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Sample records for mycobacterium avium paratuberculosis

  1. Mycobacterium avium subspecies Paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genome sequence has now defined the complete catalog of genes that make Mycobacterium avium subsp paratuberculosis what it is. Although similarity searches and bioinformatics analyses have assigned potential function to hundreds of genes in this pathogen, the future challenge is to begin to sys...

  2. Mycobacterium avium subspecies paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne’s disease in cattle and other domesticated and wild ruminant species. The organism and disease were first described over a century ago, and despite the considerable morbidity and mortality associated with Map infection...

  3. Iron Acquisition in Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Wang, Joyce; Moolji, Jalal; Dufort, Alex; Staffa, Alfredo; Domenech, Pilar; Reed, Michael B.

    2015-01-01

    ABSTRACT Mycobacterium avium subsp. paratuberculosis is a host-adapted pathogen that evolved from the environmental bacterium M. avium subsp. hominissuis through gene loss and gene acquisition. Growth of M. avium subsp. paratuberculosis in the laboratory is enhanced by supplementation of the media with the iron-binding siderophore mycobactin J. Here we examined the production of mycobactins by related organisms and searched for an alternative iron uptake system in M. avium subsp. paratuberculosis. Through thin-layer chromatography and radiolabeled iron-uptake studies, we showed that M. avium subsp. paratuberculosis is impaired for both mycobactin synthesis and iron acquisition. Consistent with these observations, we identified several mutations, including deletions, in M. avium subsp. paratuberculosis genes coding for mycobactin synthesis. Using a transposon-mediated mutagenesis screen conditional on growth without myobactin, we identified a potential mycobactin-independent iron uptake system on a M. avium subsp. paratuberculosis-specific genomic island, LSPP15. We obtained a transposon (Tn) mutant with a disruption in the LSPP15 gene MAP3776c for targeted study. The mutant manifests increased iron uptake as well as intracellular iron content, with genes downstream of the transposon insertion (MAP3775c to MAP3772c [MAP3775-2c]) upregulated as the result of a polar effect. As an independent confirmation, we observed the same iron uptake phenotypes by overexpressing MAP3775-2c in wild-type M. avium subsp. paratuberculosis. These data indicate that the horizontally acquired LSPP15 genes contribute to iron acquisition by M. avium subsp. paratuberculosis, potentially allowing the subsequent loss of siderophore production by this pathogen. IMPORTANCE Many microbes are able to scavenge iron from their surroundings by producing iron-chelating siderophores. One exception is Mycobacterium avium subsp. paratuberculosis, a fastidious, slow-growing animal pathogen whose growth

  4. Mycobacterium avium subsp. paratuberculosis in Veterinary Medicine

    PubMed Central

    Harris, N. Beth; Barletta, Raúl G.

    2001-01-01

    Mycobacterium avium subsp. paratuberculosis (basonym M. paratuberculosis) is the etiologic agent of a severe gastroenteritis in ruminants known as Johne's disease. Economic losses to the cattle industry in the United States are staggering, reaching $1.5 billion annually. A potential pathogenic role in humans in the etiology of Crohn's disease is under investigation. In this article, we review the epidemiology, pathogenesis, diagnostics, and disease control measures of this important veterinary pathogen. We emphasize molecular genetic aspects including the description of markers used for strain identification, diagnostics, and phylogenetic analysis. Recent important advances in the development of animal models and genetic systems to study M. paratuberculosis virulence determinants are also discussed. We conclude with proposals for the applications of these models and recombinant technology to the development of diagnostic, control, and therapeutic measures. PMID:11432810

  5. Disparate Host Immunity to Mycobacterium avium subsp. paratuberculosis Antigens in Calves Inoculated with M. avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii, and M. bovis

    PubMed Central

    Waters, W. R.; Bannantine, J. P.; Palmer, M. V.

    2013-01-01

    The cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and a criticism of the current tests that are used for the detection of paratuberculosis. In the present study, Mycobacterium avium subsp. paratuberculosis recombinant proteins were evaluated for antigenic specificity compared to a whole-cell sonicate preparation (MPS). Measures of cell-mediated immunity to M. avium subsp. paratuberculosis antigens were compared in calves inoculated with live M. avium subsp. paratuberculosis, M. avium subsp. avium (M. avium), Mycobacterium kansasii, or Mycobacterium bovis. Gamma interferon (IFN-γ) responses to MPS were observed in all calves that were exposed to mycobacteria compared to control calves at 4 months postinfection. Pooled recombinant M. avium subsp. paratuberculosis proteins also elicited nonspecific IFN-γ responses in inoculated calves, with the exception of calves infected with M. bovis. M. avium subsp. paratuberculosis proteins failed to elicit antigen-specific responses for the majority of immune measures; however, the expression of CD25 and CD26 was upregulated on CD4, CD8, gamma/delta (γδ) T, and B cells for the calves that were inoculated with either M. avium subsp. paratuberculosis or M. avium after antigen stimulation of the cells. Stimulation with MPS also resulted in the increased expression of CD26 on CD45RO+ CD25+ T cells from calves inoculated with M. avium subsp. paratuberculosis and M. avium. Although recombinant proteins failed to elicit specific responses for the calves inoculated with M. avium subsp. paratuberculosis, the differences in immune responses to M. avium subsp. paratuberculosis antigens were dependent upon mycobacterial exposure. The results demonstrated a close alignment in immune responses between calves inoculated with M. avium subsp. paratuberculosis and those inoculated with M. avium that were somewhat disparate from the responses in calves infected with M. bovis, suggesting

  6. Comparative Genomic Hybridizations Reveal Genetic Regions within the Mycobacterium avium Complex That Are Divergent from Mycobacterium avium subsp. paratuberculosis Isolates†

    PubMed Central

    Paustian, Michael L.; Kapur, Vivek; Bannantine, John P.

    2005-01-01

    Mycobacterium avium subsp. paratuberculosis is genetically similar to other members of the Mycobacterium avium complex (MAC), some of which are nonpathogenic and widespread in the environment. We have utilized an M. avium subsp. paratuberculosis whole-genome microarray representing over 95% of the predicted coding sequences to examine the genetic conservation among 10 M. avium subsp. paratuberculosis isolates, two isolates each of Mycobacterium avium subsp. silvaticum and Mycobacterium avium subsp. avium, and a single isolate each of both Mycobacterium intracellulare and Mycobacterium smegmatis. Genomic DNA from each isolate was competitively hybridized with DNA from M. avium subsp. paratuberculosis K10, and open reading frames (ORFs) were classified as present, divergent, or intermediate. None of the M. avium subsp. paratuberculosis isolates had ORFs classified as divergent. The two M. avium subsp. avium isolates had 210 and 135 divergent ORFs, while the two M. avium subsp. silvaticum isolates examined had 77 and 103 divergent ORFs. Similarly, 130 divergent ORFs were identified in M. intracellulare. A set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC isolates tested. Many of these ORFs are clustered together on the genome in regions with relatively low average GC content compared with the entire genome and contain mobile genetic elements. One of these regions of sequence divergence contained genes homologous to a mammalian cell entry (mce) operon. Our results indicate that closely related MAC mycobacteria can be distinguished from M. avium subsp. paratuberculosis by multiple clusters of divergent ORFs. PMID:15774884

  7. Experimental Paratuberculosis in Calves following Inoculation with a Rabbit Isolate of Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Beard, P. M.; Stevenson, K.; Pirie, A.; Rudge, K.; Buxton, D.; Rhind, S. M.; Sinclair, M. C.; Wildblood, L. A.; Jones, D. G.; Sharp, J. M.

    2001-01-01

    The role of wildlife species in the epidemiology of paratuberculosis has been the subject of increased research efforts following the discovery of natural paratuberculosis in free-living rabbits from farms in east Scotland. This paper describes the experimental inoculation of young calves with an isolate of Mycobacterium avium subsp. paratuberculosis recovered from a free-living rabbit. After a 6-month incubation period, all eight calves inoculated with the rabbit isolate had developed histopathological and/or microbiological evidence of M. avium subsp. paratuberculosis infection. Similar results were obtained from a group of calves infected with a bovine isolate of M. avium subsp. paratuberculosis. The virulence of the rabbit isolate for calves demonstrated in this study suggests that rabbits are capable of passing paratuberculosis to domestic ruminants and that wildlife reservoirs of M. avium subsp. paratuberculosis should therefore be considered when formulating control plans for the disease. PMID:11526132

  8. Cellular Interactions in Mycobacterium avium subsp. paratuberculosis Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The study of host immune responses to Mycobacterium avium subsp. paratuberculosis (MAP) is complicated by a number of factors, including the protracted nature of the disease and the stealthy nature of the pathogen. Noted as one of the more fastidious mycobacteria, infection with MAP is often chara...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Disparate host immunity to Mycobacterium avium subsp. paratuberculosis antigens in calves inoculated with M. avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii and M. bovis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and criticism of current tests for the detection of paratuberculosis. In the present study, host immune responses to antigen preparations of Mycobacterium avium subsp. paratuberculosis (MAP), consis...

  11. Development and Use of a Partial Mycobacterium avium subspecies paratuberculosis Protein Array

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As an initial step toward systematically characterizing all antigenic proteins produced by a significant veterinary pathogen, 43 recombinant Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) expression clones were constructed, cataloged and stored. Nitrocellulose filters were sp...

  12. Early Antibody Response Against Mycobacterium avium subspecies paratuberculosis Antigens in Subclinical Cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Abstract Background Our laboratories have previously reported on the experimental infection of cattle with Mycobacterium avium subsp paratuberculosis (M. paratuberculosis) using an intratonsillar infection model. In addition, we have recently developed a partial protein array representing 92 M. par...

  13. First isolation of Mycobacterium avium subsp Paratuberculosis from commercial pasteurized milk in Argentina

    PubMed Central

    Paolicchi, Fernando; Cirone, Karina; Morsella, Claudia; Gioffré, Andrea

    2012-01-01

    Mycobacterium avium subsp paratuberculosis was isolated from two out of seventy samples (2.86 %) of pasteurized and ultra-pasteurized milk. The isolates were positives to IS900 PCR and showed a C17 RFLP pattern, the most prevalent in Argentina. The present study is the first report of Mycobacterium avium subsp paratuberculosis culture from pasteurized milk in Argentina. PMID:24031925

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  15. Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes for Rapid Detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in Potable-Water Biofilms

    PubMed Central

    Lehtola, Markku J.; Torvinen, Eila; Miettinen, Ilkka T.; Keevil, C. William

    2006-01-01

    Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybridization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive and grow in drinking-water biofilms and possibly transmit via drinking water. The designed PNA probe (MAV148) specificity was tested with several bacterial species, including other mycobacteria and mycolic acid-containing bacteria. From the range of bacterial strains tested, only M. avium subsp. avium and M. avium subsp. paratuberculosis strains were hybridized. The PNA FISH method was applied successfully to detect M. avium subsp. avium spiked in water samples and biofilm established within a Propella biofilm reactor fed with potable water from a distribution supply. PMID:16391126

  16. Development and Characterization of Monoclonal Antibodies and Aptamers Against Major Antigens of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Specific antibodies, available in unlimited quantities, have not been produced against Mycobacterium avium subsp. paratuberculosis, the bacterium that causes Johne’s disease (JD). To fill this gap in JD research, monoclonal antibodies (mAbs) against M. avium subsp. paratuberculosis were produced fr...

  17. Complete genome sequence of Mycobacterium avium subsp. paratuberculosis, isolated from human breast milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp paratuberculosis is the etiologic agent of Johne’s disease. We report the draft genome sequences of six M. avium subsp paratuberculosis isolates obtained from diverse hosts including bison, cattle and sheep. These sequences will deepen our understanding of host association ...

  18. Antigenic Profiles of Recombinant Proteins from Mycobacterium avium subsp paratuberculosis in Sheep with Johne's Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods to improve the ELISA test to detect Mycobacterium avium subsp paratuberculosis have been explored over several years. Previously, selected recombinant proteins of M. avium subspecies paratuberculosis were found to be immunogenic in cattle with Johne’s disease. In the present study, antibo...

  19. The Transport of Mycobacterium avium subsp. paratuberculosis through Saturated Aquifer Materials

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne’s disease, a chronic enteric infection causing diarrhea and wasting in cattle, sheep, and other ruminants. Because Johne’s disease is spread by the ingestion of M. paratuberculosis, a good understanding...

  20. Survival of Mycobacterium avium subsp. paratuberculosis in Biofilms on Livestock Watering Trough Materials

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Despite the ubiquitous occurrence of Mycobacterium sp. in nature and the fact that Johne’s disease has been reported worldwide, little research has been done to assess the survival of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in agricultural environments. The goal of this stu...

  1. Survival of Mycobacterium avium subsp. paratuberculosis in Biofilms on Livestock Watering Trough Materials

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne’s disease, a chronic enteric infection that affects ruminants. Despite the ubiquitous occurrence of Mycobacterium sp. in nature and the fact that Johne’s disease has been reported worldwide, little rese...

  2. Development of vaccines to Mycobacterium avium subsp. paratuberculosis infection.

    PubMed

    Park, Hong-Tae; Yoo, Han Sang

    2016-07-01

    Johne's disease or paratuberculosis is a chronic debilitating disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease causes significant economic losses in livestock industries worldwide. There are no effective control measures to eradicate the disease because there are no appropriate diagnostic methods to detect subclinically infected animals. Therefore, it is very difficult to control the disease using only test and cull strategies. Vaccination against paratuberculosis has been considered as an alternative strategy to control the disease when combined with management interventions. Understanding host-pathogen interactions is extremely important to development of vaccines. It has long been known that Th1-mediated cellular immune responses are play a crucial role in protection against MAP infection. However, recent studies suggested that innate immune responses are more closely related to protective effects than adaptive immunity. Based on this understanding, several attempts have been made to develop vaccines against paratuberculosis. A variety of ideas for designing novel vaccines have emerged, and the tests of the efficacy of these vaccines are conducted constantly. However, no effective vaccines are commercially available. In this study, studies of the development of vaccines for MAP were reviewed and summarized. PMID:27489800

  3. Development of vaccines to Mycobacterium avium subsp. paratuberculosis infection

    PubMed Central

    2016-01-01

    Johne's disease or paratuberculosis is a chronic debilitating disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease causes significant economic losses in livestock industries worldwide. There are no effective control measures to eradicate the disease because there are no appropriate diagnostic methods to detect subclinically infected animals. Therefore, it is very difficult to control the disease using only test and cull strategies. Vaccination against paratuberculosis has been considered as an alternative strategy to control the disease when combined with management interventions. Understanding host-pathogen interactions is extremely important to development of vaccines. It has long been known that Th1-mediated cellular immune responses are play a crucial role in protection against MAP infection. However, recent studies suggested that innate immune responses are more closely related to protective effects than adaptive immunity. Based on this understanding, several attempts have been made to develop vaccines against paratuberculosis. A variety of ideas for designing novel vaccines have emerged, and the tests of the efficacy of these vaccines are conducted constantly. However, no effective vaccines are commercially available. In this study, studies of the development of vaccines for MAP were reviewed and summarized. PMID:27489800

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. No holes barred: Invasion of the intestinal mucosa by Mycobacterium avium subspecies paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The infection biology of Mycobacterium avium subspecies paratuberculosis (MAP) has recently crystalized with added details surrounding intestinal invasion. The involvement of pathogen-derived effector proteins such as the major membrane protein, oxidoreductase and fibronectin attachment proteins hav...

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

    PubMed Central

    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

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

    PubMed

    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

  8. Immunogenicity of Proteome-Determined Mycobacterium avium subsp. paratuberculosis-Specific Proteins in Sheep with Paratuberculosis▿

    PubMed Central

    Hughes, Valerie; Bannantine, John P.; Denham, Susan; Smith, Stuart; Garcia-Sanchez, Alfredo; Sales, Jill; Paustian, Michael L.; Mclean, Kevin; Stevenson, Karen

    2008-01-01

    Mycobacterium avium subsp. paratuberculosis causes paratuberculosis, a chronic granulomatous enteritis. Detecting animals with paratuberculosis infections is difficult because the currently available tools have low sensitivity and lack specificity; these tools are prone to generating spurious positive test results caused by exposure to environmental M. avium complex organisms. To generate candidate antigens for incorporation into a specific test for paratuberculosis, subspecies-specific proteins were determined by proteomic comparison of M. avium subsp. paratuberculosis and M. avium subsp. avium. Analysis was aimed at revealing proteins only expressed (or predominant) in the protein profile of M. avium subspecies paratuberculosis. Two-dimensional gel electrophoresis resolved approximately 1,000 protein spots from each subspecies. Proteome analysis identified protein spots whose expression profile appeared markedly increased in M. avium subsp. paratuberculosis, and 32 were identified by analysis of their tryptic peptide profile by matrix-assisted laser desorption ionization-time of flight analysis. Thirty of these proteins were cloned, and their recombinant proteins were expressed. Ovine paratuberculosis sera were used to assess their immunoreactivity by enzyme-linked immunosorbent assay (ELISA), Western blotting, and dot blot analysis. Seventeen proteins were detected in at least one of the immunoassays, and eleven proteins were detected by ELISA with an optical density in excess of the cutoff of 0.1 in four of six sera tested. The immunoreactivity of these proteins indicates their potential as unique diagnostic antigens for the development of a specific serological detection of paratuberculosis. PMID:18845834

  9. Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fecal culture is considered the gold standard for the diagnosis of paratuberculosis, however, PCR for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in fecal material is widely used today, having demonstrated great sensitivity and specificity. To insure the most efficient and rep...

  10. Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fecal culture is considered the gold standard for the diagnostics of paratuberculosis, however, PCR for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in fecal material is widely used today, having demonstrated great sensitivity and specificity. To insure the most efficient and r...

  11. Alkyl Hydroperoxide Reductases C and D Are Major Antigens Constitutively Expressed by Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Olsen, Ingrid; Reitan, Liv J.; Holstad, Gudmund; Wiker, Harald G.

    2000-01-01

    Antigens characteristic for Mycobacterium avium subspecies paratuberculosis were identified by crossed immunoelectrophoresis (CIE) and by absorbing out cross-reactive antigens by using a polyclonal and polyvalent Mycobacterium avium subspecies avium antiserum. Two antigens were present in M. avium subsp. paratuberculosis and not detected in Mycobacterium avium subsp. avium. They were identified as antigens 17 and 20 in a CIE reference system for M. avium subsp. paratuberculosis antigens. Purified antigen 20 was identified as alkyl hydroperoxide reductase C (AhpC) while the N-terminal part of purified antigen 17 showed 80% homology with alkyl hydroperoxide reductase D (AhpD) of Mycobacterium tuberculosis. AhpC had a nonreduced mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 45 kDa and is probably a homodimer linked with disulfide bridges in its native form. AhpD had a mobility corresponding to 19 kDa. Monospecific rabbit antiserum against AhpC and AhpD reacted with 9 strains of M. avium subsp. paratuberculosis but not with 20 other mycobacterial strains except for a Mycobacterium gordonae strain, against which a weak cross-reactive band was produced. Goats experimentally infected with M. avium subsp. paratuberculosis had strong gamma interferon (IFN-γ) responses toward both AhpC and AhpD, and they also had antibodies against AhpC. The ability of AhpC and AhpD to induce IFN-γ production shows that these proteins potentially could be used in future vaccines or in diagnostic assays. These results further show that AhpC and AhpD are immunologically important proteins which are constitutively and highly expressed in M. avium subsp. paratuberculosis without the bacteria being submitted to oxidative stress and that the specificities of antigens can be a matter of different levels of protein expression in various species as well as distinct structural differences. PMID:10639449

  12. Profiling Bovine Antibody Responses to Mycobacterium avium subsp. paratuberculosis Infection by Using Protein Arrays▿ †

    PubMed Central

    Bannantine, John P.; Paustian, Michael L.; Waters, W. Ray; Stabel, Judith R.; Palmer, Mitchell V.; Li, Lingling; Kapur, Vivek

    2008-01-01

    With the genome sequence of Mycobacterium avium subsp. paratuberculosis determined, technologies are now being developed for construction of protein arrays to detect the presence of antibodies against M. avium subsp. paratuberculosis in host serum. The power of this approach is that it enables a direct comparison of M. avium subsp. paratuberculosis proteins to each other in relation to their immunostimulatory capabilities. In this study, 93 recombinant proteins, produced in Escherichia coli, were arrayed and spotted onto nitrocellulose. These proteins include unknown hypothetical proteins and cell surface proteins as well as proteins encoded by large sequence polymorphisms present uniquely in M. avium subsp. paratuberculosis. Also included were previously reported or known M. avium subsp. paratuberculosis antigens to serve as a frame of reference. Sera from healthy control cattle (n = 3) and cattle infected with either M. avium subsp. avium and Mycobacterium bovis were exposed to the array to identify nonspecific or cross-reactive epitopes. These data demonstrated a degree of cross-reactivity with the M. avium subsp. avium proteins that was higher than the degree of cross-reactivity with the more distantly related M. bovis proteins. Finally, sera from naturally infected cattle (n = 3) as well as cattle experimentally infected with M. avium subsp. paratuberculosis (n = 3) were used to probe the array to identify antigens in the context of Johne's disease. Three membrane proteins were the most strongly detected in all serum samples, and they included an invasion protein, an ABC peptide transport permease, and a putative GTPase protein. This powerful combination of genomic information, molecular tools, and immunological assays has enabled the identification of previously unknown antigens of M. avium subsp. paratuberculosis. PMID:18039835

  13. Functional Characterization of Iron Dependent Regulator (IdeR) of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study we investigated an iron dependent regulator (IdeR) of Mycobacterium avium subsp. paratuberculosis (MAP). IdeR is a transcriptional factor that plays a global iron regulatory role in Mycobacterium tuberculosis (MTB) with a 19-bp recognition sequence. IdeR recognition sites within MAP ge...

  14. Expression library immunization confers protection against Mycobacterium avium subsp. paratuberculosis infection.

    PubMed

    Huntley, J F; Stabel, J R; Paustian, M L; Reinhardt, T A; Bannantine, J P

    2005-10-01

    Currently, paratuberculosis vaccines are comprised of crude whole-cell preparations of Mycobacterium avium subsp. paratuberculosis. Although effective in reducing clinical disease and fecal shedding, these vaccines have severe disadvantages as well, including seroconversion of vaccinated animals and granulomatous lesions at the site of vaccination. DNA vaccines can offer an alternative approach that may be safer and elicit more protective responses. In an effort to identify protective M. avium subsp. paratuberculosis sequences, a genomic DNA expression library was generated and subdivided into pools of clones (approximately 1,500 clones/pool). The clone pools were evaluated to determine DNA vaccine efficacy by immunizing mice via gene gun delivery and challenging them with live, virulent M. avium subsp. paratuberculosis. Four clone pools resulted in a significant reduction in the amount of M. avium subsp. paratuberculosis recovered from mouse tissues compared to mice immunized with other clone pools and nonvaccinated, infected control mice. One of the protective clone pools was further partitioned into 10 clone arrays of 108 clones each, and four clone arrays provided significant protection from both spleen and mesenteric lymph node colonization by M. avium subsp. paratuberculosis. The nucleotide sequence of each clone present in the protective pools was determined, and coding region functions were predicted by computer analysis. Comparison of the protective clone array sequences implicated 26 antigens that may be responsible for protection in mice. This study is the first study to demonstrate protection against M. avium subsp. paratuberculosis infection with expression library immunization. PMID:16177367

  15. Hidden Gems in the Mycobacterium avium subsp. paratuberculosis Genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    If 4,350 genes annotated in the M. avium subsp paratuberculosis strain K-10 genome wasn’t already enough to study, more genes have recently been uncovered, hidden deep within this genome sequence. Genomic and proteomic studies, both published and unpublished, have revealed a handful of new genes mi...

  16. Description of a novel adhesin of Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Viale, Mariana Noelia; Echeverria-Valencia, Gabriela; Romasanta, Pablo; Mon, María Laura; Fernandez, Marisa; Malchiodi, Emilio; Romano, María Isabel; Gioffré, Andrea Karina; Santangelo, María de la Paz

    2014-01-01

    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. PMID:25136616

  17. Description of a Novel Adhesin of Mycobacterium avium Subsp. paratuberculosis

    PubMed Central

    Viale, Mariana Noelia; Echeverria-Valencia, Gabriela; Romasanta, Pablo; Mon, María Laura; Fernandez, Marisa; Malchiodi, Emilio; Romano, María Isabel; Gioffré, Andrea Karina; Santangelo, María de la Paz

    2014-01-01

    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. PMID:25136616

  18. THE MYCOBACTERIUM AVIUM SUPSPECIES PARATUBERCULOSIS 35 KDA PROTEIN PLAYS A ROLE IN INVASION OF BOVINE EPITHELIAL CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) enters intestinal epithelial cells of cattle and other ruminants via a mechanism that remains to be fully elucidated. In this study, we observed that a gene encoding the M. paratuberculosis 35-kDa major membrane protein (MMP) is ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  1. Macrophage Transcriptional Response to Species-Adapted Mycobacterium avium subsp. Paratuberculosis Isolates: The Role of Pathogen Genotype in Host Response

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The transcriptional response of human and bovine macrophages to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) isolates from cattle and sheep were examined using DNA microarrays. M. paratuberculosis is the etiologic agent of Johne’s Disease, a chronic infection of ruminant anima...

  2. Survival of Mycobacterium avium subsp. paratuberculosis in bovine monocyte-derived macrophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne’s disease is a significant problem in many North American cattle herds. The efficacy of currently available vaccines is questionable. There is a need to develop efficacious vaccines and strains of Mycobacterium avium subsp. paratuberculosis (MAPTB) that could serve as potential candidates for ...

  3. Primary transcriptomes of Mycobacterium avium subsp. paratuberculosis reveal proprietary pathways in tissue and macrophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Mycobacterium avium subsp. paratuberculosis persistently infect intestines and mesenteric lymph nodes leading to a prolonged subclinical disease. We investigated the intracellular lifestyle of MAP in the intestines and lymph nodes to understand the MAP pathways that function to govern th...

  4. Unraveling the Host Response to Mycobacterium avium subsp. paratuberculosis: One Thread at a Time

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The study of host immune responses to Mycobacterium avium subsp. paratuberculosis (MAP) is complicated by a number of factors, including the protracted nature of the disease and the stealthy nature of the pathogen. Improved tools for the measurement of immunologic responses in ruminant species, par...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. Optimization of hexadecylpyridinium chloride decontamination for culture of Mycobacterium avium subsp. paratuberculosis from milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cows in advanced stages of Johne’s disease shed Mycobacterium avium subsp. paratuberculosis (MAP) into both their milk and feces, allowing for transmission of the bacteria between animals. The objective of this study was to formulate an optimized protocol for the isolation of MAP from milk and colos...

  7. Environmental contamination with Mycobacterium avium subsp. paratuberculosis in endemically infected dairy herds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Environmental contamination with Mycobacterium avium subsp. paratuberculosis (MAP) is thought to be the primary source of infection for dairy cattle. The exact link between fecal shedding of MAP by individual cows and environmental contamination levels at the herd level was explored with a cross-se...

  8. Effect of Watering Trough Chlorination on Persistence of Mycobacterium avium subsp. Paratuberculosi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The continued global increase in the number of cases of Johne’s disease suggests that more information is needed to understand the mechanisms by which the causative agent Mycobacterium avium subsp. paratuberculosis (MAP) is spread among livestock on the farm site. Livestock watering troughs are freq...

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

    EPA Science Inventory

    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 ...

  10. Immunlogic responses to Mycobacterium avium subsp. paratuberculosis protein cocktail vaccines in a mouse model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne’s disease is a chronic granulomatous enteritis characterized by severe diarrhea, wasting, and a decline in milk production caused by the bacterium Mycobacterium avium subsp. paratuberculosis (MAP). The vaccine currently on the market has some limitations including a severe injection site react...

  11. Immunologic Responses to Mycobacterium avium subsp. paratuberculosis in Neonatal Calves After Oral or Intraperitoneal Experimental Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infection models are useful for studying host responses to infection to aid in the development of diagnostic tools and vaccines. The majority of experimental models for ruminants have utilized an oral inoculation of live Mycobacterium avium subsp. paratuberculosis (MAP) in order to establish infecti...

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

    EPA Science Inventory

    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...

  13. Pathogenesis of Mycobacterium avium subsp. paratuberculosis in Neonatal Calves after Oral or Intraperitoneal Experimental Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding the infection process to Mycobacterium avium subsp. paratuberculosis is tantamount to the development of effective vaccines and therapeutics for the control of this disease in the field. The current study compared the effectiveness of oral and intraperitoneal methods of experimental in...

  14. Cytokine Secretion in Periparturient dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne's disease, cause by Mycobacterium avium subsp. paratuberculosis (MAP), has a devastating impact on the dairy industry. Cows typically are infected as neonates, and stressors, such as parturition, may induce the transition from the subclinical to a more clinical stage of disease. The objective ...

  15. Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis in a Longitudinal Study of Three Dairy Herds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to evaluate whether cows that were low shedders of Mycobacterium avium subsp. paratuberculosis (MAP) were passive shedding animals or whether they were truly infected with MAP. We also evaluated whether these MAP-infected animals could have been infected as adults by ...

  16. Osteopontin Expression in Periparturient Dairy Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne’s disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is estimated to infect more than 22% of US dairy herds. Periods of immunosuppression, typically seen at parturition, may contribute to the transition from the subclinical, or asymptomatic, to the clinical stage of inf...

  17. Evaluation of Mycobacterium avium subsp. paratuberculosis Transport in Saturated Porous Media

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne’s disease, a chronic enteric infection that affects ruminants. Eradication of Map from infected farms has been difficult and is likely due to long-term survival of the organism in the environment. The application of Ma...

  18. Parturition and Mycobacterium avium subsp. paratuberculosis: A Potential Interface for the Pathogenesis of Johne's Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne’s disease (JD), is estimated to infect more than 22% of US dairy herds and cost the industry $250 million annually. One major period of stress for dairy cows is the periparturient period, and field observations suggest ...

  19. Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) ...

  20. Assessment of Food as a Source of Exposure to Mycobacterium avium Subspecies Paratuberculosis (MAP)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The National Advisory Committee on Microbiological Criteria for Foods (NACMCF or Committee) was asked to assess the importance of food as a source of exposure to Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne’s disease, which affects primarily the small intestin...

  1. From mouth to macrophage: mechanisms of innate immune subversion by Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne’s disease (JD) is a chronic enteric infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP). The high economic cost and potential zoonotic threat of JD have driven efforts to develop tools and approaches to effectively manage this disease within livestock herds. Efforts...

  2. Characteristics of an Extensive Mycobacterium avium subspecies paratuberculosis Recombinant Protein Set

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the first step of a comprehensive large-scale antigen discovery project, 651 Mycobacterium avium subspecies paratuberculosis proteins were produced in Escherichia coli. All of these were purified by affinity chromatography, dialyzed in phosphate buffered saline, and analyzed on SDS-PAGE gels. C...

  3. Production and Characterization of Monoclonal Antibodies Against a Major Membrane Protein of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Mycobacterium avium subsp. paratuberculosis 35-kDa major membrane protein (MMP) encoded by MAP2121c has been shown to play a role in invasion of epithelial cells and is an important membrane antigen recognized by cattle with Johne’s disease. In this study, purified recombinant MMP was used to p...

  4. Antibacterial Activities of Naturally occurring Compounds against Mycobacterium avium subspecies paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibacterial activities of 19 naturally-occurring compounds (including essential oils and some of their isolated constituents, apple and green tea polyphenols and other plant extracts) against three strains of Mycobacterium avium subspecies paratuberculosis (Map), a bovine isolate NCTC 8578, a raw ...

  5. The Association of Mycobacterium avium subsp. paratuberculosis with Inflammatory Bowel Disease

    PubMed Central

    Timms, Verlaine J.; Daskalopoulos, George; Mitchell, Hazel M.; Neilan, Brett A.

    2016-01-01

    The association of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) with Crohn’s disease is a controversial issue. M. paratuberculosis is detected by amplifying the IS900 gene, as microbial culture is unreliable from humans. We determined the presence of M. paratuberculosis in patients with Crohn’s disease (CD) (n = 22), ulcerative colitis (UC) (n = 20), aphthous ulcers (n = 21) and controls (n = 42) using PCR assays validated on bovine tissue. Culture from human tissue was also performed. M. paratuberculosis prevalence in the CD and UC groups was compared to the prevalence in age and sex matched non-inflammatory bowel disease controls. Patients and controls were determined to be M. paratuberculosis positive if all three PCR assays were positive. A significant association was found between M. paratuberculosis and Crohn’s disease (p = 0.02) that was not related to age, gender, place of birth, smoking or alcohol intake. No significant association was detected between M. paratuberculosis and UC or aphthous ulcers; however, one M. paratuberculosis isolate was successfully cultured from a patient with UC. We report the resistance of this isolate to ethambutol, rifampin, clofazamine and streptomycin. Interestingly this isolate could not only survive but could grow slowly at 5°C. We demonstrate a significant association between M. paratuberculosis and CD using multiple pre-validated PCR assays and that M. paratuberculosis can be isolated from patients with UC. PMID:26849125

  6. The Association of Mycobacterium avium subsp. paratuberculosis with Inflammatory Bowel Disease.

    PubMed

    Timms, Verlaine J; Daskalopoulos, George; Mitchell, Hazel M; Neilan, Brett A

    2016-01-01

    The association of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) with Crohn's disease is a controversial issue. M. paratuberculosis is detected by amplifying the IS900 gene, as microbial culture is unreliable from humans. We determined the presence of M. paratuberculosis in patients with Crohn's disease (CD) (n = 22), ulcerative colitis (UC) (n = 20), aphthous ulcers (n = 21) and controls (n = 42) using PCR assays validated on bovine tissue. Culture from human tissue was also performed. M. paratuberculosis prevalence in the CD and UC groups was compared to the prevalence in age and sex matched non-inflammatory bowel disease controls. Patients and controls were determined to be M. paratuberculosis positive if all three PCR assays were positive. A significant association was found between M. paratuberculosis and Crohn's disease (p = 0.02) that was not related to age, gender, place of birth, smoking or alcohol intake. No significant association was detected between M. paratuberculosis and UC or aphthous ulcers; however, one M. paratuberculosis isolate was successfully cultured from a patient with UC. We report the resistance of this isolate to ethambutol, rifampin, clofazamine and streptomycin. Interestingly this isolate could not only survive but could grow slowly at 5°C. We demonstrate a significant association between M. paratuberculosis and CD using multiple pre-validated PCR assays and that M. paratuberculosis can be isolated from patients with UC. PMID:26849125

  7. Flow Cytometric Detection of Mycobacterium avium subsp. paratuberculosis-Specific Antibodies in Experimentally Infected and Naturally Exposed Calves

    PubMed Central

    Bridger, P. S.; Bulun, H.; Fischer, M.; Akineden, Ö.; Seeger, T.; Barth, S.; Henrich, M.; Doll, K.; Bülte, M.; Menge, C.; Bauerfeind, R.

    2013-01-01

    A desirable test to diagnose infections with Mycobacterium avium subsp. paratuberculosis facilitates identification of infected cattle prior to the state of M. avium subsp. paratuberculosis shedding. This study aimed at adjusting a flow cytometry (FC)-based assay, using intact M. avium subsp. paratuberculosis bacteria as the antigen, for diagnosis of M. avium subsp. paratuberculosis infections in calves. Serum samples were collected from experimentally infected (n = 12) and naturally exposed (n = 32) calves. Samples from five calves from positive dams were analyzed to determine the dynamics of maternal antibodies. Samples from adult cattle with defined infection status served as the standard (18 M. avium subsp. paratuberculosis shedders, 22 M. avium subsp. paratuberculosis free). After preadsorption with Mycobacterium phlei, sera were incubated with M. avium subsp. paratuberculosis and M. avium subsp. avium bacterial suspensions, respectively, followed by the separate detection of bovine IgG, IgG1, IgG2, and IgM attached to the bacterial surface. M. avium subsp. paratuberculosis-specific sample/positive (S/P) ratios were compared to enzyme-linked immunosorbent assay (ELISA) S/P ratios. In adult cattle, the FC assay for IgG1 had a sensitivity of 78% at a specificity of 100%. Maternally acquired antibodies could be detected in calves up to 121 days of life. While all but two sera taken at day 100 ± 10 postnatum from naturally exposed calves tested negative, elevated S/P ratios (IgG and IgG1) became detectable from 44 and 46 weeks postinoculation onwards in two calves infected experimentally. Even with the optimized FC assay, M. avium subsp. paratuberculosis-specific antibodies can only occasionally be detected in infected calves less than 12 months of age. The failure to detect such antibodies apparently reflects the distinct immunobiology of M. avium subsp. paratuberculosis infections rather than methodological constraints. PMID:23885032

  8. Evaluation of Control Points in Youngstock and Adult Dairy Cow Management to Control Transmission of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Complete a series of prospective controlled on-farm trials to critically evaluate the efficacy and cost-benefit of commonly recommended management practices for reducing the transmission of Mycobacterium avium subsp. paratuberculosis (Map) in youngstock in infected herds....

  9. Osteopontin Immunoreactivity in the Ileum and Ileoceccal Lymph Node of Dairy Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Osteopontin (Opn), a highly acidic glycoprotein, promotes cellular adhesion and recruitment and has been shown to be upregulated in the granulomas of mycobacterial infections. Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is associated with granulomatous enteritis. ...

  10. Osteopontin: A Novel Cytokine Involved in the Regulation of Mycobacterium avium subsp. paratuberculosis Infection in Periparturient Dairy Cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Osteopontin (Opn), an important mediator of the cell-mediated immune response, enhances the host immune response against mycobacterial infections. Infections caused by the intracellular bacterium, Mycobacterium avium subsp. paratuberculosis (MAP), have a devastating impact on the dairy industry. ...

  11. How does a Mycobacterium change its spots? Applying molecular tools to track diverse strains of Mycobacterium avium subspecies paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Defining genetic diversity in the wake of the release of several Mycobacterium avium subsp. paratuberculosis (MAP) genome sequences has become a major emphasis in the molecular biology and epidemiology of Johne’s disease research. These data can now be used to define the extent of strain diversity ...

  12. Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Culture of Mycobacterium avium subsp. paratuberculosis (MAP) from feces has been considered the gold standard for the diagnosis of paratuberculosis for many years. However, direct fecal PCR is becoming more widely used today, demonstrating similar sensitivity and specificity to culture. To ensure ef...

  13. Mediation of host immune responses after immunization of neonatal calves with a heat-killed Mycobacterium avium subsp. paratuberculosis vaccine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A major drawback of current whole-cell vaccines for Mycobacterium avium subsp. paratuberculosis(MAP) is the interference with diagnostic tests for bovine tuberculosis and paratuberculosis. The current study was designed to explore effects of immunization with a heat-killed whole cell vaccine (Mycop...

  14. Lymphoproliferative and Gamma Interferon Responses to Stress-Regulated Mycobacterium avium subsp. paratuberculosis Recombinant Proteins

    PubMed Central

    Gurung, Ratna B.; Begg, Douglas J.; Purdie, Auriol C.; de Silva, Kumudika; Bannantine, John P.

    2014-01-01

    Johne's disease in ruminants is a chronic infection of the intestines caused by Mycobacterium avium subsp. paratuberculosis. An important strategy to control disease is early detection, and a potentially efficient method for early detection is measurement of cell-mediated immune responses developed by the host in response to exposure or infection. One method is to measure lymphoproliferation and cytokine release from the host cells when exposed to the organism or parts of the organism. In this study, 10 recombinant M. avium subsp. paratuberculosis proteins known to be upregulated under in vitro stress conditions were evaluated by examining their ability to evoke memory as a result of exposure by vaccination or oral challenge with live Mycobacterium avium subsp. paratuberculosis. Out of 10 proteins, MAP2698c was found to induce higher cell-mediated immune responses in vaccinated and challenged sheep in comparison to healthy controls. The findings suggest that not all stress-regulated proteins have the diagnostic potential to detect cell-mediated immune responses in ovine paratuberculosis. PMID:24695774

  15. Mycobacterium avium subsp. paratuberculosis PtpA Is an Endogenous Tyrosine Phosphatase Secreted during Infection▿

    PubMed Central

    Bach, Horacio; Sun, Jim; Hmama, Zakaria; Av-Gay, Yossef

    2006-01-01

    Adaptive gene expression in prokaryotes is mediated by protein kinases and phosphatases. These regulatory proteins mediate phosphorylation of histidine or aspartate in two-component systems and serine/threonine or tyrosine in eukaryotic and eukaryote-like protein kinase systems. The genome sequence of Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, does not possess a defined tyrosine kinase. Nevertheless, it encodes for protein tyrosine phosphatases. Here, we report that Map1985, is a functional low-molecular tyrosine phosphatase that is secreted intracellularly upon macrophage infection. This finding suggests that Map1985 might contribute to the pathogenesis of Mycobacterium avium subsp. paratuberculosis by dephosphorylating essential macrophage signaling and/or adaptor molecules. PMID:16982836

  16. THE ABILITY OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS TO ENTER BOVINE EPITHELIAL CELLS IS INFLUENCED BY PREEXPOSURE TO A HYPEROSMOLAR ENVIRONMENT AND INTRACELLULAR PASSAGE IN BOVINE MAMMARY EPITHELIAL CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis is the cause of Johne’s disease in cattle and other ruminants. M. avium subsp. paratuberculosis infection of the bovine host is not well understood; however, it is assumed that crossing the bovine intestinal mucosa is important in order for M. avium subsp...

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

    PubMed

    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. PMID:25128370

  18. Virulence and Immunity Orchestrated by the Global Gene Regulator sigL in Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Ghosh, Pallab; Steinberg, Howard

    2014-01-01

    Mycobacterium avium subsp. paratuberculosis causes Johne's disease in ruminants, a chronic enteric disease responsible for severe economic losses in the dairy industry. Global gene regulators, including sigma factors are important in regulating mycobacterial virulence. However, the biological significance of such regulators in M. avium subsp. paratuberculosis rremains elusive. To better decipher the role of sigma factors in M. avium subsp. paratuberculosis pathogenesis, we targeted a key sigma factor gene, sigL, activated in mycobacterium-infected macrophages. We interrogated an M. avium subsp. paratuberculosis ΔsigL mutant against a selected list of stressors that mimic the host microenvironments. Our data showed that sigL was important in maintaining bacterial survival under such stress conditions. Survival levels further reflected the inability of the ΔsigL mutant to persist inside the macrophage microenvironments. Additionally, mouse infection studies suggested a substantial role for sigL in M. avium subsp. paratuberculosis virulence, as indicated by the significant attenuation of the ΔsigL-deficient mutant compared to the parental strain. More importantly, when the sigL mutant was tested for its vaccine potential, protective immunity was generated in a vaccine/challenge model of murine paratuberculosis. Overall, our study highlights critical role of sigL in the pathogenesis and immunity of M. avium subsp. paratuberculosis infection, a potential role that could be shared by similar proteins in other intracellular pathogens. PMID:24799632

  19. Modulation of Cytokine Gene Expression and Secretion During the Periparturient Period in Dairy Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Modulation of cytokine gene expression and secretion during the periparturient period in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis Technical abstract Johne’s disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is estimated to infect more t...

  20. Bovine Immunoinhibitory Receptors Contribute to Suppression of Mycobacterium avium subsp. paratuberculosis-Specific T-Cell Responses

    PubMed Central

    Okagawa, Tomohiro; Konnai, Satoru; Nishimori, Asami; Ikebuchi, Ryoyo; Mizorogi, Seiko; Nagata, Reiko; Kawaji, Satoko; Tanaka, Shogo; Kagawa, Yumiko; Murata, Shiro; Mori, Yasuyuki

    2015-01-01

    Johne's disease (paratuberculosis) is a chronic enteritis in cattle that is caused by intracellular infection with Mycobacterium avium subsp. paratuberculosis. This infection is characterized by the functional exhaustion of T-cell responses to M. avium subsp. paratuberculosis antigens during late subclinical and clinical stages, presumably facilitating the persistence of this bacterium and the formation of clinical lesions. However, the mechanisms underlying T-cell exhaustion in Johne's disease are poorly understood. Thus, we performed expression and functional analyses of the immunoinhibitory molecules programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) and lymphocyte activation gene 3 (LAG-3)/major histocompatibility complex class II (MHC-II) in M. avium subsp. paratuberculosis-infected cattle during the late subclinical stage. Flow cytometric analyses revealed the upregulation of PD-1 and LAG-3 in T cells in infected animals, which suffered progressive suppression of interferon gamma (IFN-γ) responses to the M. avium subsp. paratuberculosis antigen. In addition, PD-L1 and MHC-II were expressed on macrophages from infected animals, consistent with PD-1 and LAG-3 pathways contributing to the suppression of IFN-γ responses during the subclinical stages of M. avium subsp. paratuberculosis infection. Furthermore, dual blockade of PD-L1 and LAG-3 enhanced M. avium subsp. paratuberculosis-specific IFN-γ responses in blood from infected animals, and in vitro LAG-3 blockade enhanced IFN-γ production from M. avium subsp. paratuberculosis-specific CD4+ and CD8+ T cells. Taken together, the present data indicate that M. avium subsp. paratuberculosis-specific T-cell exhaustion is in part mediated by PD-1/PD-L1 and LAG-3/MHC-II interactions and that LAG-3 is a molecular target for the control of M. avium subsp. paratuberculosis-specific T-cell responses. PMID:26483406

  1. Isolation of Mycobacterium avium subsp. paratuberculosis from free-ranging birds and mammals on livestock premises.

    PubMed

    Corn, Joseph L; Manning, Elizabeth J B; Sreevatsan, Srinand; Fischer, John R

    2005-11-01

    Surveys for Mycobacterium avium subsp. paratuberculosis infection in free-ranging mammals and birds were conducted on nine dairy and beef cattle farms in Wisconsin and Georgia. Specimens were collected from 774 animals representing 25 mammalian and 22 avian species. Specimens of ileum, liver, intestinal lymph nodes, and feces were harvested from the larger mammals; a liver specimen and the gastrointestinal tract were harvested from birds and small mammals. Cultures were performed by using radiometric culture and acid-fast isolates were identified by 16S/IS900/IS1311 PCR and mycobactin dependency characteristics. M. avium subsp. paratuberculosis was cultured from tissues and feces from 39 samples from 30 animals representing nine mammalian and three avian species. The prevalence of infected wild animals by premises ranged from 2.7 to 8.3% in Wisconsin and from 0 to 6.0% in Georgia. Shedding was documented in seven (0.9%) animals: three raccoons, two armadillos, one opossum, and one feral cat. The use of two highly polymorphic short sequence repeat loci for analysis of 29 of the 39 strains identified 10 alleles. One allelic pattern broadly shared in domestic ruminants ("7,5") appeared in approximately one-third of the wildlife M. avium subsp. paratuberculosis isolates studied. Given the few cases of shedding by free-ranging animals compared to the volume of contaminated manure produced by infected domestic ruminant livestock, contamination of the farm environment by infected wildlife was negligible. Wildlife may, however, have epidemiological significance for farms where M. avium subsp. paratuberculosis recently has been eliminated or on farms free of M. avium subsp. paratuberculosis but located in the geographic vicinity of farms with infected livestock. PMID:16269731

  2. Isolation of Mycobacterium avium subsp. paratuberculosis from Free-Ranging Birds and Mammals on Livestock Premises

    PubMed Central

    Corn, Joseph L.; Manning, Elizabeth J. B.; Sreevatsan, Srinand; Fischer, John R.

    2005-01-01

    Surveys for Mycobacterium avium subsp. paratuberculosis infection in free-ranging mammals and birds were conducted on nine dairy and beef cattle farms in Wisconsin and Georgia. Specimens were collected from 774 animals representing 25 mammalian and 22 avian species. Specimens of ileum, liver, intestinal lymph nodes, and feces were harvested from the larger mammals; a liver specimen and the gastrointestinal tract were harvested from birds and small mammals. Cultures were performed by using radiometric culture and acid-fast isolates were identified by 16S/IS900/IS1311 PCR and mycobactin dependency characteristics. M. avium subsp. paratuberculosis was cultured from tissues and feces from 39 samples from 30 animals representing nine mammalian and three avian species. The prevalence of infected wild animals by premises ranged from 2.7 to 8.3% in Wisconsin and from 0 to 6.0% in Georgia. Shedding was documented in seven (0.9%) animals: three raccoons, two armadillos, one opossum, and one feral cat. The use of two highly polymorphic short sequence repeat loci for analysis of 29 of the 39 strains identified 10 alleles. One allelic pattern broadly shared in domestic ruminants (“7,5”) appeared in approximately one-third of the wildlife M. avium subsp. paratuberculosis isolates studied. Given the few cases of shedding by free-ranging animals compared to the volume of contaminated manure produced by infected domestic ruminant livestock, contamination of the farm environment by infected wildlife was negligible. Wildlife may, however, have epidemiological significance for farms where M. avium subsp. paratuberculosis recently has been eliminated or on farms free of M. avium subsp. paratuberculosis but located in the geographic vicinity of farms with infected livestock. PMID:16269731

  3. Divergent Immune Responses to Mycobacterium avium subsp. paratuberculosis Infection Correlate with Kinome Responses at the Site of Intestinal Infection

    PubMed Central

    Määttänen, Pekka; Trost, Brett; Scruten, Erin; Potter, Andrew; Kusalik, Anthony; Griebel, Philip

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease (JD) in cattle. M. avium subsp. paratuberculosis infects the gastrointestinal tract of calves, localizing and persisting primarily in the distal ileum. A high percentage of cattle exposed to M. avium subsp. paratuberculosis do not develop JD, but the mechanisms by which they resist infection are not understood. Here, we merge an established in vivo bovine intestinal segment model for M. avium subsp. paratuberculosis infection with bovine-specific peptide kinome arrays as a first step to understanding how infection influences host kinomic responses at the site of infection. Application of peptide arrays to in vivo tissue samples represents a critical and ambitious step in using this technology to understand host-pathogen interactions. Kinome analysis was performed on intestinal samples from 4 ileal segments subdivided into 10 separate compartments (6 M. avium subsp. paratuberculosis-infected compartments and 4 intra-animal controls) using bovine-specific peptide arrays. Kinome data sets clustered into two groups, suggesting unique binary responses to M. avium subsp. paratuberculosis. Similarly, two M. avium subsp. paratuberculosis-specific immune responses, characterized by different antibody, T cell proliferation, and gamma interferon (IFN-γ) responses, were also observed. Interestingly, the kinomic groupings segregated with the immune response groupings. Pathway and gene ontology analyses revealed that differences in innate immune and interleukin signaling and particular differences in the Wnt/β-catenin pathway distinguished the kinomic groupings. Collectively, kinome analysis of tissue samples offers insight into the complex cellular responses induced by M. avium subsp. paratuberculosis in the ileum and provides a novel method to understand mechanisms that alter the balance between cell-mediated and antibody responses to M. avium subsp. paratuberculosis infection. PMID

  4. Divergent immune responses to Mycobacterium avium subsp. paratuberculosis infection correlate with kinome responses at the site of intestinal infection.

    PubMed

    Määttänen, Pekka; Trost, Brett; Scruten, Erin; Potter, Andrew; Kusalik, Anthony; Griebel, Philip; Napper, Scott

    2013-08-01

    Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease (JD) in cattle. M. avium subsp. paratuberculosis infects the gastrointestinal tract of calves, localizing and persisting primarily in the distal ileum. A high percentage of cattle exposed to M. avium subsp. paratuberculosis do not develop JD, but the mechanisms by which they resist infection are not understood. Here, we merge an established in vivo bovine intestinal segment model for M. avium subsp. paratuberculosis infection with bovine-specific peptide kinome arrays as a first step to understanding how infection influences host kinomic responses at the site of infection. Application of peptide arrays to in vivo tissue samples represents a critical and ambitious step in using this technology to understand host-pathogen interactions. Kinome analysis was performed on intestinal samples from 4 ileal segments subdivided into 10 separate compartments (6 M. avium subsp. paratuberculosis-infected compartments and 4 intra-animal controls) using bovine-specific peptide arrays. Kinome data sets clustered into two groups, suggesting unique binary responses to M. avium subsp. paratuberculosis. Similarly, two M. avium subsp. paratuberculosis-specific immune responses, characterized by different antibody, T cell proliferation, and gamma interferon (IFN-γ) responses, were also observed. Interestingly, the kinomic groupings segregated with the immune response groupings. Pathway and gene ontology analyses revealed that differences in innate immune and interleukin signaling and particular differences in the Wnt/β-catenin pathway distinguished the kinomic groupings. Collectively, kinome analysis of tissue samples offers insight into the complex cellular responses induced by M. avium subsp. paratuberculosis in the ileum and provides a novel method to understand mechanisms that alter the balance between cell-mediated and antibody responses to M. avium subsp. paratuberculosis infection. PMID

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

    PubMed

    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. PMID:27127920

  6. Antigenicity of Recombinant Maltose Binding Protein-Mycobacterium avium subsp. paratuberculosis Fusion Proteins with and without Factor Xa Cleaving

    PubMed Central

    Begg, Douglas J.; Purdie, Auriol C.; Bannantine, John P.; Whittington, Richard J.

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Proteomic studies have shown that M. avium subsp. paratuberculosis expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such proteins are hypothesized to be expressed in vivo, are recognized by the host immune system, and may be of potential use in the diagnosis of JD. In this study, 50 recombinant maltose binding protein (MBP)-M. avium subsp. paratuberculosis fusion proteins were evaluated using serum samples from sheep infected with M. avium subsp. paratuberculosis, and 29 (58%) were found to be antigenic. Among 50 fusion proteins, 10 were evaluated in MBP fusion and factor Xa-cleaved forms. A total of 31 proteins (62%) were found to be antigenic in either MBP fusion or factor Xa-cleaved forms. Antigenicity after cleavage and removal of the MBP tag was marginally enhanced. PMID:24132604

  7. Peyer's Patch-Deficient Mice Demonstrate That Mycobacterium avium subsp. paratuberculosis Translocates across the Mucosal Barrier via both M Cells and Enterocytes but Has Inefficient Dissemination ▿

    PubMed Central

    Bermudez, Luiz E.; Petrofsky, Mary; Sommer, Sandra; Barletta, Raúl G.

    2010-01-01

    Mycobacterium avium subsp. paratuberculosis, the agent of Johne's disease, infects ruminant hosts by translocation through the intestinal mucosa. A number of studies have suggested that M. avium subsp. paratuberculosis interacts with M cells in the Peyer's patches of the small intestine. The invasion of the intestinal mucosa by M. avium subsp. paratuberculosis and Mycobacterium avium subsp. hominissuis, a pathogen known to interact with intestinal cells, was compared. M. avium subsp. paratuberculosis was capable of invading the mucosa, but it was significantly less efficient at dissemination than M. avium subsp. hominissuis. B-cell knockout (KO) mice, which lack Peyer's patches, were used to demonstrate that M. avium subsp. paratuberculosis enters the intestinal mucosa through enterocytes in the absence of M cells. In addition, the results indicated that M. avium subsp. paratuberculosis had equal abilities to cross the mucosa in both Peyer's patch and non-Peyer's patch segments of normal mice. M. avium subsp. paratuberculosis was also shown to interact with epithelial cells by an α5β1 integrin-independent pathway. Upon translocation, dendritic cells ingest M. avium subsp. paratuberculosis, but this process does not lead to efficient dissemination of the infection. In summary, M. avium subsp. paratuberculosis interacts with the intestinal mucosa by crossing both Peyer's patches and non-Peyer's patch areas but does not translocate or disseminate efficiently. PMID:20498259

  8. Early antibody response against Mycobacterium avium subspecies paratuberculosis antigens in subclinical cattle

    PubMed Central

    Bannantine, John P; Bayles, Darrell O; Waters, W Ray; Palmer, Mitchell V; Stabel, Judith R; Paustian, Michael L

    2008-01-01

    Background Our laboratories have previously reported on the experimental infection of cattle with Mycobacterium avium subsp paratuberculosis (M. paratuberculosis) using an intratonsillar infection model. In addition, we have recently developed a partial protein array representing 92 M. paratuberculosis coding sequences. These combined tools have enabled a unique look at the temporal analysis of M. paratuberculosis antigens within the native host. The primary objective of this study was to identify M. paratuberculosis antigens detected by cattle early during infection. A secondary objective was to evaluate the humoral immune response in cattle during the initial year of infection. Results Sera from two experimentally infected cattle, taken pre-inoculation and at day 70, 194 and 321 post infection, identified dynamic antibody reactivity among antigens with some showing an increased response over time and others showing declining levels of reactivity over the same time period. A M. paratuberculosis specific protein, encoded by MAP0862, was strongly detected initially, but the antibody response became weaker with time. The most reactive protein was a putative surface antigen encoded by MAP1087. A second protein, MAP1204, implicated in virulence, was also strongly detected by day 70 in both cattle. Subsequent experiments showed that these two proteins were detected with sera from 5 of 9 naturally infected cattle in the subclinical stage of Johne's disease. Conclusion Collectively these results demonstrate that M. paratuberculosis proteins are detected by sera from experimentally infected cattle as early as 70 days after exposure. These data further suggest at least two antigens may be useful in the early diagnosis of M. paratuberculosis infections. Finally, the construction and use of a protein array in this pilot study has led to a novel approach for discovery of M. paratuberculosis antigens. PMID:18226229

  9. Survival and Dormancy of Mycobacterium avium subsp. paratuberculosis in the Environment

    PubMed Central

    Whittington, Richard J.; Marshall, D. Jeff; Nicholls, Paul J.; Marsh, Ian B.; Reddacliff, Leslie A.

    2004-01-01

    The survival of Mycobacterium avium subsp. paratuberculosis was studied by culture of fecal material sampled at intervals for up to 117 weeks from soil and grass in pasture plots and boxes. Survival for up to 55 weeks was observed in a dry fully shaded environment, with much shorter survival times in unshaded locations. Moisture and application of lime to soil did not affect survival. UV radiation was an unlikely factor, but infrared wavelengths leading to diurnal temperature flux may be the significant detrimental component that is correlated with lack of shade. The organism survived for up to 24 weeks on grass that germinated through infected fecal material applied to the soil surface in completely shaded boxes and for up to 9 weeks on grass in 70% shade. The observed patterns of recovery in three of four experiments and changes in viable counts were indicative of dormancy, a hitherto unreported property of this taxon. A dps-like genetic element and relA, which are involved in dormancy responses in other mycobacteria, are present in the M. avium subsp. paratuberculosis genome sequence, providing indirect evidence for the existence of physiological mechanisms enabling dormancy. However, survival of M. avium subsp. paratuberculosis in the environment is finite, consistent with its taxonomic description as an obligate parasite of animals. PMID:15128561

  10. Mycobacterium avium Subspecies paratuberculosis Recombinant Proteins Modulate Antimycobacterial Functions of Bovine Macrophages.

    PubMed

    Bannantine, John P; Stabel, Judith R; Laws, Elizabeth; D Cardieri, Maria Clara; Souza, Cleverson D

    2015-01-01

    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 recombinant proteins expressed from coding sequences annotated as lipoproteins were screened for their ability to induce IL-10 expression, an indicator of MAPKp38 activation, in bovine monocyte-derived macrophages. A recombinant lipoprotein, designated as MAP3837c, was among a group of 6 proteins that strongly induced IL-10 gene transcription in bovine macrophages, averaging a 3.1-fold increase compared to non-stimulated macrophages. However, a parallel increase in expression of IL-12 and TNF-α was only observed in macrophages exposed to a subset of these 6 proteins. Selected recombinant proteins were further analyzed for their ability to enhance survival of M. avium within bovine macrophages as measured by recovered viable bacteria and nitrite production. All 6 IL-10 inducing MAP recombinant proteins along with M. paratuberculosis cells significantly enhanced phosphorylation of MAPK-p38 in bovine macrophages. Although these proteins are likely not post translationally lipidated in E. coli and thus is a limitation in this study, these results form the foundation of how the protein component of the lipoprotein interacts with the immune system. Collectively, these data reveal M. paratuberculosis proteins that might play a role in MAPK-p38 pathway activation and hence in survival of this organism within bovine macrophages. PMID:26076028

  11. Novel Feature of Mycobacterium avium subsp. paratuberculosis, Highlighted by Characterization of the Heparin-Binding Hemagglutinin Adhesin

    PubMed Central

    Lefrancois, Louise H.; Bodier, Christelle C.; Cochard, Thierry; Canepa, Sylvie; Raze, Dominique; Lanotte, Philippe; Sevilla, Iker A.; Stevenson, Karen; Behr, Marcel A.; Locht, Camille

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis comprises two genotypically defined groups, known as the cattle (C) and sheep (S) groups. Recent studies have reported phenotypic differences between M. avium subsp. paratuberculosis groups C and S, including growth rates, infectivity for macrophages, and iron metabolism. In this study, we investigated the genotypes and biological properties of the virulence factor heparin-binding hemagglutinin adhesin (HBHA) for both groups. In Mycobacterium tuberculosis, HBHA is a major adhesin involved in mycobacterium-host interactions and extrapulmonary dissemination of infection. To investigate HBHA in M. avium subsp. paratuberculosis, we studied hbhA polymorphisms by fragment analysis using the GeneMapper technology across a large collection of isolates genotyped by mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) and IS900 restriction fragment length polymorphism (RFLP-IS900) analyses. Furthermore, we analyzed the structure-function relationships of recombinant HBHA proteins of types C and S by heparin-Sepharose chromatography and surface plasmon resonance (SPR) analyses. In silico analysis revealed two forms of HBHA, corresponding to the prototype genomes for the C and S types of M. avium subsp. paratuberculosis. This observation was confirmed using GeneMapper on 85 M. avium subsp. paratuberculosis strains, including 67 strains of type C and 18 strains of type S. We found that HBHAs from all type C strains contain a short C-terminal domain, while those of type S present a long C-terminal domain, similar to that produced by Mycobacterium avium subsp. avium. The purification of recombinant HBHA from M. avium subsp. paratuberculosis of both types by heparin-Sepharose chromatography highlighted a correlation between their affinities for heparin and the lengths of their C-terminal domains, which was confirmed by SPR analysis. Thus, types C and S of M. avium subsp. paratuberculosis may be

  12. PRESENCE OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS IN ALPACAS (LAMA PACOS) INHABITING THE CHILEAN ALTIPLANO.

    PubMed

    Salgado, Miguel; Sevilla, Iker; Rios, Carolina; Crossley, Jorge; Tejeda, Carlos; Manning, Elizabeth

    2016-03-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis. The organism causes disease in both domestically managed and wild ruminant species. South American camelids have a long, shared history with indigenous people in the Andes. Over the last few decades, increasing numbers of alpacas were exported to numerous countries outside South America. No paratuberculosis surveillance has been reported for these source herds. In this study, individual fecal samples from 85 adult alpacas were collected from six separate herds in the Chilean Altiplano. A ParaTB mycobacterial growth indicator tube (MGIT) liquid culture of each individual fecal sample, followed by real-time polymerase chain reaction (PCR) protocol was used for confirmation. DNA extracts from a subset of confirmed MAP isolates were subjected to mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing. Fifteen alpaca were fecal culture test-positive. Five false-positive culture samples were negative on PCR analysis for Mycobacterium avium subsp. avium (MAA), Mycobacterium bovis (M. bovis), and the 16 S rDNA gene. Three MAP isolates subset-tested belonged to the same MIRU-VNTR type, showing four repeats for TR292 (locus 1) in contrast to the three repeats typical of the MAP reference strain K10. The number of repeats found in the remaining loci was identical to that of the K10 strain. It is not known how nor when MAP was introduced into the alpaca population in the Chilean Altiplano. The most plausible hypothesis to explain the presence of MAP in these indigenous populations is transmission by contact with infected domestic small ruminant species that may on occasion share pastures or range with alpacas. Isolation of this mycobacterial pathogen from such a remote region suggests that MAP has found its way beyond the confines of intensively managed domestic agriculture premises. PMID:27010259

  13. Is Mycobacterium avium subsp. paratuberculosis, the cause of Johne's disease in animals, a good candidate for Crohn's disease in man?

    PubMed

    Singh, A V; Singh, S V; Singh, P K; Sohal, J S

    2010-03-01

    Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease or paratuberculosis, a gastro intestinal inflammatory condition in ruminants and other animals, which is similar to Crohn's disease (CD) that occurs in man. The role of MAP in the causation of CD has been under intense investigation in the last few decades. This review summarizes the status of MAP in animals and the food chain and its association with CD in man. PMID:20443099

  14. Current perspectives on Mycobacterium avium subsp. paratuberculosis, Johne's disease, and Crohn's disease: a review.

    PubMed

    Over, Ken; Crandall, Philip G; O'Bryan, Corliss A; Ricke, Steven C

    2011-05-01

    Mycobacterium avium subsp. paratuberculosis (MAP) causes the disease of cattle, Johne's. The economic impact of this disease includes early culling of infected cattle, reduced milk yield, and weight loss of cattle sold for slaughter. There is a possible link between MAP and Crohn's disease, a human inflammatory bowel disease. MAP is also a potential human food borne pathogen because it survives current pasteurization treatments. We review the current knowledge of MAP, Johne's disease and Crohn's disease and note directions for future work with this organism including rapid and economical detection, effective management plans and preventative measures. PMID:21254832

  15. A survey to detect the presence of Mycobacterium avium subspecies paratuberculosis in Kangaroo Island macropods.

    PubMed

    Cleland, P C; Lehmann, D R; Phillips, P H; Cousins, D V; Reddacliff, L A; Whittington, R J

    2010-10-26

    The aim of this study was to determine whether Mycobacterium avium subspecies paratuberculosis (M. a. paratuberculosis) infection was present in macropods grazing with infected sheep on Kangaroo Island in 2001-2002, and to assess the likely role of such infection in the epidemiology of ovine paratuberculosis. Ileum and associated lymphatics from 482 macropods were examined using radiometric culture followed by PCR for IS900 and restriction endonuclease analysis (REA) for species identification, and isolates were strain typed using PCR for IS1311 and REA. Ileum and mesenteric lymph nodes from animals with positive tissue cultures or gross lesions suggestive of paratuberculosis were examined histologically. Faeces from a total of 840 animals were cultured in pools of 20, and individual faecal cultures were done from tissue culture positive animals, from those with microscopic lesions, and from selected animals with gross lesions. Eight animals (1.7%) yielded positive tissue cultures, and all isolates were the sheep (S) strain. Two animals that were tissue culture positive also had histopathological evidence of paratuberculosis. Twelve culture negative animals had microscopic lesions consistent with mycobacterial infection, and M. genavense was identified by PCR from a paraffin block from one of these animals. All faecal cultures were negative. These results indicate that a small proportion of macropods can become infected with M. a. paratuberculosis when grazing with infected sheep. However, excretion of large numbers of viable organisms is rare in macropods, and it is unlikely that macropods provide a wildlife reservoir of infection that would seriously compromise control efforts for paratuberculosis in sheep. PMID:20400245

  16. Mannosylated Lipoarabinomannans from Mycobacterium Avium Subsp. Paratuberculosis Alters the Inflammatory Response by Bovine Macrophages and Suppresses Killing of Mycobacterium Avium Subsp. Avium Organisms

    PubMed Central

    Souza, Cleverson; Davis, William C.; Eckstein, Torsten M.; Sreevatsan, Srinand; Weiss, Douglas J.

    2013-01-01

    Analysis of the mechanisms through which pathogenic mycobacteria interfere with macrophage activation and phagosome maturation have shown that engagement of specific membrane receptors with bacterial ligands is the initiating event. Mannosylated lipoarabinomannan (Man-LAM) has been identified as one of the ligands that modulates macrophage function. We evaluated the effects of Man-LAM derived from Mycobacterium avium subsp. paratuberculosis (MAP) on bovine macrophages. Man-LAM induced a rapid and prolonged expression of IL-10 message as well as transient expression of TNF-α. Preincubation with Man-LAM for up to 16 h did not suppress expression of IL-12 in response to interferon-γ. Evaluation of the effect of Man-LAM on phagosome acidification, phagosome maturation, and killing of Mycobacterium avium subsp. avium (MAA) showed that preincubation of macrophages with Man-LAM before addition of MAA inhibited phagosome acidification, phagolysosome fusion, and reduced killing. Analysis of signaling pathways provided indirect evidence that inhibition of killing was associated with activation of the MAPK-p38 signaling pathway but not the pathway involved in regulation of expression of IL-10. These results support the hypothesis that MAP Man-LAM is one of the virulence factors facilitating survival of MAP in macrophages. PMID:24098744

  17. [Possible association between Mycobacterium avium subsp paratuberculosis infection and Crohn's disease].

    PubMed

    Retamal, Patricio; Beltrán, Caroll; Abalos, Pedro; Quera, Rodrigo; Hermoso, Marcela

    2011-06-01

    Paratuberculosis is a chronic intestinal disease of animals caused by Mycobacterium avium subsp. paratuberculosis (MAP), which has some pathological features similar to Crohn's disease (CD) in humans. The presence of MAP in food for human consumption and in affected tissues of patients with CD has been detected. Therefore, a causal association between this microorganism and the disease in humans, has been postulated. However, several related studies have failed to confirm this hypothesis and the scientific acceptance of MAP as a zoonotic agent remains controversial. This review presents the main findings related to this issue, contrasting evidences for and against an association between MAP and CD. The need to promote national studies focusing on this area is suggested. PMID:22051762

  18. Mycobacterium avium subsp. paratuberculosis sheep strains isolated from Cyprus sheep and goats.

    PubMed

    Liapi, M; Botsaris, G; Slana, I; Moravkova, M; Babak, V; Avraam, M; Di Provvido, A; Georgiadou, S; Pavlik, I

    2015-04-01

    Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic incurable infection of intestinal tract of animals. Molecular characterization of Map isolates classifies them into two major groups, 'Cattle' or Type II and 'Sheep' or Type I/III with a different phenotype, epidemiology, virulence and pathogenesis. The aim of this study was to examine 192 Map ELISA-positive sheep and goats from Cyprus using faecal culture and genotype Map isolates using IS1311 PCR and restriction endonuclease analysis (IS1311 PCR-REA) with HinfI restriction enzyme. Map was isolated from only four (4.6%) faecal samples out of 88 sheep and 15 (14.4%) faecal samples out of 104 goats. Genotyping of the isolates using IS1311 PCR-REA revealed that sheep and goat populations on the island are infected primarily by 'Sheep' strains. Only three Map isolates from goats originated from one farm were characterized as 'Cattle' strains. PMID:23683358

  19. Association between cattle herd Mycobacterium avium subsp. paratuberculosis (MAP) infection and infection of a hare population.

    PubMed

    Salgado, Miguel; Monti, Gustavo; Sevilla, Iker; Manning, Elizabeth

    2014-10-01

    Paratuberculosis has long been considered a disease of domestic and wild ruminants only. The known host range of Mycobacterium avium subsp. paratuberculosis (MAP) was recently extended to include non-ruminant wildlife species believed to be exposed to spillover of MAP from infected domestic cattle herds. The aim of the present study was to assess the association between cattle herd MAP infection pressure level and the infection level of a hare population in two dairy farms of southern Chile. Fifty hares from a herd A and 42 hares from herd B were captured and sampled for MAP culture. The results showed a statistically significant association between the cattle herds' infection prevalence and the hare infection prevalence. PMID:25030465

  20. Detection of Mycobacterium avium subsp. paratuberculosis by a Direct In Situ PCR Method

    PubMed Central

    Delgado, Fernando; Aguilar, Diana; Garbaccio, Sergio; Francinelli, Gladys; Hernández-Pando, R.; Romano, María Isabel

    2011-01-01

    In situ detection of Mycobacterium avium subsp. paratuberculosis is useful for diagnosis and research of paratuberculosis. The aim of this paper was to detect this agent in formalin-fixed, paraffin-embedded tissue samples by a direct in situ PCR. The technique was performed on ileum or ileocaecal lymph node samples from 8 naturally infected cattle and 1 healthy calf, by using p89 and p92 primers for amplification of IS900 sequence. Moderate positive signal was detected in all positive samples and not in negative control, but tissues resulted were affected in many cases due to the enzymatic treatment and the high temperature exposition. Although the technique was useful for Map detection, the signal was lower than immunohistochemistry probably because of the fixation process. In one case, signal was higher, which might be due to the detection of spheroplasts. Thus, the described method should be recommended when others resulted negative or for spheroplasts detection. PMID:21772965

  1. Identification and Functional Characterization of the Iron Dependent Regulator (IdeR) of Mycobacterium avium subspecies paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep, has unique iron requirements in that it is mycobactin-dependent for cultivation in vitro. The iron-dependent regulator (IdeR) is a well-characterized global regulator responsible for ma...

  2. Prevention of Mycobacterium avium subsp. paratuberculosis (MAP) Infection in BALB/c Mice by Feeding Probiotic Lactobacillus acidophilus NP-51

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to examine effects of feeding Lactobacillus acidophilus strain NP51 to mice challenged with Mycobacterium avium subspecies paratuberculosis (MAP). Mice were randomized to ten treatment groups; sentinels, control, heat-killed MAP, viable MAP, heat-killed NP51, viable ...

  3. Antigenicity of recombinant maltose binding protein-Mycobacterium avium subsp. paratuberculosis fusion proteins with and without factor Xa cleaving

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp paratuberculosis (MAP) causes Johne’s disease (JD) in ruminants. Proteomic studies have shown that MAP expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such prot...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. The effects of progressing and nonprogressing Mycobacterium avium ssp. paratuberculosis infection on milk production in dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Longitudinal data from three commercial dairy herds in the northeast United States, collected from 2004 to 2011, were analyzed to determine the effect of Johne’s disease status and path on milk production. Disease status, as indicated by Mycobacterium avium subsp. paratuberculosis test results, was ...

  6. Differences in intermittent and continuous fecal shedding patterns between natural and experimental Mycobacterium avium subspecies paratuberculosis infections in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this paper is to study shedding patterns of cows infected with Mycobacterium avium subsp. paratuberculosis (MAP). While multiple single farm studies of MAP dynamics were reported, there is not large-scale meta-analysis of both natural and experimental infections. Large difference...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  8. Persistence of Mycobacterium avium subsp. paratuberculosis in soil, crops, and ensiled feed following manure spreading on infected dairy farms

    PubMed Central

    Fecteau, Marie-Eve; Hovingh, Ernest; Whitlock, Robert H.; Sweeney, Raymond W.

    2013-01-01

    The goal of this study was to determine the persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in soil, crops, and ensiled feeds following manure spreading. This bacterium was often found in soil samples, but less frequently in harvested feeds and silage. Spreading of manure on fields used for crop harvest is preferred to spreading on grazing pastures. PMID:24179246

  9. Early Immune Markers Associated with Experimental Mycobacterium avium subsp. paratuberculosis (MAP) Infection in a Neonatal Calf Model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to observe early markers of cell-mediated immunity in naïve calves infected with Mycobacterium avium subsp. paratuberculosis (MAP) and how expression of these markers evolved over the 12-month period of infection. Methods of experimental infection included: Control (n...

  10. Evaluation of survival of Mycobacterium avium subsp. paratuberculosis (Map) in ciliates isolated from Johne’s positive cow.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: Persistence of Mycobacterium avium subsp. paratuberculosis (Map) in farm environments is not well understood. Previously we examined the ability of amoebae from a cow’s watering trough to sequester and enhance growth of Map and found that one amoeba species released vesicles containin...

  11. Comparative Transcriptional Analysis of Human Macrophages Exposed to Animal and Human isolates of Mycobacterium avium subspecies paratuberculosis with Diverse Genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Interactions between Mycobacterium avium subsp. paratuberculosis and host macrophages represent critical early events in the pathogenesis of Johne’s disease. We present here a genome-wide characterization of the transcriptional changes within macrophages in responses to different genotypes of M. par...

  12. A Novel Cell Wall Lipopeptide Is Important for Biofilm Formation and Pathogenicity of Mycobacterium avium subspecies paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofilm formation by pathogenic bacteria plays a key role in their pathogenesis. Previously, the pstA gene was shown to be involved in the virulence of Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johne's disease in cattle and a potential risk factor for Crohn's d...

  13. Development and Evaluation of a Variable-number Tandem Repeat (VNTR) Method for Subtyping of Mycobacterium avium subspecies paratuberculosis Isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne’s disease in cattle and other ruminants. Because of the apparent ease by which Map can spread to susceptible animals within a dairy herd, a better understanding of the epidemiology of Map infections is required. In thi...

  14. Prevention of Mycobacterium avium subsp. paratuberculosis (MAP) infection in BALB/c mice by feeding probiotic Lactobacillus acidophilus NP-51

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to examine effects of feeding Lactobacillus acidophilus strain NP51 to mice challenged with Mycobacterium avium subspecies paratuberculosis (MAP). Mice were randomized to ten treatment groups; sentinels, control, heat-killed MAP, viable MAP, heat-killed NP51, viable ...

  15. Optimization of methods for the detection of Mycobacterium avium subsp. paratuberculosis in milk and colostrum of naturally infected dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is primarily shed into the feces but it has also been isolated from the milk and colostrum of cows. Because of this, there exists concern about transfer of the organism from dam to calf and about the prevalence of MAP in the milk supply. The prevalen...

  16. Optimization of methods for the detection of Mycobacterium avium subsp. paratuberculosis in milk and colostrum of naturally infected dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two decontamination chemicals, hexadecylpyridinium choride (HPC) and N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH), were compared for their efficacy of reducing the growth of non-specific microorganisms in milk while minimally affecting the recovery of Mycobacterium avium subsp. paratuberculosis ...

  17. Immunogenicity and protective efficacy of the Mycobacterium avium subsp. paratuberculosis attenuated mutants against challenge in a mouse model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne’s disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), results in serious economic losses worldwide especially in cattle, sheep and goats. To control the impact of JD on the animal industry, an effective vaccine with minimal adverse effects is urgently required. In order ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  20. Parturition invokes Changes in Peripheral Blood Mononuclear Cell Populations in Holstein Dairy Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty-one multiparous and two primiparous Holstein cows were grouped according to infection status with Mycobacterium avium subsp. paratuberculosis (MAP), the causative microorganism for Johne’s disease (JD). The effect of parturition and infection on the percentages of CD4+, CD8+, and T-cells, B-...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. Survival of the Causative Agent of Johne’s Disease (Mycobacterium avium subsp. paratuberculosis) in Biofilms on Trough Materials

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The continued global increase in the number of cases of Johne’s disease among dairy cattle suggests that there remain hidden sources of contamination in the farm environment where susceptible animals may be routinely exposed to Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent o...

  3. Parturition Invokes Changes in Peripheral blood Mononuclear Cell Populations in Holstein Dairy Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is estimated to infect more than 22% of US dairy herds. Once infected, cows may remain in the asymptomatic subclinical state until a period of stress, such as parturition. Parturition has a major impact on the number of ...

  4. Prevention of Mycobacterium avium subsp. paratuberculosis (MAP) Infection in Balb/c Mice by Feeding Probiotic Lactobacillus acidophilus NP-51

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to examine effects of feeding Lactobacillus acidophilus strain NP51 to mice challenged with Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne’s disease. We hypothesized that feeding NP51 would increase Th-1 responses and decrease prog...

  5. Longitudinal data collection of Mycobacterium avium subspecies Paratuberculosis infections in dairy herds. Collection and use of observational data

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Longitudinal infection data on Mycobacterium avium subspecies paratuberculosis (MAP) was collected on three dairy farms in Northeastern United States during approximately 10 years. Precise data on animal characteristics and animal location within farm were collected on these farms. Cows were followe...

  6. Prevention of Mycobacterium avium subsp. paratuberculosis Infection in BALB/c Mice by Feeding Lactobacillus acidophilus Strain NP-51

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The immune responses of 390 BALB/c mice fed the probiotic Lactobacillus acidophilus strain NP51® and infected with Mycobacterium avium subspecies paratuberculosis (MAP) were evaluated in a 6-month trial. Mice were randomized to nine treatment groups fed either viable- or heat-killed NP51 and inocula...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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,...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  9. Shedding of Mycobacterium avium subsp. paratuberculosis into milk and colostrum of naturally infected dairy cows over complete lactation cycles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The primary mode of transmission of Mycobacterium avium subsp. paratuberculosis (MAP) is fecal-oral. However, MAP is also shed into the milk and colostrum of infected cows. The objective of this study was to identify if an association exists between stage of MAP infection and days in lactation with ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  11. Identification and Functional Characterization of the Iron-dependent Regulator (IdeR) of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP), a ruminant pathogen, has unique iron requirements based on the observation that it is mycobactin dependent for successful cultivation in vitro. Thus an elucidation of iron regulation in MAP is expected to provide an understanding of its survival in ...

  12. Proteome and Differential Expression Analysis of Membrane and Cytosolic Proteins from Mycobacterium avium subsp. paratuberculosis Strains K-10 and 187.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known of the protein expression in Mycobacterium avium subspecies paratuberculosis (MAP) and how this contributes to pathogenesis. In the present study, proteins from both outer membranes and cytosol were prepared from two strains of MAP; a laboratory-adapted strain K-10 and a recent isola...

  13. NlpC/P60 domain-containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While intense research is being conducted to develop faster and more reliable methods for diagnosis of Johne’s disease, there are still significant knowledge gaps concerning the molecular function of Mycobacterium avium subspecies paratuberculosis proteins. Therefore, we describe atomic resolution ...

  14. Prevention of Mycobacterium avium subsp. paratuberculosis (MAP) infection in Balb/c mice by feeding probiotic Lactobacillus acidophilus NP-51

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to examine effects of feeding Lactobacillus acidophilus strain NP51 to mice challenged with Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne’s disease. We hypothesized that feeding NP51 would increase Th-1 responses and decrease prog...

  15. Evaluation of eight live attenuated vaccine candidates for protection against challenge with virulent Mycobacterium avium subspecies paratuberculosis in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne’s disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), which results in serious economic losses worldwide in farmed livestock such as cattle, sheep and goats. To control this disease, an effective vaccine with minimal adverse effects is needed. In order to identify a live va...

  16. Immune Responses in Mice to Mycobacterium avium subsp. paratuberculosis Following Vaccination with a Novel 74F Recombinant Polyprotein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne’s Disease (JD) is a chronic infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Here, we report the cloning and expression of a 74kDa recombinant polyprotein (Map74F) and its protective efficacy against MAP infection in mice. Map74F was generated by th...

  17. Immunological and Molecular Characterization of Susceptibility in Relationship to Bacterial Strain Differences in Mycobacterium avium subsp. paratuberculosis Infection in the Red Deer (Cervus elaphus)

    PubMed Central

    O'Brien, R.; Mackintosh, C. G.; Bakker, D.; Kopecna, M.; Pavlik, I.; Griffin, J. F. T.

    2006-01-01

    Johne's disease (JD) infection, caused by Mycobacterium avium subsp. paratuberculosis, represents a major disease problem in farmed ruminants. Although JD has been well characterized in cattle and sheep, little is known of the infection dynamics or immunological response in deer. In this study, typing of M. avium subsp. paratuberculosis isolates from intestinal lymphatic tissues from 74 JD-infected animals showed that clinical isolates of M. avium subsp. paratuberculosis from New Zealand farmed red deer were exclusively of the bovine strain genotype. The susceptibility of deer to M. avium subsp. paratuberculosis was further investigated by experimental oral-route infection studies using defined isolates of virulent bovine and ovine M. avium subsp. paratuberculosis strains. Oral inoculation with high (109 CFU/animal) or medium (107 CFU/animal) doses of the bovine strain of M. avium subsp. paratuberculosis established 100% infection rates, compared to 69% infection following inoculation with a medium dose of the ovine strain. The high susceptibility of deer to the bovine strain of M. avium subsp. paratuberculosis was confirmed by a 50% infection rate following experimental inoculation with a low dose of bacteria (103 CFU/animal). This study is the first to report experimental M. avium subsp. paratuberculosis infection in red deer, and it outlines the strong infectivity of bovine-strain M. avium subsp. paratuberculosis isolates for cervines. PMID:16714585

  18. Detection of Mycobacterium avium subsp. paratuberculosis in Infected Tissues by New Species-Specific Immunohistological Procedures

    PubMed Central

    Coetsier, Christophe; Havaux, Xavier; Mattelard, Francois; Sadatte, Sanaa; Cormont, Francoise; Buergelt, Klaus; Limbourg, Bernard; Latinne, Dominique; Bazin, Herve; Denef, Jean-Francois; Cocito, Carlo

    1998-01-01

    We have previously described the cloning and sequencing of a gene portion coding for the terminal part of a 34-kDa protein of Mycobacterium avium subsp. paratuberculosis, the etiological agent of Johne’s disease (P. Gilot, M. De Kesel, L. Machtelinckx, M. Coene, and C. Cocito, J. Bacteriol. 175:4930–4935, 1993). The recombinant polypeptide (a362) carries species-specific B-cell epitopes which do not cross-react with other mycobacterial pathogens (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol. 31:947–954, 1993). The present work describes the preparation of polyclonal and monoclonal antibodies directed against a362 and the use of these immunoglobulins for histopathological diagnosis of Johne’s disease. The new immunohistological procedures herewith detailed proved to be able to identify M. avium subsp. paratuberculosis antigens in the intestinal tissues and lymph nodes of cattle affected by either the paucibacillary or pluribacillary form of the disease. They yielded negative responses not only with healthy animals but also with those affected by tuberculosis (Mycobacterium bovis). Both immunohistological procedures proved to be as sensitive as or more sensitive than Ziehl-Neelsen staining and, in addition, to be endowed with species specificity. PMID:9665946

  19. Amplified Fragment Length Polymorphism Reveals Specific Epigenetic Distinctions between Mycobacterium avium Subspecies paratuberculosis Isolates of Various Isolation Types▿

    PubMed Central

    O'Shea, B.; Khare, S.; Klein, P.; Roussel, A.; Adams, L. G.; Ficht, T. A.; Rice-Ficht, A. C.

    2011-01-01

    Amplified fragment length polymorphism (AFLP) was employed as a genetic analysis tool for the study of the genetic relatedness of Mycobacterium avium subsp. paratuberculosis isolates harvested from bovine fecal samples and from bovine or human tissues. This analysis revealed genetic differences between these two isolate types that were confirmed through cluster analysis. Dendrogram analysis separated these two isolate types based on the isolation scheme (tissue-associated versus fecal M. avium subsp. paratuberculosis isolates). Further sequence analysis of unique genetic regions from each isolation type revealed no genetic sequence differences. However, Clustal DNA alignments identified AFLP restriction enzyme sites that were undigested in the tissue-associated isolates. AFLP analysis also disclosed that the same AFLP restriction sites were digested in all of the fecal isolates. Sequence analysis further revealed a consensus sequence upstream of the undigested restriction sites for possible methyltransferase recognition in the tissue-associated M. avium subsp. paratuberculosis isolates. PMID:21471350

  20. Effect of Feeding Heat-Treated Colostrum on Preweaning Health, Economics and Transmission of Mycobacterium avium subsp. paratuberculosis in Dairy Calves: Phase I

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction and Objectives Colostrum provides protective immunoglobulins (Ig) and nutrients essential for calf health and performance. However, colostrum may also represent an early source of pathogen exposure including Mycobacterium avium subsp. paratuberculosis (MAP). Pilot studies have suggest...

  1. Modulation of Cytokine Expression and Lymphocyte Subsets During the Periparturient Period in Dairy Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    On-farm observations suggest that dairy cows infected with Mycobacterium avium subsp. paratuberculosis (MAP) may demonstrate increased signs of clinical disease during the weeks following parturition. To date, limited research is available characterizing host immunity in periparturient dairy cows ...

  2. Treatment with antibiotics is detrimental to the recovery of viable Mycobacterium avium subsp. paratuberculosis cultured from milk and colostrum of dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibiotic cocktails are frequently used as secondary decontaminants prior to the culture of Mycobacterium avium subsp. paratuberculosis (MAP). This study investigated whether secondary incubation with an antibiotic cocktail containing vancomycin, nalidixic acid, and amphotericin B after primary exp...

  3. Early Immune Markers Associated with Mycobacterium avium subsp. paratuberculosis Infection in a Neonatal Calf Model ▿

    PubMed Central

    Stabel, J. R.; Robbe-Austerman, S.

    2011-01-01

    The objective of this study was to observe early markers of cell-mediated immunity in naïve calves infected with Mycobacterium avium subsp. paratuberculosis and how expression of these markers evolved over the 12-month period of infection. Groups for experimental infection included control (noninfected), oral (infected orally with M. avium subsp. paratuberculosis strain K-10), oral/DXM (pretreatment with dexamethasone before oral inoculation), intraperitoneal (i.p.) inoculation, and oral/M (oral inoculation with mucosal scrapings from a cow with clinical disease) groups. One of the earliest markers to emerge was antigen-specific gamma interferon (IFN-γ). Only i.p. inoculated calves had detectable antigen-specific IFN-γ responses at 7 days, with responses of the other infection groups becoming detectable at 90 and 120 days. All infection groups maintained robust IFN-γ responses for the remainder of the study. At 1 month, calves in the oral and oral/M groups had higher antigen-stimulated interleukin-10 (IL-10) levels than calves in the other treatment groups, but IL-10 secretion declined by 12 months for all calves. T-cell activation markers such as CD25, CD26, CD45RO, and CD5 were significantly upregulated in infected calves compared to noninfected controls. Oral inoculation of calves resulted in significantly increased antigen-specific lymphocyte proliferation at 9 and 12 months, as well as inducible nitric oxide synthase (iNOS) secretion at 6 and 12 months. These results demonstrate that infection of naïve calves with M. avium subsp. paratuberculosis invoked early immunologic responses characterized by robust antigen-specific IFN-γ responses and induction of CD25 and CD45RO expression on T-cell subsets. These were followed by antigen-specific lymphocyte proliferation, iNOS secretion, and expression of CD26 and CD5bright markers in the latter part of the 12-month study. PMID:21228140

  4. A murine oral model for Mycobacterium avium subsp. paratuberculosis infection and immunomodulation with Lactobacillus casei ATCC 334.

    PubMed

    Cooney, Meagan A; Steele, James L; Steinberg, Howard; Talaat, Adel M

    2014-01-01

    Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) the causative agent of Johne's disease, is one of the most serious infectious diseases in dairy cattle worldwide. Due to the chronic nature of this disease and no feasible control strategy, it is essential to have an efficient animal model which is representative of the natural route of infection as well as a viable treatment option. In this report, we evaluated the effect of different doses of M. paratuberculosis in their ability to colonize murine tissues following oral delivery and the ability of Lactobacillus casei ATCC 334, a nascent probiotic, to combat paratuberculosis. Oral inoculation of mice was able to establish paratuberculosis in a dose-dependent manner. Two consecutive doses of approximately 10(9) CFU per mouse resulted in a disseminated infection, whereas lower doses were not efficient to establish infection. All inoculated mice were colonized with M. paratuberculosis, maintained infection for up to 24 weeks post infection and generated immune responses that reflect M. paratuberculosis infection in cattle. Notably, oral administration of L. casei ATCC 334 did not reduce the level of M. paratuberculosis colonization in treated animals. Interestingly, cytokine responses and histology indicated a trend for the immunomodulation and reduction of pathology in animals receiving L. casei ATCC 334 treatment. Overall, a reproducible oral model of paratuberculosis in mice was established that could be used for future vaccine experiments. Although the L. casei ATCC 334 was not a promising candidate for controlling paratuberculosis, we established a protocol to screen other probiotic candidates. PMID:24551602

  5. A murine oral model for Mycobacterium avium subsp. paratuberculosis infection and immunomodulation with Lactobacillus casei ATCC 334

    PubMed Central

    Cooney, Meagan A.; Steele, James L.; Steinberg, Howard; Talaat, Adel M.

    2014-01-01

    Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) the causative agent of Johne's disease, is one of the most serious infectious diseases in dairy cattle worldwide. Due to the chronic nature of this disease and no feasible control strategy, it is essential to have an efficient animal model which is representative of the natural route of infection as well as a viable treatment option. In this report, we evaluated the effect of different doses of M. paratuberculosis in their ability to colonize murine tissues following oral delivery and the ability of Lactobacillus casei ATCC 334, a nascent probiotic, to combat paratuberculosis. Oral inoculation of mice was able to establish paratuberculosis in a dose-dependent manner. Two consecutive doses of approximately 109 CFU per mouse resulted in a disseminated infection, whereas lower doses were not efficient to establish infection. All inoculated mice were colonized with M. paratuberculosis, maintained infection for up to 24 weeks post infection and generated immune responses that reflect M. paratuberculosis infection in cattle. Notably, oral administration of L. casei ATCC 334 did not reduce the level of M. paratuberculosis colonization in treated animals. Interestingly, cytokine responses and histology indicated a trend for the immunomodulation and reduction of pathology in animals receiving L. casei ATCC 334 treatment. Overall, a reproducible oral model of paratuberculosis in mice was established that could be used for future vaccine experiments. Although the L. casei ATCC 334 was not a promising candidate for controlling paratuberculosis, we established a protocol to screen other probiotic candidates. PMID:24551602

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

    PubMed Central

    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

  7. A 38-kilobase pathogenicity island specific for Mycobacterium avium subsp. paratuberculosis encodes cell surface proteins expressed in the host.

    PubMed

    Stratmann, Janin; Strommenger, Birgit; Goethe, Ralph; Dohmann, Karen; Gerlach, Gerald-F; Stevenson, Karen; Li, Ling-Ling; Zhang, Qing; Kapur, Vivek; Bull, Tim J

    2004-03-01

    We have used representational difference analysis to identify a novel Mycobacterium avium subsp. paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designated mptA to -F and is immediately preceded by two putative Fur boxes. Functional genomics revealed that the mpt operon is flanked on one end by a fep cluster encoding proteins involved in the uptake of Fe(3+) and on the other end by a sid cluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kb M. avium subsp. paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110. Expression studies using Western blot analyses showed that MptC is present in the envelope fraction of M. avium subsp. paratuberculosis. The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening of Mycobacterium smegmatis transformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes an M. avium subsp. paratuberculosis pathogenicity island. PMID:14977927

  8. High-Throughput Direct Fecal PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Sheep and Cattle

    PubMed Central

    Waldron, Anna M.; Galea, Francesca; Whittington, Ann-Michele; Saunders, Vanessa F.; Begg, Douglas J.; de Silva, Kumudika; Purdie, Auriol C.; Whittington, Richard J.

    2014-01-01

    Johne's disease (JD) is a chronic enteric disease caused by Mycobacterium avium subsp. paratuberculosis that affects ruminants. Transmission occurs by the fecal-oral route. A commonly used antemortem diagnostic test for the detection of M. avium subsp. paratuberculosis in feces is liquid culture; however, a major constraint is the 2- to 3-month incubation period needed for this method. Rapid methods for the detection of M. avium subsp. paratuberculosis based on PCR have been reported, but comprehensive validation data are lacking. We describe here a new test, the high-throughput-Johnes (HT-J), to detect M. avium subsp. paratuberculosis in feces. Its diagnostic accuracy was compared with that of liquid radiometric (Bactec) fecal culture using samples from cattle (1,330 samples from 23 herds) and sheep (596 samples from 16 flocks). The multistage protocol involves the recovery of M. avium subsp. paratuberculosis cells from a fecal suspension, cell rupture by bead beating, extraction of DNA using magnetic beads, and IS900 quantitative PCR. The limit of detection of the assay was 0.0005 pg, and the limit of quantification was 0.005 pg M. avium subsp. paratuberculosis genomic DNA. Only M. avium subsp. paratuberculosis was detected from a panel of 51 mycobacterial isolates, including 10 with IS900-like sequences. Of the 549 culture-negative fecal samples from unexposed herds and flocks, 99% were negative in the HT-J test, while 60% of the bovine- and 84% of the ovine-culture-positive samples were positive in the HT-J test. As similar total numbers of samples from M. avium subsp. paratuberculosis-exposed animals were positive in culture and HT-J tests in both species, and as the results of a McNemar's test were not significant, these methods probably have similar sensitivities, but the true diagnostic sensitivities of these tests are unknown. These validation data meet the consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis and

  9. Suspicion of Mycobacterium avium subsp. paratuberculosis transmission between cattle and wild-living red deer (Cervus elaphus) by multitarget genotyping.

    PubMed

    Fritsch, Isabel; Luyven, Gabriele; Köhler, Heike; Lutz, Walburga; Möbius, Petra

    2012-02-01

    Multitarget genotyping of the etiologic agent Mycobacterium avium subsp. paratuberculosis is necessary for epidemiological tracing of paratuberculosis (Johne's disease). The study was undertaken to assess the informative value of different typing techniques and individual genome markers by investigation of M. avium subsp. paratuberculosis transmission between wild-living red deer and farmed cattle with known shared habitats. Fifty-three M. avium subsp. paratuberculosis type II isolates were differentiated by short sequence repeat analysis (SSR; 4 loci), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR; 8 loci), and restriction fragment length polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion. Isolates originated from free-living red deer (Cervus elaphus) from Eifel National Park (n = 13), six cattle herds living in the area of this park (n = 23), and five cattle herds without any contact with these red deer (n = 17). Data based on individual herds and genotypes verified that SSR G2 repeats did not exhibit sufficient stability for epidemiological studies. Two common SSR profiles (without G2 repeats), nine MIRU-VNTR patterns, and nine IS900-RFLP patterns were detected, resulting in 17 genotypes when combined. A high genetic variability was found for red deer and cattle isolates within and outside Eifel National Park, but it was revealed only by combination of different typing techniques. Results imply that within this restricted area, wild-living and farmed animals maintain a reservoir for specific M. avium subsp. paratuberculosis genotypes. No host relation of genotypes was obtained. Results suggested that four genotypes had been transmitted between and within species and that one genotype had been transmitted between cattle herds only. Use of multitarget genotyping for M. avium subsp. paratuberculosis type II strains and sufficiently stable genetic markers is essential for reliable

  10. Suspicion of Mycobacterium avium subsp. paratuberculosis Transmission between Cattle and Wild-Living Red Deer (Cervus elaphus) by Multitarget Genotyping

    PubMed Central

    Fritsch, Isabel; Luyven, Gabriele; Köhler, Heike; Lutz, Walburga

    2012-01-01

    Multitarget genotyping of the etiologic agent Mycobacterium avium subsp. paratuberculosis is necessary for epidemiological tracing of paratuberculosis (Johne's disease). The study was undertaken to assess the informative value of different typing techniques and individual genome markers by investigation of M. avium subsp. paratuberculosis transmission between wild-living red deer and farmed cattle with known shared habitats. Fifty-three M. avium subsp. paratuberculosis type II isolates were differentiated by short sequence repeat analysis (SSR; 4 loci), mycobacterial interspersed repetitive-unit–variable-number tandem-repeat analysis (MIRU-VNTR; 8 loci), and restriction fragment length polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion. Isolates originated from free-living red deer (Cervus elaphus) from Eifel National Park (n = 13), six cattle herds living in the area of this park (n = 23), and five cattle herds without any contact with these red deer (n = 17). Data based on individual herds and genotypes verified that SSR G2 repeats did not exhibit sufficient stability for epidemiological studies. Two common SSR profiles (without G2 repeats), nine MIRU-VNTR patterns, and nine IS900-RFLP patterns were detected, resulting in 17 genotypes when combined. A high genetic variability was found for red deer and cattle isolates within and outside Eifel National Park, but it was revealed only by combination of different typing techniques. Results imply that within this restricted area, wild-living and farmed animals maintain a reservoir for specific M. avium subsp. paratuberculosis genotypes. No host relation of genotypes was obtained. Results suggested that four genotypes had been transmitted between and within species and that one genotype had been transmitted between cattle herds only. Use of multitarget genotyping for M. avium subsp. paratuberculosis type II strains and sufficiently stable genetic markers is essential for reliable

  11. Mycobacterium avium ss paratuberculosis-associated diseases: piecing the Crohn's puzzle together.

    PubMed

    Gitlin, Laura; Borody, Thomas Julius; Chamberlin, William; Campbell, Jordana

    2012-09-01

    The relation of Mycobacterium avium ss paratuberculosis (MAP) to Crohn's Disease (CD) and other MAP-associated conditions remains controversial. New data, coupled with the analogous Helicobacter pylori (H. pylori) story, has permitted us to piece together the MAP puzzle and move forward with a more scientific way of treating inflammatory bowel disease, particularly CD. As infection moves centre stage in inflammatory bowel disease, the dated "aberrant reaction" etiology has lost scientific credibility. Now, our growing understanding of MAP-associated diseases demands review and articulation. We focus here on (1) the concept of MAP-associated diseases; (2) causality, Johne Disease, the "aberrant reaction" hypothesis; and (3) responses to published misconceptions questioning MAP as a pathogen in CD. PMID:22858515

  12. Genotype profiles of Mycobacterium avium subspecies paratuberculosis recovered from suspected and Crohn's disease patients in India.

    PubMed

    Singh, A V; Singh, S V; Sohal, J S; Singh, P K

    2010-06-01

    Present study aimed to genotype Mycobacterium avium subspecies paratuberculosis (MAP) recovered from suspected and Crohn' s disease patients. A total of 32 MAP and DNA (directly from clinical samples) recovered from human origin were genotyped using IS 1311 PCR-REA. Isolates were cultured from stool, biopsies and blood clots of Crohn's disease patients, and stool samples of suspected (animal attendants, lab workers etc). Of the 32 MAP isolates belonging to 28 human beings, majority (84.3%) were genotyped as 'Bison type', while 21.7% were of 'cattle' and none was 'sheep' genotype. Study first time reports distribution of 'Cattle' and 'Bison type' 'genotypes in suspected and Crohn's patients on pilot scale in India. 'Bison type' genotype was predominant in the surveyed human population. PMID:22471168

  13. Optimization of Methods for Obtaining, Extracting and Detecting Mycobacterium avium subsp. paratuberculosis in Environmental Samples using Quantitative, Real-Time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Detection of Johne’s disease, an enteric infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), has been impeded by the lack of rapid, reliable detection methods. The goal of this study was to optimize methodologies for obtaining, extracting and evaluating t...

  14. Analysis of the Immune Response to a Major Membrane Protein of Mycobacterium avium subsp. paratuberculosis in Experimentally and Naturally Infected Cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 35 kDa major membrane protein (MMP) of Mycobacterium avium subsp. paratuberculosis (Map) is implicated in the pathogenesis of paratuberculosis (Ptb) in cattle. Understanding the immune response to MMP could reveal how Map evades immune elimination and provide information needed for developing a ...

  15. Sensitivity of mycobacterium avium subsp paratuberculosis, escherichia coli and salmonella enterica serotype typhimurium to low pH, high organic acids and ensiling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability of Mycobacterium avium subsp paratuberculosis (M. paratuberculosis), Salmonella enterica serotype Typhimurium (S. Typhimurium) and a commensal Escherichia coli (E. coli) isolate to persist under low pH and high organic acid conditions was determined. Die-off rates were calculated followi...

  16. Antigen-Specific B-Cell Unresponsiveness Induced by Chronic Mycobacterium avium subsp. paratuberculosis Infection of Cattle

    PubMed Central

    Waters, W. R.; Stabel, J. R.; Sacco, R. E.; Harp, J. A.; Pesch, B. A.; Wannemuehler, M. J.

    1999-01-01

    Mycobacterium avium subsp. paratuberculosis infection of cattle results in a chronic granulomatous enteritis. Clinical disease (i.e., cachexia, diarrhea, and high fecal bacterial counts) is preceded by a lengthy subclinical stage of disease. The immunologic mechanisms associated with the progression of infected cattle from subclinical to clinical disease are unclear. In this study, a cell proliferation assay was used in combination with flow cytometry to compare peripheral blood lymphocyte responses of cattle with subclinical paratuberculosis to responses of cattle with clinical paratuberculosis. B cells from cattle with subclinical disease proliferated vigorously upon stimulation with M. avium subsp. paratuberculosis antigen, with up to 12.4% of the total B cells responding. However, B cells from cattle with clinical disease did not proliferate upon antigen stimulation despite good proliferation in response to concanavalin A stimulation. In addition, these animals had high percentages of peripheral blood B cells. B cells from noninfected animals did not proliferate upon M. avium subsp. paratuberculosis antigen stimulation. Thus, it appears that B-cell proliferation is a sensitive indicator of subclinical Johne’s disease. Furthermore, the immunologic mechanisms responsible for the antigen-specific unresponsiveness of peripheral blood B cells may be significant in the eventual progression from subclinical to clinical Johne’s disease in cattle. PMID:10084991

  17. Sensitive and specific enzyme-linked immunosorbent assay for detecting serum antibodies against Mycobacterium avium subsp. paratuberculosis in fallow deer.

    PubMed

    Prieto, José M; Balseiro, Ana; Casais, Rosa; Abendaño, Naiara; Fitzgerald, Liam E; Garrido, Joseba M; Juste, Ramon A; Alonso-Hearn, Marta

    2014-08-01

    The enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies against Mycobacterium avium subsp. paratuberculosis in wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deer M. avium subsp. paratuberculosis isolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detect M. avium subsp. paratuberculosis antibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations. PMID:24872517

  18. Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting Serum Antibodies against Mycobacterium avium subsp. paratuberculosis in Fallow Deer

    PubMed Central

    Prieto, José M.; Balseiro, Ana; Casais, Rosa; Abendaño, Naiara; Fitzgerald, Liam E.; Garrido, Joseba M.; Juste, Ramon A.

    2014-01-01

    The enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies against Mycobacterium avium subsp. paratuberculosis in wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deer M. avium subsp. paratuberculosis isolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detect M. avium subsp. paratuberculosis antibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations. PMID:24872517

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

    PubMed

    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. PMID:16244077

  20. The potential Public Health Impact of Mycobacterium avium ssp. paratuberculosis: Global Opinion Survey of Topic Specialists.

    PubMed

    Waddell, L A; Rajić, A; Stärk, K D C; McEwen, S A

    2016-05-01

    Global research knowledge has accumulated over the past few decades, and there is reasonable evidence for a positive association between Mycobacterium avium spp. paratuberculosis and Crohn's disease in humans, although its role as a human pathogen has not been entirely accepted. For this reason, management of public health risk due to M. paratuberculosis remains an important policy issue in agri-food public health arenas in many countries. Responsible authorities must decide whether existing mitigation strategies are sufficient to prevent or reduce human exposure to M. paratuberculosis. A Web-based questionnaire was administered to topic specialists to elicit empirical knowledge and opinion on the overall public health impact of M. paratuberculosis, the importance of various routes of human exposure to the pathogen, existing mitigation strategies and the need for future strategies. The questionnaire had four sections and consisted of 20 closed and five open questions. Topic specialists believed that M. paratuberculosis is likely a risk to human health (44.8%) and, given the paucity of available evidence, most frequently ranked it as a moderate public health issue (40.1%). A significant correlation was detected between topic specialists' commitment to M. paratuberculosis in terms of the number of years or proportion of work dedicated to this topic, and the likelihood of an extreme answer (high or low) to the above questions. Topic specialists identified contact with ruminants and dairy products as the most likely routes of exposure for humans. There was consensus on exposure routes for ruminants and what commodities to target in mitigation efforts. Described mandatory programmes mainly focused on culling diseased animals and voluntary on-farm prevention programmes. Despite ongoing difficulties in the identification of subclinical infections in animals, the topic specialists largely agreed that further enhancement of on-farm programmes in affected commodities by

  1. Structure determination of lipopeptides from Mycobacterium avium subspecies paratuberculosis and identification of antigenic lipopeptide probes.

    PubMed

    Mitachi, Katsuhiko; Sharma Gautam, Lekh Nath; Rice, Jeffrey H; Eda, Keiko; Wadhwa, Ashutosh; Momotani, Eiichi; Hlopak, Joseph P; Eda, Shigetoshi; Kurosu, Michio

    2016-07-15

    Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic illnesses mostly in ruminants. MAP infection of intestinal tissue triggers a fatal inflammatory disorder, Johne's disease (paratuberculosis). Development of fast and reliable diagnostic methods for Johne's disease in clinically suspected ruminants requires the discovery of MAP-specific antigens that induce immune responses. Despite a longtime interest in finding such antigens that can detect serum antibody responses with high sensitivity, the antigens currently used for a diagnosis of the MAP infections are the crude extracts from the whole cell. We performed the serum antibody response assay-guided purification of the ethanol extract from MAP isolated from an infected cow. With the results of extensive fractionations and in vitro assays, we identified that arachidyl-d-Phe-N-Me-l-Val-l-Ile-l-Phe-l-Ala-OH (named lipopeptide IIß, 3) exhibited the highest antibody binding activity in serum of a MAP-infected cattle compared with the other lipopeptides isolated from MAP. The absolute chemistry of 3 was determined unequivocally via our high-performance liquid chromatography (HPLC)-amino acid databases. α-Amino lipopeptide IIß and its fluorescent probes were synthesized and evaluated in serum antibody binding activity assays. Lipopeptide IIß-(2S)-NH2 (9) and its dansyl and fluorescein isothiocyanate (FITC) probes (10 and 11) exhibited antibody-mediated binding activity; thus, such MAP-specific lipopeptide probes can be potential biomarkers for the development of rapid and accurate diagnosis of Johne's disease. PMID:27114041

  2. Short communication: Viable Mycobacterium avium subspecies paratuberculosis in retail artisanal Coalho cheese from Northeastern Brazil.

    PubMed

    Faria, A C S; Schwarz, D G G; Carvalho, I A; Rocha, B B; De Carvalho Castro, K N; Silva, M R; Moreira, M A S

    2014-07-01

    Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis and it potentially plays a role in Crohn's disease. In humans, the main route of transmission of MAP might be the intake of contaminated milk and dairy products. Considering that MAP has already been detected in many types of cheese in different counties, and that Coalho cheese is an important dairy product in northeastern Brazil, the aim of this study was to report the first detection of MAP in retail Coalho cheese in Brazil by PCR and culture. Of 30 retail Coalho cheese samples, 3 (10%) amplified fragments of a similar size to that expected (626 bp) were obtained and viable MAP was recovered by culture from 1 (3.3%) sample. The DNA from the positive culture sample was sequenced and showed 99% identity with the insertion sequence IS900 deposited in GenBank. It was possible to identify the presence of MAP-specific DNA in the analyzed samples for the first time in Brazil, and to recover viable cells from retail Coalho cheese. PMID:24797534

  3. Host Transcriptional Profiles and Immunopathologic Response following Mycobacterium avium subsp. paratuberculosis Infection in Mice

    PubMed Central

    Shin, Min-Kyoung; Park, Hongtae; Shin, Seung Won; Jung, Myunghwan; Lee, Su-Hyung; Kim, Dae-Yong; Yoo, Han Sang

    2015-01-01

    Paratuberculosis or Johne’s disease is a chronic granulomatous enteropathy in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection. In the present study, we examined the host response to MAP infection in spleens of mice in order to investigate the host immunopathology accompanying host-pathogen interaction. Transcriptional profiles of the MAP-infected mice at 3 and 6 weeks p.i. showed severe histopathological changes, whereas those at 12 weeks p.i. displayed reduced lesion severity in the spleen and liver. MAP-infected mice at 3 and 6 weeks p.i. showed up-regulation of interferon-related genes, scavenger receptor, and complement components, suggesting an initial innate immune reaction, such as macrophage activation, bactericidal activity, and macrophage invasion of MAP. Concurrently, MAP-infected mice at 3 and 6 weeks p.i. were also suggested to express M2 macrophage phenotype with up-regulation of Mrc1, and Marco and down-regulation of MHC class II, Ccr7, and Irf5, and canonical pathways related to the T cell response including ICOS-ICOSL signaling in T helper cells, calcium-induced T lymphocyte apoptosis, and CD28 signaling in T helper cell. These results provide information which furthers the understanding of the immunopathologic response to MAP infection in mice, thereby providing insights valuable for research into the pathogenesis for MAP infection. PMID:26439498

  4. Seroprevalence and risk factors of Mycobacterium avium subspecies paratuberculosis infection in domestic sika deer in China.

    PubMed

    Meng, Qing-Feng; Li, Ying; Yang, Fan; Yao, Gui-Zhi; Qian, Ai-Dong; Wang, Wei-Li; Cong, Wei

    2015-06-01

    Paratuberculosis or Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic infectious granulomatous enteritis of ruminants and other animals, which has a worldwide occurrence, but little is known of MAP infection in domestic sika deer in Jilin Province, China. The objective of the present investigation was to examine seroprevalence and risk factors of MAP infection in Jilin Province. Serum samples collected from 1400 sika deer from 16 sika deer herds were collected in the 4 districts of the province between May 2013 and August 2014 and were tested independently for the presence of antibodies against MAP. A total of 247 (17.64 %) sika deer tested positive for MAP antibodies using a commercially available enzyme immunoassay kit. The management level of farm and collecting region of sika deer was the main risk factor associated with MAP infection. The present study revealed the seroprevalence of MAP infection in sika deer in Jilin Province, China, which provided the baseline data for taking comprehensive countermeasures and measures in effectively preventing and controlling MAP infection in sika deer. PMID:25904509

  5. Indoleamine 2,3-Dioxygenase, Tryptophan Catabolism, and Mycobacterium avium subsp. paratuberculosis: a Model for Chronic Mycobacterial Infections ▿ †

    PubMed Central

    Plain, Karren M.; de Silva, Kumudika; Earl, John; Begg, Douglas J.; Purdie, Auriol C.; Whittington, Richard J.

    2011-01-01

    Virulent mycobacterial infections progress slowly, with a latent period that leads to clinical disease in a proportion of cases. Mycobacterium avium subsp. paratuberculosis is an intracellular pathogen that causes paratuberculosis or Johne's disease (JD), a chronic intestinal disease of ruminants. Indoleamine 2,3-dioxygenase (IDO), an enzyme that regulates tryptophan metabolism, was originally reported to have a role in intracellular pathogen killing and has since been shown to have an important immunoregulatory role in chronic immune diseases. Here we demonstrate an association between increased IDO levels and progression to clinical mycobacterial disease in a natural host, characterizing gene expression, protein localization, and functional effects. IDO mRNA levels were significantly increased in M. avium subsp. paratuberculosis-infected monocytic cells. Levels of both IDO gene and protein expression were significantly upregulated within the affected tissues of sheep with JD, particularly at the site of primary infection, the ileum, of animals with severe multibacillary disease. Lesion severity was correlated with the level of IDO gene expression. IDO gene expression was also increased in the peripheral blood cells of M. avium subsp. paratuberculosis-exposed sheep and cattle. IDO breaks down tryptophan, and systemic increases were functional, as shown by decreased plasma tryptophan levels, which correlated with the onset of clinical signs, a stage well known to be associated with Th1 immunosuppression. IDO may be involved in downregulating immune responses to M. avium subsp. paratuberculosis and other virulent mycobacteria, which may be an example of the pathogen harnessing host immunoregulatory pathways to aid survival. These findings raise new questions about the host-mycobacterium interactions in the progression from latent to clinical disease. PMID:21730087

  6. Infection with Mycobacterium avium subsp. paratuberculosis Results in Rapid Interleukin-1β Release and Macrophage Transepithelial Migration

    PubMed Central

    Lamont, Elise A.; O'Grady, Scott M.; Davis, William C.; Eckstein, Torsten

    2012-01-01

    Pathogen processing by the intestinal epithelium involves a dynamic innate immune response initiated by pathogen-epithelial cell cross talk. Interactions between epithelium and Mycobacterium avium subsp. paratuberculosis have not been intensively studied, and it is currently unknown how the bacterium-epithelial cell cross talk contributes to the course of infection. We hypothesized that M. avium subsp. paratuberculosis harnesses host responses to recruit macrophages to the site of infection to ensure its survival and dissemination. We investigated macrophage recruitment in response to M. avium subsp. paratuberculosis using a MAC-T bovine macrophage coculture system. We show that M. avium subsp. paratuberculosis infection led to phagosome acidification within bovine epithelial (MAC-T) cells as early as 10 min, which resulted in upregulation of interleukin-1β (IL-1β) at transcript and protein levels. Within 10 min of infection, macrophages were recruited to the apical side of MAC-T cells. Inhibition of phagosome acidification or IL-1β abrogated this response, while MCP-1/CCL-2 blocking had no effect. IL-1β processing was dependent upon Ca2+ uptake from the extracellular medium and intracellular Ca2+ oscillations, as determined by EGTA and BAPTA-AM [1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester)] treatments. Thus, M. avium subsp. paratuberculosis is an opportunist that takes advantage of extracellular Ca2+-dependent phagosome acidification and IL-1β processing in order to efficiently transverse the epithelium and enter its niche—the macrophage. PMID:22778093

  7. Induction of B Cell Responses upon Experimental Infection of Neonatal Calves with Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Stabel, J. R.; Bannantine, J. P.; Eda, Shigetoshi; Robbe-Austerman, S.

    2011-01-01

    The objective of this study was to determine if experimental infection of neonatal calves with Mycobacterium avium subsp. paratuberculosis would invoke changes in the percentages of total B cells in the peripheral blood mononuclear cell population and of subpopulations of B cells as determined by CD5, CD25, and CD45RO markers during a 12-month period. Experimental infection groups included control (noninfected), oral (infected with M. avium subsp. paratuberculosis strain K-10), oral/DXM (pretreatment with dexamethasone before oral inoculation), i.p. (intraperitoneal inoculation), and oral/M (oral inoculation with mucosal scrapings from a cow with clinical disease) groups. Over the course of the study, the percentages of total B cells in nonstimulated and antigen-stimulated cell cultures increased for oral and i.p. group calves, with the highest percentages noted at 3 and 6 months. Oral/M group calves had increased percentages of activated B cells, as determined by CD5dim and CD5bright markers, at 9 and 12 months. Experimental infection by all methods resulted in increased expression of CD25+ and CD45RO+ B cells early in the study, but the most significant results were observed at 12 months for oral/DXM and oral/M group calves. Immunoblot analyses with a whole-cell sonicate of M. avium subsp. paratuberculosis demonstrated the most reactivity with sera from i.p. group calves and the least reactivity with sera from oral group calves. Further evidence of M. avium subsp. paratuberculosis-specific antibody responses in the i.p. group calves was demonstrated using the ethanol vortex enzyme-linked immunosorbent assay (EvELISA) method. In summary, an induction of B cell responses was noted after experimental infection with M. avium subsp. paratuberculosis, with differences in responses noted according to the method of experimental inoculation. PMID:21543587

  8. Host responses to the pathogen Mycobacterium avium subsp. paratuberculosis and beneficial microbes exhibit host sex specificity.

    PubMed

    Karunasena, Enusha; McMahon, K Wyatt; Chang, David; Brashears, Mindy M

    2014-08-01

    Differences between microbial pathogenesis in male and female hosts are well characterized in disease conditions connected to sexual transmission. However, limited biological insight is available on variances attributed to sex specificity in host-microbe interactions, and it is most often a minimized variable outside these transmission events. In this work, we studied two gut microbes-a pathogen, Mycobacterium avium subsp. paratuberculosis, and a probiotic, Lactobacillus animalis NP-51-and the interaction between each agent and the male and female gastrointestinal systems. This trial was conducted in BALB/c mice (n=5 per experimental group and per sex at a given time point), with analysis at four time points over 180 days. Host responses to M.avium subsp. paratuberculosis and L. animalis were sensitive to sex. Cytokines that were significantly different (P ≤ 0.05) betweenthe sexes included interleukin-1α/β (IL-1α/β), IL-17, IL-6, IL-10, IL-12, and gamma interferon (IFN-) and were dependent on experimental conditions. However, granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and IL-13/23 showed no sex specificity. A metabolomics study indicated a 0.5- to 2.0-fold (log2 scale) increase in short-chain fatty acids (butyrate and acetate) in males and greater increases in o-phosphocholine or histidine from female colon tissues; variances distinct to each sex were observed with age or long-term probiotic consumption. Two genera, Staphylococcus and Roseburia, were consistently overrepresented in females compared to males; other species were specific to one sex but fluctuated depending on experimental conditions. The differences observed suggest that male and female gut tissues and microbiota respond to newly introduced microorganisms differently and that gut-associated microorganisms with host immune system responses and metabolic activity are supported by biology distinct to the host sex. PMID:24814797

  9. Host Responses to the Pathogen Mycobacterium avium subsp. paratuberculosis and Beneficial Microbes Exhibit Host Sex Specificity

    PubMed Central

    McMahon, K. Wyatt; Chang, David; Brashears, Mindy M.

    2014-01-01

    Differences between microbial pathogenesis in male and female hosts are well characterized in disease conditions connected to sexual transmission. However, limited biological insight is available on variances attributed to sex specificity in host-microbe interactions, and it is most often a minimized variable outside these transmission events. In this work, we studied two gut microbes—a pathogen, Mycobacterium avium subsp. paratuberculosis, and a probiotic, Lactobacillus animalis NP-51—and the interaction between each agent and the male and female gastrointestinal systems. This trial was conducted in BALB/c mice (n = 5 per experimental group and per sex at a given time point), with analysis at four time points over 180 days. Host responses to M. avium subsp. paratuberculosis and L. animalis were sensitive to sex. Cytokines that were significantly different (P ≤ 0.05) between the sexes included interleukin-1α/β (IL-1α/β), IL-17, IL-6, IL-10, IL-12, and gamma interferon (IFN-γ) and were dependent on experimental conditions. However, granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and IL-13/23 showed no sex specificity. A metabolomics study indicated a 0.5- to 2.0-fold (log2 scale) increase in short-chain fatty acids (butyrate and acetate) in males and greater increases in o-phosphocholine or histidine from female colon tissues; variances distinct to each sex were observed with age or long-term probiotic consumption. Two genera, Staphylococcus and Roseburia, were consistently overrepresented in females compared to males; other species were specific to one sex but fluctuated depending on experimental conditions. The differences observed suggest that male and female gut tissues and microbiota respond to newly introduced microorganisms differently and that gut-associated microorganisms with host immune system responses and metabolic activity are supported by biology distinct to the host sex. PMID:24814797

  10. Results of Multiple Diagnostic Tests for Mycobacterium avium subsp. paratuberculosis in Patients with Inflammatory Bowel Disease and in Controls

    PubMed Central

    Collins, Michael T.; Lisby, Gorm; Moser, Claus; Chicks, Debra; Christensen, Steen; Reichelderfer, Mark; Høiby, Niels; Harms, Bruce A.; Thomsen, Ole Ø.; Skibsted, Ulrik; Binder, Vibeke

    2000-01-01

    Mycobacterium avium subsp. paratuberculosis has been incriminated as a cause of Crohn's disease (CD); however, studies to date have been relatively small and generally only used a single diagnostic assay. The objective of the study was to reexamine the association of M. avium subsp. paratuberculosis and CD using multiple diagnostic tests. Five methods were used to detect M. avium subsp. paratuberculosis infections in 439 inflammatory bowel disease (IBD) patients and 324 control subjects in the United States and Denmark. Most assays were adaptations of diagnostic tests for this infection performed routinely on animals. PCR for IS900, a genetic element unique to M. avium subsp. paratuberculosis, was positive significantly more often on resected bowel and lymph node tissues from CD patients (19.0%) and ulcerative colitis (UC) patients (26.2%) than from controls (6.3%) (P < 0.05). Positive IS900 PCR results occurred more often in U.S. than in Danish IBD patients, 32.0 versus 13.3% (P = 0.025). The majority of Danish patients were bacillus Calmette-Guérin (Mycobacterium bovis BCG) vaccinated (CD, 77.5%; UC, 86.6%; controls, 83.0%) whereas none of the U.S. patients with IBD and only 2% of U.S. controls were vaccinated. Among Danish IBD patients, positive PCR findings were four times more common among subjects who were not BCG vaccinated (33.3%) than among BCG vaccinates (8.8%, P = 0.02). Culture of the same tissues tested by PCR using modified BACTEC 12B medium failed to grow M. avium subsp. paratuberculosis from patients or controls. U.S. CD patients had the highest serological evidence (enzyme-linked immunosorbent assay [ELISA] for serum antibodies) of M. avium subsp. paratuberculosis infection (20.7% of patients positive) which was higher than for all UC patients studied (6.1%) or healthy controls (3.8%, P < 0.005). Among Danish patients alone, however, no significant differences in rates of ELISA-positive results among CD, UC, or control patients were found. For 181

  11. Chemical Decontamination with N-Acetyl-l-Cysteine–Sodium Hydroxide Improves Recovery of Viable Mycobacterium avium subsp. paratuberculosis Organisms from Cultured Milk

    PubMed Central

    Bradner, L.; Robbe-Austerman, S.; Beitz, D. C.

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis is shed into the milk and feces of cows with advanced Johne's disease, allowing the transmission of M. avium subsp. paratuberculosis between animals. The objective of this study was to formulate an optimized protocol for the isolation of M. avium subsp. paratuberculosis in milk. The parameters investigated included chemical decontamination with N-acetyl-l-cysteine–sodium hydroxide (NALC-NaOH), alone and in combination with antibiotics (vancomycin, amphotericin B, and nalidixic acid), and the efficacy of solid (Herrold's egg yolk medium [HEY]) and liquid (Bactec 12B and para-JEM) culture media. For each experiment, raw milk samples from a known noninfected cow were inoculated with 102 to 108 CFU/ml of live M. avium subsp. paratuberculosis organisms. The results indicate that an increased length of exposure to NALC-NaOH from 5 to 30 min and an increased concentration of NaOH from 0.5 to 2.0% did not affect the viability of M. avium subsp. paratuberculosis. Additional treatment of milk samples with the antibiotics following NALC-NaOH treatment decreased the recovery of viable M. avium subsp. paratuberculosis cells more than treatment with NALC-NaOH alone. The Bactec 12B medium was the superior medium of the three evaluated for the isolation of M. avium subsp. paratuberculosis from milk, as it achieved the lowest threshold of detection. The optimal conditions for NALC-NaOH decontamination were determined to be exposure to 1.50% NaOH for 15 min followed by culture in Bactec 12B medium. This study demonstrates that chemical decontamination with NALC-NaOH resulted in a greater recovery of viable M. avium subsp. paratuberculosis cells from milk than from samples treated with hexadecylpyridinium chloride (HPC). Therefore, it is important to optimize milk decontamination protocols to ensure that low concentrations of M. avium subsp. paratuberculosis can be detected. PMID:23637290

  12. Crohn's disease and Mycobacterium avium subsp. paratuberculosis: the need for a study is long overdue.

    PubMed

    Davis, William C; Madsen-Bouterse, Sally A

    2012-01-15

    The initial suggestion that Mycobacterium avium subsp. paratuberculosis (Map) might be involved in the pathogenesis of Crohn's disease (CD) was based on the apparent similarity of lesions in the intestine of patients with CD with those present in cattle infected with Map, the etiological agent of Johne's disease (JD). Recent investigations have now revealed the presence of Map or Map DNA in blood or lesions from adults and children with CD. Of special interest, Map has also been found in patients with other diseases as well as healthy subjects. The latter observations indicate all humans are susceptible to infection with Map and that, like with other mycobacterial pathogens such as Mycobacterium tuberculosis, infection does not invariably lead to development of clinical disease but rather development of a persistent latent stage of infection where an immune response controls but does not eliminate the pathogen. Limited information has been obtained on the immune response to Map in healthy subjects and patients with CD. Understanding how Map may be involved in the pathogenesis of CD will require a better understanding of the immune response to Map in one of its common hosts as well as healthy humans and patients with CD. PMID:22209202

  13. Survival of Mycobacterium avium subsp. paratuberculosis in biofilms on livestock watering trough materials.

    PubMed

    Cook, Kimberly L; Britt, Jenks S; Bolster, Carl H

    2010-02-24

    Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease, a chronic enteric infection that affects ruminants. Despite the ubiquitous occurrence of Mycobacterium sp. in nature and the fact that Johne's disease has been reported worldwide, little research has been done to assess its survival in agricultural environments. The goal of this 365-day study was to evaluate the ability of Map to persist in mixed-community biofilms on materials commonly used to construct livestock watering troughs. Map was inoculated into 32l of trough water containing either concrete, plastic, galvanized or stainless steel trough materials. The concentration of Map was determined by using quantitative, real-time PCR to target the IS900 sequence in DNA extracts. High concentrations of Map were detected on all trough materials after 3 days (around 1 x 10(5)cells cm(-2)). Based on the best-fit slopes, the time required for a 99% reduction (t(99)) in biofilm-associated Map cells was 144 and 115 days for plastic and stainless steel trough materials, respectively. Map concentrations did not decrease on concrete and galvanized steel trough materials. These results suggest that Map survives well in biofilms present on livestock watering trough materials. To inhibit spread of this organism and exposure of susceptible animals to Map on infected farms, best management practices aimed at maintaining biofilm-free trough surfaces should be included in any Johne's control plan. PMID:19717251

  14. Fabrication of a Novel Conductometric Biosensor for Detecting Mycobacterium avium subsp. paratuberculosis Antibodies

    PubMed Central

    Okafor, Chika; Grooms, Daniel; Alocilja, Evangelyn; Bolin, Steven

    2008-01-01

    Johne's disease (JD) is one of the most costly bacterial diseases in cattle. In the U.S., economic losses from the disease have been estimated to exceed $1,500,000,000 per year, mainly from the effects of reduced milk production. Current diagnostic tests for JD are laboratory based and many of those tests require specialized equipment and training. Development of rapid and inexpensive diagnostic assays, which are adapted for point-of-care applications, would aid in the control of JD. In this study, a polyaniline (Pani)-based conductometric biosensor, in an immunomigration format, was fabricated for the detection of serum antibody (IgG) against the causal organism of JD, Mycobacterium avium subsp. paratuberculosis (MAP). Immobilized Mycobacterium avium purified proteins in the capture membrane were used to detect MAP IgG, previously bound with Pani/anti-bovine IgG* conjugate in the conjugate membrane. After detection, the Pani in the sandwiched captured complex bridges an electrical circuit between the silver electrodes, flanking the capture membrane. The electrical conductance, caused by Pani, was measured as drop in electrical resistance. Testing of the biosensor with known JD positive and negative serum samples demonstrated a significant difference in the mean resistance observed between the groups. This proof-of-concept study demonstrated that a conductometric biosensor could detect MAP IgG in 2 minutes. The biosensor's speed of detection and the equipment involved would, among other things, support its application towards the various point-of-care opportunities aimed at JD management and control.

  15. Longitudinal Pathogenesis Study of Young Red Deer (Cervus elaphus) after Experimental Challenge with Mycobacterium avium subsp. paratuberculosis (MAP)

    PubMed Central

    Mackintosh, Colin; Clark, Gary; Tolentino, Brendan; Liggett, Simon; de Lisle, Geoff; Griffin, Frank

    2012-01-01

    Paratuberculosis progresses more quickly in young red deer than in sheep or cattle. This study describes the clinical, immunological and pathological changes over a 50-week period in fourteen 4-month-old red deer that received heavy oral challenge with Mycobacterium avium subsp. paratuberculosis (MAP). At 4 and 12 weeks post challenge they were anaesthetized and a section of jejunal lymph node was surgically removed for culture, histopathology, and genetic studies. All 14 deer became infected, none were clinically affected, and they had varying degrees of subclinical disease when killed at week 50. Week 4 biopsies showed no paratuberculosis lesions, but MAP was cultured from all animals. At weeks 12 and 50 histopathological lesions ranged from mild to severe with corresponding low-to-high antibody titres, which peaked at 12–24 weeks. IFN-γ responses peaked at 8–15 weeks and were higher in mildly affected animals than in those with severe lesions. PMID:22720193

  16. Production and proteomic characterisation of purified protein derivative from Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    2012-01-01

    Background Effective diagnosis of Johne's disease (JD), particularly at the stage of early subclinical infection, remains one of the greatest challenges for the control of JD worldwide. The IFN-γ test of cell mediated immunity is currently one of the most suitable diagnostics for subclinical infections, however a major limitation of this test is the lack of a standardised purified protein derivative (PPD) antigen (also referred to as Johnin PPD or PPDj). While attempting to replace PPDj with more specific individual antigens is an attractive proposition, bacterial culture derived PPDj remains the most effective antigen preparation for the diagnosis of subclinical JD. It may be possible to increase the reproducibility and specificity of PPDj preparations by further characterising and standardising the PPDj production. Results Using a standardised protocol, five in-house preparations of PPDj were prepared from cultures of Mycobacterium avium subsp. paratuberculosis (MAP). Compared to PPDs obtained from other institutes/laboratories, these preparations appeared to perform similarly well in the IFN-γ test. Although the broad proteomic composition of all PPDj preparations was remarkably similar, the absolute abundance of individual proteins varied markedly between preparations. All PPDj preparations contained common immunogenic proteins which were also observed in PPD preparations from Mycobacterium avium subsp. avium (PPDa) and Mycobacterium bovis (PPDb). Temporal difference in protein secretion of in vitro cultured MAP was observed between 20 and 34 weeks suggesting that the age of MAP culture used for PPDj preparations may markedly influence PPDj composition. Conclusions This study describes a protocol for the production of PPDj and its subsequent proteomic characterisation. The broad proteomic composition of different preparations of PPDj was, for the most part, highly similar. Compositional differences between PPDj preparations were found to be a direct

  17. Evidence for a Novel Gene Expression Program in Peripheral Blood Mononuclear Cells from Mycobacterium avium subsp. paratuberculosis-Infected Cattle

    PubMed Central

    Coussens, Paul M.; Colvin, Christopher J.; Rosa, Guilherme J. M.; Perez Laspiur, Juliana; Elftman, Michael D.

    2003-01-01

    A bovine-specific cDNA microarray system was used to compare gene expression profiles of peripheral blood mononuclear cells (PBMCs) from control uninfected (n = 4) and Johne's disease-positive (n = 6) Holstein cows. Microarray experiments were designed so that for each animal, a direct comparison was made between PBMCs stimulated in vitro with Mycobacterium avium subsp. paratuberculosis and PBMCs stimulated with phosphate-buffered saline (nil-stimulated PBMCs). As expected, M. avium subsp. paratuberculosis stimulation of infected cow PBMCs enhanced expression of gamma interferon transcripts. In addition, expression of 15 other genes was significantly affected (>1.25-fold change; P < 0.05) by in vitro stimulation with M. avium subsp. paratuberculosis. Similar treatment of control cow PBMCs with M. avium subsp. paratuberculosis resulted in significant changes in expression of 13 genes, only 2 of which were also affected in PBMCs from the infected cow PBMCs. To compare gene expression patterns in the two cow infection groups (infected cows and uninfected cows), a mixed-model analysis was performed with the microarray data. This analysis indicated that there were major differences in the gene expression patterns between cells isolated from the two groups of cows, regardless of in vitro stimulation. A total of 86 genes were significantly differentially expressed (P < 0.01) in M. avium subsp. paratuberculosis-stimulated PBMCs from infected cows compared to expression in similarly treated PBMCs from control cows. Surprisingly, a larger number of genes (110 genes) were also found to be significantly differentially expressed (P < 0.01) in nil-stimulated cells from the two infection groups. The expression patterns of selected genes were substantiated by quantitative real-time reverse transcriptase PCR. Flow cytometric analysis indicated that there were no gross differences in the relative populations of major immune cell types in PBMCs from infected and control cows. Thus

  18. Immunological findings associated with Argentinean strains of Mycobacterium avium subsp. paratuberculosis in bovine models.

    PubMed

    Colavecchia, Silvia B; Fernández, Bárbara; Jolly, Ana; Minatel, Leonardo; Hajos, Silvia E; Paolicchi, Fernando A; Mundo, Silvia L

    2016-08-01

    Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of ruminant paratuberculosis. The aim of this study was to evaluate the biological behavior of different Argentinean strains of MAP in two bovine infection models: macrophage (in vitro) and calf (in vivo) through the evaluation of early immune responses at the peripheral and local levels. Two MAP strains (A and C) were selected taking into account the different patterns of TNF-α and IL-10 secretion displayed by infected bovine macrophages in vitro. Two groups of calves were infected with 250mg of total wet weight live MAP: strain A infected group (MA, n=3), strain C infected group (MC, n=2). Another group of animals was mock-infected (MI, n=3). Infection was confirmed by MAP culture of feces and microscopic observation of granulomatous lesions in the gut tissue. All infected calves showed positive results in the DTH skin test. A significant increase in peripheral CD4CD25(+) cells in MC group on day 150 was detected. The specific cellular immune response developed allowed the identification of the infection as early as 30days in the MA group. However, the percentage of CD8CD25(+) cells was significantly increased on day 120 in MC group. Significant differences between groups in proliferation and cellular responses were also detected in ileocecal lymph node samples. In summary, the strains of MAP employed herein induced differential immune responses in peripheral cells, in the proliferative responses and in cell functionality at the local level. Our findings support the hypotheses that the in vitro behavior displayed by macrophages could be a tool to identify differences among MAP strains infecting bovines and that the host-pathogen interactions occurring upon infection are dependent on the strain of MAP involved. PMID:27138443

  19. Mycobacterium avium Subspecies paratuberculosis Infection Modifies Gut Microbiota under Different Dietary Conditions in a Rabbit Model

    PubMed Central

    Arrazuria, Rakel; Elguezabal, Natalia; Juste, Ramon A.; Derakhshani, Hooman; Khafipour, Ehsan

    2016-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) the causative agent of paratuberculosis, produces a chronic granulomatous inflammation of the gastrointestinal tract of ruminants. It has been recently suggested that MAP infection may be associated with dysbiosis of intestinal microbiota in ruminants. Since diet is one of the key factors affecting the balance of microbial populations in the digestive tract, we intended to evaluate the effect of MAP infection in a rabbit model fed a regular or high fiber diet during challenge. The composition of microbiota of the cecal content and the sacculus rotundus was studied in 20 New Zealand white female rabbits. The extracted DNA was subjected to paired-end Illumina sequencing of the V3-V4 hypervariable region of the 16S rRNA gene for microbiota analysis. Microbial richness (Chao1) in the cecal content was significantly increased by MAP infection in regular diet rabbits (p = 0.0043) and marginally increased (p = 0.0503) in the high fiber group. Analysis of beta-diversity showed that MAP infection produces deeper changes in the microbiota of sacculus rotundus than in the cecal content. A lower abundance of Proteobacteria in the cecal content of infected animals fed the high fiber diet and also lower abundance of Bacteroidetes in the sacculus rotundus of infected animals fed the regular diet were observed. Based on OPLS-DA analysis, we observed that some bacteria repeatedly appear to be positively associated with infection in different samples under different diets (families Dehalobacteriaceae, Coriobacteriaceae, and Mogibacteriaceae; genus Anaerofustis). The same phenomenon was observed with some of the bacteria negatively associated with MAP infection (genera Anaerostipes and Coprobacillus). However, other groups of bacteria (Enterobacteriaceae family and ML615J-28 order) were positively associated with infection in some circumstances and negatively associated with infection in others. Data demonstrate that MAP infection

  20. Detection of mycobacterium avium subsp. paratuberculosis in cheeses from small ruminants in Tuscany.

    PubMed

    Galiero, Alessia; Fratini, Filippo; Mataragka, Antonia; Turchi, Barbara; Nuvoloni, Roberta; Ikonomopoulos, John; Cerri, Domenico

    2016-01-18

    Paratuberculosis is an infectious disease which affects mainly domestic and wild ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Map has been associated with human diseases like Crohn disease, type-1 diabetes, sarcoidosis, multiple sclerosis and Hashimoto's thyroiditis. The aim of this study was to determine the level of Map positivity of cheeses produced in Tuscany (Italy) as an indication of human exposure to the specific pathogen. Sampling was focused on artisanal cheeses produced without commercial starter culture from raw sheep or goat milk, on small-scale farms. Samples were tested by quantitative PCR (qPCR) and culture. Map DNA was detected in 4/7 (57.14%) goat, and in 14/25 (56%) sheep cheeses by qPCR, whereas cultivation produced a positive result in only one case. This corresponded to a goat cheese that had also reacted positively by qPCR and yielded a viable Type S (sheep) strain of Map. The Map load of the tested samples based on qPCR ranged from 6×10 to 1.8×10(4)Map cells/g of cheese. The results indicate on average 56.57% and 66.6% positivity of cheese samples and farms, respectively. Hence, the type of cheeses that were analyzed within the context of this study seem to constitute a considerable source of human exposure to Map; although the question remains of whether the Map cells were present in a viable form, since positive results were almost exclusively recorded by qPCR. PMID:26555160

  1. Volatile Emissions from Mycobacterium avium subsp. paratuberculosis Mirror Bacterial Growth and Enable Distinction of Different Strains

    PubMed Central

    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

  2. Observed management practices in relation to the risk of infection with paratuberculosis and to the spread of Mycobacterium avium subsp. paratuberculosis in Swiss dairy and beef herds

    PubMed Central

    2014-01-01

    Background Many studies have been conducted to define risk factors for the transmission of bovine paratuberculosis, mostly in countries with large herds. Little is known about the epidemiology in infected Swiss herds and risk factors important for transmission in smaller herds. Therefore, the presence of known factors which might favor the spread of paratuberculosis and could be related to the prevalence at animal level of fecal shedding of Mycobacterium avium subsp. paratuberculosis were assessed in 17 infected herds (10 dairy, 7 beef). Additionally, the level of knowledge of herd managers about the disease was assessed. In a case–control study with 4 matched negative control herds per infected herd, the association of potential risk factors with the infection status of the herd was investigated. Results Exposure of the young stock to feces of older animals was frequently observed in infected and in control herds. The farmers’ knowledge about paratuberculosis was very limited, even in infected herds. An overall prevalence at animal level of fecal shedding of Mycobacterium avium subsp. paratuberculosis of 6.1% was found in infected herds, whereby shedders younger than 2 years of age were found in 46.2% of the herds where the young stock was available for testing. Several factors related to contamination of the heifer area with cows’ feces and the management of the calving area were found to be significantly associated with the within-herd prevalence. Animal purchase was associated with a positive herd infection status (OR = 7.25, p = 0.004). Conclusions Numerous risk factors favoring the spread of Mycobacterium avium subsp. paratuberculosis from adult animals to the young stock were observed in infected Swiss dairy and beef herds, which may be amenable to improvement in order to control the disease. Important factors were contamination of the heifer and the calving area, which were associated with higher within-herd prevalence of fecal shedding. The

  3. Monensin causes dose dependent inhibition of Mycobacterium avium subspecies paratuberculosis in radiometric culture

    PubMed Central

    Greenstein, Robert J; Su, Liya; Whitlock, Robert H; Brown, Sheldon T

    2009-01-01

    Background Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic wasting diarrheal disease in ruminants called Johne's disease, that is evocative of human inflammatory bowel disease (IBD). Agents used to treat IBD, called "anti-inflammatories", immuno-modulators" and "immuno-suppressants" inhibit MAP growth in culture. We concluded that, unknowingly, the medical profession has been treating MAP since sulfasalazine's introduction in 1942. Monensin, called a "Growth Enhancer" in cattle, ameliorates Johne's disease without a documented mechanism of action. We hypothesized that Monensin would inhibit MAP in culture. Methods Using the radiometric 14CO2 Bactec® system, that expresses mycobacterial growth in arbitrary growth index (GI) units, we studied the effect of Monensin on the growth kinetic of MAP isolated from humans with IBD ("Dominic", "Ben" & UCF-4) and cattle with Johne's disease (303 & ATCC 19698.) Results are expressed as percent inhibition of cumulative GI (%–ΔcGI). Results The positive control Clofazimine inhibits every strain tested. The negative controls Cycloheximide & Phthalimide, have no inhibition on any MAP strain. Monensin has dose dependent inhibition on every MAP strain tested. The most susceptible human isolate was UCF-4 (73% – ΔcGI at 1 μg/ml) and bovine isolate was 303 (73% – ΔcGI at 4 μg/ml.) Monensin additionally inhibits M. avium ATCC 25291 (87% – ΔcGI at 64 μg/ml) & BCG (92% – ΔcGI at 16 μg/ml). Discussion We show that in radiometric culture the "Growth Enhancer" Monensin causes dose dependent inhibition of mycobacteria including MAP. We posit that the "Growth Enhancer" effect of Monensin may, at least in part, be due to inhibition of MAP in clinical or sub-clinical Johne's disease. PMID:19338684

  4. FurA contributes to the oxidative stress response regulation of Mycobacterium avium ssp. paratuberculosis

    PubMed Central

    Eckelt, Elke; Meißner, Thorsten; Meens, Jochen; Laarmann, Kristin; Nerlich, Andreas; Jarek, Michael; Weiss, Siegfried; Gerlach, Gerald-F.; Goethe, Ralph

    2015-01-01

    The ferric uptake regulator A (FurA) is known to be involved in iron homeostasis and stress response in many bacteria. In mycobacteria the precise role of FurA is still unclear. In the presented study, we addressed the functional role of FurA in the ruminant pathogen Mycobacterium avium ssp. paratuberculosis (MAP) by construction of a furA deletion strain (MAPΔfurA). RNA deep sequencing revealed that the FurA regulon consists of repressed and activated genes associated to stress response or intracellular survival. Not a single gene related to metal homeostasis was affected by furA deletion. A decisive role of FurA during intracellular survival in macrophages was shown by significantly enhanced survival of MAPΔfurA compared to the wildtype, indicating that a principal task of mycobacterial FurA is oxidative stress response regulation in macrophages. This resistance was not associated with altered survival of mice after long term infection with MAP. Our results demonstrate for the first time, that mycobacterial FurA is not involved in the regulation of iron homeostasis. However, they provide strong evidence that FurA contributes to intracellular survival as an oxidative stress sensing regulator. PMID:25705205

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

    PubMed

    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; Tahlan, Kapil; De Buck, Jeroen

    2016-01-01

    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. PMID:26871723

  6. Molecular detection and typing of Mycobacterium avium subspecies paratuberculosis from milk samples of dairy animals.

    PubMed

    Kaur, Prabhdeep; Filia, G; Singh, S V; Patil, P K; Sandhu, K S

    2010-06-01

    Genotyping of Mycobacterium avium subspecies paratuberculosis (MAP) is important for precise classification of bacterium and for understanding the molecular epidemiology. The present study reports detection and typing of the MAP from milk. On the basis of clinical signs of diarrhea and/or weakness, the dairy animals suspected for Johne's disease were screened by Ziehl-Neelsen staining of fecal samples. The milk samples from 13 selected animals were processed for DNA extraction and direct IS900 polymerase chain reaction (PCR). MAP identified by IS900 PCR was genotyped using IS1311 PCR-restriction endonuclease analysis (REA). IS900 milk PCR revealed 30.8% animals positive for MAP, including 40% of the moderate and 50% of the heavy fecal shedders. All infected animals showed Bison type MAP in IS1311 PCR-REA. IS900 PCR can be used for screening of milk for MAP; however, the method needs to be evaluated for subclinical cases. IS1311 PCR-REA results indicated the predominance of Bison type MAP in the dairy animals of this region. PMID:20082257

  7. Assessment of food as a source of exposure to Mycobacterium avium subspecies paratuberculosis (MAP).

    PubMed

    2010-07-01

    The National Advisory Committee on Microbiological Criteria for Foods assessed the importance of food as a source of exposure to Mycobacterium avium subspecies paratuberculosis (MAP). MAP is the causative agent of Johne's disease, which affects primarily the small intestine of all ruminants. The significance of MAP as a human pathogen is unknown and is being investigated by several research groups. This document also reviews the efficacy of current detection methods, processing interventions, and MAP inactivation. Research needs related to MAP are provided. The Committee reached the following conclusions: current methods for detection of MAP have significant limitations, and a standard method for the detection of viable MAP cells is needed. Aside from MAP-infected domestic ruminant animals, the organism is found infrequently. If MAP in cattle is controlled, the source of MAP in other animals, food, and water may largely be eliminated. Milk, particularly raw milk, may be a likely food source for human exposure to MAP. Given the prevalence of MAP in U.S. cattle herds, ground beef may be a potential source of MAP. Although humans may be exposed to MAP through a variety of routes, including food and the environment, the frequency and amount of exposure will require additional research. PMID:20615354

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

    PubMed

    Capsel, Randal T; Thoen, Charles O; Reinhardt, Timothy A; Lippolis, John D; Olsen, Renee; Stabel, Judith R; Bannantine, John P

    2016-01-01

    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. PMID:27136199

  9. Genomic variations associated with attenuation in Mycobacterium avium subsp. paratuberculosis vaccine strains

    PubMed Central

    2013-01-01

    Background Mycobacterium avium subspecies paratuberculosis (MAP) whole cell vaccines have been widely used tools in the control of Johne’s disease in animals despite being unable to provide complete protection. Current vaccine strains derive from stocks created many decades ago; however their genotypes, underlying mechanisms and relative degree of their attenuation are largely unknown. Results Using mouse virulence studies we confirm that MAP vaccine strains 316 F, II and 2e have diverse but clearly attenuated survival and persistence characteristics compared with wild type strains. Using a pan genomic microarray we characterise the genomic variations in a panel of vaccine strains sourced from stocks spanning over 40 years of maintenance. We describe multiple genomic variations specific for individual vaccine stocks in both deletion (26–32 Kbp) and tandem duplicated (11–40 Kbp) large variable genomic islands and insertion sequence copy numbers. We show individual differences suitable for diagnostic differentiation between vaccine and wild type genotypes and provide evidence for functionality of some of the deleted MAP-specific genes and their possible relation to attenuation. Conclusions This study shows how culture environments have influenced MAP genome diversity resulting in large tandem genomic duplications, deletions and transposable element activity. In combination with classical selective systematic subculture this has led to fixation of specific MAP genomic alterations in some vaccine strain lineages which link the resulting attenuated phenotypes with deficiencies in high reactive oxygen species handling. PMID:23339684

  10. Mycobacterium avium ss. paratuberculosis Zoonosis – The Hundred Year War – Beyond Crohn’s Disease

    PubMed Central

    Sechi, Leonardo A.; Dow, Coad Thomas

    2015-01-01

    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. PMID:25788897

  11. Ulcerative colitis and Crohn's disease: is Mycobacterium avium subspecies paratuberculosis the common villain?

    PubMed Central

    2010-01-01

    Mycobacterium avium, subspecies paratuberculosis (MAP) causes a chronic disease of the intestines in dairy cows and a wide range of other animals, including nonhuman primates, called Johne's ("Yo-knee's") disease. MAP has been consistently identified by a variety of techniques in humans with Crohn's disease. The research investigating the presence of MAP in patients with Crohn's disease has often identified MAP in the "negative" ulcerative colitis controls as well, suggesting that ulcerative colitis is also caused by MAP. Like other infectious diseases, dose, route of infection, age, sex and genes influence whether an individual infected with MAP develops ulcerative colitis or Crohn's disease. The apparently opposite role of smoking, increasing the risk of Crohn's disease while decreasing the risk of ulcerative colitis, is explained by a more careful review of the literature that reveals smoking causes an increase in both diseases but switches the phenotype from ulcerative colitis to Crohn's disease. MAP as the sole etiologic agent of both ulcerative colitis and Crohn's disease explains their common epidemiology, geographic distribution and familial and sporadic clusters, providing a unified hypothesis for the prevention and cure of the no longer "idiopathic" inflammatory bowel diseases. PMID:21167058

  12. Mycobacterium avium subspecies paratuberculosis in the etiology of Crohn’s disease, cause or epiphenomenon?

    PubMed Central

    Liverani, Elisa; Scaioli, Eleonora; Cardamone, Carla; Dal Monte, Paola; Belluzzi, Andrea

    2014-01-01

    The origin of inflammatory bowel disease is unknown. Attempts have been made to isolate a microorganism that could explain the onset of inflammation, but no pathological agent has ever been identified. Johne’s disease is a granulomatous chronic enteritis of cattle and sheep caused by Mycobacterium avium subspecies paratuberculosis (MAP) and shows some analogies with Crohn’s disease (CD). Several studies have tried to clarify if MAP has a role in the etiology of CD. The present article provides an overview of the evidence in favor and against the “MAP-hypothesis”, analyzing the methods commonly adopted to detect MAP and the role of antimycobacterial therapy in patients with inflammatory bowel disease. Studies were identified through the electronic database, MEDLINE, and were selected based on their relevance to the objective of the review. The presence of MAP was investigated using multiple diagnostic methods for MAP detection and in different tissue samples from patients affected by CD or ulcerative colitis and in healthy controls. On the basis of their studies, several authors support a close relationship between MAP and CD. Although increasing evidence of MAP detection in CD patients is unquestionable, a clear etiological link still needs to be proven. PMID:25278700

  13. Mycobacterium avium Subspecies paratuberculosis: Human Exposure through Environmental and Domestic Aerosols

    PubMed Central

    Rhodes, Glenn; Richardson, Hollian; Hermon-Taylor, John; Weightman, Andrew; Higham, Andrew; Pickup, Roger

    2014-01-01

    Mycobacterium avium subspecies paratuberculosis (Map) causes Johne’s disease in animals and is significantly associated with Crohn’s disease (CD) in humans. Our previous studies have shown Map to be present in U.K. rivers due to land deposition from chronic livestock infection and runoff driven by rainfall. The epidemiology of CD in Cardiff showed a significant association with the River Taff, in which Map can be detected on a regular basis. We have previously hypothesized that aerosols from the river might influence the epidemiology of CD. In this preliminary study, we detected Map by quantitative PCR in one of five aerosol samples collected above the River Taff. In addition, we examined domestic showers from different regions in the U.K. and detected Map in three out of 30 independent samples. In detecting Map in river aerosols and those from domestic showers, this is the first study to provide evidence that aerosols are an exposure route for Map to humans and may play a role in the epidemiology of CD. PMID:25438013

  14. Mycobacterium avium subspecies paratuberculosis in the etiology of Crohn's disease, cause or epiphenomenon?

    PubMed

    Liverani, Elisa; Scaioli, Eleonora; Cardamone, Carla; Dal Monte, Paola; Belluzzi, Andrea

    2014-09-28

    The origin of inflammatory bowel disease is unknown. Attempts have been made to isolate a microorganism that could explain the onset of inflammation, but no pathological agent has ever been identified. Johne's disease is a granulomatous chronic enteritis of cattle and sheep caused by Mycobacterium avium subspecies paratuberculosis (MAP) and shows some analogies with Crohn's disease (CD). Several studies have tried to clarify if MAP has a role in the etiology of CD. The present article provides an overview of the evidence in favor and against the "MAP-hypothesis", analyzing the methods commonly adopted to detect MAP and the role of antimycobacterial therapy in patients with inflammatory bowel disease. Studies were identified through the electronic database, MEDLINE, and were selected based on their relevance to the objective of the review. The presence of MAP was investigated using multiple diagnostic methods for MAP detection and in different tissue samples from patients affected by CD or ulcerative colitis and in healthy controls. On the basis of their studies, several authors support a close relationship between MAP and CD. Although increasing evidence of MAP detection in CD patients is unquestionable, a clear etiological link still needs to be proven. PMID:25278700

  15. Internalization-dependent recognition of Mycobacterium avium ssp. paratuberculosis by intestinal epithelial cells.

    PubMed

    Pott, Johanna; Basler, Tina; Duerr, Claudia U; Rohde, Manfred; Goethe, Ralph; Hornef, Mathias W

    2009-12-01

    Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease, a highly prevalent chronic intestinal infection in domestic and wildlife ruminants. The microbial pathogenesis of MAP infection has attracted additional attention due to an association with the human enteric inflammatory Crohn's disease. MAP is acquired by the faecal-oral route prompting us to study the interaction with differentiated intestinal epithelial cells. MAP was rapidly internalized and accumulated in a late endosomal compartment. In contrast to other opportunistic mycobacteria or M. bovis, MAP induced significant epithelial activation as indicated by a NF-kappaB-independent but Erk-dependent chemokine secretion. Surprisingly, MAP-induced chemokine production was completely internalization-dependent as inhibition of Rac-dependent bacterial uptake abolished epithelial activation. In accordance, innate immune recognition of MAP by differentiated intestinal epithelial cells occurred through the intracellularly localized pattern recognition receptors toll-like receptor 9 and NOD1 with signal transduction via the adaptor molecules MyD88 and RIP2. The internalization-dependent innate immune activation of intestinal epithelial cells is in contrast to the stimulation of professional phagocytes by extracellular bacterial constituents and might significantly contribute to the histopathological changes observed during enteric MAP infection. PMID:19681906

  16. Mycobacterium avium subspecies Paratuberculosis and Crohn's regional ileitis: how strong is association?

    PubMed

    Singh, Sarman; Gopinath, Krishnamoorthy

    2011-07-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is a well-established etiological agent of Johne's disease in animals. In humans, similar clinical condition, first described by Crohn as regional ileitis in 1932, now known as Crohn's diseases (CD), has also been associated with this mycobacterial species. However, there are two schools of thoughts, one favoring MAP as its etiological agent while the second considers it as an immune-inflammatory condition triggered by an external factor. Onset of CD requires a series of events including predisposition of certain inherited genetic traits, associated environmental stimuli, and immune-inflammatory response. A combination of these factors probably leads to this disease. Recently, some human genes have also been identified which regulate ability to respond appropriately to the external factors. Added to these factors are concerns about the selection of clinical specimens and poor adherence to laboratory quality controls. The literature is full of contradictory findings, but there a lack of uniformity in the materials and methods used by many of these researchers. In this review, we provide our perspective under above circumstances and give our point of view which may open a platform for debate regarding the MAP as the etiological agent of human CD. PMID:22219557

  17. Application of multiple laboratory tests for Mycobacterium avium ssp. paratuberculosis detection in Crohn's disease patient specimens.

    PubMed

    Banche, Giuliana; Allizond, Valeria; Sostegni, Raffaello; Lavagna, Alessandro; Bergallo, Massimiliano; Sidoti, Francesca; Daperno, Marco; Rocca, Rodolfo; Cuffini, Anna Maria

    2015-07-01

    The difficulties involved in detecting and enumerating Mycobacterium avium subsp. paratuberculosis (MAP) as a pathogen potentially involved in Crohn's disease (CD) are well known. This study aimed to improve this situation through the application of multiple laboratory diagnostic tests to detect and isolate this bacterium from different specimens collected from CD-patients and non-CD subjects as controls. A total of 120 samples (terminal ileum and colon biopsies, blood and stool) were obtained from 19 CD-patients and from 11 individuals who did not have a clinicopathological diagnosis of CD (non-CD controls) attending for ileocolonoscopy. All samples were processed by staining techniques, culture on both solid and liquid media, and Insertion Sequence 900/F57 real-time PCR. The MAP frequency in CD-patients was found in a significantly greater proportion than in non-CD subjects; the most positive samples were biopsies from CD-patients tested by real-time PCR. MAP detection in biopsies, and in the other samples, by applying multiple and validated laboratory diagnostic tests, could be a marker of active infection, supporting MAP involvement in CD. PMID:26147146

  18. Survey of Mycobacterium avium subspecies paratuberculosis in road-killed wild carnivores in Portugal.

    PubMed

    Matos, Ana Cristina; Figueira, Luis; Martins, Maria Helena; Loureiro, Filipa; Pinto, Maria Lurdes; Matos, Manuela; Coelho, Ana Cláudia

    2014-12-01

    A survey to determine the occurrence of Mycobacterium avium subsp. paratuberculosis (MAP) in wild carnivores in Portugal was conducted by testing samples from road-killed animals between 2009 and 2012. Postmortem examinations were performed and tissues were collected from wild carnivores representing four families and six different species, with a total of 74 animals analyzed. Cultures were performed by using Löwenstein-Jensen and Middlebrook 7H11 solid media and acid-fast isolates were identified by polymerase chain reaction (PCR) and mycobactin dependency characteristics. Tissues were also screened for MAP by directly extracting DNA and testing for the MAP-specific sequences. The occurrence of infected animals (an animal had at least one tissue that was positive for culture or direct PCR) was 27.0% (n = 20). MAP was isolated from culture of 25 tissue samples (3.8%) and was detected by direct PCR in 40 (6.0%) samples. Infection was recorded in 5/6 studied species: 7/49 (14.3%) red foxes (Vulpes vulpes), 3/3 (100%) beech martens (Martes foina), 2/4 (50.0%) Eurasian otters (Lutra lutra), 7/15 (46.7%) Egyptian mongooses (Herpestes ichneumon), and 1/1 (100%) European badger (Meles meles). These species represent three different taxonomic families: Canidae (14.3% were positive), Mustelidae (75.0% were positive), and Herpestidae (46.7% were positive). The results of this study confirm the presence of MAP infection in wild carnivores in Portugal. PMID:25632662

  19. Diagnosis and Molecular Characterization of Mycobacterium avium subsp. paratuberculosis from Dairy Cows in Colombia

    PubMed Central

    Fernández-Silva, J. A.; Abdulmawjood, A.; Bülte, M.

    2011-01-01

    The objective of this study was the serological, bacteriological and molecular diagnosis, as well as the molecular characterization of Mycobacterium avium subsp. paratuberculosis (Map) in adult cows of five Colombian dairy herds. Serum samples were tested by an indirect absorbed enzyme–linked immunosorbent assay (ELISA-C). All fecal samples were tested by pooled culture. After that, fecal samples of Map positive pools were tested individually by culture and polymerase chain reaction (PCR). In one herd, slurry and tissue samples from one animal were also taken and tested by PCR and culture. Map isolates were analyzed by the Multilocus Short Sequence Repeat (MLSSR) and the Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) methods. ELISA produced positive results in 1.8% (6/329) of the animals and 40% (2/5) of the herds. Four fecal, two tissue, and two slurry samples from a herd were Map positive by culture and PCR. MLSSR and MIRU-VNTR revealed two different strain profiles among eight Map isolates recovered. This study reports the first molecular characterization of Map in one dairy herd in Colombia, the limitations for individual diagnosis of subclinical Map infections in cattle, and the usefulness of pooled fecal samples and environmental sampling for Map diagnosis. PMID:21785685

  20. Impact of the shedding level on transmission of persistent infections in Mycobacterium avium subspecies paratuberculosis (MAP).

    PubMed

    Slater, Noa; Mitchell, Rebecca Mans; Whitlock, Robert H; Fyock, Terry; Pradhan, Abani Kumar; Knupfer, Elena; Schukken, Ynte Hein; Louzoun, Yoram

    2016-01-01

    Super-shedders are infectious individuals that contribute a disproportionate amount of infectious pathogen load to the environment. A super-shedder host may produce up to 10,000 times more pathogens than other infectious hosts. Super-shedders have been reported for multiple human and animal diseases. If their contribution to infection dynamics was linear to the pathogen load, they would dominate infection dynamics. We here focus on quantifying the effect of super-shedders on the spread of infection in natural environments to test if such an effect actually occurs in Mycobacterium avium subspecies paratuberculosis (MAP). We study a case where the infection dynamics and the bacterial load shed by each host at every point in time are known. Using a maximum likelihood approach, we estimate the parameters of a model with multiple transmission routes, including direct contact, indirect contact and a background infection risk. We use longitudinal data from persistent infections (MAP), where infectious individuals have a wide distribution of infectious loads, ranging upward of three orders of magnitude. We show based on these parameters that the effect of super-shedders for MAP is limited and that the effect of the individual bacterial load is limited and the relationship between bacterial load and the infectiousness is highly concave. A 1000-fold increase in the bacterial contribution is equivalent to up to a 2-3 fold increase in infectiousness. PMID:26925966

  1. On deaf ears, Mycobacterium avium paratuberculosis in pathogenesis Crohn’s and other diseases

    PubMed Central

    Davis, William C

    2015-01-01

    The historic suggestion that Mycobacterium avium subsp. paratuberculosis (Map) might be a zoonotic pathogen was based on the apparent similarity of lesions in the intestine of patients with Crohn’s disease (CD) with those present in cattle infected with Map, the etiological agent of Johne’s disease. Reluctance to fully explore this possibility has been attributed to the difficulty in demonstrating the presence of Map in tissues from patients with CD. Advances in technology have resolved this problem and revealed the presence of Map in a significant proportion of patients with CD and other diseases. The seminal finding from recent investigations, however, is the detection of Map in healthy individuals with no clinical signs of disease. The latter observation indicates all humans are susceptible to infection with Map and lends support to the thesis that Map is zoonotic, with a latent stage of infection similar to tuberculosis, where infection leads to the development of an immune response that controls but does not eliminate the pathogen. This clarifies one of the reasons why it has been so difficult to document that Map is zoonotic and associated with the pathogenesis of CD and other diseases. As discussed in the present review, a better understanding of the immune response to Map is needed to determine how infection is usually kept under immune control during the latent stage of infection and elucidate the triggering events that lead to disease progression in the natural host and pathogenesis of CD and immune related diseases in humans. PMID:26730151

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

    PubMed Central

    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. PMID:25237653

  3. Host gene expression for Mycobacterium avium subsp. paratuberculosis infection in human THP-1 macrophages.

    PubMed

    Shin, Min-Kyoung; Shin, Seung Won; Jung, Myunghwan; Park, Hongtae; Park, Hyun-Eui; Yoo, Han Sang

    2015-07-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, which causes considerable economic loss in the dairy industry and has a possible relationship to Crohn's disease (CD) in humans. As MAP has been detected in retail pasteurized milk samples, its transmission via milk is of concern. Despite its possible role in the etiology of CD, there have been few studies examining the interactions between MAP and human cells. In the current study, we applied Ingenuity Pathway Analysis to the transcription profiles generated from a murine model with MAP infection as part of a previously conducted study. Twenty-one genes were selected as potential host immune responses, compared with the transcriptional profiles in naturally MAP-infected cattle, and validated in MAP-infected human monocyte-derived macrophage THP-1 cells. Of these, the potential host responses included up-regulation of genes related to immune response (CD14, S100A8, S100A9, LTF, HP and CHCIL3), up-regulation of Th1-polarizing factor (CCL4, CCL5, CXCL9 and CXCL10), down-regulation of genes related to metabolism (ELANE, IGF1, TCF7L2 and MPO) and no significant response of other genes (GADD45a, GPNMB, HMOX1, IFNG and NQO1) in THP-1 cells infected with MAP. PMID:25877879

  4. From mouth to macrophage: mechanisms of innate immune subversion by Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    2014-01-01

    Johne’s disease (JD) is a chronic enteric infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP). The high economic cost and potential zoonotic threat of JD have driven efforts to develop tools and approaches to effectively manage this disease within livestock herds. Efforts to control JD through traditional animal management practices are complicated by MAP’s ability to cause long-term environmental contamination as well as difficulties associated with diagnosis of JD in the pre-clinical stages. As such, there is particular emphasis on the development of an effective vaccine. This is a daunting challenge, in large part due to MAP’s ability to subvert protective host immune responses. Accordingly, there is a priority to understand MAP’s interaction with the bovine host: this may inform rational targets and approaches for therapeutic intervention. Here we review the early host defenses encountered by MAP and the strategies employed by the pathogen to avert or subvert these responses, during the critical period between ingestion and the establishment of persistent infection in macrophages. PMID:24885748

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

    PubMed Central

    Capsel, Randal T.; Thoen, Charles O.; Reinhardt, Timothy A.; Lippolis, John D.; Olsen, Renee; Stabel, Judith R.; Bannantine, John P.

    2016-01-01

    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. PMID:27136199

  6. Short communication: detection of Mycobacterium avium subspecies paratuberculosis by polymerase chain reaction in bovine milk in Brazil.

    PubMed

    Carvalho, I A; Silva, A; Campos, V E B; Moreira, M A S

    2009-11-01

    Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis, or Johne's disease, a chronic granulomatous enteritis that affects all ruminants worldwide. Since the isolation of MAP from intestinal tissue of human patients bearing Crohn's disease, there has been a debate on the possibility of this agent playing a role in the etiology of Crohn's disease. Milk could be the potential vehicle for transmission to humans. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide. In Brazil, detection of MAP is uncommon; however, it has already been detected by bacterial isolation and serological test. The aim of this study was to investigate the presence of MAP, by PCR, in raw milk samples in the region of Viçosa, Minas Gerais State, Brazil. Of 222 milk samples evaluated, 8 (3.6%) quarter milk samples amplified fragments of similar size to that expected of 626 bp. These fragments were cloned and sequenced. The genetic analysis revealed a 99% identity match between the sequences obtained in this study and the insertion sequence IS900 deposited in the GenBank. In the analyzed milk samples, MAP DNA was detected, confirming its presence in dairy cattle in the region of Viçosa. This is the first report of MAP presence in raw milk samples in Brazil. PMID:19841202

  7. Evidence of birth seasonality and clustering of Mycobacterium avium subspecies paratuberculosis infection in US dairy herds.

    PubMed

    Zare, Y; Shook, G E; Collins, M T; Kirkpatrick, B W

    2013-11-01

    Paratuberculosis (Johne's disease) is a contagious intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). In cattle, young calves are at the highest risk for acquiring the infection which occurs mainly through ingestion of MAP from contaminated milk, colostrum and feces or environmental contacts. Data consisted of birth dates and ELISA results of 8000 mature cows from 24 Jersey herds from throughout the US and 4 Wisconsin Holstein herds. Some herds also had complete fecal culture (FC) results. The first infection (case) definition (CD1) relied on only ELISA results. A second case definition (CD2) was used in which results of both ELISA and FC tests were considered: animals testing positive to either test were considered "test-positives" and cows testing negative to ELISA or to both ELISA and FC were regarded as "test-negatives". Objective one was to assess seasonality in birth of MAP-infected animals. The effects of age, breed, herd and season of birth (expressed as the sine and cosine functions of birth days within year) were examined using logistic regression. Age was significantly associated with the MAP infection status of dairy cows for both CDs (OR=1.11; 95% CI 1.09, 1.14; P<0.0001 for CD1; OR=1.16; 95% CI 1.08, 1.24; P<0.0001 for CD2). Season of birth had a significant effect on the risk of MAP infection based on CD1 (OR=0.79; 95% CI 0.71, 0.89; P<0.001 for cosine of birth days) with a peak in summer and a trough in winter based on the fitted model. Objective two was to assess whether test-positive animals were randomly distributed or were clustered by date of birth within herds. A temporal cluster analysis approach (scan statistic) implemented in SaTScan software was used for each case definition to detect clusters of birth cohorts using birthdates. Results identified significant clustering of MAP infection cases for CD1 in multiple herds (P<0.05). These results necessitate matching cases and controls of MAP

  8. Age at occurrence of Mycobacterium avium subspecies paratuberculosis in naturally infected dairy cows.

    PubMed

    Nielsen, S S; Ersbøll, A K

    2006-12-01

    Paratuberculosis is a chronic infection of ruminants and other species caused by Mycobacterium avium ssp. paratuberculosis (Map). Establishing test strategies for paratuberculosis will require insight into the temporal aspects of certainty with a given test. In this study, the age at which cows tested positive by ELISA and fecal culture (FC) was investigated by use of time-to-event analyses. The effects of herd, parity, and shedding group were evaluated at the age of test-positive ELISA and FC, respectively. Finally, the test frequency was investigated for the probability of cows being tested ELISA-positive. Milk and fecal samples were collected repeatedly over a 3-yr period from 1,776 Danish dairy cows from 8 herds. The milk samples were tested for the presence of antibodies by using an ELISA, and an FC test was used for detection of Map. Repeated ELISA testing detected 98 and 95% of cows classified as high and low shedders, respectively, suggesting that most infected cows develop antibodies. Among the high shedders, 50% were positive before 4.3 yr of age (quartiles 1 to 3: 3.4 to 5.7 yr of age). Repeated FC detected only 72% of the cows that were ELISA-positive, and 50% of the ELISA-positive cows were detected by FC at 7.6 yr of age. The age with the highest probability of testing positive was determined as the interval with the steepest slope in the survival probability plots. The highest probability of testing positive by ELISA was from 2.5 to 4.5 yr of age. The highest probability of testing positive by FC was from 2.5 to 5.5 yr of age. For both ELISA and FC, testing positive was highest in the first 300 d in milk. For cows younger than 4 yr of age, monthly testing with ELISA, compared with testing every 2 yr, could increase the probability of detecting cows with antibodies by 19%. In older cows, there were no apparent differences in the probability of testing positive by monthly sampling compared with sampling every second year. Therefore, for older animals

  9. A screening sampling plan to detect Mycobacterium avium subspecies paratuberculosis-positive dairy herds.

    PubMed

    Serraino, A; Arrigoni, N; Ostanello, F; Ricchi, M; Marchetti, G; Bonilauri, P; Bonfante, E; Giacometti, F

    2014-01-01

    Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic contagious bacterial disease primarily affecting dairy cattle. Paratuberculosis represents a dual problem for the milk production chain: in addition to economic losses to affected herds, MAP may have zoonotic potential. Infected herds must be identified in order to implement programs designed to reduce the incidence of disease within and between herds and to prevent MAP from entering the food chain. The objective of this study was to evaluate the sensitivity and specificity of a screening sampling plan (SSP) to detect MAP-positive dairy herds by repetitive analysis of bulk tank milk (BTM) samples by ELISA and in-line milk filter (ILMF) samples by PCR. Samples from BTM and ILMF were collected twice from 569 dairy herds in southern Italy. Additionally, 12,016 individual milk samples were collected: 9,509 from 102 SSP-positive herds (SSP MAP-positive) and 2,507 from 21 randomly selected SSP-negative herds (SSP MAP-negative). There was a total of 126 SSP MAP-positive herds (i.e., 21.3% SSP MAP-positive herds; 95% confidence interval=18.0-24.9); the within-herd apparent prevalence (AP) ranged between 0.00 and 22.73% (mean 6.07%). A significant difference in within-herd AP was shown between SSP MAP-positive herds and SSP MAP-negative herds. A highly significant association was shown between the median AP herd status (>5%) and positivity to at least one ILMF or BTM sample. The SSP detected a minimum of 56.25% of low AP herds (AP ≤ 2.0%) up to a maximum of 100% of herds with a within-herd AP ≥ 8.0%. Overall, the SSP detected 85.57% of herds in which at least one individual milk sample was positive by ELISA. The proposed SSP was an inexpensive and useful tool to detect MAP-positive herds with a higher risk of infection diffusion and milk contamination. Although the SSP cannot be used for MAP-free certification of herds, it could be useful to prioritize appropriate

  10. Escherichia coli, Salmonella, and Mycobacterium avium subsp. paratuberculosis in wild European starlings at a Kansas cattle feedlot.

    PubMed

    Gaukler, Shannon M; Linz, George M; Sherwood, Julie S; Dyer, Neil W; Bleier, William J; Wannemuehler, Yvonne M; Nolan, Lisa K; Logue, Catherine M

    2009-12-01

    The prevalence of Escherichia coli, Salmonella spp., and Mycobacterium avium subsp. paratuberculosis isolated from the feces of wild European starlings (Sturnus vulgaris) humanely trapped at a feedlot in central Kansas was assessed. All E. coli and Salmonella isolates recovered were tested for antimicrobial susceptibility using National Antimicrobial Resistance Monitoring System panels and the E. coli isolates were classified as to their content of genes associated with pathogenic E. coli of birds and cattle, including cvaC, iroN2, ompTp, hlyF2, eitC, iss, iutA, ireA, papC, stxI, stxII, sta, K99, F41, and eae. Escherichia coli O157:H7 and Mycobacterium avium subsp. paratuberculosis were not detected and Salmonella was isolated from only three samples, two of which displayed antimicrobial resistance. Approximately half of the E. coli isolates were resistant to antimicrobial agents with 96% showing resistance to tetracycline. Only one isolate was positive for a single gene associated with bovine pathogenic E. coli. An interesting finding of this study was that 5% of the E. coli isolates tested met the criteria established for identification as avian pathogenic E. coli (APEC). Thus these findings suggest that starlings are not a significant source of Salmonella spp., Mycobacterium avium subsp. paratuberculosis, E. coli O157, or other shiga toxin-producing E. coli in this feedlot. However, they may have the potential to spread APEC, an important pathogen of poultry and a potential pathogen to human beings. PMID:20095155

  11. Metabolic adaptation of Mycobacterium avium subsp. paratuberculosis to the gut environment.

    PubMed

    Weigoldt, Mathias; Meens, Jochen; Bange, Franz-Christoph; Pich, Andreas; Gerlach, Gerald F; Goethe, Ralph

    2013-02-01

    Knowledge on the proteome level about the adaptation of pathogenic mycobacteria to the environment in their natural hosts is limited. Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, a chronic and incurable granulomatous enteritis of ruminants, and has been suggested to be a putative aetiological agent of Crohn's disease in humans. Using a comprehensive LC-MS-MS and 2D difference gel electrophoresis (DIGE) approach, we compared the protein profiles of clinical strains of MAP prepared from the gastrointestinal tract of diseased cows with the protein profiles of the same strains after they were grown in vitro. LC-MS-MS analyses revealed that the principal enzymes for the central carbon metabolic pathways, including glycolysis, gluconeogenesis, the tricaboxylic acid cycle and the pentose phosphate pathway, were present under both conditions. Moreover, a broad spectrum of enzymes for β-oxidation of lipids, nine of which have been shown to be necessary for mycobacterial growth on cholesterol, were detected in vivo and in vitro. Using 2D-DIGE we found increased levels of several key enzymes that indicated adaptation of MAP to the host. Among these, FadE5, FadE25 and AdhB indicated that cholesterol is used as a carbon source in the bovine intestinal mucosa; the respiratory enzymes AtpA, NuoG and SdhA suggested increased respiration during infection. Furthermore higher levels of the pentose phosphate pathway enzymes Gnd2, Zwf and Tal as well as of KatG, SodA and GroEL indicated a vigorous stress response of MAP in vivo. In conclusion, our results provide novel insights into the metabolic adaptation of a pathogenic mycobacterium in its natural host. PMID:23223439

  12. Short communication: Heritability estimates for susceptibility to Mycobacterium avium subspecies paratuberculosis infection defined by ELISA and fecal culture test results in Jersey cattle.

    PubMed

    Zare, Y; Shook, G E; Collins, M T; Kirkpatrick, B W

    2014-07-01

    Paratuberculosis (Johne's disease), an enteric disorder in ruminants caused by Mycobacterium avium ssp. paratuberculosis, causes economic losses in excess of $200 million annually to the US dairy industry. Costly diagnostic testing, cumbersome control programs, incurability, and ineffective vaccination all make M. avium ssp. paratuberculosis susceptibility a good candidate for genetic studies and genetic selection a potentially useful adjunct to management-based control programs. No report has been published for heritability of susceptibility to M. avium ssp. paratuberculosis infection in Jersey cattle. The objective of this study was to estimate variance components and heritability for susceptibility to M. avium ssp. paratuberculosis infection in US Jersey cattle. Data consisted of complete serum ELISA and partial fecal culture results on a total of 2,861 Jersey cows from 23 commercial herds throughout the United States after editing. Four M. avium ssp. paratuberculosis susceptibility phenotypes were defined using (1) ELISA sample-to-positive ratios as a continuous trait, (2) ELISA results as a binary trait (positive=1, negative=0), (3) ELISA results as an ordered categorical trait, and (4) a combined test in which ELISA and fecal culture results were both taken into account in a binary analysis. Three statistical models, including linear, binary threshold, and ordered threshold sire models, were used to analyze the data. All analyses were executed using the restricted maximum likelihood method in ASReml 3 software. The heritability estimates were low to moderate and ranged from 0.08 (±0.03) to 0.27 (±0.11) based on different trait definitions. The nonzero heritability indicates that susceptibility to M. avium ssp. paratuberculosis infection in Jersey cattle is influenced by genetic factors. Therefore, selection of the least susceptible animals could decrease genetic predisposition to M. avium ssp. paratuberculosis infection in Jersey populations in future

  13. Genome-Wide Sequence Variation among Mycobacterium avium Subspecies paratuberculosis Isolates: A Better Understanding of Johne's Disease Transmission Dynamics.

    PubMed

    Hsu, Chung-Yi; Wu, Chia-Wei; Talaat, Adel M

    2011-01-01

    Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johne's disease, infects many farmed ruminants, wild-life animals, and recently isolated from 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) isolates to gain insights into genomic diversity associated with variable hosts and environments. Using Next-generation sequencing technology, all six M. ap isolates showed a high percentage of similarity (98%) to the reference genome sequence of M. ap K-10 isolated from cattle. However, two M. avium isolates (DT 78 and Env 77) showed significant sequence diversity (only 87 and 40% similarity, respectively) compared to the reference strain M. avium 104, a reflection of the wide environmental niches of this group of mycobacteria. Within the M. ap isolates, genomic rearrangements (insertions/deletions) were not detected, and only unique single nucleotide polymorphisms (SNPs) were observed among M. ap isolates. While more of the SNPs (~100) in M. ap genomes were non-synonymous, a total of ~6,000 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-genomics had a enough discriminatory power to differentiate between isolates from different hosts but yet suggesting a bovine source of infection to other animals examined in this study. Interestingly, the human isolate (M. ap 4B) was closely related to a M. ap isolate from a dairy facility, suggesting a common source of infection. Overall, the identified phylo-genomes further supported the idea of a common ancestor to both M. ap and M. avium isolates. Genome-wide analysis described here could provide a strong foundation for a population genetic structure that could be useful for the analysis of mycobacterial evolution and for the tracking of Johne

  14. Effect of various dietary regimens on oral challenge with Mycobacterium avium subsp. paratuberculosis in a rabbit model.

    PubMed

    Arrazuria, Rakel; Molina, Elena; Mateo-Abad, Maider; Arostegui, Inmaculada; Garrido, Joseba M; Juste, Ramón A; Elguezabal, Natalia

    2015-08-01

    Rabbits are susceptible to infection by Mycobacterium avium subspecies paratuberculosis (MAP) in both wildlife and experimental conditions. Under the hypotheses that nutrient balance might influence the establishment of infection, we designed an experiment where MAP intestinal colonization was assessed under three dietary regimens: high fiber, high protein, and regular diet in New Zealand white rabbits submitted to oral challenge with MAP. Lowest weight gain (F=5.17, p=0.024), higher tissue culture positivity rates (X(2)=7.43, p=0.024) and especially extended MAP-compatible lesions (F=5.78, p=0.017) were detected in the regular diet. Taken altogether, results indicate that paratuberculosis infection was achieved affecting mostly regular diet animals and showing that dietary changes may modulate the course of the infection. PMID:26267094

  15. Recovery of Mycobacterium avium subspecies paratuberculosis from the natural host for the extraction and analysis in vivo-derived RNA.

    PubMed

    Granger, Kathy; Moore, Robert J; Davies, John K; Vaughan, Jill A; Stiles, Paula L; Stewart, David J; Tizard, Mark L V

    2004-05-01

    RNA has been extracted and analysed from in vivo-derived Mycobacterium avium subspecies paratuberculosis recovered from the natural host. The bacteria were selectively extracted from the intestinal tissue of two goats exhibiting clinical signs of Johne's disease. Small intestine was rapidly removed, luminal contents washed away and the mucosa and submucosa harvested. Mycobacteria in this material were released from the macrophages by isotonic lysis and differential centrifugation. RNA was extracted and compared with RNA extracted from bacteria grown in vitro. Real-time polymerase chain reaction was used to analyse the katG gene from the bacterial messenger RNA. The katG mRNA encoding the putative catalase/peroxidase showed differential expression in the in vivo and in vitro-derived samples. We hypothesize that the increase in katG expression for in vivo-derived M. paratuberculosis may represent a response to the oxidative stress encountered within the intra-macrophage environment. PMID:15063064

  16. Immunogenicity and reactivity of novel Mycobacterium avium subsp. paratuberculosis PPE MAP1152 and conserved MAP1156 proteins with sera from experimentally and naturally infected animals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s Disease (JD) in ruminants. Development of genetic tools and completion of the MAP genome sequencing project expanded opportunities for antigen discovery. In this study, we determined the seroreactivity of two proteins encoded for at th...

  17. Association of single nucleotide polymorphisms in the ANKRA2 and CD180 genes with bovine respiratory disease and presence of Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective was to determine if single nucleotide polymorphisms (SNPs) in the ANKRA2 and CD180 genes were associated with incidence of bovine respiratory disease (BRD) and presence of Mycobacterium avium subsp. paratuberculosis (MAP) in cattle. Two independent populations were used. The first po...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In summer 2007, a randomized controlled clinical 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 subsp. paratuberculosis and on future milk production and longevity within the herd. ...

  19. Chemical decontamination with n-acetyl-l-cysteine-sodium hydroxide improves recovery of viable Mycobacterium avium subsp. paratuberculosis organisms from cultured milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is shed into milk and feces of cows with advanced Johne’s disease, allowing transmission of MAP among animals. The objective of this study was to formulate an optimized protocol for the isolation of MAP from milk. Parameters investigated included che...

  20. Evaluation of the association between fecal excretion of Mycobacterium avium subsp paratuberculosis and detection in colostrum and on teat skin surfaces of dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective—To evaluate the association between fecal excretion of Mycobacterium avium subsp paratuberculosis (MAP) by dairy cows in the periparturient period and detection of MAP DNA in colostrum specimens and on teat skin surfaces. Design—Cross-sectional study. Animals—112 Holstein cows. Procedures—...

  1. Modulation of Cytokine Gene Expression and Secretion during the Periparturient Period in Dairy cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty multiparous and two primiparous Holstein cows were grouped according to infection status with Mycobacterium avium subsp. paratuberculosis (MAP), the causative microorganism for Johne’s disease. The effect of parturition and infection on the progression of Johne’s disease was monitored by det...

  2. Predicting the role of IL-10 in the regulation of the adaptive immune responses in Mycobacterium avium subsp. paratuberculosis infections using mathematical models

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is an intracellular bacterial pathogen that causes Johne’s disease in cattle and other animals. Infection follows ingestion of the bacteria primarily through the fecal oral route and results in the colonization of the intestine and a granulomatous en...

  3. Isolation of High-Affinity Single-Chain Antibodies against Mycobacterium avium subsp. paratuberculosis Surface Proteins from Sheep with Johne's Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne’s disease, due to infection with Mycobacterium avium subsp. paratuberculosis, causes significant economic losses to the livestock farming industry. Improved investigative and diagnostic tools - necessary to understand disease processes and to identify sub-clinical infection - are much sought a...

  4. Clinical disease and stage of lactation influences shedding of Mycobacterium avium subsp. paratuberculosis into milk and colostrum of naturally infected dairy cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease (JD). One mode of transmission of MAP is through ingestion of contaminated milk and colostrum by susceptible calves. The objective of this study was to determine if the amount of MAP shed into the milk and co...

  5. Mycobacterium avium subsp. paratuberculosis PPE Protein MAP1152 and Conserved Protein MAP1156 are Antigenic in Experimentally and Naturally Infected Cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s Disease (JD) in ruminants resulting in significant production losses. An insertion mutation upstream from the MAP1152-MAP1156 region causes a change in colony morphotype and results in an attenuated phenotype in bovine monocyte derive...

  6. IDENTIFICATION OF DIFFERENCES IN THE PROTEIN CONTENT OF MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS PURIFIED PROTEIN DERIVATIVE PREPARATIONS WITH DIFFERENT SKIN TEST SPECIFICITIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subspecies paratuberculosis (Map) strain ATCC 19698 has been used to produce a purified protein derivative (PPD) that can be utilized as a diagnostic reagent. Two Map PPD lots (Johnin 9801 and Johnin 0202) were used to skin test sheep and cattle for exposure to Map. Johnin 980...

  7. A whole genome association analysis identified loci associated with Mycobacterium avium subsp. paratuberculosis infection status in U.S. Holstein cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The purpose of this study was to identify loci associated with Mycobacterium avium subspecies paratuberculosis (Map) infection status in US Holsteins using the Illumina BovineSNP50 BeadChip whole genome SNP assay. Two hundred forty-five cows from dairies in New York, Pennsylvania and Vermont were fo...

  8. Experimental Validation of a Nested Polymerase Chain Reaction Targeting the Genetic Element ISMAP02 for Detection of Mycobacterium avium subspecies paratuberculosis in Bovine Colostrum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Colostrum samples experimentally inoculated with Mycobacterium avium subsp. paratuberculosis (MAP) (strain K-10) at increasing concentrations between 1×10**1 and 1× 10**9 cells/mL were tested for recovery of MAP DNA using a modified nested ISMAP02 target PCR initially developed for detecting MAP DNA...

  9. Optimization of methods for the detection of Mycobacterium avium subsp. paratuberculosis in milk and colostrum of naturally infected dairy cows with Johne's Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s Disease (JD), a chronic enteritis that occurs in dairy cattle and other ruminants. A 2007 NAHMS Dairy Study demonstrated that over 68% of dairy herds are infected with JD so the risk of exposure within a herd is high...

  10. Survivability of Mycobacterium avium subsp paratuberculosis in grass silage after fermentation and exposure to low pH and high organic acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp paratuberculosis (Map) is a pathogen of concern in dairy production due to its ability to withstand harsh conditions and cause new infections. Infection is a result of ingesting Map cells from contaminated feed, water, or manure. The goal of this research was to evaluate th...

  11. Correlation between Herrold’s egg yolk medium culture results and quantitative real-time PCR for Mycobacterium avium subspecies paratuberculosis in pooled fecal and environmental slurry samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time PCR (qPCR) testing for Mycobacterium avium subspecies paratuberculosis (MAP) in fecal samples is a rapid alternative to culture on Herrold’s egg yolk medium (HEYM), the traditional ante-mortem reference test for MAP. Although the sensitivity and specificity of these two tests ...

  12. Modulation of Cytokine Expression and Lymphocyte Subsets during the Periparturient Period in Dairy Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to evaluate cytokine gene expression and populations of lymphocyte subsets in periparturient dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis (MAP). Blood was collected from noninfected, subclinical, and clinical MAP-infected dairy cows ...

  13. Skin test and Gamma Interferon enzyme-linked Immunosorbent assay results in Sheep exposed to dead Mycobacterium avium subspecies paratuberculosis Organisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell mediated immunity (CMI) diagnostic tests, such as the gamma interferon enzyme-linked immunosorbent assay (IFN-gamma ELISA) and the johnin skin test, have the potential to detect animals infected with Mycobacterium avium subspecies paratuberculosis (MAP) early in the course of the disease. While...

  14. Evolution of the Bovine TLR Gene Family and Member Associations with Mycobacterium avium Subspecies paratuberculosis Infection

    PubMed Central

    Fisher, Colleen A.; Bhattarai, Eric K.; Osterstock, Jason B.; Dowd, Scot E.; Seabury, Paul M.; Vikram, Meenu; Whitlock, Robert H.; Schukken, Ynte H.; Schnabel, Robert D.; Taylor, Jeremy F.; Womack, James E.; Seabury, Christopher M.

    2011-01-01

    Members of the Toll-like receptor (TLR) gene family occupy key roles in the mammalian innate immune system by functioning as sentries for the detection of invading pathogens, thereafter provoking host innate immune responses. We utilized a custom next-generation sequencing approach and allele-specific genotyping assays to detect and validate 280 biallelic variants across all 10 bovine TLR genes, including 71 nonsynonymous single nucleotide polymorphisms (SNPs) and one putative nonsense SNP. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and specialized beef and dairy breeds could not be differentiated despite an average polymorphism density of 1 marker/158 bp. Collectively, 160 tagSNPs and two tag insertion-deletion mutations (indels) were sufficient to predict 100% of the variation at 280 variable sites for both Bos subspecies and their hybrids, whereas 118 tagSNPs and 1 tagIndel predictively captured 100% of the variation at 235 variable sites for B. t. taurus. Polyphen and SIFT analyses of amino acid (AA) replacements encoded by bovine TLR SNPs indicated that up to 32% of the AA substitutions were expected to impact protein function. Classical and newly developed tests of diversity provide strong support for balancing selection operating on TLR3 and TLR8, and purifying selection acting on TLR10. An investigation of the persistence and continuity of linkage disequilibrium (r2≥0.50) between adjacent variable sites also supported the presence of selection acting on TLR3 and TLR8. A case-control study employing validated variants from bovine TLR genes recognizing bacterial ligands revealed six SNPs potentially eliciting small effects on susceptibility to Mycobacterium avium spp paratuberculosis infection in dairy cattle. The results of this study will broadly impact domestic cattle research by providing the necessary foundation to explore several

  15. Short communication: Evaluation of sampling socks for detection of Mycobacterium avium ssp. paratuberculosis on dairy farms.

    PubMed

    Wolf, R; Orsel, K; De Buck, J; Kanevets, U; Barkema, H W

    2016-04-01

    Mycobacterium avium ssp. paratuberculosis (MAP) causes Johne's disease, a production-limiting disease in cattle. Detection of infected herds is often done using environmental samples (ES) of manure, which are collected in cattle pens and manure storage areas. Disadvantages of the method are that sample accuracy is affected by cattle housing and type of manure storage area. Furthermore, some sampling locations (e.g., manure lagoons) are frequently not readily accessible. However, sampling socks (SO), as used for Salmonella spp. testing in chicken flocks, might be an easy to use and accurate alternative to ES. The objective of the study was to assess accuracy of SO for detection of MAP in dairy herds. At each of 102 participating herds, 6 ES and 2 SO were collected. In total, 45 herds had only negative samples in both methods and 29 herds had ≥1 positive ES and ≥1 positive SO. Furthermore, 27 herds with ≥1 positive ES had no positive SO, and 1 herd with no positive ES had 1 positive SO. Bayesian simulation with informative priors on sensitivity of ES and MAP herd prevalence provided a posterior sensitivity for SO of 43.5% (95% probability interval=33-58), and 78.5% (95% probability interval=62-93) for ES. Although SO were easy to use, accuracy was lower than for ES. Therefore, with improvements in the sampling protocol (e.g., more SO per farm and more frequent herd visits), as well as improvements in the laboratory protocol, perhaps SO would be a useful alternative for ES. PMID:26851860

  16. Metabolomic Profiling in Cattle Experimentally Infected with Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    De Buck, Jeroen; Shaykhutdinov, Rustem; Barkema, Herman W.; Vogel, Hans J.

    2014-01-01

    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 was a sensitive

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

    PubMed

    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. PMID:19896745

  18. In Vivo Volatile Organic Compound Signatures of Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Bergmann, Andreas; Trefz, Phillip; Fischer, Sina; Klepik, Klaus; Walter, Gudrun; Steffens, Markus; Ziller, Mario; Schubert, Jochen K.; Reinhold, Petra; Köhler, Heike; Miekisch, Wolfram

    2015-01-01

    Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of a chronic enteric disease of ruminants. Available diagnostic tests are complex and slow. In vitro, volatile organic compound (VOC) patterns emitted from MAP cultures mirrored bacterial growth and enabled distinction of different strains. This study was intended to determine VOCs in vivo in the controlled setting of an animal model. VOCs were pre-concentrated from breath and feces of 42 goats (16 controls and 26 MAP-inoculated animals) by means of needle trap microextraction (breath) and solid phase microextraction (feces) and analyzed by gas chromatography/ mass spectrometry. Analyses were performed 18, 29, 33, 41 and 48 weeks after inoculation. MAP-specific antibodies and MAP-specific interferon-γ-response were determined from blood. Identities of all marker-VOCs were confirmed through analysis of pure reference substances. Based on detection limits in the high pptV and linear ranges of two orders of magnitude more than 100 VOCs could be detected in breath and in headspace over feces. Twenty eight substances differed between inoculated and non-inoculated animals. Although patterns of most prominent substances such as furans, oxygenated substances and hydrocarbons changed in the course of infection, differences between inoculated and non-inoculated animals remained detectable at any time for 16 substances in feces and 3 VOCs in breath. Differences of VOC concentrations over feces reflected presence of MAP bacteria. Differences in VOC profiles from breath were linked to the host response in terms of interferon-γ-response. In a perspective in vivo analysis of VOCs may help to overcome limitations of established tests. PMID:25915653

  19. Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank

    PubMed Central

    Rathnaiah, Govardhan; Lamont, Elise A.; Harris, N. Beth; Fenton, Robert J.; Zinniel, Denise K.; Liu, Xiaofei; Sotos, Josh; Feng, Zhengyu; Livneh-Kol, Ayala; Shpigel, Nahum Y.; Czuprynski, Charles J.; Sreevatsan, Srinand; Barletta, Raúl G.

    2014-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated. PMID:25360421

  20. A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival.

    PubMed

    Kugadas, Abirami; Lamont, Elise A; Bannantine, John P; Shoyama, Fernanda M; Brenner, Evan; Janagama, Harish K; Sreevatsan, Srinand

    2016-01-01

    The 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 macrophages and MAC-T cells that 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 mc(2) 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. 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. PMID:27597934

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

    PubMed

    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

  2. Exploring the zoonotic potential of Mycobacterium avium subspecies paratuberculosis through comparative genomics.

    PubMed

    Wynne, James W; Bull, Tim J; Seemann, Torsten; Bulach, Dieter M; Wagner, Josef; Kirkwood, Carl D; Michalski, Wojtek P

    2011-01-01

    A comparative genomics approach was utilised to compare the genomes of Mycobacterium avium subspecies paratuberculosis (MAP) isolated from early onset paediatric Crohn's disease (CD) patients as well as Johne's diseased animals. Draft genome sequences were produced for MAP isolates derived from four CD patients, one ulcerative colitis (UC) patient, and two non-inflammatory bowel disease (IBD) control individuals using Illumina sequencing, complemented by comparative genome hybridisation (CGH). MAP isolates derived from two bovine and one ovine host were also subjected to whole genome sequencing and CGH. All seven human derived MAP isolates were highly genetically similar and clustered together with one bovine type isolate following phylogenetic analysis. Three other sequenced isolates (including the reference bovine derived isolate K10) were genetically distinct. The human isolates contained two large tandem duplications, the organisations of which were confirmed by PCR. Designated vGI-17 and vGI-18 these duplications spanned 63 and 109 open reading frames, respectively. PCR screening of over 30 additional MAP isolates (3 human derived, 27 animal derived and one environmental isolate) confirmed that vGI-17 and vGI-18 are common across many isolates. Quantitative real-time PCR of vGI-17 demonstrated that the proportion of cells containing the vGI-17 duplication varied between 0.01 to 15% amongst isolates with human isolates containing a higher proportion of vGI-17 compared to most animal isolates. These findings suggest these duplications are transient genomic rearrangements. We hypothesise that the over-representation of vGI-17 in human derived MAP strains may enhance their ability to infect or persist within a human host by increasing genome redundancy and conferring crude regulation of protein expression across biologically important regions. PMID:21799786

  3. Transcriptional Profiling of Ileocecal Valve of Holstein Dairy Cows Infected with Mycobacterium avium subsp. Paratuberculosis

    PubMed Central

    Hempel, Randy J.; Bannantine, John P.

    2016-01-01

    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 advanced clinical stage of infection, are poorly understood. This study examines gene expression in the ileocecal valve (ICV) of Holstein dairy cows at different stages of MAP infection. The ICV is known to be a primary site of MAP colonization and provides an ideal location to identify genes that are relevant to the progression of this disease. RNA was prepared from ICV tissues and RNA-Seq was used to compare gene transcription between clinical, subclinical, and uninfected control animals. Interpretation of the gene expression data was performed using pathway analysis and gene ontology categories containing multiple differentially expressed genes. Results demonstrated that many of the pathways that had strong differential gene expression between uninfected control and clinical cows were related to the immune system, such as the T- and B-cell receptor signaling, apoptosis, NOD-like receptor signaling, and leukocyte transendothelial migration pathways. In contrast, the comparison of gene transcription between control and subclinical cows identified pathways that were primarily involved in metabolism. The results from the comparison between clinical and subclinical animals indicate recruitment of neutrophils, up regulation of lysosomal peptidases, increase in immune cell transendothelial migration, and modifications of the extracelluar matrix. This study provides important insight into how cattle respond to a natural MAP infection at the gene transcription level within a key target tissue for infection. PMID:27093613

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

    PubMed Central

    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 < 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. PMID:27494144

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

    PubMed Central

    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.; Tahlan, Kapil; De Buck, Jeroen

    2016-01-01

    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. PMID:26871723

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

    PubMed Central

    2011-01-01

    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. PMID:22136728

  7. A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival

    PubMed Central

    Kugadas, Abirami; Lamont, Elise A.; Bannantine, John P.; Shoyama, Fernanda M.; Brenner, Evan; Janagama, Harish K.; Sreevatsan, Srinand

    2016-01-01

    The 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 macrophages and MAC-T cells that 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 increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. 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. PMID:27597934

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

    PubMed

    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. PMID:23587160

  9. AhpC, AhpD, and a secreted 14-kilodalton antigen from Mycobacterium avium subsp. paratuberculosis distinguish between paratuberculosis and bovine tuberculosis in an enzyme-linked immunosorbent assay.

    PubMed

    Olsen, I; Tryland, M; Wiker, H G; Reitan, L J

    2001-07-01

    Sera from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis (n = 56) and naturally (n = 4) and experimentally (n = 8) infected with Mycobacterium bovis were tested for the presence of antibodies against paratuberculosis antigens. An enzyme-linked immunosorbent assay (ELISA) was established based on absorption of M. avium subsp. paratuberculosis antigens on a hyperimmune antiserum against M. avium subsp. avium proteins in order to remove cross-reacting antigens. This absorbed-antigen ELISA recognized 66% of animals with paratuberculosis (37 of 56), while none of the animals with naturally occurring bovine tuberculosis (TB) had detectable antibodies. However, the animals with experimental bovine TB also responded in this ELISA. Similar results were found in a commercial ELISA, showing that neither of these tests was able to distinguish between paratuberculosis and bovine TB. The sera were further tested for antibody activities against purified AhpC and AhpD, which are proteins constitutively expressed by M. avium subsp. paratuberculosis, and against a secreted 14-kDa protein present in culture filtrates from the M. avium complex. Elevated antibody levels to AhpC, AhpD, and the 14-kDa antigen were found in 27% (13 of 48), 15% (7 of 48), and 27% (13 of 48), respectively, of the cattle with paratuberculosis. Together these ELISAs were positive with 35% (17 of 48) of the animals. None of the animals with bovine TB had detectable antibodies against any of the purified proteins despite their high levels of cross-reacting antibodies. These results show that purified specific antigens are needed to differentiate between paratuberculosis and bovine TB in ELISA. PMID:11427429

  10. Mycobacterium avium subsp. paratuberculosis detection in individual and bulk tank milk samples from bovine herds and caprine flocks.

    PubMed

    Favila-Humara, Lucía C; Chávez-Gris, Gilberto G; Carrillo-Casas, Erika M; Hernández-Castro, Rigoberto

    2010-04-01

    Paratuberculosis, or Johne's disease, is caused by Mycobacterium avium subsp. paratuberculosis (Map), and it generates great economic losses for the dairy industry worldwide. In humans, Map has been associated with Crohn's disease. Mexico has unknown paratuberculosis prevalence, and yet, control programs have not been applied. This study aimed to determine the presence of Map in milk samples from seropositive goats and cows and bulk tank milk samples from herds previously designated Map-infected using indirect enzyme-linked immunosorbent assay. Map DNA was detected in 100% of the bulk tank milk samples of 14 bovine herds and 3 caprine flocks using a modified insertion sequence 900 polymerase chain reaction (PCR). Additionally, Map DNA was detected in 100% of the individual milk samples from 10 cows and 8 goats. Further, based on the findings of the experimental insertion sequence 900 PCR assessment, evaluation of bulk tank and individual milk samples through a type-specific PCR was performed, which confirmed our previous findings and revealed that 56.25% cow and 63.63% goat milk had concurrent infections of the C, I, and S types. Out of 14 bulk tank milk samples, 10 had viable mycobacteria. Paratuberculosis was detected at a high frequency in cow and goat milk, which suggests that raw milk ingestion represents a potential risk of Map infection. PMID:19911881

  11. Early Induction of Humoral and Cellular Immune Responses during Experimental Mycobacterium avium subsp. paratuberculosis Infection of Calves

    PubMed Central

    Waters, W. R.; Miller, J. M.; Palmer, M. V.; Stabel, J. R.; Jones, D. E.; Koistinen, K. A.; Steadham, E. M.; Hamilton, M. J.; Davis, W. C.; Bannantine, J. P.

    2003-01-01

    Johne's disease (paratuberculosis) of cattle is widespread and causes significant economic losses for producers due to decreased production and poor health of affected animals. The chronic nature of the disease and the lack of a reproducible model of infection hinder research efforts. In the present study, instillation of Mycobacterium avium subsp. paratuberculosis into the tonsillar crypts of neonatal calves resulted in peripheral colonization as detected by antemortem culture of feces and postmortem (320 days postchallenge) culture of intestinal tissues. Antigen-specific blastogenic, gamma interferon (IFN-γ), and nitric oxide responses by blood mononuclear cells from infected calves exceeded prechallenge responses beginning 194 days postchallenge. Upon in vitro stimulation with paratuberculosis antigens, CD4+ cells from infected calves proliferated, produced IFN-γ, and increased expression of CD26 and CD45RO (indicative of an activated memory phenotype). Utilizing a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum immunoglobulin was detected as early as 134 days postchallenge and generally increased after this time point. Two antigens of ∼50 and ∼60 kDa were particularly immunodominant early in infection, as shown by immunoblot with serum collected within 2 weeks postchallenge. Findings indicate that the intratonsillar inoculation route will prove useful as an experimental model for paratuberculosis infection. Additionally, this study confirms that mycobacteria-specific antibody is detectable early in the course of experimental Johne's disease, even preceding the development of specific cell-mediated responses. PMID:12933856

  12. Genetic Structure of Mycobacterium avium subsp. paratuberculosis Population in Cattle Herds in Quebec as Revealed by Using a Combination of Multilocus Genomic Analyses

    PubMed Central

    Sohal, Jagdip Singh; Arsenault, Julie; Labrecque, Olivia; Fairbrother, Julie-Hélène; Roy, Jean-Philippe; Fecteau, Gilles

    2014-01-01

    Mycobacterium avium subsp. paratuberculosis is the etiological agent of paratuberculosis, a granulomatous enteritis affecting a wide range of domestic and wild ruminants worldwide. A variety of molecular typing tools are used to distinguish M. avium subsp. paratuberculosis strains, contributing to a better understanding of M. avium subsp. paratuberculosis epidemiology. In the present study, PCR-based typing methods, including mycobacterial interspersed repetitive units/variable-number tandem repeats (MIRU-VNTR) and small sequence repeats (SSR) in addition to IS1311 PCR-restriction enzyme analysis (PCR-REA), were used to investigate the genetic heterogeneity of 200 M. avium subsp. paratuberculosis strains from dairy herds located in the province of Quebec, Canada. The majority of strains were of the “cattle type,” or type II, although 3 strains were of the “bison type.” A total of 38 genotypes, including a novel one, were identified using a combination of 17 genetic markers, which generated a Simpson's index of genetic diversity of 0.876. Additional analyses revealed no differences in genetic diversity between environmental and individual strains. Of note, a spatial and spatiotemporal cluster was evidenced regarding the distribution of one of the most common genotypes. The population had an overall homogeneous genetic structure, although a few strains stemmed out of the consensus cluster, including the bison-type strains. The genetic structure of M. avium subsp. paratuberculosis populations within most herds suggested intraherd dissemination and microevolution, although evidence of interherd contamination was also revealed. The level of genetic diversity obtained by combining MIRU-VNTR and SSR markers shows a promising avenue for molecular epidemiology investigations of M. avium subsp. paratuberculosis transmission patterns. PMID:24829229

  13. The zoonotic potential of Mycobacterium avium ssp. paratuberculosis: a systematic review and meta-analyses of the evidence.

    PubMed

    Waddell, L A; Rajić, A; Stärk, K D C; McEWEN, S A

    2015-11-01

    This systematic review-meta-analysis appraises and summarizes all the available research (128 papers) on the zoonotic potential of Mycobacterium avium ssp. paratuberculosis. The latter has been debated for a century due to pathogenic and clinical similarities between Johne's disease in ruminants and Crohn's disease (108 studies) in humans and recently for involvement in other human diseases; human immunodeficiency virus (HIV) infection (2), sarcoidosis (3), diabetes mellitus type 1 (T1DM) (7) and type 2 (3), multiple sclerosis (5) and Hashimoto's thyroiditis (2). Meta-analytical results indicated a significant positive association, consistently across different laboratory methods for Crohn's disease [odds ratio (OR) range 4·26-8·44], T1DM (OR range 2·91-9·95) and multiple sclerosis (OR range 6·5-7·99). The latter two and the thyroiditis hypothesis require further investigation to confirm the association. Meta-regression of Crohn's disease studies using DNA detection methods indicated that choice of primers and sampling frame (e.g. general population vs. hospital-based sample) explained a significant proportion of heterogeneity. Other epidemiological studies demonstrated a lack of association between high-risk occupations and development of Crohn's disease. Due to knowledge gaps in understanding the role of M. paratuberculosis in the development or progression of human disease, the evidence at present is not strong enough to inform the potential public health impact of M. paratuberculosis exposure. PMID:25989710

  14. Susceptibility to paratuberculosis infection in cattle is associated with single nucleotide polymorphisms in Toll-like receptor 2 which modulate immune responses against Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Koets, A; Santema, W; Mertens, H; Oostenrijk, D; Keestra, M; Overdijk, M; Labouriau, R; Franken, P; Frijters, A; Nielen, M; Rutten, V

    2010-03-01

    Paratuberculosis is a chronic intestinal infection in ruminants, caused by Mycobacterium avium subspecies paratuberculosis (Map). To study the role of host genetics in disease susceptibility, the Toll-like receptor 2 (TLR2) gene, selected based on its potential role in immunity to mycobacterial infections, was analyzed for single nucleotide polymorphisms (SNP) and their potential association with disease. For SNP discovery and to study SNP association with disease, a case-control study including 24 cows from farms with paratuberculosis was conducted. Sequence analysis of the TLR2 genes from 12 paratuberculosis-infected animals and 12 age-matched healthy herd mates revealed 21 different SNP. The TLR2-1903 T/C SNP was significantly associated with resistance to Map. This and four additional TLR2 SNP were studied in a subsequent observational field study with 553 cows from farms with paratuberculosis. The allelic distribution of the TLR2-1903 T/C SNP was confirmed to be significantly different between the infected and non-infected animals. For the TLR2-1903 T/C SNP the odds ratio was calculated, and similar to the dominance model in the association study, the CT and CC genotypes were compared to the TT genotype. Cows with the TLR2-1903 T/C mutation (i.e., the CT and CC genotypes) were at 1.7 (95% CI: 1.2, 2.8) times the odds of being Map-infected compared to cows with the TT genotype. In in vitro functional assays, monocyte-derived macrophages from animals with a TLR2-1903 TT genotype produced more IL12p40 and IL1beta when stimulated with Map compared to cells derived from TLR2-1903 CT and CC genotypes. Also, T cell proliferative responses to mycobacterial antigens were higher in animals with a TLR2-1903 TT genotype. In conclusion, we have found a significant association between SNP TLR2-1903 T/C in the bovine TLR2 gene and bovine paratuberculosis infection. This SNP and other genetic markers could be useful in marker-assisted breeding strategies as an additional tool

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

    PubMed Central

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

    2014-01-01

    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

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

    PubMed

    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

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

    PubMed Central

    2012-01-01

    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 interacting associations

  18. Iron-sparing Response of Mycobacterium avium subsp. paratuberculosis is strain dependent

    PubMed Central

    2010-01-01

    Background Two genotypically and microbiologically distinct strains of Mycobacterium avium subsp. paratuberculosis (MAP) exist - S and C MAP strains that primarily infect sheep and cattle, respectively. Concentration of iron in the cultivation medium has been suggested as one contributing factor for the observed microbiologic differences. We recently demonstrated that S strains have defective iron storage systems, leading us to propose that these strains might experience iron toxicity when excess iron is provided in the medium. To test this hypothesis, we carried out transcriptional and proteomic profiling of these MAP strains under iron-replete or -deplete conditions. Results We first complemented M. smegmatisΔideR with IdeR of C MAP or that derived from S MAP and compared their transcription profiles using M. smegmatis mc2155 microarrays. In the presence of iron, sIdeR repressed expression of bfrA and MAP2073c, a ferritin domain containing protein suggesting that transcriptional control of iron storage may be defective in S strain. We next performed transcriptional and proteomic profiling of the two strain types of MAP under iron-deplete and -replete conditions. Under iron-replete conditions, C strain upregulated iron storage (BfrA), virulence associated (Esx-5 and antigen85 complex), and ribosomal proteins. In striking contrast, S strain downregulated these proteins under iron-replete conditions. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation resulted in the identification of four unannotated proteins. Two of these were upregulated by a C MAP strain in response to iron supplementation. The iron-sparing response to iron limitation was unique to the C strain as evidenced by repression of non-essential iron utilization enzymes (aconitase and succinate dehydrogenase) and upregulation of proteins of essential function (iron transport, [Fe-S] cluster biogenesis and cell division). Conclusions Taken together, our study revealed

  19. Proteome and Differential Expression Analysis of Membrane and Cytosolic Proteins from Mycobacterium avium subsp. paratuberculosis Strains K-10 and 187▿†

    PubMed Central

    Radosevich, Thomas J.; Reinhardt, Timothy A.; Lippolis, John D.; Bannantine, John P.; Stabel, Judith R.

    2007-01-01

    Little is known of protein expression in Mycobacterium avium subsp. paratuberculosis and how this contributes to pathogenesis. In the present study, proteins from both membranes and cytosol were prepared from two strains of M. avium subsp. paratuberculosis, i.e., laboratory-adapted strain K-10 and a recent isolate, strain 187, obtained from a cow exhibiting clinical signs of Johne's disease. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosol and membrane proteins from K-10 and 187 showed marked differences in protein expression. Relative levels of protein expression from both M. avium subsp. paratuberculosis strains were measured by using amine-reactive isobaric tagging reagents (iTRAQ) and tandem mass spectroscopy. Protein identification and relative expression data were obtained for 874 membrane and cytosolic proteins from the M. avium subsp. paratuberculosis proteome. These data showed a number of significant differences in protein expression between strain K-10 and clinical isolate 187. Examples of proteins expressed at higher levels in clinical isolate 187 compared to strain K-10 are AtpC, RpoA, and several proteins involved in fatty acid biosynthesis. In contrast, proteins such as AhpC and several proteins involved in nitrogen metabolism were expressed at higher levels in strain K-10 compared to strain 187. These data may provide insights into the proteins whose expression is important in natural infection but are modified once M. avium subsp. paratuberculosis is adapted to laboratory cultivation. Results from these studies will provide tools for developing a better understanding of M. avium subsp. paratuberculosis infection in the host and offer potential as diagnostic reagents and vaccine candidates. PMID:17142399

  20. Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Timms, Verlaine J; Mitchell, Hazel M; Neilan, Brett A

    2015-05-01

    The aim of this study was to investigate DNA extraction methods and PCR assays suitable for the detection of Mycobacterium paratuberculosis in bovine tissue. The majority of methods currently used to detect M. paratuberculosis have been developed using bovine samples, such as faeces, blood or tissue and, in many cases, have been based on detection from pooled samples from a herd. However most studies have not compared PCR results to culture results. In order to address this problem, four DNA extraction protocols and three PCR assays were employed to detect M. paratuberculosis in bovine tissue. Given that culture is reliable from cows, the results were then compared with the known M. paratuberculosis culture status. The following DNA extractions were included, two commercial kits, a boiling method, an in house extraction based on a published method and enrichment by sonication. The three PCR assays used included single round IS900 and f57 assays and a nested IS900 assay. In addition, another PCR assay was validated for the detection of any Mycobacterial species and a universal bacterial 16S rRNA gene assay was used to detect sample inhibition. The in-house DNA extraction was the most consistent in extracting good quality DNA compared to all other methods. The use of two PCR markers, IS900 and f57, and a universal PCR enabled the correct samples to be identified as M. paratuberculosis positive. In addition, when compared to the culture result, false-positives did not occur and PCR inhibition was readily identified. Using an in house DNA extraction coupled with the IS900 and f57 PCR markers, this study provides a reliable and simple method to detect M. paratuberculosis in both veterinary and spill over infections. PMID:25797305

  1. Comparison of the antimicrobial activities of gallium nitrate and gallium maltolate against Mycobacterium avium subsp. paratuberculosis in vitro.

    PubMed

    Fecteau, Marie-Eve; Aceto, Helen W; Bernstein, Lawrence R; Sweeney, Raymond W

    2014-10-01

    Johne's disease (JD) is an enteric infection of cattle and other ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). This study compared the antimicrobial activities of gallium nitrate (GaN) and gallium maltolate (GaM) against two field MAP isolates by use of broth culture. The concentrations that resulted in 99% growth inhibition of isolates 1 and 2 were, respectively, 636 µM and 183 µM for GaN, and 251 µM and 142 µM for GaM. For both isolates, time to detection was significantly higher for GaM than GaN. These results suggest that GaM is more efficient than GaN in inhibiting MAP growth in vitro. PMID:25155307

  2. Detection of PCR products from Mycobacterium avium subspecies Paratuberculosis using oligonucleotides containing multiple 2,4-dinitrophenyl reporter groups.

    PubMed

    Stevenson, K; Walker, C A; Grzybowski, J; Brown, T; Sharp, J M

    A pool of five oligonucleotides has been used to detect the pathogenic organism Mycobacterium avium subspecies paratuberculosis in PCR-amplified DNA from ruminants. The oligonucleotides were labelled at the 5'-end with three dinitrophenyl reporter groups and hybridised to the target DNA, which was fixed to a nylon membrane by ultraviolet irradiation. Colourimetric detection of the PCR product was carried out using an anti-DNP antibody conjugated to horseradish peroxidase or to alkaline phosphatase. Detection with alkaline phosphatase was more sensitive than with horseradish peroxidase but, in both cases, the PCR product could be easily detected. The DNP labelling system offers an economic and effective alternative to biotin, digoxigenin or fluorescein for the detection of PCR-amplified DNA. PMID:9346864

  3. Possible transmission of Mycobacterium avium subspecies paratuberculosis through potable water: lessons from an urban cluster of Crohn's disease

    PubMed Central

    Pierce, Ellen S

    2009-01-01

    A "cluster" of patients refers to the geographic proximity of unrelated patients with the same disease and suggests a common environmental cause for that disease. Clusters of patients with Crohn's disease have been linked to the presence of an infectious microorganism in unpasteurized milk and cheese, untreated water supplied by wells or springs, animal manure used as fertilizer for family vegetable gardens, and bodies of water contaminated by agricultural runoff. Mycobacterium avium subspecies paratuberculosis (MAP) is the suspected cause of Crohn's disease. MAP causes a disease in dairy cows and other animals that is similar to Crohn's disease, called Johne's ('Yo-knees') disease or paratuberculosis. Dairy cows with Johne's disease secrete MAP into their milk and excrete MAP into their feces. MAP is present in untreated water such as well water, in bodies of water contaminated by agricultural runoff, and in unpasteurized milk and cheese. The "treatment" of "tap" water to make it "drinkable" or "potable" by the processes of sedimentation, filtration and chlorination has little to no effect on MAP. MAP is so resistant to chlorine disinfection that such disinfection actually selects for its growth. Other subspecies of Mycobacterium avium grow in biofilms present on tap water pipes. Despite the documented presence of MAP in tap water and its probable growth on tap water pipes, clusters of Crohn's disease have not previously been described in relationship to tap water pipes supplying patients' homes. This report describes three unrelated individuals who lived on the same block along a street in a midwestern American city and developed Crohn's disease within four years of each other in the 1960's. A common tap water pipe supplied their homes. This is the first reported cluster of Crohn's disease possibly linked to fully treated drinking water, and is consistent with previously reported clusters of Crohn's disease linked to an infectious microorganism in water. PMID

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

    PubMed

    Leite, Fernando L; Eslabão, Livia B; Pesch, Bruce; Bannantine, John P; Reinhardt, Timothy A; Stabel, Judith R

    2015-09-15

    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 clinical state. The objective of this study was to investigate the expression of two crucial molecules in T cell function, ZAP-70 (zeta-chain-associated protein of 70 kDa) and CTLA-4 (cytotoxic T-lymphocyte antigen-4), in cows naturally infected with MAP. Peripheral blood mononuclear cells (PBMCs) isolated from control non-infected cows (n=5), and cows in subclinical (n=6) and clinical stages of paratuberculosis (n=6) were cultured alone (medium only), and with concanavalin A, and a whole cell sonicate of MAP for 24, 72 and 144 h to measure the dynamic changes of ZAP-70 and CTLA-4 expression on CD4, CD8, and gamma delta (γδ) T cells. Flow cytometry was also performed to measure ZAP-70 phosphorylation to examine proximal T cell receptor signaling in animals of different disease status. The surface expression of CTLA-4 was increased in animals in subclinical stage of infection while levels of ZAP-70 were decreased in CD4+ T cells of both subclinical and clinical animals, indicating a change in T cell phenotype with disease state. Interestingly, proximal T cell receptor signaling was not altered in infected animals. This study demonstrated changes in crucial signaling molecules in animals infected with MAP, thereby elucidating T cell alterations associated with disease progression. PMID:26163934

  5. Isolation of Mycobacterium avium Subspecies paratuberculosis Reactive CD4 T Cells from Intestinal Biopsies of Crohn's Disease Patients

    PubMed Central

    Olsen, Ingrid; Tollefsen, Stig; Aagaard, Claus; Reitan, Liv J.; Bannantine, John P.; Andersen, Peter; Sollid, Ludvig M.; Lundin, Knut E. A.

    2009-01-01

    Background Crohn's disease (CD) is a chronic granulomatous inflammation of the intestine. The etiology is unknown, but an excessive immune response to bacteria in genetically susceptible individuals is probably involved. The response is characterized by a strong Th1/Th17 response, but the relative importance of the various bacteria is not known. Methodology/Principal Findings In an attempt to address this issue, we made T-cell lines from intestinal biopsies of patients with CD (n = 11), ulcerative colitis (UC) (n = 13) and controls (n = 10). The T-cell lines were tested for responses to various bacteria. A majority of the CD patients with active disease had a dominant response to Mycobacterium avium subspecies paratuberculosis (MAP). The T cells from CD patients also showed higher proliferation in response to MAP compared to UC patients (p<0.025). MAP reactive CD4 T-cell clones (n = 28) were isolated from four CD patients. The T-cell clones produced IL-17 and/or IFN-γ, while minimal amounts of IL-4 were detected. To further characterize the specificity, the responses to antigen preparations from different mycobacterial species were tested. One T-cell clone responded only to MAP and the very closely related M. avium subspecies avium (MAA) while another responded to MAP, MAA and Mycobacterium intracellulare. A more broadly reactive T-cell clone reacted to MAP1508 which belongs to the esx protein family. Conclusions/Significance The presence of MAP reactive T cells with a Th1 or Th1/Th17 phenotype may suggest a possible role of mycobacteria in the inflammation seen in CD. The isolation of intestinal T cells followed by characterization of their specificity is a valuable tool to study the relative importance of different bacteria in CD. PMID:19479064

  6. Cytokine Gene Expression in Peripheral Blood Mononuclear Cells and Tissues of Cattle Infected with Mycobacterium avium subsp. paratuberculosis: Evidence for an Inherent Proinflammatory Gene Expression Pattern

    PubMed Central

    Coussens, Paul M.; Verman, Nitin; Coussens, Marc A.; Elftman, Michael D.; McNulty, Amanda M.

    2004-01-01

    In cattle and other ruminants, infection with the intracellular pathogen Mycobacterium avium subsp. paratuberculosis results in a granulomatous enteritis (Johne's disease) that is often fatal. The key features of host immunity to M. avium subsp. paratuberculosis infection include an appropriate early proinflammatory and cytotoxic response (Th1-like) that eventually gives way to a predominant antibody-based response (Th2-like). Clinical disease symptoms often appear subsequent to waning of the Th1-like immune response. Understanding why this shift in the immune response occurs and the underlying molecular mechanisms involved is critical to future control measures and diagnosis. Previous studies have suggested that M. avium subsp. paratuberculosis may suppress gene expression in peripheral blood mononuclear cells (PBMCs) from infected cows, despite a continued inflammatory reaction at sites of infection. In the present study, we tested the hypothesis that exposure to M. avium subsp. paratuberculosis suppresses a proinflammatory gene expression pattern in PBMCs from infected cows. To do this, we examined expression of genes encoding interleukin-1α (IL-1α), IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, IL-16, and IL-18, as well as genes encoding gamma interferon (IFN-γ), transforming growth factor β (TGF-β), and tumor necrosis factor alpha (TNF-α), in PBMCs, intestinal lesions, and mesenteric lymph nodes of cattle naturally infected with M. avium subsp. paratuberculosis. Cytokine gene expression in these cells and tissues was compared to expression in similar cells and tissues from control uninfected cattle. Our comprehensive results demonstrate that for most cytokine genes, including the genes encoding IFN-γ, TGF-β, TNF-α, IL-1α, IL-4, IL-6, IL-8, and IL-12p35, differential expression in PBMCs from infected and control cattle did not require stimulation with M. avium subsp. paratuberculosis. In fact, stimulation with M. avium subsp. paratuberculosis tended

  7. Expression of NRAMP1 and iNOS in Mycobacterium avium subsp. paratuberculosis naturally infected cattle.

    PubMed

    Delgado, F; Estrada-Chávez, C; Romano, M; Paolicchi, F; Blanco-Viera, F; Capellino, F; Chavez-Gris, G; Pereira-Suárez, A L

    2010-09-01

    Paratuberculosis (PTB) is a chronic disease caused by M. avium subsp. paratuberculosis (MAP) that affects several animal species, and some studies have suggested that there may be a relationship between Crohn's disease and PTB. Significant aspects of PTB pathogenesis are not yet completely understood, such as the role of macrophages. Natural resistance-associated macrophage protein 1 (NRAMP1) and the inducible nitric oxide synthase (iNOS) molecules have shown nonspecific effects against several intracellular pathogens residing within macrophages. However, these molecules have been scarcely studied during natural infection with MAP. In this work, changes in NRAMP1 and iNOS expression were surveyed by immunohistochemistry in tissue samples from MAP-infected cattle and healthy controls. Our findings show strong specific immunolabeling against both NRAMP1 and iNOS molecules, throughout granulomatous PTB-compatible lesions in ileum and ileocaecal lymph nodes from paratuberculous cattle compared with uninfected controls, suggesting a relationship between the expression of these molecules and the pathogenesis of PTB disease. PMID:19345998

  8. Detection of Mycobacterium avium subspecies paratuberculosis infection in two different camel species by conventional and molecular techniques

    PubMed Central

    Haghkhah, Masoud; Derakhshandeh, Abdollah; Jamshidi, Reza; Moghiseh, Asghar; Karimaghaei, Negar; Ayaseh, Mohammad; Mostafaei, Mohsen

    2015-01-01

    Paratuberculosis (John’s disease) is infectious and chronically progressive granulomatous disease which affects domestic and wild ruminants. The causative agent is Mycobacterium avium paratuberculosis (MAP), a slow growing mycobactin dependent acid-fast bacillus. We investigated the detection and frequency of MAP in apparently healthy dromedary and Bactrian camels by insertion sequence 900 (IS900) polymerase chain reaction (PCR) and acid fast staining of fecal samples in Iran. Acid fast staining results showed that 6/50 (12.0%) samples of dromedary camels and 4/26 (15.3%) samples of Bactrian camels were suspected to MAP. Although the percentage of positivity for PCR assay of fecal dromedary camel was 8.0%, no bands corresponding to MAP detect in all samples of Bactrian camels. In conclusion, Although the incidence of MAP infection was low, further studies should be conducted to get more information on MAP infection in camel population, especially in areas where camels are close to other ruminants such as dairy cow, sheep and goat. PMID:26973771

  9. The within host dynamics of Mycobacterium avium ssp. paratuberculosis infection in cattle: where time and place matter.

    PubMed

    Koets, Ad P; Eda, Shigetoshi; Sreevatsan, Srinand

    2015-01-01

    Johne's disease or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), occurs in domestic and wild animals worldwide, causing a significant economic loss to livestock industries. After a prolonged incubation time, infected cattle shed MAP bacilli into feces and spread the disease to an uninfected animal population. It is largely unknown how (or whether) the interplay between the pathogen and the host immunity determines timing of shedding after the long incubation time. Such information would provide an understanding of pathogenesis in individual animals and the epidemiology of MAP infection in animal populations. In this review, we summarize current knowledge of bovine Johne's disease pathology, pathogenesis, immunology and genetics. We discuss knowledge gaps that direly need to be addressed to provide a science-based approach to diagnostics and (immuno)prophylaxis. These knowledge gaps are related to anatomical/clinical manifestation of MAP invasion, interaction of bacteria with phagocytes, granuloma formation, shedding, establishment and kinetics of adaptive immune responses in the pathogenesis of the disease. These topics are discussed at the molecular, cellular and tissue levels with special attention to the within host dynamics including the temporal and the spatial context relevant for the various host-pathogen interactions. PMID:26092382

  10. Detection of Mycobacterium avium subspecies paratuberculosis infection in two different camel species by conventional and molecular techniques.

    PubMed

    Haghkhah, Masoud; Derakhshandeh, Abdollah; Jamshidi, Reza; Moghiseh, Asghar; Karimaghaei, Negar; Ayaseh, Mohammad; Mostafaei, Mohsen

    2015-01-01

    Paratuberculosis (John's disease) is infectious and chronically progressive granulomatous disease which affects domestic and wild ruminants. The causative agent is Mycobacterium avium paratuberculosis (MAP), a slow growing mycobactin dependent acid-fast bacillus. We investigated the detection and frequency of MAP in apparently healthy dromedary and Bactrian camels by insertion sequence 900 (IS900) polymerase chain reaction (PCR) and acid fast staining of fecal samples in Iran. Acid fast staining results showed that 6/50 (12.0%) samples of dromedary camels and 4/26 (15.3%) samples of Bactrian camels were suspected to MAP. Although the percentage of positivity for PCR assay of fecal dromedary camel was 8.0%, no bands corresponding to MAP detect in all samples of Bactrian camels. In conclusion, Although the incidence of MAP infection was low, further studies should be conducted to get more information on MAP infection in camel population, especially in areas where camels are close to other ruminants such as dairy cow, sheep and goat. PMID:26973771

  11. Monoclonal Antibodies Bind A SNP-Sensitive Epitope that is Present Uniquely in Mycobacterium avium Subspecies Paratuberculosis

    PubMed Central

    Bannantine, John P.; Stabel, Judith R.; Lamont, Elise A.; Briggs, Robert E.; Sreevatsan, Srinand

    2011-01-01

    Due to a close genetic relatedness, there is no known antibody that detects Mycobacterium avium subspecies paratuberculosis (MAP), which causes Johne’s disease in cattle and sheep, and does not cross-react with other M. avium subspecies. In the present study, a monoclonal antibody (MAb; 17A12) was identified from mice immunized with a cell membrane fraction of MAP strain K-10. This antibody is 100% specific as it detected a 25-kDa protein in all 29 MAP whole cell lysates, but did not bind to any of the 29 non-paratuberculosis strains tested in immunoblot assays. However, the antibody revealed variable reactivity levels in MAP strains as it detected higher levels in bovine isolates but comparably lower levels in ovine isolates of MAP. In order to identify the target binding protein for 17A12, a lambda phage expression library of MAP genomic fragments was screened with the MAb. Four reactive clones were identified, sequenced and all shown to be overlapping. Further analysis revealed all four clones expressed an unknown protein encoded by a sequence that is not annotated in the K-10 genome and overlapped with MAP3422c on the opposing DNA strand. The epitope of 17A12 was precisely defined to seven amino acids and was used to query the K-10 genome. Similarity searches revealed another protein, encoded by MAP1025, possessed a similar epitope (one-amino acid mismatch) that also reacted strongly to the antibody. A single nucleotide polymorphism (SNP) in MAP1025 was then identified by comparative sequence analysis, which results in a Pro28His change at residue 28, the first amino acid within the 17A12 epitope. This SNP is present in all MAP strains but absent in all non-MAP strains and accounts for the specificity of the 17A12 antibody. This new antibody is the first ever isolated that binds only to the paratuberculosis subspecies of M. avium and opens new possibilities for the specific detection of this significant ruminant pathogen. PMID:21845186

  12. NlpC/P60 domain-containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan.

    PubMed

    Bannantine, John P; Lingle, Cari K; Adam, Philip R; Ramyar, Kasra X; McWhorter, William J; Stabel, Judith R; Picking, William D; Geisbrecht, Brian V

    2016-04-01

    A subset of proteins containing NlpC/P60 domains are bacterial peptidoglycan hydrolases that cleave noncanonical peptide linkages and contribute to cell wall remodeling as well as cell separation during late stages of division. Some of these proteins have been shown to cleave peptidoglycan in Mycobacterium tuberculosis and play a role in Mycobacterium marinum virulence of zebra fish; however, there are still significant knowledge gaps concerning the molecular function of these proteins in Mycobacterium avium subspecies paratuberculosis (MAP). The MAP genome sequence encodes five NlpC/P60 domain-containing proteins. We describe atomic resolution crystal structures of two such MAP proteins, MAP_1272c and MAP_1204. These crystal structures, combined with functional assays to measure peptidoglycan cleavage activity, led to the observation that MAP_1272c does not have a functional catalytic core for peptidoglycan hydrolysis. Furthermore, the structure and sequence of MAP_1272c demonstrate that the catalytic residues normally required for hydrolysis are absent, and the protein does not bind peptidoglycan as efficiently as MAP_1204. While the NlpC/P60 catalytic triad is present in MAP_1204, changing the catalytic cysteine-155 residue to a serine significantly diminished catalytic activity, but did not affect binding to peptidoglycan. Collectively, these findings suggest a broader functional repertoire for NlpC/P60 domain-containing proteins than simply hydrolases. PMID:26799947

  13. Pathogenic 'Bison-type' Mycobacterium avium subspecies paratuberculosis genotype characterized from riverine buffalo (Bubalus bubalis) in North India.

    PubMed

    Yadav, D; Singh, S V; Singh, A V; Sevilla, I; Juste, R A; Singh, P K; Sohal, J S

    2008-07-01

    Despite low per-animal productivity of ruminants in developing countries, Johne's disease has not been investigated in buffaloes, which are primarily found in these countries. This is due to lack of expertise, diagnostic kits and priority to production diseases like Johne's disease. Presence of pathogenic Mycobacterium avium subspecies paratuberculosis (Map) was investigated by screening of target tissues (mesenteric lymph nodes and large intestine) by culture and IS 900 PCR, in 50 sacrificed buffaloes. Indigenous ELISA kit originally developed for goats and sheep was standardized in buffaloes and used to estimate sero-presence of Map in 167 serum samples representing population of buffaloes in Agra region of North India. In culture, 48.0% buffaloes were positive from 50 tissues each from mesenteric lymph nodes (34.0%) and large intestine (36.0%). IS 900 PCR was standardized using specific primers (150 C and 921) and 229 bp-amplified product was characteristic for Map. Of the 25 mesenteric lymph nodes, 40.0% were positive in IS 900 PCR. Genomic DNA from Map cultures was successfully amplified from all the 24 isolates (100.0%). Map was further genotyped as 'Bison type' using IS 1311 PCR-REA. Culture of tissues showed high presence of Map in target tissues, despite high culling rate in buffalos in view of high demand of buffalo meat. Specific tissue-PCR provided rapid confirmation of Map infection in sacrificed buffaloes. In tissue-PCR, all the cultures were positive as compared to 40.0% detected directly from tissues. ELISA kit using indigenous protoplasmic antigen was highly sensitive as compared to commercial antigen in detecting Map infection therefore, could be used as 'Herd Screening Test' in buffaloes against Johne's disease. This pilot study first time reports a highly pathogenic 'Bison-type' genotype of M. avium subspecies paratuberculosis from the riverine buffaloes (Bubalus bubalis) of Agra region in North India. PMID:17804069

  14. Increased viability but decreased culturability of Mycobacterium avium subsp. paratuberculosis in macrophages from inflammatory bowel disease patients under Infliximab treatment.

    PubMed

    Nazareth, Nair; Magro, Fernando; Appelberg, Rui; Silva, Jani; Gracio, Daniela; Coelho, Rosa; Cabral, José Miguel; Abreu, Candida; Macedo, Guilherme; Bull, Tim J; Sarmento, Amélia

    2015-12-01

    Mycobacterium avium subsp. paratuberculosis (MAP) has long been implicated as a triggering agent in Crohn's disease (CD). In this study, we investigated the growth/persistence of both M. avium subsp. hominissuis (MAH) and MAP, in macrophages from healthy controls (HC), CD and ulcerative colitis patients. For viability assessment, both CFU counts and a pre16SrRNA RNA/DNA ratio assay (for MAP) were used. Phagolysosome fusion was evaluated by immunofluorescence, through analysis of LAMP-1 colocalization with MAP. IBD macrophages were more permissive to MAP survival than HC macrophages (a finding not evident with MAH), but did not support MAP active growth. The lower MAP CFU counts in macrophage cultures associated with Infliximab treatment were not due to increased killing, but possibly to elevation in the proportion of intracellular dormant non-culturable MAP forms, as MAP showed higher viability in those macrophages. Increased MAP viability was not related to lack of phagolysosome maturation. The predominant induction of MAP dormant forms by Infliximab treatment may explain the lack of MAP reactivation during anti-TNF therapy of CD but does not exclude the possibility of MAP recrudescence after termination of therapy. PMID:25702170

  15. Mycobacterium avium subsp paratuberculosis--incidences in milk and milk products, their isolation, enumeration, characterization, and role in human health.

    PubMed

    Patel, Ami; Shah, Nihir

    2011-12-01

    Mycobacterium avium subsp paratuberculosis (MAP), excreted in the feces and milk, is reported to be not easily inactivated by pasteurization and thermal treatments as other bacteria infecting humans and animals do. The D values of all MAP strains tested were considerably higher than those published for other pathogens. Culturing techniques for this organism are labor intensive. Although an increasing amount of scientific evidence suggests that this organism can be responsible for at least some cases of Crohn's disease (CD), there is controversy about MAP being a cause of CD in humans. In general, although some studies have described an association between the presence of MAP and CD, the role of Mycobacterium species and MAP in the etiology of this human disease remains unestablished. Although published reports indicate that it may not be completely inactivated by pasteurization of milk, the effectiveness of increasing the time or temperature in the pasteurization process has not been established and hence any potential benefit to human health cannot be determined. This article summarizes the incidences of MAP in milk and milk products with respect to human health and brief discussion of various serological as well as molecular techniques used for their isolation, enumeration, and characterization. PMID:21620785

  16. Differential Changes in Heat Shock Protein-, Lipoarabinomannan-, and Purified Protein Derivative-Specific Immunoglobulin G1 and G2 Isotype Responses during Bovine Mycobacterium avium subsp. paratuberculosis Infection

    PubMed Central

    Koets, Ad P.; Rutten, Victor P. M. G.; de Boer, Masja; Bakker, Douwe; Valentin-Weigand, Peter; van Eden, Willem

    2001-01-01

    Bovine paratuberculosis is caused by infection of young calves with Mycobacterium avium subsp. paratuberculosis. In some of the chronically infected cows the long asymptomatic stage (2 to 4 years) is followed by a rapid progression to a clinical stage due to protein-losing enteropathy, which will ultimately be fatal. The current dogma is that in early stages of disease the cell-mediated responses predominate, whereas in the clinical stage of the disease the humoral responses prevail, possibly signaling a switch in immune reactivity related to disease progression. We developed immunoglobulin M (IgM)-, IgA-, and IgG1- and IgG2-isotype-specific enzyme-linked immunosorbent assays for M. avium subsp. paratuberculosis-derived antigens (heat shock proteins of 70 kDa [Hsp70] and 65 kDa [Hsp65], lipoarabinomannan [LAM], and M. avium subsp. paratuberculosis purified protein derivative PPD [PPDP]). The serological responses of cows in different stages of paratuberculosis were used to evaluate the putative shift in immune responsiveness. In the clinical stage the PPDP-specific IgG1 responses were increased compared to those in the asymptomatic stage. However, total IgG1 and IgG2 and the Hsp70-, Hsp65-, and LAM-specific isotype responses were decreased in the clinical stage were decreased compared to those in the asymptomatic stage of disease. Thus, the classical pattern was found only for PPDP antigens and the IgG1 isotype. For other antigens and isotypes and the total IgG levels, the response pattern is different and indicates that there is no uniform association with increased antibody responses during the progression from the asymptomatic stage to the clinical stage of bovine paratuberculosis. PMID:11179318

  17. LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Trangoni, Marcos D; Gioffré, Andrea K; Cerón Cucchi, María E; Caimi, Karina C; Ruybal, Paula; Zumárraga, Martín J; Cravero, Silvio L

    2015-06-01

    In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR Green(TM) allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries. PMID:26273282

  18. Seroprevalence of infection with Mycobacterium avium subspecies paratuberculosis, bovine leukemia virus, and bovine viral diarrhea virus in maritime Canada dairy cattle.

    PubMed Central

    VanLeeuwen, J A; Keefe, G P; Tremblay, R; Power, C; Wichtel, J J

    2001-01-01

    The purpose of this study was to survey the seroprevalence of infection with the agents of production-limiting diseases in dairy cattle in New Brunswick, Nova Scotia, and Prince Edward Island. In 30 randomly selected herds per province, 30 cattle per herd were randomly selected and tested for antibodies to bovine leukemia virus (BLV) and Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), while 5 unvaccinated cattle over 6 months of age were tested for antibodies to bovine viral diarrhea virus (BVDV). For BLV, 20.8% (15.8% to 27.0%) of cows were positive, and 70.0% (60.3% to 79.7%) of herds had at least one positive cow. In BLV-positive herds, the average BLV prevalence was 30.9% (24.8% to 37.2%). For M. paratuberculosis, 2.6% (1.8% to 3.9%) of cows were positive, and 16.7% (8.8% to 24.5%) of herds had at least 2 M. paratuberculosis-positive cows. In M. paratuberculosis-positive herds, the average M. paratuberculosis prevalence was 8.5% (6.9% to 10.1%). For BVDV, 46.1% (35.5% to 56.7%) of herds had at least 1 BVDV-positive animal with a titer greater than or equal to 1:64. PMID:11265187

  19. Genome-wide analysis of the emerging infection with Mycobacterium avium subspecies paratuberculosis in the Arabian camels (Camelus dromedarius).

    PubMed

    Ghosh, Pallab; Hsu, Chungyi; Alyamani, Essam J; Shehata, Maher M; Al-Dubaib, Musaad A; Al-Naeem, Abdulmohsen; Hashad, Mahmoud; Mahmoud, Osama M; Alharbi, Khalid B J; Al-Busadah, Khalid; Al-Swailem, Abdulaziz M; Talaat, Adel M

    2012-01-01

    Mycobacterium avium subspecies paratuberculosis (M. ap) is the causative agent of paratuberculosis or Johne's disease (JD) in herbivores with potential involvement in cases of Crohn's disease in humans. JD is spread worldwide and is economically important for both beef and dairy industries. Generally, pathogenic ovine strains (M. ap-S) are mainly found in sheep while bovine strains (M. ap-C) infect other ruminants (e.g. cattle, goat, deer), as well as sheep. In an effort to characterize this emerging infection in dromedary/Arabian camels, we successfully cultured M. ap from several samples collected from infected camels suffering from chronic, intermittent diarrhea suggestive of JD. Gene-based typing of isolates indicated that all isolates belong to sheep lineage of strains of M. ap (M. ap-S), suggesting a putative transmission from infected sheep herds. Screening sheep and goat herds associated with camels identified the circulation of this type in sheep but not goats. The current genome-wide analysis recognizes these camel isolates as a sub-lineage of the sheep strain with a significant number of single nucleotide polymorphisms (SNPs) between sheep and camel isolates (∼1000 SNPs). Such polymorphism could represent geographical differences among isolates or host adaptation of M. ap during camel infection. To our knowledge, this is the first attempt to examine the genomic basis of this emerging infection in camels with implications on the evolution of this important pathogen. The sequenced genomes of M. ap isolates from camels will further assist our efforts to understand JD pathogenesis and the dynamic of disease transmission across animal species. PMID:22393374

  20. Genome-wide association study of susceptibility to infection by Mycobacterium avium subspecies paratuberculosis in Holstein cattle.

    PubMed

    Alpay, Fazli; Zare, Yalda; Kamalludin, Mamat H; Huang, Xixia; Shi, Xianwei; Shook, George E; Collins, Michael T; Kirkpatrick, Brian W

    2014-01-01

    Paratuberculosis, or Johne's disease, is a chronic, granulomatous, gastrointestinal tract disease of cattle and other ruminants caused by the bacterium Mycobacterium avium, subspecies paratuberculosis (MAP). Control of Johne's disease is based on programs of testing and culling animals positive for infection with MAP while concurrently modifying management to reduce the likelihood of infection. The current study is motivated by the hypothesis that genetic variation in host susceptibility to MAP infection can be dissected and quantifiable associations with genetic markers identified. For this purpose, a case-control, genome-wide association study was conducted using US Holstein cattle phenotyped for MAP infection using a serum ELISA and/or fecal culture test. Cases included cows positive for either serum ELISA, fecal culture or both. Controls consisted of animals negative for the serum ELISA test or both serum ELISA and fecal culture when both were available. Controls were matched by herd and proximal birth date with cases. A total of 856 cows (451 cases and 405 controls) were used in initial discovery analyses, and an additional 263 cows (159 cases and 104 controls) from the same herds were used as a validation data set. Data were analyzed in a single marker analysis controlling for relatedness of individuals (GRAMMAR-GC) and also in a Bayesian analysis in which multiple marker effects were estimated simultaneously (GenSel). For the latter, effects of non-overlapping 1 Mb marker windows across the genome were estimated. Results from the two discovery analyses were generally concordant; however, discovery results were generally not well supported in analysis of the validation data set. A combined analysis of discovery and validation data sets provided strongest support for SNPs and 1 Mb windows on chromosomes 1, 2, 6, 7, 17 and 29. PMID:25473852

  1. Genome-Wide Association Study of Susceptibility to Infection by Mycobacterium avium Subspecies paratuberculosis in Holstein Cattle

    PubMed Central

    Alpay, Fazli; Zare, Yalda; Kamalludin, Mamat H.; Huang, Xixia; Shi, Xianwei; Shook, George E.; Collins, Michael T.; Kirkpatrick, Brian W.

    2014-01-01

    Paratuberculosis, or Johne's disease, is a chronic, granulomatous, gastrointestinal tract disease of cattle and other ruminants caused by the bacterium Mycobacterium avium, subspecies paratuberculosis (MAP). Control of Johne's disease is based on programs of testing and culling animals positive for infection with MAP while concurrently modifying management to reduce the likelihood of infection. The current study is motivated by the hypothesis that genetic variation in host susceptibility to MAP infection can be dissected and quantifiable associations with genetic markers identified. For this purpose, a case-control, genome-wide association study was conducted using US Holstein cattle phenotyped for MAP infection using a serum ELISA and/or fecal culture test. Cases included cows positive for either serum ELISA, fecal culture or both. Controls consisted of animals negative for the serum ELISA test or both serum ELISA and fecal culture when both were available. Controls were matched by herd and proximal birth date with cases. A total of 856 cows (451 cases and 405 controls) were used in initial discovery analyses, and an additional 263 cows (159 cases and 104 controls) from the same herds were used as a validation data set. Data were analyzed in a single marker analysis controlling for relatedness of individuals (GRAMMAR-GC) and also in a Bayesian analysis in which multiple marker effects were estimated simultaneously (GenSel). For the latter, effects of non-overlapping 1 Mb marker windows across the genome were estimated. Results from the two discovery analyses were generally concordant; however, discovery results were generally not well supported in analysis of the validation data set. A combined analysis of discovery and validation data sets provided strongest support for SNPs and 1 Mb windows on chromosomes 1, 2, 6, 7, 17 and 29. PMID:25473852

  2. Experimental infection of lambs with C and S-type strains of Mycobacterium avium subspecies paratuberculosis: immunological and pathological findings

    PubMed Central

    2014-01-01

    The two main genotypes of recognized isolates of Mycobacterium avium subsp. paratuberculosis (Map) are cattle (C) and sheep (S) strains. An experimental infection was conducted to establish the effect of Map strain on the pathogenesis of ovine paratuberculosis. Twenty-four out of thirty 1.5-month-old Assaf lambs were divided into 4 groups of 6 and infected orally with three low passage field isolates, two of S- (22G and the pigmented Ovicap49) and one of C– (764) type, and the reference K-10 strain (C type). The remaining six animals were unchallenged controls. Animals were euthanized at 150 and 390 days post-infection (dpi). Throughout the experiment, the peripheral immune response was assessed and histological and molecular (PCR) studies were conducted on samples of intestine and related lymphoid tissue. Specific antibody and IFN-γ production was significantly higher in animals infected with the C strains, while no consistent IFN- γ responses were observed in the S-type strain infected groups. A positive intradermal skin test response was detected in all infected groups. Lambs infected with S-type strains had granulomatous lesions restricted to the lymphoid tissue with no differences in the lesion intensity over time. In both C–type strain groups, lesions were more severe at 150 dpi while at 390 dpi lesions, characterized by well-demarcated granulomas with fibrosis, decreased in severity. Only infected lambs were positive to PCR. These results suggest that the strain of Map has a strong influence over the immune and pathological responses developed by the host. Lesions induced by C–type strains in lambs show a regressive character and tend to decrease as the infection progresses. PMID:24428881

  3. Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation

    PubMed Central

    O’Brien, Lorna M.; Stewart, Linda D.; Strain, Sam A. J.; Grant, Irene R.

    2016-01-01

    The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay. PMID:26815790

  4. The effects of progressing and non-progressing Mycobacterium avium subsp. paratuberculosis infection on milk production in dairy cows

    PubMed Central

    Smith, Rebecca L.; Gröhn, Y. T.; Pradhan, A.K.; Whitlock, R. H.; Van Kessel, J. S.; Smith, J. M.; Wolfgang, D.R.; Schukken, Y. H.

    2016-01-01

    Longitudinal data from 3 commercial dairy herds in the northeast United States, collected from 2004 to 2011, were analyzed to determine the effect of Mycobacterium avium subsp. paratuberculosis (MAP) infection status and progression path on milk production. Disease status, as indicated by MAP test results, was determined through quarterly ELISA serum testing, biannual fecal culture, and culture of tissues and feces at slaughter. Milk production data were collected from the Dairy Herd Information Association. Animals with positive MAP test results were categorized, based on test results over the full course of the study, as high path (at least one high-positive culture) or low path (at least one positive culture or ELISA). The cumulative number of positive ELISA and culture results were recorded. The effects of both MAP infection path, status, and number of positive tests on milk production were analyzed using a mixed linear model with an autocorrelation random effect structure. Low and high path animals produced more milk prior to their first positive test than always-negative animals, especially high path animals. While mean production decreased after a first positive test, low path animals were shown to recover some productivity. High path animals continued to exhibit a decrease in milk production, especially after their first high-positive fecal culture. These results show that not all animals that test positive for MAP will have long-term production losses. Milk production decreased significantly with each additional positive test. Ultimately, production loss appeared to be a function of MAP infection progression. PMID:26686721

  5. Growth of Mycobacterium avium subsp. paratuberculosis, Escherichia coli, and Salmonella Enteritidis during Preparation and Storage of Yogurt

    PubMed Central

    Cirone, K.; Huberman, Y.; Morsella, C.; Méndez, L.; Jorge, M.; Paolicchi, F.

    2013-01-01

    The purpose of this study was to determine the viability of Mycobacterium avium subsp. paratuberculosis (MAP), Escherichia coli (E. coli), and Salmonella Enteritidis (S. Enteritidis) during preparation and refrigerated storage of yogurt. Three yogurts were prepared using pasteurized commercial milk. Each yogurt was artificially contaminated with (1) MAP, (2) E. coli + S. Enteritidis, and (3) MAP + E. coli + S. Enteritidis. Samples were taken during and after the fermentation process until day 20 after inoculation. MAP was not detected during their preparation and short-term storage but was recuperated after starting at 180 min after inoculation storage. Live bacterial counts of E. coli, and S. Enteritidis increased during the first 24 hours, followed by a slight decrease towards the end of the study. In this study it was shown how MAP, E. coli, and S. Enteritidis resisted the acidic conditions generated during the preparation of yogurt and low storage temperatures. This work contributes to current knowledge regarding survival of MAP, E. coli, and S. Enteritidis during preparation and refrigerated storage of yogurt and emphasizes the need to improve hygiene measures to ensure the absence of these pathogenic microorganisms in dairy products. PMID:24455399

  6. Epstein Barr Virus and Mycobacterium avium subsp. paratuberculosis peptides are recognized in sera and cerebrospinal fluid of MS patients

    PubMed Central

    Mameli, Giuseppe; Cocco, Eleonora; Frau, Jessica; Marrosu, Maria Giovanna; Sechi, Leonardo Antonio

    2016-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) and Epstein-Barr virus (EBV) epitopes elicit a consistent humoral response in serum of multiple sclerosis patients, but the cross reactivity against the homologous myelin basic protein (MBP) and human interferon regulatory factor 5 (IRF5) has not been searched within the Cerebral Spinal Fluid (CSF). We evaluated in sera and CSF of patients with MS and with other neurological diseases (OND) the humoral response against EBV/MAP peptides and the IRF5/MBP. Our data showed that EBV and MAP peptides are able to induce a specific humoral immune response in MS patients compared to OND controls both in serum and in CSF. An intrathecal specific synthesis of IgG against MBP and their EBV and MAP homologous as indicated by the antibody index was observed in MS patients. The humoral response against EBV, MAP, MBP and IRF5 was significantly higher in MS patients compared to OND both in serum and in CSF. The higher presence of antibodies against MBP and their MAP and EBV homologous in CSF during relapses suggests a possible role of the pathogens in enhancing inflammation. PMID:26956729

  7. A novel cell wall lipopeptide is important for biofilm formation and pathogenicity of Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Wu, Chia-wei; Schmoller, Shelly K; Bannantine, John P; Eckstein, Torsten M; Inamine, Julia M; Livesey, Michael; Albrecht, Ralph; Talaat, Adel M

    2009-04-01

    Biofilm formation by pathogenic bacteria plays a key role in their pathogenesis. Previously, the pstA gene was shown to be involved in the virulence of Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johne's disease in cattle and a potential risk factor for Crohn's disease. Scanning electron microscopy and colonization levels of the M. ap mutant indicated that the pstA gene significantly contributes to the ability of M. ap to form biofilms. Digital measurements taken during electron microscopy identified a unique morphology for the DeltapstA mutant, which consisted of significantly shorter bacilli than the wild type. Analysis of the lipid profiles of the mycobacterial strains identified a novel lipopeptide that was present in the cell wall extracts of wild-type M. ap, but missing from the DeltapstA mutant. Interestingly, the calf infection model suggested that pstA contributes to intestinal invasion of M. ap. Furthermore, immunoblot analysis of peptides encoded by pstA identified a specific and significant level of immunogenicity. Taken together, our analysis revealed a novel cell wall component that could contribute to biofilm formation and to the virulence and immunogenicity of M. ap. Molecular tools to better control M. ap infections could be developed utilizing the presented findings. PMID:19490829

  8. Prevalence of Mycobacterium avium subsp. paratuberculosis and Escherichia coli in blood samples from patients with inflammatory bowel disease.

    PubMed

    Nazareth, Nair; Magro, Fernando; Machado, Elisabete; Ribeiro, Teresa Gonçalves; Martinho, António; Rodrigues, Pedro; Alves, Rita; Macedo, Gonçalo Nuno; Gracio, Daniela; Coelho, Rosa; Abreu, Candida; Appelberg, Rui; Dias, Camila; Macedo, Guilherme; Bull, Tim; Sarmento, Amélia

    2015-12-01

    Mycobacterium avium subsp. paratuberculosis (MAP) and adherent-invasive Escherichia coli (AIEC) have been implicated as primary triggers in Crohn's disease (CD). In this study, we evaluated the prevalence of MAP and E. coli (EC) DNA in peripheral blood from 202 inflammatory bowel disease (IBD) patients at various disease periods and compared against 24 cirrhotic patients with ascites (CIR) (non-IBD controls) and 29 healthy controls (HC). MAP DNA was detected by IS900-specific nested PCR, EC DNA by malB-specific nested PCR and AIEC identity, in selected samples, by sequencing of fimH gene. CD patients with active disease showed the highest MAP DNA prevalence among IBD patients (68 %). Infliximab treatment resulted in decreased MAP detection. CIR patients had high individual and coinfection rates (75 % MAP, 88 % EC and 67 % MAP and EC), whilst HC controls had lower MAP prevalence (38 %) and EC was undetectable in this control group. EC DNA prevalence in IBD patients was highly associated with CD, and 80 % of EC from the selected samples of CD patients analyzed carried the fimH30 allele, with a mutation strongly associated with AIEC. Our results show that coinfection with MAP and AIEC is common and persistent in CD, although the high MAP and EC detection in CIR patients suggested that colonization is, at least, partially dependent on increased gut permeability. Nevertheless, facilitative mechanisms between a susceptible host and these two potential human pathogens may allow their implication in CD pathogenesis. PMID:25994082

  9. Induction of matrix metalloproteinases and TLR2 and 6 in murine colon after oral exposure to Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Roderfeld, Martin; Koc, Arzu; Rath, Timo; Blöcher, Sonja; Tschuschner, Annette; Akineden, Ömer; Fischer, Marta; von Gerlach, Susanne; Goethe, Ralph; Eckelt, Elke; Meens, Jochen; Bülte, Michael; Basler, Tina; Roeb, Elke

    2012-06-01

    Mycobacterium avium subspecies paratuberculosis (MAP) is suspected to be a causative agent in Crohn's disease. Recent evidence suggests that MAP can induce the expression of Matrix Metalloproteinases (MMPs), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within the present study, we analysed whether oral MAP exposure can induce colonic MMP expression in vivo. In MAP exposed mice MAP and spheroplasts were visualized in intramucosal leukocyte aggregates. MAP exposed mice exhibited a higher colonic expression of Mmp-2, -9, -13, -14, Timp-1, Tlr2, Tlr6, Il-1β, and Tnf-α. Cell clusters of MMP-9 positive cells adjacent to intramucosal leukocyte aggregates and CD45(+) leukocytes were identified as the major cellular sources of MMP-9. Enhanced TLR2 expression was visualized on the luminal side of colonic enterocytes. Although MAP exposure did not lead to macroscopic intestinal inflammation, the observed MAP spheroplasts in intramucosal leukocyte aggregates together with increased colonic expression of toll-like receptors, pro-inflammatory cytokines, and MMPs upon MAP exposure represents a part of the host immune response towards MAP. PMID:22289202

  10. Johne's disease: reliability of environmental sampling to characterize Mycobacterium avium subspecies paratuberculosis (MAP) infection in beef cow-calf herds.

    PubMed

    Klawonn, W; Einax, E; Pützschel, R; Schmidt, M; Donat, K

    2016-08-01

    Environmental samples are considered to be a cost-effective method of identifying Mycobacterium avium subsp. paratuberculosis (MAP)-positive dairy herds, but evidence for beef cow-calf herds is weak. This study aims at evaluating this approach in a total of 20 German herds that were characterized by individual faecal samples (n = 2545) of all cows. For 14 MAP-positive herds having at least one MAP-positive animal, the within-herd prevalence was calculated from concurrent individual faecal culture-based testing. Six herds certified as 'MAP free' based on the negative results of previous years served as MAP-negative controls. On average, six environmental samples were taken at the end of winter from areas with high cow traffic and tested for MAP by faecal culture. According to the environmental samples, nine (64·3%) out of the 14 MAP-positive cow-calf herds were infected. The percentage of positive environmental samples and the apparent within-herd prevalence (Spearman's P = 0·73, P < 0·001) as well as the herd-level test results (positive and negative) and the herd's status based on individual testing (Fisher's exact test, P = 0·014) showed a positive association. Considering limitations in low-prevalence herds, MAP-positive beef cow-calf herds are detectable by environmental samples in temperate climate zones. PMID:27094619

  11. Molecular typing of Argentinian Mycobacterium avium subsp. paratuberculosis isolates by multiple-locus variable number-tandem repeat analysis

    PubMed Central

    Gioffré, Andrea; Correa Muñoz, Magnolia; Alvarado Pinedo, María F.; Vaca, Roberto; Morsella, Claudia; Fiorentino, María Andrea; Paolicchi, Fernando; Ruybal, Paula; Zumárraga, Martín; Travería, Gabriel E.; Romano, María Isabel

    2015-01-01

    Multiple-locus variable number-tandem repeat analysis (MLVA) of Mycobacterium avium subspecies paratuberculosis (MAP) isolates may contribute to the knowledge of strain diversity in Argentina. Although the diversity of MAP has been previously investigated in Argentina using IS900-RFLP, a small number of isolates were employed, and a low discriminative power was reached. The aim of the present study was to test the genetic diversity among MAP isolates using an MLVA approach based on 8 repetitive loci. We studied 97 isolates from cattle, goat and sheep and could describe 7 different patterns: INMV1, INMV2, INMV11, INMV13, INMV16, INMV33 and one incomplete pattern. INMV1 and INMV2 were the most frequent patterns, grouping 76.3% of the isolates. We were also able to demonstrate the coexistence of genotypes in herds and co-infection at the organism level. This study shows that all the patterns described are common to those described in Europe, suggesting an epidemiological link between the continents. PMID:26273274

  12. Serum BAFF levels, Methypredsinolone therapy, Epstein-Barr Virus and Mycobacterium avium subsp. paratuberculosis infection in Multiple Sclerosis patients

    PubMed Central

    Mameli, Giuseppe; Cocco, Eleonora; Frau, Jessica; Arru, Giannina; Caggiu, Elisa; Marrosu, Maria Giovanna; Sechi, Leonardo A.

    2016-01-01

    Elevated B lymphocyte activating factor BAFF levels have been reported in multiple sclerosis (MS) patients; moreover, disease-modifying treatments (DMT) have shown to influence blood BAFF levels in MS patients, although the significance of these changes is still controversial. In addition, BAFF levels were reported increased during infectious diseases. In our study, we wanted to investigate on the serum BAFF concentrations correlated to the antibody response against Mycobacterium avium subspecies paratuberculosis (MAP), Epstein-Barr virus (EBV) and their human homologous epitopes in MS and in patients affected with other neurological diseases (OND), divided in Inflammatory Neurological Diseases (IND), Non Inflammatory Neurological Diseases (NIND) and Undetermined Neurological Diseases (UND), in comparison to healthy controls (HCs). Our results confirmed a statistically significant high BAFF levels in MS and IND patients in comparison to HCs but not NIND and UND patients. Interestingly, BAFF levels were inversely proportional to antibodies level against EBV and MAP peptides and the BAFF levels significantly decreased in MS patients after methylprednisolone therapy. These results implicate that lower circulating BAFF concentrations were present in MS patients with humoral response against MAP and EBV. In conclusion MS patients with no IgGs against EBV and MAP may support the hypothesis that elevated blood BAFF levels could be associated with a more stable disease. PMID:27383531

  13. The structure and peroxidase activity of a 33-kDa catalase-related protein from Mycobacterium avium ssp. paratuberculosis

    PubMed Central

    Pakhomova, Svetlana; Gao, Benlian; Boeglin, William E; Brash, Alan R; Newcomer, Marcia E

    2009-01-01

    True catalases are tyrosine-liganded, usually tetrameric, hemoproteins with subunit sizes of ∼55–84 kDa. Recently characterized hemoproteins with a catalase-related structure, yet lacking in catalatic activity, include the 40–43 kDa allene oxide synthases of marine invertebrates and cyanobacteria. Herein, we describe the 1.8 Å X-ray crystal structure of a 33 kDa subunit hemoprotein from Mycobacterium avium ssp. paratuberculosis (annotated as MAP-2744c), that retains the core elements of the catalase fold and exhibits an organic peroxide-dependent peroxidase activity. MAP-2744c exhibits negligible catalatic activity, weak peroxidatic activity using hydrogen peroxide (20/s) and strong peroxidase activity (∼300/s) using organic hydroperoxides as co-substrate. Key amino acid differences significantly impact prosthetic group conformation and placement and confer a distinct activity to this prototypical member of a group of conserved bacterial “minicatalases”. Its structural features and the result of the enzyme assays support a role for MAP-2744c and its close homologues in mitigating challenge by a variety of reactive oxygen species. PMID:19827095

  14. Estimate of the direct production losses in Canadian dairy herds with subclinical Mycobacterium avium subspecies paratuberculosis infection

    PubMed Central

    Tiwari, Ashwani; VanLeeuwen, John A.; Dohoo, Ian R.; Keefe, Greg P.; Weersink, Alfons

    2008-01-01

    The objective of this study was to estimate the annual losses from Mycobacterium avium subspecies paratuberculosis (MAP) for an average, MAP-seropositive, Canadian dairy herd. A partial-budget simulation model was developed with 4 components of direct production losses (decreased milk production, premature voluntary culling, mortality, and reproductive losses). Input values were obtained primarily from a national seroprevalence survey of 373 Canadian dairy farms in 8 of 10 provinces. The model took into account the variability and uncertainty of the required input values; consequently, it produced probability distributions of the estimated losses. For an average Canadian dairy herd with 12.7% of 61 cows seropositive for MAP, the mean loss was $2992 (95% C.I., $143 to $9741) annually, or $49 per cow per year. Additional culling, decreased milk production, mortality, and reproductive losses accounted for 46%, 9%, 16%, and 29% of the losses, respectively. Canadian dairy producers should use best management practices to reduce these substantial annual losses. PMID:18624066

  15. Can Immune Response Mechanisms Explain the Fecal Shedding Patterns of Cattle Infected with Mycobacterium avium Subspecies paratuberculosis?

    PubMed Central

    Magombedze, Gesham; Eda, Shigetoshi; Koets, Ad

    2016-01-01

    Johne’s disease (JD) is a chronic disease in ruminants and is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). At late stages of the disease, MAP bacilli are shed via feces excretion and in turn create the potential for oral-fecal transmission. The role of the host immune response in MAP bacteria shedding patterns at different stages of JD is still unclear. We employed mathematical modeling to predict if the variation in MAP shedding could be correlated to the immune response in infected animals. We used a novel inverse modeling approach that assumed biological interactions among the antigen-specific lymphocyte proliferation response (cell-mediated response), antibody/humoral immune responses, and MAP bacteria. The modeling framework was used to predict and test possible biological interactions between the measured variables and returns only the essential interactions that are relevant in explaining the observed cattle MAP experimental infection data. Through confronting the models with data, we predicted observed effects (enhancement or suppression) and extents of interactions among the three variables. This analysis enabled classification of the infected cattle into three different groups that correspond to the unique predicted immune responses that are essential to explain the data from cattle within these groups. Our analysis highlights the strong and weak points of the modeling approach, as well as the key immune mechanisms predicted to be expressed in all animals and those that were different between animals, hence giving insight into how animals exhibit different disease dynamics and bacteria shedding patterns. PMID:26808389

  16. Serum BAFF levels, Methypredsinolone therapy, Epstein-Barr Virus and Mycobacterium avium subsp. paratuberculosis infection in Multiple Sclerosis patients.

    PubMed

    Mameli, Giuseppe; Cocco, Eleonora; Frau, Jessica; Arru, Giannina; Caggiu, Elisa; Marrosu, Maria Giovanna; Sechi, Leonardo A

    2016-01-01

    Elevated B lymphocyte activating factor BAFF levels have been reported in multiple sclerosis (MS) patients; moreover, disease-modifying treatments (DMT) have shown to influence blood BAFF levels in MS patients, although the significance of these changes is still controversial. In addition, BAFF levels were reported increased during infectious diseases. In our study, we wanted to investigate on the serum BAFF concentrations correlated to the antibody response against Mycobacterium avium subspecies paratuberculosis (MAP), Epstein-Barr virus (EBV) and their human homologous epitopes in MS and in patients affected with other neurological diseases (OND), divided in Inflammatory Neurological Diseases (IND), Non Inflammatory Neurological Diseases (NIND) and Undetermined Neurological Diseases (UND), in comparison to healthy controls (HCs). Our results confirmed a statistically significant high BAFF levels in MS and IND patients in comparison to HCs but not NIND and UND patients. Interestingly, BAFF levels were inversely proportional to antibodies level against EBV and MAP peptides and the BAFF levels significantly decreased in MS patients after methylprednisolone therapy. These results implicate that lower circulating BAFF concentrations were present in MS patients with humoral response against MAP and EBV. In conclusion MS patients with no IgGs against EBV and MAP may support the hypothesis that elevated blood BAFF levels could be associated with a more stable disease. PMID:27383531

  17. Mediation of Host Immune Responses after Immunization of Neonatal Calves with a Heat-Killed Mycobacterium avium subsp. paratuberculosis Vaccine ▿

    PubMed Central

    Stabel, J. R.; Waters, W. R.; Bannantine, J. P.; Lyashchenko, K.

    2011-01-01

    A major drawback of current whole-cell vaccines for Mycobacterium avium subsp. paratuberculosis is the interference with diagnostic tests for bovine tuberculosis (TB) and paratuberculosis. The current study was designed to explore the effects of immunization with a heat-killed whole-cell vaccine (Mycopar) on diagnostic test performance and to characterize host immune responses to vaccination over a 12-month period. Neonatal dairy calves were assigned to treatment groups consisting of (i) controls, not vaccinated (n = 5), and (ii) vaccinates, vaccinated with Mycopar vaccine (n = 5). The results from this study demonstrated a rapid initiation of M. avium subsp. paratuberculosis-specific gamma interferon (IFN-γ) in vaccinated calves by 7 days, with robust responses throughout the study. Vaccinated calves also had responses to M. bovis purified protein derivative tuberculin (BoPPD) but minimal reactivity to ESAT-6/CFP-10, an M. bovis recombinant fusion protein. The levels of antigen-specific interleukin-4 (IL-4) and IL-10 were markedly decreased in vaccinated calves between days 7 and 90 of the study but thereafter were similar to the levels in controls. Vaccinated calves began to seroconvert at 4 months, with 4/5 calves having detectable M. avium subsp. paratuberculosis antibody by 6 months. The responses in test platforms for bovine TB were negligible in the vaccinate group, as only one calf had a response, which was in the suspect range of the comparative cervical skin test. Serum antibody responses to M. bovis antigens ESAT-6, CFP-10, and MPB83 were negative on the Vet TB STAT-PAK, DPP VetTB, and DPP BovidTB tests. These results suggest that the Mycopar vaccine will interfere with diagnostic tools for paratuberculosis but result in low interference with the comparative cervical skin test and emerging serologic tests for M. bovis. PMID:22030370

  18. Development and validation of a liquid medium (M7H9C) for routine culture of Mycobacterium avium subsp. paratuberculosis to replace modified Bactec 12B medium.

    PubMed

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

    2013-12-01

    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

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

    PubMed Central

    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

  20. Immunogenicity and Reactivity of Novel Mycobacterium avium subsp. paratuberculosis PPE MAP1152 and Conserved MAP1156 Proteins with Sera from Experimentally and Naturally Infected Animals ▿ †

    PubMed Central

    Bannantine, John P.; Paulson, Avery L.; Chacon, Ofelia; Fenton, Robert J.; Zinniel, Denise K.; McVey, David S.; Smith, David R.; Czuprynski, Charles J.; Barletta, Raúl G.

    2011-01-01

    Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Development of genetic tools and completion of the M. avium subsp. paratuberculosis genome sequencing project have expanded the opportunities for antigen discovery. In this study, we determined the seroreactivities of two proteins encoded at the 5′ and 3′ regions of the MAP1152-MAP1156 gene cluster. MAP1152 encodes a PPE protein, and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Recombinant MAP proteins were overproduced and purified from Escherichia coli as maltose-binding protein (MBP) fusions. Immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of mice and rabbits immunized with live M. avium subsp. paratuberculosis cells and against samples from naturally infected cattle. In immunoblot assays, MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme-linked immunosorbent assay for the recombinant proteins was developed and used to test preclassified positive and negative serum samples from naturally infected and noninfected cattle. Samples, with one exception, displayed no seroreactivity against the MBP-LacZ fusion protein (P > 0.05), the negative-control antigen. MAP1152 displayed seroreactivity against all positive sera but no seroreactivity to the negative sera (P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between noninfected and infected cattle (P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, both proteins are immunogenic in mice and rabbits, and M. avium subsp. paratuberculosis-infected cattle mount a humoral response to both MAP1152 and MAP1156 cross-reactive epitopes. These findings have potential applications to diagnostics, vaccine

  1. Paratuberculosis: decrease in milk production of German Holstein dairy cows shedding Mycobacterium avium ssp. paratuberculosis depends on within-herd prevalence.

    PubMed

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

    2014-05-01

    Paratuberculosis impairs productivity of infected dairy cows because of reduced milk production and fertility and enhanced risk of culling. The magnitude of the milk yield depression in individual cows is influenced by factors such as parity, the stage of the disease and the choice of test used. The objectives of this case-control study were to substantiate the influence of the different levels of the within-herd prevalence (WHP) on individual milk yield of fecal culture (FC)-positive cows (FC+) compared with FC-negative herd-mates (FC-), and to estimate the magnitude of the deviation of the milk yield, milk components and somatic cell count (SCC) in an FC-based study. Of a total of 31 420 cows from 26 Thuringian dairy herds tested for paratuberculosis by FC, a subset of 1382 FC+ and 3245 FC- with milk recording data were selected as cases and controls, respectively. The FC- cows were matched for the same number and stage of lactation (±10 days in milk) as one FC+ from the same herd. Within a mixed model analysis using the fixed effects of Mycobacterium avium ssp. paratuberculosis (MAP) status, lactation number, days in milk, prevalence class of farm and the random effect of farm on milk yield per day (kg), the amount of fat and protein (mg/dl) and lactose (mg/dl) as well as the SCC (1000/ml) were measured. On the basis of least square means, FC+ cows had a lower test-day milk yield (27.7±0.6 kg) compared with FC- (29.0±0.6 kg), as well as a lower milk protein content and a slightly diminished lactose concentration. FC status was not associated with milk fat percentage or milk SCC. In FC+ cows, reduction in milk yield increased with increasing WHP. An interaction of FC status and farm was found for the test-day milk yield, and milk protein percentage, respectively. We conclude that the reduction in milk yield of FC+ cows compared with FC- herd-mates is significantly influenced by farm effects and depends on WHP class. Owners of MAP-positive dairy herds may

  2. Humoral response against host-mimetic homologous epitopes of Mycobacterium avium subsp. paratuberculosis in Japanese multiple sclerosis patients

    PubMed Central

    Cossu, Davide; Yokoyama, Kazumasa; Sechi, Leonardo Antonio; Otsubo, Shigeru; Tomizawa, Yuji; Momotani, Eiichi; Hattori, Nobutaka

    2016-01-01

    Several works have demonstrated the existence of a link between Mycobacterium avium subsp. paratuberculosis (MAP) and MS in Italy. In this study, we analyzed the serology of MAP in a Japanese population while looking at several markers of MAP. Fifty MS patients, 12 clinically isolated syndrome (CIS) patients, 30 other neurological disorders (OND) patients, and 50 healthy controls (HCs) were tested using ELISA for the presence of IgG antibodies toward immunodominant epitopes MAP_0106c121-132, homologues MBP85-98, homologues IRF5424-432, MAP_402718-32, and MAP_2694295-303. MAP-positive patients were also analyzed in relation to their clinical/demographic characteristics. Amongst all peptides, only antibodies against MAP_2694295-303 were more prevalent in MS patients (30%), as compared to OND patients (3%) (p = 0.009; area under roc curve (AUC) = 0.61) and HCs (2%) (p = 0.0004; AUC = 0.65) and in CIS patients (25%) compared to HCs (p = 0.023; AUC = 0.55). Logistic regression analysis showed a higher frequency of anti-MAP_2694295-303 antibodies in the sera of oligoclonal bands positive MS patients (p = 0.2; OR = 2, 95%CI: 0.55–7.7). These findings support the view that MAP could act as a risk factor or a triggering agent of MS in some Japanese patients with a genetic susceptibility to the mycobacterium. PMID:27356622

  3. Mycobacterium avium paratuberculosis and the etiology of Crohn's disease: a review of the controversy from the clinician's perspective.

    PubMed

    Rosenfeld, Greg; Bressler, Brian

    2010-10-01

    Mycobacterium avium paratuberculosis (MAP) is an obligate intracellular organism that has frequently been associated with Crohn's disease (CD). Because CD is a chronic inflammatory condition, many researchers have speculated that an infectious agent must be the cause of CD. MAP has often been proposed to be one such agent; however, despite considerable research, the evidence remains inconclusive. Higher levels of MAP have been found in the tissues and blood of CD patients than in controls, forming the foundation for much of the research into the role of MAP in CD and the primary argument in support of a causative role for MAP in CD. MAP is a slow-growing and fastidious organism that is difficult to grow in culture and, therefore, challenging to detect in patients. As a result, there has been variability in the results of studies attempting to detect the presence of MAP in CD patients, and considerable controversy over whether this organism has a causative role in the etiology of CD. Two main hypotheses exist with respect to the role of MAP in CD. The first is that MAP is a principal cause of CD, while the second is that MAP is more prevalent because of the immune dysfunction seen in CD but does not play a causative role. Clinicians are often faced with questions regarding the role of this organism and the need to treat it. The present article attempts to provide an overview of the controversy including the nature of the mycobacterium, the difficulty in detecting it, the use of antimycobacterial agents to treat it and the effect of immunosuppressive agents - all from a clinician's perspective. Although the role of MAP in CD remains controversial and an area of considerable research, it is currently only of academic interest because there is no clinically useful test to identify the presence of the organism, and no evidence to support the use of antibiotics to eradicate it for the treatment of CD. PMID:21037992

  4. Comprehensive Insights in the Mycobacterium avium subsp. paratuberculosis Genome Using New WGS Data of Sheep Strain JIII-386 from Germany

    PubMed Central

    Möbius, Petra; Hölzer, Martin; Felder, Marius; Nordsiek, Gabriele; Groth, Marco; Köhler, Heike; Reichwald, Kathrin; Platzer, Matthias; Marz, Manja

    2015-01-01

    Mycobacterium avium (M. a.) subsp. paratuberculosis (MAP)—the etiologic agent of Johne’s disease—affects cattle, sheep, and other ruminants worldwide. To decipher phenotypic differences among sheep and cattle strains (belonging to MAP-S [Type-I/III], respectively, MAP-C [Type-II]), comparative genome analysis needs data from diverse isolates originating from different geographic regions of the world. This study presents the so far best assembled genome of a MAP-S-strain: Sheep isolate JIII-386 from Germany. One newly sequenced cattle isolate (JII-1961, Germany), four published MAP strains of MAP-C and MAP-S from the United States and Australia, and M. a. subsp. hominissuis (MAH) strain 104 were used for assembly improvement and comparisons. All genomes were annotated by BacProt and results compared with NCBI (National Center for Biotechnology Information) annotation. Corresponding protein-coding sequences (CDSs) were detected, but also CDSs that were exclusively determined by either NCBI or BacProt. A new Shine–Dalgarno sequence motif (5′-AGCTGG-3′) was extracted. Novel CDSs including PE-PGRS family protein genes and about 80 noncoding RNAs exhibiting high sequence conservation are presented. Previously found genetic differences between MAP-types are partially revised. Four of ten assumed MAP-S-specific large sequence polymorphism regions (LSPSs) are still present in MAP-C strains; new LSPSs were identified. Independently of the regional origin of the strains, the number of individual CDSs and single nucleotide variants confirms the strong similarity of MAP-C strains and shows higher diversity among MAP-S strains. This study gives ambiguous results regarding the hypothesis that MAP-S is the evolutionary intermediate between MAH and MAP-C, but it clearly shows a higher similarity of MAP to MAH than to Mycobacterium intracellulare. PMID:26384038

  5. Humoral response against host-mimetic homologous epitopes of Mycobacterium avium subsp. paratuberculosis in Japanese multiple sclerosis patients.

    PubMed

    Cossu, Davide; Yokoyama, Kazumasa; Sechi, Leonardo Antonio; Otsubo, Shigeru; Tomizawa, Yuji; Momotani, Eiichi; Hattori, Nobutaka

    2016-01-01

    Several works have demonstrated the existence of a link between Mycobacterium avium subsp. paratuberculosis (MAP) and MS in Italy. In this study, we analyzed the serology of MAP in a Japanese population while looking at several markers of MAP. Fifty MS patients, 12 clinically isolated syndrome (CIS) patients, 30 other neurological disorders (OND) patients, and 50 healthy controls (HCs) were tested using ELISA for the presence of IgG antibodies toward immunodominant epitopes MAP_0106c121-132, homologues MBP85-98, homologues IRF5424-432, MAP_402718-32, and MAP_2694295-303. MAP-positive patients were also analyzed in relation to their clinical/demographic characteristics. Amongst all peptides, only antibodies against MAP_2694295-303 were more prevalent in MS patients (30%), as compared to OND patients (3%) (p = 0.009; area under roc curve (AUC) = 0.61) and HCs (2%) (p = 0.0004; AUC = 0.65) and in CIS patients (25%) compared to HCs (p = 0.023; AUC = 0.55). Logistic regression analysis showed a higher frequency of anti-MAP_2694295-303 antibodies in the sera of oligoclonal bands positive MS patients (p = 0.2; OR = 2, 95%CI: 0.55-7.7). These findings support the view that MAP could act as a risk factor or a triggering agent of MS in some Japanese patients with a genetic susceptibility to the mycobacterium. PMID:27356622

  6. Genome-Wide Sequence Variation among Mycobacterium avium Subspecies paratuberculosis Isolates: A Better Understanding of Johne’s Disease Transmission Dynamics

    PubMed Central

    Hsu, Chung-Yi; Wu, Chia-Wei; Talaat, Adel M.

    2011-01-01

    Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johne’s disease, infects many farmed ruminants, wild-life animals, and recently isolated from 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) isolates to gain insights into genomic diversity associated with variable hosts and environments. Using Next-generation sequencing technology, all six M. ap isolates showed a high percentage of similarity (98%) to the reference genome sequence of M. ap K-10 isolated from cattle. However, two M. avium isolates (DT 78 and Env 77) showed significant sequence diversity (only 87 and 40% similarity, respectively) compared to the reference strain M. avium 104, a reflection of the wide environmental niches of this group of mycobacteria. Within the M. ap isolates, genomic rearrangements (insertions/deletions) were not detected, and only unique single nucleotide polymorphisms (SNPs) were observed among M. ap isolates. While more of the SNPs (~100) in M. ap genomes were non-synonymous, a total of ~6,000 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-genomics had a enough discriminatory power to differentiate between isolates from different hosts but yet suggesting a bovine source of infection to other animals examined in this study. Interestingly, the human isolate (M. ap 4B) was closely related to a M. ap isolate from a dairy facility, suggesting a common source of infection. Overall, the identified phylo-genomes further supported the idea of a common ancestor to both M. ap and M. avium isolates. Genome-wide analysis described here could provide a strong foundation for a population genetic structure that could be useful for the analysis of mycobacterial evolution and for the tracking of Johne

  7. Short communication: Application of an N-acetyl-L-cysteine-NaOH decontamination method for the recovery of viable Mycobacterium avium subsp. paratuberculosis from milk of naturally infected cows

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is shed into the milk of cattle affected by Johne’s disease and, therefore, is a route of transmission for infection in youngstock in dairy herds. The objective of this study was to validate a decontamination and culture protocol for the recovery of ...

  8. Prevalence of Mycobacterium avium subsp. paratuberculosis in Ileocecal Lymph Nodes and on Hides and Carcasses from Cull Cows and Fed Cattle at Commercial Beef Processing Plants in the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clinical associations between Crohn’s disease in humans and Mycobacterium avium subspecies paratuberculosis (Map) have been suggested but not confirmed. Map is the causative agent for Johne’s disease in cattle. Infected cattle could be sources for Map transmission to humans via dairy and beef prod...

  9. The other way around: probiotic Lactobacillus acidophilus NP51 restrict progression of Mycobacterium avium subspecies paratuberculosis (MAP) infection in Balb/c mice via activiation of CD8 alpha+ immune cell-mediated immunity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to examine the immune-modulating effects of feeding a novel probiotic Lactobacillus acidophilus strain NP51 to specific pathogen-free Balb/c mice challenged with Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne’s disease (JD) in rumi...

  10. Quantification of the Sensitivity of Mycobacterium avium subsp paratuberculosis and Salmonella enterica subsp enterica to Low pH and High Organic Acids using Propidium Monoazide and Quantitative PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp paratuberculosis (Map) and Salmonella enterica subsp enterica (S. enterica) are two pathogens that are a concern to food and animal safety due to their ability to withstand harsh conditions encountered in the natural environment and within the host during pathogenesis. Acid...

  11. Demonstration of Allelic Exchange in the Slow-Growing Bacterium Mycobacterium avium subsp. paratuberculosis, and Generation of Mutants with Deletions at the pknG, relA and lsr2 Loci

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (Map) is the causative pathogen of Johne’s disease, a chronic inflammatory wasting disease in ruminants. The disease has been difficult to control because of the lack of an effective vaccine. To address this need, we developed an efficient allelic exchange...

  12. The other way around: Probiotic lactobacillus acidophilus NP51 restricts progression of Mycobacterium avium subspecies paratuberculosis (MAP) infection in Balb/c mice through activation of CD8+ T cell-mediated immunity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to examine immune effects of feeding novel probiotic Lactobacillus acidophilus strain NP51 to specific pathogen-free Balb/c mice challenged with Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne’s disease (JD). We hypothesized that fe...

  13. Johne's disease in a free-ranging white-tailed deer from Virginia and subsequent surveillance for Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Sleeman, Jonathan M; Manning, Elizabeth J B; Rohm, John H; Sims, Jerry P; Sanchez, Susan; Gerhold, Richard W; Keel, M Kevin

    2009-01-01

    Johne's disease (paratuberculosis) was diagnosed in a 2-yr-old, male, free-ranging white-tailed deer (Odocoileus virginianus) from Fauquier County, Virginia, USA, based on histopathology and culture for Mycobacterium avium subspecies paratuberculosis. Clinical and pathologic findings included emaciation; loss of body fat; chronic diarrhea; severe, chronic, diffuse granulomatous colitis with intrahistiocytic acid-fast bacilli; moderate, chronic granulomatous lymphadenitis with intrahistiocytic acid-fast bacilli; as well as moderate chronic, multifocal, lymphoplasmacytic hepatitis. These findings are consistent with previous reports of Johne's disease in cervids. Subsequent targeted surveillance of 10 emaciated deer with diarrhea, as well as sampling of 72 asymptomatic deer for M. avium subsp. paratuberculosis using culture of multiple tissue types, as well as serology using an enzyme-linked immunosorbent assay (ELISA) optimized for cervid antibody detection, did not reveal any additional cases of infection in this geographic region. To date, this appears to be an isolated case of Johne's disease in a free-ranging white-tailed deer, and infection with the causative agent for Johne's disease appears to be an infrequent occurrence in deer from this region. The origin of infection was most likely domestic ruminants. This is the first report of clinical Johne's disease in a free-ranging white-tailed deer outside of the Florida Keys, USA. Stressors, such as high deer population density and low selenium levels, may have contributed to the development of clinical disease in this case and warrant further investigation. PMID:19204350

  14. Mycobacterium avium subsp. paratuberculosis in the Catchment Area and Water of the River Taff in South Wales, United Kingdom, and Its Potential Relationship to Clustering of Crohn's Disease Cases in the City of Cardiff

    PubMed Central

    Pickup, R. W.; Rhodes, G.; Arnott, S.; Sidi-Boumedine, K.; Bull, T. J.; Weightman, A.; Hurley, M.; Hermon-Taylor, J.

    2005-01-01

    In South Wales, United Kingdom, a populated coastal region lies beneath hill pastures grazed by livestock in which Mycobacterium avium subsp. paratuberculosis is endemic. The Taff is a spate river running off the hills and through the principal city of Cardiff. We sampled Taff water above Cardiff twice weekly from November 2001 to November 2002. M. avium subsp. paratuberculosis was detected by IS900 PCR and culture. Thirty-one of 96 daily samples (32.3%) were IS900 PCR positive, and 12 grew M. avium subsp. paratuberculosis bovine strains. Amplicon sequences from colonies were identical to the sequence with GenBank accession no. X16293, whereas 16 of 19 sequences from river water DNA extracts had a single-nucleotide polymorphism at position 214. This is consistent with a different strain of M. avium subsp. paratuberculosis in the river, which is unculturable by the methods we used. Parallel studies showed that M. avium subsp. paratuberculosis remained culturable in lake water microcosms for 632 days and persisted to 841 days. Of four reservoirs controlling the catchment area of the Taff, M. avium subsp. paratuberculosis was present in surface sediments from three and in sediment cores from two, consistent with deposition over at least 50 years. Previous epidemiological research in Cardiff demonstrated a highly significant increase of Crohn's disease in 11 districts. These bordered the river except for a gap on the windward side. A topographical relief map shows that this gap is directly opposite a valley open to the prevailing southwesterly winds. This would influence the distribution of aerosols carrying M. avium subsp. paratuberculosis from the river. PMID:15812047

  15. Genome-Wide Association Analysis and Genomic Prediction of Mycobacterium avium Subspecies paratuberculosis Infection in US Jersey Cattle

    PubMed Central

    Zare, Yalda; Shook, George E.; Collins, Michael T.; Kirkpatrick, Brian W.

    2014-01-01

    Paratuberculosis (Johne’s disease), an enteric disorder in ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP), causes economic losses in excess of $200 million annually to the US dairy industry. To identify genomic regions underlying susceptibility to MAP infection in Jersey cattle, a case-control genome-wide association study (GWAS) was performed. Blood and fecal samples were collected from ∼5,000 mature cows in 30 commercial Jersey herds from across the US. Discovery data consisted of 450 cases and 439 controls genotyped with the Illumina BovineSNP50 BeadChip. Cases were animals with positive ELISA and fecal culture (FC) results. Controls were animals negative to both ELISA and FC tests that matched cases on birth date and herd. Validation data consisted of 180 animals including 90 cases (positive to FC) and 90 controls (negative to ELISA and FC), selected from discovery herds and genotyped by Illumina BovineLD BeadChip (∼7K SNPs). Two analytical approaches were used: single-marker GWAS using the GRAMMAR-GC method and Bayesian variable selection (Bayes C) using GenSel software. GRAMMAR-GC identified one SNP on BTA7 at 68 megabases (Mb) surpassing a significance threshold of 5×10−5. ARS-BFGL-NGS-11887 on BTA23 (27.7 Mb) accounted for the highest percentage of genetic variance (3.3%) in the Bayes C analysis. SNPs identified in common by GRAMMAR-GC and Bayes C in both discovery and combined data were mapped to BTA23 (27, 29 and 44 Mb), 3 (100, 101, 106 and 107 Mb) and 17 (57 Mb). Correspondence between results of GRAMMAR-GC and Bayes C was high (70–80% of most significant SNPs in common). These SNPs could potentially be associated with causal variants underlying susceptibility to MAP infection in Jersey cattle. Predictive performance of the model developed by Bayes C for prediction of infection status of animals in validation set was low (55% probability of correct ranking of paired case and control samples). PMID:24523889

  16. Efficacy of monensin sodium for the reduction of fecal shedding of Mycobacterium avium subsp. paratuberculosis in infected dairy cattle.

    PubMed

    Hendrick, Steven H; Kelton, David F; Leslie, Ken E; Lissemore, Kerry D; Archambault, Marie; Bagg, Randy; Dick, Paul; Duffield, Todd F

    2006-08-17

    Reducing the quantity of Mycobacterium avium subsp. paratuberculosis (MAP) being shed by cows with Johne's disease should decrease the risk of spread of this disease to young stock. Previous work has suggested that monensin sodium decreases the pathologic lesions associated with Johne's disease, but the impact on shedding of viable MAP remains unknown. After serologic screening of 32 dairy herds in southwestern Ontario, 228 cows from 13 of these herds were enrolled into a randomized clinical trial. Fecal culture and PCR were used to identify 114 cows as potential fecal shedders, while another 114 cows were enrolled as ELISA negative, herd and parity matched controls. All cows were randomized to receive either a monensin controlled release capsule (CRC) or a placebo capsule. Serial fecal and blood samples were collected for fecal culture and serum ELISA testing over a 98-day period. On day 98 of the study, treatments were switched for all cows continuing in the trial. These remaining cows were followed for another 98 days with a similar sampling protocol. Mixed effect models were used to measure the impact of treatment on the number of colony forming units identified on fecal cultures over time. During the first 98 days of the study, cows treated with a monensin CRC were found to shed 3.4cfu per tube less than placebo treated cows (P=0.05). The serum ELISA S/P ratio was reduced by 1.39 units in cows given monensin (P=0.06). However, treatment with monensin did not reduce the odds of testing positive on serology. Only the cows shedding MAP on day 0 were found to have a reduced odds of testing positive on fecal culture when treated with monensin (OR=0.27; P=0.03). Monensin sodium administered to infected animals at 335mg/day marginally reduced fecal shedding of MAP in mature dairy cattle, but the biological significance of this reduction is unknown. PMID:16631972

  17. Effects of biological and methodological factors on volatile organic compound patterns during cultural growth of Mycobacterium avium ssp. paratuberculosis.

    PubMed

    Küntzel, Anne; Fischer, Sina; Bergmann, Andreas; Oertel, Peter; Steffens, Markus; Trefz, Phillip; Miekisch, Wolfram; Schubert, Jochen K; Reinhold, Petra; Köhler, Heike

    2016-01-01

    Mycobacterium avium ssp. paratuberculosis (MAP) causes chronic granulomatous enteritis in ruminants. Bacterial growth is still the diagnostic 'gold standard', but is very time consuming. MAP-specific volatile organic compounds (VOCs) above media could accelerate cultural diagnosis. The aim of this project was to assess the kinetics of a VOC profile linked to the growth of MAP in vitro. The following sources of variability were taken into account: five different culture media, three different MAP strains, inoculation with different bacterial counts, and different periods of incubation. Needle-trap microextraction was employed for pre-concentration of VOCs, and gas chromatography-mass spectrometry for subsequent analysis. All volatiles were identified and calibrated by analysing pure references at different concentration levels. More than 100 VOCs were measured in headspaces above MAP-inoculated and control slants. Results confirmed different VOC profiles above different culture media. Emissions could be assigned to either egg-containing media or synthetic ingredients. 43 VOCs were identified as potential biomarkers of MAP growth on Herrold's Egg Yolk Medium without significant differences between the tree MAP strains. Substances belonged to the classes of alcohols, aldehydes, esters, ketones, aliphatic and aromatic hydrocarbons. With increasing bacterial density the VOC concentrations above MAP expressed different patterns: the majority of substances increased (although a few decreased after reaching a peak), but nine VOCs clearly decreased. Data support the hypotheses that (i) bacteria emit different metabolites on different culture media; (ii) different MAP strains show uniform VOC patterns; and (iii) cultural diagnosis can be accelerated by taking specific VOC profiles into account. PMID:27604146

  18. Occurrence of Mycobacterium avium subsp. paratuberculosis in milk at dairy cattle farms: a systematic review and meta-analysis.

    PubMed

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

    2012-06-15

    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 were eventually included in the meta-analysis. The apparent prevalence (AP) of MAP in BTM and IM on farm were summarized in relation to strata defined by the test used to identify MAP and the infection status of the herds/animals. There was considerable inconsistency in the reporting, resulting in missing information potentially explaining the dispersion in the estimated AP. The overall AP and 95% confidence intervals based on PCR and culture of MAP were summarized to 0.10 (0.04-0.22) in BTM and 0.20 (0.12-0.32) in IM. Quantifying the MAP load in test-positive milk samples was not possible because very few articles provided quantitative information on individual samples. PMID:22245166

  19. The effects of progressing and nonprogressing Mycobacterium avium ssp. paratuberculosis infection on milk production in dairy cows.

    PubMed

    Smith, Rebecca L; Gröhn, Y T; Pradhan, A K; Whitlock, R H; Van Kessel, J S; Smith, J M; Wolfgang, D R; Schukken, Y H

    2016-02-01

    Longitudinal data from 3 commercial dairy herds in the northeast United States, collected from 2004 to 2011, were analyzed to determine the effect of Mycobacterium avium ssp. paratuberculosis (MAP) infection status and progression path on milk production. Disease status, as indicated by MAP test results, was determined through quarterly ELISA serum testing, biannual fecal culture, and culture of tissues and feces at slaughter. Milk production data were collected from the Dairy Herd Information Association. Animals with positive MAP test results were categorized, based on test results over the full course of the study, as high path (at least one high-positive culture) or low path (at least one positive culture or ELISA). The cumulative numbers of positive ELISA and culture results were recorded. The effects of both MAP infection path, status, and number of positive tests on milk production were analyzed using a mixed linear model with an autocorrelation random effect structure. Low- and high-path animals produced more milk before their first positive test than always-negative animals, especially high-path animals. Although mean production decreased after a first positive test, low-path animals were shown to recover some productivity. High-path animals continued to exhibit a decrease in milk production, especially after their first high-positive fecal culture. These results show that not all animals that test positive for MAP will have long-term production losses. Milk production decreased significantly with each additional positive test. Ultimately, production loss appeared to be a function of MAP infection progression. PMID:26686721

  20. Prevalence and Association of Mycobacterium avium subspecies paratuberculosis with Disease Course in Patients with Ulcero-Constrictive Ileocolonic Disease

    PubMed Central

    Khan, Imteyaz Ahmad; Pilli, Sucharita; A, Surendranath; Rampal, Ritika; Chauhan, Sudhir Kumar; Tiwari, Veena; Mouli, Venigalla Pratap; Kedia, Saurabh; Nayak, Baibaswata; Das, Prasenjit; Makharia, Govind K.; Ahuja, Vineet

    2016-01-01

    Background Association of Mycobacterium avium subspecies paratuberculosis (MAP) and Crohn’s disease (CD) has been controversial due to contradictory reports. Therefore, we determined the prevalence of MAP in patients with CD and intestinal tuberculosis (ITB) and its association with clinical course. Methodology Blood and intestinal biopsies were taken from 69 CD, 32 ITB patients and 41 patients with haemorrhoidal bleed who served as controls. qPCR targeting of MAP-specific IS900 gene was used to detect the presence of MAP DNA. qPCR results were further validated by sequencing. Immunohistochemistry (IHC) was used to detect the presence of MAP antigen in biopsy specimens. CD and ITB patients were followed-up for disease course and response to therapy. Principal Findings The frequency of MAP-specific DNA in biopsies by qPCR was significantly higher in CD patients (23.2%, p = 0.03) as compared to controls (7.3%). No significant difference in intestinal MAP presence was observed between ITB patients (12.5%, p = 0.6) and controls (7.3%). MAP presence in blood of CD patients was 10.1% as compared to 4.9% in controls while no patients with ITB were found to be positive (p = 0.1). Using IHC for detection of MAP antigen, the prevalence of MAP in CD was 2.9%, 12.5% in ITB patients and 2.4% in controls. However, long-term follow-up of the patients revealed no significant associations between clinical characteristics and treatment outcomes with MAP positivity. Conclusion We report significantly high prevalence of MAP in intestinal biopsies of CD patients. However, the presence of MAP does not affect the disease course and treatment outcomes in either CD or ITB patients. PMID:27019109

  1. Associations between Mycobacterium avium subsp. paratuberculosis antibodies in bulk tank milk, season of sampling and protocols for managing infected cows

    PubMed Central

    2013-01-01

    Background The objective of this study was to identify associations between the concentration of Mycobacterium avium subsp. paratuberculosis (MAP) antibodies in bulk milk and potential risk factors in herd management and herd characteristics, explaining high MAP antibody titers in milk. An extensive questionnaire was administered to 292 organic and conventional dairy farms from New York, Wisconsin and Oregon. Bulk milk samples were taken from each farm for MAP enzyme-linked immunosorbent assay (ELISA). A general linear model was constructed with MAP ELISA value as the outcome variable and the management factors and herd characteristics as independent variables, while at the same time controlling for the study design variables of state, herd size, and production system (organic or conventional). High bulk tank MAP ELISA value may be due to either a high prevalence of MAP in a herd with many cows contributing to the antibody titer or due to a few infected cows that produce large quantities of antibodies. Results Results of the regression models indicated that bulk milk ELISA value was associated with season of sampling and the presence or absence of protocols for managing MAP-positive cows. The concentration of MAP antibodies in bulk milk varied seasonally with a peak in the summer and low concentrations in the winter months. When compared to farms that had never observed clinical Johne’s disease, keeping MAP-positive cows or only culling them after a period of delay was associated with an increase in optical density. Conclusions The seasonal variation in MAP antibody titers, with a peak in the summer, may be due to a seasonal increase in MAP-bacterial load. Additionally, seasonal calving practices may contribute to seasonal fluctuations in MAP antibody titers in bulk tank milk. Keeping MAP-positive cows increases the antibody titer in bulk milk, likely due to direct antibody production in the infected cow and indirect triggering of antibody production in herdmates

  2. Systems Biology Analysis of Gene Expression during In Vivo Mycobacterium avium paratuberculosis Enteric Colonization Reveals Role for Immune Tolerance

    PubMed Central

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

    2012-01-01

    Survival and persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in the intestinal mucosa is associated with host immune tolerance. However, the initial events during MAP interaction with its host that lead to pathogen survival, granulomatous inflammation, and clinical disease progression are poorly defined. We hypothesize that immune tolerance is initiated upon initial contact of MAP with the intestinal Peyer's patch. To test our hypothesis, ligated ileal loops in neonatal calves were infected with MAP. Intestinal tissue RNAs were collected (0.5, 1, 2, 4, 8 and 12 hrs post-infection), processed, and hybridized to bovine gene expression microarrays. By comparing the gene transcription responses of calves infected with the MAP, informative complex patterns of expression were clearly visible. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis, and genes were grouped into the specific pathways and gene ontology categories to create a holistic model. This model revealed three different phases of responses: i) early (30 min and 1 hr post-infection), ii) intermediate (2, 4 and 8 hrs post-infection), and iii) late (12 hrs post-infection). We describe here the data that include expression profiles for perturbed pathways, as well as, mechanistic genes (genes predicted to have regulatory influence) that are associated with immune tolerance. In the Early Phase of MAP infection, multiple pathways were initiated in response to MAP invasion via receptor mediated endocytosis and changes in intestinal permeability. During the Intermediate Phase, perturbed pathways involved the inflammatory responses, cytokine-cytokine receptor interaction, and cell-cell signaling. During the Late Phase of infection, gene responses associated with immune tolerance were initiated at the level of T-cell signaling. Our study provides evidence that MAP infection resulted in differentially regulated genes, perturbed pathways

  3. Cow-level association between serum 25-hydroxyvitamin D concentration and Mycobacterium avium subspecies paratuberculosis antibody seropositivity: a pilot study.

    PubMed

    Sorge, U S; Molitor, T; Linn, J; Gallaher, D; Wells, S W

    2013-02-01

    Vitamin D deficiency has been associated with various human diseases. Therefore, the objective of this study was to evaluate the cow-level association between serum 25-hydroxyvitamin D [25(OH)D] concentration and Mycobacterium avium ssp. paratuberculosis (MAP) seropositivity of dairy cows, adjusting for diet, breed, hair coat color, stage of lactation, reproductive status, and cow age. The sera of 80 MAP antibody ELISA-positive and 80 test-negative herd mates from 5 Minnesota dairy herds were analyzed for 25(OH)D and 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. The cows' age, production records, and hair coat color were recorded. Additionally, feed samples were obtained and analyzed for vitamin D(2) and vitamin D(3) content. A linear mixed model was used to identify potential predictors for serum 25(OH)D concentration, accounting for herd of origin. The majority of rations analyzed had over 22,000 IU of vitamin D/day (maximum: 52,000 I U/d) and the study cows' average serum 25(OH)D concentration was 62.5 ± 13.8 ng/mL. Serum ELISA-positive cows had, on average, 5.3 ng/mL lower 25(OH)D serum levels than test-negative herd mates. The reproductive status of cows was also associated with the 25(OH)D levels, with fresh cows having the lowest serum concentration. In this cross-sectional study, a temporal or causal association between MAP antibody ELISA status and serum 25(OH)D concentration could not be evaluated. In addition, the high levels of vitamin D in the rations of participating farms and the average 25(OH)D serum concentration suggest that additional supplementation with vitamin D in the ration is likely to be ineffective. PMID:23261386

  4. Persistence of Mycobacterium avium subsp. paratuberculosis and Other Zoonotic Pathogens during Simulated Composting, Manure Packing, and Liquid Storage of Dairy Manure

    PubMed Central

    Grewal, Sukhbir K.; Rajeev, Sreekumari; Sreevatsan, Srinand; Michel, Frederick C.

    2006-01-01

    Livestock manures contain numerous microorganisms which can infect humans and/or animals, such as Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., and Mycobacterium avium subsp. paratuberculosis (Mycobacterium paratuberculosis). The effects of commonly used manure treatments on the persistence of these pathogens have rarely been compared. The objective of this study was to compare the persistence of artificially inoculated M. paratuberculosis, as well as other naturally occurring pathogens, during the treatment of dairy manure under conditions that simulate three commonly used manure management methods: thermophilic composting at 55°C, manure packing at 25°C (or low-temperature composting), and liquid lagoon storage. Straw and sawdust amendments used for composting and packing were also compared. Manure was obtained from a large Ohio free-stall dairy herd and was inoculated with M. paratuberculosis at 106 CFU/g in the final mixes. For compost and pack treatments, this manure was amended with sawdust or straw to provide an optimal moisture content (60%) for composting for 56 days. To simulate liquid storage, water was added to the manure (to simulate liquid flushing and storage) and the slurry was placed in triplicate covered 4-liter Erlenmeyer flasks, incubated under ambient conditions for 175 days. The treatments were sampled on days 0, 3, 7, 14, 28, and 56 for the detection of pathogens. The persistence of M. paratuberculosis was also assessed by a PCR hybridization assay. After 56 days of composting, from 45 to 60% of the carbon in the compost treatments was converted to CO2, while no significant change in carbon content was observed in the liquid slurry. Escherichia coli, Salmonella, and Listeria were all detected in the manure and all of the treatments on day 0. After 3 days of composting at 55°C, none of these organisms were detectable. In liquid manure and pack treatments, some of these microorganisms were detectable up to 28 days. M

  5. DETECTION AND CHARACTERIZATION OF MYCOBACTERIUM PARATUBERCULOSIS IN ENVIRONMENTAL SAMPLES

    EPA Science Inventory

    Mycobacterium paratuberculosis, a member of the M. avium complex (MAC), is the causative agent for Johne's disease in cattle. Johne's disease is a slow, progressive infection of the intestine in cattle. M. paratuberculosis infection often results in diarrhea and wasting of cattle...

  6. Rapid and sensitive detection of Mycobacterium avium subsp. paratuberculosis in bovine milk and feces by a combination of immunomagnetic bead separation-conventional PCR and real-time PCR.

    PubMed

    Khare, Sangeeta; Ficht, Thomas A; Santos, Renato L; Romano, Juan; Ficht, Allison R; Zhang, Shuping; Grant, Irene R; Libal, Melissa; Hunter, David; Adams, L Garry

    2004-03-01

    Immunomagnetic bead separation coupled with bead beating and real-time PCR was found to be a very effective procedure for the isolation, separation, and detection of Mycobacterium avium subsp. paratuberculosis from milk and/or fecal samples from cattle and American bison. Samples were spiked with M. avium subsp. paratuberculosis organisms, which bound to immunomagnetic beads and were subsequently lysed by bead beating; then protein and cellular contaminants were removed by phenol-chloroform-isopropanol extraction prior to DNA precipitation. DNA purified by this sequence of procedures was then analyzed by conventional and real-time IS900-based PCR in order to detect M. avium subsp. paratuberculosis in feces and milk. By use of this simple and rapid technique, 10 or fewer M. avium subsp. paratuberculosis organisms were consistently detected in milk (2-ml) and fecal (200-mg) samples, making this sensitive procedure very useful and cost-effective for the diagnosis of clinical and subclinical Johne's disease (paratuberculosis) compared to bacteriological culture, which is constrained by time, labor, and expense under diagnostic laboratory conditions. PMID:15004056

  7. Rapid and Sensitive Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Milk and Feces by a Combination of Immunomagnetic Bead Separation-Conventional PCR and Real-Time PCR

    PubMed Central

    Khare, Sangeeta; Ficht, Thomas A.; Santos, Renato L.; Romano, Juan; Ficht, Allison R.; Zhang, Shuping; Grant, Irene R.; Libal, Melissa; Hunter, David; Adams, L. Garry

    2004-01-01

    Immunomagnetic bead separation coupled with bead beating and real-time PCR was found to be a very effective procedure for the isolation, separation, and detection of Mycobacterium avium subsp. paratuberculosis from milk and/or fecal samples from cattle and American bison. Samples were spiked with M. avium subsp. paratuberculosis organisms, which bound to immunomagnetic beads and were subsequently lysed by bead beating; then protein and cellular contaminants were removed by phenol-chloroform-isopropanol extraction prior to DNA precipitation. DNA purified by this sequence of procedures was then analyzed by conventional and real-time IS900-based PCR in order to detect M. avium subsp. paratuberculosis in feces and milk. By use of this simple and rapid technique, 10 or fewer M. avium subsp. paratuberculosis organisms were consistently detected in milk (2-ml) and fecal (200-mg) samples, making this sensitive procedure very useful and cost-effective for the diagnosis of clinical and subclinical Johne's disease (paratuberculosis) compared to bacteriological culture, which is constrained by time, labor, and expense under diagnostic laboratory conditions. PMID:15004056

  8. Evaluation of a Salmonella Vectored Vaccine Expressing Mycobacterium avium Subsp. paratuberculosis Antigens Against Challenge in a Goat Model

    PubMed Central

    Chen, Tsai-Tzu; Useh, Nicodemus M.; Guo, Shanguang; Yan, Weiwei; Wang, Shih-Jon; Glaser, Amy L.; McDonough, Sean P.; Singh, Bhupinder; Chang, Yung-Fu

    2013-01-01

    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. PMID:23950909

  9. Marked Differences in Mucosal Immune Responses Induced in Ileal versus Jejunal Peyer’s Patches to Mycobacterium avium subsp. paratuberculosis Secreted Proteins following Targeted Enteric Infection in Young Calves

    PubMed Central

    Facciuolo, Antonio; Gonzalez-Cano, Patricia; Napper, Scott; Griebel, Philip J.

    2016-01-01

    In cattle, Mycobacterium avium subsp. paratuberculosis infection is primarily mediated through M cells overlying Peyer’s patches (PP) in the ileum. The capacity of M. avium subsp. paratuberculosis to invade ileal PP (IPP) versus discrete PP in the jejunum (JPP) and subsequent differences in mucosal immune responses were investigated. Intestinal segments were surgically prepared in both mid-jejunum, containing two JPPs, and in terminal small intestine containing continuous IPP. M. avium subsp. paratuberculosis (109 CFU) was injected into the lumen of half of each intestinal segment when calves were 10–14 days-old and infection confirmed 1–2 months later by PCR and immunohistochemistry. Thirteen recombinant M. avium subsp. paratuberculosis proteins, previously identified as immunogenic, were used to analyze pathogen-specific B- and T-cell responses in PP and mesenteric lymph nodes. IgA plasma cell responses to 9 of 13 recombinant proteins were detected in JPP but not in IPP. Secretory IgA reacting in ELISA with 9 of the 13 recombinant proteins was detected in luminal contents from both jejunal and ileal segments. These observations support the conclusion that pathogen-specific IgA B cells were induced in JPP but not IPP early after a primary infection. The presence of secretory IgA in intestinal contents is consistent with dissemination of IgA plasma cells from the identified mucosa-associated immune induction sites. This is the first direct evidence for M. avium subsp. paratuberculosis uptake by bovine JPP and for local induction of pathogen-specific IgA plasma cell responses after enteric infection. We also provide evidence that bacterial invasion of IPP, a primary B lymphoid tissue, provides a novel strategy to evade induction of mucosal immune responses. Over 60% of PPs in the newborn calf small intestine is primary lymphoid tissue, which has significant implications when designing oral vaccines or diagnostic tests to detect early M. avium subsp

  10. Studies on pathology, cytokine gene expression and molecular typing of Mycobacterium avium subsp. paratuberculosis of naturally occurring Johne's disease in bullocks.

    PubMed

    Narnaware, S D; Periasamy, S; Tripathi, B N

    2016-06-01

    Pathology of Johne's disease (JD) in bullocks (castrated, adult male cattle) is rarely studied. Here, we report the pathology and cytokine gene expression of naturally occurring JD in bullocks. The small intestine and mesenteric lymph nodes collected from 404 bullocks, aged between 5 and 10years, were examined for JD lesions and detection of Mycobacterium avium subsp. paratuberculosis (Map). A total of 8.7% bullocks exhibited JD lesions, which were classified into multibacillary-diffuse granulomatous (n=2), paucibacillary-focal granulomatous (n=18) and paucibacillary-diffuse lymphocytic (n=15) lesions. The tissue cytokine gene expression profiles in all three forms of lesions corroborated with different immuno-pathological processes of JD in bullocks. The molecular typing and gene sequencing identified Map isolates from bullocks as bison type. PMID:27234539

  11. Lack of association between the occurrence of Crohn's disease and occupational exposure to dairy and beef cattle herds infected with Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Qual, D A; Kaneene, J B; Varty, T J; Miller, R; Thoen, C O

    2010-06-01

    A cross-sectional survey was conducted to identify associations between Crohn's disease (CD) and Mycobacterium avium ssp. paratuberculosis (Map) exposure. A questionnaire was used to collect information on exposure to cattle infected with Map, and personal and family history of CD in dairy and beef cattle producers with and without Map-infected herds, and in veterinarians who did or did not have contact with Map-infected herds. Cases of CD were selected from respondents and matched 1:4 with controls on occupation, age, and sex. Multivariable conditional logistic regression was used to assess associations between Map exposure and CD. There were 3 cases of CD in 702 producers and 4 cases in 774 veterinarians, yielding a prevalence of 0.47%. No association was found between exposure to JD and CD in any phase of the analysis. However, the number of cases of CD is not large and limits the power to detect important differences. PMID:20494145

  12. Validation and standardization of IS900 and F57 real-time quantitative PCR assays for the specific detection and quantification of Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Sidoti, Francesca; Banche, Giuliana; Astegiano, Sara; Allizond, Valeria; Cuffini, Anna Maria; Bergallo, Massimiliano

    2011-05-01

    Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease and may contribute to the onset and development of Crohn's disease in humans. Rapid detection of Map is fundamental because of its reported isolation from pasteurized milk and its potential for transmission through environmental sources. In this study, we developed two independent real-time quantitative PCR assays targeting the IS900 genetic insertion sequence and the F57 sequence, which proved capable of detecting and quantifying Map DNA. Validation and standardization of the developed methods were performed by evaluating diagnostic trueness, precision, and accuracy of the techniques. Specificity of the IS900 and F57 methods was verified in both in silico and experimental studies. The assays were found to be very accurate and precise with high repeatability and reproducibility. Moreover, the two real-time assays were very specific for Map, discriminating most of mycobacterial and nonmycobacterial species. PMID:21510779

  13. Longitudinal study of the distribution of Mycobacterium avium subsp. paratuberculosis in the environment of dairy herds in the Michigan Johne’s disease control demonstration herd project

    PubMed Central

    Pillars, Roxanne B.; Grooms, Daniel L.; Kaneene, John B.

    2009-01-01

    The objective of this study was to describe the distribution of Mycobacterium avium subsp. paratuberculosis (MAP) in the environment of infected dairy farms over time. Johne’s disease (JD) prevalence was monitored annually in 7 Michigan dairy herds. Environmental samples were collected bi-annually and cultured for MAP. Of 731 environmental samples that were cultured, 81 (11%) were positive. The lactating cow floor and manure storage areas were the areas most commonly contaminated, representing 30% and 33% of positive samples, respectively. When herd prevalence was > 2%, MAP was cultured from the lactating cow floor and/or manure storage area 75% of the time. When herd prevalence was ≤ 2%, MAP was never cultured from samples collected. For every 1 unit increase in number of positive environmental samples, within herd JD prevalence increased 1.62%. Environmental contamination with MAP is consistent over time on infected dairy farms, and management practices to reduce environmental contamination are warranted. PMID:20046602

  14. Genotype diversity in Indian isolates of Mycobacterium avium subspecies paratuberculosis recovered from domestic and wild ruminants from different agro-climatic regions.

    PubMed

    Singh, A V; Singh, S V; Singh, P K; Sohal, J S

    2010-12-01

    Fecal samples of 708 domestic and 27 wild ruminants from seven districts of three states submitted to Microbiology Laboratory from 2004 to 2008 were screened for the presence and genotype diversity of Mycobacterium avium subspecies paratuberculosis (MAP) stusing modified microscopic examination and culture tests. MAP colonies were characterized and genotyped by IS900 PCR and IS1311 PCR-REA, respectively. In microscopic examination and culture, 18.7 and 31.2% sampled were positive, respectively. Percent positivity varied in different species and agro-climatic regions. Of the 151 DNA recovered from 230 MAP isolates, 94.7% (143) and 5.2% (8) were genotyped as 'Indian Bison type' and 'Cattle type', respectively. Cattle and buffaloes were infected with both the genotypes, whereas goat and sheep were infected exclusively by 'Indian Bison type'. Study showed low diversity in MAP genotypes and 'Indian Bison type' was the predominant genotype shared by different species, breeds and agro-climatic regions. PMID:20832117

  15. Evaluation of 'cattle' and 'Indian Bison' type antigens of Mycobacterium avium subspecies paratuberculosis for diagnosis of bovine Johne's disease using 'indigenous ELISA' and AGPT.

    PubMed

    Pahangchopi, Donna; Singh, Ran Vir; Singh, Shoor Vir; Das, Paritosh; Sharma, Deepak; Sardana, Tarun; Kumar, Naveen; Chaubey, Kundan Kumar; Gupta, Saurabh

    2014-12-01

    Two antigens ('cattle' type and 'Indian Bison' type) of Mycobacterium avium subspecies paratuberculosis were evaluated for diagnosis of Johne's disease (JD) in a gaushala (cattle herd). Of the 160 cows of Sahiwal and Hariana breeds screened, 81 (50.6%) tested positive in ELISA and 66 (41.8%) in AGPT test. Using the two antigens, 33.5% tested positive in both the tests while 41.1% tested negative. Exclusively, only 8.2% tested positive in ELISA while 17.1% tested positive in AGPT. Two antigens together detected 58.9% prevalence of MAP in the gaushala. Individually, indigenous ELISA using antigen from native source of MAP proved superior to AGPT in the diagnosis of JD in cows. PMID:25651611

  16. Clinical disease and stage of lactation influence shedding of Mycobacterium avium subspecies paratuberculosis into milk and colostrum of naturally infected dairy cows.

    PubMed

    Stabel, J R; Bradner, L; Robbe-Austerman, S; Beitz, D C

    2014-10-01

    Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD). One mode of transmission of MAP is through ingestion of contaminated milk and colostrum by susceptible calves. The objective of this study was to determine if the amount of MAP shed into the milk and colostrum of infected cows was affected by severity of infection as well as the number of days in milk (DIM). Milk was collected over the 305-d lactation period from naturally infected cows in the asymptomatic subclinical (n=39) and symptomatic clinical (n=29) stages of disease, as well as 8 noninfected control cows. All milk samples were assayed for MAP by culture on Herrold's egg yolk medium and either BACTEC 12B (Becton Dickinson, Franklin Lakes, NJ) or para-JEM (Thermo Fisher Scientific, Trek Diagnostic Systems Inc., Cleveland, OH) liquid medium, and by direct PCR for the IS900 target gene. Mycobacterium avium ssp. paratuberculosis was detected in 3.8, 4.1, and 12.6% of milk samples collected from cows with subclinical JD after culture in Herrold's egg yolk medium, liquid medium, and direct PCR, respectively. The frequency of MAP positivity increased to 12.9, 18.4, and 49.2% of milk samples collected from cows with clinical JD by these same methods, respectively. None of the milk samples collected from control cows was positive for MAP by any detection method. Viable MAP was primarily isolated from milk and colostrum of subclinically and clinically infected cows collected in early lactation (DIM 0-60), with negligible positive samples observed in mid (DIM 60-240) and late (DIM 240-305) lactation. This study demonstrates that shedding of MAP into milk is affected by infection status of the cow as well as stage of lactation, providing useful information to producers to help break the cycle of infection within a herd. PMID:25064655

  17. 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.

    PubMed

    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. PMID:26805975

  18. Identification of Mycobacterium avium subsp. hominissuis Isolated From Drinking Water

    EPA Science Inventory

    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...

  19. Susceptibility of Mycobacterium avium sbsp paratuberculosis to monensin sodium or tilmicosin phosphate in vitro and resulting infectivity in a murine model.

    PubMed

    Brumbaugh, Gordon W; Simpson, R Bruce; Edwards, John F; Anders, Dwayne R; Thomson, T D

    2004-07-01

    This study was designed to determine the susceptibility in vitro and infectivity of 1 field isolate of Mycobacterium avium sbsp paratuberculosis after exposure to monensin sodium and tilmicosin phosphate. Minimum inhibitory concentrations (0.39 microg monensin sodium/mL; 1.60 microg tilmicosin phosphate/mL) were determined in quintuplicate. Organisms were then incubated with 3 different concentrations of each medication for 3 different lengths of time, then washed and resuspended in sterile physiologic saline and injected intraperitoneally into mice that were genetically susceptible to infection. Mice were euthanatized 50 d later and the number of hepatic granulomas was used as the indicator of infectivity. Neither time of incubation nor concentration of medication had any effect on the infectivity of the organisms. Monensin sodium significantly reduced the number of hepatic granulomas in genetically susceptible mice while tilmicosin phosphate did not. Antimycobacterial activity of monensin sodium suggests that the role of monensin in the control of bovine paratuberculosis should be evaluated further. PMID:15352541

  20. Susceptibility of Mycobacterium avium sbsp paratuberculosis to monensin sodium or tilmicosin phosphate in vitro and resulting infectivity in a murine model

    PubMed Central

    2004-01-01

    Abstract This study was designed to determine the susceptibility in vitro and infectivity of 1 field isolate of Mycobacterium avium sbsp paratuberculosis after exposure to monensin sodium and tilmicosin phosphate. Minimum inhibitory concentrations (0.39 μg monensin sodium/mL; 1.60 μg tilmicosin phosphate/mL) were determined in quintuplicate. Organisms were then incubated with 3 different concentrations of each medication for 3 different lengths of time, then washed and resuspended in sterile physiologic saline and injected intraperitoneally into mice that were genetically susceptible to infection. Mice were euthanatized 50 d later and the number of hepatic granulomas was used as the indicator of infectivity. Neither time of incubation nor concentration of medication had any effect on the infectivity of the organisms. Monensin sodium significantly reduced the number of hepatic granulomas in genetically susceptible mice while tilmicosin phosphate did not. Antimycobacterial activity of monensin sodium suggests that the role of monensin in the control of bovine paratuberculosis should be evaluated further. PMID:15352541

  1. Characterization of the Mycobacterium avium subsp. paratuberculosis laminin-binding/histone-like protein (Lbp/Hlp) which reacts with sera from patients with Crohn's disease.

    PubMed

    Lefrançois, Louise H; Pujol, Céline; Bodier, Christelle C; Teixeira-Gomez, Ana Paula; Drobecq, Hervé; Rosso, Marie-Laure; Raze, Dominique; Dias, André Alves; Hugot, Jean-Pierre; Chacon, Ofelia; Barletta, Raul G; Locht, Camille; Vidal Pessolani, Maria Cristina; Biet, Franck

    2011-06-01

    Mycobacterium avium subsp. paratuberculosis (Map) causes a chronic enteric disease in ruminants, called paratuberculosis or Johne's disease. The current model proposes that after ingestion by the host, Map crosses the intestinal barrier via internalization by the M cells. Experimental observations suggest, however, that Map may also transcytose the intestinal wall via the enterocytes, but the mechanisms involved in this process remain poorly understood. Cytoadherence assays performed on epithelial cells with Map revealed that the addition of laminin to the cell culture increases adhesion. A Map protein was isolated by heparin-Sepharose chromatography and identified as a laminin-binding protein like. The gene encoding this protein named Lbp/Hlp was identified in the Map genome sequence at locus MAP3024 (annotated Hup B). The deduced Map Lbp/Hlp amino acid sequence reveals 80% identity with that reported for other mycobacteria. The C-terminal domain involved in adhesion is mainly composed of arginine and lysine residues modified by methylation. In vitro tests demonstrated that recombinant Lbp/Hlp binds laminin, heparin, collagen and epithelial cells. Interestingly, we found that this adhesin corresponds to the antigen described as the target of pANCA and serum antibodies of patients with Crohn's disease. PMID:21334452

  2. Detection of Mycobacterium avium subsp. paratuberculosis in faeces using different procedures of pre-treatment for real-time PCR in comparison to culture.

    PubMed

    Sting, R; Hrubenja, M; Mandl, J; Seemann, G; Salditt, A; Waibel, S

    2014-01-01

    One of the most relevant aspects in the diagnosis of paratuberculosis (Johne's disease) in cattle is the availability of a method for the rapid and sensitive detection of Mycobacterium avium subsp. paratuberculosis (MAP) in order to facilitate the prompt removal of pathogen-shedding animals from a herd. To meet this requirement, methods for pre-treatment of bovine faecal samples and subsequent extraction of DNA for detection of MAP by real-time PCR were compared with MAP culture results. A total of 116 bovine faecal samples that showed weak (64.7%), moderate (18.1%) or strong (17.2%) growth of MAP on solid HEY medium were investigated. For PCR, supernatants, sediments or bacterial pellets were obtained from faecal samples by pre-treatment before extraction of MAP DNA based on silica membranes or magnetic particles. Samples then were tested by MAP IS900 and ISMav2 real-time PCR with an analytical sensitivity of 6 and 28 genome equivalents (GE) per mL, respectively. The best results were obtained by including a microfiltration step in the sample pre-treatment in combination with silica membrane-based mini-columns or magnetic particles for DNA extraction. This approach enhanced the detection rate of MAP in IS900 real-time PCR from 58.6% to 84.5% using silica membrane mini-columns and from 61.2% to 64.7% using magnetic particles. PMID:24280588

  3. Enhanced Expression of Interleukin-1α and Tumor Necrosis Factor Receptor-Associated Protein 1 in Ileal Tissues of Cattle Infected with Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Aho, Abraham D.; McNulty, Amanda M.; Coussens, Paul M.

    2003-01-01

    Infection with Mycobacterium avium subsp. paratuberculosis is associated with high levels of morbidity, decreased production, and early culling in dairy cattle. Clinical symptoms of Johne's disease include persistent diarrhea, inappetence, and resultant weight loss due to chronic inflammation of the small intestine. Although the presence or absence of intestinal lesions cannot be used as a definitive indicator of M. avium subsp. paratuberculosis infection, most infected cattle exhibit significant changes to intestinal mucosa, with the focus of pathology surrounding the ileal cecal junction. Typical pathology of M. avium subsp. paratuberculosis infection includes inflammation, thickening of the lumenal wall, and hyperplasia in draining lymph nodes. To further understand the pathology of Johne's disease, we compared the gene expression profiles of ileal tissues from Johne's disease-positive (n = 6), and Johne's disease-negative (n = 5) Holstein cattle. Gene expression profiles were compared with a bovine total leukocyte (BOTL-3) cDNA microarray. Genes that were expressed at significantly higher levels (>1.5-fold; P < 0.05) in tissues from Johne's disease-infected animals relative to noninfected animals included those encoding tumor necrosis factor receptor-associated protein 1 (TRAF1), interleukin-1α (IL-1α), MCP-2, N-cadherin, and β1 integrin (CD29). Dramatic upregulation of IL-1α (21.5-fold) and TRAF1 (27.5-fold) gene expression in tissues of Johne's disease-positive cows relative to tissues from control cows was confirmed by quantitative real-time PCR. Western blot analysis confirmed that IL-1α and TRAF1 mRNA levels resulted in increased protein expression in tissues of Johne's disease-positive cattle relative to tissues from control cattle. High levels of IL-1α can produce symptoms similar to those found in clinical Johne's disease. Taken together, the data presented in this report suggest that many outward symptoms of Johne's disease may be due to IL-1

  4. Isolation and detection of Mycobacterium avium subsp. paratuberculosis (MAP) from cattle in Ireland using both traditional culture and molecular based methods

    PubMed Central

    2010-01-01

    Background Mycobacterium avium subsp. paratuberculosis (MAP) causes a chronic gastroenteritis affecting many species. Johne's disease is one of the most widespread and economically important disease of ruminants. Since 1992 and the opening of the European market, the exposure and the transmission of MAP in cattle herds considerably increased. Improvements in diagnostic strategies for Ireland and elsewhere are urgently required. In total, 290 cattle from seven Irish herds with either a history or a strong likelihood of paratuberculosis infection were selected by a veterinary team over 2 years. Faecal samples (290) were collected and screened for MAP by a conventional culture method and two PCR assays. In order to further evaluate the usefulness of molecular testing, a nested PCR was also assessed. Results M. paratuberculosis was isolated and cultured from 23 faecal samples (7.9%) on solid medium. From a molecular perspective, 105 faecal samples (36%) were PCR positive for MAP specific DNA. A complete correlation (100%) was observed between the results of both molecular targets (IS900 and ISMAP02). Sensitivity was increased by ~10% with the inclusion of a nested PCR for ISMAP02 (29 further samples were positive). When culturing and PCR were retrospectively compared, every culture positive faecal sample also yielded a PCR positive result for both targets. Alternatively, however not every PCR positive sample (n = 105, 36%) produced a corresponding culture isolate. Interestingly though when analysed collectively at the herd level, the correlation between culture and PCR results was 100% (ie every herd which recorded at least 1 early PCR +ve result later yielded culture positive samples within that herd). Conclusion PCR on bovine faecal samples is a fast reliable test and should be applied routinely when screening for MAP within herds suspected of paratuberculosis. Nested PCR increases the threshold limit of detection for MAP DNA by approximately 10% but proved to be

  5. Milk yield and lactation stage are associated with positive results to ELISA for Mycobacterium avium subsp. paratuberculosis in dairy cows from Northern Antioquia, Colombia: a preliminary study.

    PubMed

    Correa-Valencia, Nathalia María; Ramírez, Nicolás Fernando; Olivera, Martha; Fernández-Silva, Jorge Arturo

    2016-08-01

    Paratuberculosis is a slow-developing infectious disease characterized by chronic granulomatous enterocolitis. This disease has a variable incubation period from 6 months to over 15 years and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Some studies have been conducted in cattle during the last decades in Colombia. However, those studies were designed using relatively small populations and were not aimed to establish prevalence. This study aimed to determine the MAP seroprevalence in selected dairy herds and to explore risk factors associated with the serology results. Serum samples and related data were collected from 696 randomly selected bovines in 28 dairy herds located in 12 different districts in one of the main dairy municipalities in Colombia (San Pedro de los Milagros). The samples were analyzed using a commercial ELISA kit. The information on risk factors was analyzed using a logistic regression. The apparent seroprevalence was 3.6 % (1/28) at the herd level and 2 % (14/696) at the animal level. The number of days in milk production between 100 and 200 days, and over 200 days as well as the daily milk production between 20 and 40 L/cow, and over 40 L/cow were associated with MAP seropositivity with odds ratios of 4.42, 3.45, 2.53, and 20.38, respectively. This study demonstrates the MAP seroprevalence in dairy herds from Antioquia, Colombia and the possible relationship between MAP seropositivity, milk yield, and lactation stage. PMID:27165342

  6. Cell-Mediated and Humoral Immune Responses after Immunization of Calves with a Recombinant Multiantigenic Mycobacterium avium subsp. paratuberculosis Subunit Vaccine at Different Ages

    PubMed Central

    Thakur, Aneesh; Aagaard, Claus; Stockmarr, Anders; Andersen, Peter

    2013-01-01

    Neonates and juvenile ruminants are very susceptible to paratuberculosis infection. This is likely due to a high degree of exposure from their dams and an immature immune system. To test the influence of age on vaccine-induced responses, a cocktail of recombinant Mycobacterium avium subsp. paratuberculosis proteins (MAP0217, MAP1508, MAP3701c, MAP3783, and MAP1609c/Ag85B) was formulated in a cationic liposome adjuvant (CAF01) and used to vaccinate animals of different ages. Male jersey calves were divided into three groups that were vaccinated at 2, 8, or 16 weeks of age and boosted twice at weeks 4 and 12 relative to the first vaccination. Vaccine-induced immune responses, the gamma interferon (IFN-γ) cytokine secretion and antibody responses, were followed for 20 weeks. In general, the specific responses were significantly elevated in all three vaccination groups after the first booster vaccination with no or only a minor effect from the second booster. However, significant differences were observed in the immunogenicity levels of the different proteins, and it appears that the older age group produced a more consistent IFN-γ response. In contrast, the humoral immune response is seemingly independent of vaccination age as we found no difference in the IgG1 responses when we compared the three vaccination groups. Combined, our results suggest that an appropriate age of vaccination should be considered in vaccination protocols and that there is a possible interference of vaccine-induced immune responses with weaning (week 8). PMID:23389934

  7. Estimation of Mycobacterium avium subsp. paratuberculosis load in raw bulk tank milk in Emilia-Romagna Region (Italy) by qPCR.

    PubMed

    Ricchi, Matteo; Savi, Roberto; Bolzoni, Luca; Pongolini, Stefano; Grant, Irene R; De Cicco, Caterina; Cerutti, Giulia; Cammi, Giuliana; Garbarino, Chiara A; Arrigoni, Norma

    2016-08-01

    Consumption of milk and dairy products is considered one of the main routes of human exposure to Mycobacterium avium subsp. paratuberculosis (MAP). Quantitative data on MAP load in raw cows' milk are essential starting point for exposure assessment. Our study provides this information on a regional scale, estimating the load of MAP in bulk tank milk (BTM) produced in Emilia-Romagna region (Italy). The survey was carried out on 2934 BTM samples (88.6% of the farms herein present) using two different target sequences for qPCR (f57 and IS900). Data about the performances of both qPCRs are also reported, highlighting the superior sensitivity of IS900-qPCR. Seven hundred and eighty-nine samples tested MAP-positive (apparent prevalence 26.9%) by IS900 qPCR. However, only 90 of these samples were quantifiable by qPCR. The quantifiable samples contained a median load of 32.4 MAP cells mL(-1) (and maximum load of 1424 MAP cells mL(-1) ). This study has shown that a small proportion (3.1%) of BTM samples from Emilia-Romagna region contained MAP in excess of the limit of detection (1.5 × 10(1) MAP cells mL(-1) ), indicating low potential exposure for consumers if the milk subsequently undergoes pasteurization or if it is destined to typical hard cheese production. PMID:26991108

  8. Molecular epidemiology of Mycobacterium avium subspecies paratuberculosis: IS900 PCR identification and IS1311 polymorphism analysis from ruminants in the Punjab region of India.

    PubMed

    Kaur, P; Filia, G; Singh, S V; Patil, P K; Ravi Kumar, G V P P S; Sandhu, K S

    2011-03-01

    Johne's disease is chronic granulomatous infectious enteritis of animals caused by Mycobacterium avium subspecies paratuberculosis. A total of 153 animals from 19 dairy farms, 2 gaushalas (unproductive-animal rehabilitation centers), 2 goat and 2 sheep farms from different districts of the Punjab region were selected on the basis of clinical signs of disease. All samples from cattle (n=86), buffalo (n=34), goat (n=25) and sheep (n=26) were subjected to Ziehl-Neelsen staining and DNA extraction by a freeze and thaw method. Ziehl-Neelsen staining detected 71% samples positive for acid-fast bacilli whereas IS900 PCR detected 55% positive for Map DNA. IS1311 PCR-REA analysis of IS900 positive samples revealed 'Bison' type as the most prevalent (82%) genotype of Map, infecting all domestic ruminants. 'Cattle' type was present in a minority of cases (15%) from cattle, buffaloes and goats. This is the first report of 'Cattle' type Map from buffalo and goat species in India. PMID:21071087

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

    PubMed

    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. PMID:26827838

  10. Evaluation of age-dependent susceptibility in calves infected with two doses of Mycobacterium avium subspecies paratuberculosis using pathology and tissue culture

    PubMed Central

    2013-01-01

    The longstanding assumption that calves of more than 6 months of age are more resistant to Mycobacterium avium subspecies paratuberculosis (MAP) infection has recently been challenged. In order to elucidate this, a challenge experiment was performed to evaluate age- and dose-dependent susceptibility to MAP infection in dairy calves. Fifty-six calves from MAP-negative dams were randomly allocated to 10 MAP challenge groups (5 animals per group) and a negative control group (6 calves). Calves were inoculated orally on 2 consecutive days at 5 ages: 2 weeks and 3, 6, 9 or 12 months. Within each age group 5 calves received either a high – or low – dose of 5 × 109 CFU or 5 × 107 CFU, respectively. All calves were euthanized at 17 months of age. Macroscopic and histological lesions were assessed and bacterial culture was done on numerous tissue samples. Within all 5 age groups, calves were successfully infected with either dose of MAP. Calves inoculated at < 6 months usually had more culture-positive tissue locations and higher histological lesion scores. Furthermore, those infected with a high dose had more severe scores for histologic and macroscopic lesions as well as more culture-positive tissue locations compared to calves infected with a low dose. In conclusion, calves to 1 year of age were susceptible to MAP infection and a high infection dose produced more severe lesions than a low dose. PMID:24099491

  11. Soluble BAFF Level Is Not Correlated to Mycobacterium avium Subspecies Paratuberculosis Antibodies and Increases After Interferon-β Therapy in Multiple Sclerosis Patients.

    PubMed

    Mameli, Giuseppe; Cossu, Davide; Caggiu, Elisa; Arru, Giannina; Niegowska, Magdalena; Cocco, Eleonora; Frau, Jessica; Marrosu, Maria Giovanna; Sechi, Leonardo A

    2016-09-01

    B cells are being recognized as one of the major players in the pathogenesis of multiple sclerosis (MS). The B cell activating factor (BAFF) system plays an essential role in B cell homeostasis and function in the periphery. Mycobacterium avium subspecies paratuberculosis (MAP) has been previously associated to MS in Sardinia. Antibodies against a MAP surface protein, MAP_2694, have been found significantly associated to MS patients, and this response was modified by interferon-β therapy. Increased BAFF levels following IFN-β therapy have been also described in MS patients. In this study, we evaluated whether soluble BAFF levels are comparable in men and women affected by MS and performed a correlation of the reported BAFF increase in MS patients under IFN-β therapy with changes of humoral response against MAP_2694. For these reasons, we investigated 44 MS patients before and after IFN-β therapy. A significant difference of BAFF levels was found between men and women with MS; moreover, we confirmed that IFN-β therapy strongly induces BAFF serum levels, but this was not related to the modification of immunological response against MAP_2694. In conclusion, our study highlights that IFN-β therapy induces the potent B cell survival factor BAFF without alterations of the humoral immune response against MAP. PMID:27370541

  12. Detection and confirmation of Mycobacterium avium subsp. paratuberculosis in direct quantitative PCR positive fecal samples by the manual fluorescent MGIT culture system.

    PubMed

    Kawaji, Satoko; Nagata, Reiko; Mori, Yasuyuki

    2014-01-01

    An efficient protocol for the manual fluorescent MGIT culture system combined with rapid confirmation of Mycobacterium avium subsp. paratuberculosis (MAP) growth in the broth culture was established and evaluated for the detection of viable MAP in direct quantitative PCR (QPCR) positive bovine feces. Manually detected fluorescence emissions from MGIT tubes were analyzed objectively using an open source software, ImageJ. For molecular confirmation of MAP growth, DNA samples harvested by simply boiling the broth, an inexpensive and time- and labor-saving DNA preparation method, yielded adequate results. The sheep strain of MAP required longer incubation time relative to the cattle strain, suggesting that the MGIT system may not support well the growth of ovine isolates as described previously. Of 61 direct QPCR positive bovine feces, the recovery rate of MAP in the MGIT system (62.3%) was significantly higher (P<0.05) than that using 7H10 agar-based slants (44.3%). The time to obtain a final result for fecal culture by the MGIT system was several weeks earlier compared to solid media. In MGIT culture positive samples, the time to detect fluorescence was correlated with the DNA quantity detected in fecal QPCR. As a positive result in the direct fecal QPCR test does not mean fecal excretion of viable MAP, bacterial isolation by fecal culture could be conducted to verify the QPCR result. For this purpose, the manual MGIT system is a sensitive and rapid culture method at least for bovine samples. PMID:24065085

  13. 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.

    PubMed

    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. PMID:24934261

  14. Gene expression profiling of Mycobacterium avium subsp. paratuberculosis in simulated multi-stress conditions and within THP-1 cells reveals a new kind of interactive intramacrophage behaviour

    PubMed Central

    2012-01-01

    Background Recent studies have identified in Mycobacterium avium subsp. paratuberculosis (MAP), already known as a pathogen in ruminants, a potential zoonotic agent of some autoimmune diseases in humans. Therefore, considering the possible risk for public health, it is necessary a thorough understanding of MAP's gene expression during infection of human host as well as the identification of its immunogenic and/or virulence factors for the development of appropriate diagnostic and therapeutic tools. Results In order to characterize MAP's transcriptome during macrophage infection, we analyzed for the first time the whole gene expression of a human derived strain of MAP in simulated intraphagosomal conditions and after intracellular infection of the human macrophage cell line THP-1 by using the DNA-microarray technology. Results showed that MAP shifts its transcriptome to an adaptive metabolism for an anoxic environment and nutrient starvation. It up-regulates several response factors to oxidative stress or intracellular conditions and allows, in terms of transcription, a passive surface peptidoglycan spoliation within the macrophage along with an intensification of the anabolic activity for lipidic membrane structures. Conclusions These results indicate a possible interactive system between MAP and its host cell based on the internal mimicry unlike other intracellular pathogens, bringing new hypothesis in the virulence and pathogenicity of MAP and its importance in human health. PMID:22646160

  15. Development of a novel DNA extraction method for identification and quantification of Mycobacterium avium subsp. paratuberculosis from tissue samples by real-time PCR.

    PubMed

    Park, Kun Taek; Allen, Andrew J; Davis, William C

    2014-04-01

    Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease in ruminants and possibly associated with human Crohn's disease. One impediment in furthering our understanding of this potential association has been the lack of an accurate method for detection of Map in affected tissues. Real time polymerase chain reaction (RT-PCR) methods have been reported to have different sensitivities in detection of Map. This is in part attributable to the difficulties of extracting Map DNA and removing PCR inhibitors from the clinical specimens. The maximum efficiency of RT-PCR can only be achieved by using high quality DNA samples. In this study, we present a novel pre-treatment method which significantly increases Map DNA recovery and decreases PCR inhibitors (p<0.05). When the pre-treatment method was combined with the DNeasy Blood and Tissue kit (Qiagen), PCR inhibition was not detected in any of three different RT-PCR methods tested in this study. The results obtained with the IS900 probe showed an excellent Kappa value (0.849) and a high correlation coefficient r (0.940) compared to the results of culture method. When used to examine unknown field samples (n=15), more positive tissues were identified with DNA extracts prepared with pre-treatment method than without (5 vs 3). This improved Map DNA extraction method from tissue samples will make RT-PCR a more powerful tool for a wide range of applications for Map identification and quantification. PMID:24534783

  16. Short communication: Herd-level prevalence of Mycobacterium avium ssp. paratuberculosis is not associated with participation in a voluntary Alberta Johne's disease control program.

    PubMed

    Ritter, C; Wolf, R; Adams, C L; Kelton, D F; Pickel, C; Mason, S; Orsel, K; De Buck, J; Barkema, H W

    2016-03-01

    Johne's disease (JD) control programs for dairy farms have the general objective of reducing both cow- and herd-level prevalence of Mycobacterium avium ssp. paratuberculosis (MAP). An important aspect of many programs is herd testing for MAP to determine the infection status of participating farms. However, it is uncertain whether MAP herd-level prevalence on farms voluntarily participating in a JD control program is different from that on nonparticipating farms. Therefore, the aim was to compare MAP infection status of participants and nonparticipants in the Alberta Johne's Disease Initiative (AJDI), a voluntary JD control program initiated in 2010 in Alberta, Canada. Between September 2012 and August 2013, environmental fecal samples were collected from 93 randomly selected farms not enrolled in the AJDI. Additionally, 81 farms that initially enrolled in the AJDI during the same time interval were also sampled. Samples were collected from 6 defined locations on each farm and cultured for MAP. Results were confirmed using conventional IS900 PCR and F 0285 quantitative PCR. Overall, 51% of participating and 51% of nonparticipating farms were identified as being MAP-infected. Furthermore, based on multivariable logistic regression, the number of MAP-positive samples was not associated with AJDI participation (taking herd size into account as a potentially modifying or confounding variable). In conclusion, there was no indication that voluntary participation in the AJDI was associated with herd-level MAP prevalence. PMID:26778309

  17. Mycobacterium avium Subspecies paratuberculosis and Bovine Leukemia Virus Seroprevalence and Associated Risk Factors in Commercial Dairy and Beef Cattle in Northern and Northeastern China

    PubMed Central

    Sun, Wu-Wen; Lv, Wen-Fa; Cong, Wei; Meng, Qing-Feng; Wang, Chun-Feng; Shan, Xiao-Feng; Qian, Ai-Dong

    2015-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) and bovine leukemia virus (BLV) are important pathogens, commonly responsible for economical loss to cattle farms all over the world, yet their epidemiology in commercial dairy and beef cattle in China is still unknown. Thus, from September 2013 to December 2014, a large-scale seroprevalence study was conducted to determine the seroprevalence and identify herd-level risk factors associated with MAP and BLV infection. The source sample was 3674 cattle from 113 herds in northern and northeastern China. Antibodies against MAP and BLV were detected using ELISA tests. At animal-level, the seroprevalence of antibodies against MAP and BLV was 11.79% (433/3674) and 18.29% (672/3674), respectively. At herd-level, the seroprevalence of antibodies against MAP and BLV was 20.35% and 21.24% (24/113), respectively. Herd size was identified to be associated with MAP infection while herd size and presence of cattle introduced from other farms were significantly associated with BLV infection. Further research is needed to confirm these findings and improve the knowledge of the epidemiology of these two pathogens in these regions and elsewhere in China. PMID:26504798

  18. Application of cattle slurry containing Mycobacterium avium subsp. paratuberculosis (MAP) to grassland soil and its effect on the relationship between MAP and free-living amoeba.

    PubMed

    Salgado, M; Alfaro, M; Salazar, F; Badilla, X; Troncoso, E; Zambrano, A; González, M; Mitchell, R M; Collins, M T

    2015-01-30

    Slurry from dairy farms is commonly used to fertilize crops and pastures. This mixture of manure, urine and water can harbor multiple microbial pathogens among which Mycobacterium avium subsp. paratuberculosis (MAP) is a major concern. Persistence of MAP in soil and infection of soil Acanthamoeba was evaluated by culture, real-time IS900 PCR, and by staining of amoeba with acid-fast and vital stains comparing soils irrigated with MAP-spiked or control dairy farm slurry. MAP DNA was detected in soil for the 8 month study duration. MAP was detected by PCR from more soil samples for plots receiving MAP-spiked slurry (n=61/66) than from soils receiving control slurry (n=10/66 samples). Vital stains verified that intracellular MAP in amoeba was viable. More MAP was found in amoeba at the end of the study than immediately after slurry application. There was no relationship between MAP presence in soil and in amoeba over time. Infection of amoeba by MAP provides a protected niche for the persistence and even possibly the replication of MAP in soils. As others have suggested, MAP-infected amoeba may act like a "Trojan horse" providing a means for persistence in soils and potentially a source of infection for grazing animals. PMID:25448447

  19. Antibodies against invasive phenotype-specific antigens increase Mycobacterium avium subspecies paratuberculosis translocation across a polarized epithelial cell model and enhance killing by bovine macrophages.

    PubMed

    Everman, Jamie L; Bermudez, Luiz E

    2015-01-01

    Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a severe chronic enteritis which affects large populations of ruminants globally. Prevention strategies to combat the spread of Johne's disease among cattle herds involve adhering to strict calving practices to ensure young susceptible animals do not come in contact with MAP-contaminated colostrum, milk, or fecal material. Unfortunately, the current vaccination options available are associated with high cost and suboptimal efficacy. To more successfully combat the spread of Johne's disease to young calves, an efficient method of protection is needed. In this study, we examined passive immunization as a mode of introducing protective antibodies against MAP to prevent the passage of the bacterium to young animals via colostrum and milk. Utilizing the infectious MAP phenotype developed after bacterial exposure to milk, we demonstrate that in vitro opsonization with serum from Johne's-positive cattle results in enhanced translocation across a bovine MDBK polarized epithelial cell monolayer. Furthermore, immune serum opsonization of MAP results in a rapid host cell-mediated killing by bovine macrophages in an oxidative-, nitrosative-, and extracellular DNA trap-independent manner. This study illustrates that antibody opsonization of MAP expressing an infectious phenotype leads to the killing of the bacterium during the initial stage of macrophage infection. PMID:26301206

  20. Evaluation of "Indigenous Absorbed ELISA Kit" for the Estimation of Seroprevalence of Mycobacterium avium Subspecies paratuberculosis Antibodies in Human Beings in North India.

    PubMed

    Singh, A V; Singh, S V; Verma, D K; Yadav, R; Singh, P K; Sohal, J S

    2011-01-01

    In present pilot study aimed to estimate, presence of Mycobacterium avium subspecies paratuberculosis (MAP) antibodies in the human serum samples originating from North India using "Indigenous absorbed ELISA kit" (ELISA kit). The phase I, "ELISA kit" was optimized using protoplasmic antigen from native isolate of MAP "Indian Bison type" recovered from the biopsies of Crohn's disease patients. The phase II, sensitivity and specificity of the kit were estimated as 40.0 and 83.3%, respectively, when evaluated in 40 human serum samples (5 Crohn's disease and 22 ulcerative colitis patients and 13 healthy human subjects) with defined MAP status with respect to stool culture. Seroprevalence of MAP antibodies was higher in CD patients (80.0%) as compared to ulcerative colitis patients (4.5%) and normal human subjects (15.3%). The phase III, seroprevalence of MAP antibodies was estimated as 23.4%, on the basis of the screening of 452 human serum samples (without history) from different geographical regions of North India. Region-wise, 34.0, 33.3, 32.8, 25.0, 23.0, 17.7, and 12.5% samples were positive from the states of Punjab, Uttarakhand, New Delhi, Himachal Pradesh, Haryana, Uttar Pradesh and Jammu and Kashmir, respectively. Study reported moderately higher presence of MAP antibodies in human population, which necessitates programs to reduce the bioburden of MAP in the environment and in animal population. PMID:23738105

  1. Comparison of three DNA preparation methods for real-time polymerase chain reaction confirmation of Mycobacterium avium subsp. paratuberculosis growth in an automated broth culture system.

    PubMed

    Sweeney, Raymond W; Whitlock, Robert H; McAdams, Susan C

    2006-11-01

    Three methods of harvesting DNA from broth culture tubes for quantitative real-time polymerase chain reaction (qrtPCR) confirmation of Mycobacterium avium subsp. paratuberculosis (MAP) were evaluated. A commercial DNA extraction kit, the boil method (boiling for 5 minutes), or direct addition of broth culture media to the PCR reaction mix were tested. Samples were evaluated at 8 or 11 days of incubation and at the time of instrument-signal culture-positive. In total, when tested at time to instrument signal positive, 10/10 (100%) of samples extracted by the commercial method were positive on qrtPCR, whereas 9/10 (90%) were positive after the boil method, and 6/10 (60%) were positive after the direct method. Increased volumes of egg-yolk emulsion added to the culture tubes prolonged the number of cycles to threshold positive for the samples that were not subjected to commercial extraction or boiling. Samples were not reliably positive when tested at 8 or 11 days of incubation. The boil method appears to represent a reasonable time- and money-saving method to harvest DNA for qrtPCR confirmation of MAP in broth culture at time to instrument signal positive. PMID:17121088

  2. Antibodies against invasive phenotype-specific antigens increase Mycobacterium avium subspecies paratuberculosis translocation across a polarized epithelial cell model and enhance killing by bovine macrophages

    PubMed Central

    Everman, Jamie L.; Bermudez, Luiz E.

    2015-01-01

    Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a severe chronic enteritis which affects large populations of ruminants globally. Prevention strategies to combat the spread of Johne's disease among cattle herds involve adhering to strict calving practices to ensure young susceptible animals do not come in contact with MAP-contaminated colostrum, milk, or fecal material. Unfortunately, the current vaccination options available are associated with high cost and suboptimal efficacy. To more successfully combat the spread of Johne's disease to young calves, an efficient method of protection is needed. In this study, we examined passive immunization as a mode of introducing protective antibodies against MAP to prevent the passage of the bacterium to young animals via colostrum and milk. Utilizing the infectious MAP phenotype developed after bacterial exposure to milk, we demonstrate that in vitro opsonization with serum from Johne's-positive cattle results in enhanced translocation across a bovine MDBK polarized epithelial cell monolayer. Furthermore, immune serum opsonization of MAP results in a rapid host cell-mediated killing by bovine macrophages in an oxidative-, nitrosative-, and extracellular DNA trap-independent manner. This study illustrates that antibody opsonization of MAP expressing an infectious phenotype leads to the killing of the bacterium during the initial stage of macrophage infection. PMID:26301206

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

    PubMed Central

    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

  4. Molecular identification and characterization of Mycobacterium avium subspecies paratuberculosis in free living non-human primate (Rhesus macaques) from North India.

    PubMed

    Singh, S V; Singh, A V; Singh, P K; Kumar, A; Singh, B

    2011-05-01

    In recent years, Mycobacterium avium subspecies paratuberculosis (MAP) has emerged as major animal pathogen with significant zoonotic concerns, worldwide. MAP infection is endemic in domestic and wild ruminant population in India. However, information on MAP infection in free ranging animal species and non human primates is limited. Present study aimed to estimate the status of MAP infection in free living Rhesus macaques suffering with multiple clinical conditions (coughing and loose stool). A total of 25 stool samples were collected from six colonies of Rhesus macaques from Mathura region (North India) and screened for the presence of MAP, using microscopic examination and IS900 PCR, directly from stool samples. PCR positive DNA samples were further genotyped using IS1311 PCR-restriction enzyme analysis. Of the 25 stool samples, 10 (40.0%) and 2 (8.0%) were positive for MAP using microscopic examination and direct IS900 PCR, respectively. IS900 PCR positive DNA samples were genotyped as 'Indian Bison type', which is a major MAP genotype infecting domestic and wild ruminant species and human beings in India. Prevalence of MAP in Rhesus macaques (Indian monkeys) was moderately high and confirmed interspecies sharing of MAP between domestic livestock and non-human primates. Presence of MAP in non-human primates, support the etiological role of MAP in inflammatory bowel disease patients. Indian monkeys may serve as model for understanding the role of non-human primates in sustenance, transmission and pathogenesis of MAP infection. PMID:21255839

  5. Antibodies Recognizing Mycobacterium avium paratuberculosis Epitopes Cross-React with the Beta-Cell Antigen ZnT8 in Sardinian Type 1 Diabetic Patients

    PubMed Central

    Masala, Speranza; Paccagnini, Daniela; Cossu, Davide; Brezar, Vedran; Pacifico, Adolfo; Ahmed, Niyaz; Mallone, Roberto; Sechi, Leonardo A.

    2011-01-01

    The environmental factors at play in the pathogenesis of type 1 diabetes (T1D) remain enigmatic. Mycobacterium avium subspecies paratuberculosis (MAP) is transmitted from dairy herds to humans through food contamination. MAP causes an asymptomatic infection that is highly prevalent in Sardinian T1D patients compared with type 2 diabetes (T2D) and healthy controls. Moreover, MAP elicits humoral responses against several mycobacterial proteins. We asked whether antibodies (Abs) against one of these proteins, namely MAP3865c, which displays a sequence homology with the β-cell protein zinc transporter 8 (ZnT8) could be cross-reactive with ZnT8 epitopes. To this end, Ab responses against MAP3865c were analyzed in Sardinian T1D, T2D and healthy subjects using an enzymatic immunoassay. Abs against MAP3865c recognized two immunodominant transmembrane epitopes in 52–65% of T1D patients, but only in 5–7% of T2D and 3–5% of healthy controls. There was a linear correlation between titers of anti-MAP3865c and anti-ZnT8 Abs targeting these two homologous epitopes, and pre-incubation of sera with ZnT8 epitope peptides blocked binding to the corresponding MAP3865c peptides. These results demonstrate that Abs recognizing MAP3865c epitopes cross-react with ZnT8, possibly underlying a molecular mimicry mechanism, which may precipitate T1D in MAP-infected individuals. PMID:22046415

  6. Evaluation of “Indigenous Absorbed ELISA Kit” for the Estimation of Seroprevalence of Mycobacterium avium Subspecies paratuberculosis Antibodies in Human Beings in North India

    PubMed Central

    Singh, A. V.; Singh, S. V.; Verma, D. K.; Yadav, R.; Singh, P. K.; Sohal, J. S.

    2011-01-01

    In present pilot study aimed to estimate, presence of Mycobacterium avium subspecies paratuberculosis (MAP) antibodies in the human serum samples originating from North India using “Indigenous absorbed ELISA kit” (ELISA kit). The phase I, “ELISA kit” was optimized using protoplasmic antigen from native isolate of MAP “Indian Bison type” recovered from the biopsies of Crohn's disease patients. The phase II, sensitivity and specificity of the kit were estimated as 40.0 and 83.3%, respectively, when evaluated in 40 human serum samples (5 Crohn's disease and 22 ulcerative colitis patients and 13 healthy human subjects) with defined MAP status with respect to stool culture. Seroprevalence of MAP antibodies was higher in CD patients (80.0%) as compared to ulcerative colitis patients (4.5%) and normal human subjects (15.3%). The phase III, seroprevalence of MAP antibodies was estimated as 23.4%, on the basis of the screening of 452 human serum samples (without history) from different geographical regions of North India. Region-wise, 34.0, 33.3, 32.8, 25.0, 23.0, 17.7, and 12.5% samples were positive from the states of Punjab, Uttarakhand, New Delhi, Himachal Pradesh, Haryana, Uttar Pradesh and Jammu and Kashmir, respectively. Study reported moderately higher presence of MAP antibodies in human population, which necessitates programs to reduce the bioburden of MAP in the environment and in animal population. PMID:23738105

  7. Type 1 Diabetes at-risk children highly recognize Mycobacterium avium subspecies paratuberculosis epitopes homologous to human Znt8 and Proinsulin

    PubMed Central

    Niegowska, Magdalena; Rapini, Novella; Piccinini, Simona; Mameli, Giuseppe; Caggiu, Elisa; Manca Bitti, Maria Luisa; Sechi, Leonardo A.

    2016-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) has been previously associated to T1D as a putative environmental agent triggering or accelerating the disease in Sardinian and Italian populations. Our aim was to investigate the role of MAP in T1D development by evaluating levels of antibodies directed against MAP epitopes and their human homologs corresponding to ZnT8 and proinsulin (PI) in 54 T1D at-risk children from mainland Italy and 42 healthy controls (HCs). A higher prevalence was detected for MAP/ZnT8 pairs (62,96% T1D vs. 7,14% HCs; p < 0.0001) compared to MAP/PI epitopes (22,22% T1D vs. 9,52% HCs) and decreasing trends were observed upon time-point analyses for most peptides. Similarly, classical ZnT8 Abs and GADA decreased in a time-dependent manner, whereas IAA titers increased by 12%. Responses in 0–9 year-old children were stronger than in 10–18 age group (75% vs. 69,1%; p < 0.04). Younger age, female sex and concomitant autoimmune disorders contributed to a stronger seroreactivity suggesting a possible implication of MAP in multiple autoimmune syndrome. Cross-reactivity of the homologous epitopes was reflected by a high correlation coefficient (r2 > 0.8) and a pairwise overlap of positivity (>83% for MAP/ZnT8). PMID:26923214

  8. Cellular composition of granulomatous lesions in gut-associated lymphoid tissues of goats during the first year after experimental infection with Mycobacterium avium subsp. paratuberculosis.

    PubMed

    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

  9. Application of IS1311 locus 2 PCR-REA assay for the specific detection of ‘Bison type’ Mycobacterium avium subspecies paratuberculosis isolates of Indian origin

    PubMed Central

    Singh, Ajay Vir; Chauhan, Devendra Singh; Singh, Abhinendra; Singh, Pravin Kumar; Sohal, Jagdip Singh; Singh, Shoor Vir

    2015-01-01

    Background & objectives: Of the three major genotypes of Mycobacterium avium subspecies paratuberculosis (MAP), ‘Bison type’ is most prevalent genotype in the domestic livestock species of the country, and has also been recovered from patients suffering from Crohn's disease. Recently, a new assay based on IS1311 locus 2 PCR- restriction endonuclease analysis (REA) was designed to distinguish between ‘Indian Bison type’ and non-Indian genotypes. The present study investigated discriminatory potential of this new assay while screening of a panel of MAP isolates of diverse genotypes and from different geographical regions. Methods: A total of 53 mycobacterial isolates (41 MAP and 12 Mycobacterium other than MAP), three MAP genomic DNA and 36 MAP positive faecal DNA samples from different livestock species (cattle, buffaloes, goat, sheep and bison) and geographical regions (India, Canada, USA, Spain and Portugal) were included in the study. The extracted DNA samples (n=92) were analyzed for the presence of MAP specific sequences (IS900, ISMav 2 and HspX) using PCR. DNA samples were further subjected to genotype differentiation using IS1311 PCR-REA and IS1311 L2 PCR-REA methods. Results: All the DNA samples (except DNA from non-MAP mycobacterial isolates) were positive for all the three MAP specific sequences based PCRs. IS1311 PCR-REA showed that MAP DNA samples of Indian origin belonged to ‘Bison type’. Whereas, of the total 19 non-Indian MAP DNA samples, 2, 15 and 2 were genotyped as ‘Bison type’, ‘Cattle type’ and ‘Sheep type’, respectively. IS1311 L2 PCR-REA method showed different restriction profiles of ‘Bison type’ genotype as compared to non-Indian DNA samples. Interpretation & conclusions: IS1311 L2 PCR-REA method successfully discriminated ‘Indian Bison type’ from other non-Indian genotypes and showed potential to be future epidemiological tool and for genotyping of MAP isolates. PMID:25857495

  10. Seroprevalence of IgG1 and IgG4 class antibodies against Mycobacterium avium subsp. paratuberculosis in Japanese population.

    PubMed

    Otsubo, Shigeru; Cossu, Davide; Eda, Shigetoshi; Otsubo, Yuriko; Sechi, Leonardo Antonio; Suzuki, Tsuyoshi; Iwao, Yumiko; Yamamoto, Shizuo; Kuribayashi, Takashi; Momotani, Eiichi

    2015-10-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the established causative agent of Johne's disease in cattle and other ruminants, and it has also been speculated to be a putative etiological agent of several human autoimmune diseases. It is acknowledged that dairy products deriving from infected animals play a role (could be vehicles) in exposing humans to MAP. MAP could stimulate the human immune system by means of their complex antigen (in the case of lipids, multivalent antigens) and may modulate it, acting as adjuvant molecules such as Freund's complete adjuvant. The immune system might be abnormally stimulated by the constant presence of MAP antigens (for example, in the dairy products), and this might be particularly relevant in genetically predisposed individuals. However, there is limited understanding about the current human exposure to MAP. The present study analyzed the antibody recognition profile of MAP lipophilic antigens in a cohort of 126 healthy Japanese. We measured the serum levels of total immunoglobulin G (IgG) and subclasses targeting MAP surface antigens through ethanol vortex indirect enzyme-linked immunosorbent assay (EVELISA) by using serum absorbed with Mycobacterium phlei. Elevated IgG (especially IgG1 and IgG4) responses were observed in 14% of the sera. To assess the specificity of EVELISA, the same samples were analyzed by means of a commercially available Johnelisa II kit. It was noteworthy that a high degree of correlation was observed when comparing the two methodologies (rs=0.7, p<0.0001). Moreover, in order to investigate the specificity of the binding, inhibition assay experiments were carried out also searching for antibodies against Bacillus Calmette-Guérin antigens, but no cross-reaction was observed. The result obtained represents the first evidence implying that the Japanese population is exposed to MAP, and additionally the existence of a foodborne chain of exposure that transmits MAP antigens to humans. PMID

  11. Cost-benefit analysis of vaccination against Mycobacterium avium ssp. paratuberculosis in dairy cattle, given its cross-reactivity with tuberculosis tests.

    PubMed

    Groenendaal, Huybert; Zagmutt, Francisco J; Patton, Elisabeth A; Wells, Scott J

    2015-09-01

    Johne's disease (JD), or paratuberculosis, is a chronic enteric disease of ruminants, caused by infection with Mycobacterium avium ssp. paratuberculosis (MAP). Johne's disease causes considerable economic losses to the US dairy industry, estimated to be over $200 million annually. Available control strategies include management measures to improve calf hygiene, test-and-cull strategies, and vaccination. Although the first 2 strategies have shown to reduce the prevalence of MAP, they require dedicated and long-term efforts from dairy producers, with often relatively slow progress. As a result, uptake of both strategies has not been as wide as expected given the economic benefits especially of improved hygiene. Vaccination has also been found to reduce the prevalence and economic losses of JD, but most economic estimates have been based on simulation of hypothetical vaccines. In addition, if an animal is vaccinated, cross-reactivity between MAP antibodies and bovine tuberculosis (BTB) antigens may occur, decreasing the specificity of BTB tests. Therefore, MAP vaccination would cause additional indirect costs to the BTB surveillance and control program. The objective of the present study was to use data from a MAP vaccine trial together with an epidemiologic and economic model to estimate the direct on-farm benefits of MAP vaccination and to estimate the indirect costs of MAP vaccination due to the cross-reactivity with BTB tests. Direct economic benefits of MAP vaccination were estimated at $8.03 (90% predictive interval: -$25.97 to $41.36) per adult animal per year, all accruing to the dairy producers. This estimate is likely an underestimation of the true direct benefits of MAP vaccination. In addition, indirect economic costs due to cross-reactivity were $2.14 per adult animal per year, making MAP vaccination economically attractive. Only in regions or states with a high frequency of BTB testing (because of, for example, Mycobacterium bovis outbreaks in a wild

  12. Dam Mycobacterium avium subspecies paratuberculosis (MAP) infection status does not predetermine calves for future shedding when raised in a contaminated environment: a cohort study.

    PubMed

    Eisenberg, Susanne W F; Rutten, Victor P M G; Koets, Ad 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 presence of MAP DNA and antibody in colostrum and that of DNA in faeces and the environment with subsequent MAP shedding of their daughters. A cohort of 117 dam-daughter pairs giving birth/being born on eight commercial dairy farms with endemic paratuberculosis was followed where colostrum, faecal and environmental samples (dust) were analysed for the presence of MAP using an IS900 real-time PCR. Antibodies in colostrum were measured by ELISA. Analysis of dust samples showed that on all farms environmental MAP exposure occurred continuously. In significantly more colostrum samples (48%) MAP DNA was detected compared to faecal samples (37%). MAP specific antibodies were present in 34% of the colostrum samples. In total MAP DNA was present in faecal samples of 41% of the daughters at least once during the sampling period. The association between faecal shedding in the offspring and the dam MAP status defined by MAP PCR on colostrum, MAP PCR on faeces or ELISA on colostrum was determined by an exact cox regression analysis for discrete data. The model indicated that the hazard for faecal shedding in daughters born to MAP positive dams was not significantly different compared to daughters born to MAP negative dams. When born to a dam with DNA positive faeces the HR was 1.05 (CI 0.6; 1.8) and with DNA positive colostrum the HR was 1.17 (CI 0.6; 2.3). When dam status was defined by a combination of both PCR outcomes (faeces and colostrum) and the ELISA outcome the HR was 1.26 (CI 0.9; 1.9). Therefore, this study indicates that neither the presence of MAP DNA in colostrum, MAP DNA in faeces nor the presence of MAP antibodies in colostrum of the dam significantly influences the hazard of

  13. Altered microRNA expression and pre-mRNA splicing events reveal new mechanisms associated with early stage Mycobacterium avium subspecies paratuberculosis infection.

    PubMed

    Liang, Guanxiang; Malmuthuge, Nilusha; Guan, Yongjuan; Ren, Yuwei; Griebel, Philip J; Guan, Le Luo

    2016-01-01

    The molecular regulatory mechanisms of host responses to Mycobacterium avium subsp. paratuberculosis (MAP) infection during the early subclinical stage are still not clear. In this study, surgically isolated ileal segments in newborn calves (n = 5) were used to establish in vivo MAP infection adjacent to an uninfected control intestinal compartment. RNA-Seq was used to profile the whole transcriptome (mRNAs) and the microRNAome (miRNAs) of ileal tissues collected at one-month post-infection. The most related function of the differentially expressed mRNAs between infected and uninfected tissues was "proliferation of endothelial cells", indicating that MAP infection may lead to the over-proliferation of endothelial cells. In addition, 46.2% of detected mRNAs displayed alternative splicing events. The pre-mRNA of two genes related to macrophage maturation (monocyte to macrophage differentiation-associated) and lysosome function (adenosine deaminase) showed differential alternative splicing events, suggesting that specific changes in the pre-mRNA splicing sites may be a mechanism by which MAP escapes host immune responses. Moreover, 9 miRNAs were differentially expressed after MAP infection. The integrated analysis of microRNAome and transcriptome revealed that these miRNAs might regulate host responses to MAP infection, such as "proliferation of endothelial cells" (bta-miR-196 b), "bacteria recognition" (bta-miR-146 b), and "regulation of the inflammatory response" (bta-miR-146 b). PMID:27102525

  14. The Features of Fecal and Ileal Mucosa-Associated Microbiota in Dairy Calves during Early Infection with Mycobacterium avium Subspecies paratuberculosis.

    PubMed

    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

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

    PubMed Central

    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

  16. Detection of Mycobacterium avium subspecies paratuberculosis in tie-stall dairy herds using a standardized environmental sampling technique and targeted pooled samples.

    PubMed

    Arango-Sabogal, Juan C; Côté, Geneviève; Paré, Julie; Labrecque, Olivia; Roy, Jean-Philippe; Buczinski, Sébastien; Doré, Elizabeth; Fairbrother, Julie H; Bissonnette, Nathalie; Wellemans, Vincent; Fecteau, Gilles

    2016-07-01

    Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of Johne's disease, a chronic contagious enteritis of ruminants that causes major economic losses. Several studies, most involving large free-stall herds, have found environmental sampling to be a suitable method for detecting MAP-infected herds. In eastern Canada, where small tie-stall herds are predominant, certain conditions and management practices may influence the survival and transmission of MAP and recovery (isolation). Our objective was to estimate the performance of a standardized environmental and targeted pooled sampling technique for the detection of MAP-infected tie-stall dairy herds. Twenty-four farms (19 MAP-infected and 5 non-infected) were enrolled, but only 20 were visited twice in the same year, to collect 7 environmental samples and 2 pooled samples (sick cows and cows with poor body condition). Concurrent individual sampling of all adult cows in the herds was also carried out. Isolation of MAP was achieved using the MGIT Para TB culture media and the BACTEC 960 detection system. Overall, MAP was isolated in 7% of the environmental cultures. The sensitivity of the environmental culture was 44% [95% confidence interval (CI): 20% to 70%] when combining results from 2 different herd visits and 32% (95% CI: 13% to 57%) when results from only 1 random herd visit were used. The best sampling strategy was to combine samples from the manure pit, gutter, sick cows, and cows with poor body condition. The standardized environmental sampling technique and the targeted pooled samples presented in this study is an alternative sampling strategy to costly individual cultures for detecting MAP-infected tie-stall dairies. Repeated samplings may improve the detection of MAP-infected herds. PMID:27408329

  17. Altered microRNA expression and pre-mRNA splicing events reveal new mechanisms associated with early stage Mycobacterium avium subspecies paratuberculosis infection

    PubMed Central

    Liang, Guanxiang; Malmuthuge, Nilusha; Guan, Yongjuan; Ren, Yuwei; Griebel, Philip J.; Guan, Le Luo

    2016-01-01

    The molecular regulatory mechanisms of host responses to Mycobacterium avium subsp. paratuberculosis (MAP) infection during the early subclinical stage are still not clear. In this study, surgically isolated ileal segments in newborn calves (n = 5) were used to establish in vivo MAP infection adjacent to an uninfected control intestinal compartment. RNA-Seq was used to profile the whole transcriptome (mRNAs) and the microRNAome (miRNAs) of ileal tissues collected at one-month post-infection. The most related function of the differentially expressed mRNAs between infected and uninfected tissues was “proliferation of endothelial cells”, indicating that MAP infection may lead to the over-proliferation of endothelial cells. In addition, 46.2% of detected mRNAs displayed alternative splicing events. The pre-mRNA of two genes related to macrophage maturation (monocyte to macrophage differentiation-associated) and lysosome function (adenosine deaminase) showed differential alternative splicing events, suggesting that specific changes in the pre-mRNA splicing sites may be a mechanism by which MAP escapes host immune responses. Moreover, 9 miRNAs were differentially expressed after MAP infection. The integrated analysis of microRNAome and transcriptome revealed that these miRNAs might regulate host responses to MAP infection, such as “proliferation of endothelial cells” (bta-miR-196 b), “bacteria recognition” (bta-miR-146 b), and “regulation of the inflammatory response” (bta-miR-146 b). PMID:27102525

  18. Association of single nucleotide polymorphisms in the ANKRA2 and CD180 genes with bovine respiratory disease and presence of Mycobacterium avium subsp. paratuberculosis(1).

    PubMed

    Casas, E; Garcia, M D; Wells, J E; Smith, T P L

    2011-12-01

    The objective was to determine whether single nucleotide polymorphisms (SNPs) in the ANKRA2 and CD180 genes are associated with incidence of bovine respiratory disease (BRD) and presence of Mycobacterium avium subsp. paratuberculosis (MAP) in cattle. Two independent populations were used. The first population (BRD-affected; N = 90) was composed of 31 half-sib progeny, from a Brahman × Angus sire, that were treated for BRD. Untreated offspring from the sire were selected to serve as controls. The second population (MAP-infected) of 330 animals of unknown parentage was evaluated for the presence of MAP in ileocecal lymph node and classified as positive or negative. Markers in both genes were assessed for association in these two populations. In the BRD-affected population, five SNPs in the ANKRA2 gene were significantly associated (P < 0.05), and two SNPs were highly associated (P < 0.01) with incidence of BRD. In addition, two SNPs in the CD180 gene were found to be associated with this trait. In the MAP-infected population, one SNP in the ANKRA2 gene was significantly associated (P < 0.05) with the presence or absence of MAP, and a SNP in the CD180 gene was highly associated (P < 0.01) with the trait. Haplotypes, using significant markers, showed a positive association with both incidence of BRD (P = 0.0001) and with the presence of MAP (P = 0.0032). Markers in the ANKRA2 and CD180 genes are associated with the ability of the animal to cope with pathogens. PMID:22034997

  19. The Features of Fecal and Ileal Mucosa-Associated Microbiota in Dairy Calves during Early Infection with Mycobacterium avium Subspecies paratuberculosis

    PubMed Central

    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

  20. The Mycobacterium avium ssp. paratuberculosis specific mptD gene is required for maintenance of the metabolic homeostasis necessary for full virulence in mouse infections

    PubMed Central

    Meiß, Thorsten; Eckelt, Elke; Basler, Tina; Meens, Jochen; Heinzmann, Julia; Suwandi, Abdulhadi; Oelemann, Walter M. R.; Trenkamp, Sandra; Holst, Otto; Weiss, Siegfried; Bunk, Boyke; Spröer, Cathrin; Gerlach, Gerald-F.; Goethe, Ralph

    2014-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease, a chronic granulomatous enteritis in ruminants. Furthermore, infections of humans with MAP have been reported and a possible association with Crohn's disease and diabetes type I is currently discussed. MAP owns large sequence polymorphisms (LSPs) that were exclusively found in this mycobacteria species. The relevance of these LSPs in the pathobiology of MAP is still unclear. The mptD gene (MAP3733c) of MAP belongs to a small group of functionally uncharacterized genes, which are not present in any other sequenced mycobacteria species. mptD is part of a predicted operon (mptABCDEF), encoding a putative ATP binding cassette-transporter, located on the MAP-specific LSP14. In the present study, we generated an mptD knockout strain (MAPΔmptD) by specialized transduction. In order to investigate the potential role of mptD in the host, we performed infection experiments with macrophages. By this, we observed a significantly reduced cell number of MAPΔmptD early after infection, indicating that the mutant was hampered with respect to adaptation to the early macrophage environment. This important role of mptD was supported in mouse infection experiments where MAPΔmptD was significantly attenuated after peritoneal challenge. Metabolic profiling was performed to determine the cause for the reduced virulence and identified profound metabolic disorders especially in the lipid metabolism of MAPΔmptD. Overall our data revealed the mptD gene to be an important factor for the metabolic adaptation of MAP required for persistence in the host. PMID:25177550

  1. Detection of Mycobacterium avium subspecies paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different regions throughout Switzerland

    PubMed Central

    Corti, Sabrina; Stephan, Roger

    2002-01-01

    Background Since Mycobacterium avium subspecies paratuberculosis (MAP) was isolated from intestinal tissue of a human patient suffering Crohn's disease, a controversial discussion exists whether MAP have a role in the etiology of Crohn's disease or not. Raw milk may be a potential vehicle for the transmission of MAP to human population. In a previous paper, we have demonstrated that MAP are found in raw milk samples obtained from a defined region in Switzerland. The aim of this work is to collect data about the prevalence of MAP specific IS900 insertion sequence in bulk-tank milk samples in different regions of Switzerland. Furthermore, we examined eventual correlation between the presence of MAP and the somatic cell counts, the total colony counts and the presence of Enterobacteriaceae. Results 273 (19.7%) of the 1384 examined bulk-tank milk samples tested IS900 PCR-positive. The prevalence, however, in the different regions of Switzerland shows significant differences and ranged from 1.7% to 49.2%. Furthermore, there were no statistically significant (p >> 0.05) differences between the somatic cell counts and the total colony counts of PCR-positive and PCR-negative milk samples. Enterobacteriaceae occur as often in IS900 PCR-positive as in PCR-negative milk samples. Conclusion This is the first study, which investigates the prevalence of MAP in bulk-tank milk samples all over Switzerland and infers the herd-level prevalence of MAP infection in dairy herds. The prevalence of 19.7% IS900 PCR-positive bulk-milk samples shows a wide distribution of subclinical MAP-infections in dairy stock in Switzerland. MAP can therefore often be transmitted to humans by raw milk consumption. PMID:12097144

  2. Dairy farms testing positive for Mycobacterium avium ssp. paratuberculosis have poorer hygiene practices and are less cautious when purchasing cattle than test-negative herds.

    PubMed

    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. PMID:26995127

  3. Host-Mycobacterium avium subsp. paratuberculosis interactome reveals a novel iron assimilation mechanism linked to nitric oxide stress during early infection

    PubMed Central

    2013-01-01

    Background The initial interaction between host cell and pathogen sets the stage for the ensuing infection and ultimately determine the course of disease. However, there is limited knowledge of the transcripts utilized by host and pathogen and how they may impact one another during this critical step. The purpose of this study was to create a host-Mycobacterium avium subsp. paratuberculosis (MAP) interactome for early infection in an epithelium-macrophage co-culture system using RNA-seq. Results Establishment of the host-MAP interactome revealed a novel iron assimilation system for carboxymycobactin. Iron assimilation is linked to nitric oxide synthase-2 production by the host and subsequent nitric oxide buildup. Iron limitation as well as nitric oxide is a prompt for MAP to enter into an iron sequestration program. This new iron sequestration program provides an explanation for mycobactin independence in some MAP strains grown in vitro as well as during infection within the host cell. Utilization of such a pathway is likely to aid MAP establishment and long-term survival within the host. Conclusions The host-MAP interactome identified a number of metabolic, DNA repair and virulence genes worthy for consideration as novel drug targets as well as future pathogenesis studies. Reported interactome data may also be utilized to conduct focused, hypothesis-driven research. Co-culture of uninfected bovine epithelial cells (MAC-T) and primary bovine macrophages creates a tolerant genotype as demonstrated by downregulation of inflammatory pathways. This co-culture system may serve as a model to investigate other bovine enteric pathogens. PMID:24112552

  4. Seroreactivity against Specific L5P Antigen from Mycobacterium avium subsp. paratuberculosis in Children at Risk for T1D

    PubMed Central

    Niegowska, Magdalena; Rapini, Novella; Biet, Frank; Piccinini, Simona; Bay, Sylvie; Lidano, Roberta; Manca Bitti, Maria Luisa; Sechi, Leonardo A.

    2016-01-01

    Aims/Hypothesis Although numerous environmental agents have been investigated over the years as possible triggers of type 1 diabetes (T1D), its causes remain unclear. We have already demonstrated an increased prevalence of antibodies against peptides derived from Mycobacterium avuim subsp. paratuberculosis (MAP) homologous to human zinc transporter 8 protein (ZnT8) and proinsulin in Italian subjects at risk for or affected by T1D. In this study, we compared titers of the previously detected antibodies with seroreactivity to MAP lipopentapetide (L5P) that recently emerged as a strong immunogenic component able to specifically distinguish MAP from other mycobacteria. Methods Plasma of 32 children and youth at risk for T1D including follow-up samples and 42 age-matched healthy controls (HC) recruited at the Tor Vergata University Hospital in Rome was analyzed by indirect ELISA for the presence of antibodies against MAP-derived epitopes MAP3865c133–141, MAP3865c125-133, MAP2404c70-85 and MAP1,4αgbp157-173 along with their ZnT8 and proinsulin homologs. The data were analyzed through two-tailed Mann-Whitney U test and relation between variables was determined by principal component analysis. Results Responses to L5P were not detectable in subjects whose initial seroreactivity to MAP peptides and their human homologs was lost in follow-up samples, whereas anti-L5P antibodies appeared constantly in individuals with a stable immunity against MAP antigens. The overall coincidence in positivity to L5P and the four MAP epitopes both in children at risk for T1D and HC exceeded 90%. Conclusions MAP-derived homologs may cross-react with ZnT8 and proinsulin peptides inducing immune responses at a young age in subjects predisposed for T1D. Thus, L5P may have a diagnostic value to immediately indicate the presence of anti-MAP seroreactivity when evaluation of a more complex antibody status is not required. Almost complete coincidence in responses to both types of antigens lends

  5. Novel Secreted Antigens of Mycobacterium paratuberculosis as Serodiagnostic Biomarkers for Johne's Disease in Cattle

    PubMed Central

    Facciuolo, Antonio; Kelton, David F.

    2013-01-01

    Johne's disease is a chronic gastroenteritis of cattle caused by Mycobacterium avium subsp. paratuberculosis that afflicts 40% of dairy herds worldwide. M. avium subsp. paratuberculosis-infected cattle can remain asymptomatic for years while transmitting the pathogen via fecal contamination and milk. Current serodiagnosis with enzyme-linked immunosorbent assays (ELISAs) fails to detect asymptomatic M. avium subsp. paratuberculosis-infected cattle due to the use of poorly defined antigens and knowledge gaps in our understanding of M. avium subsp. paratuberculosis components eliciting pathogen-specific immune responses. We set out to (i) define a subset of proteins that contain putative antigenic targets and (ii) screen these antigen pools for immunogens relevant in detecting infection. To accomplish our first objective, we captured and resolved M. avium subsp. paratuberculosis-secreted proteins using a 2-step fractionation method and reverse-phase liquid chromatography to identify 162 unique proteins, of which 66 had not been previously observed in M. avium subsp. paratuberculosis culture filtrates. Subsequent screening of M. avium subsp. paratuberculosis-secreted proteins showed four antigens, of which one or more reacted on immunoblotting with individual serum samples from 35 M. avium subsp. paratuberculosis-infected cows. Moreover, these novel antigens reacted with sera from 6 low M. avium subsp. paratuberculosis shedders and 3 fecal-culture-positive cows labeled as ELISA seronegative. The specificity of these antigens was demonstrated using negative-control sera from uninfected calves (n = 5) and uninfected cows (n = 5), which did not react to any of these antigens in immunoblotting. As three of the four antigens are novel, their characterization and incorporation into an ELISA-based format will aid in detecting asymptomatic cattle in early or subclinical stages of disease. PMID:24089453

  6. Osteopontin Immunoreactivity in Peripheral Blood Mononuclear Cells, Ileum, and Ileocecal Lymph Node of Dairy Cows Naturally Infected with Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Osteopontin (Opn), a highly acidic glycoprotein, plays an early role in initiating the innate immune response to mycobacterial infections by promoting cellular adhesion and recruitment of inflammatory cells from the peripheral blood. The formation of granulomas at the site of Mycobacterium avium s...

  7. Successful treatment of asymptomatic or clinically terminal bovine Mycobacterium avium subspecies paratuberculosis infection (Johne's disease) with the bacterium Dietzia used as a probiotic alone or in combination with dexamethasone: Adaption to chronic human diarrheal diseases.

    PubMed

    Click, Robert E

    2011-01-01

    A naturally occurring gastrointestinal disease, primarily of ruminants (Johne disease), is a chronic debilitating disease that is caused by Mycobacterium avium subspecies paratuberculosis (MAP). MAP infection occurs primarily in utero and in newborns. Outside our Dietzia probiotic treatment, there are no preventive/curative therapies for bovine paratuberculosis. Interestingly, MAP is at the center of controversy as to its role in (cause of) Crohn disease (CD) and more recently, its role in diabetes, ulcerative colitis, and irritable bowel syndrome (IBS); the latter two, like CD, are considered to be a result of chronic intestinal inflammation. Treatments, both conventional and biologic agents, which induce and maintain remission are directed at curtailing processes that are an intricate part of inflammation. Most possess side effects of varying severity, lose therapeutic value, and more importantly, none routinely result in prevention and/or cures. Based on (a) similarities of Johne disease and Crohn disease, (b) a report that Dietzia inhibited growth of MAP under specific culture conditions, and (c) findings that Dietzia when used as a probiotic, (i) was therapeutic for adult bovine paratuberculosis, and (ii) prevented development of disease in MAP-infected calves, the goal of the present investigations was to design protocols that have applicability for IBD patients. Dietzia was found safe for cattle of all ages and for normal and immunodeficient mice. The results strongly warrant clinical evaluation as a probiotic, in combination with/without dexamethasone. PMID:21460639

  8. 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.

    PubMed

    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

  9. The Identification of Circulating MiRNA in Bovine Serum and Their Potential as Novel Biomarkers of Early Mycobacterium avium subsp paratuberculosis Infection

    PubMed Central

    Britton, Louise; MacHugh, David E.; Markey, Bryan; Gordon, Stephen V.

    2015-01-01

    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. PMID:26218736

  10. Herd-level prevalence and associated risk factors for Mycobacterium avium subsp. paratuberculosis in cattle in the State of Paraíba, Northeastern Brazil.

    PubMed

    Vilar, Ana L T; Santos, Carolina S A B; Pimenta, Carla L R M; Freitas, Theonys D; Brasil, Arthur W L; Clementino, Inácio J; Alves, Clebert J; Bezerra, Camila S; Riet-Correa, Franklin; Oliveira, Taynara S; Azevedo, Sérgio S

    2015-09-01

    A cross-sectional study based on a planned sampling was carried out to determine herd-level and animal-level prevalences, and to identify risk factors associated with herd-level prevalence for bovine paratuberculosis in the State of Paraíba, Northeastern Brazil. The state was divided into three sampling groups: sampling stratum 1 (mesoregion of Sertão), sampling stratum 2 (mesoregion of Borborema), and sampling stratum 3 (mesoregions of Zona da Mata and Agreste). For each sampling stratum, herd-level and animal-level prevalences were estimated by a two-stage sampling survey. In the first stage, a pre-established number of herds (primary sampling units) were randomly selected; in the second stage, a pre-established number of cows aged ≥24 months were randomly selected (secondary sampling units). Ten animals were sampled in herds with up to 99 cows aged over 24 months; 15 animals were sampled in herds with 100 or more cows aged over 24 months; and all animals were sampled in those with up to 10 cows aged over 24 months. In total, 2504 animals were sampled from 480 herds. Enzyme-linked immunosorbant assay (ELISA) test kits were used for Mycobacterium avium subsp. paratuberculosis (MAP) antibody detection. A herd was deemed positive for the presence of MAP if it included at least one positive animal in herds of up to 24 females, and two positive animals in herds with more than 24 females. The herd-level prevalence in the State of Paraíba was 34.5% (95% CI=30.2-39.1%), 26.6% (95% CI=20.2-34.2%) in the region of Borborema, 30.5% (95% CI=23.9-38.0%) in Agreste/Mata, and 41.4% (95% CI=34.0-49.1%) in Sertão. The animal-level prevalence was 10.7% (95% CI=7.3-15.4%) in the State of Paraíba, 7.9% (95% CI=5.2-11.7%) in the region of Borborema, 9.4% (95% CI=7.3-12.1%) in Sertão, and 13.9% (95% CI=6.2--28.3%) in Agreste/Mata. The frequency of seropositive animals per herd ranged from 6.7% to 100% (median of 20%). The risk factors identified were as follows: Sertão region

  11. The Mycobacterium avium complex.

    PubMed Central

    Inderlied, C B; Kemper, C A; Bermudez, L E

    1993-01-01

    Mycobacterium avium complex (MAC) disease emerged early in the epidemic of AIDS as one of the common opportunistic infections afflicting human immunodeficiency virus-infected patients. However, only over the past few years has a consensus developed about its significance to the morbidity and mortality of AIDS. M. avium was well known to mycobacteriologists decades before AIDS, and the MAC was known to cause disease, albeit uncommon, in humans and animals. The early interest in the MAC provided a basis for an explosion of studies over the past 10 years largely in response to the role of the MAC in AIDS opportunistic infection. Molecular techniques have been applied to the epidemiology of MAC disease as well as to a better understanding of the genetics of antimicrobial resistance. The interaction of the MAC with the immune system is complex, and putative MAC virulence factors appear to have a direct effect on the components of cellular immunity, including the regulation of cytokine expression and function. There now is compelling evidence that disseminated MAC disease in humans contributes to both a decrease in the quality of life and survival. Disseminated disease most commonly develops late in the course of AIDS as the CD4 cells are depleted below a critical threshold, but new therapies for prophylaxis and treatment offer considerable promise. These new therapeutic modalities are likely to be useful in the treatment of other forms of MAC disease in patients without AIDS. The laboratory diagnosis of MAC disease has focused on the detection of mycobacteria in the blood and tissues, and although the existing methods are largely adequate, there is need for improvement. Indeed, the successful treatment of MAC disease clearly will require an early and rapid detection of the MAC in clinical specimens long before the establishment of the characteristic overwhelming infection of bone marrow, liver, spleen, and other tissue. Also, a standard method of susceptibility testing

  12. Evaluation of a Mycobacterium avium subsp. paratuberculosis leuD Mutant as a Vaccine Candidate against Challenge in a Caprine Model

    PubMed Central

    Faisal, Syed M.; Chen, Jenn-Wei; Yan, Falong; Chen, Tsai-Tzu; Useh, Nicodemus M.; Yan, Weiwei; Guo, Shanguang; Wang, Shih-Jon; Glaser, Amy L.; McDonough, Sean P.; Singh, Bhupinder; Davis, William C.; Akey, Bruce L.

    2013-01-01

    Johne's disease (JD) is prevalent worldwide and has a significant impact on the global agricultural economy. In the present study, we evaluated the protective efficacy of a leuD (Δleud) mutant and gained insight into differential immune responses after challenge with virulent M. avium subsp. paratuberculosis in a caprine colonization model. The immune response and protective efficacy were compared with those of the killed vaccine Mycopar. In vitro stimulation of peripheral blood mononuclear cells with johnin purified protein derivative showed that Mycopar and ΔleuD generated similar levels of gamma interferon (IFN-γ) but significantly higher levels than unvaccinated and challenged phosphate-buffered saline controls. However, only with ΔleuD was the IFN-γ response maintained. Flow cytometric analysis showed that the increase in IFN-γ correlated with proliferation and activation (increased expression of CD25) of CD4, CD8, and γδT cells, but this response was significantly higher in ΔleuD-vaccinated animals at some time points after challenge. Both Mycopar and ΔleuD vaccines upregulated Th1/proinflammatory and Th17 cytokines and downregulated Th2/anti-inflammatory and regulatory cytokines at similar levels at almost all time points. However, significantly higher levels of IFN-γ (at weeks 26 and 30), interleukin-2 (IL-2; week 18), IL-1b (weeks 14 and 22), IL-17 (weeks 18 and 22), and IL-23 (week 18) and a significantly lower level of IL-10 (weeks 14 and 18) and transforming growth factor β (week 18) were detected in the ΔleuD-vaccinated group. Most importantly, ΔleuD elicited an immune response that significantly limited colonization of tissues compared to Mycopar upon challenge with wild-type M. avium subsp. paratuberculosis. In conclusion, the ΔleuD mutant is a promising vaccine candidate for development of a live attenuated vaccine for JD in ruminants. PMID:23408524

  13. Detection of Mycobacterium avium subspecies paratuberculosis in environmental samples by faecal culture and real-time PCR in relation to apparent within-herd prevalence as determined by individual faecal culture.

    PubMed

    Donat, K; Kube, J; Dressel, J; Einax, E; Pfeffer, M; Failing, K

    2015-04-01

    SUMMARY Environmental samples are a cost-saving and easy-to-use approach to diagnose paratuberculosis at the herd level. Detailed knowledge concerning its uncertainties in herds with a low prevalence of Mycobacterium avium spp. paratuberculosis (MAP) is required to design sampling strategies in control programmes. This study aimed to calculate a threshold level of the apparent within-herd prevalence (WHPapp) as determined by individual mycobacterial cultivation (faecal culture; FC) of all cows thus allowing the detection of a herd as MAP-positive at a certain probability level (P d). Out of 200 environmental samples taken twice from five predefined locations in a barn, 25 were positive by FC and 60 were positive by a quantitative real-time PCR method (qPCR). A logistic regression model was used to calculate the WHPapp threshold of detection. For 50% P d, a WHPapp threshold of 2.9% was calculated for the combination of three samples (milking area, main cow alleyways, holding pen) tested simultaneously both by FC and qPCR. The threshold increased to 6.2% for 90% P d. Repeated environmental sampling did not reduce the WHPapp threshold. Depending on the particular needs for prevalence estimation or in control programmes (single or repeated sampling) the provided WHPapp thresholds at different P d will enable decisions to be made about various sampling strategies. PMID:25274140

  14. Paratuberculosis.

    PubMed Central

    Cocito, C; Gilot, P; Coene, M; de Kesel, M; Poupart, P; Vannuffel, P

    1994-01-01

    Paratuberculosis (Johne's disease) is a chronic, wasting, widespread mycobacteriosis of ruminants. It involves extensive mycobacterial shedding, which accounts for the high contagiousness, and ends with a fatal enteritis. Decreases in weight, milk production, and fertility produce severe economic loss. The DNA of the etiological agent (Mycobacterium paratuberculosis) has a base composition (66 to 67% G+C) within the range of that of mycobacteria (62 to 70% G+C), a size (4.4 x 10(6) to 4.7 x 10(6) bp) larger than that of most pathogenic mycobacteria (2.0 x 10(6) to 4.2 x 10(6) bp), and a high relatedness (> 90%) to Mycobacterium avium DNA. However, the DNAs of the two organisms can be distinguished by restriction fragment length polymorphism analysis. M. paratuberculosis genes coding for a transposase, a cell wall-associated protein (P34), and two heat shock proteins have been cloned and sequenced. Nucleic acid probes (two of which are species specific) are used, after PCR amplification, for M. paratuberculosis identification in stools and milk. As in leprosy, with disease progression, cellular immune reactions decrease and humoral immune reactions increase. Cutaneous testing with sensitins, lymphocyte proliferation assays, and cytokine tests are used to monitor cellular immune reactions in paratuberculosis, but these tests lack specificity. Complement fixation, immunodiffusion, and enzymometric tests based on antibodies to M. paratuberculosis extracts, to mycobacterial antigen complex A36, to glycolipids, and to proteins help identify affected cattle but are not species specific. The carboxyl-terminal portion of the 34-kDa cell wall-associated A36 protein (P34) carries species-specific B-cell epitopes and is the basis for an enzyme-linked immunosorbent assay. Diagnostic tests for paratuberculosis are also used in Crohn's disease, a chronic human ileitis mimicking Johne's disease, in which isolates identified as M. paratuberculosis have been found. Images PMID

  15. Roles of MAP1152 and MAP1156 Proteins in the Immunopathogenesis of Mycobacterium paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Mycobacterium avium subsp. paratuberculosis (MAP) K-10 genome is a circular chromosome of about 4.8 Mb encoding 4,344 open reading frames (ORFs). Mycobacterial genomes are characterized by the presence of gene clusters encoding conserved proteins with PE and PPE motifs. Members of these families...

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

    EPA Science Inventory

    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...

  17. Mycobacterium avium subsp. paratuberculosis Genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The completion of the MAP K-10 genome sequence has opened the doors to many new avenues of research. In the few years since the publication of the genome sequence, the manuscript describing the completed sequence has been cited in the scientific literature more than 85 times. The public availabili...

  18. LCD array and IS900 efficiency in relation to traditional diagnostic techniques for diagnosis of Mycobacterium avium subspecies paratuberculosis in cattle in Egypt.

    PubMed

    ElSayed, Mohamed Sabry Abd ElRaheam

    2014-06-01

    This study aimed to compare traditional tests (Johnin test, fecal staining and fecal culture) with advanced laboratory tests (ELISA, LCD array and IS900 PCR) for detection of Johne's disease. A total of 365 Holstein-Friesian dairy cattle (40 express profuse diarrhea unresponsive to treatment and 325 contacting them) tested with Johnin test, blood collected for ELISA and fecal samples for fecal staining as well as fecal culture, application of LCD array and PCR using IS900 on DNA extracted from Mycobacterium paratuberculosis bacilli (from feces and culture). Johnin test was 40/40 (100%) and 25/325 (7.69%), fecal staining was 13 (37.1%) and 2 (50%), ELISA was 35/40 (87.5%) and 4/25 (16%) for clinical cattle and apparently healthy contacting them respectively. Isolation was 12/13 (92.3%) of the (Johnin test +ve, ELISA +ve and Acid Fast Bacilli +ve) from the clinically positive cattle and 1/2 (50%) of the (Johnin test +ve, ELISA +ve and Acid Fast Bacilli +ve) from apparently healthy contacting them while LCD array and IS900 gave 100% confirming the isolation results. In conclusion, LCD array depending on 16S RNA and DNA hybridization with specific probes for detection of M. paratuberculosis are fast, sensitive and labor-saving when combined with IS900. PMID:26786331

  19. Analysis of culture filtrate and cell wall-associated antigens of Mycobacterium paratuberculosis with monoclonal antibodies.

    PubMed Central

    Mutharia, L M; Moreno, W; Raymond, M

    1997-01-01

    Proteins secreted by Mycobacterium species have been suggested as major immune targets in the early phase of infection. In this study, we sought to identify specific antigens in culture filtrates and in soluble cell extracts of Mycobacterium paratuberculosis. The release of antigens into the culture medium during growth of the bacilli and the distribution of specific epitopes within the Mycobacterium species were investigated by immunoblot analysis with monoclonal antibodies (MAbs) raised against M. paratuberculosis antigens. MAb B6A interacted with a cellular antigen with an apparent molecular mass of 34.5 kDa in lysates of M. paratuberculosis. MAb B6A did not interact with lysates from any other mycobacterial species, suggesting recognition of an M. paratuberculosis species-specific epitope. MAb FL1-A1 reacted with an antigen of 44.3 kDa in M. paratuberculosis and a 9-kDa antigen in Mycobacterium kansasii. MAb PII-B1 reacted with concanavalin A (ConA)-binding cellular and filtrate molecules of M. paratuberculosis and with lysates of Mycobacterium kansasii and Mycobacterium avium 18. The affinity-purified glycosylated antigens migrated as a diffuse band of between 35 and 45.6 kDa and reacted strongly with ovine and bovine paratuberculosis serum and polyclonal serum against M. tuberculosis lipoarabinomannan antigens. These glycoconjugates were the earliest antigens detected in culture filtrates of M. paratuberculosis. Deglycosylation of the ConA-binding molecules with alpha-mannosidase enzyme abolished the reaction with MAb PII-B1 and with bovine but not ovine paratuberculosis serum, suggesting selective immunogenicity in the different animal species. PMID:9009287

  20. Detection of Mycobacterium avium subspecies paratuberculosis in patients with Crohn's disease is unrelated to the presence of single nucleotide polymorphisms rs2241880 (ATG16L1) and rs10045431 (IL12B).

    PubMed

    Dalton, James P; Desmond, Alan; Shanahan, Fergus; Hill, Colin

    2014-06-01

    Mycobacterium avium subspecies paratuberculosis (MAP) has been controversially linked with Crohn's disease (CD). Detection of MAP in CD has been highly variable, and one explanation might be the genetic heterogeneity of this syndrome. Many of the single nucleotide polymorphisms (SNPs) linked with CD are contained within genes that are associated with bacterial handling in general, and some are specifically implicated in susceptibility to mycobacterial disease. We tested a cohort of IBD patients (n = 149) to determine whether the presence of MAP was associated with a selection of these SNPs. Blood samples from CD patients (n = 84), ulcerative colitis (UC, n = 65) patients and healthy controls (n = 55) were examined for the presence of MAP and SNPs in ATG16L1, IL12B, NOD2/CARD15, NKx2-3, IL23R and IRGM. Statistical analysis was then used to determine whether there was any association between the presence of MAP and these SNPs. MAP, rs2241880 (ATG16L1) and rs10045431 (IL12B) were found to be significantly associated with CD. The presence of MAP was not related to the status of the SNPs in ATG16L1 or IL12B. We have found no evidence for the contribution of these SNPs to the presence of MAP in CD patients. PMID:24522266

  1. Mycobacterium avium subsp. Paratuberculosis and the expression of selected virulence and pathogenesis genes in response to 6°C, 65°c and ph 2.0

    PubMed Central

    Pribylova, Radka; Kralik, Petr; Donnelly, Neysan; Matiasovic, Jan; Pavlik, Ivo

    2011-01-01

    The aim of this work was to study the expression of selected Mycobacterium avium subsp. paratuberculosis (MAP) genes connected with MAP virulence, adhesion and stress response. The temperature of 6°C and 65°C were chosen with regard to the food industry, storage conditions (refrigerator) and low-temperature pasteurization. A pH of 2.0, using lactic acid, was selected to mimic the natural environment of the stomach. Expression of selected genes was studied using real time reverse transcription PCR on three different MAP isolates. MAP isolates were chosen according to the number of their preceding cultivations. While isolates 8672 and 8819 were previously cultivated only once, MAP isolate 12146 went through four passages. Different expression profiles were observed in each of the three MAP isolates. However, particular similar patterns were observed. SigE, sigF and ahpC were up-regulated, while sigL was down-regulated under temperature stress. Mmp gene was found to be down-regulated under acidic conditions. Low passage isolates (8672 and 8819) showed certain level of acid resistance. PMID:24031696

  2. Subspecies Identification and Significance of 257 Clinical Strains of Mycobacterium avium

    PubMed Central

    Tran, Quynh T.

    2014-01-01

    Mycobacterium avium is abundant in the environment. It has four subspecies of three types: the human or porcine type, M. avium subsp. hominissuis; the bird type, including M. avium subsp. avium serotype 1 and serotype 2, 3 (also M. avium subsp. silvaticum); and the ruminant type, M. avium subsp. paratuberculosis. We determined the subspecies of 257 M. avium strains isolated from patients at the M.D. Anderson Cancer Center from 2001 to 2010 and assessed their clinical significance. An assay of multiplex PCR was used for the typing. Results showed M. avium subsp. hominissuis to be most common (n = 238, 92.6%), followed by M. avium subsp. avium serotype 1 (n = 12, 4.7%) and serotype 2, 3 (n = 7, 2.7%). No strains of M. avium subsp. paratuberculosis were found. Of the 238 patients with M. avium subsp. hominissuis, 65 (27.3%) showed evidence of definite or probable infections, mostly in the respiratory tract, whereas the rest had weak evidence of infection. The bird-type subspecies, despite being infrequently isolated, caused relatively more definite and probable infections (10 of 19 strains, 52.6%). Overall, women of 50 years of age or older were more prone to M. avium infection than younger women or men of all ages were. We therefore conclude that M. avium subsp. hominissuis is the dominant M. avium subspecies clinically, that the two bird-type subspecies do cause human infections, and that M. avium infects mainly postmenopausal women. The lack of human clinical isolation of the ruminant type subspecies may need further investigation. PMID:24501026

  3. Identification of seroreactive proteins in the culture filtrate antigen of Mycobacterium avium ssp. paratuberculosis human isolates to sera from Crohn's disease patients.

    PubMed

    Shin, A-Rum; Kim, Hwa-Jung; Cho, Sang Nae; Collins, Michael T; Manning, Elizabeth J B; Naser, Saleh A; Shin, Sung Jae

    2010-02-01

    The etiology of Crohn's disease (CD) is unresolved, but it is likely that an interplay of host genetic factors and environmental triggers is relevant. Mycobacterium paratuberculosis (MAP) has been focused upon as one of these triggers because it causes a similar chronic inflammatory bowel disease in animals. However, the differences among MAP antigens isolated from humans (H-MAP) and cattle (B-MAP) have not been well characterized. In this study, culture filtrate (CF) proteins from MAP isolates were tested with sera from CD patients and healthy controls in enzyme-linked immunosorbent assay (ELISA). Antibody produced by seven CD patients reacted differently according to the antigen source: strong reactivity was seen to H-MAP CF, but not to B-MAP CF. Six proteins, ModD, PepA, transaldolase, EchA9, MAP2120c, and MAP2950c, in H-MAP CF reacting specifically with CD patient sera were identified by liquid chromatography-electrospray ionization-MS. Bioinformatic analysis revealed that ModD and PepA were the same proteins reacting with sera from cattle infected with MAP. The elevated antibody responses of CD patients to rModD and rPepA were confirmed by ELISA (P<0.001). These results support previous studies showing ModD and PepA as key antigens for the diagnosis of MAP infections. The study also identified additional proteins potentially useful in the design of assays for human MAP infections. PMID:19878316

  4. Development and Characterization of Monoclonal Antibodies and Aptamers against Major Antigens of Mycobacterium avium subsp. paratuberculosis▿

    PubMed Central

    Bannantine, John P.; Radosevich, Thomas J.; Stabel, Judith R.; Sreevatsan, Srinand; Kapur, Vivek; Paustian, Michael L.

    2007-01-01

    Specific antibodies, available in unlimited quantities, have not been produced against Mycobacterium avium subsp. paratuberculosis, the bacterium that causes Johne's disease (JD). To fill this gap in JD research, monoclonal antibodies (MAbs) against M. avium subsp. paratuberculosis were produced from BALB/c mice immunized with a whole-cell extract of M. avium subsp. paratuberculosis. A total of 10 hybridomas producing MAbs to proteins ranging from 25 to 85 kDa were obtained. All MAbs showed some degree of cross-reactivity when they were analyzed against a panel of whole-cell protein lysates comprising seven different mycobacterial species. The MAbs were characterized by several methods, which included isotype analysis, specificity analysis, epitope analysis, reactivity in immunoblot assays, and electron microscopy. The identities of the antigens that bound to two selected MAbs were determined by screening an M. avium subsp. paratuberculosis lambda phage expression library. This approach revealed that MAb 9G10 detects MAP1643 (isocitrate lyase) and that MAb 11G4 detects MAP3840 (a 70-kDa heat shock protein), two proteins present in high relative abundance in M. avium subsp. paratuberculosis. The epitopes for MAb 11G4 were mapped to the N-terminal half of MAP3840, whereas MAb 9G10 bound to the C-terminal half of MAP1643. Aptamers, nucleic acids that bind to specific protein sequences, against the hypothetical protein encoded by MAP0105c were also generated and tested for their binding to M. avium subsp. paratuberculosis as well as other mycobacteria. These detection reagents may be beneficial in many JD research applications. PMID:17344350

  5. CD4⁺ T-cells, γδ T-cells and B-cells are associated with lack of vaccine protection in Mycobacterium avium subspecies paratuberculosis infection.

    PubMed

    de Silva, Kumudika; M Plain, Karren; J Begg, Douglas; C Purdie, Auriol; J Whittington, Richard

    2015-01-01

    Vaccination is one of the strategies used to control the spread of Mycobacterium avium subspecies paratuberculosis (MAP) infection in livestock. Gudair(®) is a widely-used vaccine in sheep and goats and is the only vaccine approved for use in sheep in Australia and New Zealand. This vaccine reduces mortality due to MAP-infection by up to 90% but some sheep remain infectious by shedding MAP in faeces, despite vaccination. In this study, using an experimental infection model in sheep, our aim was to assess differences in immune parameters between vaccinated MAP-exposed sheep in which the vaccine was effective compared to those in which it failed to protect against infection. We assessed immune parameters such as MAP-specific IFNγ, IL-10 and lymphocyte proliferative responses and serum antibody levels. At the end of the trial, 72% of non-vaccinated sheep and 24% of vaccinated sheep were infected, as defined by the detection of viable MAP in intestinal tissues when the trial was terminated at 49 weeks post exposure. There were significant differences in the proliferation of CD4(+), B and γδ T-cells over time in vaccinated sheep in which the vaccine failed to protect against infection compared to the non-infected vaccinated sheep. There were no significant differences in the IFNγ response or serum antibody levels between the vaccinated infected and vaccinated non-infected sheep. These results emphasise the importance of specific lymphocyte subsets in protecting against MAP-infection, especially in vaccinated sheep, and that immune parameters other than the commonly used IFNγ and antibody tests are required when assessing vaccine efficacy. PMID:25444806

  6. 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.

    PubMed

    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 (<20%) sequence similarity to, and significantly different catalytic functions from, BLC. cAOS transforms 8R-hydroperoxy-eicosatetraenoic acid to an allene epoxide, whereas the MAP protein is a putative organic peroxide-dependent peroxidase. To elucidate factors influencing the functions of these and related heme proteins, we have investigated the heme iron coordination properties of these tyrosinate-ligated heme 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. PMID:22104301

  7. TLR4, IL10RA, and NOD2 mutation in paediatric Crohn's disease patients: an association with Mycobacterium avium subspecies paratuberculosis and TLR4 and IL10RA expression.

    PubMed

    Wagner, Josef; Skinner, Narelle A; Catto-Smith, Anthony G; Cameron, Donald J S; Michalski, Wojtek P; Visvanathan, Kumar; Kirkwood, Carl D

    2013-08-01

    Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated in the pathogenesis of Crohn's disease (CD). The role of CD susceptibility genes in association with these microbes is not known. Sixty-two early onset paediatric CD patients and 46 controls with known MAP status were analysed for an association with 34 single nucleotide polymorphisms (SNPs) from 18 CD susceptibility genes. Functional studies on peripheral blood mononuclear cells (PBMCs) were conducted on 17 CD patients with known CD mutations to assess IL-6, IL-10, and TNF-α expression upon stimulation with MAP precipitated protein derivative (PPD) and lipopolysaccharide (LPS). In addition, surface expression of IL10R and TLR4 on resting B cells, NK cells, T cells, and monocytes was assessed. A mutation in TLR4 (rs4986790) and IL10RA (rs22291130) was significantly associated with MAP-positive CD patients compared to MAP-negative CD patients (27.6 vs. 6.1 %, p = 0.021, and 62.1 vs. 33.3 %, p = 0.024, respectively). PPD and LPS significantly increased IL-6, IL-10, and TNF-α production in PBMCs. IL-10 and TNF-α production were significantly lower in a subgroup of CD patients (5/12) with a known NOD2 mutation. Receptor for IL-10 was significantly higher expressed on NK cells (CD56low) and on NK T cells harbouring a NOD2 mutations compared to wildtype cells (p = 0.031 and 0.005, respectively). TLR4 was significantly higher expressed on NK cells (CD56high) harbouring a NOD2 mutations compared to wildtype cells (p = 0.038). PMID:23455702

  8. Blocking the mitogen activated protein kinase-p38 pathway is associated with increase expression of nitric oxide synthase and higher production of nitric oxide by bovine macrophages infected with Mycobacterium avium subsp paratuberculosis.

    PubMed

    Souza, Cleverson D

    2015-03-15

    This study evaluated the role of the mitogen-activated protein kinase (MAPK)-p38 pathway in the nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by bovine monocyte-derived macrophages ingesting Mycobacterium avium subsp. paratuberculosis (MAP) organisms in vitro. Bovine monocyte-derived macrophages were incubated with MAP organisms with or without a specific inhibitor of the MAPKp38 pathway and activation of the MAPKp38, interleukin - (IL) IL-10, IL-12, iNOS mRNA expression and NO production were evaluated. Incubation of macrophages with MAP organisms activates the MAPKp38 pathway at early time points post infection. Chemically inhibition of MAPKp38 before incubation of bovine macrophages with MAP resulted in increased expression of IL-12 mRNA at 2, 6 and 24h, decreased expression of IL-10 mRNA at 2, 6 and 24h and increased expression of iNOS mRNA at 2 and 6h. Nitric oxide was evaluated to indirectly determine the effects of MAPKp38 pathway on the anti-microbial activity of bovine macrophages. Incubation of bovine macrophages with MAP resulted in modest increased production of NO at 4 and 6h post infection. Pretreatment of bovine macrophages with the MAPKp38 inhibitor SB203580 before addition of MAP organisms resulted in increased production of NO at 2, 4, 6 and 24h post infection. This study expanded our knowledge of the importance of the MAPKp38 pathway in limiting an appropriate macrophage response to MAP and suggested how activation of MAPKp38 pathway may be a target of this organism to disrupt earlier antimicrobial mechanisms of macrophages. These findings raises the interesting possibility that the cellular manipulation of MAPKp38 may be useful in designing novel vaccines against MAP. PMID:25700780

  9. Coordination modes of tyrosinate-ligated 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

    PubMed Central

    Bandara, D. M. Indika; Sono, Masanori; Bruce, Grant S.; Brash, Alan R.; Dawson, John H.

    2012-01-01

    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 (< 20%) sequence similarity to, and significantly different catalytic functions from, BLC. cAOS transforms 8R-hydroperoxy-eicosatetraenoic acid to an allene epoxide, whereas the MAP protein is an organic peroxide-dependent peroxidase. To shed light on the functional differences among these three proteins, we have investigated the heme iron coordination properties of these tyrosinate-ligated heme proteins 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 His93Tyr Mb, which may be attributed to the presence of an Arg+-Nω-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)-O2 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+-Nω-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. PMID:22104301

  10. Reducing human exposure to Mycobacterium avium.

    PubMed

    Falkinham, Joseph O

    2013-08-01

    In light of the increasing prevalence of Mycobacterium avium pulmonary disease and the challenges of treating patients with M. avium infection, consideration of measures to reduce exposure is warranted. Because M. avium inhabits water and soil, humans are surrounded by that opportunistic pathogen. Because infection has been linked to the presence of M. avium in household plumbing, increasing hot water temperature, reducing aerosol (mist) exposures in bathrooms and showers, and installing filters that prevent the passage of mycobacteria will likely reduce M. avium exposure. Granular activated carbon (charcoal) filters support the growth of M. avium and should be avoided. When gardening, avoid the inhalation of soil dusts by using a mask or wetting the soil because peat-rich potting soils have high numbers of mycobacteria. PMID:23952861

  11. Identification of Immune Parameters To Differentiate Disease States among Sheep Infected with Mycobacterium avium subsp. paratuberculosis▿

    PubMed Central

    Gillan, Sonia; O'Brien, Rory; Hughes, Alan D.; Griffin, J. Frank T.

    2010-01-01

    Johne's disease, a chronic enteritis of ruminants, is caused by infection with Mycobacterium avium subsp. paratuberculosis. Three distinct forms have been observed in sheep: paucibacillary disease (PB), multibacillary disease (MB), and asymptomatic infection (AS). In this study, immune parameters for animals naturally infected with M. avium subsp. paratuberculosis and identified postmortem as having PB, MB, or AS were compared to provide a further understanding of the immunological reactivity contributing to or resulting from these different disease states in sheep. PB was associated with strong ex vivo M. avium subsp. paratuberculosis antigen-stimulated gamma interferon responses, pronounced increases in CD25+ T-cell frequencies in circulation, antibody production, and a B-cell population that expanded significantly upon ex vivo antigenic stimulation. The MB group featured the highest antibody levels and a lack of cellular immune responsiveness to the M. avium subsp. paratuberculosis antigen. The AS group expressed an immunological phenotype intermediate between that for noninfected control animals and that for the PB group. The relationship between immune responses and disease severity within the PB group was investigated more closely; significant positive correlations were observed between disease severity and both the CD8+ population in the circulating blood and the expression of interleukin-4 mRNA in antigen-stimulated blood samples ex vivo. Together, these data point toward distinct immune profiles in sheep that correspond to different Johne's disease states, which can be determined from circulating blood and/or from localized intestinal tract tissue samples. PMID:19923568

  12. Thiopurine drugs azathioprine and 6-mercaptopurine inhibit Mycobacterium paratuberculosis growth in vitro.

    PubMed

    Shin, Sung Jae; Collins, Michael T

    2008-02-01

    The in vitro susceptibility of human- and bovine-origin Mycobacterium paratuberculosis to the thioupurine drugs 6-mercaptopurine (6-MP) and azathioprine (AZA) was established using conventional plate counting methods and the MGIT 960 ParaTB culture system. Both 6-MP and AZA had antibacterial activity against M. paratuberculosis; isolates from Crohn's disease patients tended to be more susceptible than were bovine-origin isolates. Isolates of Mycobacterium avium, used as controls, were generally resistant to both AZA and 6-MP, even at high concentrations (> or =64.0 microg/ml). Among rapidly growing mycobacteria, Mycobacterium phlei was susceptible to 6-MP and AZA whereas Mycobacterium smegmatis strains were not. AZA and 6-MP limited the growth of, but did not kill, M. paratuberculosis in a dose-dependent manner. Anti-inflammatory drugs in the sulfonamide family (sulfapyridine, sulfasalazine, and 5-aminosalycilic acid [mesalamine]) had little or no antibacterial activity against M. paratuberculosis. The conventional antibiotics azithromycin and ciprofloxacin, used as control drugs, were bactericidal for M. paratuberculosis, exerting their killing effects on the organism relatively quickly. Simultaneous exposure of M. paratuberculosis to 6-MP and ciprofloxacin resulted in significantly higher CFU than use of ciprofloxacin alone. These data may partially explain the paradoxical response of Crohn's disease patients infected with M. paratuberculosis to treatment with immunosuppressive thiopurine drugs, i.e., they do not worsen with anti-inflammatory treatment as would be expected with a microbiological etiologic pathogen. These findings also should influence the design of therapeutic trials to evaluate antibiotic treatments of Crohn's disease: AZA drugs may confound interpretation of data on therapeutic responses for both antibiotic-treated and control groups. PMID:18070971

  13. Cloning and expression of portions of the 34-kilodalton-protein gene of Mycobacterium paratuberculosis: its application to serological analysis of Johne's disease.

    PubMed Central

    De Kesel, M; Gilot, P; Misonne, M C; Coene, M; Cocito, C

    1993-01-01

    Paratuberculosis (Johne's disease), an endemic mycobacteriosis of cattle that is caused by Mycobacterium paratuberculosis, is characterized by incoercible diarrhea and fecal shedding of bacteria. The present work aimed at developing a specific serological test for this disease. We have recently shown that a 34-kDa protein belonging to the major antigen complex A36 of M. paratuberculosis is immunodominant and contains epitopes specific with respect to all mycobacteria tested, including Mycobacterium bovis and the closely related species Mycobacterium avium. From a lambda gt11 genomic library of M. paratuberculosis, three portions of the gene coding for this 34-kDa protein have been isolated. Two of them expressed cross-reacting mycobacterial epitopes. One portion (in clone a362) expressed a polypeptide which cross-reacted with all tested M. paratuberculosis strains but not with 20 other bacteria tested, including many strains of the M. avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum group. The occurrence at the M. paratuberculosis surface of epitopes corresponding to the a362 polypeptide was shown by immune electron microscopy. The recombinant a362 polypeptide was used as reagent for an enzyme-linked immunoassay for paratuberculosis. This assay correctly diagnosed all the tested blood samples from infected cattle at all stages of the disease. Images PMID:7681851

  14. Detection of Mycobacterium avium in pet birds

    PubMed Central

    Godoy, Silvia Neri; Sakamoto, Sidnei Miyoshi; de Paula, Cátia Dejuste; Catão-Dias, José Luiz; Matushima, Eliana Reiko

    2009-01-01

    The present study is a report on the presence of Mycobacterium avium in four birds of the psittaciform order kept as pets. Anatomopathological diagnosis showed lesions suggestive of the agent and presence of alcohol-acid resistant bacilli (AARB) shown by the Ziehl-Neelsen staining. The identification of Mycobacterium avium was performed by means of PRA (PCR Restriction Analysis). DNA was directly extracted from tissue of the lesions and blocked in paraffin. The role of this agent in pet bird infection is discussed, as well as its zoonotic potential. PMID:24031356

  15. Investigation on the occurrence and pathology of paratuberculosis (Johne's disease) in apparently healthy cattle in Jordan

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Paratuberculosis (Johne’s disease) is infectious, incurable, chronically progressive granulomatous enteritis which affects domestic and exotic ruminants. The causative agent is Mycobacterium avium paratuberculosis (M. Johnei), a slow growing mycobactin-dependent acid-fast bacillus. We investigated t...

  16. 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

    PubMed Central

    Riepema, Karel; Bakker, Douwe; Gordon, Stephen V.

    2015-01-01

    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

  17. 'Bio-load' and bio-type profiles of Mycobacterium avium subspecies paratuberculosis infection in the domestic livestock population endemic for Johne's disease: a survey of 28 years (1985-2013) in India.

    PubMed

    Singh, S V; Singh, P K; Singh, A V; Sohal, J S; Kumar, N; Chaubey, K K; Gupta, S; Rawat, K D; Kumar, A; Bhatia, A K; Srivastav, A K; Dhama, K

    2014-08-01

    Bio-load and bio-profile of Mycobacterium avium subspecies paratuberculosis was studied in the domestic livestock population of the country. Of the 23,429 farm and farmer's animals screened, average bio-load was 23.3% (Period of study; 28 years for goats; 13 years for sheep, cattle and buffaloes). Species-wise, bio-load was 20.1, 32.7, 39.3 and 28.3% in goats, sheep, cattle and buffaloes, respectively. Bio-load was significantly lower in time period A (P < 0.001) and B (P < 0.03), compared with period C. Geographical zone-wise, bio-load of MAP was significantly higher (P < 0.05) in Central zone compared with South, West, East and North zones. Bio-load in 11 states ranged from 16.2 to 87.8%. Of 8450, 5643, 8185 and 1151 samples screened by microscopy, culture, indigenous ELISA and IS900 blood PCR, 20.0, 10.6, 35.1 and 26.6% samples were positive, respectively. Bio-load was 32.8 and 31.6% in farm and farmer's goats and sheep, respectively, and 62.1% in farmer's cattle. MAP bio-load was also monitored in four farm units (three goats and one sheep) for breed improvement and three farm goats units for experimental purposes at Central Institute for Research on Goats in Mathura district. Of the 8025 goats and 1525 sheep that died from 1988 to 2013, 10.9 and 3.0% deaths were due to JD, respectively. On the basis of JD and suspected JD, 10.0 and 28.4% goats and 2.2 and 40.9% sheep, respectively were culled from the farm units in 25 years. Microscopic examination of 214 tissues (mesenteric lymph nodes and intestines) of 107 animals, it was observed that bio-load of MAP was high (25.0-60.0%) in farm animals. 'Indian Bison Type' was the dominant biotype, irrespective of domestic livestock species and the geographical zone. PMID:25135463

  18. PARATUBERCULOSIS: AN UPDATE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Paratuberculosis (Johne’s disease) is a chronic, progressive enteric disease of ruminants caused by infection with Mycobacterium avium subsp. paratuberculosis. Economic losses from this disease are estimated to be $200US/infected cow/year and are the result of animal culling, reduced milk productio...

  19. ELECTROPHORETIC MOBILITY OF MYCOBACTERIUM AVIUM COMPLEX ORGANISMS

    EPA Science Inventory

    The electrophoretic mobilities (EPMs) of thirty Mycobacterium avium Complex (MAC) organisms isolated from clinical and environmental sources were measured in 9.15 mM KH2PO4 buffered water. The EPMs of fifteen clinical isolates ranged from -1.9 to -5.0 µm cm V-1 ...

  20. ELECTROPHORETIC MOBILITY OF MYCOBACTERIUM AVIUM COMPLEX ORGANISMS

    EPA Science Inventory

    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...

  1. Sero-prevalence of bovine Johne's disease in buffaloes and cattle population of North India using indigenous ELISA kit based on native Mycobacterium avium subspecies paratuberculosis 'Bison type' genotype of goat origin.

    PubMed

    Singh, S V; Singh, A V; Singh, R; Sharma, S; Shukla, N; Misra, S; Singh, P K; Sohal, J S; Kumar, H; Patil, P K; Misra, P; Sandhu, K S

    2008-09-01

    Present pilot study is the first attempt in the country to estimate sero-prevalence of Bovine Johne's disease (BJD) by screening cattle and buffaloes representing large population belonging to farmer's and farm herds in the home tracts (Uttar Pradesh (UP) and Punjab) of Hariana cattle and Murrah buffaloes in North India. Indigenous and in-house plate ELISA kit (using protoplasmic antigen from native Mycobacterium avium subsp. paratuberculosis 'Bison type' strain of goat origin), originally developed for goats and sheep was standardized in bovines and used for screening. For this study, 33 villages of south and west UP were randomly selected and surveyed from 2001 to 2003. There were 7943 farmer's families having 38,251 livestock, including cattle, buffaloes, goats and sheep (per family 4.8% livestock). Numerically, buffaloes and cattle were 54.7% and 22.1%, respectively. Serum samples were collected from 726 animals (4.2% of 16, 981 livestock with 4375 farmer's families) located in 33 randomly surveyed villages. Serum samples (699), submitted to Epidemiology Department of Veterinary College (Punjab Agricultural University, Ludhiana), in the year 2004 by farmer's and organized farm herds (Buffaloes, 372, Cattle, 327), were screened by this ELISA kit. Soluble protoplasmic antigen was prepared from Map (S 5) 'Bison type' strain isolated from a terminally sick goat with Johne's disease. Of the total 1425 bovine (Buffaloes and cattle) serum samples screened using indigenous ELISA kit, sero-prevalence of Johne's disease was 29.0% (28.6% in buffalo and 29.8% in cattle) in Northern India. State-wise sero-prevalence was 31.9% and 23.3% in UP and Punjab, respectively. In UP, of the 601 randomly sampled buffaloes, sero-prevalence was 40.3% (16.6% in young and 40.9% adults) and 25.5% (10.5% in young and 26.3% adults) in south and west UP, respectively. Of the 125 cattle screened, sero-prevalence was 42.6% (nil in young and 44.4% adults) and 30.0% (nil in young and 30.6% adults

  2. MYCOBACTERIUM AVIUM AND DRINKING WATER WHAT ARE THE CONNECTIONS?

    EPA Science Inventory

    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...

  3. New probes used for IS1245 and IS1311 restriction fragment length polymorphism of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis isolates of human and animal origin in Norway

    PubMed Central

    Johansen, Tone Bjordal; Olsen, Ingrid; Jensen, Merete Rusås; Dahle, Ulf R; Holstad, Gudmund; Djønne, Berit

    2007-01-01

    Background Mycobacterium avium is an environmental mycobacterium that can be divided into the subspecies avium, hominissuis, paratuberculosis and silvaticum. Some M. avium subspecies are opportunistic pathogens for animals and humans. They are ubiquitous in nature and can be isolated from natural sources of water, soil, plants and bedding material. Isolates of M. avium originating from humans (n = 37), pigs (n = 51) and wild birds (n = 10) in Norway were examined by IS1245 and IS1311 RFLP using new and specific probes and for the presence of IS901 and ISMpa1 by PCR. Analysis and generation of a dendrogram were performed with the software BioNumerics. Results IS1311 RFLP provided clear results that were easy to interpret, while IS1245 RFLP generated more complex patterns with a higher discriminatory power. The combination of the two methods gave additional discrimination between isolates. All avian isolates except one were M. avium subsp. avium with two copies of IS1311 and one copy of IS1245, while the isolates of human and porcine origin belonged to M. avium subsp.hominissuis. The isolates from human patients were distributed randomly among the clusters of porcine isolates. There were few identical isolates. However, one isolate from a human patient was identical to a porcine isolate. Regional differences were detected among the porcine isolates, while there was no clustering of human isolates according to type of clinical symptoms or geographical location of the patient's home addresses. Conclusion The results demonstrate that a wide range of M. avium subsp.hominissuis are present in pigs and humans in Norway, and that some of these isolates are very similar. It remains to be determined whether humans are infected from pigs or if they are infected from common environmental sources. PMID:17335590

  4. Methylation of GPLs in Mycobacterium smegmatis and Mycobacterium avium

    PubMed Central

    Jeevarajah, Dharshini; Patterson, John H.; Taig, Ellen; Sargeant, Tobias; McConville, Malcolm J.; Billman-Jacobe, Helen

    2004-01-01

    Several species of mycobacteria express abundant glycopeptidolipids (GPLs) on the surfaces of their cells. The GPLs are glycolipids that contain modified sugars including acetylated 6-deoxy-talose and methylated rhamnose. Four methyltransferases have been implicated in the synthesis of the GPLs of Mycobacterium smegmatis and Mycobacterium avium. A rhamnosyl 3-O-methytransferase and a fatty acid methyltransferase of M. smegmatis have been previously characterized. In this paper, we characterize the methyltransferases that are responsible for modifying the hydroxyl groups at positions 2 and 4 of rhamnose and propose the biosynthetic sequence of GPL trimethylrhamnose formation. The analysis of M. avium genes through the creation of specific mutants is technically difficult; therefore, an alternative approach to determine the function of putative methyltransferases of M. avium was undertaken. Complementation of M. smegmatis methyltransferase mutants with M. avium genes revealed that MtfC and MtfB of the latter species have 4-O-methyltransferase activity and that MtfD is a 3-O-methyltransferase which can modify rhamnose of GPLs in M. smegmatis. PMID:15466031

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

    PubMed

    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. PMID:26964909

  6. Inactivation of Mycobacterium paratuberculosis in cows' milk at pasteurization temperatures.

    PubMed Central

    Grant, I R; Ball, H J; Neill, S D; Rowe, M T

    1996-01-01

    The thermal inactivation of 11 strains of Mycobacterium paratuberculosis at pasteurization temperatures was investigated. Cows' milk inoculated with M. paratuberculosis at two levels (10(7) and 10(4) CFU/ml) was pasteurized in the laboratory by (i) a standard holder method (63.5 degrees C for 30 min) and (ii) a high-temperature, short-time (HTST) method (71.7 degrees C for 15 s). Additional heating times of 5, 10, 15, 20, and 40 min at 63.5 degrees C were included to enable the construction of a thermal death curve for the organism. Viability after pasteurization was assessed by culture on Herrold's egg yolk medium containing mycobactin J (HEYM) and in BACTEC Middlebrook 12B radiometric medium supplemented with mycobactin J and sterile egg yolk emulsion. Confirmation of acid-fast survivors of pasteurization as viable M. paratuberculosis cells was achieved by subculture on HEYM to indicate viability coupled with PCR using M. paratuberculosis-specific 1S900 primers. When milk was initially inoculated with 10(6) to 10(7) CFU of M. paratuberculosis per ml, M. paratuberculosis cells were isolated from 27 of 28 (96%) and 29 of 34 (85%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. Correspondingly, when 10(3) to 10(4) CFU of M. paratuberculosis per ml of milk were present before heat treatment, M. paratuberculosis cells were isolated from 14 of 28 (50%) and 19 of 33 (58%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. The thermal death curve for M. paratuberculosis was concave in shape, exhibiting a rapid initial death rate followed by significant "tailing." Results indicate that when large numbers of M. paratuberculosis cells are present in milk, the organism may not be completely inactivated by heat treatments simulating holder and HTST pasteurization under laboratory conditions. PMID:8593064

  7. DETECTION,QUANTIFICATION AND CHARACTERIZATION OF MYCOBACTERIUM AVIUM COMPLEX (MAC) ORGANISMS IN DRINKING WATER.

    EPA Science Inventory

    Bacteria belonging to the Mycobacterium avium complex (MAC), including Mycobacterium avium and M. intracellulare, are clinically relevant and cause a myriad of opportunistic infections. Children, the elderly, and persons with previous lung conditions or immune system dysfunction...

  8. The associations between culling due to clinical Johne's disease or the detection of Mycobacterium avium subsp. paratuberculosis fecal shedding and the diagnosis of clinical or subclinical diseases in two dairy herds in Minnesota, USA.

    PubMed

    Raizman, Eran A; Wells, Scott J; Godden, Sandra M; Fetrow, John; Oakes, J Michael

    2007-07-16

    Our objectives were to identify associations between clinical or subclinical diseases and subsequent culling because of clinical Johne's disease (JD) or the detection of fecal shedding of Mycobacterium paratuberculosis (Map) after 305 days in milk (DIM). A total of 1297 cows from two Minnesota dairies were enrolled in the study. From study cows, fecal samples were obtained prior to calving (close-up period) and after at least 305 DIM or at the time of leaving the herd (sold/dead). Between 3 and 21 DIM, blood samples were obtained for serum betahydroxybutyrate (BHB) and serum total-protein testing. Body-condition score (BCS) was evaluated during the close-up period, between 3 and 21 DIM, and at the end of lactation. The diagnosis time (DIM) of clinical disease events (culling because of JD clinical signs, ketosis, lameness, mastitis, displacement abomasum, injury, metritis, milk fever, pneumonia, and retained placenta) was recorded. Sixty-six cows were culled because of JD clinical signs (CCDJ) with average DIM of 209. CCDJ was associated with event of pneumonia (n=131) (OR=3.0, 95% CI=1.0-6.0) and level of fecal shedding (light: OR=13.0, 95% CI=5.3-30.0; moderate: OR=34.0, 95% CI=13.0-89.0; heavy: OR=66.0, 95% CI=26.0-171.0). Detection of fecal shedding at the end of the lactation (n=79) was associated only with event of pneumonia (OR=2.0, 95% CI=1.0-4.0). PMID:17368838

  9. A rabbit model for study of Mycobacterium paratuberculosis infection.

    PubMed Central

    Mokresh, A H; Czuprynski, C J; Butler, D G

    1989-01-01

    Of 21 newborn rabbits inoculated orally with Mycobacterium paratuberculosis ATCC 19698, 13 (62%) became infected, as determined by histopathology and culture. Of the 21 inoculated rabbits, 14 (67%) experienced episodes of intermittent diarrhea, sometimes as early as 5 months after inoculation. Feces varied in consistency from soft-semisolid to watery. The organism was isolated from the sacculus rotundus, vermiform appendix of the cecum, ileum, mesenteric lymph node, and feces of 9 of 21 (43%) M. paratuberculosis-inoculated rabbits 8 to 10 months after inoculation. One infected rabbit gradually became severely emaciated; advanced paratuberculosis was confirmed by culture and histopathology. Of 21 rabbits, 9 (43%) developed multifocal, well-demarcated granulomatous enteritis in the sacculus rotundus and the vermiform appendix of the cecum. There was no significant difference in the rate of infection when the organisms were administered daily for 5 or 10 days in cow milk or broth. There was no discernible effect of pregnancy, parturition, or lactation on the severity of intestinal lesions, clinical signs, or the number of rabbits infected. Complement fixation and delayed-type hypersensitivity skin tests failed to detect infection. The results of this study suggest that newborn rabbits inoculated orally with M. paratuberculosis constitute a useful animal model for the study of paratuberculosis infection. Images PMID:2807547

  10. Differential Immune Responses of Red Deer (Cervus elaphus) following Experimental Challenge with Mycobacterium avium subsp. paratuberculosis▿

    PubMed Central

    Robinson, Mark; O'Brien, Rory; Mackintosh, Colin; Griffin, Frank

    2008-01-01

    Immune responses of red deer (Cervus elaphus) that presented with different levels of paucibacillary pathology were profiled to detail immune changes during the progression of Johne's disease. Immune responses were monitored using an immunoglobulin G1 (IgG1) antibody enzyme-linked immunosorbent assay (ELISA), a gamma interferon (IFN-γ) ELISA, and flow cytometry. Animals in the study were divided into outcome groups postmortem according to disease severity. All animals mounted IgG1 antibody and IFN-γ responses to both the vaccination and experimental challenges. The Mycobacterium avium subsp. paratuberculosis-specific IgG1 antibody responses in the challenged group showed marked differences between infected and severely diseased animals. Slightly higher IFN-γ responses were seen in infected animals compared with severely diseased animals. No significant changes were seen in the phenotype of lymphocyte populations investigated. Vaccination with killed M. avium subsp. paratuberculosis in mineral oil adjuvant reduced the level of severe disease; however, it obscured immunological differences between the infected and severely diseased groups. This suggests protection is not exclusively mediated via the presence of a type 1 response and, furthermore, the presence of a type 2 response is compatible with protection. These profiles provide information on the different immune processes in Johne's disease progression. PMID:18400974

  11. Endophthalmitis due to Mycobacterium avium in a patient with AIDS.

    PubMed

    Cohen, J I; Saragas, S J

    1990-02-01

    A 27-year-old man with acquired immunodeficiency syndrome [AIDS] had endophthalmitis OS four years after a trabeculectomy was done. The patient had a history of disseminated infection with Mycobacterium avium. Examination showed an intact filtering bleb with inflammation and hypopyon formation in the anterior chamber OS. The M. avium was cultured from an anterior chamber paracentesis. The patient responded to treatment with gentamicin, cefazolin, and prednisolone. Infection with M. avium should be included in the differential diagnosis of endophthalmitis in patients with AIDS. PMID:2316950

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

    PubMed

    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. PMID:24738057

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

    PubMed Central

    Sapierzyński, Rafał; Szeleszczuk, Piotr

    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. PMID:24738057

  14. Proposal to elevate Mycobacterium avium complex ITS sequevar MAC-Q to Mycobacterium vulneris sp. nov.

    PubMed

    van Ingen, J; Boeree, M J; Kösters, K; Wieland, A; Tortoli, E; Dekhuijzen, P N R; van Soolingen, D

    2009-09-01

    The Mycobacterium avium complex (MAC) consists of four recognized species, Mycobacterium avium, Mycobacterium colombiense, Mycobacterium intracellulare and Mycobacterium chimaera, and a variety of other strains that may be members of undescribed taxa. We report on two isolates of a scotochromogenic, slowly growing, non-tuberculous Mycobacterium species within the M. avium complex from a lymph node and an infected wound after a dogbite of separate patients in The Netherlands. The extrapulmonary infections in immunocompetent patients suggested a high level of virulence. These isolates were characterized by a unique nucleotide sequence in the 16S rRNA gene, 99% similar to Mycobacterium colombiense, and the MAC-Q 16S-23S internal transcribed spacer (ITS) sequence. Sequence analyses of the hsp65 gene revealed 97% similarity to M. avium. The rpoB gene sequence was 98% similar to M. colombiense. Phenotypically, the scotochromogenicity, positive semi-quantitative catalase and heat-stable catalase tests, negative tellurite reductase and urease tests and susceptibility to hydroxylamine and oleic acid set these isolates apart from related species. High-performance liquid chromatography analysis of cell-wall mycolic acid content revealed a unique pattern, related to that of M. avium and M. colombiense. Together, these findings supported a separate species status within the Mycobacterium avium complex. We propose elevation of scotochromogenic M. avium complex strains sharing this 16S gene and MAC-Q ITS sequence to separate species status, for which the name Mycobacterium vulneris sp. nov. is proposed. The type strain is NLA000700772T (=DSM 45247T=CIP 109859T). PMID:19620376

  15. Nucleotide sequence analysis and seroreactivities of the 65K heat shock protein from Mycobacterium paratuberculosis.

    PubMed Central

    el-Zaatari, F A; Naser, S A; Engstrand, L; Burch, P E; Hachem, C Y; Whipple, D L; Graham, D Y

    1995-01-01

    Mycobacterium paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. It has also been implicated as a possible cause of Crohn's disease, an inflammatory bowel disease of unknown etiology. The mycobacterial 65K heat shock proteins (hsp-65K) are among the most extensively studied mycobacterial proteins, and their immunogenic characteristics have been suggested to be the basis for autoimmunization in chronic inflammatory diseases. In this context, we isolated and sequenced the hsp-65K-encoding gene from our M. paratuberculosis PTB65K genomic library. A high degree of identity was found between the open reading frame (ORF) of the PTB65K gene and those of Mycobacterium tuberculosis (89.6%), Mycobacterium leprae (86.6%), and Mycobacterium avium 18 (98.8%). The amino acid sequence alignment of the PTB65K protein with the hsp-65K homologs revealed that the M. tuberculosis and M. leprae proteins each differed by 36 amino acid residues and that the M. avium 18 protein differed by 8 residues. We also investigated the humoral immune responses of animals with Johne's disease and patients with Crohn's disease against the recombinant PTB65K antigen. Immunoblot analysis showed that sera from only 3 of 10 clinically ill and 5 of 25 subclinically ill cows reacted with PTB65K. In addition, sera from two of two sheep and one of two goats with clinical symptoms of Johne's disease also reacted with PTB65K; 0 samples from 10 normal cows reacted. In humans, sera from 7 of 13 patients with Crohn's disease, 3 of 4 with tuberculosis, 5 of 6 with leprosy, 5 of 12 with non-inflammatory bowel disease, and 0 of 4 with ulcerative colitis reacted with the recombinant PTB65K antigen. These results indicate that this PTB65K heat shock protein is uninformative when used for serodiagnosis of Johne's disease in animals. However, in humans, the high intensity of antibody reactions of some sera from Crohn's disease patients compared with that from noninflammatory

  16. REGIONAL ASSESSMENT OF EXPOSURE TO MYCOBACTERIUM AVIUM COMPLEX(MAC)

    EPA Science Inventory

    There is accumulating evidence that potable water is a source of Mycobacterium avium complex (MAC). The linkage of mycobacteriosis to drinking water has been shown in AIDS populations where up to 8% of deaths in this group is attributed to MAC. Infection with these organisms ha...

  17. Interaction of Mycobacterium avium with environmental amoebae enhances virulence.

    PubMed Central

    Cirillo, J D; Falkow, S; Tompkins, L S; Bermudez, L E

    1997-01-01

    Environmental mycobacteria are a common cause of human infections. Recently, contaminated domestic water supplies have been suggested as a potential environmental source of several mycobacterial diseases. Since many of these mycobacterial species replicate best intracellularly, environmental hosts have been sought. In the present study, we examined the interaction of Mycobacterium avium with a potential protozoan host, the water-borne amoeba Acanthamoeba castellanii. We found that M. avium enters and replicates in A. castellanii. In addition, similar to that shown for mycobacteria within macrophages, M. avium inhibits lysosomal fusion and replicates in vacuoles that are tightly juxtaposed to the bacterial surfaces within amoebae. In order to determine whether growth of M. avium in amoebae plays a role in human infections, we tested the effects of this growth condition on virulence. We found that growth of M. avium in amoebae enhances both entry and intracellular replication compared to growth of bacteria in broth. Furthermore, amoeba-grown M. avium was also more virulent in the beige mouse model of infection. These data suggest a role for protozoa present in water environments as hosts for pathogenic mycobacteria, particularly M. avium. PMID:9284149

  18. Modification of a bovine ELISA to detect camelid antibodies to Mycobacterium paratuberculosis.

    PubMed

    Kramsky, J A; Miller, D S; Hope, A; Collins, M T

    2000-12-20

    Mycobacterium avium subsp. paratuberculosis infection, or Johne's disease, reportedly has a low prevalence in South American camelid populations. Recently, however, single cases in the United States as well as an outbreak of the disease in Australian alpacas (Lama pacos) have been described. To provide a rapid and cost-effective method of diagnosing Johne's disease in this species, the bovine Parachek((R)) Johne's Absorbed EIA (CSL, Vic., Australia) was modified to create a camelid-specific serum antibody assay. An anti-llama IgG conjugated to horseradish peroxidase replaced the anti-bovine immunoglobulin. Checkerboard titration of principal reagents was performed using serum from nine tissue and/or fecal culture-positive camelids. Optimal dilutions of key components were determined in order to provide clear discrimination between positive and negative controls. Completion of a kinetic assay determined the optical density at which the enzyme-substrate reaction should be stopped. A herd of 100 camelids with no history of disease or exposure to M. a. paratuberculosis, a subset of which were tissue and/or fecal culture-negative, was tested to establish a cut-off value. Sample results were expressed as a percentage of the results for control sera by transforming optical density values to ELISA values (EV%). A preliminary EV% cut-off of 20 was established. Using this prototype assay, culture-positive animals showed significantly different antibody responses from culture-negative animals. These results indicate that this camelid-specific ELISA, once refined, may be a useful tool for screening camelid herds for M. a. paratuberculosis infection. PMID:11118718

  19. GENETIC FINGERPRINTING OF MYCOBACTERIUM AVIUM COMPLEX (MAC) ORGANISMS ISOLATED FROM HOSPITAL PATIENTS AND THE ENVIRONMENT

    EPA Science Inventory

    A particularly pathogenic group of mycobacteria belong to the Mycobacterium avium complex (MAC), which includes M. avium and M. intracellulare. MAC organisms cause disease in children, the elderly, and immuno-compromised individuals. A critical step in preventing MAC infections...

  20. Paratuberculosis Infection of Nonruminant Wildlife in Scotland

    PubMed Central

    Beard, P. M.; Daniels, M. J.; Henderson, D.; Pirie, A.; Rudge, K.; Buxton, D.; Rhind, S.; Greig, A.; Hutchings, M. R.; McKendrick, I.; Stevenson, K.; Sharp, J. M.

    2001-01-01

    Recent reports of natural paratuberculosis (or Johne's disease) in rabbits, foxes, and stoats has focused debate on the presence and importance of wildlife reservoirs in the epidemiology of this disease. This paper describes an extensive study investigating 18 nonruminant wildlife species for evidence of paratuberculosis. Using both culture and histopathological analysis, fox, stoat, weasel, crow, rook, jackdaw, rat, wood mouse, hare, and badger were found to harbor Mycobacterium avium subsp. paratuberculosis, the causative organism of paratuberculosis, suggesting that the epidemiology of this disease is more complex than previously realized. PMID:11283080

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

    PubMed

    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. PMID:24283140

  2. Biochemical characteristics of various strains of Mycobacterium paratuberculosis.

    PubMed

    Chiodini, R J

    1986-07-01

    Biochemical activities of 20 wild-type strains and of 2 laboratory strains of Mycobacterium paratuberculosis were evaluated. Biochemical activities evaluated were growth at 30 C, 37 C, and 42 C; production of urease, niacin, pyrazinamidase, arylsulfatase, and catalase; hydrolyzation of Tween 80; reduction of nitrate and tellurite; and growth in 5% NaCl. Antimicrobial susceptibility to thiophene-2-carboxylic acid hydrazide (10 micrograms/ml), neotetrazolium chloride (1:40,000), streptomycin (2 micrograms/ml), rifampin (0.25 micrograms/ml), and isoniazid (10 micrograms/ml) also was determined. Generally, M paratuberculosis was biochemically inactive, with only a few strains producing pyrazinamidase and maintaining catalase activity after heating. All strains grew optimally at 37 C, grew slightly at 30 C, and did not grow at 42 C. Wild-type strains did not grow in the presence of neotetrazolium chloride, streptomycin, and rifampin, and grew in the presence of thiophene-2-carboxylic acid hydrazide and isoniazid. Although biochemical evaluation can be used as an aid in the identification of M paratuberculosis, growth rate, and mycobactin dependency remain major criteria for positive identification. PMID:3740613

  3. Genetic IS901 RFLP diversity among Mycobacterium avium subsp. avium isolates from four pheasant flocks.

    PubMed

    Moravkova, Monika; Lamka, Jiri; Slany, Michal; Pavlik, Ivo

    2013-01-01

    IS901 RFLP analysis of 36 Mycobacterium avium subsp. avium (MAA) isolates from 15 pheasants (Phasianus colchicus) and two goshawks (Accipiter gentilis) from four pheasant farms was performed. Using this method, six different IS901 RFLP types (E, F, G, M, Q, and V) were identified. The distribution of IS901 RFLP profiles was tightly linked to individual flocks. Matching IS901 RFLP profiles observed in the present study indicate MAA transmission between pheasants and goshawks in the same locality. In two flocks, different pheasants within a flock as well as in various organs of five individual pheasants were found to have two distinct IS901 RFLP profiles. PMID:23388436

  4. Induction of Mycobacterium avium proteins upon infection of human macrophages.

    PubMed

    Brunori, Lara; Giannoni, Federico; Bini, Luca; Liberatori, Sabrina; Frota, Cristiane; Jenner, Peter; Thoresen, Ove Fredrik; Orefici, Graziella; Fattorini, Lanfranco

    2004-10-01

    Induction of Mycobacterium avium proteins labelled with [35S]methionine and mRNAs upon infection of the human macrophage cell line THP-1 was investigated by two-dimensional gel electrophoresis-mass spectrometry and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. M. avium overexpressed proteins within the macrophages that are involved in fatty acids metabolism (FadE2, FixA), cell wall synthesis (KasA), and protein synthesis (EF-tu). The correlation of differential protein and mRNA expression varied between good and no correlation. Overall, these four proteins may be involved in the adaptation and survival of M. avium within human macrophages. PMID:15378697

  5. Isolation of Mycobacterium avium from waterfowl with polycystic livers.

    USGS Publications Warehouse

    Roffe, Thomas J.

    1989-01-01

    An unusual gross appearance of avian tuberculosis, where fluid-filled thin-walled cysts are produced and grossly apparent in preference to granulomas, is presented. Histopathology confirmed the granulomatous nature of the lesions and the presence of intracellular acid-fast organisms. Mycobacterium avium complex was cultured from affected organs. The unusual gross presentation in these cases indicates the need to consider tuberculosis in the differential of cystic diseases of avian livers.

  6. Characterization of a specific Mycobacterium paratuberculosis recombinant clone expressing 35,000-molecular-weight antigen and reactivity with sera from animals with clinical and subclinical Johne's disease.

    PubMed Central

    El-Zaatari, F A; Naser, S A; Graham, D Y

    1997-01-01

    Johne's disease is a chronic enteritis of ruminants associated with enormous worldwide economic losses for the dairy cow- and goat-rearing industries. Management limitations and eradication programs for this disease have been hampered by the lack of a simple and specific diagnostic test for the detection of subclinical cases. We used a recombinant clone expressing a 35,000-molecular-weight Mycobacterium paratuberculosis antigen (p35 antigen) from a previously constructed expression library of M. paratuberculosis in Escherichia coli. The DNA fragment encoding the p35 gene hybridized only to DNA from Mycobacterium avium complex, but not to DNAs from other mycobacteria and nonmycobacterial organisms. The seroreactivity of p35 was evaluated by immunoblotting against 57 reference serum samples obtained from infected and uninfected animals. p35 was recognized by sera from 100% of animals with advanced Johne's disease (clinical stage) (12 cattle, 2 goats, and 2 sheep) and by sera from 75% of 20 cattle with early infection (subclinical stage). None of the sera from 15 M. paratuberculosis-free cows, 3 Mycobacterium bovis BCG-infected tuberculous cattle, or 3 cows artificially inoculated with multiple doses of viable M. paratuberculosis reacted with p35. The overall sensitivity, specificity, positive predictive value, and negative predictive value were 86, 100, 100, and 75%, respectively. The accuracy of p35 immunoblotting was superior to those of commercially available diagnostic tests for Johne's disease. These results suggest that the p35 recombinant protein has potential for use in the serodiagnosis of animals with Johne's disease at all stages of infection. The DNA fragment encoding p35 may also serve as a probe for identification of M. avium complex infection. PMID:9196196

  7. Pathology of subclinical paratuberculosis (Johne's Disease) in Awassi sheep with reference to its occurrence in Jordan

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Paratuberculosis (Johne’s disease) is an incurable infectious, chronically progressive enteric disease affecting domestic and exotic ruminants. The causative agent is Mycobacterium Avium Paratuberculosis (M Johnei). In this study, the occurrence of subclinical Johne’s disease in Awassi sheep is inve...

  8. Comparative Genomic Analysis of Mycobacterium avium subspecies Obtained from Multiple Host Species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A comparative genomic approach was used to identify large sequence polymorphisms among Mycobacterium avium (M. avium) subspecies obtained from a variety of host animals. DNA microarrays were used as a platform for comparing mycobacterial isolates with the sequenced bovine isolate M. avium subsp. p...

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

    PubMed Central

    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

  10. Apoptosis of human monocytes and macrophages by Mycobacterium avium sonicate.

    PubMed Central

    Hayashi, T; Catanzaro, A; Rao, S P

    1997-01-01

    Mycobacterium avium is an intracellular organism which multiplies predominantly within human macrophages. This organism has previously been shown to induce apoptosis in human macrophages. With a view to identifying M. avium components that induce cell death in infected host cells, sonicated extracts of M. avium as well as individual components isolated from the M. avium sonicate were tested in various assays with a human monocytic cell line (THP-1). THP-1 cells incubated with M. avium sonicate showed significantly reduced viability after a 2-day exposure compared to control cells incubated with media alone. This effect was dose dependent, with only 6.6% +/- 5.2% and 48.8% +/- 10.3% of the cells being viable by trypan blue exclusion at 600 and 300 microg/ml, respectively. Control cells, on the other hand, exhibited a viability of 98.8% +/- 1.0%. In addition, an 80% ammonium sulfate fraction of the M. avium sonicate and the previously characterized 68-kDa protein were found to have similar effects on THP-1 cells. In both cases, the reduction in viability was due to apoptosis characterized by chromatin condensation, DNA fragmentation by agarose gel electrophoresis, or terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL) and release of nuclear matrix protein (NMP) into the culture medium. M. avium sonicate-induced apoptosis of THP-1 cells was completely inhibited by the commonly used antioxidants pyrrolidinedithiocarbamate (PDTC) and butylated hydroxyanisole (BHA), indicating that the generation of free oxygen radicals may be responsible for inducing cell death. M. avium sonicate was found to induce apoptosis of monocyte-derived macrophages (MDMs) as well. This effect was not reversed in the presence of PDTC and was not accompanied with DNA fragmentation when determined by agarose gel electrophoresis, as seen in the case of THP-1 cells. However, these MDMs were found to contain fragmented DNA by TUNEL. These findings suggest that the mechanism

  11. Draft genome sequence of a Mycobacterium avium complex isolate from a broadbill bird

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report the draft genome sequences of ten Mycobacterium avium complex isolates obtained from diverse hosts. This collection includes isolates obtained from deer, pig, elephant, ruddy duck and Red-tailed hawk species. The type strain of Mycobacterium avium subspecies silvaticum (ATCC 49884) is also...

  12. Avian mycobacteriosis caused by Mycobacterium avium subspecies avium in four ornamental birds and in vitro drug sensitivity testing of isolates.

    PubMed

    Stepień-Pyśniak, Dagmara; Puk, Krzysztof; Guz, Leszek; Wawrzyniak, Agata; Marek, Agnieszka; Kosikowska, Urszula

    2016-01-01

    Avian tuberculosis, one of the most important diseases affecting various species of birds, is most often caused by Mycobacterium (M.) avium. This report describes cases of M. avium subsp. avium (MAA) infection in a white-crested Holland dwarf rooster, a male and a female golden pheasant and a male peacock. We also investigated the prevalence of mycobacteria in 60 other birds and 40 alpacas. Tissue samples of necropsied birds were cultured for mycobacteria. From non-necropsied 60 other birds and alpacas only faecal samples were collected. Clinical signs in the affected white-crested Holland cock included gradual loss of body weight and hoarse attempts at crowing during its last 3 weeks, with a dramatic loss of body condition and depression over the final week. Only slight weakening was observed in the peacock just before its death, and the golden pheasants died suddenly. Diagnosis was confirmed by microbiological, molecular and pathological results. Mycobacterium avium subsp. avium strains were isolated from the internal organs of the affected birds. Only one faecal sample from 60 other birds was culture- and PCR-positive for M. avium subsp. avium, while another one was only PCR-positive for M. chelonae. We did not isolate any Mycobacterium spp. from faecal samples of alpacas and all of them were PCR-negative. All 18 isolated M. avium strains were resistant to rifampicin, isoniazid, ethambutol, ethionamide, capreomycin and ofloxacin, and susceptible to cycloserine and streptomycin. PMID:26904899

  13. Mycobacterium simiae and Mycobacterium avium-M. intracellulare mixed infection in acquired immune deficiency syndrome.

    PubMed Central

    Lévy-Frébault, V; Pangon, B; Buré, A; Katlama, C; Marche, C; David, H L

    1987-01-01

    Acquired immune deficiency syndrome was diagnosed in a 43-year-old man, born and living in Congo. The patient presented a disseminated infection caused by mycobacteria which were recovered from blood, jejunal fluid, and duodenal and rectal biopsies. Identification, according to conventional tests and mycolate profile determination, showed that Mycobacterium avium-M. intracellulare and M. simiae were both involved. Images PMID:3793869

  14. Immunology of Paratuberculosis Infection and Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The study of host immune responses to Mycobacterium avium subsp. paratuberculosis (MAP) is complicated by a number of factors, including the protracted nature of the disease and the stealthy nature of the pathogen. Improved tools for the measurement of immunologic responses in ruminant species, par...

  15. Pathology of Paratuberculosis (Johne's Disease) in Clinically Healthy Awassi Sheep with Reference to its Prevalence in Jordan

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Paratuberculosis (Johne’s disease) is an incurable infectious, chronically progressive enteric disease affecting domestic and exotic ruminants. The causative agent is Mycobacterium Avium Paratuberculosis. In this study the occurrence of subclinical Johne’s disease in Awassi sheep is investigated. Ou...

  16. Distinctive western blot antibody patterns induced by infection of mice with individual strains of the Mycobacterium avium complex.

    PubMed Central

    Elsaghier, A; Nolan, A; Allen, B; Ivanyi, J

    1992-01-01

    Systemic infection of mice with organisms of the Mycobacterium avium complex (MAC) induced antibody responses, characteristic for each of the three tested individual strains. The influence of host genetic factors was reflected up to 3 months after infection by the finding of generally oligobanded and multibanded Western blot patterns in C57B1/6 and BALB/c mice, respectively. Nevertheless, more bands developed at 6 months in C57BL/6 mice. The response to three antigens of 18,000, 38,000 and 24,000 MW was analysed in greater detail. Antibodies to a protease-resistant 18,000 MW band produced only by BALB/c mice were either strain specific, following infection with M. avium, strain Maa-B2, or cross-reactive within MAC, following infection with M. avium strain Maa-A6 and M. paratuberculosis, strain Map-203. Another protease-resistant antigen of 38,000 MW was immunogenic only in Maa-B2 infected mice. This constituent was found to be related to the protease-sensitive antigen of corresponding molecular weight from M. tuberculosis. Two 24,000 MW proteins of M. paratuberculosis were separated by two-dimensional gel electrophoresis: antibodies to the anodic band were induced by Map-203 infection, whilst the cathodic band was revealed by heteroclitic antibodies from Maa-B2-infected mice. The latter antigen is apparently expressed during in vivo replication, but not during in vitro culture of Maa-B2 bacteria. We generally conclude, that the selective antibody patterns after live infection, could be attributed to differences in the release of native antigens within mycobacterial lesions. In view of a high degree of species specificity, some of the immunogenic constituents identified may also be useful for serodiagnostic application. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:1526646

  17. Pulmonary Mycobacterium avium infection demonstrating unusual lobar caseous pneumonia.

    PubMed

    Okuzumi, Shinichi; Minematsu, Naoto; Sasaki, Mamoru; Ohsawa, Kazuma; Murakami, Marohito

    2016-09-01

    Mycobacterium avium complex (MAC) infection is a major medical concern in Japan because of its increased prevalence and associated mortality. A common radiological feature in pulmonary MAC infection is a mixture of two basic patterns: fibrocavitary and nodular bronchiectatic; however, lobar consolidation is rare. We report an 83-year-old man with lobar caseous pneumonia caused by pulmonary MAC infection. Radiological findings were predominantly composed of dense lobar consolidation and ground-glass opacity. A diagnosis was made in accordance with the clinical and microbiological criteria set by the American Thoracic Society. A histological examination of lung specimens obtained by using a bronchoscope revealed a caseous granulomatous inflammation with an appearance of Langhans cells. The patient was treated using combined mycobacterium chemotherapy with an initial positive response for 6 months; however, the disease progressed later. We suggest that an awareness of lobar pneumonic consolidation as a rare radiological finding in pulmonary MAC infection is important. PMID:27516892

  18. Characterization of IS1245 for Strain Typing of Mycobacterium avium

    PubMed Central

    Pestel-Caron, Martine; Arbeit, Robert D.

    1998-01-01

    IS1245 is an insertion element widely prevalent among isolates of Mycobacterium avium. We used PvuII Southern blots to analyze IS1245 polymorphisms among 159 M. avium isolates (141 clinical isolates from 40 human immunodeficiency virus-infected patients plus 18 epidemiologically related environmental isolates) that represented 40 distinct M. avium strains, as resolved by previous studies by pulsed-field gel electrophoresis (PFGE). All 40 strains carried DNA homologous to IS1245 and thus were typeable. Twenty-five (63%) strains had ≥10 copies of the element, 6 (15%) had 4 to 9 copies, and 9 (23%) had only 1 to 3 copies. Among the last group of nine strains (each of which was distinct by PFGE analysis), IS1245 typing resolved only four patterns and thus provided poor discriminatory power. To evaluate the in vivo stability of IS1245, we analyzed 32 strains for which sets of 2 to 19 epidemiologically related isolates were available. For 19 (59%) of these sets, all isolates representing the same strain had indistinguishable IS1245 patterns. Within eight (25%) sets, one or more isolates had IS1245 patterns that differed by one or two fragments from the modal pattern for the isolates of that strain. Five (16%) sets included isolates whose patterns differed by three or more fragments; on the basis of IS1245 typing those isolates would have been designated distinct strains. IS1245 was stable during in vitro passage, suggesting that the variations observed represented natural translocations of the element. IS1245 provides a useful tool for molecular strain typing of M. avium but may have limitations for analyzing strains with low copy numbers or for resolving extended epidemiologic relationships. PMID:9650925

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

    EPA Science Inventory

    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...

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

    EPA Science Inventory

    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...

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

    EPA Science Inventory

    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...

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

    EPA Science Inventory

    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...

  3. HOST RESPONSES TO MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS: A COMPLEX ARSENAL

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The immune system is not always successful in recognizing and destroying pathogens it may encounter. Host immunity to mycobacteria is characterized by a very complex series of events, designed to clear the infection. The first line of defense is uptake and processing of the pathogen by macrophages...

  4. Proteome and Antigens of Mycobacterium avium subspecies Paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This chapter will review what is known about the proteome of this significant veterinary pathogen by discussing the use of various whole cell preparations and fractionated components thereof. With improvements in technology, many new modifications to the traditional ELISA assay have been introduced...

  5. Mycobacterium paratuberculosis detection in cow's milk in Argentina by immunomagnetic separation-PCR.

    PubMed

    Gilardoni, Liliana Rosa; Fernández, Bárbara; Morsella, Claudia; Mendez, Laura; Jar, Ana María; Paolicchi, Fernando Alberto; Mundo, Silvia Leonor

    2016-01-01

    The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900-PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 10(1) CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina. PMID:26991290

  6. Diagnostic testing patterns of natural Mycobacterium paratuberculosis infection in pygmy goats

    PubMed Central

    Manning, Elizabeth J.B.; Steinberg, Howard; Krebs, Vernon; Collins, Michael T.

    2003-01-01

    Thirteen pygmy goats (Capra hircus) from a herd naturally infected with Mycobacterium avium ss. paratuberculosis (MPTB) were monitored with 4 diagnostic assays for 2 to 15 mo. Cellular and humoral immune responses to the infection were assessed with assays of gamma interferon (IFNγ), serum antibody [enzyme-linked immunosorbent assay (ELISA) and agar gel diffusion (AGID)], and radiometric fecal culture. Microscopic examination and radiometric culture of tissue from 12 sites were performed at necropsy. Goats were considered infected if MPTB was isolated from any tissue sample collected at necropsy. Mycobacterial isolates were confirmed as MPTB with an IS900 polymerase chain reaction assay. Ten goats whose antemortem tests indicated infection carried heavy organism burdens at necropsy, both within and beyond the gastrointestinal system. False-negative ELISA, AGID, and/or culture results were obtained in 5 of the 10 confirmed cases during the study period. In 3 goats with sporadic fecal shedding of MPTB or detectable IFNγ response, or both, no abnormalities were detected at necropsy and no MPTB was isolated from the tissue samples; the antemortem fecal-culture and IFNγ results were thus considered false-positive. Diagnosticians should be alert to the possibility of both false-positive and false-negative test results for Johne's disease in goats. False-positive fecal-culture results may occur when a high prevalence of infection exists in the herd and the premises are likely to be heavily contaminated. The diverse antemortem testing patterns seen in these goats underscore the importance of using varied diagnostic assays serially or in parallel to increase the likelihood of identifying all infected goats. PMID:12889728

  7. Immunohistochemical methods for the visualization of Mycobacterium paratuberculosis in bovine tissues.

    PubMed

    Massone, A R; Martín, A A; Ibargoyen, G S; Gimeno, E J

    1990-06-01

    The peroxidase-antiperoxidase (PAP), streptavidin-biotin (SB), and avidin-biotin-complex (ABC) techniques have been evaluated for the visualization of Mycobacterium paratuberculosis (Mp) in formalin-fixed, paraffin-embedded bovine tissues. The used immunoperoxidase techniques were comparatively better than the Ziehl-Neelsen stain, specially for the demonstration of small number of mycobacteria in tissue sections. PMID:2382517

  8. Rapid discrimination of Mycobacterium avium strains from AIDS patients by randomly amplified polymorphic DNA analysis.

    PubMed Central

    Matsiota-Bernard, P; Waser, S; Tassios, P T; Kyriakopoulos, A; Legakis, N J

    1997-01-01

    A randomly amplified polymorphic DNA (RAPD) analysis was performed for the molecular typing of Mycobacterium avium strains. This method was applied to epidemiologically unrelated M. avium strains isolated from the blood of 10 different AIDS patients and to strains that were considered epidemiologically related, as they had been isolated from the same patient but from different body locations (4 patients, 10 strains). Three oligonucleotide primers among the six tested were found to generate RAPD profiles with DNA from all M. avium strains and to successfully type them. This method for the typing of M. avium strains is rapid and easy to perform. PMID:9163488

  9. [Isolation of Mycobacterium avium-intracellulare from a hepatic biopsy].

    PubMed

    Ruiz, Aroldo; Mederos, Lilian; Capó, Virginia

    2002-01-01

    A 64 years-old patient, who was a farmer suffering from chronic fever for two years, loss of weight and acute asthenia, was studied. He was admitted to "Pedro Kourí" Tropical Medicine Institute where the studies were conducted and revealed a globular sedimentation rate of 116 mm in 2 hours, and anemia of 9,8g% hemoglobin. The laparoscopic study indicated hepatic granulomatosis that was confirmed by hepatic biopsy in which a sample was taken from the liver to be microbiologically and cytologically examined. By microbiological methods, a non-pigmented slowly-growing strain was isolated, which was classified by conventional diagnostic techniques for the non-tuberculous mycobacteria classification and the alternative diagnosing technique known as bidimensional thin layer chromatography to confirm the previous classification and set the mycolic acid patterns. The isolated strain belonged to group III of Rynyon and was identified as Mycobacterium avium-intracellulare. PMID:15849945

  10. Inactivation of Mycobacterium paratuberculosis and Mycobacterium tuberculosis in fresh soft cheese by gamma radiation

    NASA Astrophysics Data System (ADS)

    Badr, Hesham M.

    2011-11-01

    The effectiveness of gamma irradiation on the inactivation of Mycobacterium paratuberculosis, Mycobacterium bovis and Mycobacterium tuberculosis in fresh soft cheese that prepared from artificially inoculated milk samples was studied. Irradiation at dose of 2 kGy was sufficient for the complete inactivation of these mycobacteria as they were not detected in the treated samples during storage at 4±1 °C for 15 days. Moreover, irradiation of cheese samples, that were prepared from un-inoculated milk, at this effective dose had no significant effects on their gross composition and contents from riboflavin, niacin and pantothenic acid, while significant decreases in vitamin A and thiamin were observed. In addition, irradiation of cheese samples had no significant effects on their pH and nitrogen fractions contents, except for the contents of ammonia, which showed a slight, but significant, increases due to irradiation. The analysis of cheese fats indicated that irradiation treatment induced significant increase in their oxidation parameters and contents from free fatty acids; however, the observed increases were relatively low. On the other hand, irradiation of cheese samples induced no significant alterations on their sensory properties. Thus, irradiation dose of 2 kGy can be effectively applied to ensure the safety of soft cheese with regards to these harmful mycobacteria.

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

    PubMed Central

    Parvandar-Asadollahi, Kaveh; Mosavari, Nader; Mayahi, Mansoor

    2015-01-01

    Background and Objectives: Avian tuberculosis is one of the most important infections affecting most species of birds. Mycobacterium avium can not only infect all species of birds, but also infect some domesticated mammals. The most crucial aspect of control and eradication scheme is identification of infection sources and transmission routs. Molecular techniques such as restriction fragment length polymorphism and pulse field gel electrophoresis have been shown to be much more discriminatory and suitable for use in the epidemiological study. Materials and Methods: Eighty suspected pigeons to avian tuberculosis based on their clinical signs, were subjected to the study. Forty Mycobacterium avium subsp. avium isolates out of a total of 51 identified isolates were subjected to the test. Results: IS901-RFLP using Pvu II was successfully conducted and produced 7 patterns. The majority of isolates (60%) were RFLP type PI.1. This type was the most similar type to standard strain. However, all the patterns obtained in this study were different from the standard strain. Conclusion: The result of this study indicate that these isolates probably are limited to Khuzestan region. We recommend DNA fingerprinting differentiation of non tuberculous Mycobacteria particularly Mycobacterium avium complex isolated from infected birds and human to possibly find source of infections. PMID:26719782

  12. High-Density Lipoprotein Binds to Mycobacterium avium and Affects the Infection of THP-1 Macrophages.

    PubMed

    Ichimura, Naoya; Sato, Megumi; Yoshimoto, Akira; Yano, Kouji; Ohkawa, Ryunosuke; Kasama, Takeshi; Tozuka, Minoru

    2016-01-01

    High-density lipoprotein (HDL) is involved in innate immunity toward various infectious diseases. Concerning bacteria, HDL is known to bind to lipopolysaccharide (LPS) and to neutralize its physiological activity. On the other hand, cholesterol is known to play an important role in mycobacterial entry into host cells and in survival in the intracellular environment. However, the pathogenicity of Mycobacterium avium (M. avium) infection, which tends to increase worldwide, remains poorly studied. Here we report that HDL indicated a stronger interaction with M. avium than that with other Gram-negative bacteria containing abundant LPS. A binding of apolipoprotein (apo) A-I, the main protein component of HDL, with a specific lipid of M. avium might participate in this interaction. HDL did not have a direct bactericidal activity toward M. avium but attenuated the engulfment of M. avium by THP-1 macrophages. HDL also did not affect bacterial killing after ingestion of live M. avium by THP-1 macrophage. Furthermore, HDL strongly promoted the formation of lipid droplets in M. avium-infected THP-1 macrophages. These observations provide new insights into the relationship between M. avium infection and host lipoproteins, especially HDL. Thus, HDL may help M. avium to escape from host innate immunity. PMID:27516907

  13. High-Density Lipoprotein Binds to Mycobacterium avium and Affects the Infection of THP-1 Macrophages

    PubMed Central

    Ichimura, Naoya; Sato, Megumi; Yoshimoto, Akira; Yano, Kouji; Ohkawa, Ryunosuke; Kasama, Takeshi

    2016-01-01

    High-density lipoprotein (HDL) is involved in innate immunity toward various infectious diseases. Concerning bacteria, HDL is known to bind to lipopolysaccharide (LPS) and to neutralize its physiological activity. On the other hand, cholesterol is known to play an important role in mycobacterial entry into host cells and in survival in the intracellular environment. However, the pathogenicity of Mycobacterium avium (M. avium) infection, which tends to increase worldwide, remains poorly studied. Here we report that HDL indicated a stronger interaction with M. avium than that with other Gram-negative bacteria containing abundant LPS. A binding of apolipoprotein (apo) A-I, the main protein component of HDL, with a specific lipid of M. avium might participate in this interaction. HDL did not have a direct bactericidal activity toward M. avium but attenuated the engulfment of M. avium by THP-1 macrophages. HDL also did not affect bacterial killing after ingestion of live M. avium by THP-1 macrophage. Furthermore, HDL strongly promoted the formation of lipid droplets in M. avium-infected THP-1 macrophages. These observations provide new insights into the relationship between M. avium infection and host lipoproteins, especially HDL. Thus, HDL may help M. avium to escape from host innate immunity. PMID:27516907

  14. Clinical Significance and Epidemiologic Analyses of Mycobacterium avium and Mycobacterium intracellulare among Patients without AIDS

    PubMed Central

    Han, Xiang Y.; Tarrand, Jeffrey J.; Infante, Rosa; Jacobson, Kalen L.; Truong, Mylene

    2005-01-01

    The clinical significance and prevalence of Mycobacterium avium and Mycobacterium intracellulare were analyzed in a cohort of 7,472 patients who, from 1999 to 2003, sought care at the University of Texas M.D. Anderson Cancer Center, Houston, and had cultures performed for mycobacteria. Patients were stratified for age, sex, and underlying diseases, and bacteria were identified by 16S rRNA gene sequencing. M. avium was isolated in 62 (0.83%) of 7,472 patients and M. intracellulare in 65 (0.87%). Clinically, only 10 of the 62 (16.2%) patients with M. avium had probable to definite evidence of infection, whereas the majority (83.8%) had weak evidence of infection. Sex and age did not affect the isolation or infection of M. avium. Hematological tumors predisposed to M. avium colonization but not infection. In contrast, 41 of the 65 (63.1%) patients with M. intracellulare had probable to definite infection, a level much higher than those with M. avium (P < 0.001). M. intracellulare was more prevalent in women (1.33% of 3,311) than in men (0.50% of 4,161) (P < 0.001), and underlying diseases had no effect in women. Men with lung cancer had a higher prevalence (1.37%) than men without (0.34%) (4.0-fold; P < 0.001), but it was similar to that in women. A marked age trend for the isolation of M. intracellulare among women was noted: 0.27% (1-fold) for ages of <50 years, 0.85% (3.1-fold) for ages 50 to 59 years, 1.50% (5.6-fold) for ages 60 to 69 years, and 3.74% (13.9-fold) for ages ≥70 years (trend, P < 0.001). The combined rate for women ≥50 was 1.86% (95% confidence interval [1.30 to 2.42%]) (6.9-fold). Together, these results suggest that, among non-AIDS patients, M. intracellulare is more pathogenic and tends to infect women increasingly beyond menopause (age ≥50 years) regardless of underlying disease. The prevalence rate of 1.86% in postmenopausal women suggests the need to further investigate the public health significance of M. intracellulare. PMID:16145084

  15. Assessing the effectiveness of low-pressure ultraviolet light for inactivating Mycobacterium avium complex (MAC) micro-organisms

    EPA Science Inventory

    Aims: To assess low-pressure ultraviolet light (LP-UV) inactivation kinetics of Mycobacterium avium complex (MAC) strains in a water matrix using collimated beam apparatus. Methods and Results: Strains of M. avium (n = 3) and Mycobacterium intracellulare (n = 2) were exposed t...

  16. Ammonium Ion Requirement for the Cell Cycle of Mycobacterium avium

    PubMed Central

    McCarthy, Charlotte

    1978-01-01

    Mycobacterium avium has a defined cell cycle in which small cells elongate to about five times their original length and then divide by fragmentation. The nitrogen requirement for production of maximal number of colony-forming units was assessed by varying concentrations and kinds of nitrogen source in the medium. Ferric ammonium citrate at a concentration in 7H10 medium of 0.17 μmol/ml or ammonium chloride at 0.25 μmol/ml as the nitrogen source permitted the cells to elongate and to undergo limited division, with the final culture at 4 × 107 colony-forming units per ml. Ammonium chloride at 2.5 μmol/ml or glutamine at 1.37 μmol/ml supported completion of the cell cycle with final colony-forming units at about 5 × 108/ml. Other amino acids, including glutamic acid, at 2.5 μmol/ml did not support completion of the cell cycle, although in most cases an intermediate number of colony-forming units per milliliter were formed. Limited uptake of [14C]glutamic acid and uptake of [14C]glutamine were not detectable until cell fission began. Cells not limited for nitrogen took up five times as much 35S during fission as limited cells did during the same time. The nonlimited cells contained 10 times as much sulfolipid as the nitrogen-limited cells at the end of the cell cycle. These results demonstrate that rapidly dividing cells of M. avium utilize amino acids and sulfur and also synthesize sulfolipids in events that are apparently separable from metabolic functions of elongating cells. The results are contrasted with those found for other mycobacteria in which no cell cycle has been demonstrated. Images PMID:624592

  17. Characterization of Mouse Models of Mycobacterium avium Complex Infection and Evaluation of Drug Combinations

    PubMed Central

    Almeida, Deepak V.; Tyagi, Sandeep; Converse, Paul J.; Ammerman, Nicole C.; Grosset, Jacques H.

    2015-01-01

    The Mycobacterium avium complex is the most common cause of nontuberculous mycobacterial lung disease worldwide; yet, an optimal treatment regimen for M. avium complex infection has not been established. Clarithromycin is accepted as the cornerstone drug for treatment of M. avium lung disease; however, good model systems, especially animal models, are needed to evaluate the most effective companion drugs. We performed a series of experiments to evaluate and use different mouse models (comparing BALB/c, C57BL/6, nude, and beige mice) of M. avium infection and to assess the anti-M. avium activity of single and combination drug regimens, in vitro, ex vivo, and in mice. In vitro, clarithromycin and moxifloxacin were most active against M. avium, and no antagonism was observed between these two drugs. Nude mice were more susceptible to M. avium infection than the other mouse strains tested, but the impact of treatment was most clearly seen in M. avium-infected BALB/c mice. The combination of clarithromycin-ethambutol-rifampin was more effective in all infected mice than moxifloxacin-ethambutol-rifampin; the addition of moxifloxacin to the clarithromycin-containing regimen did not increase treatment efficacy. Clarithromycin-containing regimens are the most effective for M. avium infection; substitution of moxifloxacin for clarithromycin had a negative impact on treatment efficacy. PMID:25624335

  18. Polyclonal Mycobacterium avium infections in patients with AIDS: variations in antimicrobial susceptibilities of different strains of M. avium isolated from the same patient.

    PubMed Central

    von Reyn, C F; Jacobs, N J; Arbeit, R D; Maslow, J N; Niemczyk, S

    1995-01-01

    Broth microdilution MICs were determined for pairs of strains isolated from five AIDS patients with polyclonal Mycobacterium avium infection. Four (80%) of the five patients were infected simultaneously with strains having different antimicrobial susceptibility patterns. These findings have implications for the interpretation of susceptibility data in M. avium prophylaxis and treatment trials. PMID:7790424

  19. Mycobacterium avium-intracellulare brain abscess in HIV-positive patient

    PubMed Central

    Karne, Sampada S.; Sangle, Shashikala A.; Kiyawat, Dilip S.; Dharmashale, Sujata N.; Kadam, Dilip B.; Bhardwaj, Renu S.

    2012-01-01

    Mycobacterial opportunistic infections are a major cause of morbidity and mortality among patients living with HIV (PLHIV) worldwide. Nontuberculous mycobacterial (NTM) infection is one of the leading causes of opportunistic infection in patients with advanced acquired immunodeficiency syndrome i.e., with CD4 count less than 50/cu.mm. Mycobacterium avium complex (MAC) is among the most common opportunistic bacterial infections in those patients with advanced immunodeficiency apart from cryptococcal meningitis, progressive multifocal leukoencephalopathy, etc. Common presentations of mycobacterium avium complex are fever, lymphadenitis and respiratory disease. Immune reconstitution disease is also known to manifest with MAC infections in PLHIV on highly active antiretroviral therapy. Very few cases of central nervous system involvement due to NTM infection have been described. We are reporting a case of advanced acquired immunodeficiency who presented with brain abscess due to Mycobacterium avium intracellulare. PMID:22412276

  20. Stability of Insertion Sequence IS1245, a Marker for Differentiation of Mycobacterium avium Strains

    PubMed Central

    Bauer, Jeanett; Andersen, Åse Bengård

    1999-01-01

    Recently a novel insertion element, IS1245, has been described and suggested for use as a probe in restriction fragment length polymorphism studies of Mycobacterium avium strains. An important issue in this context is the stability of the insertion element. We analyzed single colonies of M. avium cultures and found frequent small one- to two-band changes. However, following repeated in vitro passages over 1 year, similar one- to two-band changes were observed in the IS1245 patterns of only six M. avium strains investigated. PMID:9889238

  1. Mycobacterium avium in pygmy rabbits (Brachylagus idahoensis): 28 cases.

    PubMed

    Harrenstien, Lisa A; Finnegan, Mitchell V; Woodford, Nina L; Mansfield, Kristin G; Waters, W Ray; Bannantine, John P; Paustian, Michael L; Garner, Michael M; Bakke, Antony C; Peloquin, Charles A; Phillips, Terry M

    2006-12-01

    The Columbia basin subpopulation of pygmy rabbit Brachylagus idahoensis was listed as endangered by the United States Fish and Wildlife Service in November 2001, and no pygmy rabbits have been seen in the wild since spring 2002. Captive propagation efforts have attempted to increase population size in preparation for reintroduction of animals into central Washington. Disseminated mycobacteriosis due to Mycobacterium avium has been the most common cause of death of adult captive pygmy rabbits. Between June 2002 and September 2004, mycobacteriosis was diagnosed in 28 captive adult pygmy rabbits (representing 29% of the captive population), in contrast to 18 adult pygmy rabbits dying of all other causes in the same time period. Antemortem and postmortem medical records were evaluated retrospectively to describe the clinical course of mycobacteriosis in pygmy rabbits, physical examination findings, and diagnostic test results in the diagnosis of mycobacteriosis in pygmy rabbits. Various treatment protocols, possible risk factors for mortality, and recommendations for prevention of mycobacteriosis were evaluated also. Compromised cell-mediated immunity appears to be the best explanation at this time for the observed high morbidity and mortality from mycobacterial infections in pygmy rabbits. PMID:17315435

  2. Scavenging of reactive oxygen species by a glycolipid fraction of Mycobacterium avium serovar 2.

    PubMed

    Scherer, T A; Lauredo, I T; Abraham, W M

    1997-01-01

    Previous experiments indicated that MIF-A3, a peptidoglycolipid extracted from Mycobacterium avium serovar 2 (Mycobacterium paratuberculosis 18), inhibits the killing of Candida albicans by activated bovine peripheral blood-derived macrophages and murine thioglycollate-elicited peritoneal macrophages in vitro. Subsequent in vitro data from our laboratory indicated that this reduction in killing may be related to the ability of MIF-A3 to scavenge reactive oxygen species (ROS). In this study we examined this hypothesis directly by determining if MIF-A3 reduced exogenous H2O2-induced candidacidal activity. When Candida albicans was incubated with H2O2 (4 mM) alone, colony-forming units/ml x 10(4) (CFU/ml) were 0.4 +/- 0.1 (mean +/- SE, n = 4) as compared to 11.3 +/- 2.0 CFU/ml in control (untreated) cultures (p < .05). The addition of catalase at concentrations > or = 6.8 U/ml, completely blocked the fungicidal effect of H2O2. However, reducing the amount of catalase from 6.8 U/ml to 3.4 U/ml resulted in a loss of scavenging activity, which was associated with a 50% increase in H2O2-mediated killing. Substituting MIF-A3 (400 micrograms/ml) for catalase, also reduced H2O2-induced fungicidal activity. In the absence of MIF-A3, H2O2 reduced Candida albicans to less than 10(3) CFU/ml. However, in the presence of MIF-A3 the CFU/ml of Candida albicans increased 7.5-fold. Based on concentration-response curves of H2O2 inhibition vs. increasing amounts of catalase we determined that the relative inhibitory capacity of the MIF-A3 (400 micrograms/ml) was approximately 1.0 U/ml "catalase equivalents." These findings provide direct evidence that MIF-A3 can scavenge H2O2, and reduce H2O2-induced killing of Candida albicans. PMID:8981049

  3. Mycobacterium avium Genes Associated with the Ability To Form a Biofilm

    PubMed Central

    Yamazaki, Yoshitaka; Danelishvili, Lia; Wu, Martin; MacNab, Molly; Bermudez, Luiz E.

    2006-01-01

    Mycobacterium avium is widely distributed in the environment, and it is chiefly found in water and soil. M. avium, as well as Mycobacterium smegmatis, has been recognized to produce a biofilm or biofilm-like structure. We screened an M. avium green fluorescent protein (GFP) promoter library in M. smegmatis for genes involved in biofilm formation on polyvinyl chloride (PVC) plates. Clones associated with increased GFP expression ≥2.0-fold over the baseline were sequenced. Seventeen genes, most encoding proteins of the tricarboxylic acid (TCA) cycle and GDP-mannose and fatty acid biosynthesis, were identified. Their regulation in M. avium was confirmed by examining the expression of a set of genes by real-time PCR after incubation on PVC plates. In addition, screening of 2,000 clones of a transposon mutant bank constructed using M. avium strain A5, a mycobacterial strain with the ability to produce large amounts of biofilm, revealed four mutants with an impaired ability to form biofilm. Genes interrupted by transposons were homologues of M. tuberculosis 6-oxodehydrogenase (sucA), enzymes of the TCA cycle, protein synthetase (pstB), enzymes of glycopeptidolipid (GPL) synthesis, and Rv1565c (a hypothetical membrane protein). In conclusion, it appears that GPL biosynthesis, including the GDP-mannose biosynthesis pathway, is the most important pathway involved in the production of M. avium biofilm. PMID:16391123

  4. Associations between Mycobacterium paratuberculosis sero-status, milk quality parameters, and reproduction in dairy cows.

    PubMed

    Pesqueira, María N; Factor, Camino; Mato, Ivan; Sanjuán, María L; Macias, Laura; Eiras, Carmen; Arnaiz, Ignacio; Camino, Fernando; Yus, Eduardo; Diéguez, Francisco J

    2015-01-01

    The objective of this study was to investigate the influence of Mycobocterium avium subsp. paratuberculosis (MAP) sero-status of dairy cows on different milk production variables and reproductive traits. The study was carried out on 40 herds from the region of Galicia (North-West Spain). These herds were randomly selected from a larger group that had taken part in a voluntary paratuberculosis control program since 2005, which involves regular serum sampling of every adult animal to run antibody-ELISA tests. Milk production and reproductive data were obtained from the "Dairy Herd Improvement Program (DHIP) of Galicia". All the gathered data were processed following a linear regression model. Results indicated that there was no significant effect of MAP sero-status on individual milk production variables. However, a significant difference was observed at the calving-to-first-insemination interval, with an average increase of 14 days in positive animals compared to negatives. It has to be taken into consideration that the paratuberculosis status was only defined by the serological status. Since para tb-infected animals may have antbodies or may not, para tb-positive animals can also be included in the sero-negative group of animals, which may bias the results. PMID:26591382

  5. Rapid susceptibility testing of Mycobacterium avium complex and Mycobacterium tuberculosis isolated from AIDS patients

    NASA Technical Reports Server (NTRS)

    Dhople, Arvind M.

    1994-01-01

    In ominous projections issued by both U.S. Public Health Service and the World Health Organization, the epidemic of HIV infection will continue to rise more rapidly worldwide than predicted earlier. The AIDS patients are susceptible to diseases called opportunistic infections of which tuberculosis and Mycobacterium avium complex (MAC) infection are most common. This has created an urgent need to uncover new drugs for the treatment of these infections. In the seventies, NASA scientists at Goddard Space Flight Center, Greenbelt, MD, had adopted a biochemical indicator, adenosine triphosphate (ATP), to detect presence of life in extraterrestrial space. We proposed to develop ATP assay technique to determine sensitivity of antibacterial compounds against MAC and M. tuberculosis.

  6. Thioridazine as Chemotherapy for Mycobacterium avium Complex Diseases

    PubMed Central

    Deshpande, Devyani; Srivastava, Shashikant; Musuka, Sandirai

    2016-01-01

    Mycobacterium avium-intracellulare complex (MAC) causes an intractable intracellular infection that presents as chronic pulmonary disease. Currently, therapy consists of ethambutol and macrolides and takes several years to complete. The neuroleptic phenothiazine thioridazine kills mycobacteria by inhibiting the electron transport chain. In several experiments with bacterial populations of up to 1012 CFU/ml, we failed to isolate any bacteria resistant to 3 times the MIC of thioridazine, suggesting the absence of resistant mutants at bacterial burdens severalfold higher than those encountered in patients. In the hollow-fiber model of intracellular MAC (HFS-MAC), thioridazine achieved an extracellular half-life of 16.8 h and an intracellular half-life of 19.7 h. Thioridazine concentrations were >28,000-fold higher inside infected macrophages than in the HFS-MAC central compartment (equivalent to plasma). Thioridazine maximal kill was 5.20 ± 0.75 log10 CFU/ml on day 7 (r2 = 0.96) and 7.19 ± 0.31 log10 CFU/ml on day 14 (r2 = 0.99), the highest seen with any drug in the system. Dose fractionation studies revealed that thioridazine efficacy and acquired drug resistance were driven by the peak concentation-to-MIC ratio, with a 50% effective concentration (EC50) of 2.78 ± 0.44 for microbial killing. Acquired drug resistance was encountered by day 21 with suboptimal doses, demonstrating that fluctuating drug concentrations drive evolution faster than static concentrations in mutation frequency studies. However, the thioridazine EC50 changed 16.14-fold when the concentration of fetal bovine serum was changed from 0% to 50%, suggesting that intracellular potency could be heavily curtailed by protein binding. Efficacy in patients will depend on the balance between trapping of the drug in the pulmonary system and the massive intracellular concentrations versus very high protein binding of thioridazine. PMID:27216055

  7. Thioridazine as Chemotherapy for Mycobacterium avium Complex Diseases.

    PubMed

    Deshpande, Devyani; Srivastava, Shashikant; Musuka, Sandirai; Gumbo, Tawanda

    2016-08-01

    Mycobacterium avium-intracellulare complex (MAC) causes an intractable intracellular infection that presents as chronic pulmonary disease. Currently, therapy consists of ethambutol and macrolides and takes several years to complete. The neuroleptic phenothiazine thioridazine kills mycobacteria by inhibiting the electron transport chain. In several experiments with bacterial populations of up to 10(12) CFU/ml, we failed to isolate any bacteria resistant to 3 times the MIC of thioridazine, suggesting the absence of resistant mutants at bacterial burdens severalfold higher than those encountered in patients. In the hollow-fiber model of intracellular MAC (HFS-MAC), thioridazine achieved an extracellular half-life of 16.8 h and an intracellular half-life of 19.7 h. Thioridazine concentrations were >28,000-fold higher inside infected macrophages than in the HFS-MAC central compartment (equivalent to plasma). Thioridazine maximal kill was 5.20 ± 0.75 log10 CFU/ml on day 7 (r(2) = 0.96) and 7.19 ± 0.31 log10 CFU/ml on day 14 (r(2) = 0.99), the highest seen with any drug in the system. Dose fractionation studies revealed that thioridazine efficacy and acquired drug resistance were driven by the peak concentation-to-MIC ratio, with a 50% effective concentration (EC50) of 2.78 ± 0.44 for microbial killing. Acquired drug resistance was encountered by day 21 with suboptimal doses, demonstrating that fluctuating drug concentrations drive evolution faster than static concentrations in mutation frequency studies. However, the thioridazine EC50 changed 16.14-fold when the concentration of fetal bovine serum was changed from 0% to 50%, suggesting that intracellular potency could be heavily curtailed by protein binding. Efficacy in patients will depend on the balance between trapping of the drug in the pulmonary system and the massive intracellular concentrations versus very high protein binding of thioridazine. PMID:27216055

  8. Development and Characterization of attenuated Mutant Candidate Vaccines for Control of Paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (Map) is the causative pathogen of Johne’s disease, a chronic inflammatory wasting disease in ruminants. The disease has been difficult to control because of the lack of an effective vaccine. To develop a live attenuated vaccine for Map, as well as for the...

  9. MYCOBACTERIUM AVIUM COMPLEX IN DRINKING WATER: DETECTION, DISTRIBUTION, AND ROUTES OF EXPOSURE

    EPA Science Inventory

    Organisms of the Mycobacterium avium complex (MAC) are an increasingly prevalent cause of clinical disease and are known to be widespread in drinking water supplies. However, there are significant gaps in our current knowledge. Available methods for the detection of MAC in wat...

  10. Shared Mycobacterium avium genotypes observed among unlinked clinical and environmental isolates

    EPA Science Inventory

    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, ...

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

    EPA Science Inventory

    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...

  12. Immunologic responses to Mycobacterium avium subsp. paratuberculois protein cocktail vaccines in a mouse model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Johne’s disease is a chronic granulomatous enteritis characterized by severe diarrhea, wasting and a decline in milk production caused by the bacterium Mycobacterium avium subsp. paratuberculois (MAP). The vaccine currently on the market has some limitations including a severe injection site reactio...

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

    PubMed

    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

    2014-03-01

    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 <0.05). By further analysis of logistic regression with a stepwise variable-selection, we constructed the highest likelihood multivariate model, adjusted for age, to predict a therapeutic response, using 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

  14. Detection of Mycobacterium tuberculosis and