-VNTR analysis was applied at 10 loci of which six showed variation. Within the group of 52 isolates, 17 different MIRU-VNTR patterns were detected. One MIRU-VNTR pattern was found in 29 isolates, one pattern in four isolates, one pattern in three isolates, two times one MIRU-VNTR pattern was found occurring in two isolates, and 12 patterns were found only once. Eleven herds provided multiple isolates. In five herds a single MIRU-VNTR pattern was detected among multiple isolates whereas in six herds more than one pattern was found. This study confirms that between dairy farms as well as within dairy farms, infected animals shed MAP with different MIRU-VNTR patterns. Analysis of trading history and age within herds indicated that cows born within the same birth cohort can be infected with MAP strains exhibiting variations in the number of MIRU-VNTR repeats. These data indicate that such multiple genotypes of MAP can coexist within one herd. Copyright © 2010 Elsevier B.V. All rights reserved.

[Disinfection efficiency of peracetic acid, alone and in combination with hypochlorite, against Mycobacterium avium in drinking water].

Science.gov (United States)

Schiavano, G F; Sisti, M; De Santi, M; Brandi, G

2006-01-01

Peracetic acid (PAA) is a disinfectant with a wide spectrum of antimicrobial activity, but little is known about the feasibility of using it in the field of drinking water treatment. The aim of this study has been assess disinfectant efficacy of PAA, alone or in combination with hypochlorite, against M. avium in drinking water M. avium is a common opportunistic pathogen in immunocompromised subjects that is able to survive and grow in drinking water distribution systems. In this study PAA did not show appreciable activity against the greater number of tested strains (16/21) up to 5 ppm of PAA, a weak activity was seen on 4 strains, while a significant reduction in viable cells (about 50%) was seen only on 1 strain after 48 h of treatment with 5 ppm of PAA. We also evidenced that M. avium was unaffected by chlorine concentration usually present in drinking water distribution system. Finally, the combination of PAA and sodium hypochlorite did not promote enhanced antimicrobial efficacy respect to the single disinfectants. In conclusion, our result would indicate that PAA is an unlikely candidate for the disinfection of drinking water from M. avium and further strategies are required to eliminate M. avium from drinking water system.

Limited value of transbronchial lung biopsy for diagnosing Mycobacterium avium complex lung disease.

Science.gov (United States)

Sekine, Akimasa; Saito, Takefumi; Satoh, Hiroaki; Morishita, Yukio; Tsunoda, Yoshiya; Tanaka, Toru; Yatagai, Yohei; Lin, Shih-Yuen; Miyazaki, Kunihiko; Miura, Yukiko; Hayashihara, Kenji

2017-11-01

It remains unclear whether transbronchial lung biopsy (TBLB) is useful for diagnosing Mycobacterium avium complex (MAC) lung disease. Thirty-eight consecutive patients with MAC lung disease, who were evaluated with TBLB tissue culture between June 2006 and May 2010, were included. Bronchial washing (BW) and histopathological evaluation were performed in all patients. The positivity rates of BW and TBLB tissue culture, and typical histopathological findings for MAC disease were investigated. Furthermore, all patients were divided into two groups according to the presence of intrabronchial purulent or mucopurulent secretion and the clinical, bacteriological and pathological characteristics were compared between the two groups. The positive culture rates of BW and TBLB specimens for MAC were 100% (38 patients) and 28.9% (11 patients). BW materials were much more sensitive for culture positivity than TBLB specimens (P present in the TBLB specimens of only 11 patients (28.9%). Intrabronchial secretion was identified in 15 patients (39.5%, secretion-positive group) and absent in 23 patients (60.5%, secretion-negative group). Typical histopathological findings for MAC disease were more common in the secretion-positive group than in the secretion-negative group (53.3% vs 13.0%, P = 0.01), although the radiological classification and smear positivity of BW were not different between the two groups. TBLB for pathological and bacterial investigations would provide only a limited value for MAC diagnosis. Moreover, the presence of intrabronchial secretion may be an important manifestation of ongoing airway damage, which would require early treatment. © 2016 John Wiley & Sons Ltd.

Preparation and Purification of Polyclonal Antibodies against Mycobacterium Avium Paratuberculosis Antigens in Rabbit

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Hafezeh Alizadeh

2012-12-01

Full Text Available Background and Objective: Johne’s disease is the chronic granulomatous enteritis of ruminants, and a major health hazard worldwide. In recent years, researchers have focused on mycobacterium avium subsp. paratuberculosis (MAP antigens in diagnostic tests. Identification of antibodies against MAP antigens is, therefore, effective for the diagnosis or preparation of vaccine. The aim of this study was to prepare and purify polyclonal antibodies against MAP antigens. Materials and Methods: A New Zealand white rabbit was immunized at a certain time period with MAP antigens and Freund’s adjuvant. After the immunization of the animal, the rabbit was bled to obtain enriched serum. Immunoglobulins were obtained via sedimentation with ammonium sulfate 35% and then IgG was purified by ion exchange (DEAE-cellulose chromatography. Serologic test was used to evaluate the interaction of antigens and antibodies. Results: Ion exchange chromatography of IgG showed one peak, and SDS_PAGE of IgG showed a single band. Serologic test was applied and clear precipitation lines were appeared up to 1:16 dilution, which indicated the high quality of the product. Conclusion: In this study, the humoral immune response was induced well by immunization with MAP antigens in a New Zealand white rabbit and polyclonal antibodies were produced in high titers. Polyclonal antibodies are relatively inexpensive and easy to produce in large quantities and can connect to the more connective sites, resulting in better sensitivity. Identification of polyclonal antibodies via immunological tests can play a significant role in studying MAP disorders.

Dual skin tests with Mycobacterium avium sensitin and PPD to detect misdiagnosis of latent tuberculosis infection.

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Larson, E M; O'Donnell, M; Chamblee, S; Horsburgh, C R; Marsh, B J; Moreland, J D; Johnson, L S; von Reyn, C Fordham

2011-11-01

A positive tuberculin skin test (TST) may indicate cross-reacting immunity to non-tuberculous mycobacteria (NTM) and not latent tuberculosis infection (LTBI). To assess misclassification of LTBI, as assessed by skin testing with Mycobacterium avium sensitin (MaS), and to determine how this misclassification affects the analysis of risk factors for LTBI. In a population-based survey, participants underwent skin testing with M. tuberculosis purified protein derivative (PPD) and MaS. A PPD-dominant skin test was a reaction that was ≥ 3 mm larger than the MaS reaction; a MaS-dominant skin test was a reaction that was ≥ 3 mm larger than the PPD reaction. Of 447 randomly selected persons, 135 (30%) had a positive PPD test. Of these, 21 (16%) were MaS- dominant, and were therefore attributable to NTM and misclassified as LTBI. PPD reactions of 5-14 mm were more likely to be misclassified than those ≥ 15 mm (OR = 5.0, 95%CI 1.9-13.2). Adjusting for misclassification had only a small impact on the analysis of risk factors for LTBI. A substantial number of individuals who are diagnosed with LTBI are actually sensitized to NTM. Using dual skin testing would reduce misdiagnosis and prevent unnecessary treatment.

MIRU-VNTR genotype diversity and indications of homoplasy in M. avium strains isolated from humans and slaughter pigs in Latvia.

Science.gov (United States)

Kalvisa, Adrija; Tsirogiannis, Constantinos; Silamikelis, Ivars; Skenders, Girts; Broka, Lonija; Zirnitis, Agris; Jansone, Inta; Ranka, Renate

2016-09-01

Diseases which are caused by non-tuberculous mycobacteria (NTM) are an increasing problem in the developed countries. In Latvia, one of the most clinically important members of NTM is Mycobacterium avium (M. avium), an opportunistic pathogen which has been isolated from several lung disease patients and tissue samples of slaughter pigs. This study was designed to characterize the genetic diversity of the M. avium isolates in Latvia and to compare the distribution of genotypic patterns among humans and pigs. Eleven (Hall and Salipante, 2010) clinical M. avium samples, isolated from patients of Center of Tuberculosis and Lung Diseases (years 2003-2010), and 32 isolates from pig necrotic mesenterial lymph nodes in different regions (years 2003-2007) were analyzed. The majority (42 of 43) of samples were identified as M. avium subsp. hominissuis; one porcine isolate belonged to M. avium subsp. avium. MIRU-VNTR genotyping revealed 13 distinct genotypes, among which nine genotype patterns, including M. avium subsp. avium isolate, were newly identified. IS1245 RFLP fingerprinting of 25 M. avium subsp. hominissuis samples yielded 17 different IS1245 RFLP patterns, allowing an efficient discrimination of isolates. Clusters of identical RFLP profiles were observed within host species, geographical locations and time frame of several years. Additional in silico analysis on simulated MIRU-VNTR genotype population datasets showed that the MIRU-VNTR pattern similarity could partly arise due to probabilistic increase of acquiring homoplasy among subpopulations, thus the similar MIRU-VNTR profiles of M. avium strains even in close geographical proximity should be interpreted with caution. Copyright © 2016 Elsevier B.V. All rights reserved.

MRP8/14 induces autophagy to eliminate intracellular Mycobacterium bovis BCG.

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Wang, Jinli; Huang, Chunyu; Wu, Minhao; Zhong, Qiu; Yang, Kun; Li, Miao; Zhan, Xiaoxia; Wen, Jinsheng; Zhou, Lin; Huang, Xi

2015-04-01

To explore the role of myeloid-related protein 8/14 in mycobacterial infection. The mRNA and protein expression levels of MRP8 or MRP14 were measured by real-time PCR and flow cytometry, respectively. Role of MRP8/14 was tested by overexpression or RNA interference assays. Flow cytometry and colony forming unit were used to test the phagocytosis and the survival of intracellular Mycobacterium bovis BCG (BCG), respectively. Autophagy mediated by MRP8/14 was detected by Western blot and immunofluorescence. The colocalization of BCG phagosomes with autophagosomes or lysosomes was by detected by confocal microscopy. ROS production was detected by flow cytometry. MRP8/14 expressions were up-regulated in human monocytic THP1 cells and primary macrophages after mycobacterial challenge. Silencing of MRP8/14 suppressed bacterial killing, but had no influence on the phagocytosis of BCG. Importantly, silencing MRP8/14 decreased autophagy and BCG phagosome maturation in THP1-derived macrophages, thereby increasing the BCG survival. Additionally, we demonstrated that MRP8/14 promoted autophagy in a ROS-dependent manner. The present study revealed a novel role of MRP8/14 in the autophagy-mediated elimination of intracellular BCG by promoting ROS generation, which may provide a promising therapeutic target for tuberculosis and other intracellular bacterial infectious diseases. Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Mycobacterium avium complex (MAC isolated from AIDS patients and the criteria required for its implication in disease Complexo Mycobacterium avium (MAC isolado de pacientes com AIDS e os critérios exigidos para sua implicação em doença

Directory of Open Access Journals (Sweden)

David Jamil Hadad

1995-10-01

Full Text Available Before the AIDS pandemia, the Mycobacterium avium complex (MAC was responsible in most cases for the pneumopathies that attack patients with basic chronic pulmonary diseases such as emphysema and chronic bronchitis36. In 1981, with the advent of the acquired immunodeficiency syndrome (AIDS, MAC started to represent one of the most frequent bacterial diseases among AIDS patients, with the disseminated form of the disease being the major clinical manifestation of the infection8. Between January 1989 and February 1991, the Section of Mycobacteria of the Adolfo Lutz Institute, São Paulo, isolated MAC from 103 patients by culturing different sterile and no-sterile processed specimens collected from 2304 patients seen at the AIDS Reference and Training Center and/or Emilio Ribas Infectology Institute. Disseminated disease was diagnosed in 29 of those patients on the basis of MAC isolation from blood and/or bone marrow aspirate. The other 74 patients were divided into categories highly (5, moderately (26 and little suggestive of disease (43 according to the criteria of DAVIDSON (198910. The various criteria for MAC isolation from sterile and non-sterile specimens are discussed.Anterior a pandemia de AIDS, o Complexo Mycobacterium avium (MAC era responsável pela maioria das vezes, por pneumopatias acometendo pacientes com doença pulmonar crônica de base como enfisema e bronquite crônica36. Em 1981, com o advento da síndrome de imunodeficiência adquirida (SIDA, o MAC passou a representar uma das doenças bacterianas mais frequentes em pacientes com esta síndrome, sendo a doença disseminada a principal forma de manifestação clínica da infecção8. Entre Janeiro de 1989 e Fevereiro de 1991, no Setor de Micobactérias do Instituto Adolfo Lutz em São Paulo, o MAC foi isolado de 103 pacientes a partir do cultivo de diferentes espécimes estéreis e não estéreis processados, coletados de 2.304 pacientes atendidos no Centro de Referência e

Current status of Mycobacterium avium subspecies paratuberculosis infection in animals & humans in India: What needs to be done?

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Ajay Vir Singh

2016-01-01

Full Text Available Mycobacterium avium subspecies paratuberculosis (MAP has emerged as a major health problem for domestic livestock and human beings. Reduced per animal productivity of domestic livestock seriously impacts the economics of dairy farming globally. High to very high bioload of MAP in domestic livestock and also in the human population has been reported from north India. Presence of live MAP bacilli in commercial supplies of raw and pasteurized milk and milk products indicates its public health significance. MAP is not inactivated during pasteurization, therefore, entering into human food chain daily. Recovery of MAP from patients with inflammatory bowel disease or Crohn's disease and animal healthcare workers suffering with chronic gastrointestinal problems indicate a close association of MAP with a number of chronic and other diseases affecting human health. Higher bioload of MAP in the animals increases the risk of exposure to the human population with MAP. This review summarizes the current status of MAP infection in animals as well as in human beings and also highlights the prospects of effective management and control of disease in animals to reduce the risk of exposure to human population.

Application of a Mycobacterium tuberculosis protein array for antigen discovery in Johne's disease

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Mycobacterium avium subspecies paratuberculosis (Map), the bacterium that causes Johne’s disease, is a major health concern in farmed ruminant livestock including sheep and cattle. Diagnosis of Map infections, particularly of subclinical animals remains challenging, and we lack effective vaccines f...

Disseminated Mycobacterium intracellulare infection in a broad-snouted caiman Caiman latirostris.

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Kik, Marja J L

2013-11-25

A 10 yr old broad-snouted caiman Caiman latirostris from a small Dutch animal park was presented with long-term variable periods of anorexia and weight loss. Blood chemistry showed slightly elevated uric acid levels and low ionised calcium concentration. Ultrasonographical thickening of the intestinal wall in the region of the duodenum was evident. Pathological changes were a thickening of the wall of 90% of the small intestines, enlarged spleen with multifocal white foci and an enlarged light-brown liver. Histopathological lesions consisted of disseminated granulomas in the intestinal wall, the liver and the spleen. Multinucleated giant cells and epitheloid macrophages were abundant. Ziehl-Neelsen staining showed numerous intralesional acid-fast bacteria. Polymerase chain reaction for Mycobacterium intracellulare was positive.

Profiling of Intracellular Metabolites: An Approach to Understanding the Characteristic Physiology of Mycobacterium leprae.

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Miyamoto, Yuji; Mukai, Tetsu; Matsuoka, Masanori; Kai, Masanori; Maeda, Yumi; Makino, Masahiko

2016-08-01

Mycobacterium leprae is the causative agent of leprosy and also known to possess unique features such as inability to proliferate in vitro. Among the cellular components of M. leprae, various glycolipids present on the cell envelope are well characterized and some of them are identified to be pathogenic factors responsible for intracellular survival in host cells, while other intracellular metabolites, assumed to be associated with basic physiological feature, remain largely unknown. In the present study, to elucidate the comprehensive profile of intracellular metabolites, we performed the capillary electrophoresis-mass spectrometry (CE-MS) analysis on M. leprae and compared to that of M. bovis BCG. Interestingly, comparison of these two profiles showed that, in M. leprae, amino acids and their derivatives are significantly accumulated, but most of intermediates related to central carbon metabolism markedly decreased, implying that M. leprae possess unique metabolic features. The present study is the first report demonstrating the unique profiles of M. leprae metabolites and these insights might contribute to understanding undefined metabolism of M. leprae as well as pathogenic characteristics related to the manifestation of the disease.

Metabolomic profiling in cattle experimentally infected with Mycobacterium avium subsp. paratuberculosis.

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Jeroen De Buck

Full Text Available The sensitivity of current diagnostics for Johne's disease, a slow, progressing enteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP, is too low to reliably detect all infected animals in the subclinical stage. The objective was to identify individual metabolites or metabolite profiles that could be used as biomarkers of early MAP infection in ruminants. In a monthly follow-up for 17 months, calves infected at 2 weeks of age were compared with aged-matched controls. Sera from all animals were analyzed by 1H nuclear magnetic resonance spectrometry. Spectra were acquired, processed, and quantified for analysis. The concentration of many metabolites changed over time in all calves, but some metabolites only changed over time in either infected or non-infected groups and the change in others was impacted by the infection. Hierarchical multivariate statistical analysis achieved best separation between groups between 300 and 400 days after infection. Therefore, a cross-sectional comparison between 1-year-old calves experimentally infected at various ages with either a high- or a low-dose and age-matched non-infected controls was performed. Orthogonal Projection to Latent Structures Discriminant Analysis (OPLS DA yielded distinct separation of non-infected from infected cattle, regardless of dose and time (3, 6, 9 or 12 months after infection. Receiver Operating Curves demonstrated that constructed models were high quality. Increased isobutyrate in the infected cattle was the most important agreement between the longitudinal and cross-sectional analysis. In general, high- and low-dose cattle responded similarly to infection. Differences in acetone, citrate, glycerol and iso-butyrate concentrations indicated energy shortages and increased fat metabolism in infected cattle, whereas changes in urea and several amino acids (AA, including the branched chain AA, indicated increased protein turnover. In conclusion, metabolomics

Characterization of the Apa antigen from M. avium subsp. paratuberculosis: a conserved Mycobacterium antigen that elicits a strong humoral response in cattle.

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Gioffré, A; Echeverría-Valencia, G; Arese, A; Morsella, C; Garbaccio, S; Delgado, F; Zumárraga, M; Paolicchi, F; Cataldi, A; Romano, M I

2009-12-15

Johne's disease or paratuberculosis is widespread in almost all countries and remains difficult to eradicate. Nowadays, diagnosis of Mycobacterium avium subsp. paratuberculosis (MPTB) infection is one of the main concerns. In this work, we evaluated the expression, biochemical properties and antigenicity of the Apa antigen, encoded by the gene annotated as MAP1569, in the MPTB genome. We confirmed its expression in MPTB and its glycosylation by the ConA binding assay. Although the MPTB-Apa is not an immunodominant antigen, MPTB-infected cattle showed a strong humoral response to recombinant Apa by Western blot and ELISA. Milk was also a suitable sample to be tested by ELISA. We comparatively analysed the humoral cross-reactivity to the Apa from MPTB (MPTB-Apa) and the orthologue from Mycobacterium tuberculosis (MT-Apa, identical to that from Mycobacterium bovis) in both infected and control cows. Response of M. bovis- and MPTB-infected animals against MT-Apa was similar (P=0.6985) but the response of the M. bovis-infected ones to MPTB-Apa was differential, being significantly diminished (PApa stimulation in the IFNgamma release assay, we found no significant differences when compared infected herds with non-infected ones (P=0.34). This antigen, in contrast to bovine Purified Protein Derivative (PPDb), was strongly represented in avian PPD (PPDa), as shown by the recognition of BALB/c mice hyperimmune sera against MPTB-Apa by Dot-blot immunoassay. We therefore demonstrated the antigenicity of Apa in MPTB-infected animals and a differential response to the recombinant antigen when compared to M. bovis-infected animals. These traits herein described, added to the usefulness of milk samples to detect IgG anti-Apa, could be important for routine screening in dairy cattle, considering a multiantigenic approach to overcome the lack of immunodominance.

STING-Dependent 2'-5' Oligoadenylate Synthetase-Like Production Is Required for Intracellular Mycobacterium leprae Survival.

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de Toledo-Pinto, Thiago Gomes; Ferreira, Anna Beatriz Robottom; Ribeiro-Alves, Marcelo; Rodrigues, Luciana Silva; Batista-Silva, Leonardo Ribeiro; Silva, Bruno Jorge de Andrade; Lemes, Robertha Mariana Rodrigues; Martinez, Alejandra Nóbrega; Sandoval, Felipe Galvan; Alvarado-Arnez, Lucia Elena; Rosa, Patrícia Sammarco; Shannon, Edward Joseph; Pessolani, Maria Cristina Vidal; Pinheiro, Roberta Olmo; Antunes, Sérgio Luís Gomes; Sarno, Euzenir Nunes; Lara, Flávio Alves; Williams, Diana Lynn; Ozório Moraes, Milton

2016-07-15

Cytosolic detection of nucleic acids elicits a type I interferon (IFN) response and plays a critical role in host defense against intracellular pathogens. Herein, a global gene expression profile of Mycobacterium leprae-infected primary human Schwann cells identified the genes differentially expressed in the type I IFN pathway. Among them, the gene encoding 2'-5' oligoadenylate synthetase-like (OASL) underwent the greatest upregulation and was also shown to be upregulated in M. leprae-infected human macrophage cell lineages, primary monocytes, and skin lesion specimens from patients with a disseminated form of leprosy. OASL knock down was associated with decreased viability of M. leprae that was concomitant with upregulation of either antimicrobial peptide expression or autophagy levels. Downregulation of MCP-1/CCL2 release was also observed during OASL knock down. M. leprae-mediated OASL expression was dependent on cytosolic DNA sensing mediated by stimulator of IFN genes signaling. The addition of M. leprae DNA enhanced nonpathogenic Mycobacterium bovis bacillus Calmette-Guerin intracellular survival, downregulated antimicrobial peptide expression, and increased MCP-1/CCL2 secretion. Thus, our data uncover a promycobacterial role for OASL during M. leprae infection that directs the host immune response toward a niche that permits survival of the pathogen. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Genome-Wide Diversity and Phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian Dairy Cattle.

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Christina Ahlstrom

Full Text Available Mycobacterium avium subsp. paratuberculosis (MAP is the causative bacterium of Johne's disease (JD in ruminants. The control of JD in the dairy industry is challenging, but can be improved with a better understanding of the diversity and distribution of MAP subtypes. Previously established molecular typing techniques used to differentiate MAP have not been sufficiently discriminatory and/or reliable to accurately assess the population structure. In this study, the genetic diversity of 182 MAP isolates representing all Canadian provinces was compared to the known global diversity, using single nucleotide polymorphisms identified through whole genome sequencing. MAP isolates from Canada represented a subset of the known global diversity, as there were global isolates intermingled with Canadian isolates, as well as multiple global subtypes that were not found in Canada. One Type III and six "Bison type" isolates were found in Canada as well as one Type II subtype that represented 86% of all Canadian isolates. Rarefaction estimated larger subtype richness in Québec than in other Canadian provinces using a strict definition of MAP subtypes and lower subtype richness in the Atlantic region using a relaxed definition. Significant phylogeographic clustering was observed at the inter-provincial but not at the intra-provincial level, although most major clades were found in all provinces. The large number of shared subtypes among provinces suggests that cattle movement is a major driver of MAP transmission at the herd level, which is further supported by the lack of spatial clustering on an intra-provincial scale.

[Study on the effects of HTST pasteurization temperatures on Mycobacterium avium subsp. paratuberculosis in an industrial fluid milk-processing system].

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Igimi, Shizunobu; Iriguchi, Shoichi; Monden, Shuko; Okada, Yumiko; Yamamoto, Shigeki; Mori, Yasuyuki

2010-01-01

Johne disease is ruminant chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP). The domestic animals infected with this pathogen present severe weight loss due to chronic diarrhea and a reduction in lactation yield. These result in enormous economic loss since the affected animals are subsequently subject to artificial selections and disinfection of the environment are absolutely necessary. Furthermore, MAP has been suspected to have pathological relationship to Crohn's disease, human chronic granulomatous enteritis. The bacterium grows slower on solid culture and its colony becomes visible after two months of culture. In Japan, there has been almost no investigation on pasteurization temperature of commercial milk using MAP. It comes from the fact that the growth rate of MAP is very slow and that MAP is a related species to Mycobacterium tuberculosis, which pasteurization condition has been well defined. The studies on the pasteurization conditions of commercial milk have been mainly targeted to reduce the risk of infection to Coxiella and Mycobacterium tuberculosis. However, there has been a concern about the possibility that MAP is remained in pasteurized milk because MAPs form an aggregate and the bacterium at its center may not receive enough heat to get pasteurized. From these reasons, the present study aims to investigate validity of the current pasteurization conditions of commercial milk by implementing experimental pasteurization at various pasteurization temperatures using milk experimentally infected with MAP, and to clarify if MAP is eliminated at these temperatures in order to achieve smooth enforcement of the current ministry order. We conducted plant pasteurization experiment at four pasteurization conditions (high temperature, short time (HTST); 82, 77, 72 degrees C for 15 seconds and low temperature, long time (LTLT); 63 degrees C for 30 minutes) using two MAP strains, ATCC19698 and OKY-20. In conclusion

Predicting the Role of IL-10 in the Regulation of the Adaptive Immune Responses in Mycobacterium avium Subsp. paratuberculosis Infections Using Mathematical Models

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Magombedze, Gesham; Eda, Shigetoshi; Stabel, Judy

2015-01-01

Mycobacterium avium subsp. paratuberculosis (MAP) is an intracellular bacterial pathogen that causes Johne’s disease (JD) in cattle and other animals. The hallmark of MAP infection in the early stages is a strong protective cell-mediated immune response (Th1-type), characterized by antigen-specific γ-interferon (IFN-γ). The Th1 response wanes with disease progression and is supplanted by a non-protective humoral immune response (Th2-type). Interleukin-10 (IL-10) is believed to play a critical role in the regulation of host immune responses to MAP infection and potentially orchestrate the reversal of Th1/Th2 immune dominance during disease progression. However, how its role correlates with MAP infection remains to be completely deciphered. We developed mathematical models to explain probable mechanisms for IL-10 involvement in MAP infection. We tested our models with IL-4, IL-10, IFN-γ, and MAP fecal shedding data collected from calves that were experimentally infected and followed over a period of 360 days in the study of Stabel and Robbe-Austerman (2011). Our models predicted that IL-10 can have different roles during MAP infection, (i) it can suppress the Th1 expression, (ii) can enhance Th2 (IL-4) expression, and (iii) can suppress the Th1 expression in synergy with IL-4. In these predicted roles, suppression of Th1 responses was correlated with increased number of MAP. We also predicted that Th1-mediated responses (IFN-γ) can lead to high expression of IL-10 and that infection burden regulates Th2 suppression by the Th1 response. Our models highlight areas where more experimental data is required to refine our model assumptions, and further test and investigate the role of IL-10 in MAP infection. PMID:26619346

Predicting the Role of IL-10 in the Regulation of the Adaptive Immune Responses in Mycobacterium avium Subsp. paratuberculosis Infections Using Mathematical Models.

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Gesham Magombedze

Full Text Available Mycobacterium avium subsp. paratuberculosis (MAP is an intracellular bacterial pathogen that causes Johne's disease (JD in cattle and other animals. The hallmark of MAP infection in the early stages is a strong protective cell-mediated immune response (Th1-type, characterized by antigen-specific γ-interferon (IFN-γ. The Th1 response wanes with disease progression and is supplanted by a non-protective humoral immune response (Th2-type. Interleukin-10 (IL-10 is believed to play a critical role in the regulation of host immune responses to MAP infection and potentially orchestrate the reversal of Th1/Th2 immune dominance during disease progression. However, how its role correlates with MAP infection remains to be completely deciphered. We developed mathematical models to explain probable mechanisms for IL-10 involvement in MAP infection. We tested our models with IL-4, IL-10, IFN-γ, and MAP fecal shedding data collected from calves that were experimentally infected and followed over a period of 360 days in the study of Stabel and Robbe-Austerman (2011. Our models predicted that IL-10 can have different roles during MAP infection, (i it can suppress the Th1 expression, (ii can enhance Th2 (IL-4 expression, and (iii can suppress the Th1 expression in synergy with IL-4. In these predicted roles, suppression of Th1 responses was correlated with increased number of MAP. We also predicted that Th1-mediated responses (IFN-γ can lead to high expression of IL-10 and that infection burden regulates Th2 suppression by the Th1 response. Our models highlight areas where more experimental data is required to refine our model assumptions, and further test and investigate the role of IL-10 in MAP infection.

Effect of days in milk and milk yield on testing positive in milk antibody ELISA to Mycobacterium avium subsp. paratuberculosis in dairy cattle

DEFF Research Database (Denmark)

Nielsen, Søren Saxmose; Toft, Nils

2012-01-01

Milk samples are becoming more used as a diagnostic specimen for assessment of occurrence of antibodies to Mycobacterium avium subsp. paratuberculosis (MAP). This study assessed the effect of days in milk (DIM) and milk yield on testing positive in a commercial MAP specific milk antibody ELISA...... from the first couple of DIM should be excluded from MAP testing until further information on their significance is established. Milk yield also had a significant effect on odds of testing positive due to its diluting effect. Inclusion of milk yield in the interpretation of test results could improve...... among 222,774 Danish Holstein cows. Results showed that odds of testing positive on 1-2 DIM were 9-27 times higher than the rest of lactation, where the chance of testing positive varied less. The reason is most likely a high concentration of non-specific antibodies in colostrum. Consequently, samples...

Detection of Mycobacterium avium subspecies paratuberculosis in formula milk from Bogor using PCR IS 900

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Widagdo S. Nugroho

2008-09-01

Full Text Available Crohn’s disease (CD that becomes a public health concern in developed countries shows similarities in clinical signs and pathological features with Johne’s disease (JD in ruminants infected by Mycobacterium avium subspecies paratuberculosis (MAP. Few researches conducted in Europe, the USA, and Australia showed relationships between MAP, CD, JD and dairy products. Indonesians consume milk and diary products from domestic and imported source. Adji in 2004 found some domestic dairy cows that were seropositive for MAP, and this could be a serious problem in dairy farm animals and human health in the future. The aim of this study was to detect MAP in the growing up formula milk. Fifty samples from five established factories were taken from supermarkets in Bogor. Polymerase chain reaction method (PCR with insertion sequence (IS 900 as primer and culture in Herrold’s egg yolk media with mycobactin J (HEYM J as a gold standard were used in this study. Neither MAP grew up in HEYM J medium after 20 weeks of culture period nor positive samples by PCR IS 900 were found. Although there were no positive samples found in this study, further extensive and comprehensive studies on MAP should be done with more and varied samples, as well as in human to provide data on MAP in Indonesia. (Med J Indones 2008; 17: 183-7Keywords: Crohn’s disease, dairy cow, growing up formula milk

Effects of fractionated colostrum replacer and vitamins A, D, and E on haptoglobin and clinical health in neonatal Holstein calves challenged with Mycobacterium avium ssp. paratuberculosis.

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Krueger, L A; Reinhardt, T A; Beitz, D C; Stuart, R L; Stabel, J R

2016-04-01

Thirty Holstein calves were obtained from 2 dairy farms in central Iowa at birth and randomly assigned to 1 of 6 treatment groups: (1) colostrum deprived (CD), no vitamins; (2) colostrum replacer (CR), no vitamins; (3) CR, vitamin A; (4) CR, vitamin D3; (5) CR, vitamin E; and (6) CR, vitamins A, D3, E, with 5 calves per treatment in a 14-d study. Calves were fed pasteurized whole milk (CD) or fractionated colostrum replacer (CR) at birth (d 0) and injected with vitamins according to treatment group. From d 1 through d 14 of the study, all calves were fed pasteurized whole milk (PWM) supplemented with vitamins as assigned. All calves were inoculated with Mycobacterium avium ssp. paratuberculosis on d 1 and 3 of age. Calves fed CR acquired IgG1 and haptoglobin in serum within 24 h of birth, whereas CD calves did not. The CR-fed calves were 2.5 times less likely to develop scours, and CR calves supplemented with vitamins D3 and E also demonstrated a decreased incidence of scours. Serum vitamin levels of A, D, and E increased within treatment group by d 7 and 14 of the study. Interestingly, synergistic effects of supplemental vitamins A, D3, and E on serum 25-(OH)-vitamin D were observed at d 7, resulting in higher levels than in calves administered vitamin D only. Further, vitamin D3 deficiency was observed in CD and CR calves fed a basal diet of pasteurized whole milk and no supplemental vitamins. Colonization of tissues with Mycobacterium avium ssp. paratuberculosis was negligible and was not affected by colostrum feeding or vitamin supplementation. Results demonstrated passive transfer of haptoglobin to neonatal calves, and potential health benefits of supplemental vitamins D3 and E to calves fed pasteurized whole milk. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Detection of Mycobacterium avium subsp. paratuberculosis in bovine manure using Whatman FTA card technology and Lightcycler real-time PCR.

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Jaravata, Carmela V; Smith, Wayne L; Rensen, Gabriel J; Ruzante, Juliana M; Cullor, James S

2006-01-01

A modified forensic DNA extraction and real-time fluorescent polymerase chain reaction assay has been evaluated for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine fecal samples using primers and fluorescent resonance energy transfer (FRET) probes targeting the IS900 gene sequence of MAP. DNA was successfully extracted from manure samples by utilizing the Whatman FTA card technology, which allows for simple processing and storage of samples at room temperature. The FTA cards were washed and subjected to a Chelex-100 incubation to remove any remaining polymerase chain reaction (PCR) inhibitors and to elute the DNA from the FTA card. This isolated DNA was then subjected to direct real time fluorescent PCR analysis. Detection of MAP DNA from bovine fecal samples spiked with known concentrations of viable MAP cells was obtained. The detection limits of the assay was consistently found to be between 10(2) and 10(4) colony forming units [CFU]/g, with some samples containing as low as 10 CFU/g, yielding positive assay results. This cost-efficient assay allows reporting of results as early as 4 h after fecal collection, which can be particularly useful in highthroughput herd screening.

Mycobacterium Lysine ε-aminotransferase is a novel alarmone metabolism related persister gene via dysregulating the intracellular amino acid level.

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Duan, Xiangke; Li, Yunsong; Du, Qinglin; Huang, Qinqin; Guo, Siyao; Xu, Mengmeng; Lin, Yanping; Liu, Zhidong; Xie, Jianping

2016-01-25

Bacterial persisters, usually slow-growing, non-replicating cells highly tolerant to antibiotics, play a crucial role contributing to the recalcitrance of chronic infections and treatment failure. Understanding the molecular mechanism of persister cells formation and maintenance would obviously inspire the discovery of new antibiotics. The significant upregulation of Mycobacterium tuberculosis Rv3290c, a highly conserved mycobacterial lysine ε-aminotransferase (LAT) during hypoxia persistent model, suggested a role of LAT in persistence. To test this, a lat deleted Mycobacterium smegmatis was constructed. The expression of transcriptional regulator leucine-responsive regulatory protein (LrpA) and the amino acids abundance in M. smegmatis lat deletion mutants were lowered. Thus, the persistence capacity of the deletion mutant was impaired upon norfloxacin exposure under nutrient starvation. In summary, our study firstly reported the involvement of mycobacterium LAT in persister formation, and possibly through altering the intracellular amino acid metabolism balance.

Identification of ISMyo2, a novel insertion sequence element of IS21 family and its diagnostic potential for detection of Mycobacterium yongonense.

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Kim, Byoung-Jun; Kim, Kijeong; Kim, Bo-Ram; Kook, Yoon-Hoh; Kim, Bum-Joon

2015-10-15

Mycobacterium yongonense, as a novel member of the M. avium complex (MAC), was recently reported to be isolated from human specimens in South Korea and Italy. Due to its close relatedness to other MAC members, particularly M. intracellulare in taxonomic aspects, the development of a novel diagnostic method for its specific detection is necessary for clinical or epidemiologic purposes. Using the Mycobacterium yongonense genome information, we have identified a novel IS-element, ISMyo2. Targeting the ISMyo2 sequence, we developed a real-time PCR method and applied the technique to Mycobacterial genomic DNA. To identify proper nucleic acid targets for the diagnosis, comparisons of all insertion sequence (IS) elements of 3 M. intracellulare and 3 M. yongonense strains, whose complete genome sequences we reported recently, led to the selection of a novel target gene, the M. yongonense-specific IS element, ISMyo2 (2,387 bp), belonging to the IS21 family. Next, we developed a real-time PCR method using SYBR green I for M. yongonense-specific detection targeting ISMyo2, producing a 338-bp amplicon. When this assay was applied to 28 Mycobacterium reference strains and 63 MAC clinical isolates, it produced amplicons in only the 6 M. yongonense strains, showing a sensitivity of 100 fg of genomic DNA, suggesting its feasibility as a diagnostic method for M. yongonense strains. We identified a novel ISMyo2 IS element belonging to the IS21 family specific to M. yongonense strains via genome analysis, and a real-time PCR method based on its sequences was developed.

Dysbiosis of the Fecal Microbiota in Cattle Infected with Mycobacterium avium subsp. paratuberculosis.

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Fecteau, Marie-Eve; Pitta, Dipti W; Vecchiarelli, Bonnie; Indugu, Nagaraju; Kumar, Sanjay; Gallagher, Susan C; Fyock, Terry L; Sweeney, Raymond W

2016-01-01

Johne's disease (JD) is a chronic, intestinal infection of cattle, caused by Mycobacterium avium subsp. paratuberculosis (MAP). It results in granulomatous inflammation of the intestinal lining, leading to malabsorption, diarrhea, and weight loss. Crohn's disease (CD), a chronic, inflammatory gastrointestinal disease of humans, has many clinical and pathologic similarities to JD. Dysbiosis of the enteric microbiota has been demonstrated in CD patients. It is speculated that this dysbiosis may contribute to the intestinal inflammation observed in those patients. The purpose of this study was to investigate the diversity patterns of fecal bacterial populations in cattle infected with MAP, compared to those of uninfected control cattle, using phylogenomic analysis. Fecal samples were selected to include samples from 20 MAP-positive cows; 25 MAP-negative herdmates; and 25 MAP-negative cows from a MAP-free herd. The genomic DNA was extracted; PCR amplified sequenced on a 454 Roche platform, and analyzed using QIIME. Approximately 199,077 reads were analyzed from 70 bacterial communities (average of 2,843 reads/sample). The composition of bacterial communities differed between the 3 treatment groups (P Permanova test). Taxonomic assignment of the operational taxonomic units (OTUs) identified 17 bacterial phyla across all samples. Bacteroidetes and Firmicutes constituted more than 95% of the bacterial population in the negative and exposed groups. In the positive group, lineages of Actinobacteria and Proteobacteria increased and those of Bacteroidetes and Firmicutes decreased (P < 0.001). Actinobacteria was highly abundant (30% of the total bacteria) in the positive group compared to exposed and negative groups (0.1-0.2%). Notably, the genus Arthrobacter was found to predominate Actinobacteria in the positive group. This study indicates that MAP-infected cattle have a different composition of their fecal microbiota than MAP-negative cattle.

Dysbiosis of the Fecal Microbiota in Cattle Infected with Mycobacterium avium subsp. paratuberculosis.

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Marie-Eve Fecteau

Full Text Available Johne's disease (JD is a chronic, intestinal infection of cattle, caused by Mycobacterium avium subsp. paratuberculosis (MAP. It results in granulomatous inflammation of the intestinal lining, leading to malabsorption, diarrhea, and weight loss. Crohn's disease (CD, a chronic, inflammatory gastrointestinal disease of humans, has many clinical and pathologic similarities to JD. Dysbiosis of the enteric microbiota has been demonstrated in CD patients. It is speculated that this dysbiosis may contribute to the intestinal inflammation observed in those patients. The purpose of this study was to investigate the diversity patterns of fecal bacterial populations in cattle infected with MAP, compared to those of uninfected control cattle, using phylogenomic analysis. Fecal samples were selected to include samples from 20 MAP-positive cows; 25 MAP-negative herdmates; and 25 MAP-negative cows from a MAP-free herd. The genomic DNA was extracted; PCR amplified sequenced on a 454 Roche platform, and analyzed using QIIME. Approximately 199,077 reads were analyzed from 70 bacterial communities (average of 2,843 reads/sample. The composition of bacterial communities differed between the 3 treatment groups (P < 0.001; Permanova test. Taxonomic assignment of the operational taxonomic units (OTUs identified 17 bacterial phyla across all samples. Bacteroidetes and Firmicutes constituted more than 95% of the bacterial population in the negative and exposed groups. In the positive group, lineages of Actinobacteria and Proteobacteria increased and those of Bacteroidetes and Firmicutes decreased (P < 0.001. Actinobacteria was highly abundant (30% of the total bacteria in the positive group compared to exposed and negative groups (0.1-0.2%. Notably, the genus Arthrobacter was found to predominate Actinobacteria in the positive group. This study indicates that MAP-infected cattle have a different composition of their fecal microbiota than MAP-negative cattle.

Identification of novel seroreactive antigens in Johne’s disease cattle using the Mycobacterium tuberculosis protein array

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Johne’s disease, a chronic gastrointestinal inflammatory disease caused by Mycobacterium avium subspecies paratuberculosis (Map), is endemic in dairy cattle and other ruminants worldwide and remains a challenge to diagnose using traditional serological methods. Given the close phylogenetic relations...

Culture phenotypes and molecular characterization of Mycobacterium avium subsp. paratuberculosis isolates from small ruminants.

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Dimareli-Malli, Z; Mazaraki, K; Stevenson, K; Tsakos, P; Zdragas, A; Giantzi, V; Petridou, E; Heron, I; Vafeas, G

2013-08-01

In this study the suitability of different solid media was investigated for the isolation of Mycobacterium avium subsp. paratuberculosis (Map) in order to identify the optimum single or combination of media to permit the isolation of all strain types from small ruminants. A subset of these Map strains was then further characterized by molecular typing methods to assess the genetic diversity of Map strains in the study area (Northern Greece). Map strains were isolated from tissues and faeces of infected goats (n=52) and sheep (n=8) and were analysed for polymorphisms in IS1311 to classify the strain type as Type C or S. The study found that M7H11 supplemented with mycobactin j, OADC and new born calf serum (M7H11+Mj) is the best single choice of medium for the primary isolation of Map of both Type C and S from small ruminants. The combination of M7H11+Mj and Herrolds egg yolk medium supplemented with mycobactin j and sodium pyruvate allowed the detection of all Map isolates in this study. Nineteen Map isolates were characterised by pulsed-field gel electrophoresis and the isolates demonstrated significant genetic diversity. Twelve different SnaBI and 16 distinct SpeI profiles were detected of which 25 have not been described previously and are new profiles. The combination of both enzyme profiles gave 13 different multiplex profiles. Ten different multiplex profiles were detected in goats and three in sheep. One ovine isolate gave the same multiplex profile as a caprine isolate and two different profiles were found within a single goat herd. Copyright © 2013 Elsevier Ltd. All rights reserved.

Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA.

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Zozaya-Valdés, Enrique; Porter, Jessica L; Coventry, John; Fyfe, Janet A M; Carter, Glen P; Gonçalves da Silva, Anders; Schultz, Mark B; Seemann, Torsten; Johnson, Paul D R; Stewardson, Andrew J; Bastian, Ivan; Roberts, Sally A; Howden, Benjamin P; Williamson, Deborah A; Stinear, Timothy P

2017-06-01

Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium - M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico -predicted specificity for M. chimaera Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera . Copyright © 2017 American Society for Microbiology.

Mycobacterium avium subesp. paratuberculosis: uma preocupação para a indústria de laticínios

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Márcio Ferraz Cunha

2009-04-01

Full Text Available Mycobacterium avium subesp. paratuberculosis é conhecida como o agente etiológico da doença de Johne, ou paratuberculose, que afeta principalmente animais ruminantes. É integrante da família Mycobacteriaceae, da qual também fazem parte a M. tuberculosis e a M. bovis, responsáveis pela tuberculose humana e bovina, respectivamente. Foi sugerido que a M. paratuberculosis poderia estar envolvida na patogênese da doença de Crohn, a qual possui sintomas similares à paratuberculose, mas afeta seres humanos. Como o microrganismo pode ser excretado no leite de animais infectados, o primeiro passo foi avaliar a sua termoresistência. Alguns estudos indicaram que a bactéria sobrevive ao tratamento térmico da pasteurização HTST (72ºC/15 s. Entretanto, os estudos existentes na literatura científica até o momento não permitem afirmar que M. paratuberculosis seja responsável pela doença de Crohn, bem como apresentam dúvidas sobre a termoresistência dessa bactéria. A realização de mais pesquisas sobre este microrganismo é de fundamental importância, com o objetivo de orientar a produção de produtos lácteos isentos de contaminação por M. paratuberculosis.

The formation of lipid droplets favors intracellular Mycobacterium leprae survival in SW-10, non-myelinating Schwann cells

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Jin, Song-Hyo; An, Sung-Kwan; Lee, Seong-Beom

2017-01-01

Leprosy is a chronic infectious disease that is caused by the obligate intracellular pathogen Mycobacterium leprae (M.leprae), which is the leading cause of all non-traumatic peripheral neuropathies worldwide. Although both myelinating and non-myelinating Schwann cells are infected by M.leprae in patients with lepromatous leprosy, M.leprae preferentially invades the non-myelinating Schwann cells. However, the effect of M.leprae infection on non-myelinating Schwann cells has not been elucidate...

Comparison of rapid diagnostic tests to detect Mycobacterium avium subsp. paratuberculosis disseminated infection in bovine liver.

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Zarei, Mehdi; Ghorbanpour, Masoud; Tajbakhsh, Samaneh; Mosavari, Nader

2017-08-01

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, a chronic enteritis in cattle and other domestic and wild ruminants. The presence of MAP in tissues other than intestines and associated lymph nodes, such as meat and liver, is a potential public health concern. In the present study, the relationship between the results of rapid diagnostic tests of the Johne's disease, such as serum ELISA, rectal scraping PCR, and acid-fast staining, and the presence of MAP in liver was evaluated. Blood, liver, and rectal scraping samples were collected from 200 slaughtered cattle with unknown Johne's disease status. ELISA was performed to determine the MAP antibody activity in the serum. Acid-fast staining was performed on rectal scraping samples, and PCR was performed on rectal scraping and liver samples. PCR-positive liver samples were used for mycobacterial culture. Overall, the results of this study demonstrated that MAP can be detected and cultured from liver of slaughtered cattle and rapid diagnostic tests of Johne's disease have limited value in detecting cattle with MAP infection in liver. These findings show that the presence of MAP in liver tissue may occur in cows with negative results for rapid diagnostic tests and vice versa. Hence, liver might represent another possible risk of human exposure to MAP. Given concerns about a potential zoonotic role for MAP, these results show the necessity to find new methods for detecting cattle with MAP disseminated infection.

Viable Mycobacterium avium ssp. paratuberculosis isolated from calf milk replacer.

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Grant, Irene R; Foddai, Antonio C G; Tarrant, James C; Kunkel, Brenna; Hartmann, Faye A; McGuirk, Sheila; Hansen, Chungyi; Talaat, Adel M; Collins, Michael T

2017-12-01

When advising farmers on how to control Johne's disease in an infected herd, one of the main recommendations is to avoid feeding waste milk to calves and instead feed calf milk replacer (CMR). This advice is based on the assumption that CMR is free of viable Mycobacterium avium ssp. paratuberculosis (MAP) cells, an assumption that has not previously been challenged. We tested commercial CMR products (n = 83) obtained from dairy farms around the United States by the peptide-mediated magnetic separation (PMS)-phage assay, PMS followed by liquid culture (PMS-culture), and direct IS900 quantitative PCR (qPCR). Conventional microbiological analyses for total mesophilic bacterial counts, coliforms, Salmonella, coagulase-negative staphylococci, streptococci, nonhemolytic Corynebacterium spp., and Bacillus spp. were also performed to assess the overall microbiological quality of the CMR. Twenty-six (31.3%) of the 83 CMR samples showed evidence of the presence of MAP. Seventeen (20.5%) tested positive for viable MAP by the PMS-phage assay, with plaque counts ranging from 6 to 1,212 pfu/50 mL of reconstituted CMR (average 248.5 pfu/50 mL). Twelve (14.5%) CMR samples tested positive for viable MAP by PMS-culture; isolates from all 12 of these samples were subsequently confirmed by whole-genome sequencing to be different cattle strains of MAP. Seven (8.4%) CMR samples tested positive for MAP DNA by IS900 qPCR. Four CMR samples tested positive by both PMS-based tests and 5 CMR samples tested positive by IS900 qPCR plus one or other of the PMS-based tests, but only one CMR sample tested positive by all 3 MAP detection tests applied. All conventional microbiology results were within current standards for whole milk powders. A significant association existed between higher total bacterial counts and presence of viable MAP indicated by either of the PMS-based assays. This represents the first published report of the isolation of viable MAP from CMR. Our findings raise concerns

Clinical Concentrations of Thioridazine Kill Intracellular Multidrug-Resistant Mycobacterium tuberculosis

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Ordway, Diane; Viveiros, Miguel; Leandro, Clara; Bettencourt, Rosário; Almeida, Josefina; Martins, Marta; Kristiansen, Jette E.; Molnar, Joseph; Amaral, Leonard

2003-01-01

The phenothiazines chlorpromazine (CPZ) and thioridazine (TZ) have equal in vitro activities against antibiotic-sensitive and -resistant Mycobacterium tuberculosis. These compounds have not been used as anti-M. tuberculosis agents because their in vitro activities take place at concentrations which are beyond those that are clinically achievable. In addition, chronic administration of CPZ produces frequent severe side effects. Because CPZ has been shown to enhance the killing of intracellular M. tuberculosis at concentrations in the medium that are clinically relevant, we have investigated whether TZ, a phenothiazine whose negative side effects are less frequent and serious than those associated with CPZ, kills M. tuberculosis organisms that have been phagocytosed by human macrophages, which have nominal killing activities against these bacteria. Both CPZ and TZ killed intracellular antibiotic-sensitive and -resistant M. tuberculosis organisms when they were used at concentrations in the medium well below those present in the plasma of patients treated with these agents. These concentrations in vitro were not toxic to the macrophage, nor did they affect in vitro cellular immune processes. TZ thus appears to be a serious candidate for the management of a freshly diagnosed infection of pulmonary tuberculosis or as an adjunct to conventional antituberculosis therapy if the patient originates from an area known to have a high prevalence of multidrug-resistant M. tuberculosis isolates. Nevertheless, we must await the outcomes of clinical trials to determine whether TZ itself may be safely and effectively used as an antituberculosis agent. PMID:12604522

Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rondonia, Brazil

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Cleoni Alves Mendes de Lima

2013-06-01

Full Text Available The main cause of pulmonary tuberculosis (TB is infection with Mycobacterium tuberculosis (MTB. We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.

The Essential Role of Cholesterol Metabolism in the Intracellular Survival of Mycobacterium leprae Is Not Coupled to Central Carbon Metabolism and Energy Production.

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Marques, Maria Angela M; Berrêdo-Pinho, Marcia; Rosa, Thabatta L S A; Pujari, Venugopal; Lemes, Robertha M R; Lery, Leticia M S; Silva, Carlos Adriano M; Guimarães, Ana Carolina R; Atella, Georgia C; Wheat, William H; Brennan, Patrick J; Crick, Dean C; Belisle, John T; Pessolani, Maria Cristina V

2015-12-01

Mycobacterium leprae induces the formation of lipid droplets, which are recruited to pathogen-containing phagosomes in infected macrophages and Schwann cells. Cholesterol is among the lipids with increased abundance in M. leprae-infected cells, and intracellular survival relies on cholesterol accumulation. The present study investigated the capacity of M. leprae to acquire and metabolize cholesterol. In silico analyses showed that oxidation of cholesterol to cholest-4-en-3-one (cholestenone), the first step of cholesterol degradation catalyzed by the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD), is apparently the only portion of the cholesterol catabolic pathway seen in Mycobacterium tuberculosis preserved by M. leprae. Incubation of bacteria with radiolabeled cholesterol confirmed the in silico predictions. Radiorespirometry and lipid analyses performed after incubating M. leprae with [4-(14)C]cholesterol or [26-(14)C]cholesterol showed the inability of this pathogen to metabolize the sterol rings or the side chain of cholesterol as a source of energy and carbon. However, the bacteria avidly incorporated cholesterol and, as expected, converted it to cholestenone both in vitro and in vivo. Our data indicate that M. leprae has lost the capacity to degrade and utilize cholesterol as a nutritional source but retains the enzyme responsible for its oxidation to cholestenone. Thus, the essential role of cholesterol metabolism in the intracellular survival of M. leprae is uncoupled from central carbon metabolism and energy production. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies. Our study focused on the obligate intracellular pathogen Mycobacterium leprae and its capacity to metabolize cholesterol. The data make an important contribution for those interested in understanding the

Insights from the Genome Sequence of Mycobacterium lepraemurium: Massive Gene Decay and Reductive Evolution

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Andrej Benjak

2017-10-01

Full Text Available Mycobacterium lepraemurium is the causative agent of murine leprosy, a chronic, granulomatous disease similar to human leprosy. Due to the similar clinical manifestations of human and murine leprosy and the difficulty of growing both bacilli axenically, Mycobacterium leprae and M. lepraemurium were once thought to be closely related, although it was later suggested that M. lepraemurium might be related to Mycobacterium avium. In this study, the complete genome of M. lepraemurium was sequenced using a combination of PacBio and Illumina sequencing. Phylogenomic analyses confirmed that M. lepraemurium is a distinct species within the M. avium complex (MAC. The M. lepraemurium genome is 4.05 Mb in length, which is considerably smaller than other MAC genomes, and it comprises 2,682 functional genes and 1,139 pseudogenes, which indicates that M. lepraemurium has undergone genome reduction. An error-prone repair homologue of the DNA polymerase III α-subunit was found to be nonfunctional in M. lepraemurium, which might contribute to pseudogene formation due to the accumulation of mutations in nonessential genes. M. lepraemurium has retained the functionality of several genes thought to influence virulence among members of the MAC.

Low genetic diversity of bovine Mycobacterium avium subspecies paratuberculosis isolates detected by MIRU-VNTR genotyping.

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de Kruijf, Marcel; Lesniak, Olga N; Yearsley, Dermot; Ramovic, Elvira; Coffey, Aidan; O'Mahony, Jim

2017-05-01

Mycobacterial interspersed repetitive unit and variable number tandem repeat (MIRU-VNTR) has been developed as a simple, rapid and cost efficient molecular typing method to differentiate Mycobacterium avium subspecies paratuberculosis (MAP) isolates. The aim of this study was to determine the genomic diversity of MAP across the Republic of Ireland by utilising the MIRU-VNTR typing method on a large collection of MAP isolates. A total of 114 MAP isolates originated from 53 herds across 19 counties in the Republic of Ireland were genotyped based on eight established MIRU-VNTR loci. Four INMV groups were observed during this study. INMV 1 was found in 67 MAP isolates (58.8%) and INMV 2 was observed in 45 isolates (39.4%). INMV 3 and INMV 116 recorded only one isolate each (0.9%). The unique INMV 116 group has never been reported among herds thus far and the molecular pattern of the MAP isolate classified in INMV 116 showed a difference at the MIRU-VNTR X3 locus compared to the other three INMV groups observed. INMV 1, INMV 2 and INMV 3 are observed frequently in Europe and comprised 99.1% of the total MAP isolates characterised in this study, indicating that MAP exhibited low level of genetic diversity across the Republic of Ireland using the MIRU-VNTR method. By the implementation of SNP analysis or MLSSR as an additional typing method, MAP genetic diversity would increase. INMV 3 is unique to Ireland and whereas INMV 116 has never been previously reported among herds by MIRU-VNTR typing. Copyright © 2017 Elsevier B.V. All rights reserved.

Serological, culture and molecular survey of Mycobacterium avium paratuberculosis in a goat flock in Tuscany.

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Galiero, Alessia; Turchi, Barbara; Pedonese, Francesca; Nuvoloni, Roberta; Cantile, Carlo; Colombani, Giuseppe; Forzan, Mario; Cerri, Domenico; Bandecchi, Patrizia; Fratini, Filippo

2017-11-01

Mycobacterium avium paratuberculosis (Map) is a pathogen which causes a chronic progressive granulomatous enteritis known as paratuberculosis or Johne's disease and it primarily affects wild and domestic ruminants. The aim of this research was to examine a flock which consisted of 294 goats and was located in Garfagnana district (Tuscany, Italy) performing ELISA tests, culture and IS900 PCR assay; direct diagnostic methods were carried out not only on bulk tank milk and cheese samples but also on individual milk and tissue specimens collected from nine subjects positive to ELISA tests. Out of 294 animals, 20 goats (6.8%) were positive to ELISA surveys. Bulk tank milk samples were negative to culture and to PCR assay carried out on the DNA extracted directly from them, while, with respect to cheese, Map was detected by culture in 2/12 (16.66%) cheeses ripened for 3-7 days, and by PCR in 2/12 (16.66%) cheeses ripened for 3-7 days and in 3/12 (25%) cheeses ripened for 45 days. Regarding individual milk samples, Map was detected by culture in 2/9 (22.22%) specimens and by PCR in 5/9 (55.55%) samples. Furthermore, Map was isolated from the intestine in 9/9 (100%) animals, from the mesenteric lymph nodes in 8/9 (88.88%) subjects, from the liver in 4/9 (44.44%) goats, from the spleen in 5/9 (55.55%) animals, while Map DNA was found in all the tissue samples analyzed.The results demonstrated the presence of paratuberculosis in a goat flock located in Garfagnana district (Tuscany, Italy).

Genetic loci involved in antibody response to Mycobacterium avium ssp. paratuberculosis in cattle.

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Giulietta Minozzi

Full Text Available BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP causes chronic enteritis in a wide range of animal species. In cattle, MAP causes a chronic disease called Johne's disease, or paratuberculosis, that is not treatable and the efficacy of vaccine control is controversial. The clinical phase of the disease is characterised by diarrhoea, weight loss, drop in milk production and eventually death. Susceptibility to MAP infection is heritable with heritability estimates ranging from 0.06 to 0.10. There have been several studies over the last few years that have identified genetic loci putatively associated with MAP susceptibility, however, with the availability of genome-wide high density SNP maker panels it is now possible to carry out association studies that have higher precision. METHODOLOGY/PRINCIPAL FINDINGS: The objective of the current study was to localize genes having an impact on Johne's disease susceptibility using the latest bovine genome information and a high density SNP panel (Illumina BovineSNP50 BeadChip to perform a case/control, genome-wide association analysis. Samples from MAP case and negative controls were selected from field samples collected in 2007 and 2008 in the province of Lombardy, Italy. Cases were defined as animals serologically positive for MAP by ELISA. In total 966 samples were genotyped: 483 MAP ELISA positive and 483 ELISA negative. Samples were selected randomly among those collected from 119 farms which had at least one positive animal. CONCLUSION/SIGNIFICANCE: THE ANALYSIS OF THE GENOTYPE DATA IDENTIFIED SEVERAL CHROMOSOMAL REGIONS ASSOCIATED WITH DISEASE STATUS: a region on chromosome 12 with high significance (P<5x10(-6, while regions on chromosome 9, 11, and 12 had moderate significance (P<5x10(-5. These results provide evidence for genetic loci involved in the humoral response to MAP. Knowledge of genetic variations related to susceptibility will facilitate the incorporation of this information

Mycobacterium avium subspecies paratuberculosis causes Crohn's disease in some inflammatory bowel disease patients.

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Naser, Saleh A; Sagramsingh, Sudesh R; Naser, Abed S; Thanigachalam, Saisathya

2014-06-21

Crohn's disease (CD) is a chronic inflammatory condition that plagues millions all over the world. This debilitating bowel disease can start in early childhood and continue into late adulthood. Signs and symptoms are usually many and multiple tests are often required for the diagnosis and confirmation of this disease. However, little is still understood about the cause(s) of CD. As a result, several theories have been proposed over the years. One theory in particular is that Mycobacterium avium subspecies paratuberculosis (MAP) is intimately linked to the etiology of CD. This fastidious bacterium also known to cause Johne's disease in cattle has infected the intestines of animals for years. It is believed that due to the thick, waxy cell wall of MAP it is able to survive the process of pasteurization as well as chemical processes seen in irrigation purification systems. Subsequently meat, dairy products and water serve as key vehicles in the transmission of MAP infection to humans (from farm to fork) who have a genetic predisposition, thus leading to the development of CD. The challenges faced in culturing this bacterium from CD are many. Examples include its extreme slow growth, lack of cell wall, low abundance, and its mycobactin dependency. In this review article, data from 60 studies showing the detection and isolation of MAP by PCR and culture techniques have been reviewed. Although this review may not be 100% comprehensive of all studies, clearly the majority of the studies overwhelmingly and definitively support the role of MAP in at least 30%-50% of CD patients. It is very possible that lack of detection of MAP from some CD patients may be due to the absence of MAP role in these patients. The latter statement is conditional on utilization of methodology appropriate for detection of human MAP strains. Ultimately, stratification of CD and inflammatory bowel disease patients for the presence or absence of MAP is necessary for appropriate and effective

Improved Culture Medium (TiKa) for Mycobacterium avium Subspecies Paratuberculosis (MAP) Matches qPCR Sensitivity and Reveals Significant Proportions of Non-viable MAP in Lymphoid Tissue of Vaccinated MAP Challenged Animals

DEFF Research Database (Denmark)

Bull, Tim J.; Munshil, Tulika; Melvang, Heidi Mikkelsen

2017-01-01

The quantitative detection of viable pathogen load is an important tool in determining the degree of infection in animals and contamination of foodstuffs. Current conventional culture methods are limited in their ability to determine these levels in Mycobacterium avium subspecies paratuberculosis......Ka culture equates well with qPCR and provides important evidence that accuracy in estimating viable MAP load using DNA tests alone may vary significantly between samples of mucosal and lymphatic origin....... (MAP) due to slow growth, clumping and low recoverability issues. The principle goal of this study was to evaluate a novel culturing process (TiKa) with unique ability to stimulate MAP growth from low sample loads and dilutions. We demonstrate it was able to stimulate a mean 29-fold increase...

Pharmacokinetic-Pharmacodynamic modelling of intracellular Mycobacterium tuberculosis growth and kill rates is predictive of clinical treatment duration.

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Aljayyoussi, Ghaith; Jenkins, Victoria A; Sharma, Raman; Ardrey, Alison; Donnellan, Samantha; Ward, Stephen A; Biagini, Giancarlo A

2017-03-29

Tuberculosis (TB) treatment is long and complex, typically involving a combination of drugs taken for 6 months. Improved drug regimens to shorten and simplify treatment are urgently required, however a major challenge to TB drug development is the lack of predictive pre-clinical tools. To address this deficiency, we have adopted a new high-content imaging-based approach capable of defining the killing kinetics of first line anti-TB drugs against intracellular Mycobacterium tuberculosis (Mtb) residing inside macrophages. Through use of this pharmacokinetic-pharmacodynamic (PK-PD) approach we demonstrate that the killing dynamics of the intracellular Mtb sub-population is critical to predicting clinical TB treatment duration. Integrated modelling of intracellular Mtb killing alongside conventional extracellular Mtb killing data, generates the biphasic responses typical of those described clinically. Our model supports the hypothesis that the use of higher doses of rifampicin (35 mg/kg) will significantly reduce treatment duration. Our described PK-PD approach offers a much needed decision making tool for the identification and prioritisation of new therapies which have the potential to reduce TB treatment duration.

Avaliação sorológica e de fatores de risco para a infecção por Mycobacterium avium subsp. paratuberculosis em rebanhos leiteiros da Microrregião de Garanhuns, Pernambuco Serological evaluation and risk factors for Mycobacterium avium subsp. paratuberculosis infection in dairy herds of Microregion Garanhuns, Pernambuco

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Luenda de M. e Sá

2013-03-01

Full Text Available Objetivou-se com esse trabalho realizar um inquérito epidemiológico da infecção por Mycobacterium avium subsp. paratuberculosis (MAP em bovinos leiteiros da microrregião de Garanhuns, Pernambuco, Brasil. Para este estudo foram coletadas amostras sanguíneas de 408 animais, provenientes de 19 rebanhos localizados em 15 municípios. O exame sorológico foi realizado por Ensaio Imunoenzimático (ELISA indireto para detecção de anticorpos frente ao MAP. Em todas as propriedades, foi aplicado um questionário investigativo para análise dos fatores de risco, e as coordenadas geográficas coletadas por um aparelho de Global Position System (GPS para realização da distribuição espacial. A prevalência da infecção por MAP foi de 2,7% (11/408; I.C. 1,4-4,9. O número de focos foi 47,4% (9/19. Na análise de regressão logística foi identificado como fator de risco a taxa anual de nascimentos superior a 51 bezerros/ano (OR 3,8; I.C. 1,1-13,1. Desta forma, conclui-se que a infecção por MAP encontra-se presente nos rebanhos bovinos leiteiros da microrregião estudada e que medidas de controle baseadas nos fatores de risco identificados devem ser implementadas com o objetivo de reduzir o número de focos da infecção.The present study aimed to conduct an epidemiological investigation of Mycobacterium avium subsp. paratuberculosis (MAP infection in dairy cattle of the Garanhuns microregion, in Pernambuco, Brazil. Blood samples were collected from 408 animals from 19 herds located in 15 cities. Serological tests were performed by indirect immunoenzymatic assay (ELISA for antibodies against MAP. In all farms, a questionnaire to investigate risk factors was used, and Global Position System (GPS receivers were used to collect geographic coordinates to show the spatial distribution of the animals. The prevalence of MAP infected cattle was 2.7% (11/408; I.C. 1.4-4.9. The rate of infection was 47.4% (9/19. An annual birth rate over 51 calves

Economic analysis of Mycobacterium avium subspecies paratuberculosis vaccines in dairy herds.

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Cho, J; Tauer, L W; Schukken, Y H; Gómez, M I; Smith, R L; Lu, Z; Grohn, Y T

2012-04-01

Johne's disease, or paratuberculosis, is a chronic infectious enteric disease of ruminants, caused by infection with Mycobacterium avium ssp. paratuberculosis (MAP). Given the absence of a fail-safe method of prevention or a cure, Johne's disease can inflict significant economic loss on the US dairy industry, with an estimated annual cost of over $200 million. Currently available MAP control strategies include management measures to improve hygiene, culling MAP serologic- or fecal-positive adult cows, and vaccination. Although the 2 first control strategies have been reported to be effective in reducing the incidence of MAP infection, the changes in herd management needed to conduct these control strategies require significant effort on the part of the dairy producer. On the other hand, vaccination is relatively simple to apply and requires minor changes in herd management. Despite these advantages, only 5% of US dairy operations use vaccination to control MAP. This low level of adoption of this technology is due to limited information on its cost-effectiveness and efficacy and some important inherent drawbacks associated with current MAP vaccines. This study investigates the epidemiological effect and economic values of MAP vaccines in various stages of development. We create scenarios for the potential epidemiological effects of MAP vaccines, and then estimate economically justifiable monetary values at which vaccines become economically beneficial to dairy producers such that a net present value (NPV) of a farm's net cash flow can be higher than the NPV of a farm using no control or alternative nonvaccine controls. Any vaccination with either low or high efficacy considered in this study yielded a higher NPV compared with a no MAP control. Moreover, high-efficacy vaccines generated an even higher NPV compared with alternative controls, making vaccination economically attractive. Two high-efficacy vaccines were particularly effective in MAP control and NPV

Are we overlooking infections owing to non-tuberculous mycobacteria during routine conventional laboratory investigations?

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Kushal Garima

2012-01-01

Full Text Available A large number of potentially pathogenic non-tuberculous mycobacteria (NTM encountered in the clinical laboratory makes it necessary to identify their species to ensure appropriate treatment. However, labor-intensive conventional methods of speciation are not used in every laboratory, and hence NTM infections are often ignored. Polymerase chain reaction (PCR restriction analysis (PRA was applied in this study for early identification and speciation of mycobacterial species on 306 cultures of acid-fast bacilli isolated from patients suspected of suffering from tuberculosis. Mycobacterium tuberculosis was identified in 85.6% of the isolates. The NTM isolated most commonly was Mycobacterium kansasii/gastri group (3.5%, followed by Mycobacterium fortuitum (3.2%. Four of the M. fortuitum were grown from cultures obtained on the same day, but from samples from different patients and were probably laboratory contaminants. Mycobacterium intracellulare and Mycobacterium avium were identified in 2.94% and 2.28% of the isolates, respectively. Three isolates of M. avium and two isolates of M. intracellulare were obtained in repeated cultures from sputum samples of the same patients and were thus pathogenic. A single isolate of Mycobacterium abscessus was obtained from a breast abscess. A rare pathogen Mycobacterium phocaicum was isolated from one patient with epididymitis. However, whether it was the causative agent of epididymitis in this patient remains doubtful. The results of this study highlight the importance of speciation of mycobacteria for appropriate diagnosis and the importance of including molecular assays to augment conventional methods of diagnosis of mycobacterial diseases for rapid identification of NTM so that these potential pathogens are not overlooked in routine diagnostic procedures.

Loperamide Restricts Intracellular Growth of Mycobacterium tuberculosis in Lung Macrophages.

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Juárez, Esmeralda; Carranza, Claudia; Sánchez, Guadalupe; González, Mitzi; Chávez, Jaime; Sarabia, Carmen; Torres, Martha; Sada, Eduardo

2016-12-01

New approaches for improving tuberculosis (TB) control using adjunct host-directed cellular and repurposed drug therapies are needed. Autophagy plays a crucial role in the response to TB, and a variety of autophagy-inducing drugs that are currently available for various medical conditions may serve as an adjunct treatment in pulmonary TB. Here, we evaluated the potential of loperamide, carbamazepine, valproic acid, verapamil, and rapamycin to enhance the antimicrobial immune response to Mycobacterium tuberculosis (Mtb). Human monocyte-derived macrophages (MDMs) and murine alveolar cells (MACs) were infected with Mtb and treated with loperamide, carbamazepine, valproic acid, verapamil, and rapamycin in vitro. Balb/c mice were intraperitoneally administered loperamide, valproic acid, and verapamil, and MACs were infected in vitro with Mtb. The induction of autophagy, the containment of Mtb within autophagosomes and the intracellular Mtb burden were determined. Autophagy was induced by all of the drugs in human and mouse macrophages, and loperamide significantly increased the colocalization of microtubule-associated protein 1 light chain 3 with Mtb in MDMs. Carbamazepine, loperamide, and valproic acid induced microtubule-associated protein 1 light chain 3 and autophagy related 16- like protein 1 gene expression in MDMs and in MACs. Loperamide also induced a reduction in TNF-α production. Loperamide and verapamil induced autophagy, which was associated with a significant reduction in the intracellular growth of Mtb in MACs and alveolar macrophages. The intraperitoneal administration of loperamide and valproic acid induced autophagy in freshly isolated MACs. The antimycobacterial activity in MACs was higher after loperamide treatment and was associated with the degradation of p62. In conclusion, loperamide shows potential as an adjunctive therapy for the treatment of TB.

Clinical study of pulmonary infection caused by mycobacterium avium complex. Evaluation of radiographic features on the primary pulmonary infection

International Nuclear Information System (INIS)

Harada, Yasuko; Harada, Susumu; Kitahara, Yoshinari; Kajiki, Akira; Maruyama, Masao; Takamoto, Masahiro; Ishibashi, Tsuneo

1996-01-01

During the 13 year period of 1982 to 1994 we had 103 patients with pulmonary Mycobacterium avium complex (MAC) infections. All met the criteria of atypical mycobacteriosis (Japanese Mycobacteriosis Research Group of the National Chest Hospitals). Of 103 patients 70 had no underlying pulmonary diseases and classified as primary type. Radiographic features of chest X-rays or computed tomography (CT) of primary infection were evaluated. Results obtained were as follows: Primary infection of MAC was classified into two types. One was localized type. This type was further classified into three patterns; tuberculosis-like pattern, pneumonia pattern in the lingual and/or middle lobe and pneumonia pattern in other lobes. Another one was diffuse type. Tuberculosis-like pattern was most common in males. On the other hand, the pneumonia pattern and the diffuse type were most common in females. Four characteristic features were seen as follows (Type 1-4) in the chest CT examination of diffuse pattern. Type 1: Nodules near the pleura. Type 2: Nodules with subpleural thickening. Type 3: Bronchial wall thickening and ectatic change of the draining bronchi. Type 4: Cystic bronchiectatic change associated with atelectasis of the segment or the lobe. Bronchiectatic changes became severe and widespreaded in all lung fields as the disease progressed slowly. These findings were more prevalent in the lingual and/or middle lobe than the other lobes. (author)

Genome sequence of Mycobacterium yongonense RT 955-2015 isolate from a patient misdiagnosed with multi-drug resistant tuberculosis: first clinical isolate in Tanzania.

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Mnyambwa, Nicholaus Peter; Kim, Dong-Jin; Ngadaya, Esther; Chun, Jongsik; Ha, Sung-Min; Petrucka, Pammla; Addo, Kennedy Kwasi; Kazwala, Rudovick R; Mfinanga, Sayoki G

2018-04-24

Mycobacterium yongonense is a recently described novel species belonging to Mycobacterium avium complex which is the most prevalent etiology of non-tuberculous mycobacteria associated with pulmonary infections, and posing tuberculosis diagnostic challenges in high-burden, resource-constrained settings. We used whole genome shotgun sequencing and comparative microbial genomic analyses to characterize the isolate from a patient diagnosed with multi-drug resistant tuberculosis (MDR-TB) after relapse. We present a genome sequence of the first case of M. yongonense (M. yongonense RT 955-2015) in Tanzania. Sequence analysis revealed that the RT 955-2015 strain had a high similarity to M. yongonense 05-1390(T) (98.74%) and M. chimaera DSM 44623(T) (98%). Its 16S rRNA showed similarity to M. paraintracellulare KCTC 290849(T) (100%); M. intracellulare ATCC 13950(T) (100%); M. chimaera DSM 44623(T) (99.9%); and M. yongonense 05-1390(T) (98%). The strain had a substantially different rpoB sequence from that of M. yongonense 05-1390 (95.16%) but exhibited a sequence closely related to M. chimaera DSM 44623(T) (99.86%), M. intracellulare ATCC 13950(T) (99.53%), and M. paraintracellulare KCTC 290849(T) (99.53%). In light of the OrthoANI algorithm, and phylogenetic analysis, we conclude that the isolate was M. yongonense Type II genotype, which is an indication that the patient was misdiagnosed with TB/MDR-TB and received inappropriate treatment. Copyright © 2018. Published by Elsevier Ltd.

Short communication: Passive shedding of Mycobacterium avium ssp. paratuberculosis in commercial dairy goats in Brazil.

Science.gov (United States)

Schwarz, D G G; Lima, M C; Barros, M; Valente, F L; Scatamburlo, T M; Rosado, N; Oliveira, C T S A M; Oliveira, L L; Moreira, M A S

2017-10-01

Goat farming is a low-cost alternative to dairy production in developing countries. In Brazil, goat production has increased in recent years due in part to the implementation of programs encouraging this activity. Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a disease that causes chronic granulomatous enteritis in ruminants, but MAP transmission dynamics are still poorly understood in goats. In a previously published study of our research group, 10 dairy goat farms (467 animals) from Minas Gerais state were analyzed for MAP detection; 2 fecal cultures and 11 milk samples tested positive for MAP by conventional PCR and were confirmed by sequencing. Because no clinical signs were observed over 1 yr of monitoring, we hypothesized that these MAP-positive goats could be passive shedders. Thus, in the present study, 4 positive goats (4/13) from the previous study were purchased and feces and milk samples were collected for evaluation (twice, with an interval of 3 mo between tests) by culture of MAP, IS900 PCR, or both. All analyses were negative for MAP. At the last time point, blood samples were collected for ELISA, the animals were killed, and tissues collected for tissue culture and histopathology. At necropsy, no macroscopic lesions related to paratuberculosis were observed. Similarly, no histological changes were observed and MAP in samples stained by Ziehl-Neelsen was not detected. These animals were characterized as potential passive shedders with upward contamination of the teat canal by MAP. This is the first report of the passive shedding phenomenon in goats in Brazil and it highlights the importance of identifying these animals for control programs and to ensure the quality of dairy products. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

MicroRNA 27a-3p Regulates Antimicrobial Responses of Murine Macrophages Infected by Mycobacterium avium subspecies paratuberculosis by Targeting Interleukin-10 and TGF-β-Activated Protein Kinase 1 Binding Protein 2

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Tariq Hussain

2018-01-01

Full Text Available Mycobacterium avium subspecies paratuberculosis (MAP persistently survive and replicate in mononuclear phagocytic cells by adopting various strategies to subvert host immune response. Interleukin-10 (IL-10 upregulation via inhibition of macrophage bactericidal activity is a critical step for MAP survival and pathogenesis within the host cell. Mitogen-activated protein kinase p38 signaling cascade plays a crucial role in the elevation of IL-10 and progression of MAP pathogenesis. The contribution of microRNAs (miRNAs and their influence on the activation of macrophages during MAP pathogenesis are still unclear. In the current study, we found that miRNA-27a-3p (miR-27a expression is downregulated during MAP infection both in vivo and in vitro. Moreover, miR-27a is also downregulated in toll-like receptor 2 (TLR2-stimulated murine macrophages (RAW264.7 and bone marrow-derived macrophage. ELISA and real-time qRT-PCR results confirm that overexpression of miR-27a inhibited MAP-induced IL-10 production in macrophages and upregulated pro-inflammatory cytokines, while miR-27a inhibitor counteracted these effects. Luciferase reporter assay results revealed that IL-10 and TGF-β-activated protein kinase 1 binding protein 2 (TAB 2 are potential targets of miR-27a. In addition, we demonstrated that miR-27a negatively regulates TAB 2 expression and diminishes TAB 2-dependent p38/JNK phosphorylation, ultimately downregulating IL-10 expression in MAP-infected macrophages. Furthermore, overexpression of miR-27a significantly inhibited the intracellular survival of MAP in infected macrophages. Our data show that miR-27a augments antimicrobial activities of macrophages and inhibits the expression of IL-10, demonstrating that miR-27a regulates protective innate immune responses during MAP infection and can be exploited as a novel therapeutic target in the control of intracellular pathogens, including paratuberculosis.

Codon optimisation to improve expression of a Mycobacterium avium ssp. paratuberculosis-specific membrane-associated antigen by Lactobacillus salivarius.

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Johnston, Christopher; Douarre, Pierre E; Soulimane, Tewfik; Pletzer, Daniel; Weingart, Helge; MacSharry, John; Coffey, Aidan; Sleator, Roy D; O'Mahony, Jim

2013-06-01

Subunit and DNA-based vaccines against Mycobacterium avium ssp. paratuberculosis (MAP) attempt to overcome inherent issues associated with whole-cell formulations. However, these vaccines can be hampered by poor expression of recombinant antigens from a number of disparate hosts. The high G+C content of MAP invariably leads to a codon bias throughout gene expression. To investigate if the codon bias affects recombinant MAP antigen expression, the open reading frame of a MAP-specific antigen MptD (MAP3733c) was codon optimised for expression against a Lactobacillus salivarius host. Of the total 209 codons which constitute MAP3733c, 172 were modified resulting in a reduced G+C content from 61% for the native gene to 32.7% for the modified form. Both genes were placed under the transcriptional control of the PnisA promoter; allowing controlled heterologous expression in L. salivarius. Expression was monitored using fluorescence microscopy and microplate fluorometry via GFP tags translationally fused to the C-termini of the two MptD genes. A > 37-fold increase in expression was observed for the codon-optimised MAP3733synth variant over the native gene. Due to the low cost and improved expression achieved, codon optimisation significantly improves the potential of L. salivarius as an oral vaccine stratagem against Johne's disease. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Clinical efficacy of anti-glycopeptidolipid-core IgA test for diagnosing Mycobacterium avium complex infection in lung.

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Numata, Takanori; Araya, Jun; Yoshii, Yutaka; Shimizu, Kenichiro; Hara, Hiromichi; Nakayama, Katsutoshi; Kuwano, Kazuyoshi

2015-11-01

It is difficult to verify the bacteriological diagnosis of Mycobacterium avium complex (MAC) infection. The anti-glycopeptidolipid (GPL)-core IgA antibody test was recently developed as a diagnostic method for MAC pulmonary disease. Only a few studies evaluate its clinical efficacy. We conducted retrospective evaluations of clinical characteristics of patients suspected of MAC infection to explore the usefulness of the anti-GPL-core IgA antibody test. We retrospectively evaluated 296 patients who were suspected to have MAC infection and underwent anti-GPL-core IgA antibody test between March 2013 and July 2014 in Jikei University hospital. A total of 29 patients were diagnosed with 'definite MAC' based on the American Thoracic Society (ATS) criteria with multiple identifications of MAC. On the other hand, 106 patients were diagnosed with other pulmonary diseases than MAC. The sensitivity and specificity of anti-GPL-core IgA antibody test for MAC diagnosis were 58.6% and 98.1%, respectively. The definite MAC group showed no significant differences in strains, treatment history or number of segments involved. The duration of MAC disease in the positive-antibody group was significantly longer than in the negative-antibody group (P = 0.046). A significant increase in the false-negative rate was observed in patients with malignant disease (P = 0.029). The anti-GPL-core IgA antibody test demonstrated high sensitivity and specificity for the diagnosis of MAC infection especially in patients without malignant diseases. © 2015 Asian Pacific Society of Respirology.

Occurrence of Mycobacterium avium subspecies paratuberculosis and Neospora caninum in Alberta cow-calf operations.

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Pruvot, M; Kutz, S; Barkema, H W; De Buck, J; Orsel, K

2014-11-01

Mycobacterium avium subsp. paratuberculosis (MAP) and Neospora caninum (NC) are two pathogens causing important production limiting diseases in the cattle industry. Significant impacts of MAP and NC have been reported on dairy cattle herds, but little is known about the importance, risk factors and transmission patterns in western Canadian cow-calf herds. In this cross-sectional study, the prevalence of MAP and NC infection in southwest Alberta cow-calf herds was estimated, risk factors for NC were identified, and the reproductive impacts of the two pathogens were assessed. Blood and fecal samples were collected from 840 cows on 28 cow-calf operations. Individual cow and herd management information was collected by self-administered questionnaires and one-on-one interviews. Bayesian estimates of the true prevalence of MAP and NC were computed, and bivariable and multivariable statistical analysis were done to assess the association between the NC serological status and ranch management risk factors, and the clinical effects of the two pathogens. Bayesian estimates of true prevalence indicated that 20% (95% probability interval: 8-38%) of herds had at least one MAP-positive cow, with a within-herd prevalence in positive herds of 22% (8-45%). From the Bayesian posterior distributions of NC prevalence, the median herd-level prevalence was 66% (33-95%) with 10% (4-21%) cow-level prevalence in positive herds. Multivariable analysis indicated that introducing purchased animals in the herd might increase the risk of NC. The negative association of NC with proper carcass disposal and presence of horses on ranch (possibly in relation to herd monitoring and guarding activities), may suggest the importance of wild carnivores in the dynamics of this pathogen in the study area. We also observed an association between MAP and NC serological status and the number of abortions. Additional studies should be done to further examine specific risk factors for MAP and NC, assess the

Thermal inactivation profiles of Mycobacterium avium subsp. paratuberculosis in lamb skeletal muscle homogenate fluid.

Science.gov (United States)

Whittington, Richard J; Waldron, Anna; Warne, Darian

2010-01-31

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in livestock and there is a debate about its role in humans in chronic inflammatory bowel disorders such as Crohn's disease, but the relationship remains unproven. Nevertheless livestock health authorities in many countries aim to lower the prevalence of this infection to reduce potential contamination of the human food supply. MAP may occur in bovine milk and data on thermal inactivation suggest pasteurisation is an effective process. Recently MAP has been identified in skeletal muscle of cattle and sheep but there are no data on its thermal inactivation in these substrates. In this study the inactivation of MAP was studied in a fluid homogenate of lamb skeletal muscle at temperatures previously identified as being relevant to cooking processes applied by domestic consumers. A PCR thermocycler was used to ensure accurate temperatures and rapid heat exchange, while radiometric culture was used to ensure sensitive detection of viable MAP for determination of D and z values. Among the two predominant strains of MAP, S and C, D(55) ranged from 56 to 89 min, D(60) was 8 to 11 min, D(65) was 26 to 35s while D(70) was 1.5 to 1.8s. Values for z were 4.21C degrees for the S strain and 4.51C degrees for the C strain. At temperatures of 65-70 degrees C, MAP appeared to be less heat tolerant in skeletal muscle fluid than in previous reports using milk as the medium. The total thermal exposure of MAP during baking of a sample of 16 leg-of-lamb roasts in domestic ovens was determined to result in more than 20 log reductions in most cases, that is the product was microbiologically safe. Based on the models used in this study, there is a low probability of survival of MAP provided that red meat is cooked to recommended standards. Crown Copyright 2009. Published by Elsevier B.V. All rights reserved.

Sero-Surveillance of Mycobacterium avium subspecies paratuberculosis Infection in Domestic Livestock in North India Using Indigenous Absorbed Elisa Test

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S. V. Singh

2018-06-01

Full Text Available A total of 829 serum samples belonging to domestic livestock (Cattle, buffaloes, goat and sheep and driven from different parts of North India between 2005 to 2008, were screened to estimate the seroprevalence of Mycobacterium avium subspecies paratuberculosis (MAP infection using 'indigenous absorbed ELISA kit'. Seroprevalence of MAP in the domestic livestock was 23.1%. Prevalence was higher in large ruminants (24.1% as compared to small ruminants (22.5%. Highest seropositivity was in cattle (26.9%, followed by goats (23.9%, buffaloes (20.2%, and sheep (19.0%. In cattle region-wise, 25.8, 29.1 and 30.7% animals were positive from Mathura (UP, Rohtak (Haryana, and Bareilly (UP regions, respectively. In buffaloes, the highest prevalence was found at Bareilly (26.6% followed by Rohtak (20.0% and Bhaghpat (18.4% regions. In goats, 19.6, 37.5, 40.0 and 21.9% animals were positive from Mathura (farm herd, Etawah, Agra and Ajmer (farmers herd regions, respectively. In sheep, prevalence of MAP was 25.5 and 16.3% in Mathura and Mannavanur regions, respectively. In sheep, prevalence was higher in Northern region as compared to the Southern region of the country. The present study showed that the prevalence of MAP in domestic livestock was moderately higher; therefore there is an urgent need to control the disease at National level in order to improve per animal productivity in the country.

Evasión molecular de la activación del macrófago bovino por Mycobacterium avium subespecie paratuberculosis

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René Ramírez G.

2013-11-01

Full Text Available El Mycobacterium avium subespecie paratuberculosis (MAP es el agente causal de una enfermedad granulomatosica crónica, que afecta el tracto gastrointestinal de rumiantes domesticos y salvajes, conocida como la enfermedad de Johne o paratuberculosis. MAP es un microorganismo de crecimiento lento en cultivo, no obstante sobrevive in vivo en células fagocíticas mononucleares de los rumiantes, bajo condiciones de susceptibilidad individual, virulencia de la cepa infectante y estado inmune del individuo afectado. Una vez MAP es fagocitado por el macrófago bovino, tanto el macrófago como MAP activan: el uno para tratar de destruir a MAP y luego sufrir apoptosis y el otro para evadir su destrucción dentro del fagolisosoma del macrófago. El balance de dicha confrontación molecular determina el curso inicial de la infección hacia la eliminación eficiente del microorganismo o hacia el establecimiento de la infección, que culminará en los estadios III (clínico intermitente y IV (clínica terminal de la enfermedad de Johne. En la presente revisión se discuten los diferentes mecanismos moleculares por los cuales MAP evade la respuesta inmune, con énfasis en su comportamiento dentro de la vacuola fagocítica y como el agente establece mecanismos de sobrevivencia intracelular y altera la activación de los macrófagos del hospedero y de la respuesta inmune específica.

AIDS: radiologic findings in the thorax and abdomen

International Nuclear Information System (INIS)

Langer, M.; Langer, R.

1993-01-01

Thoracic manifestation of AIDS are initially diagnosed on plain film studies. Pneumocystis carinii infections are characterised by bi-hilar streaky, interstitial and diffuse micronodular infiltrations. These findings are best seen on thoracic CT examinations. Thoracic Kaposi sarcoma is characterized by larger, rounded interstitial infiltrations. In abdominal AIDS ultrasound is the first screening method. It detects equally well lymph nodes in Kaposi sarcoma as in all lymphomas and opportunistic infections. CT can differentiate between mycobacterium tuberculosis and mycobacterium avium intracellulare infections, because of the central low-density lymphoma in tuberculosis. (orig.)

Heritability estimates for Mycobacterium avium subspecies paratuberculosis status of German Holstein cows tested by fecal culture.

Science.gov (United States)

Küpper, J; Brandt, H; Donat, K; Erhardt, G

2012-05-01

The objective of this study was to estimate genetic manifestation of Mycobacterium avium ssp. paratuberculosis (MAP) infection in German Holstein cows. Incorporated into this study were 11,285 German Holstein herd book cows classified as MAP-positive and MAP-negative animals using fecal culture results and originating from 15 farms in Thuringia, Germany involved in a paratuberculosis voluntary control program from 2008 to 2009. The frequency of MAP-positive animals per farm ranged from 2.7 to 67.6%. The fixed effects of farm and lactation number had a highly significant effect on MAP status. An increase in the frequency of positive animals from the first to the third lactation could be observed. Threshold animal and sire models with sire relationship were used as statistical models to estimate genetic parameters. Heritability estimates of fecal culture varied from 0.157 to 0.228. To analyze the effect of prevalence on genetic parameter estimates, the total data set was divided into 2 subsets of data into farms with prevalence rates below 10% and those above 10%. The data set with prevalence above 10% show higher heritability estimates in both models compared with the data set with prevalence below 10%. For all data sets, the sire model shows higher heritabilities than the equivalent animal model. This study demonstrates that genetic variation exists in dairy cattle for paratuberculosis infection susceptibility and furthermore, leads to the conclusion that MAP detection by fecal culture shows a higher genetic background than ELISA test results. In conclusion, fecal culture seems to be a better trait to control the disease, as well as an appropriate feature for further genomic analyses to detect MAP-associated chromosome regions. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Mycobacterium avium and purified protein derivative-specific cytotoxicity mediated by CD4+ lymphocytes from healthy HIV-seropositive and-seronegative individuals

DEFF Research Database (Denmark)

Ravn, P; Pedersen, B K

1996-01-01

mycobacteria. Our objective was to investigate the M.tuberculosis-and M. avium-specific cytotoxic capacity of T cells from healthy, bacille Calmette-Guérin-vaccinated, HIV-seropositive individuals. Blood mononuclear cells were obtained from 10 healthy HIV-seropositive and 10 healthy seronegative persons...... with no history of previous or active mycobacterial infection. Antigen-specific killing of macrophages presenting mycobacterial antigens (purified protein derivative or M. avium culture filtrate) was conducted. The phenotype of the killer cells was determined by a fluorescence-activated cell sorter after antigen...

Evaluation of PMS-PCR technology for detection of Mycobacterium avium subsp. paratuberculosis directly from bovine fecal specimens.

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Salgado, M; Steuer, P; Troncoso, E; Collins, M T

2013-12-27

Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis, or Johne's disease, in animals. Diagnosis of MAP infection is challenging because of the pathogen's fastidious in vitro growth requirements and low-level intermittent shedding in feces during the preclinical phase of the infection. Detection of these "low-shedders" is important for effective control of paratuberculosis as these animals serve as sources of infection for susceptible calves. Magnetic separation technology, used in combination with culture or molecular methods for the isolation and detection of pathogenic bacteria, enhances the analytical sensitivity and specificity of detection methods. The aim of the present study was to evaluate peptide-mediated magnetic separation (PMS) capture technology coupled with IS900 PCR using the Roche real-time PCR system (PMS-PCR), in comparison with fecal culture using BACTEC-MGIT 960 system, for detection of MAP in bovine fecal samples. Among the 351 fecal samples 74.9% (263/351) were PMS-PCR positive while only 12.3% (43/351) were MGIT culture-positive (p=0.0001). All 43 MGIT culture-positive samples were also positive by PMS-PCR. Mean PMS-PCR crossing-point (Cp) values for the 13 fecal samples with the highest number of MAP, based on time to detection, (26.3) were significantly lower than for the 17 fecal samples with technology provided results in a shorter time and yielded a higher number of positive results than MGIT culture. Earlier and faster detection of animals shedding MAP by PMS-PCR should significantly strengthen control efforts for MAP-infected cattle herds by helping to limit infection transmission at earlier stages of the infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Identification of new antigen candidates for the early diagnosis of Mycobacterium avium subsp. paratuberculosis infection in goats.

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Souriau, Armel; Freret, Sandrine; Foret, Benjamin; Willemsen, Peter T J; Bakker, Douwe; Guilloteau, Laurence A

2017-12-01

Currently Mycobacterium avium subsp. paratuberculosis (MAP) infection is diagnosed through indirect tests based on the immune response induced by the infection. The antigens commonly used in IFN-γ release assays (IGRA) are purified protein derivative tuberculins (PPD). However, PPDs, lack both specificity (Sp) and sensitivity (Se) in the early phase of infection. This study investigated the potential of 16 MAP recombinant proteins and five lipids to elicit the release of IFN-γ in goats from herds with or without a history of paratuberculosis. Ten recombinant proteins were selected as potential candidates for the detection of MAP infection in young goats. They were found to detect 25 to 75% of infected shedder (IS) and infected non-shedder (INS) kids younger than 10months of age. In comparison, PPD was shown to detect only 10% of INS and no IS kids. For seven antigens, Se (21-33%) and Sp (≥90%) of IGRA were shown to be comparable with PPD at 20months old. Only three antigens were suitable candidates to detect IS adult goats, although Se was lower than that obtained with PPD. In paratuberculosis-free herds, IGRA results were negative in 97% of indoor goats and 86% of outdoor goats using the 10 antigens. However, 22 to 44% of one-year-old outdoor goats were positive suggesting that they may be infected. In conclusion, this study showed that ten MAP recombinant proteins are potential candidates for early detection of MAP infected goats. Combining these antigens could form a possible set of MAP antigens to optimize the Se of caprine IGRA. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of the efficacy of valproic acid and suberoylanilide hydroxamic acid (vorinostat in enhancing the effects of first-line tuberculosis drugs against intracellular Mycobacterium tuberculosis

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Martin Rao

2018-04-01

Full Text Available Background: New tuberculosis (TB drug treatment regimens are urgently needed. This study evaluated the potential of the histone deacetylase inhibitors (HDIs valproic acid (VPA and suberoylanilide hydroxamic acid (SAHA to enhance the effects of first-line anti-TB drugs against intracellular Mycobacterium tuberculosis. Methods: M. tuberculosis H37Rv cultures were exposed to VPA or SAHA over 6 days, in the presence or absence of isoniazid (INH and rifampicin (RIF. The efficacy of VPA and SAHA against intracellular M. tuberculosis with and without INH or RIF was tested by treating infected macrophages. Bactericidal activity was assessed by counting mycobacterial colony-forming units (CFU. Results: VPA treatment exhibited superior bactericidal activity to SAHA (2-log CFU reduction, while both HDIs moderately improved the activity of RIF against extracellular M. tuberculosis. The bactericidal effect of VPA against intracellular M. tuberculosis was greater than that of SAHA (1-log CFU reduction and equalled that of INH (1.5-log CFU reduction. INH/RIF and VPA/SAHA combination treatment inhibited intracellular M. tuberculosis survival in a shorter time span than monotherapy (3 days vs. 6 days. Conclusions: VPA and SAHA have adjunctive potential to World Health Organization-recommended TB treatment regimens. Clinical evaluation of the two drugs with regard to reducing the treatment duration and improving treatment outcomes in TB is warranted. Keywords: Mycobacterium tuberculosis, Adjunct host-directed therapy, Tuberculosis, Histone deacetylase inhibitors, Repurposed drugs

Effect of feeding heat-treated colostrum on risk for infection with Mycobacterium avium ssp. paratuberculosis, milk production, and longevity in Holstein dairy cows.

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Godden, S M; Wells, S; Donahue, M; Stabel, J; Oakes, J M; Sreevatsan, S; Fetrow, J

2015-08-01

In summer 2007, a randomized controlled field trial was initiated on 6 large Midwest commercial dairy farms to investigate the effect of feeding heat-treated (HT) colostrum on transmission of Mycobacterium avium ssp. paratuberculosis (MAP) and on future milk production and longevity within the herd. On each farm, colostrum was collected daily from fresh cows, pooled, divided into 2 aliquots, and then 1 aliquot was heat-treated in a commercial batch pasteurizer at 60°C for 60min. A sample from each batch of colostrum was collected for PCR testing (MAP-positive vs. MAP-negative). Newborn heifer calves were removed from the dam within 30 to 60min of birth and systematically assigned to be fed 3.8 L of either fresh (FR; n=434) or heat-treated (HT; n=490) colostrum within 2h of birth. After reaching adulthood (>2 yr old), study animals were tested once annually for 3 yr (2010, 2011, 2012) for infection with MAP using serum ELISA and fecal culture. Lactation records describing milk production data and death or culling events were collected during the 3-yr testing period. Multivariable model logistic and linear regression was used to investigate the effect of feeding HT colostrum on risk for testing positive to MAP during the 3-yr testing period (positive/negative; logistic regression) and on first and second lactation milk yield (kg/cow; linear regression), respectively. Cox proportional hazards regression was used to investigate the effect of feeding HT colostrum on risk and time to removal from the herd. Fifteen percent of all study animals were fed PCR-positive colostrum. By the end of the 3-yr testing period, no difference was noted in the proportion of animals testing positive for MAP, with either serum ELISA or fecal culture, when comparing the HT group (10.5%) versus the FR group (8.1%). There was no effect of treatment on first- (HT=11.797kg; FR=11,671kg) or second-lactation (HT=11,013kg; FR=11,235kg) milk production. The proportion of cows leaving the herd by

Long-term detection of Mycobacterium avium subspecies paratuberculosis in individual and bulk tank milk from a dairy herd with a low prevalence of Johne's disease.

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Khol, J L; Wassertheurer, M; Sodoma, E; Revilla-Fernández, S; Damoser, J; Osterreicher, E; Dünser, M; Kleb, U; Baumgartner, W

2013-06-01

Mycobacterium avium ssp. paratuberculosis (MAP) causes Johne's disease (JD) in ruminants and is shed into the milk of infected cows, which contributes to the controversial discussion about a possible link between MAP and Crohn's disease in humans. The aim of the study was to investigate the risk for the entry of MAP in the food chain via milk from dairy farms with subclinical JD. Therefore, the occurrence of MAP in the milk of a dairy herd with a low prevalence of JD was studied in single and bulk tank milk samples over a period of 23 mo and compared with MAP shedding into feces. Milk, fecal, and blood samples were taken from all cows older than 1.5 yr of age at the beginning and the end of the trial and analyzed for MAP or specific antibodies. In addition, 63 cows (33 MAP infected and 30 MAP noninfected) were selected for monthly sampling. Raw and pasteurized bulk tank milk samples were collected on a monthly basis. The milk samples were tested for MAP by real-time quantitative PCR (qPCR), and the fecal samples were tested for bacterial shedding by qPCR or solid culture. Based on the results of the herd investigations, the prevalence of cows shedding MAP was around 5%; no cases of clinical JD were observed during the study period. The results of the ELISA showed high variation, with 2.1 to 5.1% positive milk samples and 14.9 to 18.8% ELISA-positive blood samples. Monthly milk sampling revealed low levels of MAP shedding into the individual milk samples of both MAP-infected and noninfected cows, with only 13 cows shedding the bacterium into milk during the study period. Mycobacterium avium ssp. paratuberculosis was not detected by qPCR in any raw or pasteurized bulk tank milk sample throughout the study. A significant positive association could be found between MAP shedding into milk and feces. From the results of the present study, it can be concluded that MAP is only shed via milk in a small proportion of cows with subclinical JD for a limited period of time and

Increasing the ex vivo antigen-specific IFN-γ production in subpopulations of T cells and NKp46+ cells by anti-CD28, anti-CD49d and recombinant IL-12 costimulation in cattle vaccinated with recombinant proteins from Mycobacterium avium subspecies paratuberculosis

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Thakur, Aneesh; Riber, Ulla; Davis, William C.

2013-01-01

-γ secretion by CD4, CD8, γδ T cells and NK cells. Age matched male jersey calves, experimentally infected with Mycobacterium avium subsp. paratuberculosis (MAP), were vaccinated with a cocktail of recombinant MAP proteins or left unvaccinated. Vaccine induced ex vivo recall responses were measured through Ag......T cells, which encounter specific antigen (Ag), require additional signals to mount a functional immune response. Here, we demonstrate activation of signal 2, by anti-CD28 mAb (aCD28) and other costimulatory molecules (aCD49d, aCD5), and signal 3, by recombinant IL-12, enhance Ag-specific IFN...

Impact of Mycobacterium avium subspecies paratuberculosis on profit efficiency in semi-extensive dairy sheep and goat farms of Apulia, southern Italy.

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Sardaro, Ruggiero; Pieragostini, Elisa; Rubino, Giuseppe; Petazzi, Ferruccio

2017-01-01

A recent study on paratubercolosis in semi-extensive dairy sheep and goat farms in Apulia revealed a flock positivity of 60.5% and a seroprevalence of 3.0% for sheep and 14.5% for goat, with peaks of 50%. In such a context, providing detailed economic information is crucial for the implementation of a suitable control plan. In this paper we investigated the impact of Mycobacterium avium subspecies paratuberculosis (MAP) on profit efficiency of the Apulian dairy sheep and goat farms. Empirical results through a stochastic frontier model showed that the uninfected farms had a mean level of profit efficiency of 84%, which dropped to 64% in the presence of paratubercolosis as it negatively affected the productivity of feeding, veterinary and labour factors. Structural, managerial and production aspects were involved in the greater inefficiency of the infected farms compared to the uninfected ones: lower experience and schooling of farmers, no access to credit, fewer family members (women in particular) participating in the farming activities, high density of animals per hectare, small flocks, high number of goats in mixed flocks, no confinement practices for young and purchased animals and no pasture rotation. Hence, targeted interventions on these factors by decision makers can ensure effectiveness and efficiency to veterinary and economic action plans. Copyright © 2016 Elsevier B.V. All rights reserved.

Evidence of a pro-apoptotic effect of specific antibodies in a bovine macrophage model of infection with Mycobacterium avium subsp. paratuberculosis.

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Jolly, Ana; Lompardía, Silvina; Hajos, Silvia E; Mundo, Silvia L

2016-01-01

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic granulomatous enteritis in ruminants. Understanding the protective immune response following infection is crucial to improve the diagnosis and the development of vaccines against this disease. The goal of this work was to assess whether specific antibodies were able to modulate the macrophage response to MAP infection by evaluating apoptosis and TNF-α secretion in an in vitro model. Sera from healthy (n=2), MAP-infected (n=3) and lipoarabinomannan (LAM)-immunized (n=3) bovines were evaluated. LAM was chosen as immunogen due to its relevant role in mycobacterial pathogenesis. We demonstrated by two different techniques (Acridine Orange/Ethidium Bromide microscopy and Annexin V/7-Amino-Actinomycin D flow cytometry) that the immune sera from both, MAP-infected and LAM-immunized bovines, significantly increased macrophage apoptosis in infected cultures. Comparable levels of apoptosis were detected when MAP was pre-incubated with purified specific antibodies instead of whole serum. Furthermore, this effect was accompanied by a significantly higher secretion of TNF-α. These results strongly suggest that specific antibodies could limit the impact of MAP on the apoptosis of bovine cells. This work would contribute to elucidate the role of the specific antibody response in bovine JD and its prevention. Copyright © 2015 Elsevier B.V. All rights reserved.

Revival and emended description of 'Mycobacterium paraffinicum' Davis, Chase and Raymond 1956 as Mycobacterium paraffinicum sp. nov., nom. rev.

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Toney, Nadege; Adekambi, Toidi; Toney, Sean; Yakrus, Mitchell; Butler, W Ray

2010-10-01

The omission of the name 'Mycobacterium paraffinicum' from the Approved Lists of Bacterial Names was due to phenotypic confusion surrounding a close relationship with Mycobacterium scrofulaceum. Correspondingly, 'M. paraffinicum' strains grew slowly in > 7 days, stained acid-alcohol-fast and produced yellow-pigmented, smooth, waxy colonies in the dark at an optimal temperature of 35°C. However, 'M. paraffinicum' strains demonstrated no activity for urease, nicotinamidase or pyrazinamidase and lacked growth at 42°C, unlike M. scrofulaceum. The mycolic acid pattern, as determined by HPLC, clustered 'M. paraffinicum' with M. scrofulaceum, Mycobacterium avium and Mycobacterium parascrofulaceum. Strains were fully susceptible to linezolid, rifabutin, clarithromycin and amikacin. Examination of the historical reference strain of 'M. paraffinicum', ATCC 12670, and five additional isolates using comparative studies with 16S rRNA, hsp65 and rpoB gene and concatenated sequences showed that they formed a tight taxonomic group that was distinct from similar non-tuberculous mycobacteria. Multilocus enzyme electrophoresis (MEE) analysis confirmed a close association of the five additional isolates with the reference strain of 'M. paraffinicum' with a genetic distance of 0.12 and showed that all six strains were distinct from other closely related species. These genetic results provided unambiguous evidence of the uniqueness of this slowly growing, scotochromogenic species and supported the revival of the name as Mycobacterium paraffinicum (ex Davis, Chase and Raymond 1956) sp. nov., nom. rev. We propose the previously deposited reference strain ATCC 12670(T) =DSM 44181(T) =NCIMB 10420(T), located in collections worldwide, as the type strain.

Linking chronic infection and autoimmune diseases: Mycobacterium avium subspecies paratuberculosis, SLC11A1 polymorphisms and type-1 diabetes mellitus.

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Daniela Paccagnini

2009-09-01

Full Text Available The etiology of type 1 diabetes mellitus (T1DM is still unknown; numerous studies are performed to unravel the environmental factors involved in triggering the disease. SLC11A1 is a membrane transporter that is expressed in late endosomes of antigen presenting cells involved in the immunopathogenic events leading to T1DM. Mycobacterium avium subsp. paratuberculosis (MAP has been reported to be a possible trigger in the development of T1DM.Fifty nine T1DM patients and 79 healthy controls were genotyped for 9 polymorphisms of SLC11A1 gene, and screened for the presence of MAP by PCR. Differences in genotype frequency were evaluated for both T1DM patients and controls. We found a polymorphism in the SLC11A1 gene (274C/T associated to type 1 diabetic patients and not to controls. The presence of MAP DNA was also significantly associated with T1DM patients and not with controls.The 274C/T SCL11A1 polymorphism was found to be associated with T1DM as well as the presence of MAP DNA in blood. Since MAP persists within macrophages and it is also processed by dendritic cells, further studies are necessary to evaluate if mutant forms of SLC11A1 alter the processing or presentation of MAP antigens triggering thereby an autoimmune response in T1DM patients.

Characterization of tuberculous granulomas in different stages of progression and associated tertiary lymphoid tissue in goats experimentally infected with Mycobacterium avium subsp. hominissuis.

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Schinköthe, Jan; Köhler, Heike; Liebler-Tenorio, Elisabeth M

2016-08-01

Oral infection of goats with Mycobacterium avium subsp. hominissuis (MAH) resulted in a large variety of granulomas in organized gut-associated lymphatic tissues and intestinal lymph nodes. To characterize the cellular composition of granulomas, CD4(+), CD8(+), γδ, B lymphocytes and plasma, CD25(+), CD68(+), MHC-II(+), Ki67(+) and endothelial cells were labeled in consecutive frozen sections by immunohistochemistry and acid fast bacilli (AFB) by Kinyoun stain. Granulomas with extensive necrosis, little mineralization and variable numbers of AFB surrounded by many CD4(+) T cells, but only few epitheloid macrophages were observed in severely sick goats at 2-3mpi. They were interpreted as exuberant immune reaction. Organized granulomas with very few AFB were seen in clinically healthy goats at 13mpi. The necrotic cores were surrounded by a zone of granulomatous infiltrate with many epitheloid macrophages and few lymphocytes. This zone was initially wide and highly vascularized and became progressively smaller. It was enclosed by an increasing layer of connective tissue. All organized granulomas were surrounded by compartimentalized tertiary lymphoid tissue. The granulomas in experimental infection of goats with MAH reflect the heterogeneity of lesions seen in mycobacterial infections of humans and ruminants and are therefore valuable for comparative research. Copyright © 2016 Elsevier Ltd. All rights reserved.

Polymorphisms in the gene encoding bovine interleukin-10 receptor alpha are associated with Mycobacterium avium ssp. paratuberculosis infection status

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Kelton David F

2010-04-01

Full Text Available Abstract Background Johne's disease is a chronic inflammatory bowel disease (IBD of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP. Since this pathogen has been implicated in the pathogenesis of human IBDs, the goal of this study was to assess whether single nucleotide polymorphism (SNPs in several well-known candidate genes for human IBD are associated with susceptibility to MAP infection in dairy cattle. Methods The bovine candidate genes, interleukin-10 (IL10, IL10 receptor alpha/beta (IL10RA/B, transforming growth factor beta 1 (TGFB1, TGFB receptor class I/II (TGFBR1/2, and natural resistance-associated macrophage protein 1 (SLC11A1 were sequenced for SNP discovery using pooled DNA samples, and the identified SNPs were genotyped in a case-control association study comprised of 242 MAP negative and 204 MAP positive Holstein dairy cattle. Logistic regression was used to determine the association of SNPs and reconstructed haplotypes with MAP infection status. Results A total of 13 SNPs were identified. Four SNPs in IL10RA (984G > A, 1098C > T, 1269T > C, and 1302A > G were tightly linked, and showed a strong additive and dominance relationship with MAP infection status. Haplotypes AGC and AAT, containing the SNPs IL10RA 633C > A, 984G > A and 1185C > T, were associated with an elevated and reduced likelihood of positive diagnosis by serum ELISA, respectively. Conclusions SNPs in IL10RA are associated with MAP infection status in dairy cattle. The functional significance of these SNPs warrants further investigation.

In vitro bioassessment of the immunomodulatory activity of Saccharomyces cerevisiae components using bovine macrophages and Mycobacterium avium ssp. paratuberculosis.

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Li, Z; Kang, H; You, Q; Ossa, F; Mead, P; Quinton, M; Karrow, N A

2018-04-11

The yeast Saccharomyces cerevisiae and its components are used for the prevention and treatment of enteric disease in different species; therefore, they may also be useful for preventing Johne's disease, a chronic inflammatory bowel disease of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP). The objective of this study was to identify potential immunomodulatory S. cerevisiae components using a bovine macrophage cell line (BOMAC). The BOMAC phagocytic activity, reactive oxygen species production, and immune-related gene (IL6, IL10, IL12p40, IL13, IL23), transforming growth factor β, ARG1, CASP1, and inducible nitric oxide synthase expression were investigated when BOMAC were cocultured with cell wall components from 4 different strains (A, B, C, and D) and 2 forms of dead yeast from strain A. The BOMAC phagocytosis of mCherry-labeled MAP was concentration-dependently attenuated when BOMAC were cocultured with yeast components for 6 h. Each yeast derivative also induced a concentration-dependent increase in BOMAC reactive oxygen species production after a 6-h exposure. In addition, BOMAC mRNA expression of the immune-related genes was investigated after 6 and 24 h of exposure to yeast components. All yeast components were found to regulate the immunomodulatory genes of BOMAC; however, the response varied among components and over time. The in vitro bioassessment studies reported here suggest that dead yeast and its cell wall components may be useful for modulating macrophage function before or during MAP infection. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Description of the Infection Status in a Norwegian Cattle Herd Naturally Infected by Mycobacterium avium subsp. paratuberculosis

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Nyberg O

2005-03-01

Full Text Available The Norwegian surveillance and control programme for paratuberculosis revealed 8 seroreactors in a single dairy cattle herd that had no clinical signs of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis infection. Paratuberculosis had been a clinical problem in goats several years previously in this herd. All 45 cattle were culled and a thorough investigation of the infection status was conducted by the use of interferon-γ (IFN-γ immunoassay, measurement of antibodies, and pathological and bacteriological examination. In the IFN-γ immunoassay, 9 animals gave positive results, and 13 were weakly positive, while 19 animals were negative. In the serological test,10 animals showed positive reactions, and 5 were doubtful, while 30 animals gave negative reactions. There appeared to be a weak trend toward younger animals having raised IFN-γ and older animals having raised serological tests. Histopathological lesions compatible with paratuberculosis were diagnosed in 4 animals aged between 4 and 9 years. Three of these animals had positive serological reaction and one animal gave also positive results in the IFN-γ immunoassay. Infection was confirmed by isolation of M. a. paratuberculosis from 2 of these 4 animals. One single bacterial isolate examined by restriction fragment length polymorphism (RFLP had the same profile, B-C1, as a strain that had been isolated from a goat at the same farm several years previously. Despite many animals being positive in one or both of the immunological tests, indicative of a heavily infected herd, none of the animals showed clinical signs and only one cow was shown to be shedding bacteria. A cross-reaction with other mycobacteria might have caused some of the immunoreactions in these animals. It is also possible that the Norwegian red cattle breed is resistant to clinical infection with M. a. paratuberculosis.

The modification and evaluation of an ELISA test for the surveillance of Mycobacterium avium subsp. paratuberculosis infection in wild ruminants

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Pruvot Mathieu

2013-01-01

Full Text Available Abstract Background Enzyme-linked immunosorbent assay (ELISA is often used to test wildlife samples for Mycobacterium avium subsp. paratuberculosis (MAP infection. However, commercially available kits are only validated for use with domestic ruminant species. A literature review was performed to document the current use of MAP serum ELISA in wild and semi-domestic ruminants. We then modified and evaluated a commercial ELISA kit (IDEXX Mycobacterium paratuberculosis Antibody Test Kit for use with species for which it was not originally developed: elk (Cervus elaphus, bison (Bison bison and caribou (Rangifer tarandus. We tested the affinity of different conjugates for immunoglobulin G (IgG isolated from these species, performed checkerboard tests to determine the optimal dilutions of samples and conjugates, and established cut-off values using two different methods: a Receiver Operational Curve on a panel of known samples for elk, and an alternate method involving a panel of unknown serum samples for the three species. Results We found that the anti-bovine conjugate included in the IDEXX ELISA kit has limited affinity for elk, bison, and caribou IgG. Protein G showed good affinity for IgG of all three species, while anti-deer conjugate also bound elk and caribou IgG. Using Protein G with elk serum, a cut-off sample-to-positive (S/P value of 0.22 was selected, resulting in a sensitivity and specificity of 73% and 90%, respectively, whereas, using an anti-deer conjugate with elk serum, an S/P cut-off value of 0.29 gave a sensitivity of 68%, with 100% specificity. Cut-off values for bison and caribou using the Protein G conjugate were 0.17 and 0.25 respectively. Conclusions Due to incomplete reporting and a lack of test validation, it is difficult to critically appraise results of many sero-surveys that have previously been done for MAP in wildlife. Commercial ELISA kits may have limited or no capacity to detect antibodies from species other than for

Culture-Independent Identification of Mycobacterium avium Subspecies paratuberculosis in Ovine Tissues: Comparison with Bacterial Culture and Histopathological Lesions

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Kamal R. Acharya

2017-12-01

Full Text Available Johne’s disease is a chronic debilitating enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP. Current abattoir surveillance programs detect disease via examination of gross lesions and confirmation by histopathological and/or tissue culture, which is time-consuming and has relatively low sensitivity. This study aimed to investigate whether a high-throughput quantitative PCR (qPCR test is a viable alternative for tissue testing. Intestine and mesenteric lymph nodes were sourced from sheep experimentally infected with MAP and the DNA extracted using a protocol developed for tissues, comprised enzymatic digestion of the tissue homogenate, chemical and mechanical lysis, and magnetic bead-based DNA purification. The extracted DNA was tested by adapting a previously validated qPCR for fecal samples, and the results were compared with culture and histopathology results of the corresponding tissues. The MAP tissue qPCR confirmed infection in the majority of sheep with gross lesions on postmortem (37/38. Likewise, almost all tissue culture (61/64 or histopathology (52/58 positives were detected with good to moderate agreement (Cohen’s kappa statistic and no significant difference to the reference tests (McNemar’s Chi-square test. Higher MAP DNA quantities corresponded to animals with more severe histopathology (odds ratio: 1.82; 95% confidence interval: 1.60, 2.07. Culture-independent strain typing on tissue DNA was successfully performed. This MAP tissue qPCR method had a sensitivity equivalent to the reference tests and is thus a viable replacement for gross- and histopathological examination of tissue samples in abattoirs. In addition, the test could be validated for testing tissue samples intended for human consumption.

Delayed onset Mycobacterium intracellulare keratitis after laser in situ keratomileusis: A case report and literature review.

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Ko, JaeSang; Kim, Se Kyung; Yong, Dong Eun; Kim, Tae-Im; Kim, Eung Kweon

2017-12-01

Infectious keratitis is a relatively uncommon but potentially sight-threatening complication of laser in situ keratomileusis (LASIK). Mycobacterial keratitis is usually regarded as late onset keratitis among post-LASIK keratitis. There has been no documented case of Mycobacterium intracellulare post-LASIK keratitis of a long-latent period. A 36-year-old man was referred to our out-patient clinic, for persistent corneal epithelial defect with intrastromal infiltration. He had undergone uneventful bilateral LASIK procedure 4 years before. He complained decreased vision, accompanied by ocular pain, photophobia, and redness in his left eye for 7 months. Lamellar keratectomy was taken using femtosecond laser. Bacterial culture with sequenced bacterial 16s ribosomal DNA confirmed the organism to be M intracellulare. After 3 months of administration of topical clarithromycin, amikacin, and moxifloxacin, the corneal epithelial defect was resolved and the infiltration was much improved. However, newly developed diffuse haziness with surrounding granular infiltration in the central cornea was noted. Drug toxicity was suspected and topical moxifloxacin was discontinued, resulting in resolution of the diffuse haze with infiltration. The patient was followed up regularly without medication thereafter and recurrence was not found for 7 years. This case presents the first case of M intracellulare keratitis after LASIK. LASIK surgeons should aware that post-LASIK keratitis can develop long after the operation and careful suspicion of infectious disease with meticulous diagnostic test is needed. Copyright © 2017 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.

Cellular and humoral immune responses in sheep vaccinated with candidate antigens MAP2698c and MAP3567 from Mycobacterium avium subspecies paratuberculosis

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Gurung, Ratna B.; Purdie, Auriol C.; Whittington, Richard J.; Begg, Douglas J.

2014-01-01

Control of Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP) in ruminants using commercially available vaccine reduces production losses, mortality, fecal shedding and histopathological lesions but does not provide complete protection from infection and interferes with serological diagnosis of Johne's disease and bovine tuberculosis. At this time no recombinant antigens have been found to provide superior protection compared to whole killed or live-attenuated MAP vaccines. Therefore, there is a need to evaluate more candidate MAP antigens. In this study recombinant MAP antigens MAP2698c and MAP3567 were formulated with four different MONTANIDE™ (ISA 50V2, 61VG, 71VG, and 201VG) adjuvants and evaluated for their ability to produce specific immune responses in vaccinated sheep. The cellular immune response was measured with an interferon-gamma (IFN-γ) release assay and the humoral immune response was measured by antibody detection enzyme linked immunosorbent assay. Recombinant vaccine formulation with the antigen MAP2698c and MONTANIDE™ ISA 201VG adjuvant produced strong whole-MAP as well as MAP2698c-specific IFN-γ responses in a high proportion of the vaccinated sheep. The formulation caused less severe injection site lesions in comparison to other formulations. The findings from this study suggest that the MAP2698c + 201VG should be evaluated in a challenge trial to determine the efficacy of this vaccine candidate. PMID:25077074

IDENTIFICATION OF MYCOBACTERIUM GENAVENSE IN A DIANA MONKEY (CERCOPITHECUS DIANA) BY POLYMERASE CHAIN REACTION AND HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY.

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Kelly, Kathleen M; Wack, Allison N; Bradway, Dan; Simons, Brian W; Bronson, Ellen; Osterhout, Gerard; Parrish, Nicole M; Montali, Richard J

2015-06-01

A 25-yr-old Diana monkey (Cercopithecus diana) with a 1.5-yr history of chronic colitis and diarrhea was found to have disseminated granulomatous disease with intralesional acid fast bacilli. Bacilli were identified as Mycobacterium genavense by polymerase chain reaction, sequencing of the 16S-23S ribosomal RNA intergenic spacer (ITS) gene, and mycolic acid analysis by high-performance liquid chromatography. Mycobacterium genavense is a common cause of mycobacteriosis in free-ranging and captive birds. In addition, recognition of opportunistic infection in human immunodeficiency virus-positive patients is increasing. Disease manifestations of M. genavense are similar to Mycobacterium avium complex (MAC) and include fever, wasting, and diarrhea with disseminated disease. Similar clinical signs and lesions were observed in this monkey. Mycobacterium genavense should be considered as a differential for disseminated mycobacterial disease in nonhuman primates as this agent can mimic MAC and related mycobacteria.

Repeated cycles of chemical and physical disinfection and their influence on Mycobacterium avium subsp. paratuberculosis viability measured by propidium monoazide F57 quantitative real time PCR.

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Kralik, Petr; Babak, Vladimir; Dziedzinska, Radka

2014-09-01

Mycobacterium avium subsp. paratuberculosis (MAP) has a high degree of resistance to chemical and physical procedures frequently used for the elimination of other bacteria. Recently, a method for the determination of viability by exposure of MAP to propidium monoazide (PMA) and subsequent real time quantitative PCR (qPCR) was established and found to be comparable with culture. The aim of this study was to apply the PMA qPCR method to determine the impact of increasing concentration or time and repeated cycles of the application of selected disinfectants on MAP viability. Different MAP isolates responded to the same type of stress in different ways. The laboratory strain CAPM 6381 had the highest tolerance, while the 8819 low-passage field isolate was the most sensitive. Ultraviolet exposure caused only a partial reduction in MAP viability; all MAP isolates were relatively resistant to chlorine. Only the application of peracetic acid led to the total elimination of MAP. Repeated application of the treatments resulted in more significant decreases in MAP viability compared to single increases in the concentration or time of exposure to the disinfectant. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lactase persistence, NOD2 status and Mycobacterium avium subsp. paratuberculosis infection associations to Inflammatory Bowel Disease

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Elguezabal Natalia

2012-06-01

Full Text Available Abstract Background Inflammatory Bowel Disease (IBD, which includes both Crohn’s disease (CD and ulcerative colitis (UC, is caused by a complex interplay involving genetic predisposition, environmental factors and an infectious agent. Mycobacterium avium subsp. paratuberculosis (MAP is a promising pathogen candidate since it produces a chronic intestinal inflammatory disease in ruminants that resembles CD in humans. MAP is a ubiquitous microorganism, although its presence in the food chain, especially in milk from infected animals, is what made us think that there could be an association between lactase persistence (LP and IBD. The LCT mutation has brought adaptation to dairy farming which in turn would have increased exposure of the population to infection by MAP. NOD2 gene mutations are highly associated to CD. Methods In our study, CD and UC patients and controls from the North of Spain were genotyped for the lactase gene (LCT and for three NOD-2 variants, R702W, G908R and Cins1007fs. MAP PCR was carried out in order to assess MAP infection status and these results were correlated with LCT and NOD2 genotypes. Results As for LP, no association was found with IBD, although UC patients were less likely to present the T/T−13910 variant compared to controls, showing a higher C-allele frequency and a tendency to lactase non-persistence (LNP. NOD2 mutations were associated to CD being the per-allele risk higher for the Cins1007fs variant. MAP infection was more extended among the healthy controls (45.2% compared to CD patients (21.38% and UC patients (19.04% and this was attributed to therapy. The Asturian CD cohort presented higher levels of MAP prevalence (38.6% compared to the Basque CD cohort (15.5%, differences also attributed to therapy. No interaction was found between MAP infection and LCT or NOD2 status. Conclusions We conclude that LP is not significantly associated with IBD, but that MAP infection and NOD2 do show not mutually

Volatile emissions from Mycobacterium avium subsp. paratuberculosis mirror bacterial growth and enable distinction of different strains.

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Phillip Trefz

Full Text Available Control of paratuberculosis in livestock is hampered by the low sensitivity of established direct and indirect diagnostic methods. Like other bacteria, Mycobacterium avium subsp. paratuberculosis (MAP emits volatile organic compounds (VOCs. Differences of VOC patterns in breath and feces of infected and not infected animals were described in first pilot experiments but detailed information on potential marker substances is missing. This study was intended to look for characteristic volatile substances in the headspace of cultures of different MAP strains and to find out how the emission of VOCs was affected by density of bacterial growth. One laboratory adapted and four field strains, three of MAP C-type and one MAP S-type were cultivated on Herrold's egg yolk medium in dilutions of 10(-0, 10(-2, 10(-4 and 10(-6. Volatile substances were pre-concentrated from the headspace over the MAP cultures by means of Solid Phase Micro Extraction (SPME, thermally desorbed from the SPME fibers and separated and identified by means of GC-MS. Out of the large number of compounds found in the headspace over MAP cultures, 34 volatile marker substances could be identified as potential biomarkers for growth and metabolic activity. All five MAP strains could clearly be distinguished from blank culture media by means of emission patterns based on these 34 substances. In addition, patterns of volatiles emitted by the reference strain were significantly different from the field strains. Headspace concentrations of 2-ethylfuran, 2-methylfuran, 3-methylfuran, 2-pentylfuran, ethyl acetate, 1-methyl-1-H-pyrrole and dimethyldisulfide varied with density of bacterial growth. Analysis of VOCs emitted from mycobacterial cultures can be used to identify bacterial growth and, in addition, to differentiate between different bacterial strains. VOC emission patterns may be used to approximate bacterial growth density. In a perspective volatile marker substances could be used to

Volatile emissions from Mycobacterium avium subsp. paratuberculosis mirror bacterial growth and enable distinction of different strains.

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Trefz, Phillip; Koehler, Heike; Klepik, Klaus; Moebius, Petra; Reinhold, Petra; Schubert, Jochen K; Miekisch, Wolfram

2013-01-01

Control of paratuberculosis in livestock is hampered by the low sensitivity of established direct and indirect diagnostic methods. Like other bacteria, Mycobacterium avium subsp. paratuberculosis (MAP) emits volatile organic compounds (VOCs). Differences of VOC patterns in breath and feces of infected and not infected animals were described in first pilot experiments but detailed information on potential marker substances is missing. This study was intended to look for characteristic volatile substances in the headspace of cultures of different MAP strains and to find out how the emission of VOCs was affected by density of bacterial growth. One laboratory adapted and four field strains, three of MAP C-type and one MAP S-type were cultivated on Herrold's egg yolk medium in dilutions of 10(-0), 10(-2), 10(-4) and 10(-6). Volatile substances were pre-concentrated from the headspace over the MAP cultures by means of Solid Phase Micro Extraction (SPME), thermally desorbed from the SPME fibers and separated and identified by means of GC-MS. Out of the large number of compounds found in the headspace over MAP cultures, 34 volatile marker substances could be identified as potential biomarkers for growth and metabolic activity. All five MAP strains could clearly be distinguished from blank culture media by means of emission patterns based on these 34 substances. In addition, patterns of volatiles emitted by the reference strain were significantly different from the field strains. Headspace concentrations of 2-ethylfuran, 2-methylfuran, 3-methylfuran, 2-pentylfuran, ethyl acetate, 1-methyl-1-H-pyrrole and dimethyldisulfide varied with density of bacterial growth. Analysis of VOCs emitted from mycobacterial cultures can be used to identify bacterial growth and, in addition, to differentiate between different bacterial strains. VOC emission patterns may be used to approximate bacterial growth density. In a perspective volatile marker substances could be used to diagnose MAP

Sero-diagnosis of Mycobacterium avium complex lung disease using serum immunoglobulin A antibody against glycopeptidolipid antigen in Taiwan.

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Chin-Chung Shu

Full Text Available BACKGROUND: Lung disease (LD due to non-tuberculous mycobacteria is an important clinical concern. Mycobacterium avium complex (MAC is one of the most common causative agents but the diagnosis of MAC-LD remains challenging. Detection of serum IgA antibody against MAC glycopeptidolipid (GPL has recently been shown to improve the diagnosis of MAC-LD, but has yet to be validated worldwide. METHODS: This prospective study was conducted in a tertiary referral center in northern Taiwan and enrolled patients with MAC-LD, MAC contamination, other lung diseases, and control subjects. Serum immunoglobulin A (IgA antibody against MAC-GPL was detected in the participants and its specificity and sensitivity was assessed. RESULTS: There were 56 patients with MAC-LD, 11 with MAC contamination, 13 M. kansasii-LD, 26 LD due to rapidly-growing mycobacteria (RGM, 48 pulmonary tuberculosis, and 42 household contacts of patients with TB. Patients with MAC-LD were older and 32% of them had an underlying co-morbidity. By logistic regression, serum MAC-GPL IgA level was an independent predictor of MAC-LD among the study subjects and those with culture-positive specimens for MAC. By the receiver operating characteristic curve, serum MAC-GPL IgA had a good power to discriminate MAC-LD from MAC contamination. Under the optimal cut-off value of 0.73 U/mL, its sensitivity and specificity were 60% and 91%, respectively. Among MAC-LD patients, presence of co-morbidity was associated with MAC-GPL <0.73 U/ml in logistic regression analysis. CONCLUSIONS: Measurement of serum anti-MAC-GPL IgA level is useful for the diagnosis of MAC-LD. However, its implement in clinical practice for immuno-compromised hosts needs careful consideration.

Specific immunoassays confirm association of Mycobacterium avium Subsp. paratuberculosis with type-1 but not type-2 diabetes mellitus.

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Valentina Rosu

Full Text Available Mycobacterium avium subspecies paratuberculosis (MAP is a versatile pathogen with a broad host range. Its association with type-1 diabetes mellitus (T1DM has been recently proposed. Rapid identification of infectious agents such as MAP in diabetic patients at the level of clinics might be helpful in deciphering the role of chronic bacterial infection in the development of autoimmune diseases such as T1DM.We describe use of an ELISA method to identify live circulating MAP through the detection of a cell envelope protein, MptD by a specific M13 phage--fMptD. We also used another ELISA format to detect immune response to MptD peptide. Both the methods were tested with blood plasma obtained from T1DM, type-2 diabetes (T2DM patients and non-diabetic controls. Our results demonstrate MptD and fMptD ELISA assays to be accurate and sensitive to detect MAP bacilli in a large fraction (47.3% of T1DM patients as compared to non-diabetic controls (12.6% and those with confirmed T2DM (7.7%. Comparative analysis of ELISA assays performed here with 3 other MAP antigen preparations, namely HbHA, Gsd and whole cell MAP lysates confirmed comparable sensitivity of the MptD peptide and the fMptD based ELISA assays. Moreover, we were successful in demonstrating positive bacterial culture in two of the clinical specimen derived from T1DM patients.The MptD peptide/fMptD based ELISA or similar tests could be suggested as rapid and specific field level diagnostic tests for the identification of MAP in diabetic patients and for finding the explanations towards the occurrence of type-1 or type-2 diabetes in the light of an active infectious trigger.

Assessment of the relative sensitivity of milk ELISA for detection of Mycobacterium avium ssp. paratuberculosis infectious dairy cows.

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Laurin, Emilie L; Sanchez, Javier; Chaffer, Marcelo; McKenna, Shawn L B; Keefe, Greg P

2017-01-01

Milk ELISA are commonly used for detection of Mycobacterium avium ssp. paratuberculosis (MAP) antibodies in dairy cows, due to low cost and quick processing for large numbers of samples. However, low sensitivity and variations from host and environmental factors can impede detection of MAP antibodies at early disease stages. The objectives of our study were to assess the sensitivity of milk ELISA in comparison with fecal tests and to evaluate how detectable antibody concentrations in milk vary with changes in fecal shedding of MAP, cow age, cow parity, days in milk, and time of year. To compare the sensitivity of a commercial milk ELISA with solid and broth fecal culture and with fecal real-time PCR, a longitudinal study was performed for the identification of MAP-infectious animals as determined by prior fecal testing for MAP shedding. In addition, associations between variation in milk MAP ELISA score and changes in fecal MAP shedding, host age, days in milk, and season were evaluated. Monthly milk and fecal samples were collected over 1 yr from 46 cows that were previously shedding MAP in their feces. Sensitivity of milk ELISA was 29.9% (95% CI: 24.8 to 35.1%), compared with 46.7% (40.7 to 52.7%) for fecal solid culture, 55.0% (49.3 to 60.7%) for fecal broth culture, and 78.4% (73.3 to 83.1%) for fecal direct real-time PCR. The effect of stage of lactation could not be separated from the effect of season, with increased milk ELISA scores at greater days in milk in winter. However, unpredictable monthly variations in results were observed among the 3 assays for individual cow testing, which highlights the importance of identifying patterns in pathogen and antibody detection over time in MAP-positive herds. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Sero-Diagnosis of Mycobacterium avium Complex Lung Disease Using Serum Immunoglobulin A Antibody against Glycopeptidolipid Antigen in Taiwan

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Wang, Jann-Tay; Jou, Ruwen; Wang, Jann-Yuan; Kobayashi, Kazuo; Lai, Hsin-Chih; Yu, Chong-Jen; Lee, Li-Na; Luh, Kwen-Tay

2013-01-01

Background Lung disease (LD) due to non-tuberculous mycobacteria is an important clinical concern. Mycobacterium avium complex (MAC) is one of the most common causative agents but the diagnosis of MAC-LD remains challenging. Detection of serum IgA antibody against MAC glycopeptidolipid (GPL) has recently been shown to improve the diagnosis of MAC-LD, but has yet to be validated worldwide. Methods This prospective study was conducted in a tertiary referral center in northern Taiwan and enrolled patients with MAC-LD, MAC contamination, other lung diseases, and control subjects. Serum immunoglobulin A (IgA) antibody against MAC-GPL was detected in the participants and its specificity and sensitivity was assessed. Results There were 56 patients with MAC-LD, 11 with MAC contamination, 13 M. kansasii-LD, 26 LD due to rapidly-growing mycobacteria (RGM), 48 pulmonary tuberculosis, and 42 household contacts of patients with TB. Patients with MAC-LD were older and 32% of them had an underlying co-morbidity. By logistic regression, serum MAC-GPL IgA level was an independent predictor of MAC-LD among the study subjects and those with culture-positive specimens for MAC. By the receiver operating characteristic curve, serum MAC-GPL IgA had a good power to discriminate MAC-LD from MAC contamination. Under the optimal cut-off value of 0.73 U/mL, its sensitivity and specificity were 60% and 91%, respectively. Among MAC-LD patients, presence of co-morbidity was associated with MAC-GPL <0.73 U/ml in logistic regression analysis. Conclusions Measurement of serum anti-MAC-GPL IgA level is useful for the diagnosis of MAC-LD. However, its implement in clinical practice for immuno-compromised hosts needs careful consideration. PMID:24260398

Correlation of antigen-specific IFN-γ responses of fresh blood samples from Mycobacterium avium subsp. paratuberculosis infected heifers with responses of day-old samples co-cultured with IL-12 or anti-IL-10 antibodies

DEFF Research Database (Denmark)

Mikkelsen, Heidi; Aagaard, Claus; Nielsen, Søren Saxmose

2012-01-01

Paratuberculosis is a chronic infection of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Early stage MAP infection can be detected by measuring cell-mediated immune responses using the interferon gamma (IFN-γ) assay. Whole blood samples are cultured...... to enhance IFN-γ responses of cultures stimulated with Johnin purified protein derivative (PPDj). Here we examined the correlation of IFN-γ production in response to PPDj and 15 recombinant antigens in day-old blood samples from heifers 10–21 months of age from a MAP infected herd with addition of either...... overnight with specific MAP antigens followed by quantification of IFN-γ by ELISA. It is recommended that the time interval from sampling to culture does not exceed eight hours but addition of the co-stimulating cytokine interleukin 12 (IL-12) or anti-IL-10 antibodies to culture have been demonstrated...

NCBI nr-aa BLAST: CBRC-XTRO-01-3068 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-XTRO-01-3068 ref|NP_960250.1| AppC [Mycobacterium avium subsp. paratuberculosis... K-10] gb|AAS03633.1| AppC [Mycobacterium avium subsp. paratuberculosis K-10] NP_960250.1 2.1 28% ...

Siderocalin inhibits the intracellular replication of Mycobacterium tuberculosis in macrophages

DEFF Research Database (Denmark)

Johnson, Erin E; Srikanth, Chittur V; Sandgren, Andreas

2010-01-01

Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis (M.tb) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show that sideroc......Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis (M.tb) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show...... findings are consistent with an important role for siderocalin in protection against M.tb infection and suggest that exogenously administered siderocalin may have therapeutic applications in tuberculosis....

NCBI nr-aa BLAST: CBRC-CREM-01-1284 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-CREM-01-1284 ref|NP_962383.1| SugI [Mycobacterium avium subsp. paratuberculosis... K-10] gb|AAS05999.1| SugI [Mycobacterium avium subsp. paratuberculosis K-10] NP_962383.1 2e-85 55% ...

Gene expression profiles of immune-regulatory genes in whole blood of cattle with a subclinical infection of Mycobacterium avium subsp. paratuberculosis.

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Hyun-Eui Park

Full Text Available Johne's disease is a chronic wasting disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP, resulting in inflammation of intestines and persistent diarrhea. The initial host response against MAP infections is mainly regulated by the Th1 response, which is characterized by the production of IFN-γ. With the progression of disease, MAP can survive in the host through the evasion of the host's immune response by manipulating the host immune response. However, the host response during subclinical phases has not been fully understood. Immune regulatory genes, including Th17-derived cytokines, interferon regulatory factors, and calcium signaling-associated genes, are hypothesized to play an important role during subclinical phases of Johne's disease. Therefore, the present study was conducted to analyze the expression profiles of immune regulatory genes during MAP infection in whole blood. Different expression patterns of genes were identified depending on the infection stages. Downregulation of IL-17A, IL-17F, IL-22, IL-26, HMGB1, and IRF4 and upregulation of PIP5K1C indicate suppression of the Th1 response due to MAP infection and loss of granuloma integrity. In addition, increased expression of IRF5 and IRF7 suggest activation of IFN-α/β signaling during subclinical stages, which induced indoleamine 2,3-dioxygenase mediated depletion of tryptophan metabolism. Increased expression of CORO1A indicate modulation of calcium signaling, which enhanced the survival of MAP. Taken together, distinct host gene expression induced by MAP infection indicates enhanced survival of MAP during subclinical stages.

Immune response induced by Epstein-Barr virus and Mycobacterium avium subsp. paratuberculosis peptides in current and past infectious mononucleosis: a risk for multiple sclerosis?

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Mameli, G; Madeddu, G; Cossu, D; Galleri, G; Manetti, R; Babudieri, S; Mura, M Stella; Sechi, L A

2016-01-01

Infectious mononucleosis (IM) caused by Epstein-Barr virus (EBV) has been associated with increased risk of multiple sclerosis (MS). However, the mechanism linking these pathologies is unclear. Different reports indicate the association of EBV, and recently Mycobacterium avium subsp. paratuberculosis (MAP), with MS. For a better understanding of the role of these pathogens, the host response induced by selected antigenic peptides in subjects with a history of IM that significantly increases the risk of MS was investigated. Both humoral and cell-mediated response against peptides able to induce a specific immune activation in MS patients deriving from lytic and latent EBV antigens BOLF1(305-320), EBNA1(400-413), from MAP MAP_4027(18-32), MAP_0106c(121-132) and from human proteins IRF5(424-434) and MBP(85-98) in subjects with current and past IM were examined. EBNA1 and MAP_0106c peptides were able to induce a humoral immune response in subjects with a history of clinical IM in an independent manner. Moreover, these peptides were capable of inducing pro-inflammatory cytokine interferon γ by CD4+ and CD8+ T lymphocytes and interleukin 6 and tumour necrosis factor α by CD14+ monocyte cells. Our results highlight that EBV and MAP may be involved independently in the same causal process leading to MS in subjects with a history of IM. © 2015 EAN.

Demographics of cattle positive for Mycobacterium avium subspecies paratuberculosis by faecal culture, from submissions to the Cork Regional Veterinary Laboratory

Science.gov (United States)

2009-01-01

The demography of bovine infections caused by Mycobacterium avium subspecies paratuberculosis (MAP) in Ireland is poorly defined. The objective of this study was to describe the demographics of cattle positive to MAP on faecal culture, based on submissions to the Cork Regional Veterinary Laboratory (Cork RVL) from 1994 to 2006. The study focused on all available faecal samples from adult cattle with non-responsive chronic diarrhoea that were submitted by private veterinary practitioners to Cork RVL for MAP culture. For each MAP-positive by faecal culture animal, data were collated from Cork RVL and Cattle Movement Monitoring Scheme (CMMS) records. Johne's disease (JD) was confirmed in 110 animals from 86 herds by the Cork RVL between 1994 and 2006, with a rate of positive cases between 15% and 18% over last four years of the study. Two breeds (Holstein/Friesian or Limousin) made up 78% of submissions. Movements were assessed for the 57 study animals with available movement information, 90% died within one year of the test and 26% tested positive in the herd they were born into. The study provides preliminary information about movement trends and demographics of animals with MAP positive submissions. Although the study area is restricted, it includes the most intensive (and economically-important) dairy region in Ireland. The demographics of JD infection from the study area are in agreement with international reports. Further work is required to determine demographic trends, incidence and prevalence of JD throughout Ireland. It is hoped this work may contribute to the development of a surveillance strategy for MAP by regional veterinary laboratories. PMID:21851736

Presence of intestinal Mycobacterium avium subspecies paratuberculosis (MAP DNA is not associated with altered MMP expression in ulcerative colitis

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Halwe Jörg M

2011-04-01

Full Text Available Abstract Background Mycobacterium avium subspecies paratuberculosis (MAP is suspected to be a causative agent in human Crohn's disease (CD. Recent evidence suggests that pathogenic mycobacteria and MAP can induce the expression of Matrix Metalloproteinases (MMP, which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD. Within this study we assessed the prevalence of intestinal MAP specific DNA in patients with Crohn's disease, ulcerative colitis (UC, and healthy controls. We further analysed regulation patterns of MMPs in mucosal tissues of UC patients with and without intestinal MAP DNA detection. Methods Colonic biopsy samples were obtained from 63 Norwegian and German IBD patients and 21 healthy controls. RNA was quantified by quantitative real-time polymerase chain reaction (PCR to study MMP gene expression in both pathological and healthy mucosal specimens. The presence of MAP DNA in colonic mucosa was examined using MAP specific PCR. Results MAP DNA was detected in 20% of UC patients and 33% of healthy controls but only in 7% of patients with CD. UC patients treated with corticosteroids exhibited a significantly increased frequency of intestinal MAP DNA compared to those not receiving corticosteroids. Expression of MMP-1, -2, -7, -9, -13, -19, -28 and TNF-α did not differ between UC patients with presence of intestinal MAP DNA compared to those without. MMP-2, MMP-9 and MMP-13 were significantly decreased in UC patients receiving corticosteroids. Conclusions The presence of intestinal MAP specific DNA is not associated with altered MMP expression in UC in vivo. Corticosteroids are associated with increased detection of intestinal MAP DNA and decreased expression of certain MMPs. Frequent detection of MAP DNA in healthy controls might be attributable to the wide environmental distribution of MAP and its presence in the food-chain.

Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species.

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Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun; Koh, Won-Jung; Shin, Sung Jae

2017-09-01

The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis ( M. tuberculosis ) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680 , and (iv) simultaneously detect five clinically important NTM ( M. avium , M. intracellulare , M. abscessus , M. massiliense , and M. kansasii ) by targeting IS 1311 , DT1, mass_3210 , and mkan_rs12360 An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 10 3 and 10 4 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis , M. tuberculosis Beijing genotype, and major NTM species. Copyright © 2017 American Society for Microbiology.

Bayesian estimation of sensitivity and specificity of a commercial serum/milk ELISA against the Mycobacterium avium subsp. Paratuberculosis (MAP) antibody response for each lactation stage in Greek dairy sheep.

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Angelidou, Elisavet; Kostoulas, Polychronis; Leontides, Leonidas

2016-02-01

A total of 854 paired milk and blood samples were collected from ewes of a Greek flock and used to estimate the sensitivity and specificity of a commercial ELISA for detection of Mycobacterium avium subsp. paratuberculosis (MAP) specific antibodies in each stage of lactation. We implemented Bayesian mixture models to derive the distributions of the test response for the healthy and the infected ewes. In the colostrum period, early, mid and late lactation stage the median values of the area under the curves (AUC) were 0.61 (95% credible interval: 0.50; 0.84), 0.61 (0.51;0.84), 0.65 (0.51;0.91), 0.65(0.51;0.89) for the serum ELISA and and 0.60 (0.50; 0.84), 0.61 (0.50; 0.84), 0.67(0.51; 0.91), 0.66(0.50; 0.90) for the milk ELISA, respectively. Both serum and milk ELISA had low to average overall discriminatory ability as measured by the area under the curves and comparable sensitivities and specificities at the recommended cutoffs. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparison of fecal pooling strategies for detection of Mycobacterium avium ssp. paratuberculosis in cattle.

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McKenna, S L B; Ritter, C; Dohoo, I; Keefe, G P; Barkema, H W

2018-05-23

In herds with typical moderate to low within-herd prevalence, testing for Mycobacterium avium ssp. paratuberculosis (MAP), the infectious agent of Johne's disease, will be more cost-effective if individual fecal samples are cultured in composite pools. However, sensitivity to classify a pool containing 1 or more positive individual samples as positive may depend on pool size and number of individual positive samples within a pool. Fecal samples collected from 994 dairy cows sampled at slaughter were cultured to detect MAP. Culturing was done both individually and as composite pooled samples using the TREK ESP Culture System II broth medium (Thermo Fisher Scientific, Trek Diagnostic Systems Inc., Cleveland, OH). Composite samples consisted of pools containing feces from 3, 5, 8, 10, or 15 cows. The number of individual fecal culture-positive cows within each pool ranged from 0 to 4. Culture of individual fecal samples detected MAP in 36 (3.6%) of the 994 cows. Individual samples that were detected within the first 50 d by TREK ESP Culture System II were more likely to lead to a positive pool result. In total, 840 pooled fecal samples were examined for presence of MAP, and of those, 272 pools actually contained feces from fecal culture-positive cows. The crude sensitivity (proportion of pools that contained at least 1 fecal-positive cow that tested positive) for pools of 3, 5, 8, 10, and 15 was 47, 67, 44, 59, and 39%, respectively. Across pools, an increase of the number of fecal culture-positive samples from 1 to 2 enhanced overall crude sensitivity from 44 to 71%. However, sensitivity did not further increase for pools with 3 or 4 fecal culture-positive samples (63 and 60%, respectively). Additionally, a simulation analysis assessing probability of pooled fecal samples being positive in herds of 50 and 100 cows was conducted. The simulation assumed that 1, 2, or 5 cows per herd were MAP fecal culture-positive and that pools of 5 and 10 were used. This low

The Features of Fecal and Ileal Mucosa-Associated Microbiota in Dairy Calves during Early Infection with Mycobacterium avium Subspecies paratuberculosis.

Science.gov (United States)

Derakhshani, Hooman; De Buck, Jeroen; Mortier, Rienske; Barkema, Herman W; Krause, Denis O; Khafipour, Ehsan

2016-01-01

Current diagnostic tests for Johne's disease (JD), a chronic granulomatous inflammation of the gastrointestinal tract of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP), lack the sensitivity to identify infected animals at early (asymptomatic) stages of the disease. The objective was to determine the pattern of MAP-associated dysbiosis of intestinal microbiota as a potential biomarker for early detection of infected cattle. To that end, genomic DNA was extracted from ileal mucosa and fecal samples collected from 28 MAP-positive and five control calves. High-throughput Illumina sequencing of the V4 hypervariable region of the 16S rRNA gene was used for community profiling of ileal mucosa-associated (MAM) or fecal microbiota. The PERMANOVA analysis of unweighted UniFrac distances revealed distinct clustering of ileal MAM (P = 0.049) and fecal microbiota (P = 0.068) in MAP-infected vs. control cattle. Microbiota profile of MAP-infected animals was further investigated by linear discriminant analysis effective size (LEfSe); several bacterial taxa within the phylum Proteobacteria were overrepresented in ileal MAM of control calves. Moreover, based on reconstructed metagenomes (PICRUSt) of ileal MAM, functional pathways associated with MAP infection were inferred. Enrichment of lysine and histidine metabolism pathways, and underrepresentation of glutathione metabolism and leucine and isoleucine degradation pathways in MAP-infected calves suggested potential contributions of ileal MAM in development of intestinal inflammation. Finally, simultaneous overrepresentation of families Planococcaceae and Paraprevotellaceae, as well as underrepresentation of genera Faecalibacterium and Akkermansia in the fecal microbiota of infected cattle, served as potential biomarker for identifying infected cattle during subclinical stages of JD. Collectively, based on compositional and functional shifts in intestinal microbiota of infected cattle, we inferred that

The features of fecal and ileal mucosa-associated microbiota in dairy calves during early infection with Mycobacterium avium subspecies paratuberculosis

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Hooman eDerakhshani

2016-03-01

Full Text Available Current diagnostic tests for Johne's disease (JD, a chronic granulomatous inflammation of the gastrointestinal tract of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP, lack the sensitivity to identify infected animals at early (asymptomatic stages of the disease. The objective was to determine the pattern of MAP-associated dysbiosis of intestinal microbiota as a potential biomarker for early detection of infected cattle. To that end, genomic DNA was extracted from ileal mucosa and fecal samples collected from 28 MAP-positive and five control calves. High-throughput Illumina sequencing of the V4 hypervariable region of the 16S rRNA gene was used for community profiling of ileal mucosa-associated (MAM or fecal microbiota. The PERMANOVA analysis of unweighted UniFrac distances revealed distinct clustering of ileal MAM (P = 0.049 and fecal microbiota (P = 0.068 in MAP-infected versus control cattle. Microbiota profile of MAP infected animals was further investigated by linear discriminant analysis effective size (LEfSe; several bacterial taxa within the phylum Proteobacteria were overrepresented in ileal MAM of control calves. Moreover, based on reconstructed metagenomes (PICRUSt of ileal MAM, functional pathways associated with MAP infection were inferred. Enrichment of lysine and histidine metabolism pathways, and underrepresentation of glutathione metabolism and leucine and isoleucine degradation pathways in MAP-infected calves suggested potential contributions of ileal MAM in development of intestinal inflammation. Finally, simultaneous overrepresentation of families Planococcaceae and Paraprevotellaceae, as well as underrepresentation of genera Faecalibacterium and Akkermansia in the fecal microbiota of infected cattle, served as potential biomarker for identifying infected cattle during subclinical stages of JD. Collectively, based on compositional and functional shifts in intestinal microbiota of infected cattle, we

The Features of Fecal and Ileal Mucosa-Associated Microbiota in Dairy Calves during Early Infection with Mycobacterium avium Subspecies paratuberculosis

Science.gov (United States)

Derakhshani, Hooman; De Buck, Jeroen; Mortier, Rienske; Barkema, Herman W.; Krause, Denis O.; Khafipour, Ehsan

2016-01-01

Current diagnostic tests for Johne's disease (JD), a chronic granulomatous inflammation of the gastrointestinal tract of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP), lack the sensitivity to identify infected animals at early (asymptomatic) stages of the disease. The objective was to determine the pattern of MAP-associated dysbiosis of intestinal microbiota as a potential biomarker for early detection of infected cattle. To that end, genomic DNA was extracted from ileal mucosa and fecal samples collected from 28 MAP-positive and five control calves. High-throughput Illumina sequencing of the V4 hypervariable region of the 16S rRNA gene was used for community profiling of ileal mucosa-associated (MAM) or fecal microbiota. The PERMANOVA analysis of unweighted UniFrac distances revealed distinct clustering of ileal MAM (P = 0.049) and fecal microbiota (P = 0.068) in MAP-infected vs. control cattle. Microbiota profile of MAP-infected animals was further investigated by linear discriminant analysis effective size (LEfSe); several bacterial taxa within the phylum Proteobacteria were overrepresented in ileal MAM of control calves. Moreover, based on reconstructed metagenomes (PICRUSt) of ileal MAM, functional pathways associated with MAP infection were inferred. Enrichment of lysine and histidine metabolism pathways, and underrepresentation of glutathione metabolism and leucine and isoleucine degradation pathways in MAP-infected calves suggested potential contributions of ileal MAM in development of intestinal inflammation. Finally, simultaneous overrepresentation of families Planococcaceae and Paraprevotellaceae, as well as underrepresentation of genera Faecalibacterium and Akkermansia in the fecal microbiota of infected cattle, served as potential biomarker for identifying infected cattle during subclinical stages of JD. Collectively, based on compositional and functional shifts in intestinal microbiota of infected cattle, we inferred that

The Impact of the Antimicrobial Compounds Produced by Lactic Acid Bacteria on the Growth Performance of Mycobacterium avium subsp. paratuberculosis

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Petr Kralik

2018-04-01

Full Text Available Cell-free supernatants (CFSs extracted from various lactic acid bacteria (LAB cultures were applied to Mycobacterium avium subsp. paratuberculosis (MAP cells to determine their effect on MAP viability. In addition, 5% lactic acid (LA; pH 3 and commercially synthetized nisin bacteriocin were also tested. This procedure was chosen in order to mimic the influence of LAB compounds during the production and storage of fermented milk products, which can be contaminated by MAP. Its presence in milk and milk products is of public concern due to the possible ingestion of MAP by consumers and the discussed role of MAP in Crohn’s disease. Propidium monoazide real-time PCR (PMA qPCR was used for viability determination. Although all CFS showed significant effects on MAP viability, two distinct groups of CFS – effective and less effective – could be distinguished. The effective CFSs were extracted from various lactobacilli cultures, their pH values were mostly lower than 4.5, and their application resulted in >2 log10 reductions in MAP viability. The group of less effective CFS were filtered from Lactococcus and enterococci cultures, their pH values were higher than 4.5, and their effect on MAP viability was <2 log10. LA elicited a reduction in MAP viability that was similar to that of the group of less effective CFS. Almost no effect was found when using commercially synthetized nisin at concentrations of 0.1–1000 μg/ml. A combination of the influence of the type of bacteriocin, the length of its action, bacteriocin production strain, and pH are all probably required for a successful reduction in MAP viability. However, certain bacteriocins and their respective LAB strains (Lactobacillus sp. appear to play a greater role in reducing the viability of MAP than pH.

Testing of milk replacers for Mycobacterium avium subsp. paratuberculosis by PCR and bacterial culture as a possible source for Johne's disease (paratuberculosis) in calves.

Science.gov (United States)

Khol, Johannes Lorenz; Braun, Anna Lena; Slana, Iva; Kralik, Petr; Wittek, Thomas

2017-09-01

Johne's disease (paratuberculosis) is caused by Mycobacterium avium subsp. paratuberculosis (MAP) and can lead to severe economic losses in the affected cattle herds. The transmission of the disease occurs mainly orally, by the ingestion of MAP, which is shed in the feces and milk of infected animals. Calves show a high susceptibility for the infection compared to adult animals. The use of milk replacers can, therefore, contribute to the prevention of the transmission of the disease to calves in MAP-positive herds by preventing the ingestion of the bacterium with milk from infected animals. The objective of this study was to test milk replacers for calves for the presence of MAP by bacteriological culture and PCR. Therefore, commercially available milk replacers for calves were purchased from 15 different companies. All of the products were tested for MAP by solid culture and real time quantitative PCR (qPCR) targeting IS900 and F57. During the present study, MAP could not be detected by qPCR or solid culture in commercially available milk replacers for calf rearing. The results of the present study underpins that the use of milk replacers for calf rearing might contribute to the reduction of MAP intake by calves in JD positive herds. Additional studies, including more products with a higher diversity, are needed to further elucidate the presence or absence of MAP in milk replacers for calves. Copyright © 2017 Elsevier B.V. All rights reserved.

Cytomegalovirus implicated in a case of progressive outer retinal necrosis (PORN).

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Sfeir, Maroun

2015-08-01

Progressive outer retinal necrosis, also known as PORN, has been described as a variant of necrotizing herpetic retinopathy, occurring particularly in patients with acquired immune deficiency syndrome (AIDS). Although the etiologic organism has been reported to be Varicella-zoster virus, cytomegalovirus (CMV) can be an etiologic agent. Our case illustrates the occurrence of two opportunistic infections: PORN associated with CMV and Mycobacterium avium intracellulare duodenitis in a patient with uncontrolled HIV infection. Copyright © 2015 Elsevier B.V. All rights reserved.

Prevalencia de anticuerpos IgG específicos dirigidos a Mycobacterium avium en caprinos y ovinos en el sur de la Guajira y norte del Cesar

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María Fernanda Cepeda

2016-06-01

Full Text Available Introducción: Los departamentos de la Guajira y Cesar son considerados los principales productores de ovinos y caprinos del país, según censo del Instituto Colombiano Agropecuario (ICA en el año 2015. Estas especies animales revisten interés desde el punto de vista sanitario, no solo por los productos obtenidos de ellos, sino también por la capacidad que tienen de ser reservorio de diferentes agentes infecciosos. A la fecha en Colombia no se encuentran reportes relacionados con la prevalencia de agentes infecciosos, como es el caso de Mycobacterium avium en estas poblaciones animales, razón por la cual, en 2015 se comienza a desarrollar un trabajo inter-institucional titulado: “Proyecto Piloto de Excelencia Sanitaria en Ganadería para la Guajira y el Cesar”, proyecto en el que participan entidades públicas y privadas, el cual, anida el presente trabajo. Objetivo: Determinar la prevalencia de M. avium en caprinos y ovinos, con el propósito de diseñar e implementar un perfil sanitario en los municipios del sur de la Guajira y el Norte del Cesar. Materiales y métodos: El presente trabajo corresponde a un estudio descriptivo de corte transversal, que tuvo como universo de estudio una población de 15.300 ovinos y caprinos del sur de la Guajira y norte del Cesar definida por Censo realizado en 2015 por el ICA. Tomado como base esta población, se calculó un tamaño muestral de 998 individuos mediante la herramienta WinEpi versión 2.0 (Zaragoza, España. La determinación de la prevalencia de M. avium se realizará mediante la determinación de la presencia de IgG sérica específica para este agente infeccioso mediante pruebas comerciales de ELISA indirecta, según las recomendaciones del fabricante. Las pruebas serán realizadas a sueros provenientes de muestras sanguíneas de animales incluidos en la muestra calculada, tomadas por veterinarios entrenados en municipios del sur de la Guajira y norte del Cesar, los cuales, fueron

Siderocalin inhibits the intracellular replication of Mycobacterium tuberculosis in macrophages

DEFF Research Database (Denmark)

Johnson, Erin E; Srikanth, Chittur V; Sandgren, Andreas

2010-01-01

Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis (M.tb) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show that sideroc...

High throughput phenotypic selection of Mycobacterium tuberculosis mutants with impaired resistance to reactive oxygen species identifies genes important for intracellular growth.

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Olga Mestre

Full Text Available Mycobacterium tuberculosis has the remarkable capacity to survive within the hostile environment of the macrophage, and to resist potent antibacterial molecules such as reactive oxygen species (ROS. Thus, understanding mycobacterial resistance mechanisms against ROS may contribute to the development of new anti-tuberculosis therapies. Here we identified genes involved in such mechanisms by screening a high-density transposon mutant library, and we show that several of them are involved in the intracellular lifestyle of the pathogen. Many of these genes were found to play a part in cell envelope functions, further strengthening the important role of the mycobacterial cell envelope in protection against aggressions such as the ones caused by ROS inside host cells.

Bacteriological and virulence study of a Mycobacterium chimaera isolate from a patient in China.

Science.gov (United States)

Liu, Guan; Chen, Su-Ting; Yu, Xia; Li, Yu-Xun; Ling, Ying; Dong, Ling-Ling; Zheng, Su-Hua; Huang, Hai-Rong

2015-04-01

A clinical isolate from a patient was identified as Mycobacterium chimaera, a recently identified species of nontuberculous Mycobacteria. The biochemical and molecular identity, drug sensitivity and virulence of this isolated strain were investigated. 16S rRNA, the 16S-23S ITS, hsp65 and rpoB were amplified, and their sequence similarities with other mycobacteria were analyzed. The minimum inhibitory concentrations of 22 anti-microbial agents against this isolate were established, and the virulence of the isolate was evaluated by intravenous injection into C57BL/6 mice using Mycobacterium tuberculosis H37Rv as a control strain. Growth and morphological characteristics and mycolic acid profile analysis revealed that this isolated strain was a member of the Mycobacterium avium complex. BLAST analysis of the amplified sequences showed that the isolated strain was closely related to M. chimaera. Susceptibility testing showed that the isolate was sensitive to rifabutin, rifapentine, clarithromycin, azithromycin, imipenem and cefoxitin. Bacterial load determination and tissue histopathology of the infected mice indicated that the isolate was highly virulent. The first case of M. chimaera infection in China was evaluated. The information derived from this case may offer valuable guidance for clinical diagnosis and treatment.

Evaluation of a Salmonella vectored vaccine expressing Mycobacterium avium subsp. paratuberculosis antigens against challenge in a goat model.

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Syed M Faisal

Full Text Available Johnes disease (JD, caused by Mycobacterium avium subsp paratuberculosis (MAP, occurs worldwide as chronic granulomatous enteritis of domestic and wild ruminants. To develop a cost effective vaccine, in a previous study we constructed an attenuated Salmonella strain that expressed a fusion product made up of partial fragments of MAP antigens (Ag85A, Ag85B and SOD that imparted protection against challenge in a mouse model. In the current study we evaluated the differential immune response and protective efficacy of the Sal-Ag vaccine against challenge in a goat model as compared to the live attenuated vaccine MAP316F. PBMCs from goats vaccinated with Sal-Ag and challenged with MAP generated significantly lower levels of IFN-γ, following in vitro stimulation with either Antigen-mix or PPD jhonin, than PBMC from MAP316F vaccinated animals. Flow cytometric analysis showed the increase in IFN-γ correlated with a significantly higher level of proliferation of CD4, CD8 and γδT cells and an increased expression of CD25 and CD45R0 in MAP316F vaccinated animals as compared to control animals. Evaluation of a range of cytokines involved in Th1, Th2, Treg, and Th17 immune responses by quantitative PCR showed low levels of expression of Th1 (IFN-γ, IL-2, IL-12 and proinflammatory cytokines (IL-6, IL-8, IL-18, TNF-α in the Sal-Ag immunized group. Significant levels of Th2 and anti-inflammatory cytokines transcripts (IL-4, IL-10, IL-13, TGF-β were expressed but their level was low and with a pattern similar to the control group. Over all, Sal-Ag vaccine imparted partial protection that limited colonization in tissues of some animals upon challenge with wild type MAP but not to the level achieved with MAP316F. In conclusion, the data indicates that Sal-Ag vaccine induced only a low level of protective immunity that failed to limit the colonization of MAP in infected animals. Hence the Sal-Ag vaccine needs further refinement to increase its efficacy.

Crystal Structure of a UDP-glucose-specific Glycosyltransferase from a Mycobacterium Species

Energy Technology Data Exchange (ETDEWEB)

Fulton, Zara; McAlister, Adrian; Wilce, Matthew C.J.; Brammananth, Rajini; Zaker-Tabrizi, Leyla; Perugini, Matthew A.; Bottomley, Stephen P.; Coppel, Ross L.; Crellin, Paul K.; Rossjohn, Jamie; Beddoe, Travis (Monash); (Melbourne)

2008-10-24

Glycosyltransferases (GTs) are a large and ubiquitous family of enzymes that specifically transfer sugar moieties to a range of substrates. Mycobacterium tuberculosis contains a large number of GTs, many of which are implicated in cell wall synthesis, yet the majority of these GTs remain poorly characterized. Here, we report the high resolution crystal structures of an essential GT (MAP2569c) from Mycobacterium avium subsp. paratuberculosis (a close homologue of Rv1208 from M. tuberculosis) in its apo- and ligand-bound forms. The structure adopted the GT-A fold and possessed the characteristic DXD motif that coordinated an Mn{sup 2+} ion. Atypical of most GTs characterized to date, MAP2569c exhibited specificity toward the donor substrate, UDP-glucose. The structure of this ligated complex revealed an induced fit binding mechanism and provided a basis for this unique specificity. Collectively, the structural features suggested that MAP2569c may adopt a 'retaining' enzymatic mechanism, which has implications for the classification of other GTs in this large superfamily.

WC1+ γδ T cells from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis respond differentially to stimulation with PPD-J.

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Albarrak, S M; Waters, W R; Stabel, J R; Hostetter, J M

2017-08-01

A role for γδ T cells in protection against mycobacterial infections including Johne's disease (JD) has been suggested. In neonatal calves where the risk to infection with Mycobacterium avium subsp. paratuberculosis (MAP) is high, the majority of circulating CD3 + lymphocytes are γδ TCR + . Bovine γδ T cells are divided into two major subsets based on the surface expression of workshop cluster 1 (WC1). The WC1 + subset, the predominant subset in periphery, is further divided into WC1.1 + and WC1.2 + subpopulations. The ability of γδ T cells to produce IFN-γ prior to CD4 + αβ T cell activation could be crucial to the outcome of MAP infection. In the current study, cattle were naturally infected with MAP and were classified as either in the subclinical or clinical stage of infection. Compared to the control non-infected group, γδ T cell frequency in circulating lymphocytes was significantly lower in the clinical group. The observed decline in frequency was restricted to the WC1.2 + subset, and was not associated with preferential migration to infection sites (distal-ileum). γδ T cells proliferated significantly in recall responses to stimulation with purified protein derivative from MAP (PPD-J) only in subclinically infected cattle. These responses were a heterogeneous mixture of WC1.1 and WC1.2 subsets. Proliferation and IFN-γ production by the WC1.1 + γδ T cell subset was significantly higher in the subclinical group compared to the control and clinical groups. Our data indicates differences in MAP-specific ex-vivo responses of peripheral WC1 + γδ T cells of cattle with the subclinical or clinical form of JD. Copyright © 2017 Elsevier B.V. All rights reserved.

Pathogenic mechanisms of intracellular bacteria.

Science.gov (United States)

Niller, Hans Helmut; Masa, Roland; Venkei, Annamária; Mészáros, Sándor; Minarovits, Janos

2017-06-01

We wished to overview recent data on a subset of epigenetic changes elicited by intracellular bacteria in human cells. Reprogramming the gene expression pattern of various host cells may facilitate bacterial growth, survival, and spread. DNA-(cytosine C5)-methyltransferases of Mycoplasma hyorhinis targeting cytosine-phosphate-guanine (CpG) dinucleotides and a Mycobacterium tuberculosis methyltransferase targeting non-CpG sites methylated the host cell DNA and altered the pattern of gene expression. Gene silencing by CpG methylation and histone deacetylation, mediated by cellular enzymes, also occurred in M. tuberculosis-infected macrophages. M. tuberculosis elicited cell type-specific epigenetic changes: it caused increased DNA methylation in macrophages, but induced demethylation, deposition of euchromatic histone marks and activation of immune-related genes in dendritic cells. A secreted transposase of Acinetobacter baumannii silenced a cellular gene, whereas Mycobacterium leprae altered the epigenotype, phenotype, and fate of infected Schwann cells. The 'keystone pathogen' oral bacterium Porphyromonas gingivalis induced local DNA methylation and increased the level of histone acetylation in host cells. These epigenetic changes at the biofilm-gingiva interface may contribute to the development of periodontitis. Epigenetic regulators produced by intracellular bacteria alter the epigenotype and gene expression pattern of host cells and play an important role in pathogenesis.

Mycobacterium kansasii Isolated from Tuberculinpositive Rhesus Macaques (Macaca mulatta) in the Absence of Disease.

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Shipley, Steven T; Johnson, David K; Roodgar, Morteza; Smith, David Glenn; Montgomery, Charles A; Lloyd, Steven M; Higgins, James A; Kriel, Edwin H; Klein, Hilton J; Porter, William P; Nazareno, Jerome B; Houghton, Paul W; Panda, Aruna; DeTolla, Louis J

2017-08-01

Mycobacterial infections are of primary health concern in NHP colonies in biomedical research. NHP are constantly monitored and screened for Mycobacterium spp. We report 6 Chinese-origin rhesus macaques infected with Mycobacterium kansasii that exhibited positive tuberculin skin tests in the absence of disease. Two of these macaques were being used for research purposes; the remaining 4 macaques were residing at the contract quarantine company. Histopathology and acid-fast staining of fixed tissues from all macaques showed that all were free of disease. Thoracic radiographs were negative for any signs of disease or infection. Samples from bronchial lavage and tissues including lung, spleen, hilar and mesenteric lymph nodes tested negative by PCR assay for Mycobacterium spp. One of the research macaques tested culture-positive for M. kansasii and a poorly characterized M. avium complex organism. One macaque from the contract quarantine facility tested culture positive for M. kansasii. Genomic testing and target gene RNA expression analysis of the 2 M. kansasii isolates were performed to evaluate possible kinship and affected genes that might contribute to susceptibility to mycobacterial infection. Genotyping of the 2 isolates revealed 2 genetically distinct strains (strains 1 and 4). The presence of positive tuberculin skin tests in the absence of disease raises serious concerns regarding diagnostic methods used for infected NHP.

Dehalogenation of Haloalkanes by Mycobacterium tuberculosis H37Rv and Other Mycobacteria

Science.gov (United States)

Jesenská, Andrea; Sedlác̆ek, Ivo; Damborský, Jir̆í

2000-01-01

Haloalkane dehalogenases convert haloalkanes to their corresponding alcohols by a hydrolytic mechanism. To date, various haloalkane dehalogenases have been isolated from bacteria colonizing environments that are contaminated with halogenated compounds. A search of current databases with the sequences of these known haloalkane dehalogenases revealed the presence of three different genes encoding putative haloalkane dehalogenases in the genome of the human parasite Mycobacterium tuberculosis H37Rv. The ability of M. tuberculosis and several other mycobacterial strains to dehalogenate haloaliphatic compounds was therefore studied. Intact cells of M. tuberculosis H37Rv were found to dehalogenate 1-chlorobutane, 1-chlorodecane, 1-bromobutane, and 1,2-dibromoethane. Nine isolates of mycobacteria from clinical material and four strains from a collection of microorganisms were found to be capable of dehalogenating 1,2-dibromoethane. Crude extracts prepared from two of these strains, Mycobacterium avium MU1 and Mycobacterium smegmatis CCM 4622, showed broad substrate specificity toward a number of halogenated substrates. Dehalogenase activity in the absence of oxygen and the identification of primary alcohols as the products of the reaction suggest a hydrolytic dehalogenation mechanism. The presence of dehalogenases in bacterial isolates from clinical material, including the species colonizing both animal tissues and free environment, indicates a possible role of parasitic microorganisms in the distribution of degradation genes in the environment. PMID:10618227

The formation of lipid droplets favors intracellular Mycobacterium leprae survival in SW-10, non-myelinating Schwann cells.

Science.gov (United States)

Jin, Song-Hyo; An, Sung-Kwan; Lee, Seong-Beom

2017-06-01

Leprosy is a chronic infectious disease that is caused by the obligate intracellular pathogen Mycobacterium leprae (M.leprae), which is the leading cause of all non-traumatic peripheral neuropathies worldwide. Although both myelinating and non-myelinating Schwann cells are infected by M.leprae in patients with lepromatous leprosy, M.leprae preferentially invades the non-myelinating Schwann cells. However, the effect of M.leprae infection on non-myelinating Schwann cells has not been elucidated. Lipid droplets (LDs) are found in M.leprae-infected Schwann cells in the nerve biopsies of lepromatous leprosy patients. M.leprae-induced LD formation favors intracellular M.leprae survival in primary Schwann cells and in a myelinating Schwann cell line referred to as ST88-14. In the current study, we initially characterized SW-10 cells and investigated the effects of LDs on M.leprae-infected SW-10 cells, which are non-myelinating Schwann cells. SW-10 cells express S100, a marker for cells from the neural crest, and NGFR p75, a marker for immature or non-myelinating Schwann cells. SW-10 cells, however, do not express myelin basic protein (MBP), a marker for myelinating Schwann cells, and myelin protein zero (MPZ), a marker for precursor, immature, or myelinating Schwann cells, all of which suggests that SW-10 cells are non-myelinating Schwann cells. In addition, SW-10 cells have phagocytic activity and can be infected with M. leprae. Infection with M. leprae induces the formation of LDs. Furthermore, inhibiting the formation of M. leprae-induced LD enhances the maturation of phagosomes containing live M.leprae and decreases the ATP content in the M. leprae found in SW-10 cells. These facts suggest that LD formation by M. leprae favors intracellular M. leprae survival in SW-10 cells, which leads to the logical conclusion that M.leprae-infected SW-10 cells can be a new model for investigating the interaction of M.leprae with non-myelinating Schwann cells.

The Use Of Rap-PCR In Studying Mycobacterium tuberculosis ...

African Journals Online (AJOL)

Mycobacterium tuberculosis is the second leading cause of death from infectious agent. This study sought to detect M. tuberculosis genes, which were specifically expressed, or upregulated during intracellular infection of. J774 murine macrophages; as such genes may be potential targets for novel drug action. J774 murine ...

Optimisation of decontamination method and influence of culture media on the recovery of Mycobacterium avium subspecies paratuberculosis from spiked water sediments.

Science.gov (United States)

Aboagye, G; Rowe, M T

2018-07-01

The recovery of Mycobacterium avium subspecies paratuberculosis (Map) from the environment can be a laborious process - owing to Map being fastidious, its low number, and also high numbers of other microbial populations in such settings. Protocols i.e. filtration, decontamination and modified elution were devised to recover Map from spiked water sediments. Three culture media: Herrold's Egg Yolk Media (HEYM), Middlebrook 7H10 (M-7H10) and Bactec 12B were then employed to grow the organism following its elution. In the sterile sediment samples the recovery of Map was significant between the time of exposure for each of HEYM and M-7H10, and insignificant between both media (P < 0.05). However, in the non-sterile sediment samples, the HEYM grew other background microflora including moulds at all the times of exposure whilst 4 h followed by M-7H10 culture yielded Map colonies without any background microflora. Using sterile samples only for the Bactec 12B, the recovery of Map decreased as time of exposure increased. Based on these findings, M-7H10 should be considered for the recovery of Map from the natural environment including water sediments where the recovery of diverse microbial species remains a challenge. Map is a robust pathogen that abides in the environment. In water treatment operations, Map associates with floccules and other particulate matter including sediments. It is also a fastidious organism, and its detection and recovery from the water environment is a laborious process and can be misleading within the abundance of other mycobacterial species owing to their close resemblance in phylogenetic traits. In the absence of a reliable recovery method, Map continues to pose public health risks through biofilm in household water tanks, hence the need for the development of a reliable recovery protocol to monitor the presence of Map in water systems in order to curtail its public health risks. Copyright © 2018 Elsevier B.V. All rights reserved.

Host genetics in granuloma formation: human-like lung pathology in mice with reciprocal genetic susceptibility to M. tuberculosis and M. avium.

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Elena Kondratieva

2010-05-01

Full Text Available Development of lung granulomata is a hallmark of infections caused by virulent mycobacteria, reflecting both protective host response that restricts infection spreading and inflammatory pathology. The role of host genetics in granuloma formation is not well defined. Earlier we have shown that mice of the I/St strain are extremely susceptible to Mycobacterium tuberculosis but resistant to M. avium infection, whereas B6 mice show a reversed pattern of susceptibility. Here, by directly comparing: (i characteristics of susceptibility to two infections in vivo; (ii architecture of lung granulomata assessed by immune staining; and (iii expression of genes encoding regulatory factors of neutrophil influx in the lung tissue, we demonstrate that genetic susceptibility of the host largely determines the pattern of lung pathology. Necrotizing granuloma surrounded by hypoxic zones, as well as a massive neutrophil influx, develop in the lungs of M. avium-infected B6 mice and in the lungs of M. tuberculosis-infected I/St mice, but not in the lungs of corresponding genetically resistant counterparts. The mirror-type lung tissue responses to two virulent mycobacteria indicate that the level of genetic susceptibility of the host to a given mycobacterial species largely determines characteristics of pathology, and directly demonstrate the importance of host genetics in pathogenesis.

Correlation of bacterial viability with uptake of (14C) acetate into phenolic glycolipid-1 of Mycobacterium leprae within Schwannoma cells

International Nuclear Information System (INIS)

Mistry, Y.; Antia, N.H.; Mukherjee, R.

1989-01-01

The viability of Mycobacterium leprae, maintained within 33B Schwannoma cells, was estimated in terms of incorporation of ( 14 C) acetate into its specific phenolic glycolipid-1. This measure of viability was correlated with two other assays, viz., fluorescein diacetate/ethidium bromide staining and mouse footpad growth. Observation of a 2-fold increase in the number of intracellular Mycobacterium leprae over an experimental period of 12 days also corroborated this contention. Furthermore, on addition of anti-leprosy drugs to these intracellular Mycobacterium leprae there was significant decrease in phenolic glycolipid-1 synthesis indicative of loss of viability of the organisms. This study also established the importance of the host cell for active bacillary metabolism, as Mycobacterium leprae maintained in cell-free conditions showed no incorporation into phenolic glycolipid-1. Moreover, compromising the host's protein synthesis capacity with cycloheximide, also led to reduction in bacillary metabolism. As this system measures the metabolic synthesis of a unique Mycobacterium leprae component, it would be useful for development and screening of compounds acting against specific bacillary targets. (author). 19 refs., 5 tabs

Dairy farms testing positive for Mycobacterium avium ssp. paratuberculosis have poorer hygiene practices and are less cautious when purchasing cattle than test-negative herds.

Science.gov (United States)

Wolf, R; Barkema, H W; De Buck, J; Orsel, K

2016-06-01

Mycobacterium avium ssp. paratuberculosis (MAP), the causative agent of Johne's disease, is present on most dairy farms in Alberta, causing economic losses and presenting a potential public health concern. The objective of this cross-sectional study was to identify risk factors for Alberta dairy herds being MAP-positive based on environmental samples (ES). Risk assessments were conducted and ES were collected on 354 Alberta dairy farms (62% of eligible producers) voluntarily participating in the Alberta Johne's Disease Initiative. In univariate logistic regression, risk factors addressing animal and pen hygiene, as well as the use of feeding equipment to remove manure and manure application on pastures, were all associated with the number of positive ES. Furthermore, based on factor analysis, risk factors were clustered and could be summarized as 4 independent factors: (1) animal, pen, and feeder contamination; (2) shared equipment and pasture contamination; (3) calf diet; and (4) cattle purchase. Using these factor scores as independent variables in multivariate logistic regression models, a 1-unit increase in animal, pen, and feeder contamination resulted in 1.31 times higher odds of having at least 1 positive ES. Furthermore, a 1-unit increase in cattle purchase also resulted in 1.31 times the odds of having at least 1 positive ES. Finally, a 100-cow increase in herd size resulted in an odds ratio of 2.1 for having at least 1 positive ES. In conclusion, cleanliness of animals, pens, and feeders, as well as cattle purchase practices, affected risk of herd infection with MAP. Therefore, improvements in those management practices should be the focus of effective tools to control MAP on dairy farms. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The effect of Cerasus avium stalk extract on albumin glycation reaction

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Mohadeseh Abdoli

2014-10-01

Full Text Available Background: Non-enzymatic glycosylation of proteins is the major cause of diabetic complications. The inhibition of glycation process can reduce complications of diabetes. In the Iranian traditional medicine, the decoction (boiled extraction of Cerasus avium stalk is used as a hypoglycemic agent. The aim of this study was to investigate the in vitro inhibitory effects of decoction and ethanolic and aqueous extracts of Cerasus avium stalk on albumin glycation reaction. Methods: In this experimental study, first, the ethanolic, aqueous and decoction extracts of Cerasus avium stalk were prepared. Then, different concentrations of these extracts were prepared and added to albumin and glucose solutions. Finally, compared to control group that was not treated with any extracts, the albumin glycation rate in the groups treated with various concentrations of extracts was evaluated using TBA (thio-barbituric acid method. Results: The results showed that compared to control group, decoction of Cerasus avium stalk in the concentrations of 20, 10 and 2 mg/dl could reduce albumin glycation to 85.10±1.55, 72.35±1.75 and 51.25±1.22 %, respectively (P>0.001. Moreover, in the concentration of 20 mg/dl, the inhibitory effect of decoction of Cerasus avium stalk on the albumin glycation reaction was higher than those of aqueous (P=0.021 and ethanolic (P=0.009 extracts. Conclusion: The findings showed that the extracs of Cerasus avium stalk, in particular in the decoction form, could significantly reduce the rate of albumin glycation; therefore, it can be used for decreasing diabetes mellitus complications.

The Identification of Circulating MiRNA in Bovine Serum and Their Potential as Novel Biomarkers of Early Mycobacterium avium subsp paratuberculosis Infection.

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Damien Farrell

Full Text Available Mycobacterium avium subspecies paratuberculosis (MAP is the aetiological agent of Johne's disease (JD, a chronic enteritis in ruminants that causes substantial economic loses to agriculture worldwide. Current diagnostic assays are hampered by low sensitivity and specificity that seriously complicate disease control; a new generation of diagnostic and prognostic assays are therefore urgently needed. Circulating microRNAs (miRNAs have been shown to have significant potential as novel biomarkers for a range of human diseases, but their potential application in the veterinary sphere has been less well characterised. The aim of this study was therefore to apply RNA-sequencing approaches to serum from an experimental JD infection model as a route to identify novel diagnostic and prognostic miRNA biomarkers. Sera from experimental MAP-challenged calves (n = 6 and age-matched controls (n = 6 were used. We identified a subset of known miRNAs from bovine serum across all samples, with approximately 90 being at potentially functional abundance levels. The majority of known bovine miRNAs displayed multiple isomiRs that differed from the canonical sequences. Thirty novel miRNAs were identified after filtering and were found within sera from all animals tested. No significant differential miRNA expression was detected when comparing sera from MAP-challenged animals to their age-matched controls at six-month's post-infection. However, comparing sera from pre-infection bleeds to six-month's post-infection across all 12 animals did identify increased miR-205 (2-fold and decreased miR-432 (2-fold within both challenged and control groups, which suggests changes in circulating miRNA profiles due to ageing or development (P<0.00001. In conclusion our study has identified a range of novel miRNA in bovine serum, and shown the utility of small RNA sequencing approaches to explore the potential of miRNA as novel biomarkers for infectious disease in cattle.

Multiplexed Quantitation of Intraphagocyte Mycobacterium tuberculosis Secreted Protein Effectors

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Fadel Sayes

2018-04-01

Full Text Available Summary: The pathogenic potential of Mycobacterium tuberculosis largely depends on ESX secretion systems exporting members of the multigenic Esx, Esp, and PE/PPE protein families. To study the secretion and regulation patterns of these proteins while circumventing immune cross-reactions due to their extensive sequence homologies, we developed an approach that relies on the recognition of their MHC class II epitopes by highly discriminative T cell receptors (TCRs of a panel of T cell hybridomas. The latter were engineered so that each expresses a unique fluorescent reporter linked to specific antigen recognition. The resulting polychromatic and multiplexed imaging assay enabled us to measure the secretion of mycobacterial effectors inside infected host cells. We applied this novel technology to a large panel of mutants, clinical isolates, and host-cell types to explore the host-mycobacteria interplay and its impact on the intracellular bacterial secretome, which also revealed the unexpected capacity of phagocytes from lung granuloma to present mycobacterial antigens via MHC class II. : Sayes et al. develop an approach to express distinct fluorescent reporters that is based on the recognition of specific Mycobacterium tuberculosis MHC class II epitopes by highly discriminative T cell hybridomas. This multiplexed technology allows the study of secretion, subcellular location, and regulation patterns of these instrumental protein members. Keywords: mycobacterium tuberculosis, type VII secretion systems, intracellular bacteria, T-cell hybridomas, mycobacterial virulence factors, bacterial antigen presentation, lentiviral vectors, reporter T cells, in vivo antigen presentation, protein localization

[Pulmonary Mycobacterium Avium-Complex (MAC) Disease Differentially Diagnosed from Metastasis of Testicular Cancer : A Case Report].

Science.gov (United States)

Mori, Kohei; Teranishi, Jyn-Ichi; Yoneyama, Shuko; Ishida, Hiroaki; Hattori, Yusuke; Yumura, Yasushi; Miyoshi, Yasuhide; Kondo, Keiichi; Uemura, Hiroji; Noguchi, Kazumi

2017-01-01

A 45 year-old-man was admitted to our hospital because of discomfort in his left scrotum. He had a left testicular tumor. We performed high orchiectomy and pathological findings revealed testicular cancer. He was treated with bleomycin, etoposide and cisplatin. Computed tomography showed a new mass in the left lung after 3 cycles of the chemotherapy. Because of its rapid growth, the tumor was thought to be a metastasis lesion of testicular cancer or pulmonary infection. Transbronchial lung biopsy showed an invasion of multinucleated giant cells and granuloma. The culture and polymerase chain reaction of the bronchial sputum were positive for myobacterium avium-complex (MAC). From these findings, the left lung tumor was diagnosed as pulmonary MAC disease. He received partial resection of the left lung and the lesion was diagnosed as granuloma. There was no recurrence of testicular cancer or pulmonary disease after the surgery.

Mycobacterium grossiae sp. nov., a rapidly growing, scotochromogenic species isolated from human clinical respiratory and blood culture specimens.

Science.gov (United States)

Paniz-Mondolfi, Alberto Enrique; Greninger, Alexander L; Ladutko, Lynn; Brown-Elliott, Barbara A; Vasireddy, Ravikiran; Jakubiec, Wesley; Vasireddy, Sruthi; Wallace, Richard J; Simmon, Keith E; Dunn, Bruce E; Jackoway, Gary; Vora, Surabhi B; Quinn, Kevin K; Qin, Xuan; Campbell, Sheldon

2017-11-01

A previously undescribed, rapidly growing, scotochromogenic species of the genus Mycobacterium (represented by strains PB739 T and GK) was isolated from two clinical sources - the sputum of a 76-year-old patient with severe chronic obstructive pulmonary disease, history of tuberculosis exposure and Mycobacterium avium complex isolated years prior; and the blood of a 15-year-old male with B-cell acute lymphoblastic leukaemia status post bone marrow transplant. The isolates grew as dark orange colonies at 25-37 °C after 5 days, sharing features in common with other closely related species. Analysis of the complete 16S rRNA gene sequence (1492 bp) of strain PB739 T demonstrated that the isolate shared 98.8 % relatedness with Mycobacterium wolinskyi. Partial 429 bp hsp65 and 744 bp rpoB region V sequence analyses revealed that the sequences of the novel isolate shared 94.8 and 92.1 % similarity with those of Mycobacterium neoaurum and Mycobacterium aurum, respectively. Biochemical profiling, antimicrobial susceptibility testing, HPLC/gas-liquid chromatography analyses and multilocus sequence typing support the taxonomic status of these isolates (PB739 T and GK) as representatives of a novel species. Both isolates were susceptible to the Clinical and Laboratory Standards Institute recommended antimicrobials for susceptibility testing of rapidly growing mycobacteria including amikacin, ciprofloxacin, moxifloxacin, doxycycline/minocycline, imipenem, linezolid, clarithromycin and trimethropin/sulfamethoxazole. Both isolates PB739 T and GK showed intermediate susceptibility to cefoxitin. We propose the name Mycobacterium grossiae sp. nov. for this novel species and have deposited the type strain in the DSMZ and CIP culture collections. The type strain is PB739 T (=DSM 104744 T =CIP 111318 T ).

Two-Component Systems of Mycobacterium tuberculosis—Structure-Based Approaches

DEFF Research Database (Denmark)

Tucker, Paul; Nowak, Elzbieta; Morth, Jens Preben

2007-01-01

Mycobacterium tuberculosis contains few two‐component systems compared to many other bacteria, possibly because it has more serine/threonine signaling pathways. Even so, these two‐component systems appear to play an important role in early intracellular survival of the pathogen as well as in aspe...

Localization of CORO1A in the Macrophages Containing Mycobacterium leprae

International Nuclear Information System (INIS)

Suzuki, Koichi; Takeshita, Fumihiko; Nakata, Noboru; Ishii, Norihisa; Makino, Masahiko

2006-01-01

Mycobacteria have acquired an intracellular lifestyle within the macrophage, which is best exemplified by the enlarged infected histiocytes seen in lepromatous leprosy. To survive within the cell, mycobacteria must escape intracellular bactericidal mechanisms. In a study of Mycobacterium bovis Bacille Calmette-Guérin (M. bovis BCG) infection, it was shown that the host protein, CORO1A, also known as tryptophan aspartate-containing coat protein (TACO), accumulates on the phagosomal membrane, resulting in inhibition of phagosome-lysosome fusion, and thus augmenting intracellular survival. In this study, we show that CORO1A strongly localizes on the membrane of phagosomes that contain Mycobacterium leprae (M. leprae), where Toll-like receptor 2 was also visualized by immunostaining. When cultured macrophages were infected with M. leprae, CORO1A recruitment from the plasma membrane to the phagosomal membrane was observed. Moderate to strong CORO1A retention was observed in late lesions that contained foamy histiocytes, in which M. leprae were difficult to detect by acid-fast staining. These results suggest that components accumulating within the phagosome rather than viable bacilli are responsible for the retention of CORO1A, and that there is also a bactericidal mechanism in the macrophage that might counter the effects of CORO1A

Designing a risk-based surveillance program for Mycobacterium avium ssp. paratuberculosis in Norwegian dairy herds using multivariate statistical process control analysis.

Science.gov (United States)

Whist, A C; Liland, K H; Jonsson, M E; Sæbø, S; Sviland, S; Østerås, O; Norström, M; Hopp, P

2014-11-01

Surveillance programs for animal diseases are critical to early disease detection and risk estimation and to documenting a population's disease status at a given time. The aim of this study was to describe a risk-based surveillance program for detecting Mycobacterium avium ssp. paratuberculosis (MAP) infection in Norwegian dairy cattle. The included risk factors for detecting MAP were purchase of cattle, combined cattle and goat farming, and location of the cattle farm in counties containing goats with MAP. The risk indicators included production data [culling of animals >3 yr of age, carcass conformation of animals >3 yr of age, milk production decrease in older lactating cows (lactations 3, 4, and 5)], and clinical data (diarrhea, enteritis, or both, in animals >3 yr of age). Except for combined cattle and goat farming and cattle farm location, all data were collected at the cow level and summarized at the herd level. Predefined risk factors and risk indicators were extracted from different national databases and combined in a multivariate statistical process control to obtain a risk assessment for each herd. The ordinary Hotelling's T(2) statistic was applied as a multivariate, standardized measure of difference between the current observed state and the average state of the risk factors for a given herd. To make the analysis more robust and adapt it to the slowly developing nature of MAP, monthly risk calculations were based on data accumulated during a 24-mo period. Monitoring of these variables was performed to identify outliers that may indicate deviance in one or more of the underlying processes. The highest-ranked herds were scattered all over Norway and clustered in high-density dairy cattle farm areas. The resulting rankings of herds are being used in the national surveillance program for MAP in 2014 to increase the sensitivity of the ongoing surveillance program in which 5 fecal samples for bacteriological examination are collected from 25 dairy herds

Prevalencia de micobacteriosis y de tuberculosis en pacientes de un hospital de referencia de la provincia de Córdoba Prevalence of mycobacteriosis and tuberculosis in a reference hospital, Cordoba province

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A.I. Barnes

2004-12-01

Full Text Available Las micobacterias ambientales (MA constituyen un importante grupo de especies bacterianas que se encuentran en el medio ambiente, pueden colonizar y ocasionalmente producir enfermedad enel hombre. En este trabajo se investigó la frecuencia de casos de micobacteriosis en relación con los de tuberculosis durante un período de diez años (1.991-2.000. Se estudiaron 16.700 muestras de 9.300 pacientes adultos de ambos sexos asistidos en el Hospital Regional de Tuberculosis de la Provincia de Córdoba, por consulta espontánea. Los aislamientos se realizaron por cultivo en los medios de Lowenstein Jensen y Stonebrink. Las colonias de bacilos ácidoalcohol resistentes (BAAR se identificaron por pruebas bioquímicas y moleculares. El total de casos diagnosticados fue de 716, de los cuales 684 (95,5% correspondieron a al complejo Mycobacterium tuberculosis y a micobacterias ambientales 32 (4,5%. Los casos de micobacteriosis se definieron por reiterados aislamientos con desarrollo representativo de una micobacteria ambiental, sospecha clínica y radiológica. De los 32 casos de micobacteriosis, el 75% del total correspondió aMycobacterium avium-intracellulare,15,6% a Mycobacterium fortuitum, 3,1% a Mycobacterium kansasii y 6,3% a Mycobacterium chelonae.Los casos de tuberculosis fueron 94,5% de localización pulmonar y 5,5% extrapulmonar.Environmental mycobacteria (EM constitute an important group of bacteria species found in the environment. They can colonize and occasionally produce disease in man. Sixteen thousand three hundred samples from 9300 adult symptomatic patients from the Hospital Regional of Tuberculosis in Cordoba were bacteriolocally investigated. The isolations were performed by culture on Lowenstein Jensen and Stonebrink culture media. The colonies of acid fast bacilli (AFB were identified by biochemical and molecular tests. Among 716 culture positive cases, 684 (95.5% were due to Mycobacterium tuberculosis complex and 32 to

Intracellular bacillary burden reflects a burst size for Mycobacterium tuberculosis in vivo.

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Teresa Repasy

2013-02-01

Full Text Available We previously reported that Mycobacterium tuberculosis triggers macrophage necrosis in vitro at a threshold intracellular load of ~25 bacilli. This suggests a model for tuberculosis where bacilli invading lung macrophages at low multiplicity of infection proliferate to burst size and spread to naïve phagocytes for repeated cycles of replication and cytolysis. The current study evaluated that model in vivo, an environment significantly more complex than in vitro culture. In the lungs of mice infected with M. tuberculosis by aerosol we observed three distinct mononuclear leukocyte populations (CD11b(- CD11c(+/hi, CD11b(+/lo CD11c(lo/-, CD11b(+/hi CD11c(+/hi and neutrophils hosting bacilli. Four weeks after aerosol challenge, CD11b(+/hi CD11c(+/hi mononuclear cells and neutrophils were the predominant hosts for M. tuberculosis while CD11b(+/lo CD11c(lo/- cells assumed that role by ten weeks. Alveolar macrophages (CD11b(- CD11c(+/hi were a minority infected cell type at both time points. The burst size model predicts that individual lung phagocytes would harbor a range of bacillary loads with most containing few bacilli, a smaller proportion containing many bacilli, and few or none exceeding a burst size load. Bacterial load per cell was enumerated in lung monocytic cells and neutrophils at time points after aerosol challenge of wild type and interferon-γ null mice. The resulting data fulfilled those predictions, suggesting a median in vivo burst size in the range of 20 to 40 bacilli for monocytic cells. Most heavily burdened monocytic cells were nonviable, with morphological features similar to those observed after high multiplicity challenge in vitro: nuclear condensation without fragmentation and disintegration of cell membranes without apoptotic vesicle formation. Neutrophils had a narrow range and lower peak bacillary burden than monocytic cells and some exhibited cell death with release of extracellular neutrophil traps. Our studies suggest

Role of genotype® mycobacterium common mycobacteria/additional species assay for rapid differentiation between Mycobacterium tuberculosis complex and different species of non-tuberculous mycobacteria

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Amresh Kumar Singh

2013-01-01

Full Text Available Background: Mycobacterium tuberculosis complex (MTBC and non-tuberculous mycobacteria (NTM may or may not have same clinical presentations, but the treatment regimens are always different. Laboratory differentiation between MTBC and NTM by routine methods are time consuming and cumbersome to perform. We have evaluated the role of GenoType® Mycobacterium common mycobacteria/additional species (CM/AS assay for differentiation between MTBC and different species of NTM in clinical isolates from tuberculosis (TB cases. Materials and Methods: A total of 1080 clinical specimens were collected from January 2010 to June 2012. Diagnosis was performed by Ziehl-Neelsen staining followed by culture in BacT/ALERT 3D system (bioMerieux, France. A total of 219 culture positive clinical isolates (BacT/ALERT® MP cultures were selected for differentiation by p-nitrobenzoic acid (PNB sensitivity test as and BIO-LINE SD Ag MPT64 TB test considering as the gold standard test. Final identification and differentiation between MTBC and different species of NTM were further confirmed by GenoType® Mycobacterium CM/AS assay (Hain Lifescience, Nehren, Germany. Results: Out of 219 BacT/ALERT® MP culture positive isolates tested by PNB as 153 MTBC (69.9% and by GenoType® Mycobacterium CM/AS assay as 159 (72.6% MTBC and remaining 60 (27.4% were considered as NTM species. The GenoType® Mycobacterium CM/AS assay was proved 99.3% sensitive and 98.3% specific for rapid differentiation of MTBC and NTM. The most common NTM species were; Mycobacterium fortuitum 20 (33.3% among rapid growing mycobacteria and Mycobacterium intracellulare 11 (18.3% among slow growing mycobacteria. Conclusion: The GenoType® Mycobacterium assay makes rapid and accurate identification of NTM species as compared with different phenotypic and molecular diagnostic tool and helps in management of infections caused by different mycobacteria.

Immunopathological changes and apparent recovery from infection revealed in cattle in an experimental model of Johne's disease using a lyophilised culture of Mycobacterium avium subspecies paratuberculosis.

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Begg, Douglas J; Plain, Karren M; de Silva, Kumudika; Gurung, Ratna; Gunn, Alison; Purdie, Auriol C; Whittington, Richard J

2018-06-01

Johne's disease (JD) or paratuberculosis is an economically significant, chronic enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Experimental models of JD in cattle are logistically challenging due to the need for long term monitoring, because the clinical disease can take years to manifest. Three trials were undertaken, the largest involving 20 cattle exposed orally to a low dose of C strain MAP and 10 controls studied for 4.75 years. Frequent blood and faecal sampling was used to monitor immunological and infection parameters, and intestinal biopsies were performed at two time points during the subclinical disease phase. Although clinical disease was not seen, there was evidence of infection in 35% of the animals and at necropsy 10% had histopathological lesions consistent with JD, similar to the proportions expected in naturally infected herds. Faecal shedding occurred in two distinct phases: firstly there was intermittent shedding <∼9 months post-exposure that did not correlate with disease outcomes; secondly, in a smaller cohort of animals, this was followed by more consistent shedding of increasing quantities of MAP, associated with intestinal pathology. There was evidence of regression of histopathological lesions in the ileum of one animal, which therefore had apparently recovered from the disease. Both cattle with histopathological lesions of paratuberculosis at necropsy had low MAP-specific interferon-gamma responses at 4 months post-exposure and later had consistently shed viable MAP; they also had the highest loads of MAP DNA in faeces 4.75 year s post-exposure. In a trial using a higher dose of MAP, a higher proportion of cattle developed paratuberculosis. The information derived from these trials provides greater understanding of the changes that occur during the course of paratuberculosis in cattle. Copyright © 2018 Elsevier B.V. All rights reserved.

Direct molecular mass determination of trehalose monomycolate from 11 species of mycobacteria by MALDI-TOF mass spectrometry.

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Fujita, Yukiko; Naka, Takashi; Doi, Takeshi; Yano, Ikuya

2005-05-01

Direct estimation of the molecular mass of single molecular species of trehalose 6-monomycolate (TMM), a ubiquitous cell-wall component of mycobacteria, was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. When less than 1 microg TMM was analysed by MALDI-TOF mass spectrometry, quasimolecular ions [M+Na]+ of each molecular species were demonstrated and the numbers of carbons and double bonds (or cyclopropane rings) were determined. Since the introduction of oxygen atoms such as carbonyl, methoxy and ester groups yielded the appropriate shift of mass ions, the major subclasses of mycolic acid (alpha, methoxy, keto and wax ester) were identified without resorting to hydrolytic procedures. The results showed a marked difference in the molecular species composition of TMM among mycobacterial species. Unexpectedly, differing from other mycoloyl glycolipids, TMM from Mycobacterium tuberculosis showed a distinctive mass pattern, with abundant odd-carbon-numbered monocyclopropanoic (or monoenoic) alpha-mycolates besides dicyclopropanoic mycolate, ranging from C75 to C85, odd- and even-carbon-numbered methoxymycolates ranging from C83 to C94 and even- and odd-carbon-numbered ketomycolates ranging from C83 to C90. In contrast, TMM from Mycobacterium bovis (wild strain and BCG substrains) possessed even-carbon-numbered dicyclopropanoic alpha-mycolates. BCG Connaught strain lacked methoxymycolates almost completely. These results were confirmed by MALDI-TOF mass analysis of mycolic acid methyl esters liberated by alkaline hydrolysis and methylation of the original TMM. Wax ester-mycoloyl TMM molecular species were demonstrated for the first time as an intact form in the Mycobacterium avium-intracellulare group, M. phlei and M. flavescens. The M. avium-intracellulare group possessed predominantly C85 and C87 wax ester-mycoloyl TMM, while M. phlei and the rapid growers tested contained C80, C81, C82 and C83 wax ester

Dual Targeting of Intracellular Pathogenic Bacteria with a Cleavable Conjugate of Kanamycin and an Antibacterial Cell-Penetrating Peptide.

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Brezden, Anna; Mohamed, Mohamed F; Nepal, Manish; Harwood, John S; Kuriakose, Jerrin; Seleem, Mohamed N; Chmielewski, Jean

2016-08-31

Bacterial infection caused by intracellular pathogens, such as Mycobacterium, Salmonella, and Brucella, is a burgeoning global health epidemic that necessitates urgent action. However, the therapeutic value of a number of antibiotics, including aminoglycosides, against intracellular pathogenic bacteria is compromised due to their inability to traverse eukaryotic membranes. For this significant problem to be addressed, a cleavable conjugate of the antibiotic kanamycin and a nonmembrane lytic, broad-spectrum antimicrobial peptide with efficient mammalian cell penetration, P14LRR, was prepared. This approach allows kanamycin to enter mammalian cells as a conjugate linked via a tether that breaks down in the reducing environment within cells. Potent antimicrobial activity of the P14KanS conjugate was demonstrated in vitro, and this reducible conjugate effectively cleared intracellular pathogenic bacteria within macrophages more potently than that of a conjugate lacking the disulfide moiety. Notably, successful clearance of Mycobacterium tuberculosis within macrophages was observed with the dual antibiotic conjugate, and Salmonella levels were significantly reduced in an in vivo Caenorhabditis elegans model.

Antimycobacterial Efficacy of Andrographis paniculata Leaf Extracts Under Intracellular and Hypoxic Conditions.

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Bhatter, Purva; Gupta, Pooja; Daswani, Poonam; Tetali, Pundarikakshudu; Birdi, Tannaz

2015-01-01

The inhibition of the growth of Mycobacterium tuberculosis by the extracts of Andrographis paniculata has been studied using intracellular and axenic hypoxic conditions. The inhibition (confirmed using the gold standard colony forming unit assay) was found to increase with "double stimuli" or higher concentration of the extract. Organic solvent extracts were found to inhibit bacterial growth more than the aqueous extracts under microaerophilic conditions mimicked through axenic and intracellular assays. This could be further explored to evaluate the potential of the plant to be used against nonreplicating/dormant bacilli. © The Author(s) 2014.

Reduced pyrazinamidase activity and the natural resistance of Mycobacterium kansasii to the antituberculosis drug pyrazinamide.

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Sun, Z; Zhang, Y

1999-03-01

Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug that requires conversion to the bactericidal compound pyrazinoic acid (POA) by the bacterial pyrazinamidase (PZase) activity of nicotinamidase to show activity against Mycobacterium tuberculosis. Mutations leading to a loss of PZase activity cause PZA resistance in M. tuberculosis. M. kansasii is naturally resistant to PZA and has reduced PZase activity along with an apparently detectable nicotinamidase activity. The role of the reduction in PZase activity in the natural PZA resistance of M. kansasii is unknown. The MICs of PZA and POA for M. kansasii were determined to be 500 and 125 micrograms/ml, respectively. Using [14C]PZA and [14C]nicotinamide, we found that M. kansasii had about 5-fold-less PZase activity and about 25-fold-less nicotinamidase activity than M. tuberculosis. The M. kansasii pncA gene was cloned on a 1.8-kb BamHI DNA fragment, using M. avium pncA probe. Sequence analysis showed that the M. kansasii pncA gene encoded a protein with homology to its counterparts from M. tuberculosis (69.9%), M. avium (65.6%), and Escherichia coli (28.5%). Transformation of naturally PZA-resistant M. bovis BCG with M. kansasii pncA conferred partial PZA susceptibility. Transformation of M. kansasii with M. avium pncA caused functional expression of PZase and high-level susceptibility to PZA, indicating that the natural PZA resistance in M. kansasii results from a reduced PZase activity. Like M. tuberculosis, M. kansasii accumulated POA in the cells at an acidic pH; however, due to its highly active POA efflux pump, the naturally PZA-resistant species M. smegmatis did not. These findings suggest the existence of a weak POA efflux mechanism in M. kansasii.

Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level.

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Ahlstrom, Christina; Barkema, Herman W; Stevenson, Karen; Zadoks, Ruth N; Biek, Roman; Kao, Rowland; Trewby, Hannah; Haupstein, Deb; Kelton, David F; Fecteau, Gilles; Labrecque, Olivia; Keefe, Greg P; McKenna, Shawn L B; De Buck, Jeroen

2015-03-08

Mycobacterium avium subsp. paratuberculosis (MAP), the causative bacterium of Johne's disease in dairy cattle, is widespread in the Canadian dairy industry and has significant economic and animal welfare implications. An understanding of the population dynamics of MAP can be used to identify introduction events, improve control efforts and target transmission pathways, although this requires an adequate understanding of MAP diversity and distribution between herds and across the country. Whole genome sequencing (WGS) offers a detailed assessment of the SNP-level diversity and genetic relationship of isolates, whereas several molecular typing techniques used to investigate the molecular epidemiology of MAP, such as variable number of tandem repeat (VNTR) typing, target relatively unstable repetitive elements in the genome that may be too unpredictable to draw accurate conclusions. The objective of this study was to evaluate the diversity of bovine MAP isolates in Canadian dairy herds using WGS and then determine if VNTR typing can distinguish truly related and unrelated isolates. Phylogenetic analysis based on 3,039 SNPs identified through WGS of 124 MAP isolates identified eight genetically distinct subtypes in dairy herds from seven Canadian provinces, with the dominant type including over 80% of MAP isolates. VNTR typing of 527 MAP isolates identified 12 types, including "bison type" isolates, from seven different herds. At a national level, MAP isolates differed from each other by 1-2 to 239-240 SNPs, regardless of whether they belonged to the same or different VNTR types. A herd-level analysis of MAP isolates demonstrated that VNTR typing may both over-estimate and under-estimate the relatedness of MAP isolates found within a single herd. The presence of multiple MAP subtypes in Canada suggests multiple introductions into the country including what has now become one dominant type, an important finding for Johne's disease control. VNTR typing often failed to

Esters of pyrazinoic acid are active against pyrazinamide-resistant strains of Mycobacterium tuberculosis and other naturally resistant mycobacteria in vitro and ex vivo within macrophages.

KAUST Repository

Pires, David

2015-10-05

Pyrazinamide (PZA) is active against major Mycobacterium tuberculosis species (M. tuberculosis, M. africanum, and M. microti), but not against M. bovis and M. avium. The latter two are mycobacteria species involved in human and cattle tuberculosis and in HIV co-infections, respectively. PZA is a first-line agent for the treatment of human tuberculosis and requires activation by a mycobacterial pyrazinamidase to form the active metabolite pyrazinoic acid (POA). As a result of this mechanism, resistance to PZA as often found in tuberculosis patients is caused by point mutations in pyrazinamidase. In previous work, we have shown that POA esters and amides synthesized in our laboratory were stable in plasma. Although the amides did not present significant activity, the esters were active against sensitive mycobacteria at concentrations 5-to-10 fold lower than those of PZA. Here, we report that these POA derivatives possess antibacterial efficacy in vitro and ex vivo against several species and strains of Mycobacterium with natural or acquired resistance to PZA, including M. bovis and M. avium. Our results indicate that the resistance was probably overcome by cleavage of the prodrugs into POA and a long-chain alcohol. Although it is not possible to rule out that the esters may have intrinsic activity per se, we bring evidence here that long-chain fatty alcohols possess a significant anti-mycobacterial effect against PZA-resistant species and strains and are not mere inactive promoieties. These findings may lead to candidate dual-drugs having enhanced activity against both PZA-susceptible and PZA-resistant isolates and being suitable for clinical development.

Development of growth rate measuring method for intracellular, parasitic acid-fast bacteria using radioisotopes

International Nuclear Information System (INIS)

Nakata, Noboru; Fukutomi, Yasuo

1998-01-01

To prevent and treat infections diseases caused by pathogenic acid-fast bacteria such as Mycobacterium leprae, Tubercle bacillus, it is important to elucidate the mechanisms of intracellular proliferations of these bacteria. This research project was started to make DNA library using a new constructed shuttle vector. Development of in vitro evaluation method for intracellular proliferation of mycobacterium and its transformed cells was attempted on the basis of Buddemeyer method. This method was able to precisely determine the metabolic activities as low as those in leprae and its modified method using 14 C-palmitic acid was highly sensitive and the results were obtainable in a shorter period. The generated CO 2 was satisfactorily absorbed into scintillator without using a filter paper. A new culture medium from which arginine, a NO-producing compound was eliminated was used to repress the sterilizing effects of NO, but the metabolic activities of leprae was not enhanced. (M.N.)

Development of growth rate measuring method for intracellular, parasitic acid-fast bacteria using radioisotopes

Energy Technology Data Exchange (ETDEWEB)

Nakata, Noboru; Fukutomi, Yasuo [National Inst. of Infectious Deseases, Tokyo (Japan)

1998-02-01

To prevent and treat infections diseases caused by pathogenic acid-fast bacteria such as Mycobacterium leprae, Tubercle bacillus, it is important to elucidate the mechanisms of intracellular proliferations of these bacteria. This research project was started to make DNA library using a new constructed shuttle vector. Development of in vitro evaluation method for intracellular proliferation of mycobacterium and its transformed cells was attempted on the basis of Buddemeyer method. This method was able to precisely determine the metabolic activities as low as those in leprae and its modified method using {sup 14}C-palmitic acid was highly sensitive and the results were obtainable in a shorter period. The generated CO{sub 2} was satisfactorily absorbed into scintillator without using a filter paper. A new culture medium from which arginine, a NO-producing compound was eliminated was used to repress the sterilizing effects of NO, but the metabolic activities of leprae was not enhanced. (M.N.)

East and Central Afiican Journal of Pharmaceutical Sciences ...

African Journals Online (AJOL)

herbarium specimens were deposited (NIPRD. 5131). Test Organisms. Four species of Mycobacrerium were used for the study namely Mycobacterium tuberculosis,. Mycobacrerium bovis, Mycobacterium avium- complex and Mycobacrerium smegmatis ATCC. 607. The first three species were isolated from sputum samples ...

Molecular Survey of the Occurrence of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa, and Amoeba Hosts in Two Chloraminated Drinking Water Distribution Systems

Science.gov (United States)

Wang, Hong; Edwards, Marc; Falkinham, Joseph O.

2012-01-01

The spread of opportunistic pathogens via public water systems is of growing concern. The purpose of this study was to identify patterns of occurrence among three opportunistic pathogens (Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa) relative to biotic and abiotic factors in two representative chloraminated drinking water distribution systems using culture-independent methods. Generally, a high occurrence of Legionella (≥69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (≤20%) and M. avium (≤33.3%), and rare detection of Pseudomonas aeruginosa (≤13.3%) were observed in both systems according to quantitative PCR. Also, Hartmanella vermiformis was more prevalent than Acanthamoeba, both of which are known hosts for opportunistic pathogen amplification, the latter itself containing pathogenic members. Three-minute flushing served to distinguish distribution system water from plumbing in buildings (i.e., premise plumbing water) and resulted in reduced numbers of copies of Legionella, mycobacteria, H. vermiformis, and 16S rRNA genes (P Legionella and H. vermiformis, were noted, emphasizing potential microbial ecological relationships. Overall, the results provide insight into factors that may aid in controlling opportunistic pathogen proliferation in real-world water systems. PMID:22752174

The oriented development of antituberculotics: salicylanilides.

Science.gov (United States)

Waisser, Karel; Matyk, Josef; Divisová, Hana; Husáková, Petra; Kunes, Jirí; Klimesová, Vera; Kaustová, Jarmila; Möllmann, Ute; Dahse, Hans-Martin; Miko, Milan

2006-11-01

On the basis of our previous results 22 salicylanilides were synthesized. The compounds were tested for in vitro antimycobacterial activity against Mycobacterium tuberculosis, Mycobacterium kansasii, and Mycobacterium avium. The Free-Wilson method was used to evaluate structure-antimycobacterial activity relationships. 4-Chloro-N-(4-propylphenyl)salicylamide and 5-chloro-N-(4-propylphenyl)salicylamide were selected for preclinical studies.

Optimized nested polymerase chain reaction for antemortem detection of Mycobacteria in Amazon parrots (Amazona aestiva) and orange-winged Amazons (Amazona amazonica).

Science.gov (United States)

Baquião, Arianne Costa; Luna, Janaina Oliveira; Medina, Aziz Orro; Sanfilippo, Luiz Francisco; de Faria, Maria Jacinta; dos Santos, Manuel Armando Azevedo

2014-03-01

The objectives of this study were to optimize nested polymerase chain reaction (PCR) for Mycobacterium avium complex and Mycobacterium tuberculosis complex and apply them on samples from parrots. Results were negative for the presence of these Mycobacterium in the samples, and nested PCR was specific, faster, and more sensitive than other tests, thereby justifying its use in antemortem diagnosis.

Sensibilidad in vitro de micobacterias a dos péptidos sintéticos híbridos con actividad antimicrobiana In-vitro activity of two hybrid synthetic peptides having antimicrobial activity against mycobacteria

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E. Zerbini

2006-12-01

Full Text Available El aumento de aislamientos clínicos de Mycobacterium tuberculosis resistentes a las drogas esenciales y de casos de micobacteriosis diseminadas debidas al complejo Mycobacterium avium hacen necesario investigar nuevos agentes antimicobacterianos. Los péptidos antimicrobianos son un nuevo grupo de antibióticos que poseen un mecanismo de acción particular. Algunos de ellos, como la cecropina y la melitina, han sido aislados de insectos y han demostrado buena actividad in vitro contra bacterias gram positivas y gram negativas. Híbridos sintéticos de esos péptidos han presentado mayor actividad que los péptidos individuales. En este trabajo se evaluó la actividad in vitro de dos péptidos híbridos sintéticos de melitina y cecropina contra M. tuberculosis, complejo M. avium, Mycobacterium fortuitum y Mycobacterium smegmatis. Se determinó la concentración inhibitoria mínima empleando la técnica de macrodilución en caldo. Luego se estableció la concentración bactericida mínima en medio Lowenstein Jensen. Los péptidos evaluados mostraron ser activos in vitro contra M. smegmatis, mientras que no presentaron ninguna actividad contra las otras micobacterias estudiadas.The increase in both Mycobacterium tuberculosis human clinical isolates resistant to the essential drugs and cases of disseminated micobacteriosis due to Mycobacterium avium Complex, underlines the need to investigate new antimicobacterial agents. The antimicrobial peptides are a new group of active antibiotics with a particular mechanism of action. Some of them, like cecropin and melittin, isolated from insects, have demonstrated good in vitro activity against Gram-positive and Gram-negative bacteria. Synthetic hybrids of those peptides have been more active than individual peptides. In this study, the in vitro activity of two hybrid synthetic peptides from melittin and cecropin against M. tuberculosis, M. avium Complex, Mycobacterium fortuitum and Mycobacterium smegmatis

Longitudinal relationship between fecal culture, fecal quantitative PCR, and milk ELISA in Mycobacterium avium ssp. paratuberculosis-infected cows from low-prevalence dairy herds.

Science.gov (United States)

Beaver, A; Sweeney, R W; Hovingh, E; Wolfgang, D R; Gröhn, Y T; Schukken, Y H

2017-09-01

Mycobacterium avium ssp. paratuberculosis (MAP), the causative agent of ruminant Johne's disease, presents a particular challenge with regard to infection mitigation on dairy farms. Diagnostic testing strategies to identify and quantify MAP and associated antibodies are imperfect, and certain facets of the relationship between diagnostic tests remain to be explored. Additional repeated-measures data from known infected animals are needed to complement the body of cross-sectional research on Johne's disease-testing methods. Statistical models that accurately account for multiple diagnostic results while adjusting for the effects of individual animals and herds over time can provide a more detailed understanding of the interplay between diagnostic outcomes. Further, test results may be considered as continuous wherever possible so as to avoid the information loss associated with dichotomization. To achieve a broader understanding of the relationship between diagnostic tests, we collected a large number of repeated fecal and milk samples from 14 infected cows, in addition to bulk milk samples, from 2 low-prevalence dairy herds in the northeast United States. Predominately through the use of mixed linear modeling, we identified strong associations between milk ELISA optical density, fecal quantitative PCR, and fecal culture in individual animals while concurrently adjusting for variables that could alter these relationships. Notably, we uncovered subtleties in the predictive abilities of fecal shedding level on milk ELISA results, with animals categorized as disease progressors reaching higher ELISA optical density levels. Moreover, we observed that spikes in fecal shedding could predict subsequent high ELISA values up to 2 mo later. We also investigated the presence of MAP in individual milk samples via PCR and noted an association between poor udder hygiene and MAP positivity in milk, suggesting some level of environmental contamination. The paucity of positive milk

Phenotypic assays for Mycobacterium tuberculosis infection.

Science.gov (United States)

Song, Ok-Ryul; Deboosere, Nathalie; Delorme, Vincent; Queval, Christophe J; Deloison, Gaspard; Werkmeister, Elisabeth; Lafont, Frank; Baulard, Alain; Iantomasi, Raffaella; Brodin, Priscille

2017-10-01

Tuberculosis (TB) is still a major global threat, killing more than one million persons each year. With the constant increase of Mycobacterium tuberculosis strains resistant to first- and second-line drugs, there is an urgent need for the development of new drugs to control the propagation of TB. Although screenings of small molecules on axenic M. tuberculosis cultures were successful for the identification of novel putative anti-TB drugs, new drugs in the development pipeline remains scarce. Host-directed therapy may represent an alternative for drug development against TB. Indeed, M. tuberculosis has multiple specific interactions within host phagocytes, which may be targeted by small molecules. In order to enable drug discovery strategies against microbes residing within host macrophages, we developed multiple fluorescence-based HT/CS phenotypic assays monitoring the intracellular replication of M. tuberculosis as well as its intracellular trafficking. What we propose here is a population-based, multi-parametric analysis pipeline that can be used to monitor the intracellular fate of M. tuberculosis and the dynamics of cellular events such as phagosomal maturation (acidification and permeabilization), zinc poisoning system or lipid body accumulation. Such analysis allows the quantification of biological events considering the host-pathogen interplay and may thus be derived to other intracellular pathogens. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Detection of serum antibodies cross-reacting with Mycobacterium avium subspecies paratuberculosis and beta-cell antigen zinc transporter 8 homologous peptides in patients with high-risk proliferative diabetic retinopathy.

Science.gov (United States)

Pinna, Antonio; Masala, Speranza; Blasetti, Francesco; Maiore, Irene; Cossu, Davide; Paccagnini, Daniela; Mameli, Giuseppe; Sechi, Leonardo A

2014-01-01

MAP3865c, a Mycobacterium avium subspecies paratuberculosis (MAP) cell membrane protein, has a relevant sequence homology with zinc transporter 8 (ZnT8), a beta-cell membrane protein involved in Zn++ transportation. Recently, antibodies recognizing MAP3865c epitopes have been shown to cross-react with ZnT8 in type 1 diabetes patients. The purpose of this study was to detect antibodies against MAP3865c peptides in patients with high-risk proliferative diabetic retinopathy and speculate on whether they may somehow be involved in the pathogenesis of this severe retinal disorder. Blood samples were obtained from 62 type 1 and 80 type 2 diabetes patients with high-risk proliferative diabetic retinopathy and 81 healthy controls. Antibodies against 6 highly immunogenic MAP3865c peptides were detected by indirect ELISA. Type 1 diabetes patients had significantly higher rates of positive antibodies than controls. Conversely, no statistically significant differences were found between type 2 diabetes patients and controls. After categorization of type 1 diabetes patients into two groups, one with positive, the other with negative antibodies, we found that they had similar mean visual acuity (∼ 0.6) and identical rates of vitreous hemorrhage (28.6%). Conversely, Hashimoto's thyroiditis prevalence was 4/13 (30.7%) in the positive antibody group and 1/49 (2%) in the negative antibody group, a statistically significant difference (P = 0.016). This study confirmed that type 1 diabetes patients have significantly higher rates of positive antibodies against MAP/ZnT8 peptides, but failed to find a correlation between the presence of these antibodies and the severity degree of high-risk proliferative diabetic retinopathy. The significantly higher prevalence of Hashimoto's disease among type 1 diabetes patients with positive antibodies might suggest a possible common environmental trigger for these conditions.

Detection of Mycobacterium avium subsp. paratuberculosis from cattle and buffaloes in Egypt using traditional culture, serological and molecular based methods

Directory of Open Access Journals (Sweden)

G. S. Abdellrazeq

2014-08-01

Full Text Available Background: Johne's disease (JD caused by Mycobacterium avium subsp. paratuberculosis (MAP represents a real threat to the agriculture and dairy food industries and believed to be a potential public health problem. Signs of infection in ruminant include weight loss, diarrhea, decreased milk production, and eventually death. The definition of an infected animal based either on the presence of anti-MAP antibodies, or positive bacterial culture. No treatment for the disease exists and controlling the disease is difficult due to its long latent period. JD is a worldwide problem and multiple studies in many countries have been carried out to determine the prevalence of MAP infections. Although some primary non intensive studies confirm presence of JD in Egypt, the disease is currently neglected by the official Egyptian veterinary agencies. There is no official data, no national control program, and no used vaccine. Aim: This study aimed to evaluate three conventional diagnostic methods for MAP under the Egyptian circumstances with a general aim to determine the appropriate strategy to develop a JD control program. These methods were pooled fecal culture, humoral response and insertion sequence IS900 targets polymerase chain reaction (IS900 PCR. Materials and Methods: Fecal and serum samples (500 each were collected from Holstein-Friesian cattle and buffaloes housed in five Egyptian governorates. Fecal samples were examined for MAP on the basis of a strategic pooling procedure and performed on Herrold's Egg Yolk Agar Medium (HEYM. Smears were prepared from developed colonies and stained using a Ziehl-Neelsen (ZN technique. The identity of developed colonies was further confirmed by PCR analysis of IS900 sequence. Sera from both culture-positive and culture-negative animals were evaluated individually for humoral response. Results: Out of 50 pooled specimens, 34 (68% fecal cultures were positive for MAP. Serum positive samples of culture

Radiometric studies on the oxidation of (I-14C) fatty acids by drug-susceptible and drug-resistant mycobacteria

International Nuclear Information System (INIS)

Camargo, E.E.; Kopajtic, T.M.; Hopkins, G.K.; Cannon, N.P.; Wagner Junior, H.N.

1987-01-01

A radiometric assay system has been used to study oxidation patterns of (l - 14 C) fatty acids by drug-susceptible and drug-resistant organisms of the genus Mycobacterium (M. tuberculosis - H 37 Rv and Erdman, M. bovis, M. avium, M. intracellulare, M.Kansasii and M. chelonei). The organisms were inoculated in sterile reaction vials containing liquid 7H9 medium, 10% ADC enrichment and 1.0 uli of one of the (l- 14 C) fatty acids (butyric, hexanoic, octanoic, decanoic, lauric, myristic, palmitic, stearic, oleic, linoleic, linolenic). Vials were incubated at 37 0 C and the 14 CO 2 envolved was measured daily for 3 days with a Bactec R-301 instrument. (M.A.C.) [pt

A docking study of enhanced intracellular survival protein from Mycobacterium tuberculosis with human DUSP16/MKP-7

International Nuclear Information System (INIS)

Yoon, Hye-Jin; Kim, Kyoung Hoon; Yang, Jin Kuk; Suh, Se Won; Kim, Hyunsik; Jang, Soonmin

2013-01-01

A docking study of Mtb Eis with its substrate DUSP16/MKP-7 was performed. The docking model suggests dissociation of hexameric Mtb Eis into dimers or monomers. The intracellular pathogen Mycobacterium tuberculosis (Mtb) causes tuberculosis, and one of its secreted effector proteins, called enhanced intracellular survival (Eis) protein, enhances its survival in macrophages. Mtb Eis activates JNK-specific dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase-7 (MKP-7) through the acetylation on Lys55, thus inactivating JNK by dephosphorylation. Based on the recently reported crystal structure of Mtb Eis, a docking model for the binding of Mtb Eis to DUSP16/MKP-7 was generated. In the docking model, the substrate helix containing Lys55 of DUSP16/MKP-7 fits nicely into the active-site cleft of Mtb Eis; the twisted β-sheet of Eis domain II embraces the substrate helix from one side. Most importantly, the side-chain of Lys55 is inserted toward acetyl-CoA and the resulting distance is 4.6 Å between the NZ atom of Lys55 and the carbonyl carbon of the acetyl group in acetyl-CoA. The binding of Mtb Eis and DUSP16/MKP-7 is maintained by strong electrostatic interactions. The active-site cleft of Mtb Eis has a negatively charged surface formed by Asp25, Glu138, Asp286, Glu395 and the terminal carboxylic group of Phe396. In contrast, DUSP16/MKP-7 contains five basic residues, Lys52, Lys55, Arg56, Arg57 and Lys62, which point toward the negatively charged surface of the active-site pocket of Mtb Eis. Thus, the current docking model suggests that the binding of DUSP16/MKP-7 to Mtb Eis should be established by charge complementarity in addition to a very favorable geometric arrangement. The suggested mode of binding requires the dissociation of the hexameric Mtb Eis into dimers or monomers. This study may be useful for future studies aiming to develop inhibitors of Mtb Eis as a new anti-tuberculosis drug candidate

A docking study of enhanced intracellular survival protein from Mycobacterium tuberculosis with human DUSP16/MKP-7

Energy Technology Data Exchange (ETDEWEB)

Yoon, Hye-Jin, E-mail: yoonhj@snu.ac.kr; Kim, Kyoung Hoon [Seoul National University, Seoul 151-747 (Korea, Republic of); Yang, Jin Kuk [Soongsil University, Seoul 156-743 (Korea, Republic of); Suh, Se Won [Seoul National University, Seoul 151-747 (Korea, Republic of); Seoul National University, Seoul 151-747 (Korea, Republic of); Kim, Hyunsik; Jang, Soonmin, E-mail: yoonhj@snu.ac.kr [Sejong University, Seoul 143-747 (Korea, Republic of)

2013-11-01

A docking study of Mtb Eis with its substrate DUSP16/MKP-7 was performed. The docking model suggests dissociation of hexameric Mtb Eis into dimers or monomers. The intracellular pathogen Mycobacterium tuberculosis (Mtb) causes tuberculosis, and one of its secreted effector proteins, called enhanced intracellular survival (Eis) protein, enhances its survival in macrophages. Mtb Eis activates JNK-specific dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase-7 (MKP-7) through the acetylation on Lys55, thus inactivating JNK by dephosphorylation. Based on the recently reported crystal structure of Mtb Eis, a docking model for the binding of Mtb Eis to DUSP16/MKP-7 was generated. In the docking model, the substrate helix containing Lys55 of DUSP16/MKP-7 fits nicely into the active-site cleft of Mtb Eis; the twisted β-sheet of Eis domain II embraces the substrate helix from one side. Most importantly, the side-chain of Lys55 is inserted toward acetyl-CoA and the resulting distance is 4.6 Å between the NZ atom of Lys55 and the carbonyl carbon of the acetyl group in acetyl-CoA. The binding of Mtb Eis and DUSP16/MKP-7 is maintained by strong electrostatic interactions. The active-site cleft of Mtb Eis has a negatively charged surface formed by Asp25, Glu138, Asp286, Glu395 and the terminal carboxylic group of Phe396. In contrast, DUSP16/MKP-7 contains five basic residues, Lys52, Lys55, Arg56, Arg57 and Lys62, which point toward the negatively charged surface of the active-site pocket of Mtb Eis. Thus, the current docking model suggests that the binding of DUSP16/MKP-7 to Mtb Eis should be established by charge complementarity in addition to a very favorable geometric arrangement. The suggested mode of binding requires the dissociation of the hexameric Mtb Eis into dimers or monomers. This study may be useful for future studies aiming to develop inhibitors of Mtb Eis as a new anti-tuberculosis drug candidate.

Exploring Anti-Bacterial Compounds against Intracellular Legionella

Science.gov (United States)

Harrison, Christopher F.; Kicka, Sébastien; Trofimov, Valentin; Berschl, Kathrin; Ouertatani-Sakouhi, Hajer; Ackermann, Nikolaus; Hedberg, Christian; Cosson, Pierre; Soldati, Thierry; Hilbi, Hubert

2013-01-01

Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an ‘accidental’ human pathogen and cause a severe pneumonia known as Legionnaires’ disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoeba castellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target. PMID:24058631

Exploring anti-bacterial compounds against intracellular Legionella.

Directory of Open Access Journals (Sweden)

Christopher F Harrison

Full Text Available Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an 'accidental' human pathogen and cause a severe pneumonia known as Legionnaires' disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoebacastellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target.

Nontuberculous mycobacteria: incidence in Southwest Ireland from 1987 to 2000.

LENUS (Irish Health Repository)

Kennedy, M P

2012-02-03

SETTING: The Southwest of Ireland (Counties Cork and Kerry) 1987-2000, average population 549,500. OBJECTIVE: Nontuberculous mycobacteria (NTM) cause significant morbidity worldwide and the study of epidemiology and characteristics helps in their prevention and treatment. This study was performed to determine the incidence of NTM disease in comparison to Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium bovis (M. bovis) in Southwest Ireland, over the above time period. DESIGN: A retrospective study was carried out in all human isolates of NTM, M. tuberculosis and M. bovis between 1987 and 2000, in the Southwest Region of Ireland. RESULTS: The mean incidence of NTM (0.4\\/100,000 population) has risen since 1995, principally of pulmonary Mycobacterium avium intracellulare complex (MAC). The annual incidence of M. tuberculosis in humans over 14 years in the same region was 971\\/100,000 population with a significant reduction since 1994 and M. bovis remained constant at 0.5\\/100,000 population. CONCLUSION: The increasing incidence of disease causing NTM noted in Southwest Ireland reflects global data and is surmised to be due to an ageing population, increased incidence related to chronic fibrotic lung disease, and environmental mycobacterial factors.

Bovine NK cells can produce gamma interferon in response to the secreted mycobacterial proteins ESAT-6 and MPP14 but not in response to MPB70

DEFF Research Database (Denmark)

Olsen, Ingrid; Boysen, P.; Kulberg, S.

2005-01-01

to the Mycobacterium tuberculosis complex-specific protein ESAT-6, MPP14 from Mycobacterium avium subsp. paratuberculosis, and purified protein derivative (PPD) from M. tuberculosis. In contrast, no response was induced by MPB70, which is another M. tuberculosis complex-specific secreted antigen. The production of IFN...

Detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacterial isolates by real-time PCR.

Science.gov (United States)

Shrestha, Nabin K; Tuohy, Marion J; Hall, Gerri S; Reischl, Udo; Gordon, Steven M; Procop, Gary W

2003-11-01

Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. Despite advances in molecular diagnostics, the ability to rapidly diagnose M. tuberculosis infections by PCR is still inadequate, largely because of the possibility of false-negative reactions. We designed and validated a real-time PCR for mycobacteria by using the LightCycler system with 18 reference strains and 168 clinical mycobacterial isolates. All clinically significant mycobacteria were detected; the mean melting temperatures (with 99.9% confidence intervals [99.9% CI] in parentheses) for the different mycobacteria were as follows: M. tuberculosis, 64.35 degrees C (63.27 to 65.42 degrees C); M. kansasii, 59.20 degrees C (58.07 to 60.33 degrees C); M. avium, 57.82 degrees C (57.05 to 58.60 degrees C); M. intracellulare, 54.46 degrees C (53.69 to 55.23 degrees C); M. marinum, 58.91 degrees C (58.28 to 59.55 degrees C); rapidly growing mycobacteria, 53.09 degrees C (50.97 to 55.20 degrees C) or 43.19 degrees C (42.19 to 44.49 degrees C). This real-time PCR assay with melting curve analysis consistently accurately detected and differentiated M. tuberculosis from NTM. Detection of an NTM helps ensure that the negative result for M. tuberculosis is a true negative. The specific melting temperature also provides a suggestion of the identity of the NTM present, when the most commonly encountered mycobacterial species are considered. In a parallel comparison, both the LightCycler assay and the COBAS Amplicor M. tuberculosis assay correctly categorized 48 of 50 specimens that were proven by culture to contain M. tuberculosis, and the LightCycler assay correctly characterized 3 of 3 specimens that contained NTM.

Alternate radiolabeled markers for detecting metabolic activity of Mycobacterium leprae residing in murine macrophages

International Nuclear Information System (INIS)

Prasad, H.K.; Hastings, R.C.

1985-01-01

This study demonstrated the utility of using 4% NaOH as a murine macrophage cell-solubilizing agent to discriminate between host macrophage metabolism and that of intracellular Mycobacterium leprae. A 4% concentration of NaOH had no deleterious effect on labeled mycobacteria. Thereby, alternate radiolabeled indicators of the metabolic activity of intracellular M. leprae could be experimented with. Significant incorporation of 14 C-amino acid mixture, [ 14 C]leucine, [ 14 C]uridine, and carrier-free 32 P was observed in cultures containing freshly extracted (''live'') strains of M. leprae as compared with control cultures containing autoclaved bacilli

Evaluation of fecal culture and fecal RT-PCR to detect Mycobacterium avium ssp. paratuberculosis fecal shedding in dairy goats and dairy sheep using latent class Bayesian modeling.

Science.gov (United States)

Bauman, Cathy A; Jones-Bitton, Andria; Jansen, Jocelyn; Kelton, David; Menzies, Paula

2016-09-20

The study's objective was to evaluate the ability of fecal culture (FCUL) and fecal PCR (FPCR) to identify dairy goat and dairy sheep shedding Mycobacterium avium ssp. paratuberculosis. A cross-sectional study of the small ruminant populations was performed in Ontario, Canada between October 2010 and August 2011. Twenty-nine dairy goat herds and 21 dairy sheep flocks were visited, and 20 lactating females > two years of age were randomly selected from each farm resulting in 580 goats and 397 sheep participating in the study. Feces were collected per rectum and cultured using the BD BACTEC™ MGIT™ 960 system using a standard (49 days) and an extended (240 days) incubation time, and underwent RT-PCR based on the hsp-X gene (Tetracore®). Statistical analysis was performed using a 2-test latent class Bayesian hierarchical model for each species fitted in WinBUGS. Extending the fecal culture incubation time statistically improved FCUL sensitivity from 23.1 % (95 % PI: 15.9-34.1) to 42.7 % (95 % PI: 33.0-54.5) in dairy goats and from 5.8 % (95 % PI: 2.3-12.4) to 19.0 % (95 % PI: 11.9-28.9) in dairy sheep. FPCR demonstrated statistically higher sensitivity than FCUL (49 day incubation) with a sensitivity of 31.9 % (95 % PI: 22.4-43.1) in goats and 42.6 % (95 % PI: 28.8-63.3) in sheep. Fecal culture demonstrates such low sensitivity at the standard incubation time it cannot be recommended as a screening test to detect shedding of MAP in either goats or sheep. Extending the incubation time resulted in improved sensitivity; however, it is still disappointingly low for screening purposes. Fecal PCR should be the screening test of choice in both species; however, it is important to recognize that control programs should not be based on testing alone when they demonstrate such low sensitivity.

Coordination modes of tyrosinate-ligated catalase-type heme enzymes: magnetic circular dichroism studies of Plexaura homomalla allene oxide synthase, Mycobacterium avium ssp. paratuberculosis protein-2744c, and bovine liver catalase in their ferric and ferrous states.

Science.gov (United States)

Bandara, D M Indika; Sono, Masanori; Bruce, Grant S; Brash, Alan R; Dawson, John H

2011-12-01

Bovine liver catalase (BLC), catalase-related allene oxide synthase (cAOS) from Plexaura homomalla, and a recently isolated protein from the cattle pathogen Mycobacterium avium ssp. paratuberculosis (MAP-2744c (MAP)) are all tyrosinate-ligated heme enzymes whose crystal structures have been reported. cAOS and MAP have low (enzymes in their ferric and ferrous states using magnetic circular dichroism and UV-visible absorption spectroscopy. The MAP protein shows remarkable spectral similarities to cAOS and BLC in its native Fe(III) state, but clear differences from ferric proximal heme ligand His93Tyr Mb (myoglobin) mutant, which may be attributed to the presence of an Arg(+)-N(ω)-H···¯O-Tyr (proximal heme axial ligand) hydrogen bond in the first three heme proteins. Furthermore, the spectra of Fe(III)-CN¯, Fe(III)-NO, Fe(II)-NO (except for five-coordinate MAP), Fe(II)-CO, and Fe(II)-O(2) states of cAOS and MAP, but not H93Y Mb, are also similar to the corresponding six-coordinate complexes of BLC, suggesting that a tyrosinate (Tyr-O¯) is the heme axial ligand trans to the bound ligands in these complexes. The Arg(+)-N(ω)-H to ¯O-Tyr hydrogen bond would be expected to modulate the donor properties of the proximal tyrosinate oxyanion and, combined with the subtle differences in the catalytic site structures, affect the activities of cAOS, MAP and BLC. Copyright © 2011 Elsevier Inc. All rights reserved.

Nanoparticles as Antituberculosis Drugs Carriers: Effect on Activity Against Mycobacterium tuberculosis in Human Monocyte-Derived Macrophages

International Nuclear Information System (INIS)

Anisimova, Y.V.; Gelperina, S.I.; Peloquin, C.A.; Heifets, L.B.

2000-01-01

This is the first report evaluating the nanoparticle delivery system for three antituberculosis drugs: isoniazid, rifampin, and streptomycin. The typical particle size is 250 nm. We studied accumulation of these drugs in human monocytes as well as their antimicrobial activity against Mycobacterium tuberculosis residing in human monocyte-derived macrophages. Nanoparticle encapsulation increased the intracellular accumulation (cell-association) of all three tested drugs, but it enhanced the antimicrobial activity of isoniazid and streptomycin only. On the other hand, the activity of encapsulated rifampin against intracellular bacteria was not higher than that of the free drug

Unanticipated Mycobacterium tuberculosis complex culture inhibition by immune modulators, immune suppressants, a growth enhancer, and vitamins A and D: clinical implications.

Science.gov (United States)

Greenstein, Robert J; Su, Liya; Shahidi, Azra; Brown, William D; Clifford, Anya; Brown, Sheldon T

2014-09-01

The development of novel antibiotics to treat multidrug-resistant (MDR) tuberculosis is time-consuming and expensive. Multiple immune modulators, immune suppressants, anti-inflammatories, and growth enhancers, and vitamins A and D, inhibit Mycobacterium avium subspecies paratuberculosis (MAP) in culture. We studied the culture inhibition of Mycobacterium tuberculosis complex by these agents. Biosafety level two M. tuberculosis complex (ATCC 19015 and ATCC 25177) was studied in radiometric Bactec or MGIT culture. Agents evaluated included clofazimine, methotrexate, 6-mercaptopurine, cyclosporine A, rapamycin, tacrolimus, monensin, and vitamins A and D. All the agents mentioned above caused dose-dependent inhibition of the M. tuberculosis complex. There was no inhibition by the anti-inflammatory 5-aminosalicylic acid, which causes bacteriostatic inhibition of MAP. We conclude that, at a minimum, studies with virulent M. tuberculosis are indicated with the agents mentioned above, as well as with the thioamide 5-propothiouricil, which has previously been shown to inhibit the M. tuberculosis complex in culture. Our data additionally emphasize the importance of vitamins A and D in treating mycobacterial diseases. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

'Nano-immuno test' for the detection of live Mycobacterium avium subspecies paratuberculosis bacilli in the milk samples using magnetic nano-particles and chromogen.

Science.gov (United States)

Singh, Manju; Singh, Shoor Vir; Gupta, Saurabh; Chaubey, Kundan Kumar; Stephan, Bjorn John; Sohal, Jagdip Singh; Dutta, Manali

2018-04-26

Early rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP) bacilli in milk samples is the major challenge since traditional culture method is time consuming and laboratory dependent. We report a simple, sensitive and specific nano-technology based 'Nano-immuno test' capable of detecting viable MAP bacilli in the milk samples within 10 h. Viable MAP bacilli were captured by MAP specific antibody-conjugated magnetic nano-particles using resazurin dye as chromogen. Test was optimized using true culture positive (10-bovine and 12-goats) and true culture negative (16-bovine and 25-goats) raw milk samples. Domestic livestock species in India are endemically infected with MAP. After successful optimization, sensitivity and specificity of the 'nano-immuno test' in goats with respect to milk culture was 91.7% and 96.0%, respectively. Whereas, it was 90.0% (sensitivity) and 92.6% (specificity) with respect to IS900 PCR. In bovine milk samples, sensitivity and specificity of 'nano-immuno test' with respect to milk culture was 90.0% and 93.7%, respectively. However, with respect to IS900 PCR, the sensitivity and specificity was 88.9% and 94.1%, respectively. Test was validated with field raw milk samples (goats-258 and bovine-138) collected from domestic livestock species to detect live/viable MAP bacilli. Of 138 bovine raw milk samples screened by six diagnostic tests, 81 (58.7%) milk samples were positive for MAP infection in one or more than one diagnostic tests. Of 81 (58.7%) positive bovine raw milk samples, only 24 (17.4%) samples were detected positive for the presence of viable MAP bacilli. Of 258 goats raw milk samples screened by six diagnostic tests, 141 (54.6%) were positive for MAP infection in one or more than one test. Of 141 (54.6%) positive raw milk samples from goats, only 48 (34.0%) were detected positive for live MAP bacilli. Simplicity and efficiency of this novel 'nano-immuno test' makes it suitable for wide-scale screening of milk

ORF Alignment: NC_002944 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available h = 155 ... Query: 200 HTQMAPAASKPPEVANLATKMFQALMPSRSGAIQKPAGSQTIGRATDNDIVIQDVLASRH 259 ... HTQMAPA...ASKPPEVANLATKMFQALMPSRSGAIQKPAGSQTIGRATDNDIVIQDVLASRH Sbjct: 5 ... HTQMAPAASKPPEVANLATK... ... protein MAP3465 [Mycobacterium avium subsp. ... paratuberculosis str. k10] ... Lengt

Different Transcriptional Profiles of Human Monocyte-Derived Dendritic Cells Infected with Distinct Strains of Mycobacterium tuberculosis and Mycobacterium bovis Bacillus Calmette-Guérin

Directory of Open Access Journals (Sweden)

Nunzia Sanarico

2011-01-01

Full Text Available In order to analyze dendritic cells (DCs activation following infection with different mycobacterial strains, we studied the expression profiles of 165 genes of human monocyte-derived DCs infected with H37Rv, a virulent Mycobacterium tuberculosis (MTB laboratory strain, CMT97, a clinical MTB isolate, Mycobacterium bovis bacillus Calmette-Guérin (BCG, Aventis Pasteur, and BCG Japan, both employed as vaccine against tuberculosis. The analysis of the gene expression reveals that, despite a set of genes similarly modulated, DCs response resulted strain dependent. In particular, H37Rv significantly upregulated EBI3 expression compared with BCG Japan, while it was the only strain that failed to release a significant IL-10 amount. Of note, BCG Japan showed a marked increase in CCR7 and TNF-α expression regarding both MTB strains and it resulted the only strain failing in exponential intracellular growth. Our results suggest that DCs display the ability to elicit a tailored strain-specific immune response.

The Changing Pattern of Nontuberculous Mycobacterial Disease

Directory of Open Access Journals (Sweden)

Joseph O Falkinham

2003-01-01

Full Text Available Nontuberculous mycobacteria are human opportunistic pathogens whose source of infection is the environment. These include both slow-growing (eg, Mycobacterium kansasii and Mycobacterium avium and rapid-growing (eg, Mycobacterium abscessus and Mycobacterium fortuitum species. Transmission is through ingestion or inhalation of water, particulate matter or aerosols, or through trauma. The historic presentation of pulmonary disease in older individuals with predisposing lung conditions and in children has been changing. Pulmonary disease in elderly individuals who lack the classic predisposing lung conditions is increasing. Pulmonary disease and hypersensitivity pneumonitis have been linked with occupational or home exposures to nontuberculous mycobacteria. There has been a shift from Mycobacterium scrofulaceum to M avium in children with cervical lymphadenitis. Further, individuals who are immunosuppressed due to therapy or HIV-infection are at a greatly increased risk for nontuberculous mycobacterial infection. The changing pattern of nontuberculous mycobacterial disease is due in part to the ability of these pathogens to survive and proliferate in habitats that they share with humans, such as drinking water. The advent of an aging population and an increase in the proportion of immunosuppressed individuals suggest that the prevalence of nontuberculous mycobacterial disease will increase.

Alternate radiolabeled markers for detecting metabolic activity of Mycobacterium leprae residing in murine macrophages

Energy Technology Data Exchange (ETDEWEB)

Prasad, H.K.; Hastings, R.C.

1985-05-01

This study demonstrated the utility of using 4% NaOH as a murine macrophage cell-solubilizing agent to discriminate between host macrophage metabolism and that of intracellular Mycobacterium leprae. A 4% concentration of NaOH had no deleterious effect on labeled mycobacteria. Thereby, alternate radiolabeled indicators of the metabolic activity of intracellular M. leprae could be experimented with. Significant incorporation of /sup 14/C-amino acid mixture, (/sup 14/C)leucine, (/sup 14/C)uridine, and carrier-free /sup 32/P was observed in cultures containing freshly extracted (''live'') strains of M. leprae as compared with control cultures containing autoclaved bacilli.

ORF Alignment: NC_002944 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available ... protein MAP4001 [Mycobacterium avium subsp. ... paratuberculosis str. k10] ... Length = 80 ... Query: 10 VELLTRDGCTIC...ERIHARLVELAAELGFELSTTDVDAAAAAGNPGLRTEFGDRLPVILLD 69 ... VELLTRDGCTICE...RIHARLVELAAELGFELSTTDVDAAAAAGNPGLRTEFGDRLPVILLD Sbjct: 1 ... VELLTRDGCTICERIHARLVELAAELGFELSTTDVDAAAAAGNPGLRTEFGDRLPVILLD 60 ...

Mycobacterium paratuberculosis Zoonosis-The Hundred Year War –Beyond Crohn’s Disease

Directory of Open Access Journals (Sweden)

Leonardo A Sechi

2015-03-01

Full Text Available The factitive role of Mycobacterium avium ss. paratuberculosis (MAP in Crohn’s disease has been debated for more than a century. The controversy is due to the fact that Crohn’s disease is so similar to a disease of MAP-infected ruminant animals, Johne’s disease; and, though MAP can be readily detected in the infected ruminants, it is much more difficult to detect in humans. Molecular techniques that can detect MAP in pathologic Crohn’s specimens as well as dedicated specialty labs successful in culturing MAP from Crohn’s patients have provided strong argument for MAP’s role in Crohn’s disease. Perhaps more incriminating for MAP as a zoonotic agent is the increasing number of diseases with which MAP has been related: Blau syndrome, type 1 diabetes, Hashimoto thyroiditis and multiple sclerosis. In this article we debate about genetic susceptibility to mycobacterial infection and human exposure to MAP; moreover, it suggests that molecular mimicry between protein epitopes of MAP and human proteins is a likely bridge between infection and these autoimmune disorders.

Isolamento de micobactérias em Felis concolor em cativeiro

Directory of Open Access Journals (Sweden)

María Julia Traversa

2009-02-01

Full Text Available Este trabalho foi realizado em uma reserva natural da Argentina com antecedentes de tuberculose em uma suçuarana adulta. O objetivo foi identificar por meio de técnicas bacteriológicas e de biologia molecular as espécies isoladas da orofaringe de cinco suçuaranas que apresentavam sinais clínicos inespecíficos. As amostras foram colhidas das suçuaranas após sedação. Posteriormente foram processadas para obtenção do isolamento e identificação por meio de provas bioquímicas do gênero Mycobacterium pela técnica de PCR. Investigou-se a presença das seqüências de inserção IS6110 e IS1081 e hsp65. Obtiveram-se resultados positivos à coloração de Ziehl-Neelsen de quatro amostras, isolando cinco cepas de crescimento lento. As cepas foram classificadas como M. gordonae em dois casos e M. simiae, M scrofulaceum e M. avium/intracellulare em um. Por PRA, identificou-se o padrão de M. gordonae em três cepas e M. avium III ou M. simiae em dois.

MYCOBACTERIOSIS IN CAPTIVE PSITTACINES: A BRIEF REVIEW AND CASE SERIES IN COMMON COMPANION SPECIES (ECLECTUS RORATUS, AMAZONA ORATRIX, AND PIONITES MELANOCEPHALA).

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McRee, Anna Elizabeth; Higbie, Christine T; Nevarez, Javier G; Rademacher, Nathalie T; Tully, Thomas N

2017-09-01

In 2015, three psittacines were presented within 30 days, each with differing clinical signs and patient histories. A 13-yr-old male eclectus parrot (Eclectus roratus) was presented for weakness, depression, and acute anorexia. On presentation it was determined to have a heart murmur, severely elevated white blood cell count (93.9 10 3 /μl) with a left shift (2.8 10 3 /μl bands), and anemia (30%). Severe hepatomegaly was noted on radiographs, ultrasonography, and computed tomography. A cytological sample of the liver obtained through a fine needle aspirate revealed intracellular acid-fast bacilli identified as Mycobacterium avium. A 20-yr-old female double yellow-headed Amazon parrot (Amazona oratrix) was presented for a 1-mo history of lethargy and weight loss despite a good appetite. The parrot's total white blood cell count was 16.8 10 3 /μl and the PCV was 35%. Following its death, a necropsy revealed a generalized granulomatous condition that involved the small intestines, lungs, liver, spleen, and medullary cavities of the long bones, with intracellular acid-fast bacilli identified as Mycobacterium genavense. The third case, an 18-mo-old female black-headed caique (Pionites melanocephala), was presented with a 1-day history of lethargy and depression. On presentation, the caique had a heart murmur, distended coelom, palpable thickening of the coelomic organs, and increased lung sounds. Following the caique's death, a complete necropsy revealed mycobacteriosis of the liver, spleen, small intestines, pericardial fat, and bone marrow. The infection was identified as Mycobacterium genavense. The importance of advances in Mycobacterium spp. identification, continued presence of this organism in captive avian populations, difficulty in obtaining a definitive antemortem diagnosis, and conflicting recommendations regarding treatment are thought-provoking areas of focus in this case series.

Mycobacterium Avium Complex (MAC)

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... sweat, and saliva red-orange (may stain contact lenses); can interfere with birth control pills. Many drug interactions. CAN MAC BE PREVENTED? The bacteria that cause MAC are very common. It is ...

Evaluation of testing strategies to identify infected animals at a single round of testing within dairy herds known to be infected with Mycobacterium avium ssp. paratuberculosis.

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More, S J; Cameron, A R; Strain, S; Cashman, W; Ezanno, P; Kenny, K; Fourichon, C; Graham, D

2015-08-01

As part of a broader control strategy within herds known to be infected with Mycobacterium avium ssp. paratuberculosis (MAP), individual animal testing is generally conducted to identify infected animals for action, usually culling. Opportunities are now available to quantitatively compare different testing strategies (combinations of tests) in known infected herds. This study evaluates the effectiveness, cost, and cost-effectiveness of different testing strategies to identify infected animals at a single round of testing within dairy herds known to be MAP infected. A model was developed, taking account of both within-herd infection dynamics and test performance, to simulate the use of different tests at a single round of testing in a known infected herd. Model inputs included the number of animals at different stages of infection, the sensitivity and specificity of each test, and the costs of testing and culling. Testing strategies included either milk or serum ELISA alone or with fecal culture in series. Model outputs included effectiveness (detection fraction, the proportion of truly infected animals in the herd that are successfully detected by the testing strategy), cost, and cost-effectiveness (testing cost per true positive detected, total cost per true positive detected). Several assumptions were made: MAP was introduced with a single animal and no management interventions were implemented to limit within-herd transmission of MAP before this test. In medium herds, between 7 and 26% of infected animals are detected at a single round of testing, the former using the milk ELISA and fecal culture in series 5 yr after MAP introduction and the latter using fecal culture alone 15 yr after MAP introduction. The combined costs of testing and culling at a single round of testing increases with time since introduction of MAP infection, with culling costs being much greater than testing costs. The cost-effectiveness of testing varied by testing strategy. It was also

Cellular composition of granulomatous lesions in gut-associated lymphoid tissues of goats during the first year after experimental infection with Mycobacterium avium subsp. paratuberculosis.

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Krüger, C; Köhler, H; Liebler-Tenorio, E M

2015-01-15

Mycobacterium avium subsp. paratuberculosis (MAP) causes lesions in naturally and experimentally infected ruminants which greatly differ in severity, cellular composition and number of mycobacteria. Morphologically distinct lesions are already found during the clinically inapparent phase of infection. The complex local host response and number of MAP were characterized at the initial sites of lesions, organized gut-associated lymphoid tissue, in experimentally infected goats. Tissues were collected at 3, 6, 9 and 12 month post-inoculation (mpi) from goat kids that had orally received 10 times 10mg of bacterial wet mass of MAP (JII-1961). The cellular composition of lesions in Peyer's patches in the jejunum and next to the ileocecal valve was evaluated in 21 MAP-inoculated goats, where lesions were compared with unaltered tissue of six control goats. CD68+, CD4+, CD8+, γδ T lymphocytes, B lymphocytes and plasma cells, MHC class II+ and CD25+ cells were demonstrated by immunohistochemistry in serial cryostat sections. At 3 mpi, extensive granulomatous infiltrates predominated, consisting of numerous epitheloid cells admixed with many CD4 and γδ T lymphocytes. Only single MAP were detected. This indicates a strong cellular immune reaction able to control MAP infection. γδ T lymphocytes were markedly increased in this type of lesion which may reflect their important role early in the pathogenesis of paratuberculosis. At 9 and 12 mpi, divergent lesions were observed which may reflect different outcomes of host-pathogen interactions. In five goats, minimal granulomatous lesions were surrounded by extensive lymphoplasmacytic infiltrates and no MAP were detected by immunohistochemistry. This was interpreted as effective host response that was able to eliminate MAP locally. In three goats, decreased numbers of lymphocytes, but extensive granulomatous infiltrates with numerous epitheloid cells containing increased numbers of mycobacteria were seen. This shift of the

Increasing Recovery of Nontuberculous Mycobacteria from Respiratory Specimens over a 10-Year Period in a Tertiary Referral Hospital in South Korea.

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Koh, Won-Jung; Chang, Boksoon; Jeong, Byeong-Ho; Jeon, Kyeongman; Kim, Su-Young; Lee, Nam Yong; Ki, Chang-Seok; Kwon, O Jung

2013-11-01

The number of patients with pulmonary disease caused by nontuberculous mycobacteria (NTM) has been increasing worldwide. The aim of this study was to evaluate long-term trends in the NTM recovery rate from respiratory specimens over a 10-year period in a tertiary referral hospital in South Korea. We retrospectively reviewed the records of mycobacterial cultures of respiratory specimens at Samsung Medical Center from January 2001 to December 2011. During the study period, 32,841 respiratory specimens from 10,563 patients were found to be culture-positive for mycobacteria. These included 12,619 (38%) Mycobacterium tuberculosis and 20,222 (62%) NTM isolates. The proportion of NTM among all positive mycobacterial cultures increased from 43% (548/1,283) in 2001 to 70% (3,341/4,800) in 2011 (ptrend). The recovery rate of NTM isolates from acid-fast bacilli smear-positive specimens increased from 9% (38/417) in 2001 to 64% (1,284/1,997) in 2011 (ptrend). The proportion of positive liquid cultures was higher for NTM than for M. tuberculosis (p<0.001). The most frequently isolated NTM were Mycobacterium avium-intracellulare complex (53%) and Mycobacterium abscessus-massiliense complex (25%). The recovery rate of NTM from respiratory specimens in South Korea has increased steadily.

Complete genome sequence and comparative genomic analysis of Mycobacterium massiliense JCM 15300 in the Mycobacterium abscessus group reveal a conserved genomic island MmGI-1 related to putative lipid metabolism.

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Tsuyoshi Sekizuka

Full Text Available Mycobacterium abscessus group subsp., such as M. massiliense, M. abscessus sensu stricto and M. bolletii, are an environmental organism found in soil, water and other ecological niches, and have been isolated from respiratory tract infection, skin and soft tissue infection, postoperative infection of cosmetic surgery. To determine the unique genetic feature of M. massiliense, we sequenced the complete genome of M. massiliense type strain JCM 15300 (corresponding to CCUG 48898. Comparative genomic analysis was performed among Mycobacterium spp. and among M. abscessus group subspp., showing that additional ß-oxidation-related genes and, notably, the mammalian cell entry (mce operon were located on a genomic island, M. massiliense Genomic Island 1 (MmGI-1, in M. massiliense. In addition, putative anaerobic respiration system-related genes and additional mycolic acid cyclopropane synthetase-related genes were found uniquely in M. massiliense. Japanese isolates of M. massiliense also frequently possess the MmGI-1 (14/44, approximately 32% and three unique conserved regions (26/44; approximately 60%, 34/44; approximately 77% and 40/44; approximately 91%, as well as isolates of other countries (Malaysia, France, United Kingdom and United States. The well-conserved genomic island MmGI-1 may play an important role in high growth potential with additional lipid metabolism, extra factors for survival in the environment or synthesis of complex membrane-associated lipids. ORFs on MmGI-1 showed similarities to ORFs of phylogenetically distant M. avium complex (MAC, suggesting that horizontal gene transfer or genetic recombination events might have occurred within MmGI-1 among M. massiliense and MAC.

Cryptosporidium avium n. sp (Apicomplexa: Cryptosporidiidae) in birds

Czech Academy of Sciences Publication Activity Database

Holubová, Nikola; Sak, Bohumil; Horčičková, Michaela; Hlásková, Lenka; Květoňová, Dana; Menchaca, S.; McEvoy, J.; Kváč, Martin

2016-01-01

Roč. 115, č. 6 (2016), s. 2243-2251 ISSN 0932-0113 R&D Projects: GA ČR GA15-01090S Institutional support: RVO:60077344 Keywords : Cryptosporidium avium * morphology * molecular analyses * transmission studies * Cryptosporidium avian genotype V Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine Impact factor: 2.329, year: 2016

Polymorphisms of twenty regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis

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Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans or animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and the other members o...

The Mycobacterium tuberculosis homologue of the Mycobacterium ...

African Journals Online (AJOL)

With the completion of genome sequencing of Mycobacterium tuberculosis and upsurge in the incidence of M. tuberculosis infection worldwide partly as a result of HIV pandemic, there is need for rationale approach to vaccine and chemotherapy discoveries for M. tuberculosis. The homologue of mig gene of. Mycobacterium ...

Combating highly resistant emerging pathogen Mycobacterium abscessus and Mycobacterium tuberculosis with novel salicylanilide esters and carbamates.

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Baranyai, Zsuzsa; Krátký, Martin; Vinšová, Jarmila; Szabó, Nóra; Senoner, Zsuzsanna; Horváti, Kata; Stolaříková, Jiřina; Dávid, Sándor; Bősze, Szilvia

2015-08-28

In the Mycobacterium genus over one hundred species are already described and new ones are periodically reported. Species that form colonies in a week are classified as rapid growers, those requiring longer periods (up to three months) are the mostly pathogenic slow growers. More recently, new emerging species have been identified to lengthen the list, all rapid growers. Of these, Mycobacterium abscessus is also an intracellular pathogen and it is the most chemotherapy-resistant rapid-growing mycobacterium. In addition, the cases of multidrug-resistant Mycobacterium tuberculosis infection are also increasing. Therefore there is an urgent need to find new active molecules against these threatening strains. Based on previous results, a series of salicylanilides, salicylanilide 5-chloropyrazinoates and carbamates was designed, synthesized and characterised. The compounds were evaluated for their in vitro activity on M. abscessus, susceptible M. tuberculosis H37Rv, multidrug-resistant (MDR) M. tuberculosis MDR A8, M. tuberculosis MDR 9449/2006 and on the extremely-resistant Praha 131 (XDR) strains. All derivatives exhibited a significant activity with minimum inhibitory concentrations (MICs) in the low micromolar range. Eight salicylanilide carbamates and two salicylanilide esters exhibited an excellent in vitro activity on M. abscessus with MICs from 0.2 to 2.1 μM, thus being more effective than ciprofloxacin and gentamicin. This finding is potentially promising, particularly, as M. abscessus is a threateningly chemotherapy-resistant species. M. tuberculosis H37Rv was inhibited with MICs from 0.2 μM, and eleven compounds have lower MICs than isoniazid. Salicylanilide esters and carbamates were found that they were effective also on MDR and XDR M. tuberculosis strains with MICs ≥1.0 μM. The in vitro cytotoxicity (IC50) was also determined on human MonoMac-6 cells, and selectivity index (SI) of the compounds was established. In general, salicylanilide

Reduction of Mycobacterium avium ssp. paratuberculosis in colostrum: Development and validation of 2 methods, one based on curdling and one based on centrifugation.

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Verhegghe, M; Rasschaert, G; Herman, L; Goossens, K; Vandaele, L; De Bleecker, K; Vlaemynck, G; Heyndrickx, M; De Block, J

2017-05-01

The aim of this study was to develop and validate 2 protocols (for use on-farm and at a central location) for the reduction of Mycobacterium avium ssp. paratuberculosis (MAP) in colostrum while preserving beneficial immunoglobulins (IgG). The on-farm protocol was based on curdling of the colostrum, where the IgG remain in the whey and the MAP bacteria are trapped in the curd. First, the colostrum was diluted with water (2 volumes colostrum to 1 volume water) and 2% rennet was added. After incubation (1 h at 32°C), the curd was cut and incubated again, after which whey and curd were separated using a cheesecloth. The curd was removed and milk powder was added to the whey. Approximately 1 log reduction in MAP counts was achieved. A reduction in total proteins and IgG was observed due to initial dilution of the colostrum. After curd formation, more than 95% of the immunoglobulins remained in the whey fraction. The semi-industrial protocol was based on centrifugation, which causes MAP to precipitate, while the IgG remain in the supernatant. This protocol was first developed in the laboratory. The colostrum was diluted with skimmed colostrum (2 volumes colostrum to 1 volume skimmed colostrum), then skimmed and centrifuged (at 15,600 × g for 30 min at room temperature). We observed on average 1.5 log reduction in the MAP counts and a limited reduction in proteins and IgG in the supernatant. To obtain a semi-industrial protocol, dairy pilot appliances were evaluated and the following changes were applied to the protocol: after 2:1 dilution as above, the colostrum was skimmed and subsequently clarified, after which the cream was heat treated and added to the supernatant. To investigate the effect of the colostrum treatment on the nutritional value and palatability of the colostrum and the IgG transfer, an animal experiment was conducted with 24 calves. Six received the dam's colostrum, 6 were given untreated purchased colostrum (control), and 2 groups of 6 calves

Mycobacterium saopaulense sp. nov., a rapidly growing mycobacterium closely related to members of the Mycobacterium chelonae--Mycobacterium abscessus group.

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Nogueira, Christiane Lourenço; Whipps, Christopher M; Matsumoto, Cristianne Kayoko; Chimara, Erica; Droz, Sara; Tortoli, Enrico; de Freitas, Denise; Cnockaert, Margo; Palomino, Juan Carlos; Martin, Anandi; Vandamme, Peter; Leão, Sylvia Cardoso

2015-12-01

Five isolates of non-pigmented, rapidly growing mycobacteria were isolated from three patients and,in an earlier study, from zebrafish. Phenotypic and molecular tests confirmed that these isolates belong to the Mycobacterium chelonae-Mycobacterium abscessus group, but they could not be confidently assigned to any known species of this group. Phenotypic analysis and biochemical tests were not helpful for distinguishing these isolates from other members of the M. chelonae–M.abscessus group. The isolates presented higher drug resistance in comparison with other members of the group, showing susceptibility only to clarithromycin. The five isolates showed a unique PCR restriction analysis pattern of the hsp65 gene, 100 % similarity in 16S rRNA gene and hsp65 sequences and 1-2 nt differences in rpoB and internal transcribed spacer (ITS) sequences.Phylogenetic analysis of a concatenated dataset including 16S rRNA gene, hsp65, and rpoB sequences from type strains of more closely related species placed the five isolates together, as a distinct lineage from previously described species, suggesting a sister relationship to a group consisting of M. chelonae, Mycobacterium salmoniphilum, Mycobacterium franklinii and Mycobacterium immunogenum. DNA–DNA hybridization values .70 % confirmed that the five isolates belong to the same species, while values ,70 % between one of the isolates and the type strains of M. chelonae and M. abscessus confirmed that the isolates belong to a distinct species. The polyphasic characterization of these isolates, supported by DNA–DNA hybridization results,demonstrated that they share characteristics with M. chelonae–M. abscessus members, butconstitute a different species, for which the name Mycobacterium saopaulense sp. nov. is proposed. The type strain is EPM10906T (5CCUG 66554T5LMG 28586T5INCQS 0733T).

Capsular glucan and intracellular glycogen of Mycobacterium tuberculosis: biosynthesis and impact on the persistence in mice

DEFF Research Database (Denmark)

Sambou, Tounkang; Dinadayala, Premkumar; Stadthagen, Gustavo

2008-01-01

Mycobacterium tuberculosis and other pathogenic mycobacterial species produce large amounts of a glycogen-like alpha-glucan that represents the major polysaccharide of their outermost capsular layer. To determine the role of the surface-exposed glucan in the physiology and virulence of these bact......Mycobacterium tuberculosis and other pathogenic mycobacterial species produce large amounts of a glycogen-like alpha-glucan that represents the major polysaccharide of their outermost capsular layer. To determine the role of the surface-exposed glucan in the physiology and virulence...... of these bacteria, orthologues of the glg genes involved in the biosynthesis of glycogen in Escherichia coli were identified in M. tuberculosis H37Rv and inactivated by allelic replacement. Biochemical analyses of the mutants and complemented strains indicated that the synthesis of glucan and glycogen involves...... the alpha-1,4-glucosyltransferases Rv3032 and GlgA (Rv1212c), the ADP-glucose pyrophosphorylase GlgC (Rv1213) and the branching enzyme GlgB (Rv1326c). Disruption of glgC reduced by half the glucan and glycogen contents of M. tuberculosis, whereas the inactivation of glgA and Rv3032 affected the production...

Mycobacterial and nonbacterial pulmonary complications in hospitalized patients with human immunodeficiency virus infection: A prospective, cohort study

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Afessa Bekele

2001-09-01

Full Text Available Abstract Background A prospective observational study was done to describe nonbacterial pulmonary complications in hospitalized patients with human immunodeficiency virus (HIV infection. Methods The study included 1,225 consecutive hospital admissions of 599 HIV-infected patients treated from April 1995 through March 1998. Data included demographics, risk factors for HIV infection, Acute Physiology and Chronic Health Evaluation (APACHE II score, pulmonary complications, CD4+ lymphocyte count, hospital stay and case-fatality rate. Results Patient age (mean ± SD was 38.2 ± 8.9 years, 62% were men, and 84% were African American. The median APACHE II score was 14, and median CD4+ lymphocyte count was 60/μL. Pulmonary complications were Pneumocystis carinii pneumonia (85 in 78 patients, Mycobacterium avium complex (51 in 38, Mycobacterium tuberculosis (40 in 35, Mycobacterium gordonae (11 in 11, Mycobacterium kansasii (10 in 9, Cytomegalovirus (10 in 10, Nocardia asteroides (3 in 3, fungus ball (2 in 2, respiratory syncytial virus (1, herpes simplex virus (1, Histoplasma capsulatum (1, lymphoma (3 in 3, bronchogenic carcinoma (2 in 2, and Kaposi sarcoma (1. The case-fatality rate of patients was 11% with Pneumocystis carinii pneumonia; 5%, Mycobacterium tuberculosis; 6%, Mycobacterium avium complex; and 7%, noninfectious pulmonary complications. Conclusion Most pulmonary complications in hospitalized patients with HIV are from Pneumocystis and mycobacterial infection.

Getting “Inside” Type I IFNs: Type I IFNs in Intracellular Bacterial Infections

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Deann T. Snyder

2017-01-01

Full Text Available Type I interferons represent a unique and complex group of cytokines, serving many purposes during innate and adaptive immunity. Discovered in the context of viral infections, type I IFNs are now known to have myriad effects in infectious and autoimmune disease settings. Type I IFN signaling during bacterial infections is dependent on many factors including whether the infecting bacterium is intracellular or extracellular, as different signaling pathways are activated. As such, the repercussions of type I IFN induction can positively or negatively impact the disease outcome. This review focuses on type I IFN induction and downstream consequences during infection with the following intracellular bacteria: Chlamydia trachomatis, Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, Francisella tularensis, Brucella abortus, Legionella pneumophila, and Coxiella burnetii. Intracellular bacterial infections are unique because the bacteria must avoid, circumvent, and even co-opt microbial “sensing” mechanisms in order to reside and replicate within a host cell. Furthermore, life inside a host cell makes intracellular bacteria more difficult to target with antibiotics. Because type I IFNs are important immune effectors, modulating this pathway may improve disease outcomes. But first, it is critical to understand the context-dependent effects of the type I IFN pathway in intracellular bacterial infections.

Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

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Gentry-Weeks, C R; Hultsch, A L; Kelly, S M; Keith, J M; Curtiss, R

1992-01-01

Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the

The Warburg effect in mycobacterial granulomas is dependent on the recruitment and activation of macrophages by interferon-γ

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Appelberg, Rui; Moreira, Diana; Barreira-Silva, Palmira; Borges, Margarida; Silva, Letícia; Dinis-Oliveira, Ricardo Jorge; Resende, Mariana; Correia-Neves, Margarida; Jordan, Michael B; Ferreira, Nuno C; Abrunhosa, Antero J; Silvestre, Ricardo

2015-01-01

Granulomas are the hallmark of mycobacterial disease. Here, we demonstrate that both the cell recruitment and the increased glucose consumption in granulomatous infiltrates during Mycobacterium avium infection are highly dependent on interferon-γ (IFN-γ). Mycobacterium avium-infected mice lacking IFN-γ signalling failed to developed significant inflammatory infiltrations and lacked the characteristic uptake of the glucose analogue fluorine-18-fluorodeoxyglucose (FDG). To assess the role of macrophages in glucose uptake we infected mice with a selective impairment of IFN-γ signalling in the macrophage lineage (MIIG mice). Although only a partial reduction of the granulomatous areas was observed in infected MIIG mice, the insensitivity of macrophages to IFN-γ reduced the accumulation of FDG. In vivo, ex vivo and in vitro assays showed that macrophage activated by IFN-γ displayed increased rates of glucose uptake and in vitro studies showed also that they had increased lactate production and increased expression of key glycolytic enzymes. Overall, our results show that the activation of macrophages by IFN-γ is responsible for the Warburg effect observed in organs infected with M. avium. PMID:25807843

Photodynamic inactivation of the models Mycobacterium phlei and Mycobacterium smegmatis in vitro

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Bruce-Micah, R.; Gamm, U.; Hüttenberger, D.; Cullum, J.; Foth, H.-J.

2009-07-01

Photodynamic inactivation (PDI) of bacterial strains presents an attractive potential alternative to antibiotic therapies. Success is dependent on the effective accumulation in bacterial cells of photochemical substances called photosensitizers, which are usually porphyrins or their derivatives. The kinetics of porphyrin synthesis after treatment with the precursor ALA and the accumulation of the Chlorin e6 and the following illumination were studied. The goal was to estimate effectivity of the destructive power of these PS in vitro in respect of the physiological states of Mycobacteria. So the present results examine the cell destruction by PDI using ALA-induced Porphyrins and Chlorin e6 accumulated in Mycobacterium phlei and Mycobacterium smegmatis, which serve as models for the important pathogens Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium bovis. We could show that both Mycobacterium after ALA and Chlorin e6 application were killed by illumination with light of about 662 nm. A reduction of about 97% could be reached by using a lightdose of 70 mW/cm2.

Mycobacterium tuberculosis Rv3402c enhances mycobacterial survival within macrophages and modulates the host pro-inflammatory cytokines production via NF-kappa B/ERK/p38 signaling.

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Wu Li

Full Text Available Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis, a process which depends on an array of virulence factors to colonize and replicate within the host. The M. tuberculosis iron regulated open reading frame (ORF rv3402c, encoding a conserved hypothetical protein, was shown to be up-regulated upon infection in both human and mice macrophages. To explore the function of this ORF, we heterologously expressed the rv3402c gene in the non-pathogenic fast-growing Mycobacterium smegmatis strain, and demonstrated that Rv3402c, a cell envelope-associated protein, was able to enhance the intracellular survival of recombinant M. smegmatis. Enhanced growth was not found to be the result of an increased resistance to intracellular stresses, as growth of the Rv3402c expressing strain was unaffected by iron depletion, H2O2 exposure, or acidic conditions. Colonization of macrophages by M. smegmatis expressing Rv3402c was associated with substantial cell death and significantly greater amount of TNF-α and IL-1β compared with controls. Rv3402c-induced TNF-α and IL-1β production was found to be mediated by NF-κB, ERK and p38 pathway in macrophages. In summary, our study suggests that Rv3402c delivered in a live M. smegmatis vehicle can modify the cytokines profile of macrophage, promote host cell death and enhance the persistence of mycobacterium within host cells.

Expression of Mycobacterium smegmatis pyrazinamidase in Mycobacterium tuberculosis confers hypersensitivity to pyrazinamide and related amides.

Science.gov (United States)

Boshoff, H I; Mizrahi, V

2000-10-01

A pyrazinamidase (PZase)-deficient pncA mutant of Mycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 microg/ml), in accordance with the well-established role of pncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662-667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809-5814, 1998) or the M. tuberculosis pncA gene into the pncA mutant complemented its PZase/nicotinamidase defect. In both pzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. The pzaA-complemented strain was hypersensitive to PZA (MIC, /=20 microg/ml) and was also sensitive to benzamide (MIC, 20 microg/ml), unlike the wild-type and pncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 microg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 microg/ml in all cases) and rendered Escherichia coli hypersensitive for growth at low pH.

In vitro Inhibition of Mycobacterium smegmatis and Mycobacterium ...

African Journals Online (AJOL)

Some Nigerian plants used in traditional medicine to treat tuberculosis and/or some of its symptoms were screened for in vitro activity against Mycobacterium smegmatis and a clinical isolate of Mycobacterium tuberculosis. Only 3 of the 6 crude methanolic extracts of the 6 plant species exhibited inhibitory activities against ...

Real-time monitoring of mycobacterium genomic DNA with target-primed rolling circle amplification by a Au nanoparticle-embedded SPR biosensor.

Science.gov (United States)

Xiang, Yang; Zhu, Xiaoyan; Huang, Qing; Zheng, Junsong; Fu, Weiling

2015-04-15

In this study, we developed a surface plasmon resonance (SPR) DNA biosensor array based on target-primed rolling circle amplification (RCA) for isothermal and rapid detection of two pathogenic mycobacteria, Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium complex (MAC).The species-specific padlock probe (PLP) was designed to target the sequence in 16S-23S rRNA gene internal transcribed spacer (ITS). After ligation, the circularized PLP could be primed by the target sequence to initial RCA. The RCA performed simultaneously with the cleavage reaction to produce small fragments of single strand DNA which immediately hybridized with the probe immobilized on the sensor chip without denaturation. This process caused SPR angle changes on the chip surface, which made the detection for analysis from the solution achievable, and dynamic real-time RCA monitoring of mycobacterium possible. Besides, Au nanoparticles (AuNPs) were directly assembled onto the surface of the sensor chip via hexanedithiol (HDT) for the enhancement of sensitivity as a label-free detection system. Experimental results show that the signal enhancement by the target-primed RCA together with AuNPs-embedded surface caused at least10-fold increased sensitivity as compared with conventional RCA on bare SPR chip method. Within 40min amplification duration as low as 20amol of synthetic targets and 10(4)CFUmL(-1) of genomic DNA from clinical samples can be detected. The proposed method not only provides a simple design idea for liquid-phase amplification monitoring, but also apply it in clinical pathogen detection, which holds great promise in ultrasensitive bioassay in the future. Copyright © 2014. Published by Elsevier B.V.

Chlamydia psittaci and C. avium in feral pigeon (Columba livia domestica) droppings in two cities in the Netherlands.

Science.gov (United States)

Burt, Sara A; Röring, Romy E; Heijne, Marloes

2018-06-05

Feral pigeons (Columba livia domestica) live and breed in many city centres and contact with their droppings can be a hazard for human health if the birds carry Chlamydia psittaci. The aim of this study was to establish whether pigeon droppings in two Dutch cities (Utrecht and Haarlem) contain C. psittaci and/or C. avium, which could be a potential hazard for transmission to humans. In May 2017 seven feral pigeon 'hot spots' with between 5 and 40+ pigeons present were identified in two cities by visual observations over two days. During the following ten days fresh droppings were collected at these hot spots and the samples were pooled per three droppings to achieve 40-41 samples per city. Samples were analysed for Chlamydia DNA with a broad range 23S Chlamydiaceae Real-Time PCR and positive samples were tested with a specific C. psittaci and C. avium Real-Time PCR. Positive C. psittaci samples were genotyped. C. psittaci and C. avium were detected in both cities. For C. psittaci the prevalences in Utrecht and Haarlem were 2.4% and 7.5%, respectively; for C. avium 36.6% and 20.0%, respectively. One sample contained both species. All C. psittaci samples belonged to genotype B. C. psittaci and C. avium are present in feral pigeon droppings in Utrecht and Haarlem. Human contact with droppings from infected pigeons or inhalation of dust from dried droppings represent a potential hazard to public health.

Iron in intracellular infection: to provide or to deprive?

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Sandro eSilva-Gomes

2013-12-01

Full Text Available Due to their chemical versatility, transition metals were incorporated as cofactors for several basic metabolic pathways in living organisms. This same characteristic makes them potentially harmful, since they can be engaged in deleterious reactions like Fenton chemistry. As such, organisms have evolved highly specialized mechanisms to supply their own metal needs while keeping their toxic potential in check.This dual character comes into play in host-pathogen interactions, given that the host can either deprive the pathogen of these key nutrients or exploit them to induce toxicity towards the invading agent. Iron stands as the prototypic example of how a metal can be used to limit the growth of pathogens by nutrient deprivation, a mechanism widely studied in Mycobacterium infections. However, the host can also take advantage of iron-induced toxicity to control pathogen proliferation, as observed in infections caused by Leishmania. Whether we may harness either of the two pathways for therapeutical purposes is still ill-defined.In this review, we discuss how modulation of the host iron availability impacts the course of infections, focusing on those caused by two relevant intracellular pathogens, Mycobacterium and Leishmania.

A Negative Feedback Loop Between Autophagy and Immune Responses in Mycobacterium leprae Infection.

Science.gov (United States)

Ma, Yuelong; Zhang, Li; Lu, Jie; Shui, Tiejun; Chen, Jia; Yang, Jun; Yuan, Joanna; Liu, Yeqiang; Yang, Degang

2017-01-01

The obligate intracellular bacterium Mycobacterium leprae is the causative agent of leprosy and primarily infects macrophages, leading to irreversible nerve damage and deformities. So far, the underlying reasons allowing M. leprae to persist and propagate in macrophages, despite the presence of cellular immunity, are still a mystery. Here, we investigated the role of autophagy, a cellular process that degrades cytosolic materials and intracellular pathogens, in M. leprae infection. We found that live M. leprae infection of macrophages resulted in significantly elevated autophagy level. However, macrophages with high autophagy levels preferentially expressed lower levels of proinflammatory cytokines, including interleukin (IL)-1β, IL-6, IL-12, and tumor necrosis factor-α, and preferentially primed anti-inflammatory T cells responses, characterized by high IL-10 and low interferon-γ, granzyme B, and perforin responses. These anti-inflammatory T cells could suppress further induction of autophagy, leading to improved survival of intracellular M. leprae in infected macrophages. Therefore, these data demonstrated that although autophagy had a role in eliminating intracellular pathogens, the induction of autophagy resulted in anti-inflammatory immune responses, which suppressed autophagy in a negative feedback loop and allowed the persistence of M. leprae.

Mycobacterium persicum sp. nov., a novel species closely related to Mycobacterium kansasii and Mycobacterium gastri.

Science.gov (United States)

Shahraki, Abdolrazagh Hashemi; Trovato, Alberto; Mirsaeidi, Mehdi; Borroni, Emanuele; Heidarieh, Parvin; Hashemzadeh, Mohamad; Shahbazi, Narges; Cirillo, Daniela M; Tortoli, Enrico

2017-06-01

Four strains isolated in Iran from pulmonary specimens of unrelated patients are proposed as representative of a novel Mycobacterium species. Similarity, at the phenotypic level, with Mycobacterium kansasii is remarkable with the photochromogenic yellow pigmentation of the colonies being the salient feature. They differ, however, genotypically from this species and present unique sequences in 16S rRNA, hsp65 and rpoB genes. The average nucleotide identity and the genome-to-genome distance fully support the status of an independent species. The name proposed for this species is Mycobacterium persicum sp. nov. with AFPC-000227T (=DSM 104278T=CIP 111197T) as the type strain.

THE PERSISTENCE OF MYCOBACTERIUM AVIUM IN A DRINKING WATER DISTRIBUTION SYSTEM AFTER THE ADDITION OF FILTRATION TREATMENT

Science.gov (United States)

There is evidence that drinking water may be a source of pathogenic nontuberculous mycobacteria (NTM) infections in humans. One method by which NTM are believed to enter drinking water distribution systems is by their intracellular location within protozoa. Our goal was to determ...

The investigation of the truncated mbtA gene within the mycobactin cluster of Mycobacterium avium subspecies paratuberculosis as a novel diagnostic marker for real-time PCR.

Science.gov (United States)

de Kruijf, Marcel; Coffey, Aidan; O'Mahony, Jim

2017-05-01

The inability of Mycobacterium avium subspecies paratuberculosis (MAP) to produce endogenous mycobactin in-vitro is most likely due to the presence of a truncated mbtA gene within the mycobactin cluster of MAP. The main goal of this study was to investigate this unique mbtA truncation as a potential novel PCR diagnostic marker for MAP. Novel primers were designed that were located within the truncated region and the contiguous MAP2179 gene. Primers were evaluated against non-MAP isolates and no amplicons were generated. The detection limit of this mbtA-MAP2179 target was evaluated using a range of MAP DNA concentrations, MAP inoculated faecal material and 20 MAP isolates. The performance of mbtA-MAP2179 was compared to the established f57 target. The detection limits recorded for MAP K-10 DNA and from MAP K-10 inoculated faecal samples were 0.34pg and 10 4 CFU/g respectively for both f57 and mbtA-MAP2179. A detection limit of 10 3 CFU/g was recorded for both targets, but not achieved consistently. The detection limit of MAP from inoculated faecal material was successful at 10 3 CFU/g for mbtA-MAP2179 when FAM probe real-time PCR was used. A MAP cell concentration of 10 2 CFU/g was detected successfully, but again not consistently achieved. All 20 mycobacterial isolates were successfully identified as MAP by f57 and mbtA-MAP2179. Interestingly, the mbtA-MAP2179 real-time PCR assay resulted in the formation of a unique melting curve profile that contained two melting curve peaks rather than one single peak. This melting curve phenomenon was attributed towards the asymmetrical GC% distribution within the mbtA-MAP2179 amplicon. This study investigated the implementation of the mbtA-MAP2179 target as a novel diagnostic marker and the detection limits obtained with mbtA-MAP2179 were comparable to the established f57 target, making the mbtA-MAP2179 an adequate confirmatory target. Moreover, the mbtA-MAP2179 target could be implemented in multiplex real-time PCR assays and

Tuberculosis

Science.gov (United States)

Friend, Milton

1999-01-01

Avian tuberculosis is usually caused by the bacterium Mycobacterium avium. At least 20 different types of M. avium have been identified, only three of which are known to cause disease in birds. Other types of Mycobacterium rarely cause tuberculosis in most avian species; however, parrots, macaws, and other large perching birds are susceptible to human and bovine types of tuberculosis bacilli. Avian tuberculosis generally is transmitted by direct contact with infected birds, ingestion of contaminated feed and water, or contact with a contaminated environment. Inhalation of the bacterium can cause respiratory tract infections. Wild bird studies in the Netherlands disclosed tuberculosis-infected puncture-type injuries in birds of prey that fight at the nest site (kestrels) or on the ground (buteo-type buzzards), but tuberculosisinfected injuries were not found in accipiters (falco

Development of Piezoelectric DNA-Based Biosensor for Direct Detection of Mycobacterium Tuberculosis in Clinical Specimens

Directory of Open Access Journals (Sweden)

Thongchai KAEWPHINIT

2010-02-01

Full Text Available This study was focused on establishment of piezoelectric biosensor for direct detection of Mycobacterium tuberculosis (MTB in clinical specimens. The quartz crystal immobilized via 3-mercaptopropionic acid (MPA/avidin/DNA biotinylated probe on gold surface and hybridization of the DNA target to DNA biotinylated probe. The optimal concentration of MPA, avidin and 5’-biotinylated DNA probe for immobilization of specific DNA probe on gold surface were 15 mM, 0.1 mg/ml and 1.5 μM, respectively. The detection of genomic DNA digestion in the range from 0.5 to 30 μg/ml. The fabricated biosensor was evaluated through an examination of 200 samples. No cross hybridization were observed against M. avium complex (MAC and other microorganism. This target DNA preparation without amplification will reduce time consuming, costs, and the tedious step of amplification. This study can be extended to develop the new method which is high sensitivity, specificity, cheap, easy to use, and rapid for detection of MTB in many fields.

Growth and fruit bearing of the sweet cherry (Prunus avium L

African Journals Online (AJOL)

Radunic

2011-06-06

Jun 6, 2011 ... Modern intensive production of sweet cherry (Prunus avium L.) tends to planting of high ... the highest was recorded on "V", while the smallest was in Spanish bush. Training system and density did not affect the fruit weight.

Mycobacterium canettii Infection of Adipose Tissues.

Science.gov (United States)

Bouzid, Fériel; Brégeon, Fabienne; Poncin, Isabelle; Weber, Pascal; Drancourt, Michel; Canaan, Stéphane

2017-01-01

Adipose tissues were shown to host Mycobacterium tuberculosis which is persisting inside mature adipocytes. It remains unknown whether this holds true for Mycobacterium canettii , a rare representative of the M. tuberculosis complex responsible for lymphatic and pulmonary tuberculosis. Here, we infected primary murine white and brown pre-adipocytes and murine 3T3-L1 pre-adipocytes and mature adipocytes with M. canettii and M. tuberculosis as a positive control. Both mycobacteria were able to infect 18-22% of challenged primary murine pre-adipocytes; and to replicate within these cells during a 7-day experiment with the intracellular inoculums being significantly higher in brown than in white pre-adipocytes for M. canettii ( p = 0.02) and M. tuberculosis ( p = 0.03). Further in-vitro infection of 3T3-L1 mature adipocytes yielded 9% of infected cells by M. canettii and 17% of infected cells by M. tuberculosis ( p = 0.001). Interestingly, M. canettii replicated and accumulated intra-cytosolic lipid inclusions within mature adipocytes over a 12-day experiment; while M. tuberculosis stopped replicating at day 3 post-infection. These results indicate that brown pre-adipocytes could be one of the potential targets for M. tuberculosis complex mycobacteria; and illustrate differential outcome of M. tuberculosis complex mycobacteria into adipose tissues. While white adipose tissue is an unlikely sanctuary for M. canettii , it is still an open question whether M. canettii and M. tuberculosis could persist in brown adipose tissues.

Mycobacterium franklinii sp. nov., a species closely related to members of the Mycobacterium chelonae-Mycobacterium abscessus group.

Science.gov (United States)

Lourenço Nogueira, Christiane; Simmon, Keith E; Chimara, Erica; Cnockaert, Margo; Carlos Palomino, Juan; Martin, Anandi; Vandamme, Peter; Brown-Elliott, Barbara A; Wallace, Richard; Cardoso Leão, Sylvia

2015-07-01

Two isolates from water, D16Q19 and D16R27, were shown to be highly similar in their 16S rRNA, 16S-23S internal transcribed spacer (ITS), hsp65 and rpoB gene sequences to 'Mycobacterium franklinii' DSM 45524, described in 2011 but with the name not validly published. They are all nonpigmented rapid growers and are related phenotypically and genetically to the Mycobacterium chelonae-Mycobacterium abscessus group. Extensive characterization by phenotypic analysis, biochemical tests, drug susceptibility testing, PCR restriction enzyme analysis of the hsp65 gene and ITS, DNA sequencing of housekeeping genes and DNA-DNA hybridization demonstrated that 'M. franklinii' DSM 45524, D16Q19 and D16R27 belong to a single species that is separated from other members of the M. chelonae-M. abscessus group. On the basis of these results we propose the formal recognition of Mycobacterium franklinii sp. nov. Strain DSM 45524(T) ( = ATCC BAA-2149(T)) is the type strain.

Non-tuberculous mycobacterial soft tissue swelling in an immunocompetent patient

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Virendra S Athavale

2014-01-01

Full Text Available Non-tuberculosis mycobacteria (NTM include those mycobacterium species that are not members of the Mycobacterium tuberculosis complex. They assumed significance with the growing pandemic of the acquired immune deficiency syndrome (AIDS since the 1980s, when Mycobacterium avium infections were found to be associated with AIDS. However, the epidemiology of NTM disease among patients without Human immunodeficiency virus infections is not well documented. We report a case of NTM soft tissue swelling in an immunocompetent 18-year-old male who responded well to local excision and antitubercular treatment.

Immune reconstitution inflammatory syndrome due to Mycobacterium avium complex successfully followed up using 18 F-fluorodeoxyglucose positron emission tomography-computed tomography in a patient with human immunodeficiency virus infection: A case report

International Nuclear Information System (INIS)

Namkoong, Ho; Fujiwara, Hiroshi; Ishii, Makoto; Yagi, Kazuma; Haraguchi, Mizuha; Matsusaka, Masako; Suzuki, Shoji; Asakura, Takanori; Asami, Takahiro; Saito, Fumitake; Fukunaga, Koichi; Tasaka, Sadatomo; Betsuyaku, Tomoko; Hasegawa, Naoki

2015-01-01

In human immunodeficiency virus (HIV)-infected patients, immune reconstitution inflammatory syndrome (IRIS) due to nontuberculous mycobacteria (NTM) infection is one of the most difficult types of IRIS to manage. 18 F-fluorodeoxyglucose positron emission tomography/computed tomography ( 18 F-FDG PET/CT) has been suggested as a useful tool for evaluating the inflammatory status of HIV-infected patients. We present the first case of Mycobacterium avium complex (MAC)-associated IRIS (MAC-IRIS) that was successfully followed up using 18 F-FDG PET/CT. A 44-year-old homosexual Japanese man was referred to our hospital with fever and dyspnea. He was diagnosed with Pneumocystis jiroveci pneumonia and found to be HIV positive. After the initiation of combined antiretroviral therapy (cART), the patient’s mediastinal and bilateral hilar lymphadenopathy gradually enlarged, and bilateral infiltrates appeared in the upper lung fields. 18 F-FDG PET/CT was performed five months after the initiation of cART and showed intense accumulation of fluorodeoxyglucose (FDG) corresponding to the lesions of infiltration as well as the mediastinal and bilateral hilar lymphadenopathy. A bronchial wash culture and pathology findings led to a diagnosis of MAC-IRIS. Anti-mycobacterial chemotherapy with rifampicin, ethambutol, clarithromycin, and levofloxacin was started. One year after the chemotherapy was initiated, there was a significant reduction in FDG uptake in the area of the lesions except in the mediastinal lymph node. This implied incomplete resolution of the MAC-IRIS-related inflammation. Anti-mycobacterial chemotherapy was continued because of the residual lesion. To date, the patient has not experienced a recurrence of MAC-IRIS, a period of nine months. We present a case of MAC-IRIS in an HIV-infected patient whose disease activity was successfully followed up using 18 F-FDG PET/CT. Our data suggest that 18 F-FDG PET/CT is useful for evaluating the disease activity of NTM-IRIS and

The microRNA miR-29 controls innate and adaptive immune responses to intracellular bacterial infection by targeting interferon-γ.

Science.gov (United States)

Ma, Feng; Xu, Sheng; Liu, Xingguang; Zhang, Qian; Xu, Xiongfei; Liu, Mofang; Hua, Minmin; Li, Nan; Yao, Hangping; Cao, Xuetao

2011-07-24

Interferon-γ (IFN-γ) has a critical role in immune responses to intracellular bacterial infection. MicroRNAs (miRNAs) are important in the regulation of innate and adaptive immunity. However, whether miRNAs can directly target IFN-γ and regulate IFN-γ production post-transcriptionally remains unknown. Here we show that infection of mice with Listeria monocytogenes or Mycobacterium bovis bacillus Calmette-Guérin (BCG) downregulated miR-29 expression in IFN-γ-producing natural killer cells, CD4(+) T cells and CD8(+) T cells. Moreover, miR-29 suppressed IFN-γ production by directly targeting IFN-γ mRNA. We developed mice with transgenic expression of a 'sponge' target to compete with endogenous miR-29 targets (GS29 mice). We found higher serum concentrations of IFN-γ and lower L. monocytogenes burdens in L. monocytogenes-infected GS29 mice than in their littermates. GS29 mice had enhanced T helper type 1 (T(H)1) responses and greater resistance to infection with BCG or Mycobacterium tuberculosis. Therefore, miR-29 suppresses immune responses to intracellular pathogens by targeting IFN-γ.

Disease caused by non-tuberculous mycobacteria: diagnostic procedures and treatment evaluation in the North of Buenos Aires Province

Directory of Open Access Journals (Sweden)

Belén Imperiale

2012-03-01

Full Text Available Non-tuberculous mycobacteria (NTM have emerged as pathogens frequently associated to HIV co-infection. The aims of this study were to describe the clinical importance of NTM in patients from the North of Buenos Aires Province and the drug-susceptibility patterns in relation with the therapy used. A total of 23,624 clinical specimens were investigated during the period 2004-2010. Ziehl-Neelsen stain and cultures were used for diagnosis. Molecular and biochemical tests were performed to identify the mycobacteria. TB and mycobacterioses cases were 2 118 and 108 respectively. Sixteen NTM species were found: Mycobacterium avium and Mycobacterium intracellulare as the main causative agents. Infections produced by more than one species at the same time were confirmed (4 cases. Macrolides and fluoroquinolones were the most active in vitro drugs. Treatment evaluation showed that 68.0 % of the cases completed the therapy, 20 % died; and 12 % were relapses. The cases in which the treatment outcome was evaluated received an individual tailor-made therapeutic scheme including those drugs showing in vitro activity and presumed in vivo usefulness. More than a quarter of the patients had HIV co-infection and the majority of the deaths were associated with this co-infection.Enfermedad causada por micobacterias no tuberculosas: diagnóstico y evaluación del tratamiento en el norte del Gran Buenos Aires. Las micobacterias no tuberculosas (MNT emergieron como patógenos frecuentemente asociados a la co-infección con el HIV. EL objetivo del estudio fue describir la importancia clínica de las MNT en pacientes de la región norte de la provincia de Buenos Aires y los patrones de drogo-sensibilidad en relación con la terapia empleada. Se investigó un total de 23.624 especímenes clínicos durante, el período 2004-2010. La tinción de Ziehl-Neelsen y los cultivos se utilizaron para diagnóstico. Las micobacterias fueron identificadas mediante pruebas bioqu

Analysis of Biobanked Serum from a Mycobacterium avium subsp paratuberculosis Bovine Infection Model Confirms the Remarkable Stability of Circulating miRNA Profiles and Defines a Bovine Serum miRNA Repertoire.

Directory of Open Access Journals (Sweden)

Ronan G Shaughnessy

Full Text Available Johne's Disease (JD is a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP. Current disease control strategies are hampered by the lack of sensitive and specific diagnostic modalities. Therefore, novel diagnostic and prognostic tools are needed, and circulating microRNAs (miRNAs may hold potential in this area. The aims of this study were twofold: (i to address the stability of miRNA in bovine sera from biobanked samples, and (ii to assess the potential of miRNAs as biomarkers for JD disease progression. To address these aims we used bovine sera from an experimental MAP infection model that had been stored at -20°C for over a decade, allowing us to also assess the stability of miRNA profiles in biobanked serum samples through comparison with fresh sera. Approximately 100-200 intact miRNAs were identified in each sample with 83 of these being consistently detected across all 57 samples. The miRNA profile of the biobanked sera stored at -20°C for over 10 years was highly similar to the profile of <1 year-old sera stored at -80°C, with an overlap of 73 shared miRNAs. IsomiR analysis also indicated a distinct bovine serum-specific isomiR profile as compared to previously reported bovine macrophage miRNA profiles. To explore the prognostic potential of miRNA profiles cattle defined as seropositive for anti-MAP antibodies (n = 5 were compared against seronegative cattle (n = 7. No significant differential expressed miRNAs were detected at either the early (6 months or late (43, 46 and 49 months intervals (FDR≤0.05, fold-change≥1.5 across seropositive or seronegative animals. However, comparing pre-infection sera to the early and late time-points identified increased miR-29a and miR-92b abundance (2-fold that may be due to blood-cell population changes over time (P<0.001. In conclusion our study has demonstrated that bovine circulating miRNAs retain their integrity under long-term sub-optimal storage

Structural analysis of biofilm formation by rapidly and slowly growing nontuberculous mycobacteria

Science.gov (United States)

Mycobacterium avium complex (MAC) and rapidly growing mycobacteria (RGM) such as M. abscessus, M. mucogenicum, M. chelonae and M. fortuitum, implicated in healthcare-associated infections, are often isolated from potable water supplies as part of the microbial flora. To understa...

Resistance mechanisms and drug susceptibility testing of nontuberculous mycobacteria.

NARCIS (Netherlands)

Ingen, J. van; Boeree, M.J.; Soolingen, D. van; Mouton, J.W.

2012-01-01

Nontuberculous mycobacteria (NTM) are increasingly recognized as causative agents of opportunistic infections in humans. For most NTM infections the therapy of choice is drug treatment, but treatment regimens differ by species, in particular between slow (e.g. Mycobacterium avium complex,

Mycobacterium bovis and Other Uncommon Members of the Mycobacterium tuberculosis Complex.

Science.gov (United States)

Esteban, Jaime; Muñoz-Egea, Maria-Carmen

2016-12-01

Since its discovery by Theobald Smith, Mycobacterium bovis has been a human pathogen closely related to animal disease. At present, M. bovis tuberculosis is still a problem of importance in many countries and is considered the main cause of zoonotic tuberculosis throughout the world. Recent development of molecular epidemiological tools has helped us to improve our knowledge about transmission patterns of this organism, which causes a disease indistinguishable from that caused by Mycobacterium tuberculosis. Diagnosis and treatment of this mycobacterium are similar to those for conventional tuberculosis, with the important exceptions of constitutive resistance to pyrazinamide and the fact that multidrug-resistant and extremely drug-resistant M. bovis strains have been described. Among other members of this complex, Mycobacterium africanum is the cause of many cases of tuberculosis in West Africa and can be found in other areas mainly in association with immigration. M. bovis BCG is the currently available vaccine for tuberculosis, but it can cause disease in some patients. Other members of the M. tuberculosis complex are mainly animal pathogens with only exceptional cases of human disease, and there are even some strains, like "Mycobacterium canettii," which is a rare human pathogen that could have an important role in the knowledge of the evolution of tuberculosis in the history.

The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae

KAUST Repository

Phelan, Jody; Maitra, Arundhati; McNerney, Ruth; Nair, Mridul; Gupta, Antima; Coll, Francesc; Pain, Arnab; Bhakta, Sanjib; Clark, Taane G.

2015-01-01

Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02 Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.

The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae

KAUST Repository

Phelan, Jody

2015-06-04

Mycobacterium aurum (M. aurum) is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02 Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related) to 96.2% for rrs (streptomycin, capreomycin). We observed two homologous genes encoding the catalase-peroxidase enzyme (katG) that is associated with resistance to isoniazid. Similarly, two embB homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum, this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.

The draft genome of Mycobacterium aurum , a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae

Directory of Open Access Journals (Sweden)

Jody Phelan

2015-01-01

Full Text Available Mycobacterium aurum (M. aurum is an environmental mycobacteria that has previously been used in studies of anti-mycobacterial drugs due to its fast growth rate and low pathogenicity. The M. aurum genome has been sequenced and assembled into 46 contigs, with a total length of 6.02 Mb containing 5684 annotated protein-coding genes. A phylogenetic analysis using whole genome alignments positioned M. aurum close to Mycobacterium vaccae and Mycobacterium vanbaalenii, within a clade related to fast-growing mycobacteria. Large-scale genomic rearrangements were identified by comparing the M. aurum genome to those of Mycobacterium tuberculosis and Mycobacterium leprae. M. aurum orthologous genes implicated in resistance to anti-tuberculosis drugs in M. tuberculosis were observed. The sequence identity at the DNA level varied from 68.6% for pncA (pyrazinamide drug-related to 96.2% for rrs (streptomycin, capreomycin. We observed two homologous genes encoding the catalase-peroxidase enzyme (katG that is associated with resistance to isoniazid. Similarly, two emb B homologues were identified in the M. aurum genome. In addition to describing for the first time the genome of M. aurum , this work provides a resource to aid the use of M. aurum in studies to develop improved drugs for the pathogenic mycobacteria M. tuberculosis and M. leprae.

Alternaria cerasidanica sp nov., isolated in Denmark from drupes of Prunus avium

DEFF Research Database (Denmark)

Roberts, R. G.; Reymond, S. T.; Andersen, Birgitte

2010-01-01

The ex-type strain of Alternaria cerasidanica was isolated in 2001 from an immature, asymptomatic drupe of Prunus avium collected at a commercial cherry orchard near Skaelskor, Denmark. Cultural morphology, sporulation pattern and cluster analyses of combined RAPD, RAMS (microsatellite), and AFLP...

MycoCAP - Mycobacterium Comparative Analysis Platform.

Science.gov (United States)

Choo, Siew Woh; Ang, Mia Yang; Dutta, Avirup; Tan, Shi Yang; Siow, Cheuk Chuen; Heydari, Hamed; Mutha, Naresh V R; Wee, Wei Yee; Wong, Guat Jah

2015-12-15

Mycobacterium spp. are renowned for being the causative agent of diseases like leprosy, Buruli ulcer and tuberculosis in human beings. With more and more mycobacterial genomes being sequenced, any knowledge generated from comparative genomic analysis would provide better insights into the biology, evolution, phylogeny and pathogenicity of this genus, thus helping in better management of diseases caused by Mycobacterium spp.With this motivation, we constructed MycoCAP, a new comparative analysis platform dedicated to the important genus Mycobacterium. This platform currently provides information of 2108 genome sequences of at least 55 Mycobacterium spp. A number of intuitive web-based tools have been integrated in MycoCAP particularly for comparative analysis including the PGC tool for comparison between two genomes, PathoProT for comparing the virulence genes among the Mycobacterium strains and the SuperClassification tool for the phylogenic classification of the Mycobacterium strains and a specialized classification system for strains of Mycobacterium abscessus. We hope the broad range of functions and easy-to-use tools provided in MycoCAP makes it an invaluable analysis platform to speed up the research discovery on mycobacteria for researchers. Database URL: http://mycobacterium.um.edu.my.

Milk quality assurance for paratuberculosis: simulation of within-herd infection dynamics and economicsof within-herd infection dynamics and economics

NARCIS (Netherlands)

Weber, M.F.; Nielen, M.; Velthuis, A.G.J.; Roermund, van H.J.W.

2008-01-01

bulk milk quality assurance programme for Mycobacterium avium subsp. paratuberculosis (Map) in dairy herds was simulated with a stochastic simulation model (JohneSSim). The aim of this study was to evaluate the epidemiological and economic effects of preventive management measures and various test

Progressive bovine paratuberculosis is associated with local loss of CD4(+) T cells, increased frequency of gamma delta T cells, and related changes in T-cell function

NARCIS (Netherlands)

Koets, A.; Rutten, V.; Hoek, van A.; Mil, van F.; Muller, K.; Bakker, D.; Gruys, E.; Eden, van W.

2002-01-01

Bovine paratuberculosis is caused by the infection of young calves with Mycobacterium avium subsp. paratuberculosis, resulting in a chronic granulomatous infection of predominantly the ileum. After an incubation period of 2 to 5 years, the disease becomes progressive in some of the chronically

Can chemoprophylaxis against opportunistic infections be discontinued after an increase in CD4 cells induced by highly active antiretroviral therapy?

DEFF Research Database (Denmark)

Kirk, O; Lundgren, Jens Dilling; Pedersen, C

1999-01-01

/100 PY follow-up (95% confidence interval, 0.0-3.2). No cases of cerebral toxoplasmosis, cytomegalovirus chorioretinitis, or disseminated Mycobacterium avium infection were observed. Follow-up time for these was, however, limited. CONCLUSION: PCP-chemoprophylaxis can be safely discontinued after HAART...

Methods for detecting pathogens in the beef food chain: detecting particular pathogens

Science.gov (United States)

The main food-borne pathogens of concern in the beef food chain are Shiga toxin-producing Escherichia coli (STEC) and Salmonella spp.; however, the presence of other pathogens, including Listeria monocytogenes, Campylobacter spp., Clostridium spp., Bacillus cereus, and Mycobacterium avium subsp. par...

ACANTHAMOEBA SP.S-11 PHAGOCYTOTIC ACTIVITY ON MYCOBACTERIUM LEPRAE IN DIFFERENT NUTRIENT CONDITIONS.

Science.gov (United States)

Paling, Sepling; Wahyuni, Ratna; Ni'matuzahroh; Winarni, Dwi; Iswahyudi; Astari, Linda; Adriaty, Dinar; Agusni, Indropo; Izumi, Shinzo

2018-01-01

Mycobacterium leprae ( M. leprae ) is a pathogenic bacterium that causes leprosy. The presence of M. leprae in the environment is supported by microorganisms that act as the new host for M. leprae . Acanthamoeba 's potential to be a host of M. leprae in the environment. Acanthamoeba sp. is Free Living Amoeba (FLA) that classified as holozoic, saprophytic, and saprozoic. The existence of nutrients in the environment influence Acanthamoeba ability to phagocytosis or pinocytosis. This study is aimed to determine Acanthamoeba sp.S-11 phagocytic activity to Mycobacterium leprae ( M. leprae ) which cultured in non-nutrient media and riched-nutrient media. This research conducted by culturing Acanthamoeba sp.S-11 and M. leprae on different nutrient media conditions. M. leprae intracellular DNA were isolated and amplified by M. leprae specific primers through Real Time PCR (Q-PCR). The results showed that Acanthamoeba co-cultured on non-nutrient media were more active to phagocyte M. leprae than on rich-nutrient media. The use of non-nutrient media is recommended to optimize Acanthamoeba sp. phagocytic activity to M. leprae .

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

Directory of Open Access Journals (Sweden)

V. N. Zimina

2017-01-01

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