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Sample records for mycobacterium avium paratuberculosis

  1. 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

  2. Immunogenicity of Proteome-determined Mycobacterium avium subsp. paratuberculosis-specific Proteins in Sheep with Paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subspecies 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 being prone to spurious positive test results ca...

  3. 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

  4. First identification of Mycobacterium avium paratuberculosis sheep strain in Argentina.

    PubMed

    Travería, G E; Zumarraga, M; Etchechoury, I; Romano, M I; Cataldi, A; Pinedo, M F Alvarado; Pavlik, I; Pribylova, R; Romero, J R

    2013-01-01

    We here identified for the first time the presence of Mycobacterium avium paratuberculosis (MAP) sheep (S) strain in Argentina. IS900 polymerase chain reaction (PCR) was positive. The S strain was compared with MAP cattle (C) strains by using IS1311 PCR-restriction endonuclease analysis (PCR-REA), multiplex PCR and restriction fragment length polymorphism (RFLP) analysis.

  5. 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...

  6. 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...

  7. 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...

  8. 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...

  9. Evaluation of Subspecies-specific Proteins for the Diagnosis of Mycobacterium avium subspecies paratuberculosis Infections

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subspecies paratuberculosis (Map)-specific proteins (35) were identified by comparing the proteomes of Map isolates with those of the genetically similar subspecies IS901+ Mycobacterium avium subspecies avium or silvaticum. This approach identified subspecies-specific proteins in...

  10. Mycobacterium avium subsp. paratuberculosis: pathogen, pathogenesis and diagnosis.

    PubMed

    Manning, E J; Collins, M T

    2001-04-01

    Johne's disease, or paratuberculosis, is a chronic intestinal infection caused by Mycobacterium avium subsp. paratuberculosis. The usually fatal disease is characterised by cachexia, and in some species diarrhoea, after a long pre-clinical phase. Treatment is ineffective and economically impracticable. The infection primarily affects domestic and free-ranging ruminants, but has also been reported in primates, rabbits, stoats and foxes. Since paratuberculosis is often subclinical, under-reporting is suspected, even though the disease is notifiable in numerous countries. Herd prevalence of bovine paratuberculosis in Europe ranges from 7% to 55%. In the United States of America, herd prevalence is strongly associated with herd size; 40% of herds of more than 300 head were found to be infected. In Australia, reported dairy herd infection rates range between 9% and 22%. Paratuberculosis in domestic livestock entails significant economic losses due to several factors (e.g. reduced production, premature culling and increased veterinary costs). Free-ranging and captive wildlife are also at risk from paratuberculosis.

  11. 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...

  12. 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 ...

  13. Profiling Host Antibody Responses to Mycobacterium avium subspecies paratuberculosis Infection Using Protein Arrays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Along with the complete genome sequence of Mycobacterium avium subspecies paratuberculosis, technologies are now developed for the 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 enable...

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

    PubMed Central

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

    2016-01-01

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

  15. 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...

  16. 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

  17. 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...

  18. 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...

  19. Host responses to persistent Mycobacterium avium subspecies paratuberculosis infection in surgically isolated bovine ileal segments.

    PubMed

    Charavaryamath, Chandrashekhar; Gonzalez-Cano, Patricia; Fries, Patrick; Gomis, Susantha; Doig, Kimberley; Scruten, Erin; Potter, Andrew; Napper, Scott; Griebel, Philip J

    2013-02-01

    A lack of appropriate disease models has limited our understanding of the pathogenesis of persistent enteric infections with Mycobacterium avium subsp. paratuberculosis. A model was developed for the controlled delivery of a defined dose of M. avium subsp. paratuberculosis to surgically isolated ileal segments in newborn calves. The stable intestinal segments enabled the characterization of host responses to persistent M. avium subsp. paratuberculosis infections after a 9-month period, including an analysis of local mucosal immune responses relative to an adjacent uninfected intestinal compartment. M. avium subsp. paratuberculosis remained localized at the initial site of intestinal infection and was not detected by PCR in the mesenteric lymph node. M. avium subsp. paratuberculosis-specific T cell proliferative responses included both CD4 and γδ T cell receptor (γδTcR) T cell responses in the draining mesenteric lymph node. The levels of CD8(+) and γδTcR(+) T cells increased significantly (P < 0.05) in the lamina propria, and M. avium subsp. paratuberculosis-specific tumor necrosis factor alpha (TNF-α) and gamma interferon secretion by lamina propria leukocytes was also significantly (P < 0.05) increased. There was a significant (P < 0.05) accumulation of macrophages and dendritic cells (DCs) in the lamina propria, but the expression of mucosal toll-like receptors 1 through 10 was not significantly changed by M. avium subsp. paratuberculosis infection. In conclusion, surgically isolated ileal segments provided a model system for the establishment of a persistent and localized enteric M. avium subsp. paratuberculosis infection in cattle and facilitated the analysis of M. avium subsp. paratuberculosis-specific changes in mucosal leukocyte phenotype and function. The accumulation of DC subpopulations in the lamina propria suggests that further investigation of mucosal DCs may provide insight into host responses to M. avium subsp. paratuberculosis infection and

  20. 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

  1. Control of Mycobacterium avium subsp. paratuberculosis infection in agricultural species.

    PubMed

    Kennedy, D J; Benedictus, G

    2001-04-01

    Paratuberculosis or Johne's disease is a chronic intestinal disease caused by Mycobacterium avium subsp. paratuberculosis, which continues to spread in agricultural species. Control of paratuberculosis is challenging and should not be underestimated. Due to the long incubation period of the infection, disease is largely subclinical in domesticated livestock. Hence, direct effects on animal productivity and welfare are often masked and may appear insufficient to justify large investments in control programmes by individual farmers, livestock industries or governments. Furthermore, in some countries the main effects of the disease are indirect, resulting from the impact of market discrimination against herds and flocks known to be infected, or from the control measures enforced to reduce transmission. In such circumstances, producers may be unwilling to co-operate with surveillance that may detect infection in herds or flocks. As control programmes are rarely successful in eliminating the infection from a herd or flock in the short term without an aggressive and costly programme, financial and community support assists producers to deal with the challenge. Successful prevention and control depends on animal health authorities and livestock industries acquiring a good understanding of the nature and epidemiology of infection, and of the application of tools for diagnosis and control. Building support for control programmes under the leadership of the affected livestock industries is critical, as programmes are unlikely to be successful without ongoing political will, supported by funding for research, surveillance and control.

  2. Mycobacterium avium subspecies paratuberculosis, Crohn's disease and the Doomsday scenario

    PubMed Central

    Hermon-Taylor, John

    2009-01-01

    Johne's disease is chronic inflammation of the intestine caused by Mycobacterium avium subspecies paratuberculosis. Infection and disease are mainly in domestic livestock but can affect many species including primates. Johne's is a new disease which emerged at the turn of the 19th and 20th centuries and principally involved Europe and North America. It has since spread to former low incidence regions to become a global problem. Crohn's disease is a chronic inflammation of the intestine in humans which emerged in Europe and North America mid 20th century and increased to become a major healthcare problem. It has now spread to former low incidence regions. Infected animals shed Mycobacterium avium subspecies paratuberculosis in milk and into the environment. Human populations are widely exposed. Outcomes maybe influenced by microbial phenotype. Exposure to extracellular forms of these pathogens may confer some natural protection; exposure to intracellular forms which have passaged through milk macrophages or environmental protists may pose a greater threat to humans particularly individuals with an inherited or acquired susceptibility. Hot spots of human disease such as in Winnipeg which sits on rock at the junction of two rivers may result from local exposure to high levels of waterborne pathogens brought down from farmland. When appropriate methods are used most people with Crohn's disease are found to be infected. There are no data which demonstrate that these pathogens are harmless to humans. An overwhelming balance of probability and Public health risk favours the conclusion that Mycobacterium avium subspecies paratuberculosis is also pathogenic for people. A two tier co-operative pathogenic mechanism is proposed in Crohn's disease. Intracellular infection with the primary pathogen widely distributed throughout the gut causes an immune dysregulation and a specific chronic enteric neuropathy with loss of mucosal integrity. Segments of gross inflammatory disease

  3. Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis on Dairy Farms.

    PubMed

    Li, Lingling; Katani, Robab; Schilling, Megan; Kapur, Vivek

    2016-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of severe chronic intestinal inflammatory disease in ruminants, termed Johne's disease, and can infect many other animal species, including humans. MAP has a long incubation period prior to manifestation of clinical signs including diarrhea, weight loss, and loss of production. MAP has a high prevalence in dairy herds and results in considerable adverse impacts on animal health and productivity throughout the world. Recent investigations have leveraged the characterization of the MAP genome for the development of powerful new molecular techniques for MAP strain differentiation. These approaches are providing key insights into the epidemiology and transmission of MAP on and between dairy herds. We summarize the state of the art for MAP diagnostics and strain differentiation and our current knowledge of mechanisms of within- and between-herd transmission of MAP, along with future needs for the development of rational MAP infection control programs.

  4. 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...

  5. 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...

  6. Maximizing Capture Efficiency and Specificity of Magnetic Separation for Mycobacterium avium subsp. paratuberculosis Cells ▿

    PubMed Central

    Foddai, Antonio; Elliott, Christopher T.; Grant, Irene R.

    2010-01-01

    In order to introduce specificity for Mycobacterium avium subsp. paratuberculosis prior to a phage amplification assay, various magnetic-separation approaches, involving either antibodies or peptides, were evaluated in terms of the efficiency of capture (expressed as a percentage) of M. avium subsp. paratuberculosis cells and the percentage of nonspecific binding by other Mycobacterium spp. A 50:50 mixture of MyOne Tosylactivated Dynabeads coated with the chemically synthesized M. avium subsp. paratuberculosis-specific peptides biotinylated aMp3 and biotinylated aMptD (i.e., peptide-mediated magnetic separation [PMS]) proved to be the best magnetic-separation approach for achieving 85 to 100% capture of M. avium subsp. paratuberculosis and minimal (<1%) nonspecific recovery of other Mycobacterium spp. (particularly if beads were blocked with 1% skim milk before use) from broth samples containing 103 to 104 CFU/ml. When PMS was coupled with a recently optimized phage amplification assay and used to detect M. avium subsp. paratuberculosis in 50-ml volumes of spiked milk, the mean 50% limit of detection (LOD50) was 14.4 PFU/50 ml of milk (equivalent to 0.3 PFU/ml). This PMS-phage assay represents a novel, rapid method for the detection and enumeration of viable M. avium subsp. paratuberculosis organisms in milk, and potentially other sample matrices, with results available within 48 h. PMID:20851966

  7. Identification of immunoreactive proteins of Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Piras, Cristian; Soggiu, Alessio; Bonizzi, Luigi; Greco, Viviana; Ricchi, Matteo; Arrigoni, Norma; Bassols, Anna; Urbani, Andrea; Roncada, Paola

    2015-02-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of a chronic enteritis of ruminants (bovine paratuberculosis (PTB)--Johne's disease) that is associated with enormous worldwide economic losses for the animal production. Diagnosis is based on observation of clinical signs, the detection of antibodies in milk or serum, or evaluation of bacterial culture from feces. The limit of these methods is that they are not able to detect the disease in the subclinical stage and are applicable only when the disease is already advanced. For this reason, the main purpose of this study is to use the MAP proteome to detect novel immunoreactive proteins that may be helpful for PTB diagnoses. 2DE and 2D immunoblotting of MAP proteins were performed using sera of control cattle and PTB-infected cattle in order to highlight the specific immunoreactive proteins. Among the assigned identifiers to immunoreactive spots it was found that most of them correspond to surface-located proteins while three of them have never been described before as antigens. The identification of these proteins improves scientific knowledge that could be useful for PTB diagnoses. The sequence of the identified protein can be used for the synthesis of immunoreactive peptides that could be screened for their immunoreaction against bovine sera infected with MAP. All MS data have been deposited in the ProteomeXchange consortium with identifier PXD001159 and DOI 10.6019/PXD001159.

  8. 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...

  9. Genetic relatedness among Mycobacterium paratuberculosis and M. avium complex.

    PubMed

    Labidi, A; Thoen, C O

    1989-01-01

    Total DNA was extracted from M. paratuberculosis (ATCC 19698) and from M. avium complex (ATCC 25291) cultivated on RVB-10 enriched liquid media. Restriction endonuclease analysis of total DNA was performed with 34 enzymes and DNA digestion profiles were compared. Fifteen enzymes revealed important differences between the two species. Two pairs of enzymes (EcoRII, BstNI) and (MboI, Sau3AI) provide evidence for the presence of dcmI and dam methylation in DNA of M. avium complex and M. paratuberculosis. The differences in DNA fragments of these two species could be of potential value in differentiating these clinically significant mycobacteria.

  10. Occurrence of Mycobacterium avium subsp. paratuberculosis in untreated water in Northern Ireland.

    PubMed

    Whan, Lynne; Ball, Hywel J; Grant, Irene R; Rowe, Michael T

    2005-11-01

    Mycobacterium avium subsp. paratuberculosis is the known cause of Johne's disease of both domestic and wild ruminants and has been implicated as a possible cause of Crohn's disease in humans. The organism is shed in the feces of infected animals and can survive for protracted periods in the environment and hence could be present in catchment areas receiving agricultural runoff. A limited survey was undertaken in Northern Ireland to test for M. avium subsp. paratuberculosis in untreated water entering nine water treatment works (WTWs) over a 1-year period. Three detection methods were employed, viz., immunomagnetic separation-PCR and culture on Herrold's egg yolk medium (HEYM) and BACTEC 12B medium, the latter both supplemented with mycobactins. Of the 192 untreated water samples tested, 15 (8%) tested M. avium subsp. paratuberculosis positive by one or more of the three detection methods. M. avium subsp. paratuberculosis was successfully isolated from eight untreated water samples, three by BACTEC culture and five by culture on HEYM. Although the highest incidence of M. avium subsp. paratuberculosis was found in spring, overall, there was no statistically significant difference between the seasons. No significant correlation was found between numbers of coliforms or fecal coliforms and the presence of M. avium subsp. paratuberculosis. In general, a higher incidence of M. avium subsp. paratuberculosis was found in untreated water entering those WTWs that had a high mean water pH value over the sampling period. This work indicates the need to determine the efficacy of water treatment processes to either kill or remove M. avium subsp. paratuberculosis from untreated water and the possible risks posed by contact with recreational water sources.

  11. Peptides specific for Mycobacterium avium subspecies paratuberculosis infection: diagnostic potential.

    PubMed

    Casey, J L; Sanalla, A M; Tamvakis, D; Thalmann, C; Carroll, E L; Parisi, K; Coley, A M; Stewart, D J; Vaughan, J A; Michalski, W P; Luke, R; Foley, M

    2011-08-01

    Mycobacterium avium subspecies paratuberculosis (Map) is the causative agent of Johne's disease (JD). Current serological diagnostic tests for JD are limited by their sensitivity when used in sub-clinical stages of the disease. Our objective was to identify peptides that mimic diagnostically important Map epitopes that might be incorporated into a new-generation JD diagnostic. Four peptides were isolated from a phage-displayed random peptide library by screening on antibodies derived from Map-infected goats. The peptides were recognised by antibodies from Map-infected goats but not by antibodies from uninfected goats. The peptides elicited immune responses in rabbits, which reacted strongly with bona fide Map antigens proving the peptides were true epitope mimics. To assess the diagnostic value a panel of goat sera was screened for reactivity's with peptides. The peptides were recognised by antibodies from a proportion of goats infected with Map compared with control animals with a diagnostic specificity of 100% and the sensitivity ranged from 50 to 75%. Combinations of any two peptides improved sensitivity 62.5-87.5% and 100% sensitivity was achieved with three of the four peptides in combination. These data suggest peptides representing diagnostically important Map epitopes could be incorporated into a sensitive diagnostic test.

  12. 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...

  13. 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...

  14. Culture and molecular method for detection of Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis in milk and dairy products.

    PubMed

    Messelhäusser, U; Kämpf, P; Hörmansdorfer, S; Wagner, B; Schalch, B; Busch, U; Höller, C; Wallner, P; Barth, G; Rampp, A

    2012-01-01

    A combined molecular and cultural method for the detection of the Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium subsp. paratuberculosis was developed and tested with artificially contaminated milk and dairy products. Results indicate that the method can be used for a reliable detection as a basis for first risk assessments.

  15. 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...

  16. 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...

  17. 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...

  18. Iron-sparing Response of Mycobacterium avium subsp. paratuberculosis is Strain Dependent

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Two genotypically and microbiologically distinct strains of Mycobacterium avium subsp. paratuberculosis (MAP) exist – the type I and type II strains that primarily infect sheep and cattle, respectively. Concentration of iron in the cultivation medium has been suggested as one contributin...

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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 ...

  4. 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) ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  11. Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis Isolates Recovered from Wild Animal Species

    PubMed Central

    Motiwala, Alifiya S.; Amonsin, Alongkorn; Strother, Megan; Manning, Elizabeth J. B.; Kapur, Vivek; Sreevatsan, Srinand

    2004-01-01

    Mycobacterial isolates were obtained by radiometric culture from 33 different species of captive or free-ranging animals (n = 106) and environmental sources (n = 3) from six geographic zones within the United States. The identities of all 109 isolates were confirmed by using mycobactin J dependence and characterization of five well-defined molecular markers, including two integration loci of IS900 (loci L1 and L9), one Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis)-specific sequence (locus 251), and one M. avium subsp. avium-specific marker (IS1245), as well as hsp65 and IS1311 restriction endonuclease analyses. Seventy-six acid-fast isolates were identified as M. paratuberculosis, 15 were identified as belonging to the M. avium-M. intracellulare complex (but not M. paratuberculosis), and the remaining 18 were identified as mycobacteria outside the M. avium-M. intracellulare complex. Fingerprinting by multiplex PCR for IS900 integration loci clustered 67 of the 76 M. paratuberculosis strains into a single clade (designated clade A18) and had a Simpson's diversity index (D) of 0.53. In contrast, sequence-based characterization of a recently identified M. paratuberculosis short sequence repeat (SSR) region enabled the differentiation of the M. paratuberculosis isolates in clade A18 into seven distinct alleles (D = 0.75). The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Taken together, the results of our analyses demonstrate that SSR analysis enables the genetic characterization of M. paratuberculosis isolates from different host species and provide evidence for the host specificity of some M. paratuberculosis strains as well as sharing of strains between wild and domesticated animal species. PMID:15071028

  12. 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.

  13. Novel Single Nucleotide Polymorphism-Based Assay for Genotyping Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Goldstone, Robert J.; McLuckie, Joyce; Smith, David G. E.

    2015-01-01

    Typing of Mycobacterium avium subspecies paratuberculosis strains presents a challenge, since they are genetically monomorphic and traditional molecular techniques have limited discriminatory power. The recent advances and availability of whole-genome sequencing have extended possibilities for the characterization of Mycobacterium avium subspecies paratuberculosis, and whole-genome sequencing can provide a phylogenetic context to facilitate global epidemiology studies. In this study, we developed a single nucleotide polymorphism (SNP) assay based on PCR and restriction enzyme digestion or sequencing of the amplified product. The SNP analysis was performed using genome sequence data from 133 Mycobacterium avium subspecies paratuberculosis isolates with different genotypes from 8 different host species and 17 distinct geographic regions around the world. A total of 28,402 SNPs were identified among all of the isolates. The minimum number of SNPs required to distinguish between all of the 133 genomes was 93 and between only the type C isolates was 41. To reduce the number of SNPs and PCRs required, we adopted an approach based on sequential detection of SNPs and a decision tree. By the analysis of 14 SNPs Mycobacterium avium subspecies paratuberculosis isolates can be characterized within 14 phylogenetic groups with a higher discriminatory power than mycobacterial interspersed repetitive unit–variable number tandem repeat assay and other typing methods. Continuous updating of genome sequences is needed in order to better characterize new phylogenetic groups and SNP profiles. The novel SNP assay is a discriminative, simple, reproducible method and requires only basic laboratory equipment for the large-scale global typing of Mycobacterium avium subspecies paratuberculosis isolates. PMID:26677250

  14. 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. ...

  15. 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. ...

  16. Spread of Mycobacterium avium subsp. paratuberculosis through soil and grass on a mouflon (Ovis aries) pasture.

    PubMed

    Kaevska, Marija; Lvoncik, S; Lamka, J; Pavlik, I; Slana, I

    2014-10-01

    The aims of this study were to describe spatial contamination of the environment on a mouflon pasture, as well as to assess the contamination of grass and roots after surface contamination and in depth contamination with feces and buried tissues from animals infected with Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis). Samples of soil, roots, and aerial parts of plants were collected from different locations inside the mouflon pasture, and one control sample site was chosen outside the area where the animals are living. M. a. paratuberculosis DNA was present in all the examined sites and was more often detected in roots than in soil. DNA was detected at up to 80 cm of depth and was spatially more widespread than the initial hypothesis of M. a. paratuberculosis leaching vertically into deeper layers of soil. This study broadens our knowledge of the spread and persistence of M. a. paratuberculosis in an environment with highly infected animals.

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

    PubMed

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

    2014-07-15

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

  18. 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 ...

  19. Ecology and genomic features of infection with Mycobacterium avium subspecies paratuberculosis in Egypt.

    PubMed

    Amin, Adel S; Hsu, Chung-Yi; Darwish, Samah F; Ghosh, Pallab; AbdEl-Fatah, Eman M; Behour, Tahani S; Talaat, Adel M

    2015-04-01

    Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of paratuberculosis, or Johne's disease, in cattle, with potential involvement in cases of Crohn's disease in humans. Johne's disease is found worldwide and is economically important for both beef and dairy industries. In an effort to characterize this important infection in Egypt, we analysed the ecological and genomic features of recent isolates of M. paratuberculosis. In this report, we examined 26 Holstein dairy herds distributed throughout Egypt, from 2010 to 2013. Using PCR analysis of faecal samples, we estimated a mean herd-level prevalence of 65.4 %, with animal-level infection that reached a mean of 13.6 % among animals suffering from diarrhoea. Whole genome sequencing of field isolates identified numerous single nucleotide polymorphisms among field isolates relative to the standard M. paratuberculosis K10 genome. Interestingly, the virulence of M. paratuberculosis isolates from Egypt revealed diverse virulence phenotypes in the murine model of paratuberculosis, with significant differences in tissue colonization, particularly during the chronic stage of infection. Overall, our analysis confirmed that Johne's disease is a newly identified problem in Egypt and indicated that M. paratuberculosis has potentially diverse genotypes that impact its virulence. Further ecological mapping and genomic analysis of M. paratuberculosis will enhance our understanding of the transmission and evolutionary dynamics of this pathogen under natural field conditions.

  20. Antibody recognition to secreted proteins of Mycobacterium avium subsp. paratuberculosis in sera from infected ruminants.

    PubMed

    Pradenas, M; Jara, M C; Hernández, N; Zambrano, A; Collins, M T; Kruze, J

    2009-09-18

    Two liquid culture media to obtain secreted proteins of Mycobacterium avium subsp. paratuberculosis at different incubation periods were evaluated. Middlebrook 7H9-OADC (7H9) and Watson-Reid (WR) broths were inoculated with a field strain of M. paratuberculosis and growth curves determined using nonlinear regression analysis. Most culture filtrate (CF) proteins were of low molecular weight and reacted strongly against sera from cultured-positive cases of paratuberculosis. CF proteins obtained in WR yielded a higher number of bands and were detected earlier than those obtained from 7H9. A high degree of variability in CF protein immunoreactivity was seen among infected animals. Sera from cattle with clinical paratuberculosis or heavy fecal shedders of M. paratuberculosis reacted more intensively and to more CF proteins than did sera from other infected cattle. Immunoblots showed differences in antibody binding to CF proteins when sera were absorbed with M. avium but not with others environmental mycobacteria. Immunoblots with sera from infected goats and a sheep showed reactivity with proteins of 32, 33 and 46kDa both before and after the sera were absorbed with M. phlei. Antibodies found in serum of infected deer reacted with CF proteins in a similar way as did for cattle. These results suggest that a pool of CF proteins of M. paratuberculosis could be good candidates as antigens for serodiagnosis of paratuberculosis.

  1. 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...

  2. 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...

  3. Immunoreactivity of the Mycobacterium avium subsp. paratuberculosis 19-kDa lipoprotein

    PubMed Central

    Huntley, Jason FJ; Stabel, Judith R; Bannantine, John P

    2005-01-01

    Background The Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne's disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed. Results MAP0261c is conserved in mycobacteria, showing a 95% amino acid identity in M. avium subspecies avium, 84% in M. intracellulare and 76% in M. bovis and M. tuberculosis. MAP0261c was cloned, expressed, and purified as a fusion protein with the maltose-binding protein (MBP-19 kDa) in Escherichia coli. IFN-γ production was measured from 21 naturally infected and 9 control cattle after peripheral blood mononuclear cells (PBMCs) were stimulated with a whole cell lysate (WCL) of M. avium subsp. paratuberculosis or the recombinant MBP-19 kDa. Overall, the mean response to MBP-19 kDa was not as strong as the mean response to the WCL. By comparison, cells from control, non-infected cattle did not produce IFN-γ after stimulation with either WCL or MBP-19 kDa. To assess the humoral immune response to the 19-kDa protein, sera from cattle with clinical Johne's disease were used in immunoblot analysis. Reactivity to MBP-19 kDa protein, but not MBP alone, was observed in 9 of 14 infected cattle. Antibodies to the 19-kDa protein were not observed in 8 of 9 control cows. Conclusions Collectively, these results demonstrate that while the 19-kDa protein from M. avium subsp. paratuberculosis stimulates a humoral immune response and weak IFN-γ production in infected cattle, the elicited responses are not strong enough to be used in a sensitive diagnostic assay. PMID:15663791

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

    PubMed Central

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

    2016-01-01

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

  5. 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.

  6. Virulence and immunity orchestrated by the global gene regulator sigL in Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Ghosh, Pallab; Steinberg, Howard; Talaat, Adel M

    2014-07-01

    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.

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

    PubMed

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

    2015-10-19

    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.

  8. 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

  9. 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.

  10. Disseminated Mycobacterium avium subsp. paratuberculosis infection in two wild Eurasian otters (Lutra lutra L.) from Portugal.

    PubMed

    Matos, Ana Cristina; Figueira, Luis; Martins, Maria Helena; Matos, Manuela; Alvares, Sofia; Pinto, Maria Lurdes; Coelho, Ana Cláudia

    2013-03-01

    Disseminated Mycobacterium avium subsp. paratuberculosis (MAP) infections were found in two Eurasian otters (Lutra lutra, L. 1758) killed by vehicular trauma in February and March 2010 in Castelo Branco, Portugal. At postmortem examination, the organs showed no significant gross alterations; however, microscopically, both animals had diffuse lymphadenitis with macrophage infiltration and deposition of hyaline material in the center of the lymphoid follicles. Acid-fast organisms were isolated from gastrointestinal tissue samples via bacteriologic culture. These organisms were identified as M. avium subsp. paratuberculosis by IS900 polymerase chain reaction (PCR). Additionally, direct IS900 PCR-positive results were obtained for multiple organs of both animals. This is the first report of MAP infection of otters in Portugal.

  11. Attenuated strains of Mycobacterium avium subspecies paratuberculosis as vaccine candidates against Johne's disease.

    PubMed

    Settles, Erik W; Kink, John A; Talaat, Adel

    2014-04-11

    Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease in ruminants. Johne's disease has a severe economic impact on the dairy industry in the USA and worldwide. In an effort to combat this disease, we screened several transposon mutants that were attenuated in the murine model of paratuberculosis for the potential use as live attenuated vaccines. Using the murine model, two vaccine candidates (pgs1360, pgs3965 with mutations of fabG2_2 and umaA1, respectively) were at or below the limit of detection for tissue colonization suggesting their low level persistence and hence safety. Prior to challenge, both candidates induced a M. paratuberculosis-specific IFN-γ, an indication of eliciting cell-mediated immunity. Following challenge with a virulent strain of M. paratuberculosis, the two vaccine candidates significantly reduced bacterial colonization in organs with reduced histological scores compared to control animals. In addition, one of the vaccine candidates (pgs3965) also induced IL-17a, a cytokine associated with protective immunity in mycobacterial infection. Our analysis suggested that the pgs3965 vaccine candidate is a potential live-attenuated vaccine that could be tested further in ruminant models of paratuberculosis. The analysis also validated our screening strategy to identify effective vaccine candidates against intracellular pathogens.

  12. A Gene Specific to Mycobacterium avium subsp. paratuberculosis, But Only at the Transcription-translation Level

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is no known antibody that detects M. avium subsp paratuberculosis and does not cross react with other M. avium subspecies. In the present study, a monoclonal antibody was identified from mice immunized with a cell membrane fraction of M. avium subsp paratuberculosis strain K-10. This antibod...

  13. 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

  14. Contrasting results of culture-dependent and molecular analyses of Mycobacterium avium subsp. paratuberculosis from wood bison.

    PubMed

    Forde, Taya; De Buck, Jeroen; Elkin, Brett; Kutz, Susan; van der Meer, Frank; Orsel, Karin

    2013-07-01

    Reduced to near extinction in the late 1800s, a number of wood bison populations (Bison bison athabascae) have been re-established through reintroduction initiatives. Although an invaluable tool for conservation, translocation of animals can spread infectious agents to new areas or expose animals to pathogens in their new environment. Mycobacterium avium subsp. paratuberculosis, a bacterium that causes chronic enteritis in ruminants, is among the pathogens of potential concern for wood bison management and conservation. In order to inform translocation decisions, our objectives were to determine the M. avium subsp. paratuberculosis infection status of wood bison herds in Canada and to culture and genetically characterize the infective strain(s). We tested fecal samples from bison (n = 267) in nine herds using direct PCR for three M. avium subsp. paratuberculosis-specific genetic targets with different copy numbers within the M. avium subsp. paratuberculosis genome. Restriction enzyme analysis (REA) and sequencing of IS1311 were performed on seven samples from five different herds. We also evaluated a panel of different culture conditions for their ability to support M. avium subsp. paratuberculosis growth from feces and tissues of direct-PCR-positive animals. Eighty-one fecal samples (30%) tested positive using direct IS900 PCR, with positive samples from all nine herds; of these, 75% and 21% were also positive using ISMAP02 and F57, respectively. None of the culture conditions supported the growth of M. avium subsp. paratuberculosis from PCR-positive samples. IS1311 REA and sequencing indicate that at least two different M. avium subsp. paratuberculosis strain types exist in Canadian wood bison. The presence of different M. avium subsp. paratuberculosis strains among wood bison herds should be considered in the planning of translocations.

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

    PubMed

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

    2007-07-01

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

  16. Mycobacterium avium subsp. paratuberculosis antibody response, fecal shedding, and antibody cross-reactivity to Mycobacterium bovis in M. avium subsp. paratuberculosis-infected cattle herds vaccinated against Johne's disease.

    PubMed

    Tewari, Deepanker; Hovingh, Ernest; Linscott, Rick; Martel, Edmond; Lawrence, John; Wolfgang, David; Griswold, David

    2014-05-01

    Vaccination for Johne's disease with killed inactivated vaccine in cattle herds has shown variable success. The vaccine delays the onset of disease but does not afford complete protection. Johne's disease vaccination has also been reported to interfere with measurements of cell-mediated immune responses for the detection of bovine tuberculosis. Temporal antibody responses and fecal shedding of Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, were measured in 2 dairy cattle herds using Johne's disease vaccine (Mycopar) over a period of 7 years. Vaccination against Johne's disease resulted in positive serum M. avium subsp. paratuberculosis antibody responses in both herds, and the responses persisted in vaccinated cattle up to 7 years of age. Some vaccinated animals (29.4% in herd A and 36.2% in herd B) showed no serological reactivity to M. avium subsp. paratuberculosis. M. avium subsp. paratuberculosis-specific antibody responses were also detected in milk from Johne's disease-vaccinated animals, but fewer animals (39.3% in herd A and 49.4% in herd B) had positive results with milk than with serum samples. With vaccination against M. avium subsp. paratuberculosis, fecal shedding in both dairy herds was reduced significantly (P < 0.001). In addition, when selected Johne's disease-vaccinated and -infected animals were investigated for serological cross-reactivity to Mycobacterium bovis, no cross-reactivity was observed.

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

    PubMed Central

    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

  18. 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.

  19. Molecular identification of Mycobacterium avium subspecies paratuberculosis in oral biopsies of Crohn’s disease patients

    PubMed Central

    2013-01-01

    Oral lesions may be found in patients with Crohn’s disease (CD), in a percentage up to 20%. The aim of this study was to investigate a possible relationship between Mycobacterium avium subsp. paratuberculosis (MAP) and oral lesions in CD patients. 23 oral biopsies were examined performing IS900 Nested PCR; 9 of them were positive: 8 from CD patients and 1 from a control. Our purpose is to go on with this study, amplifying the number of subjects examined and testing subjects with oral lesions related to diseases other than CD to verify the specific association between MAP and oral lesions in CD patients. PMID:23842143

  20. Molecular identification of Mycobacterium avium subspecies paratuberculosis in oral biopsies of Crohn's disease patients.

    PubMed

    Molicotti, Paola; Scanu, Antonio M; Lumbau, Aurea; Cannas, Sara; Bua, Alessandra; Lugliè, Pietrina; Zanetti, Stefania

    2013-07-10

    Oral lesions may be found in patients with Crohn's disease (CD), in a percentage up to 20%. The aim of this study was to investigate a possible relationship between Mycobacterium avium subsp. paratuberculosis (MAP) and oral lesions in CD patients. 23 oral biopsies were examined performing IS900 Nested PCR; 9 of them were positive: 8 from CD patients and 1 from a control. Our purpose is to go on with this study, amplifying the number of subjects examined and testing subjects with oral lesions related to diseases other than CD to verify the specific association between MAP and oral lesions in CD patients.

  1. What Role Does Mycobacterium avium subsp. paratuberculosis Play in Crohn's Disease?

    PubMed

    Bach, Horacio

    2015-02-01

    Crohn's disease (CD) is a chronic, debilitating inflammatory bowel disease with no etiological agent yet identified. Studies have demonstrated that the bacterium Mycobacterium avium subsp. paratuberculosis (MAP) is present in a high percentage of CD patients. Although MAP has been isolated from human specimens, current techniques fail to show the presence of MAP in 100 % of tissues or biopsies obtained from CD patient lesions, and thus MAP cannot meet Koch's postulate as the etiological agent of CD. In this report, the effect of genetic and immune factors as well as the presence of MAP as a potential environmental factor is analyzed.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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,...

  7. 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...

  8. 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...

  9. 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...

  10. 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 ...

  11. 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 ...

  12. 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...

  13. 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...

  14. 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...

  15. 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 ...

  16. 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...

  17. 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...

  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. 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 ...

  20. 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...

  1. 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 ...

  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), the causative agent of Johne’s disease. We hypothesized that feeding NP51 would increase Th-1 responses and decrease prog...

  3. 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 ...

  4. 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...

  5. 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 ...

  6. 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 ...

  7. 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...

  8. 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...

  9. Identification of Mycobacterium avium subsp. paratuberculosis in Biopsy Specimens from Patients with Crohn's Disease Identified by In Situ Hybridization

    PubMed Central

    Sechi, Leonardo A.; Manuela, Mura; Francesco, Tanda; Amelia, Lissia; Antonello, Solinas; Giovanni, Fadda; Stefania, Zanetti

    2001-01-01

    Crohn's disease is a chronic inflammatory disease of the gastrointestinal tract of unknown etiology. We report on the presence of cell wall-deficient Mycobacterium avium subsp. paratuberculosis in 35 of 48 paraffin-embedded tissue specimens from 33 patients with Crohn's disease by in situ hybridization with IS900 as a probe. PMID:11724871

  10. 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...

  11. 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

  12. Production and Evaluation of an Improved Mycobacterium avium subsp. paratuberculosis Purified Protein Derivative for Use in In-Vivo and In-Vitro Diagnostic Testing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Purified protein derivatives (PPD’s) were prepared from the cultured filtrate of Mycobacterium avium subsp. paratuberculosis (MAP) ATCC strain 19698. Production of PPD has historically been problematic for maintaining optimal floating cultures yielding defined immunogenic components. To obtain mor...

  13. 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...

  14. 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...

  15. Evaluation of immunodominant proteins of Mycobacterium avium paratuberculosis cell wall by Western blot analysis.

    PubMed

    Hashemi, Maryam; Madani, Rasool; Razmi, Nematollah

    2014-04-01

    Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow growing mycobactin, whose dependence on mycobacterial species is known to be the causative agent of Johne's disease (paratuberculosis) in all species of domestic ruminants worldwide. The organism is transmitted via close contact, ingestion, or transplacentally from mother to fetus and occurs commonly in grazing domestic animals. Johne's disease (JD) is characterized by gradual weight loss, decreased milk production, and diarrhea due to the chronic, progressive, granulomatous enteritis and lymphadenitis. The disease can cause serious economic damage to the dairy industry due to the loss of milk production and early culling of infected animals. In recent years, researchers have focused on the identification of a specific antigen of M. paratuberculosis to use in diagnosis test and preparation of effective vaccine. The goal of this study is evaluation of the immunodominant proteins of M. paratuberculosis cell wall. The amount of protein was determined with a Lowry assay (22.68 μg/100 μL). For production of polyclonal antibody against proteins of M. paratuberculosis cell wall, a New Zealand white rabbit was immunized with antigen and Freund's adjuvant. After immunization, the rabbit was bled to produce enriched serum. Antibodies were purified from serum with ion exchange chromatography. In the Ouchterlony test, the reactions between antigen and antibodies were seen in dilutions of one quarter for serum, one quarter for Ig, and one half for IgG by clear precipitation lines due to the well immunization of the rabbit. Electrophoresis and Western blot analysis were used and subsequently a sharp band appeared in nitrocellulose paper; these bands were about 25, 37, 50, 75, and 150 kDa molecular weight, which indicated immunodominant proteins.

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

    PubMed Central

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

    2017-01-01

    Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic inflammatory bowel disease of cattle characterized by intermittent to chronic diarrhea. In addition, MAP has been isolated from Crohn's disease (CD) patients. The impact of MAP on severity of clinical symptoms in JD as well as its role in CD are yet unknown. We have previously shown that MAP is able to colonize inflamed enteric tissue and to exacerbate the inflammatory tissue response (Suwandi et al., 2014). In the present study, we analyzed how repeated MAP administration influences the course of dextran sulfate sodium (DSS)-induced colitis. In comparison to mice exposed to DSS or MAP only, repeated exposure of DSS-treated mice to MAP (DSS/MAP) revealed a significantly enhanced clinical score, reduction of colon length as well as severe CD4+ T cell infiltration into the colonic lamina propria. Functional analysis identified a critical role of CD4+ T cells in the MAP-induced disease exacerbation. Additionally, altered immune responses were observed when closely related mycobacteria species such as M. avium ssp. avium and M. avium ssp. hominissuis were administered. These data reveal the specific ability of MAP to aggravate intestinal inflammation and clinical symptoms. Overall, this phenotype is compatible with similar disease promoting capabilites of MAP in JD and CD. PMID:28361039

  17. 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.

  18. Proteome-determined type-specific proteins of Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Hughes, Valerie; Garcia-Sanchez, Alfredo; Smith, Stuart; Mclean, Kevin; Lainson, Alex; Nath, Mintu; Stevenson, Karen

    2012-07-06

    Mycobacterium avium subspecies paratuberculosis (M. a. paratuberculosis) is a pathogen of ruminants, causing paratuberculosis (characterized by severe emaciation). The disease is endemic in many countries including the UK and places a severe economic burden on the global livestock industry. Two types of M. a. paratuberculosis can be classified by pulsed-field electrophoresis (I/III and II), which are phenotypically distinct and appear to have different host preferences. Proteomes of Type I and Type II M. a. paratuberculosis were analyzed by 2-D gel electrophoresis to determine if any significant differences existed between the subtypes. Seven different strains of Type I and 18 strains of Type II were analyzed and compared to detect type-specific differences. These 'type-specific' differences existed regardless of growth phase and were also exhibited in cells isolated directly from pathogenic lesions. Twenty-three spots predominated on the Type I profile, from which 17 proteins were identified. Twenty-one spots predominated on the Type II profile, from which 16 proteins were identified. None of the proteins identified as differentially represented on the profiles of Type I or Type II corresponded to open reading frames of the defining genomic regions as previously described for the Type I (sheep) and Type II (cattle). Sequence polymorphisms existing in Type I and II strains were identified in some open reading frames or regulatory regions of genes that correspond to proteins expressed in a type-specific fashion. The consequence of these is discussed in relation to protein expression and their impact on the type phenotype is discussed.

  19. 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

  20. Optimization of a phage amplification assay to permit accurate enumeration of viable Mycobacterium avium subsp. paratuberculosis cells.

    PubMed

    Foddai, Antonio; Elliott, Christopher T; Grant, Irene R

    2009-06-01

    A commercially available phage amplification assay, FASTPlaqueTB (Biotec Laboratories, Ipswich, United Kingdom), when used according to the manufacturer's instructions, does not permit accurate enumeration of Mycobacterium avium subsp. paratuberculosis. The aim of this study was to optimize the phage amplification assay conditions to permit accurate quantification of viable M. avium subsp. paratuberculosis cells. The burst time for M. avium subsp. paratuberculosis was initially determined to inform decisions about optimal incubation time before plating, and then other test parameters were altered to evaluate how the correlation between plaque and colony counts was affected. The D29 mycobacteriophage replicates more slowly in M. avium subsp. paratuberculosis than in Mycobacterium smegmatis (used to optimize the commercial test originally), and the mean burst time for four M. avium subsp. paratuberulosis strains was 210 +/- 36.8 min at 37 degrees C compared to 63 +/- 17.5 min for M. smegmatis mc(2) 155. To achieve 100% correlation between plaque and colony counts, the optimized phage assay includes the following: (i) resuspension of the samples to be tested in Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase and 2 mM calcium chloride, followed by overnight incubation at 37 degrees C before performance of the phage assay; (ii) a 2-h incubation of the sample with D29 mycobacteriophage before viricide treatment; and (iii) a further 90-min incubation after viricide treatment and neutralization up to the burst time (total incubation time, 210 min) before plating with M. smegmatis mc(2) 155 in 7H9 agar. The optimized phage amplification assay was able to detect 1 to 10 CFU/ml of M. avium subsp. paratuberculosis in spiked milk or broth within 48 h, as demonstrated by the results of several blind trials.

  1. 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

  2. High-throughput direct fecal PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in sheep and cattle.

    PubMed

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

    2014-03-01

    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

  3. 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

  4. Presence of PPE proteins in Mycobacterium avium subsp. paratuberculosis isolates and their immunogenicity in cattle.

    PubMed

    Newton, Victoria; McKenna, Shawn L; De Buck, Jeroen

    2009-03-30

    Johne's disease or paratuberculosis in cattle is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Although the stages of infection have been well described, very few virulence factors of MAP have been studied in detail. We aimed to study the localization and immunogenicity of members of the polymorphic PPE protein family which is unique to Mycobacteria and has been linked to virulence in Mycobacterium tuberculosis (Mtb). The presence of PPE proteins in the cell wall was investigated by enzymatic digest of surface exposed proteins of live MAP bacteria and analysis by LC-MS/MS. Polyclonal antisera were generated against a recombinant fragment of one PPE protein and a synthetic peptide of the other to confirm their surface exposure. Sera from naturally infected cows were investigated for the presence of specific antibodies against the recombinant PPE protein. Two PPE proteins, Map3420c and Map1506, were detected by mass spectrometry and confirmed to be surface exposed on live MAP cells by immunohistochemistry. The sera from naturally infected animals contained specific antibodies against recombinantly expressed Map3420c as demonstrated by western blotting. These findings show the in vitro expression of two PPE proteins. Additionally the surface exposure and immunogenicity of PPE proteins of MAP was demonstrated.

  5. Seroprevalence of Mycobacterium avium subspecies paratuberculosis in Korean black goats (Capra hircus aegagrus).

    PubMed

    Lee, Kyung Woo; Jung, Byeong Yeal; Moon, Oun Kyoung; Yang, Dong Kun; Lee, Su Hwa; Kim, Ji Yeon; Kweon, Chang Hee

    2006-12-01

    In total, 582 sera from 116 black goat herds were analyzed by a commercially available ELISA kit to monitor the seroprevalence of Mycobacterium avium subspecies paratuberculosis (Mpt) in Korean black goats (Capra hircus aegagrus). The mean number of goats sampled per herd was 5.11, 4.66, and 5.38 for the northern, central, and southern regions of Korea, respectively. The apparent regional prevalence of Mpt was estimated at 18.2-38.2% and 4.6-15.3% for herds and goats, respectively. The Mpt-positive goats were predominantly detected in the south (n=28), compared to either the northern (n=9) or central (n=11) regions (chi=14.459, P<0.05). Our findings indicate that Mpt is prevalent among the goat population, but regional variation exists.

  6. Interferon gamma response to Mycobacterium avium subsp. paratuberculosis specific lipopentapeptide antigen L5P in cattle.

    PubMed

    Holbert, Sébastien; Branger, Maxime; Souriau, Armel; Lamoureux, Bérénice; Ganneau, Christelle; Richard, Gaëlle; Cochard, Thierry; Tholoniat, Christophe; Bay, Sylvie; Winter, Nathalie; Moyen, Jean Louis; Biet, Franck

    2015-10-01

    After Mycobacterium avium subsp. paratuberculosis (Map) infection the cell-mediated immune (CMI) response indicative of early Th1 activation may be detected using interferon-gamma release assay (IGRA). Currently, the purified protein derivatives (PPDs), i.e., the total extract of mycobacteria antigens are used to recall CMI responses against Map. This study aimed to assess the ability of the chemically synthesized Map specific cell wall lipopentapeptide L5P to induce CMI response in cows infected by Map compared to PPD. L5P and PPD elicited an IFN-γ response in 12 and 35 animals from two Map infected herds respectively, but IFN-γ was not detected in the 13 cows recruited from a non-infected herd. Levels of IFN-γ detected were higher with PPD than with L5P. There was no correlation between the IFN-γ response and the humoral response to Map or faecal culture.

  7. 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.

  8. 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.

  9. Inter- and Intra-subtype genotypic differences that differentiate Mycobacterium avium subspecies paratuberculosis strains

    PubMed Central

    2012-01-01

    Background Mycobacterium avium subspecies paratuberculosis (Map) is the aetiological agent of Johne’s disease or paratuberculosis and is included within the Mycobacterium avium complex (MAC). Map strains are of two major types often referred to as ‘Sheep’ or ‘S-type’ and ‘Cattle’ or ‘C-type’. With the advent of more discriminatory typing techniques it has been possible to further classify the S-type strains into two groups referred to as Type I and Type III. This study was undertaken to genotype a large panel of S-type small ruminant isolates from different hosts and geographical origins and to compare them with a large panel of well documented C-type isolates to assess the genetic diversity of these strain types. Methods used included Mycobacterial Interspersed Repetitive Units - Variable-Number Tandem Repeat analysis (MIRU-VNTR), analysis of Large Sequence Polymorphisms by PCR (LSP analysis), Single Nucleotide Polymorphism (SNP) analysis of gyr genes, Pulsed-Field Gel Electrophoresis (PFGE) and Restriction Fragment Length Polymorphism analysis coupled with hybridization to IS900 (IS900-RFLP) analysis. Results The presence of LSPA4 and absence of LSPA20 was confirmed in all 24 Map S-type strains analysed. SNPs within the gyr genes divided the S-type strains into types I and III. Twenty four PFGE multiplex profiles and eleven different IS900-RFLP profiles were identified among the S-type isolates, some of them not previously published. Both PFGE and IS900-RFLP segregated the S-type strains into types I and III and the results concurred with those of the gyr SNP analysis. Nine MIRU-VNTR genotypes were identified in these isolates. MIRU-VNTR analysis differentiated Map strains from other members of Mycobacterium avium Complex, and Map S-type from C-type but not type I from III. Pigmented Map isolates were found of type I or III. Conclusion This is the largest panel of S-type strains investigated to date. The S-type strains could be further divided

  10. 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

  11. Serologic tests for detecting antibodies against Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis in Eurasian wild boar (Sus scrofa scrofa).

    PubMed

    Boadella, Mariana; Lyashchenko, Konstantin; Greenwald, Reena; Esfandiari, Javan; Jaroso, Raquel; Carta, Tania; Garrido, Joseba M; Vicente, Joaquín; de la Fuente, José; Gortázar, Christian

    2011-01-01

    New tools to detect exposure of free-range Eurasian wild boar (Sus scrofa scrofa) to pathogenic mycobacteria would be valuable for improved disease surveillance and wildlife management. Two hundred sera from wild boar of known Mycobacterium bovis infection status were used to evaluate test suitability for the detection of antibodies against M. bovis and Mycobacterium avium subsp. paratuberculosis (or cross-reacting members of the M. avium complex). Two traditional enzyme-linked immunosorbent assays were evaluated using M. bovis purified protein derivative (bPPD) and paratuberculosis protoplasmatic antigen 3 (PPA3) as antigens, respectively, and a new point-of-care test format for bovine tuberculosis (bTB) that uses the innovative dual-path platform (DPP TB) test. The effect of individual factors (sex, age, lesions) on the diagnostic performance of the serologic tests was also determined. Although the DPP had a sensitivity of 89.6% and a specificity of 90.4%, for bPPD, the sensitivity was 79.2% and the specificity 100%. Both tests had a kappa agreement of 0.80. Sixty-five of 68 (95.6%) wild boar sera with antibodies against the PPA3 antigen corresponded to known M. bovis-infected wild boar. Significant differences were not observed in the bPPD and DPP readings among lesion categories or between age classes. A slight sex-related difference in sensitivity toward males in the DPP was found, but it was not detected in the bPPD enzyme-linked immunosorbent assay. The results support the use of antibody-based diagnostic tests for both large-scale and individual bTB testing of Eurasian wild boar and suggest that wild boar cannot be used as sentinels for infections caused by M. avium complex members.

  12. Culture- and quantitative IS900 real-time PCR-based analysis of the persistence of Mycobacterium avium subsp. paratuberculosis in a controlled dairy cow farm environment.

    PubMed

    Moravkova, M; Babak, V; Kralova, A; Pavlik, I; Slana, I

    2012-09-01

    The aim of this study was to monitor the persistence of Mycobacterium avium subsp. paratuberculosis in environmental samples taken from a Holstein farm with a long history of clinical paratuberculosis. A herd of 606 head was eradicated, and mechanical cleaning and disinfection with chloramine B with ammonium (4%) was carried out on the farm; in the surrounding areas (on the field and field midden) lime was applied. Environmental samples were collected before and over a period of 24 months after destocking. Only one sample out of 48 (2%) examined on the farm (originating from a waste pit and collected before destocking) was positive for M. avium subsp. paratuberculosis by cultivation on solid medium (Herrold's egg yolk medium). The results using real-time quantitative PCR (qPCR) showed that a total of 81% of environmental samples with an average mean M. avium subsp. paratuberculosis cell number of 3.09 × 10(3) were positive for M. avium subsp. paratuberculosis before destocking compared to 43% with an average mean M. avium subsp. paratuberculosis cell number of 5.86 × 10(2) after 24 months. M. avium subsp. paratuberculosis-positive samples were detected in the cattle barn as well as in the calf barn and surrounding areas. M. avium subsp. paratuberculosis was detected from different matrices: floor and instrument scrapings, sediment, or scraping from watering troughs, waste pits, and cobwebs. M. avium subsp. paratuberculosis DNA was also detected in soil and plants collected on the field midden and the field 24 months after destocking. Although the proportion of positive samples decreased from 64% to 23% over time, the numbers of M. avium subsp. paratuberculosis cells were comparable.

  13. Mycobacterium avium subspecies paratuberculosis confirmed following serological surveillance of small ruminants in Grenada, West Indies.

    PubMed

    Kumthekar, Sachin; Manning, Elizabeth J B; Ghosh, Pallab; Tiwari, Keshaw; Sharma, Ravindra N; Hariharan, Harry

    2013-07-01

    Surveillance for Mycobacterium avium subsp. paratuberculosis (Map) infection in small ruminants of Grenada was undertaken using a commercial enzyme-linked immunosorbent assay (ELISA). Among the 479 sheep tested, 11 (2.3%) were ELISA positive while only 1 out of 260 goats (0.3%) was ELISA positive. Five of the 12 ELISA-positive animals were also positive in a commercial agar gel immunodiffusion (AGID) assay, and 4 of these showed acid-fast rods consistent with Map in fecal smears. Two sheep that were test-positive by ELISA, AGID, and fecal smears were euthanized and necropsied. Both had gross and histological lesions of paratuberculosis affecting the ileocecal area of small intestines and adjacent lymph nodes. These tissues were successfully cultured in 2 of 3 variants of Middlebrook 7H10 medium. The identity of acid-fast organisms isolated from the tissues was confirmed as Map by multiplex conventional polymerase chain reaction. Using IS1311 amplification and Hinf I restriction digest analysis, isolates were identified as cattle (C) strains of Map. The current study describes Map infection in Grenada and confirms the presence of C type in sheep on the island of Carriacou. The low seroprevalence in clinically normal animals on the islands of Grenada and Carriacou suggests that control measures implemented in the near future may have a good chance of preventing spread of the infection.

  14. The role of IL-10 in Mycobacterium avium subsp. paratuberculosis infection.

    PubMed

    Hussain, Tariq; Shah, Syed Zahid Ali; Zhao, Deming; Sreevatsan, Srinand; Zhou, Xiangmei

    2016-12-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is an intracellular pathogen and is the causative agent of Johne's disease of domestic and wild ruminants. Johne's disease is characterized by chronic granulomatous enteritis leading to substantial economic losses to the livestock sector across the world. MAP persistently survives in phagocytic cells, most commonly in macrophages by disrupting its early antibacterial activity. MAP triggers several signaling pathways after attachment to pathogen recognition receptors (PRRs) of phagocytic cells. MAP adopts a survival strategy to escape the host defence mechanisms via the activation of mitogen-activated protein kinase (MAPK) pathway. The signaling mechanism initiated through toll like receptor 2 (TLR2) activates MAPK-p38 results in up-regulation of interleukin-10 (IL-10), and subsequent repression of inflammatory cytokines. The anti-inflammatory response of IL-10 is mediated through membrane-bound IL-10 receptors, leading to trans-phosphorylation and activation of Janus Kinase (JAK) family receptor-associated tyrosine kinases (TyKs), that promotes the activation of latent transcription factors, signal transducer and activators of transcription 3 (STAT3). IL-10 is an important inhibitory cytokine playing its role in blocking phagosome maturation and apoptosis. In the current review, we describe the importance of IL-10 in early phases of the MAP infection and regulatory mechanisms of the IL-10 dependent pathways in paratuberculosis. We also highlight the strategies to target IL-10, MAPK and STAT3 in other infections caused by intracellular pathogens.

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

    PubMed

    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.

  16. 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.

  17. 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

  18. 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.

  19. Comparative transcriptional analysis of human macrophages exposed to animal and human isolates of Mycobacterium avium subspecies paratuberculosis with diverse genotypes.

    PubMed

    Motiwala, Alifiya S; Janagama, Harish K; Paustian, Michael L; Zhu, Xiaochun; Bannantine, John P; Kapur, Vivek; Sreevatsan, Srinand

    2006-11-01

    Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in animals and has been hypothesized to be associated with Crohn's disease in humans. Recently, M. avium subsp. paratuberculosis isolates recovered from Crohn's disease patients were shown to have limited diversity, implying the existence of human disease-associated genotypes and strain sharing with animals (A. H. Ghadiali et al., J. Clin. Microbiol. 42:5345-5348, 2004). To explore whether these genotypic differences or similarities among human and animal isolates translated to functionally significant attributes such as variance in host preference and/or difference in magnitude of infections, we performed a global scale analysis of M. avium subsp. paratuberculosis isolates that were representative of different genotypes and host species using DNA microarrays. Genome-wide characterization of the transcriptional changes was carried out using a human monocytic cell line (THP-1 cells) in response to different genotypes of M. avium subsp. paratuberculosis isolates recovered from various hosts. We identified several differentially expressed genes during early intracellular infection, including those involved in common canonical pathways such as NF-kappaB, interleukin-6 (IL-6), mitogen-activated protein kinase/extracellular signal-regulated kinase, and Jun N-terminal protein kinase signaling, as well as genes involved in T helper type 1 (Th1) responses (such as CCL5 ligand) and those that encode several proinflammatory cytokines and chemokine receptors. The cattle and human isolates of M. avium subsp. paratuberculosis, regardless of their short sequence repeat (SSR) genotype, induced similar global gene expression patterns in THP-1 cells. They differentially regulated genes necessary for cell survival without causing major alterations in proinflammatory genes. In contrast, the sheep isolates representing diverse SSR genotypes closely resembled the global gene expression pattern of an M

  20. Rapid assessment of the viability of Mycobacterium avium subsp. paratuberculosis cells after heat treatment, using an optimized phage amplification assay.

    PubMed

    Foddai, Antonio; Elliott, Christopher T; Grant, Irene R

    2010-03-01

    Thermal inactivation experiments were carried out to assess the utility of a recently optimized phage amplification assay to accurately enumerate viable Mycobacterium avium subsp. paratuberculosis cells in milk. Ultra-heat-treated (UHT) whole milk was spiked with large numbers of M. avium subsp. paratuberculosis organisms (10(6) to 10(7) CFU/ml) and dispensed in 100-microl aliquots in thin-walled 200-microl PCR tubes. A Primus 96 advanced thermal cycler (Peqlab, Erlangen, Germany) was used to achieve the following time and temperature treatments: (i) 63 degrees C for 3, 6, and 9 min; (ii) 68 degrees C for 20, 40, and 60 s; and (iii) 72 degrees C for 5, 10, 15, and 25 s. After thermal stress, the number of surviving M. avium subsp. paratuberculosis cells was assessed by both phage amplification assay and culture on Herrold's egg yolk medium (HEYM). A high correlation between PFU/ml and CFU/ml counts was observed for both unheated (r(2) = 0.943) and heated (r(2) = 0.971) M. avium subsp. paratuberculosis cells. D and z values obtained using the two types of counts were not significantly different (P > 0.05). The D(68 degrees C), mean D(63 degrees C), and D(72 degrees C) for four M. avium subsp. paratuberculosis strains were 81.8, 9.8, and 4.2 s, respectively, yielding a mean z value of 6.9 degrees C. Complete inactivation of 10(6) to 10(7) CFU of M. avium subsp. paratuberculosis/ml milk was not observed for any of the time-temperature combinations studied; 5.2- to 6.6-log(10) reductions in numbers were achieved depending on the temperature and time. Nonlinear thermal inactivation kinetics were consistently observed for this bacterium. This study confirms that the optimized phage assay can be employed in place of conventional culture on HEYM to speed up the acquisition of results (48 h instead of a minimum of 6 weeks) for inactivation experiments involving M. avium subsp. paratuberculosis-spiked samples.

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

    PubMed Central

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

    2016-01-01

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

  2. 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

  3. Susceptibility to and diagnosis of Mycobacterium avium subspecies paratuberculosis infection in dairy calves: A review.

    PubMed

    Mortier, Rienske A R; Barkema, Herman W; De Buck, Jeroen

    2015-10-01

    The primary objectives of paratuberculosis control programs are reducing exposure of calves to Mycobacterium avium subspecies paratuberculosis (MAP), reducing herd infection pressure and regular testing of cattle >36 months of age. Although control programs based on these principles have reduced prevalence of MAP infection in dairy herds, they have generally not eliminated the infection. Recent infection trial(s) have yielded new knowledge regarding diagnostic testing and age- and dose-dependent susceptibility to MAP infection. Calves up to 1 year of age are still susceptible to MAP infection; therefore, control programs should refrain from referring to specific ages with respect to susceptibility and prevention of new infections. Notwithstanding, lesions were more severe when calves were inoculated at 2 weeks versus 1 year of age. Furthermore, a high inoculation dose resulted in more pronounced lesions than a low inoculation dose, especially in young calves. Consequently, keeping infection pressure low should decrease the incidence of new MAP infections and severity of JD in cattle that do acquire the infection. It was also evident that early diagnosis of MAP infection was possible and could improve efficacy of control programs. Although its use will still need to be validated in the field, a combination of antibody ELISA and fecal culture in young stock, in addition to testing cattle >36 months of age when screening a herd for paratuberculosis, was expected to improve detection of dairy cattle infected with MAP. Although calves were inoculated using a standardized method in a controlled environment, there were substantial differences among calves with regards to immune response, shedding and pathology. Therefore, we inferred there were genetic differences in susceptibility. Important insights were derived from experimental infection trials. Therefore, it was expected that these could improve paratuberculosis control programs by reducing severity and incidence of

  4. Polymerase chain reaction-restriction fragment length polymorphism of the rpoB gene for identification of Mycobacterium avium subsp. paratuberculosis and differentiation of Mycobacterium avium subspecies.

    PubMed

    Whang, Jake; Lee, Byung Soo; Choi, Go-Eun; Cho, Sang-Nae; Kil, Park Young; Collins, Michael T; Shin, Sung Jae

    2011-05-01

    Mycobacterial speciation by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) of the rpoB gene was evaluated for identification of Mycobacterium avium subsp. paratuberculosis (MAP) and other Mycobacterium avium complex (MAC) members to the species or subspecies level by comparison with conventional methods including hsp65 sequencing, high-performance liquid chromatography, and PCR for accepted species- or subspecies-specific genomic targets. A total of 185 type and clinical mycobacterial strains from humans, animals, and environments were tested. A 360-bp PCR product was subsequently digested with MspI, HaeIII, and SmaI restriction enzymes. The PRA using SmaI restriction showed a unique digestion pattern for MAP distinguishing it from other MAC members and other Mycobacterium spp. Moreover, HaeIII and MspI restriction of the rpoB gene enabled MAC-species and -subspecies discrimination. The rpoB-PRA using SmaI or MspI and HaeIII restriction of the rpoB gene is a simple, convenient, and reliable confirmatory assay for simultaneous identification of MAP and other MAC members.

  5. Apa antigen of Mycobacterium avium subsp. paratuberculosis as a target for species-specific immunodetection of the bacteria in infected tissues of cattle with paratuberculosis.

    PubMed

    Souza, Giliane S; Rodrigues, Ana Bárbara F; Gioffré, Andrea; Romano, Maria I; Carvalho, Eulógio C Q; Ventura, Thatiana L B; Lasunskaia, Elena B

    2011-09-15

    Comparative genomics of Mycobacterium spp. have revealed conservative genes and respective proteins differently expressed in mycobacteria that could be used as targets for the species-specific immunodiagnostics. The alanine and proline-rich antigen Apa is a mycobacterial protein that present significant variability in primary sequence length and composition between members of M. avium and M. tuberculosis complexes. In this study, the recombinant Apa protein encoded by the MAP1569/ModD gene of M. avium subsp. paratuberculosis (Map) was used to generate a panel of monoclonal antibodies which were shown to recognize the most important veterinary pathogens of the M. avium complex, specifically Map and M. avium subsp. hominissuis, and which did not cross-react with M. bovis or M. tuberculosis. The produced antibodies were demonstrated to be a useful tool for the species-specific immunofluorescence or immunohistochemical detection of Map in experimentally infected cell cultures or intestinal tissues from cattle with bovine paratuberculosis and, additionally, they may be employed for the discrimination of pathogenic M. avium subspecies via Western blotting.

  6. Application of the C(18)-carboxypropylbetaine specimen processing method to recovery of Mycobacterium avium subsp. paratuberculosis from ruminant tissue specimens.

    PubMed

    Thornton, Charles G; MacLellan, Kerry M; Stabel, Judith R; Carothers, Christine; Whitlock, Robert H; Passen, Selvin

    2002-05-01

    The causative agent of Johne's disease is Mycobacterium avium subsp. paratuberculosis. This is a chronic, debilitating gastrointestinal disorder that affects ruminants and is responsible for significant economic loss. The specimen processing method that combines C(18)-carboxypropylbetaine (CB-18) treatment and lytic enzyme decontamination has been shown to improve the diagnosis of mycobacterioses. This processing method was applied to the isolation of M. avium subsp. paratuberculosis from ruminant tissue samples. The BACTEC 12B liquid culture system was used but was supplemented with 1% egg yolk emulsion, 4 microg of mycobactin J, and 0.5% pyruvate (12B/EMP) for use in conjunction with this method. The final concentration of antibiotics used was 10 microg of vancomycin, 30 microg of amphotericin B, and 20 microg of nalidixic acid (VAN) per ml. A 7H10-based solid medium was also used that included mycobactin J, pyruvate, and VAN but excluded the egg yolk emulsion (7H10/MPV). Several M. avium subsp. paratuberculosis isolates were examined during the evaluation of this processing method. It was observed that treatment with lytic enzymes stimulated the growth of M. avium subsp. paratuberculosis; however, the growth of one isolate was markedly inhibited due to the presence of vancomycin. Subsequently, the vancomycin concentration in the VAN formulation was reduced to 2 microg/ml. A blinded panel of 60 previously characterized tissue samples from bovine and bison were then processed and analyzed by smear and culture. Historically, 31 and 37 specimens were classified as positive by histology and culture, respectively. The overall sensitivity and specificity of smear relative to culture following CB-18 processing were 97.6 and 89.5%, respectively. The 12B/EMP/VAN liquid culture system recovered M. avium subsp. paratuberculosis from 39 specimens, whereas 7H10/MPV and Herrold's egg yolk media recovered M. avium subsp. paratuberculosis from 26 and 16 specimens, respectively

  7. 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.

  8. 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

  9. Evaluation of a PCR assay on overgrown environmental samples cultured for Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Arango-Sabogal, Juan C; Labrecque, Olivia; Paré, Julie; Fairbrother, Julie-Hélène; Roy, Jean-Philippe; Wellemans, Vincent; Fecteau, Gilles

    2016-11-01

    Culture of Mycobacterium avium subsp. paratuberculosis (MAP) is the definitive antemortem test method for paratuberculosis. Microbial overgrowth is a challenge for MAP culture, as it complicates, delays, and increases the cost of the process. Additionally, herd status determination is impeded when noninterpretable (NI) results are obtained. The performance of PCR is comparable to fecal culture, thus it may be a complementary detection tool to classify NI samples. Our study aimed to determine if MAP DNA can be identified by PCR performed on NI environmental samples and to evaluate the performance of PCR before and after the culture of these samples in liquid media. A total of 154 environmental samples (62 NI, 62 negative, and 30 positive) were analyzed by PCR before being incubated in an automated system. Growth was confirmed by acid-fast bacilli stain and then the same PCR method was again applied on incubated samples, regardless of culture and stain results. Change in MAP DNA after incubation was assessed by converting the PCR quantification cycle (Cq) values into fold change using the 2(-ΔCq) method (ΔCq = Cq after culture - Cq before culture). A total of 1.6% (standard error [SE] = 1.6) of the NI environmental samples had detectable MAP DNA. The PCR had a significantly better performance when applied after culture than before culture (p = 0.004). After culture, a 66-fold change (SE = 17.1) in MAP DNA was observed on average. Performing a PCR on NI samples improves MAP culturing. The PCR method used in our study is a reliable and consistent method to classify NI environmental samples.

  10. Short communication: Survival of Mycobacterium avium ssp. paratuberculosis in tissues of cows following low-dose exposure to electron beam irradiation.

    PubMed

    Bode, John F; Thoen, Charles O

    2016-08-01

    This investigation was designed to determine the effects of low-dose electron beam irradiation on the survival of Mycobacterium avium ssp. paratuberculosis in tissue samples collected at necropsy from clinically affected cows. Mycobacterium avium ssp. paratuberculosis was isolated from the ileum and ileocecal valve of one cow and from the ileum of another cow irradiated at 4.0 kGy, but was not isolated from the ileum, ileocecal valve, or mesenteric lymph node of 11 other cows irradiated at 4 kGy.

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

    PubMed

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

    2005-06-01

    The effect of various pasteurization time-temperature conditions with and without homogenization on the viability of Mycobacterium avium subsp. paratuberculosis was investigated using a pilot-scale commercial high-temperature, short-time (HTST) pasteurizer and raw milk spiked with 10(1) to 10(5) M. avium subsp. paratuberculosis cells/ml. Viable M. avium subsp. paratuberculosis was cultured from 27 (3.3%) of 816 pasteurized milk samples overall, 5 on Herrold's egg yolk medium and 22 by BACTEC culture. Therefore, in 96.7% of samples, M. avium subsp. paratuberculosis had been completely inactivated by HTST pasteurization, alone or in combination with homogenization. Heat treatments incorporating homogenization at 2,500 lb/in2, applied upstream (as a separate process) or in hold (at the start of a holding section), resulted in significantly fewer culture-positive samples than pasteurization treatments without homogenization (P < 0.001 for those in hold and P < 0.05 for those upstream). Where colony counts were obtained, the number of surviving M. avium subsp. paratuberculosis cells was estimated to be 10 to 20 CFU/150 ml, and the reduction in numbers achieved by HTST pasteurization with or without homogenization was estimated to be 4.0 to 5.2 log10. The impact of homogenization on clump size distribution in M. avium subsp. paratuberculosis broth suspensions was subsequently assessed using a Mastersizer X spectrometer. These experiments demonstrated that large clumps of M. avium subsp. paratuberculosis cells were reduced to single-cell or "miniclump" status by homogenization at 2,500 lb/in2. Consequently, when HTST pasteurization was being applied to homogenized milk, the M. avium subsp. paratuberculosis cells would have been present as predominantly declumped cells, which may possibly explain the greater inactivation achieved by the combination of pasteurization and homogenization.

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

    PubMed Central

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

    2005-01-01

    The effect of various pasteurization time-temperature conditions with and without homogenization on the viability of Mycobacterium avium subsp. paratuberculosis was investigated using a pilot-scale commercial high-temperature, short-time (HTST) pasteurizer and raw milk spiked with 101 to 105 M. avium subsp. paratuberculosis cells/ml. Viable M. avium subsp. paratuberculosis was cultured from 27 (3.3%) of 816 pasteurized milk samples overall, 5 on Herrold's egg yolk medium and 22 by BACTEC culture. Therefore, in 96.7% of samples, M. avium subsp. paratuberculosis had been completely inactivated by HTST pasteurization, alone or in combination with homogenization. Heat treatments incorporating homogenization at 2,500 lb/in2, applied upstream (as a separate process) or in hold (at the start of a holding section), resulted in significantly fewer culture-positive samples than pasteurization treatments without homogenization (P < 0.001 for those in hold and P < 0.05 for those upstream). Where colony counts were obtained, the number of surviving M. avium subsp. paratuberculosis cells was estimated to be 10 to 20 CFU/150 ml, and the reduction in numbers achieved by HTST pasteurization with or without homogenization was estimated to be 4.0 to 5.2 log10. The impact of homogenization on clump size distribution in M. avium subsp. paratuberculosis broth suspensions was subsequently assessed using a Mastersizer X spectrometer. These experiments demonstrated that large clumps of M. avium subsp. paratuberculosis cells were reduced to single-cell or “miniclump” status by homogenization at 2,500 lb/in2. Consequently, when HTST pasteurization was being applied to homogenized milk, the M. avium subsp. paratuberculosis cells would have been present as predominantly declumped cells, which may possibly explain the greater inactivation achieved by the combination of pasteurization and homogenization. PMID:15932977

  13. New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Herthnek, David; Bölske, Göran

    2006-01-01

    Background Johne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples. Results Our confirmatory PCR systems on IS900 were found sensitive and specific, only yielding weak false positive reactions in one strain for each system. The PCR system on F57 did not elicit any false positives and was only slightly less sensitive than our primary IS900-system. DNA from both naturally infected and spiked faeces that tested positive with our primary system could be confirmed with all new systems, except one low-level infected sample that tested negative with the F57 system. Conclusion We recommend using the newly constructed DH3 PCR system on the F57 gene as the primary confirmatory test for PCR positives, but should it fail due to its lower sensitivity, the DH1 and DH2 PCR systems should be used. PMID:17020599

  14. Transcriptional analysis of diverse strains Mycobacterium avium subspecies paratuberculosis in primary bovine monocyte derived macrophages.

    PubMed

    Zhu, Xiaochun; Tu, Zheng J; Coussens, Paul M; Kapur, Vivek; Janagama, Harish; Naser, Saleh; Sreevatsan, Srinand

    2008-10-01

    In this study we analyzed the macrophage-induced gene expression of three diverse genotypes of Mycobacterium avium subsp. paratuberculosis (MAP). Using selective capture of transcribed sequences (SCOTS) on three genotypically diverse MAP isolates from cattle, human, and sheep exposed to primary bovine monocyte derived macrophages for 48 h and 120 h we created and sequenced six cDNA libraries. Sequence annotations revealed that the cattle isolate up-regulated 27 and 241 genes; the human isolate up-regulated 22 and 53 genes, and the sheep isolate up-regulated 35 and 358 genes, at the two time points respectively. Thirteen to thirty-three percent of the genes identified did not have any annotated function. Despite variations in the genes identified, the patterns of expression fell into overlapping cellular functions as inferred by pathway analysis. For example, 10-12% of the genes expressed by all three strains at each time point were associated with cell-wall biosynthesis. All three strains of MAP studied up-regulated genes in pathways that combat oxidative stress, metabolic and nutritional starvation, and cell survival. Taken together, this comparative transcriptional analysis suggests that diverse MAP genotypes respond with similar modus operandi for survival in the host.

  15. First report of MIRU-VNTR genotyping of Mycobacterium avium subsp. paratuberculosis isolates from Egypt.

    PubMed

    Fawzy, A; Fayed, A; Youssef, H; El-Sayed, A; Zschöck, M

    2016-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, an economically important disease in ruminants worldwide. It was first isolated in Egypt in 2005. Since then, the pathogen has been detected in different Egyptian provinces. In order to trace the source of infection, genotyping using simple methods of high discriminatory power such as mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR) were carried out in different countries. Until now there is no published information about MIRU-VNTR genotyping of MAP isolates in Egypt. To address that point, 100 faecal samples were collected and cultivated from 3 different suspected dairy farms. Fourteen isolates belonging to one farm were identified as MAP and subjected to genotyping using 8 different MIRU-VNTR loci PCRs. Two different genotypes were recognized based on size polymorphism observed in one locus (VNTR-7) that was confirmed by sequencing. Our work provides a preliminary basis of constructing a MIRU-VNTR genotyping database of MAP in Egypt.

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

    PubMed

    Pierce, Ellen S

    2010-12-17

    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.

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

    Magombedze, Gesham; Shiri, Tinevimbo; Eda, Shigetoshi; Stabel, Judy R.

    2017-01-01

    Available diagnostic assays for Mycobacterium avium subsp. paratuberculosis (MAP) have poor sensitivities and cannot detect early stages of infection, therefore, there is need to find new diagnostic markers for early infection detection and disease stages. We analyzed longitudinal IFN-γ, ELISA-antibody and fecal shedding experimental sensitivity scores for MAP infection detection and disease progression. We used both statistical methods and dynamic mathematical models to (i) evaluate the empirical assays (ii) infer and explain biological mechanisms that affect the time evolution of the biomarkers, and (iii) predict disease stages of 57 animals that were naturally infected with MAP. This analysis confirms that the fecal test is the best marker for disease progression and illustrates that Th1/Th2 (IFN-γ/ELISA antibodies) assays are important for infection detection, but cannot reliably predict persistent infections. Our results show that the theoretical simulated macrophage-based assay is a potential good diagnostic marker for MAP persistent infections and predictor of disease specific stages. We therefore recommend specifically designed experiments to test the use of a based assay in the diagnosis of MAP infections. PMID:28317944

  19. 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

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

    NASA Astrophysics Data System (ADS)

    Magombedze, Gesham; Shiri, Tinevimbo; Eda, Shigetoshi; Stabel, Judy R.

    2017-03-01

    Available diagnostic assays for Mycobacterium avium subsp. paratuberculosis (MAP) have poor sensitivities and cannot detect early stages of infection, therefore, there is need to find new diagnostic markers for early infection detection and disease stages. We analyzed longitudinal IFN-γ, ELISA-antibody and fecal shedding experimental sensitivity scores for MAP infection detection and disease progression. We used both statistical methods and dynamic mathematical models to (i) evaluate the empirical assays (ii) infer and explain biological mechanisms that affect the time evolution of the biomarkers, and (iii) predict disease stages of 57 animals that were naturally infected with MAP. This analysis confirms that the fecal test is the best marker for disease progression and illustrates that Th1/Th2 (IFN-γ/ELISA antibodies) assays are important for infection detection, but cannot reliably predict persistent infections. Our results show that the theoretical simulated macrophage-based assay is a potential good diagnostic marker for MAP persistent infections and predictor of disease specific stages. We therefore recommend specifically designed experiments to test the use of a based assay in the diagnosis of MAP infections.

  1. Mycobacterium avium subsp. paratuberculosis as a trigger of type-1 diabetes: destination Sardinia, or beyond?

    PubMed

    Rani, Pittu Sandhya; Sechi, Leonardo A; Ahmed, Niyaz

    2010-03-29

    Type 1 diabetes mellitus (T1DM) is a multifactorial autoimmune disease in which the insulin producing beta cell population is destroyed by the infiltrated T lymphocytes. Even though the exact cause of T1DM is yet to be ascertained, varying degree of genetic susceptibility and environmental factors have been linked to the disease progress and outcome. Mycobacterium avium subsp. paratuberculosis (MAP) is an obligate zoonotic pathogen that causes chronic infection of intestines in ruminants, the Johne's disease. MAP that can even survive pasteurization and chlorination has also been implicated to cause similar type of enteritis in humans called Crohn's disease. With the increasing recognition of the link between MAP and Crohn's disease, it has been postulated that MAP is an occult antigen which besides Crohn's could as well be thought to trigger T1DM. Epitope homologies between mycobacterial proteins (Hsp 65) and pancreatic glutamic acid decarboxylase (GAD 65) and infant nutrition studies implicate MAP as one of the triggers for T1DM. PCR and ELISA analyses in diabetic patients from Sardinia suggest that MAP acts as a possible trigger for T1DM. Systematic mechanistic insights are needed to prove this link. Unfortunately, no easy animal model(s) or in-vitro systems are available to decipher the complex immunological network that is triggered in MAP infection leading to T1DM.

  2. 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

    2016-02-29

    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.

  3. 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.

  4. 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

  5. Macrophage polarization in cattle experimentally exposed to Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Thirunavukkarasu, Shyamala; de Silva, Kumudika; Begg, Douglas J; Whittington, Richard J; Plain, Karren M

    2015-12-01

    Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease (JD) in cattle, has significant impacts on the livestock industry and has been implicated in the etiology of Crohn's disease. Macrophages play a key role in JD pathogenesis, which is driven by the manipulation of host immune mechanisms by MAP. A change in the macrophage microenvironment due to pathogenic or host-derived stimuli can lead to classical (M1) or alternative (M2) polarization of macrophages. In addition, prior exposure to antigenic stimuli has been reported to alter the response of macrophages to subsequent stimuli. However, macrophage polarization in response to MAP exposure and its possible implications have not been previously addressed. In this study, we have comprehensively examined monocyte/macrophage polarization and responsiveness to antigens from MAP-exposed and unexposed animals. At 3 years post-exposure, there was a heterogeneous macrophage activation pattern characterized by both classical and alternate phenotypes. Moreover, subsequent exposure of macrophages from MAP-exposed cattle to antigens from MAP and other mycobacterial species led to significant variation in the production of nitric oxide, interleukin-10 and tumour necrosis factor α. These results indicate the previously unreported possibility of changes in the activation state and responsiveness of circulating monocytes/macrophages from MAP-exposed cattle.

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

    PubMed

    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.

  7. 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.

  8. Detection of Mycobacterium avium subsp. paratuberculosis in bovine milk from the state of Pernambuco, Brazil.

    PubMed

    Albuquerque, Pedro Paulo Feitosa de; Santos, André de Souza; Souza Neto, Orestes Luiz de; Kim, Pomy de Cássia Peixoto; Cavalcanti, Erika Fernanda Torres Samico Fernandes; Oliveira, Júnior Mário Baltazar de; Mota, Rinaldo Aparecido; Júnior, José Wilton Pinheiro

    The aim of this study was to detect the IS900 region of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine milk samples using real-time polymerase chain reaction (qPCR) and conventional PCR, and to study the agreement between these tests. A total of 121 bovine milk samples were collected from herds considered positive for MAP, from the State of Pernambuco, Brazil. MAP DNA was detected in 20 samples (16.5%) using conventional PCR and in 34 samples (28.1%) using qPCR. MAP DNA was detected in all of the 6 animal farms studied. Moderate agreement was found between qPCR and conventional PCR results, where the sensitivity and specificity of conventional PCR in relation to qPCR were 50% and 96.6%, respectively. Thus, the IS900 region of MAP was found in bovine milk samples from the State of Pernambuco. To the best of our knowledge, this is the first report of MAP DNA found in bovine milk in Northeast Brazil. We also demonstrated the qPCR technique is more sensitive than conventional PCR with respect to detection of MAP in milk samples.

  9. 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.

  10. Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Hansen, Sören; Schäfer, Jenny; Fechner, Kim; Czerny, Claus-Peter; Abd El Wahed, Ahmed

    2016-01-01

    Background The detection of Mycobacterium avium subsp. paratuberculosis (MAP) infections in ruminants is crucial to control spread among animals and to humans. Cultivation of MAP is seen as the gold standard for detection, although it is very time consuming and labour intensive. In addition, several PCR assays have been developed to detect MAP in around 90 minutes, but these assays required highly sophisticated equipment as well as lengthy and complicated procedure. Methodology/Principal Findings In this study, we have developed a rapid assay for the detection of MAP based on the recombinase polymerase amplification (RPA) assay targeting a MAP specific region, the IS900 gene. The detection limit was 16 DNA molecules in 15 minutes as determined by the probit analysis on eight runs of the plasmid standard. Cross reactivity with other mycobacterial and environmentally associated bacterial strains was not observed. The clinical performance of the MAP RPA assay was tested using 48 MAP-positive and 20 MAP-negative blood, sperm, faecal and tissue samples. All results were compared with reads of a highly sensitive real-time PCR assay. The specificity of the MAP RPA assay was 100%, while the sensitivity was 89.5%. Conclusions/Significance The RPA assay is quicker and much easier to handle than real-time PCR. All RPA reagents were cold-chain independent. Moreover, combining RPA assay with a simple extraction protocol will maximize its use at point of need for rapid detection of MAP. PMID:27992571

  11. 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

  12. 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.

  13. Mycobacterium avium subsp. paratuberculosis as a trigger of type-1 diabetes: destination Sardinia, or beyond?

    PubMed Central

    2010-01-01

    Type 1 diabetes mellitus (T1DM) is a multifactorial autoimmune disease in which the insulin producing β cell population is destroyed by the infiltrated T lymphocytes. Even though the exact cause of T1DM is yet to be ascertained, varying degree of genetic susceptibility and environmental factors have been linked to the disease progress and outcome. Mycobacterium avium subsp. paratuberculosis (MAP) is an obligate zoonotic pathogen that causes chronic infection of intestines in ruminants, the Johne's disease. MAP that can even survive pasteurization and chlorination has also been implicated to cause similar type of enteritis in humans called Crohn's disease. With the increasing recognition of the link between MAP and Crohn's disease, it has been postulated that MAP is an occult antigen which besides Crohn's could as well be thought to trigger T1DM. Epitope homologies between mycobacterial proteins (Hsp 65) and pancreatic glutamic acid decarboxylase (GAD 65) and infant nutrition studies implicate MAP as one of the triggers for T1DM. PCR and ELISA analyses in diabetic patients from Sardinia suggest that MAP acts as a possible trigger for T1DM. Systematic mechanistic insights are needed to prove this link. Unfortunately, no easy animal model(s) or in-vitro systems are available to decipher the complex immunological network that is triggered in MAP infection leading to T1DM. PMID:20350307

  14. 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

  15. Proteomic changes in the ileum of sheep infected with Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Pisanu, S; Cubeddu, T; Uzzau, S; Rocca, S; Addis, M F

    2017-01-01

    Johne's disease (JD) is a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). To identify the processes activated in the sheep intestine during natural MAP infection, and to provide a panel of differential host and pathogen proteins with diagnostic and prognostic potential, a differential shotgun proteomics workflow, including mass spectrometry, label-free quantisation and pathway analysis, was applied to ileal tissues of ewes with and without JD. Out of 2889 total proteins identified, 384 were differentially expressed and 341 were expressed at a higher level in JD. On the basis of Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis, these proteins were involved in numerous relevant biological networks and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including inhibition of phagosome acidification (such as V-ATPase), bacterial invasion, leucocyte recruitment and activation, and antimicrobial activity (such as haptoglobin, lactoferrin, cathelicidins, calgranulins and interleukins). A total of 28 MAP proteins were identified, including bacterioferritin, β-lactamase and heparin-binding haemagglutinin (HBHA), a mycobacterial adhesin crucial for dissemination of infection.

  16. 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

  17. A new compartmental model of Mycobacterium avium subsp. paratuberculosis infection dynamics in cattle

    PubMed Central

    Smith, Rebecca L.; Schukken, Ynte H.; Gröhn, Yrjö T.

    2015-01-01

    Models of Mycobacterium avium subsp. paratuberculosis (MAP), a chronic infectious agent of cattle, are used to identify effective control programs. However, new biological findings show that adult infections occur and that infected animals can be separated into 2 paths: animals that will become high-shedding and, eventually, experience clinical disease (high-path); and animals that will shed only small quantities of MAP and will remain subclinical (low-path). Longitudinal data analysis found that high-path animals progress more quickly than previously believed. A standard model of MAP transmission in dairy herds was modified to include adult low-path infections and 2 infection pathways for infected calves. Analysis of this model showed that adult infection may play an important role in MAP dynamics on a dairy farm, and that the increased rate of progression for high-path animals influences both the prevalence and the persistence of MAP on a dairy farm. This new model will be able to determine the effectiveness of MAP control programs more accurately than previous models. PMID:26520176

  18. 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

  19. Macrophage polarization in cattle experimentally exposed to Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Thirunavukkarasu, Shyamala; de Silva, Kumudika; Begg, Douglas J; Whittington, Richard J; Plain, Karren M

    2015-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease (JD) in cattle, has significant impacts on the livestock industry and has been implicated in the etiology of Crohn's disease. Macrophages play a key role in JD pathogenesis, which is driven by the manipulation of host immune mechanisms by MAP. A change in the macrophage microenvironment due to pathogenic or host-derived stimuli can lead to classical (M1) or alternative (M2) polarization of macrophages. In addition, prior exposure to antigenic stimuli has been reported to alter the response of macrophages to subsequent stimuli. However, macrophage polarization in response to MAP exposure and its possible implications have not been previously addressed. In this study, we have comprehensively examined monocyte/macrophage polarization and responsiveness to antigens from MAP-exposed and unexposed animals. At 3 years post-exposure, there was a heterogeneous macrophage activation pattern characterized by both classical and alternate phenotypes. Moreover, subsequent exposure of macrophages from MAP-exposed cattle to antigens from MAP and other mycobacterial species led to significant variation in the production of nitric oxide, interleukin-10 and tumour necrosis factor α. These results indicate the previously unreported possibility of changes in the activation state and responsiveness of circulating monocytes/macrophages from MAP-exposed cattle. PMID:26454271

  20. European hares in Chile: a different lagomorph reservoir for Mycobacterium avium subsp. paratuberculosis?

    PubMed

    Salgado, Miguel; Manning, Elizabeth J B; Monti, Gustavo; Bölske, Göran; Söderlund, Robert; Ruiz, Manuel; Paredes, Enrique; Leiva, Sergio; Van Kruningen, Herbert; Kruze, Juan

    2011-07-01

    Ruminants are the principal host for infection by Mycobacterium avium subsp. paratuberculosis (Map), the cause of Johne's disease. Based on studies of a Map-infected population of European rabbits (Oryctolagus cuniculus) in Scotland, lagomorphs as a broad taxonomic order were proposed as potential nonruminant reservoirs for Map. To determine whether a different lagomorph species may serve as a wildlife reservoir, we investigated Map infection in European hares (Lepus europaeus) sharing habitat with known Map-infected dairy cattle in southern Chile. Fecal, mesenteric lymph node, and ileal samples were aseptically collected from 385 wild hares for liquid culture and real-time polymerase chain reaction identification of acid-fast isolates. All tissue samples were also acid-fast stained and examined microscopically. We isolated Map from at least one tissue from 48 hares (12.6%) and fecal samples from 16 hares (4.2%). No Map was found in tissues of eight of the fecal-culture-positive hares. Histologically, all tissues from all hares were within normal limits, and no acid-fast organisms were observed in any sample. Active infection, implying amplification of the organism secondary to resultant disease, was not evident. With this report Map isolations on a population versus incidental detection have now been made from two lagomorph species. However, although the rabbit population studied in Scotland appears to function as a Map reservoir, the hares studied in Chile appear to be a dead-end host, serving only as potential mechanical vectors for the organism.

  1. 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

  2. Serological and molecular detection of Mycobacterium avium subsp. paratuberculosis in cattle of dairy herds in Colombia.

    PubMed

    Fernández-Silva, Jorge Arturo; Abdulmawjood, Amir; Akineden, Omer; Bülte, Michael

    2011-12-01

    The objective of this study is the detection of Mycobacterium avium subsp. paratuberculosis (MAP) by serum enzyme-linked immunosorbent assay (ELISA), fecal polymerase chain reaction (PCR), and fecal culture in Colombian dairy herds. Serum and fecal samples from asymptomatic cows (n = 307) of 14 dairy herds were tested for MAP by an unabsorbed ELISA test (ELISA-A). Serum and fecal samples from positive ELISA-A animals (n = 31) were further tested by an absorbed ELISA test (ELISA-B) and PCR. Fecal samples from animals of herds positive by ELISA-A and PCR (n = 105) were inoculated onto three different culture media. ELISA-A produced positive results in 10% of the serum samples and 71% of the herds. ELISA-B and PCR results were positive in two and six serum and fecal samples from positive ELISA-A animals, respectively. Fecal samples were negative for MAP on all culture media. The results of this study confirmed the presence of MAP in local dairy herds and the difficulties of MAP detection in asymptomatic animals by ELISA, PCR, and fecal culture.

  3. Persistence of Mycobacterium avium subspecies paratuberculosis in endangered Florida Key deer and Key deer habitat.

    PubMed

    Murray, Heidi L; Yabsley, Michael J; Keel, M Kevin; Manning, Elizabeth J B; Wilmers, Thomas J; Corn, Joseph L

    2014-04-01

    Mycobacterium avium subsp. paratuberculosis (MAP) was first reported in the endangered Key deer (Odocoileus virginianus clavium) in 1996 on Big Pine Key, Florida, USA. By 2008, eight additional MAP-positive Key deer had been identified on Big Pine Key and the nearby Newfound Harbor Keys. This study was conducted to determine if MAP was still present in Key deer and whether natural or man-made freshwater sources were contaminated with MAP. Between November 2009 and September 2012, MAP was isolated from 36/369 (10%) fecal samples collected from the ground throughout the Key deer range on Big Pine Key and the Newfound Harbor Keys, but all 36 positive samples were from Little Palm Island (36/142 [25%]). Only 1/729 (0.1%) environmental samples was positive; this was from the garden fountain on Little Palm Island (1/81 [1%]). In addition, MAP was detected in 3/43 (7%) necropsied Key deer, all from Little Palm Island (3/3 [100%]). Of these three Key deer, pooled samples from the ileum, cecum, and ileocecal lymph node from two were MAP-culture positive and feces from one of these were culture-positive. The third deer was only PCR-positive. Evidence of MAP was only detected on Little Palm Island during this sampling period and environmental contamination was limited.

  4. 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

  5. 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

  6. Detection of Mycobacterium avium subsp. paratuberculosis by buoyant density centrifugation, sequence capture PCR and dot blot hybridisation.

    PubMed

    Halldórsdóttir, Stefania; Englund, Stina; Nilsen, Sigrun Fredsvold; Olsaker, Ingrid

    2002-07-22

    Detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) by polymerase chain reaction (PCR) is often hampered by the lack of efficient methods for sample treatment. We report a protocol for analysis of faecal samples based on buoyant density centrifugation in Percoll and IS900 sequence capture PCR combined with a dot blot assay for detection of low-grade infection of M. paratuberculosis. Serial dilutions of M. paratuberculosis genomic DNA and M. paratuberculosis bacteria were used to assess the sensitivity of the method. The final evaluation was performed with spiked faecal samples, which also were analysed by culture. The presence of PCR inhibitory substances in processed faecal samples was evaluated by including a PCR internal control. By using buoyant density centrifugation, sequence capture PCR, and dot blot hybridisation, we achieved a sensitivity of 10(3)CFU (colony forming units)/g of faeces. The detection limit by culture was assessed to 10(2)CFU/g of faeces. We conclude that the described protocol is a fast and sensitive alternative to bacterial culture of faecal samples.

  7. Pasteurization of milk and the heat resistance of Mycobacterium avium subsp. paratuberculosis: a critical review of the data.

    PubMed

    Lund, Barbara M; Gould, Grahame W; Rampling, Anita M

    2002-07-25

    Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) causes Johne's disease in ruminants (including cattle, sheep and goats) and other animals, and may contribute to Crohn's disease in humans. This possibility, and the fact that M. paratuberculosis may be present in raw milk, make it important to ensure that the heat treatment specified for pasteurization of milk will give acceptable inactivation of this bacterium, with an adequate margin of safety. Published studies of the heat resistance of this bacterium in milk have given widely differing results. Possible reasons for these differences, and the technical problems involved in the work, are reviewed. It is concluded that there is a need (i) for the adoption of an agreed Performance Criterion for pasteurization of milk in relation to this bacterium, (ii) a need for definitive laboratory experiments to understand and determine the heat resistance of M. paratuberculosis, and (iii) a need for an assessment of whether the minimum heat treatments specified at present for pasteurization of milk (Process Criteria) will meet the Performance Criterion for M. paratuberculosis. Measures are also required to ensure that commercial processes deliver continually the specified heat treatment, and to ensure that post-pasteurization contamination is avoided.

  8. 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

  9. 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.

  10. PCR-based detection of Mycobacterium avium subsp. paratuberculosis infection in cattle in South Korea using fecal samples

    PubMed Central

    PARK, Hong-Tae; SHIN, Min-Kyoung; PARK, Hyun-Eui; CHO, Yong-Il; YOO, Han Sang

    2016-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of bovine paratuberculosis (PTB). The first step in the control of PTB is the identification and isolation of sub-clinical fecal shedders from the herd. In the current study, real-time and nested PCR targeting MAP-specific genetic elements (IS900 and ISMAP02) DNA isolated from fecal samples were used to detect MAP infection in cattle. Of the 1,562 fecal samples obtained from 37 herds, regardless of diarrhea, 35 samples tested positive in both IS900-targeted real-time and ISMAP02-targeted nested PCR. At the herd level, 12 of the 37 herds were found to be positive for MAP. Detection rates of the PCR tests were similar to those reported for ELISA-based methods. These results suggest that PCR can be used to detect sub-clinical fecal shedders of MAP. PMID:27301582

  11. 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—...

  12. 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...

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

    PubMed Central

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

    2010-01-01

    This study investigated the susceptibility of 1- to 2-year-old cattle to Mycobacterium avium subsp. paratuberculosis (MAP) on pasture previously grazed by infected cattle. The exposure of yearling cattle to pastures contaminated with MAP resulted in infection with MAP, showing that age resistance to infection can be overcome by pressure of infection. PMID:20436867

  14. 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...

  15. 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...

  16. 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. ...

  17. 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...

  18. 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...

  19. 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...

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

    PubMed

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

    2012-04-01

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

  1. Proteome-scale identification and characterization of mitochondria targeting proteins of Mycobacterium avium subspecies paratuberculosis: Potential virulence factors modulating host mitochondrial function.

    PubMed

    Rana, Aarti; Kumar, Devender; Rub, Abdur; Akhter, Yusuf

    2015-07-01

    Mycobacterium avium subsp. paratuberculosis is the etiological agent of Johne's Disease among ruminants. During the course of infection, it expresses a number of proteins for its successful persistence inside the host that cause variety of physiological abnormalities in the host. Mitochondrion is one of the attractive targets for pathogenic bacteria. Employing a proteome-wide sequence and structural signature based approach we have identified 46 M. avium subsp. paratuberculosis proteins as potential targets for the host mitochondrial targeting. These may act as virulence factors modulating mitochondrial physiology for bacterial survival and immune evasion inside the host cells.

  2. 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

  3. 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

  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. Where Are All the Mycobacterium avium Subspecies paratuberculosis in Patients with Crohn's Disease?

    PubMed Central

    Pierce, Ellen S.

    2009-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic granulomatous inflammation of the intestines, Johne's disease, in dairy cows and every other species of mammal in which it has been identified. MAP has been identified in the mucosal layer and deeper bowel wall in patients with Crohn's disease by methods other than light microscopy, and by direct visualization in small numbers by light microscopy. MAP has not been accepted as the cause of Crohn's disease in part because it has not been seen under the microscope in large numbers in the intestines of patients with Crohn's disease. An analysis of the literature on the pathology of Crohn's disease and on possible MAP infection in Crohn's patients suggests that MAP might directly infect endothelial cells and adipocytes and cause them to proliferate, causing focal obstruction within already existing vessels (including granuloma formation), the development of new vessels (neoangiogenesis and lymphangiogenesis), and the “creeping fat” of the mesentery that is unique in human pathology to Crohn's disease but also occurs in bovine Johne's disease. Large numbers of MAP might therefore be found in the mesentery attached to segments of intestine affected by Crohn's disease rather than in the bowel wall, the blood and lymphatic vessels running through the mesentery, or the mesenteric fat itself. The walls of fistulas might result from the neoangiogenesis or lymphangiogenesis that occurs in the bowel wall in Crohn's disease and therefore are also possible sites of large numbers of MAP. The direct visualization of large numbers of MAP organisms in the tissues of patients with Crohn's disease will help establish that MAP causes Crohn's disease. PMID:19325887

  6. 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

  7. SNP genotyping of animal and human derived isolates of Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Wynne, James W; Beller, Christie; Boyd, Victoria; Francis, Barry; Gwoźdź, Jacek; Carajias, Marios; Heine, Hans G; Wagner, Josef; Kirkwood, Carl D; Michalski, Wojtek P

    2014-08-27

    Mycobacterium avium subsp. paratuberculosis (MAP) is the aetiological agent of Johne's disease (JD), a chronic granulomatous enteritis that affects ruminants worldwide. While the ability of MAP to cause disease in animals is clear, the role of this bacterium in human inflammatory bowel diseases remains unresolved. Previous whole genome sequencing of MAP isolates derived from human and three animal hosts showed that human isolates were genetically similar and showed a close phylogenetic relationship to one bovine isolate. In contrast, other animal derived isolates were more genetically diverse. The present study aimed to investigate the frequency of this human strain across 52 wild-type MAP isolates, collected predominantly from Australia. A Luminex based SNP genotyping approach was utilised to genotype SNPs that had previously been shown to be specific to the human, bovine or ovine isolate types. Fourteen SNPs were initially evaluated across a reference panel of isolates with known genotypes. A subset of seven SNPs was chosen for analysis within the wild-type collection. Of the seven SNPs, three were found to be unique to paediatric human isolates. No wild-type isolates contain these SNP alleles. Interestingly, and in contrast to the paediatric isolates, three additional adult human isolates (derived from adult Crohn's disease patients) also did not contain these SNP alleles. Furthermore we identified two SNPs, which demonstrate extensive polymorphism within the animal-derived MAP isolates. One of which appears unique to ovine and a single camel isolate. From this study we suggest the existence of genetic heterogeneity between human derived MAP isolates, some of which are highly similar to those derived from bovine hosts, but others of which are more divergent.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. Antemortem and postmortem examinations of the cattle calf naturally infected with Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Appana, Gangadhararao; Das, Dipankar; Veerasami, Maroudam; Senthilkumar, Ramachandran Lakshmikanthan; Durishetty, Munishkumar; Ramalakshmi, B.; Bahekar, Vijay; Mukherjee, Falguni; Chandran, Dev; Kumar, P. Uday; Sesikeran, B.

    2013-01-01

    A male cattle calf was detected as subclinically and naturally infected with Mycobacterium avium subspecies paratuberculosis (MAP) by a series of antemortem and postmortem tests. The MAP infection was identified by strong antibody and cell-mediated immune (CMI) response by a commercial ELISA kit and an intradermal Johnin test, respectively, in the initial antemortem examination. The antemortem status of the calf was further confirmed by MAP-specific interferon gamma (IFN-γ) response. For detection of IFN-γ response, MAP-specific IFN-γ release assays (IGRAs): (a) immuno capture ELISA (IC-ELISA) and (b) ELISPOT was employed. In addition, the presence of intracellular cytokine IFN-γ was detected by flow cytometry. For all cytokine assays, MAP-specific recombinant antigens HSP65 and 35 kDa were employed to overcome the poor sensitivity and specificity resulting from the use of Johnin, the crude protein purified derivative of MAP. Postmortem examination of the MAP-infected/suspected cattle calf did not reveal any pathognomonic gross lesions in the gastro-intestinal tract. Histopathological examination of multiple organs showed the presence of epithelioid cells/macrophages and edematous lesions in the mesenteric lymph nodes suggestive of MAP; however, no granulomas were observed in the intestinal tract. The necropsy samples of rectum and mesenteric lymph nodes were positive for isolation of MAP by culture in the BACTEC™ MGIT™ 960 system, and acid fast bacilli were demonstrated by fluorescence microscopy confirming the infection. Due to differential and complex expression patterns of MAP antigens reported in literature, a combination of assays such as those based on IGRAs and antibody detection is essential. Therefore, the current experimental evidence confirms the efficacy of the approach adopted. However, further studies will be needed to understand the optimal combination MAP-specific antigens for use in IGRAs or antibody assays that can be used for detecting

  13. A rational framework for evaluating the next generation of vaccines against Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Bannantine, John P; Hines, Murray E; Bermudez, Luiz E; Talaat, Adel M; Sreevatsan, Srinand; Stabel, Judith R; Chang, Yung-Fu; Coussens, Paul M; Barletta, Raúl G; Davis, William C; Collins, Desmond M; Gröhn, Yrjö T; Kapur, Vivek

    2014-01-01

    Since the early 1980s, several investigations have focused on developing a vaccine against Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep. These studies used whole-cell inactivated vaccines that have proven useful in limiting disease progression, but have not prevented infection. In contrast, modified live vaccines that invoke a Th1 type immune response, may improve protection against infection. Spurred by recent advances in the ability to create defined knockouts in MAP, several independent laboratories have developed modified live vaccine candidates by transpositional mutation of virulence and metabolic genes in MAP. In order to accelerate the process of identification and comparative evaluation of the most promising modified live MAP vaccine candidates, members of a multi-institutional USDA-funded research consortium, the Johne's disease integrated program (JDIP), met to establish a standardized testing platform using agreed upon protocols. A total of 22 candidates vaccine strains developed in five independent laboratories in the United States and New Zealand voluntarily entered into a double blind stage gated trial pipeline. In Phase I, the survival characteristics of each candidate were determined in bovine macrophages. Attenuated strains moved to Phase II, where tissue colonization of C57/BL6 mice were evaluated in a challenge model. In Phase III, five promising candidates from Phase I and II were evaluated for their ability to reduce fecal shedding, tissue colonization and pathology in a baby goat challenge model. Formation of a multi-institutional consortium for vaccine strain evaluation has revealed insights for the implementation of vaccine trials for Johne's disease and other animal pathogens. We conclude by suggesting the best way forward based on this 3-phase trial experience and challenge the rationale for use of a macrophage-to-mouse-to native host pipeline for MAP vaccine development.

  14. 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

  15. 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

  16. Surveillance system sensitivities and probability of freedom from Mycobacterium avium subsp. paratuberculosis infection in Swedish cattle.

    PubMed

    Frössling, Jenny; Wahlström, Helene; Agren, Estelle Carina Constance; Cameron, Angus; Lindberg, Ann; Sternberg Lewerin, Susanna

    2013-01-01

    Previous investigations suggest that the prevalence of Mycobacterium avium subsp. paratuberculosis (MAP) in Swedish cattle is low and all recent cases have been linked to imported animals. The aim of this study was to evaluate the surveillance system for MAP infection in Swedish cattle and to estimate the probability that the Swedish cattle population is free from this infection. Calculations of surveillance sensitivities and probability of freedom were made using stochastic scenario-tree modelling, which allows inclusion of information from several different sources, of complex surveillance data including results from non-representative sampling, as well as of documentations of differences in risk of being infected. The surveillance components included in the model were: (1) clinical surveillance, (2) fallen stock investigations, (3) the national surveillance programme (mainly beef herds), (4) a survey involving dairy herds and (5) a risk-based survey targeting herds with imported cattle. Previous or current presence of imported animals and participation in the on-going control programme was specified for each tested herd, in order to adjust for differences in risk. Calculations were made for each year from the start of 2005 to the end of 2008, and this formed the basis for a final estimate covering the whole study period and predictions of future probabilities of freedom from MAP. Results show that when applying a design prevalence of one animal in 0.1% of the herds, the probability of freedom at the end of 2008 was 0.63. At the design prevalence of one animal in 0.5% of herds, the estimated probability is >95% and it is demonstrated that the prevalence of MAP in Swedish cattle is below this level or absent. In order to increase the annual surveillance sensitivity in the future and thereby improve the probability of freedom, new surveillance activities or an intensification of current ones are needed.

  17. Inactivation of Mycobacterium avium subsp. paratuberculosis during cooking of hamburger patties.

    PubMed

    Hammer, Philipp; Walte, Hans-Georg C; Matzen, Sönke; Hensel, Jann; Kiesner, Christian

    2013-07-01

    The role of Mycobacterium avium subsp. paratuberculosis (MAP) in Crohn's disease in humans has been debated for many years. Milk and milk products have been suggested as possible vectors for transmission since the beginning of this debate, whereas recent publications show that slaughtered cattle and their carcasses, meat, and organs can also serve as reservoirs for MAP transmission. The objective of this study was to generate heat-inactivation data for MAP during the cooking of hamburger patties. Hamburger patties of lean ground beef weighing 70 and 50 g were cooked for 6, 5, 4, 3, and 2 min, which were sterilized by irradiation and spiked with three different MAP strains at levels between 10² and 10⁶ CFU/ml. Single-sided cooking with one flip was applied, and the temperatures within the patties were recorded by seven thermocouples. Counting of the surviving bacteria was performed by direct plating onto Herrold's egg yolk medium and a three-vial most-probable-number method by using modified Dubos medium. There was considerable variability in temperature throughout the patties during frying. In addition, the log reduction in MAP numbers showed strong variations. In patties weighing 70 g, considerable bacterial reduction of 4 log or larger could only be achieved after 6 min of cooking. For all other cooking times, the bacterial reduction was less than 2 log. Patties weighing 50 g showed a 5-log or larger reduction after cooking times of 5 and 6 min. To determine the inactivation kinetics, a log-linear regression model was used, showing a constant decrease of MAP numbers over cooking time.

  18. A rational framework for evaluating the next generation of vaccines against Mycobacterium avium subspecies paratuberculosis

    PubMed Central

    Bannantine, John P.; Hines, Murray E.; Bermudez, Luiz E.; Talaat, Adel M.; Sreevatsan, Srinand; Stabel, Judith R.; Chang, Yung-Fu; Coussens, Paul M.; Barletta, Raúl G.; Davis, William C.; Collins, Desmond M.; Gröhn, Yrjö T.; Kapur, Vivek

    2014-01-01

    Since the early 1980s, several investigations have focused on developing a vaccine against Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep. These studies used whole-cell inactivated vaccines that have proven useful in limiting disease progression, but have not prevented infection. In contrast, modified live vaccines that invoke a Th1 type immune response, may improve protection against infection. Spurred by recent advances in the ability to create defined knockouts in MAP, several independent laboratories have developed modified live vaccine candidates by transpositional mutation of virulence and metabolic genes in MAP. In order to accelerate the process of identification and comparative evaluation of the most promising modified live MAP vaccine candidates, members of a multi-institutional USDA-funded research consortium, the Johne's disease integrated program (JDIP), met to establish a standardized testing platform using agreed upon protocols. A total of 22 candidates vaccine strains developed in five independent laboratories in the United States and New Zealand voluntarily entered into a double blind stage gated trial pipeline. In Phase I, the survival characteristics of each candidate were determined in bovine macrophages. Attenuated strains moved to Phase II, where tissue colonization of C57/BL6 mice were evaluated in a challenge model. In Phase III, five promising candidates from Phase I and II were evaluated for their ability to reduce fecal shedding, tissue colonization and pathology in a baby goat challenge model. Formation of a multi-institutional consortium for vaccine strain evaluation has revealed insights for the implementation of vaccine trials for Johne's disease and other animal pathogens. We conclude by suggesting the best way forward based on this 3-phase trial experience and challenge the rationale for use of a macrophage-to-mouse-to native host pipeline for MAP vaccine development

  19. Detection of Mycobacterium avium subsp. paratuberculosis in kidney samples of red deer (Cervus elaphus) in Portugal: Evaluation of different methods

    PubMed Central

    MATOS, Ana C.; FIGUEIRA, Luis; MARTINS, Maria H.; MATOS, Manuela; ÁLVARES, Sofia; MENDES, Andreia; PINTO, Maria L.; COELHO, Ana C.

    2017-01-01

    Paratuberculosis or Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic granulomatous enteritis affecting both domestic and wild ruminants. The present work is part of a wider set of studies designed to assess the prevalence of paratuberculosis in free ranging red deer (Cervus elaphus). With that purpose, 877 free-ranging red deer legally hunted in the Centre-eastern Portugal were submitted to necropsy and sampled for molecular methods, microbiology and histopathology. Thirty-seven (4.2%) kidneys revealed acid-fast bacilli when screened with the Ziehl-Neelsen technique. Map was detected by IS900 polymerase chain reaction (PCR) in thirty (81.1%) of the Ziehl-Neelsen positive kidneys. Subsequent PCR and/or culture from the different organs of the 37 examined animals allowed us to detect 86.4% (32 animals) infected red deer. Our results suggest that renal involvement in Map infected deer may be underdiagnosed and thus the routine examination of this organ and its inclusion in PCR techniques designed for Map detection could substantially improve the diagnostic of paratuberculosis in red deer. PMID:28132959

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

    PubMed Central

    2010-01-01

    Background Mycobacterium avium subsp. paratuberculosis (MAP) persistently infects intestines and mesenteric lymph nodes leading to a prolonged subclinical disease. The MAP genome sequence was published in 2005, yet its transcriptional organization in natural infection is unknown. While prior research analyzed regulated gene sets utilizing defined, in vitro stress related or advanced surgical methods with various animal species, we investigated the intracellular lifestyle of MAP in the intestines and lymph nodes to understand the MAP pathways that function to govern this persistence. Results Our transcriptional analysis shows that 21%, 8% and 3% of the entire MAP genome was represented either inside tissues, macrophages or both, respectively. Transcripts belonging to latency and cell envelope biogenesis were upregulated in the intestinal tissues whereas those belonging to intracellular trafficking and secretion were upregulated inside the macrophages. Transcriptomes of natural infection and in vitro macrophage infection shared genes involved in transcription and inorganic ion transport and metabolism. MAP specific genes within large sequence polymorphisms of ancestral M. avium complex were downregulated exclusively in natural infection. Conclusions We have unveiled common and unique MAP pathways associated with persistence, cell wall biogenesis and virulence in naturally infected cow intestines, lymph nodes and in vitro infected macrophages. This dichotomy also suggests that in vitro macrophage models may be insufficient in providing accurate information on the events that transpire during natural infection. This is the first report to examine the primary transcriptome of MAP at the local infection site (i.e. intestinal tissue). Regulatory pathways that govern the lifecycle of MAP appear to be specified by tissue and cell type. While tissues show a "shut-down" of major MAP metabolic genes, infected macrophages upregulate several MAP specific genes along with a

  1. Genetic structure of Mycobacterium avium subsp. paratuberculosis population in cattle herds in Quebec as revealed by using a combination of multilocus genomic analyses.

    PubMed

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

    2014-08-01

    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.

  2. Effect of Turbulent-Flow Pasteurization on Survival of Mycobacterium avium subsp. paratuberculosis Added to Raw Milk

    PubMed Central

    Pearce, Lindsay E.; Truong, H. Tuan; Crawford, Robert A.; Yates, Gary F.; Cavaignac, Sonia; de Lisle, Geoffrey W.

    2001-01-01

    A pilot-scale pasteurizer operating under validated turbulent flow (Reynolds number, 11,050) was used to study the heat sensitivity of Mycobacterium avium subsp. paratuberculosis added to raw milk. The ATCC 19698 type strain, ATCC 43015 (Linda, human isolate), and three bovine isolates were heated in raw whole milk for 15 s at 63, 66, 69, and 72°C in duplicate trials. No strains survived at 72°C for 15 s; and only one strain survived at 69°C. Means of pooled D values (decimal reduction times) at 63 and 66°C were 15.0 ± 2.8 s (95% confidence interval) and 5.9 ± 0.7 s (95% confidence interval), respectively. The mean extrapolated D72°C was <2.03 s. This was equivalent to a >7 log10 kill at 72°C for 15 s (95% confidence interval). The mean Z value (degrees required for the decimal reduction time to traverse one log cycle) was 8.6°C. These five strains showed similar survival whether recovery was on Herrold's egg yolk medium containing mycobactin or by a radiometric culture method (BACTEC). Milk was inoculated with fresh fecal material from a high-level fecal shedder with clinical Johne's disease. After heating at 72°C for 15 s, the minimum M. avium subsp. paratuberculosis kill was >4 log10. Properly maintained and operated equipment should ensure the absence of viable M. avium subsp. paratuberculosis in retail milk and other pasteurized dairy products. An additional safeguard is the widespread commercial practice of pasteurizing 1.5 to 2° above 72°C. PMID:11525992

  3. Induction of B Cell Responses Upon Experimental Infection of Neonatal Calves with Mycobacterium avium subsp. paratuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Animal models are useful for studying host responses to infection and aid in the development of diagnostic tools and vaccines. The current study was designed to compare the effects of different methods of experimental infection: Oral (Mycobacterium avium subsp. parauberculosis (MAP) strain K-10; Or...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Animal models are useful for studying host responses to infection and aid in the development of diagnostic tools and vaccines. The current study was designed to compare the effects of different methods of experimental infection: Oral (Mycobacterium avium subsp. parauberculosis (MAP) strain K-10; Or...

  5. Evaluation of quantitative polymerase chain reaction assays targeting Mycobacterium avium, M. intracellulare, and M. avium subspecies paratuberculosis in drinking water biofilms.

    PubMed

    Chern, Eunice C; King, Dawn; Haugland, Richard; Pfaller, Stacy

    2015-03-01

    Mycobacterium avium (MA), Mycobacterium intracellulare (MI), and Mycobacterium avium subsp. paratuberculosis (MAP) are difficult to culture due to their slow growing nature. A quantitative polymerase chain reaction (qPCR) method for the rapid detection of MA, MI, and MAP can be used to provide data supporting drinking water biofilms as potential sources of human exposure. The aim of this study was to characterize two qPCR assays targeting partial 16S rRNA gene sequences of MA and MI and use these assays, along with two previously reported MAP qPCR assays (IS900 and Target 251), to investigate Mycobacterium occurrence in kitchen faucet biofilms. MA and MI qPCR assays demonstrated 100% specificity and sensitivity when evaluated against 18 non-MA complex, 76 MA, and 17 MI isolates. Both assays detected approximately 1,000 cells from a diluted cell stock inoculated on a sampling swab 100% of the time. DNA analysis by qPCR indicated that 35.3, 56.9 and 11.8% of the 51 kitchen faucet biofilm samples collected contained MA, MI, and MAP, respectively. This study introduces novel qPCR assays designed to specifically detect MA and MI in biofilm. Results support the use of qPCR as an alternative to culture for detection and enumeration of MA, MI, and MAP in microbiologically complex samples.

  6. Genetic variation of toll-like receptor genes and infection by Mycobacterium avium ssp. paratuberculosis in Holstein-Friesian cattle.

    PubMed

    Ruiz-Larrañaga, O; Manzano, C; Iriondo, M; Garrido, J M; Molina, E; Vazquez, P; Juste, R A; Estonba, A

    2011-07-01

    Toll-like receptors (TLR) are membrane proteins that play a key role in innate immunity, by recognizing pathogens and subsequently activating appropriate responses. Mutations in TLR genes are associated with susceptibility to inflammatory and infectious diseases in humans. In cattle, 3 members of the TLR family, TLR1, TLR2, and TLR4, are associated with Mycobacterium avium ssp. paratuberculosis infection, although the extent of this association for the TLR1 and TLR4 receptors has not yet been determined. Moreover, the causal variant in the TLR2 gene has not yet been unequivocally established. In this study, 24 single nucleotide polymorphisms (SNP) in the bovine TLR1, TLR2, and TLR4 genes were selected from the literature, databases, and in silico searches, for a population-based genetic association study of a Spanish Holstein-Friesian sample. Whereas previous results regarding the TLR1 gene were not corroborated, a risk haplotype was detected in TLR2; however, its low frequency indicates that this detected association should be interpreted with caution. In the case of the TLR4 gene, 3 tightly linked SNP were found to be associated with susceptibility to M. avium ssp. paratuberculosis infection. Moreover, one of these SNP, the SNP c.-226G>C, which is localized in the 5'UTR region of the TLR4 gene, has been reported to be able to alter TLR4 expression, raising the possibility that this mutation may contribute to the response of the individual to infection.

  7. The effect of Protexin on prevention of ileocecal infection by Mycobacterium avium subspecies paratuberculosis in dairy calves.

    PubMed

    Badiei, A; Moosakhani, F; Hamidi, A; Sami, M

    2013-10-01

    The objective of this study was to evaluate the effect of Protexin (Probiotics International Ltd., South Petherton, UK) in the prevention of ileocecal infection by Mycobacterium avium ssp. paratuberculosis (MAP) in dairy calves in the field situation. Forty Holstein bull calves whose dams were paratuberculosis negative (confirmed by serum ELISA test and fecal nested PCR) were randomly selected in 2 groups. All calves were fed raw milk collected from the bulk tank in a paratuberculosis-infected dairy farm, which was confirmed by PCR. The treatment group (20 calves) was given 2 g of Protexin from birth until weaning (90 d). The control group (20 calves) did not consume Protexin. The calves were culled at 12 mo of age and the ileocecal lymph nodes were sampled. The lymph nodes were tested by nested PCR to evaluate MAP infection. In the treatment group, 2 out of 20 calf (10%) ileoceca were infected by MAP, whereas in the control group, 8 out of 20 calf (40%) ileoceca were infected by MAP. A significant difference existed between ileocecal infection by MAP in treatment and control groups. Thus, Protexin showed a significant effect in decreasing the ileocecal infection by MAP.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. Sampling location, herd size, and season influence Mycobacterium avium ssp. paratuberculosis environmental culture results.

    PubMed

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

    2015-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP), the etiologic agent of Johne's disease, a chronic progressive enteritis, is a common pathogen on dairy farms. Environmental sampling is frequently used to detect MAP-infected herds, because it does not require sample collection from individual animals. The objectives were to determine (1) location-specific odds of MAP-positive environmental sampling results and whether season or herd size affect results; (2) whether season and herd size affect the odds of collection of samples from certain locations; and (3) whether sample-set composition affects the odds of a positive set. Herd veterinarians, producer organization staff, and University of Calgary staff collected 5,588 samples on dairy farms in Alberta and Saskatchewan. Samples from sick-cow and calving pens and samples from dry-cow housing had lower odds of testing MAP-positive than lactating cow-pen samples (odds ratio=0.3 and 0.4, respectively). Samples collected from bedding packs and manure piles were less frequently MAP-positive than those collected from alleyways and lagoons, whereas samples collected in spring and summer more often tested MAP-positive than those collected in winter. Sample sets collected in summer more often included samples from all locations than samples collected in winter; therefore, we inferred that collectors had difficulties accessing certain areas in winter. Substitution of sample locations with others had minor effect on the sensitivity of sample sets containing 6 samples. However, set composition had an effect on the sensitivity of sample sets containing only 2 samples. In that regard, whereas sets with 2 manure-storage-area samples detected 81% of farms with at least one positive environmental sample, sets with only dry, sick, or calving-pen samples detected only 59%. Environmental samples should be collected from areas where manure from numerous cows accumulates and can be well mixed (e.g., alleyways and manure lagoons

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

    PubMed

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

    2011-10-01

    Environmental contamination with Mycobacterium avium subsp. paratuberculosis (MAP) is thought to be one of the primary sources 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-sectional analysis of longitudinally collected samples on 3 dairy farms. Composite samples from multiple environmental sites in 3 commercial dairy herds in the Northeast US were cultured quarterly for MAP, providing 1131 samples (133 (11.8%) were culture-positive), and all adult animals in the herds were tested biannually by fecal culture (FC), for 6 years. Of the environmental sites sampled, manure storage areas and shared alleyways were most likely to be culture-positive. Environmental sample results were compared to FC results from either the concurrent or previous sampling date at both the herd and the pen level. At the herd level, a 1 log unit increase in average fecal shedding increased the odds of a positive non-pen environmental sample by a factor of 6 and increased the average amount of MAP in non-pen samples by 2.9 cfu/g. At the pen level, a 1 log unit increase in average fecal shedding in the pen increased the odds of a positive environment by a factor of 2.4 and the average amount of MAP was increased by 3.5 cfu/g. We were not able to model the relationship between non-pen environmental sample status and the distance between shedding animals and the sample's location, and neighboring pens did not significantly affect the results of the pen-level analysis. The amount of MAP in pen-level samples and the probability of a pen testing positive for MAP were both positively but non-significantly correlated with the number of animals in the pen shedding >30 cfu/g of MAP. At least 6 environmental samples met the criteria for the U.S. Voluntary Bovine Johne's Disease Control Program on 47 of the 72 sampling dates; of these, 19 of the 47 FC-positive sampling dates

  14. 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

  15. 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.

  16. 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.

  17. Bayesian kriging of seroprevalence to Mycobacterium avium subspecies paratuberculosis and Neospora caninum in Alberta beef and dairy cattle.

    PubMed

    Thompson, James A; Scott, H Morgan

    2007-12-01

    Identifying spatial patterns of risk is important in the study of diseases with ecologic causes. Furthermore, relatively complex hierarchical modeling is required to determine how factors that are organized across levels interact, such as how an ecologic cause interacts with farm management and with animal characteristics. The objective of this study was to map the risk for Mycobacterium avium subspecies paratuberculosis (MAP - the causative agent of Johne's disease) and Neospora caninum (NC - the cause of neosporosis) infections in Alberta beef and dairy cattle. This objective utilized Bayesian generalized linear kriging to partition herd effects into a portion attributable to location and a portion that was independent of location. Seropositivity to NC in beef cattle showed strong support for spatial covariance, suggesting that ecologic causes were important for beef cattle but not dairy cattle. There was little evidence of spatial covariance for MAP seropositivity in either beef or dairy cattle.

  18. Bayesian kriging of seroprevalence to Mycobacterium avium subspecies paratuberculosis and Neospora caninum in Alberta beef and dairy cattle

    PubMed Central

    Thompson, James A.; Scott, H. Morgan

    2007-01-01

    Identifying spatial patterns of risk is important in the study of diseases with ecologic causes. Furthermore, relatively complex hierarchical modeling is required to determine how factors that are organized across levels interact, such as how an ecologic cause interacts with farm management and with animal characteristics. The objective of this study was to map the risk for Mycobacterium avium subspecies paratuberculosis (MAP — the causative agent of Johne’s disease) and Neospora caninum (NC — the cause of neosporosis) infections in Alberta beef and dairy cattle. This objective utilized Bayesian generalized linear kriging to partition herd effects into a portion attributable to location and a portion that was independent of location. Seropositivity to NC in beef cattle showed strong support for spatial covariance, suggesting that ecologic causes were important for beef cattle but not dairy cattle. There was little evidence of spatial covariance for MAP seropositivity in either beef or dairy cattle. PMID:18189052

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

    PubMed

    Pierce, Ellen S

    2009-09-23

    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.

  20. 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.

  1. Development and Evaluation of a Novel Multicopy-Element-Targeting Triplex PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Feces

    PubMed Central

    Garrido, Joseba M.; Molina, Elena; Geijo, María V.; Elguezabal, Natalia; Vázquez, Patricia; Juste, Ramón A.

    2014-01-01

    The enteropathy called paratuberculosis (PTB), which mainly affects ruminants and has a worldwide distribution, is caused by Mycobacterium avium subsp. paratuberculosis. This disease significantly reduces the cost-effectiveness of ruminant farms, and therefore, reliable and rapid detection methods are needed to control the spread of the bacterium in livestock and in the environment. The aim of this study was to identify a specific and sensitive combination of DNA extraction and amplification to detect M. avium subsp. paratuberculosis in feces. Negative bovine fecal samples were inoculated with increasing concentrations of two different bacterial strains (field and reference) to compare the performance of four extraction and five amplification protocols. The best results were obtained using the JohnePrep and MagMax extraction kits combined with an in-house triplex real-time PCR designed to detect IS900, ISMap02 (an insertion sequence of M. avium subsp. paratuberculosis present in 6 copies per genome), and an internal amplification control DNA simultaneously. These combinations detected 10 M. avium subsp. paratuberculosis cells/g of spiked feces. The triplex PCR detected 1 fg of genomic DNA extracted from the reference strain K10. The performance of the robotized version of the MagMax extraction kit combined with the IS900 and ISMap02 PCR was further evaluated using 615 archival fecal samples from the first sampling of nine Friesian cattle herds included in a PTB control program and followed up for at least 4 years. The analysis of the results obtained in this survey demonstrated that the diagnostic method was highly specific and sensitive for the detection of M. avium subsp. paratuberculosis in fecal samples from cattle and a very valuable tool to be used in PTB control programs. PMID:24727272

  2. Development and evaluation of a novel multicopy-element-targeting triplex PCR for detection of Mycobacterium avium subsp. paratuberculosis in feces.

    PubMed

    Sevilla, Iker A; Garrido, Joseba M; Molina, Elena; Geijo, María V; Elguezabal, Natalia; Vázquez, Patricia; Juste, Ramón A

    2014-06-01

    The enteropathy called paratuberculosis (PTB), which mainly affects ruminants and has a worldwide distribution, is caused by Mycobacterium avium subsp. paratuberculosis. This disease significantly reduces the cost-effectiveness of ruminant farms, and therefore, reliable and rapid detection methods are needed to control the spread of the bacterium in livestock and in the environment. The aim of this study was to identify a specific and sensitive combination of DNA extraction and amplification to detect M. avium subsp. paratuberculosis in feces. Negative bovine fecal samples were inoculated with increasing concentrations of two different bacterial strains (field and reference) to compare the performance of four extraction and five amplification protocols. The best results were obtained using the JohnePrep and MagMax extraction kits combined with an in-house triplex real-time PCR designed to detect IS900, ISMap02 (an insertion sequence of M. avium subsp. paratuberculosis present in 6 copies per genome), and an internal amplification control DNA simultaneously. These combinations detected 10 M. avium subsp. paratuberculosis cells/g of spiked feces. The triplex PCR detected 1 fg of genomic DNA extracted from the reference strain K10. The performance of the robotized version of the MagMax extraction kit combined with the IS900 and ISMap02 PCR was further evaluated using 615 archival fecal samples from the first sampling of nine Friesian cattle herds included in a PTB control program and followed up for at least 4 years. The analysis of the results obtained in this survey demonstrated that the diagnostic method was highly specific and sensitive for the detection of M. avium subsp. paratuberculosis in fecal samples from cattle and a very valuable tool to be used in PTB control programs.

  3. Evaluation of four commercial serum ELISAs for detection of Mycobacterium avium subsp. paratuberculosis infection in dairy cows.

    PubMed

    Diéguez, Francisco J; González, Ana M; Menéndez, Santiago; Vilar, María J; Sanjuán, María L; Yus, Eduardo; Arnaiz, Ignacio

    2009-05-01

    During the first 3 months of 2006, a study was performed on four dairy cattle herds with a history of clinical paratuberculosis, to evaluate different enzyme-linked immunosorbent assays (ELISAs). Serum samples obtained from 326 animals were analysed using four ELISAs to detect antibodies to Mycobacterium avium subsp. paratuberculosis (MAP). Kappa (kappa) concordance coefficients in pairwise comparisons of the ELISA outcomes ranged up to 0.22 (linear kappa) and 0.25 (quadratic kappa). When the borderline positives obtained were considered as negatives, kappa values remained low (kappa up to 0.19). Having performed the serological tests, faecal samples were then obtained from 55 animals (including all animals testing positive in two or more ELISAs) from the same herds. Faecal culture and faecal polymerase chain reaction (PCR) for the detection of MAP were negative in all cases. The results indicate that neither the currently available serum ELISAs nor faecal culture and PCR are effective for the early detection of MAP in dairy cattle.

  4. 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

    2015-06-19

    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.

  5. Dose-dependent interferon-gamma release in dairy calves experimentally infected with Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Mortier, Rienske A R; Barkema, Herman W; Wilson, Todd A; Sajobi, Tolulope T; Wolf, Robert; De Buck, Jeroen

    2014-10-15

    The interferon-gamma (IFN-γ) release assay is considered useful for diagnosis of subclinical paratuberculosis. However, interpretation can be subjective and complex; therefore, additional information regarding the course of the cellular immune response and effects of age and dose at infection would be helpful. Thirty-three calves were randomly allocated to 10 challenge groups and a negative control group. Calves were inoculated orally at 2 weeks or at 3, 6, 9, or 12 months of age. Within each age group, calves received either a high or low dose of Mycobacterium avium subspecies paratuberculosis (MAP). Monthly blood samples were collected, stimulated with Purified Protein Derivative (PPD) Johnin in vitro, and the subsequent release of IFN-γ measured. Calves inoculated with a high dose had earlier and stronger IFN-γ responses than low-dose calves. Furthermore, calves inoculated at 2 weeks of age produced less IFN-γ compared to those inoculated later in life. The IFN-γ response peaked (on average) 4 months after exposure; therefore, this would be an optimal interval to test cattle for MAP-infection (although the timing of field-based infections is unknown and clearance of infection a possibility). To conclude, the IFN-γ release assay could be a valuable diagnostic test on herd-level to indicate exposure to MAP.

  6. Early Foetal Loss Correlates Positively with Seroconversion against Mycobacterium avium paratuberculosis in High-Producing Dairy Cows.

    PubMed

    Garcia-Ispierto, I; López-Gatius, F

    2016-04-01

    This study was designed to examine (i) the seroprevalence of Mycobacterium avium subs paratuberculosis (MAP) in a high-producing dairy herd with clinical symptoms of bovine paratuberculosis, (ii) MAP seroconversion and seronegativation dynamics in the herd and (iii) possible relationships between MAP infection status and herd reproductive performance. One single blood test per cow was performed early post-partum on a monthly basis from day 10-40 post-partum during the first year of the study in 519 cows belonging to a commercial dairy herd. A subset of 111 cows that became pregnant during the study was tested again 60-200 days later during the early foetal period, immediately after the first confirmation of gestation at 58-64 days post-AI. Logistic regression analysis indicated no effect of any independent variable on MAP seropositivity and conception rate 28-34 days post-AI. MAP seropositivity was not a factor affecting the anoestrous, subfertility and early foetal loss rates. In the subset of 111 cows, animals that seroconverted had a 3.9 times greater risk of suffering from early foetal loss (30.3%, 10/33) than the remaining pregnant animals (10.3%, 8/78), (95% confidence interval: 1.11-13.4; p = 0.003). In conclusion, early foetal loss was positively correlated with seroconversion to MAP. Reproductive performance was not impaired by MAP infection.

  7. Genetic diversity of Mycobacterium avium subspecies paratuberculosis and the influence of strain type on infection and pathogenesis: a review.

    PubMed

    Stevenson, Karen

    2015-06-19

    Mycobacterium avium subspecies paratuberculosis (Map) is an important pathogen that causes a chronic, progressive granulomatous enteritis known as Johne's disease or paratuberculosis. The disease is endemic in many parts of the world and responsible for considerable losses to the livestock and associated industries. Diagnosis and control are problematic, due mostly to the long incubation period of the disease when infected animals show no clinical signs and are difficult to detect, and the ability of the organism to survive and persist in the environment. The existence of phenotypically distinct strains of Map has been known since the 1930s but the genetic differentiation of Map strain types has been challenging and only recent technologies have proven sufficiently discriminative for strain comparisons, tracing the sources of infection and epidemiological studies. It is important to understand the differences that exist between Map strains and how they influence both development and transmission of disease. This information is required to develop improved diagnostics and effective vaccines for controlling Johne's disease. Here I review the current classification of Map strain types, the sources of the genetic variability within strains, growth characteristics and epidemiological traits associated with strain type and the influence of strain type on infection and pathogenicity.

  8. Mycobacterium avium subsp. paratuberculosis survival during fermentation of soured milk products detected by culture and quantitative real time PCR methods.

    PubMed

    Klanicova, B; Slana, I; Roubal, P; Pavlik, I; Kralik, P

    2012-07-02

    Mycobacterium avium paratuberculosis (MAP), etiological agent of paratuberculosis in ruminants, is able to survive extreme conditions like very low pH (stomach), high temperature (pasteurization) or low temperature (refrigerated storage). Cheese, infant powder milk, cream and other milk and dairy products might thus be considered as possible sources of MAP for humans. The aim of this study was to investigate the survival of two MAP field isolates during fermentation of three different types of soured milk products (SMP; yogurt, acidophilus milk and kefir) under laboratory conditions. Pasteurized MAP-free milk was artificially contaminated with 10(6)MAPcells/mL and survival and absolute numbers of MAP were monitored during fermentation (4 or 16 h) and after six weeks of storage at 4°C by culture and quantitative real time PCR (qPCR). Viability of MAP was determined by culture using Herrold's egg yolk medium and Middlebrook 7H10 with antibiotics, supplemented with Mycobactin J and incubated at 37°C for up to 12 weeks. The absolute numbers of MAP were quantified by previously published qPCR assays targeting F57 and IS900 loci in MAP genome. We herein confirm that MAP can survive pH reduction, however, longer exposure to pH below 4 in SMP seems to be critical because it inhibits growth. Therefore, it is suggested that probiotic cultures that can decrease pH below 4 during fermentation could provide better inactivation of MAP in SMP.

  9. First isolation of mycobacterium avium subsp. Paratuberculosis from wild guanacos (Lama guanicoe) on Tierra del Fuego Island.

    PubMed

    Salgado, M; Herthnek, D; Bölske, G; Leiva, S; Kruze, J

    2009-04-01

    The aim of this study was to search for Mycobacterium avium subsp. paratuberculosis (Map) infection in a free-ranging wild animal species in a region where Johnes's disease has yet to be reported and to classify Map isolates using a genomic typing method. Fecal samples were obtained from 501 wild guanacos (Lama guanicoe) from Tierra del Fuego Island, Chile, in August 2006. Samples were cultured using Herrold's egg yolk medium with and without mycobactin J. After 9 mo of incubation, suspected Map colonies showing mycobactin dependence were confirmed by real-time polymerase chain reaction (PCR) based on IS900 and F57. Isolates were further tested using IS1311 PCR with restriction endonuclease analysis in order to type the guanaco Map strains. Twenty-one of 501 (4.2%) animals were fecal culture-positive for Map; identity was confirmed by real-time PCR and isolates were classified as cattle-type. Most culture-positive animals were located in four contiguous geographic areas, and the infection was most commonly found among adult animals. Prevalence was higher in females (5.9%) than males (3.1%) but the difference was not statistically significant. This represents the first isolation of Map from a free-ranging wildlife species in Chile. It expands the geographic range of paratuberculosis and the diversity of wildlife species that can become infected with Map.

  10. Detection of antibody responses against Mycobacterium avium subsp. paratuberculosis stress-associated proteins within 30 weeks after infection in cattle.

    PubMed

    Kawaji, Satoko; Nagata, Reiko; Whittington, Richard J; Mori, Yasuyuki

    2012-11-15

    In this study, humoral immune responses in cattle against Mycobacterium avium subsp. paratuberculosis (MAP) stress-associated recombinant proteins were assessed longitudinally by ELISA during the first 30 weeks after MAP infection. A total of 11 MAP genes previously identified by proteomic analysis were selected for cloning and expression. These included possible general stress-associated proteins of MAP and proteins expressed in vivo in MAP-infected sheep at an early stage of infection. An increase in the antibody levels against 5 recombinant antigen preparations (MAP1027c, MAP1339, MAP1588c, MAP1589c and MAP2411) was seen in MAP-infected calves (n=16) but not in control calves (n=3) over the time examined. Antibody responses were recorded as early as two weeks post-inoculation, and 87.5% of the inoculated cattle responded to at least one of the five immunogenic antigen preparations within the first 30 weeks of infection, suggesting that these proteins identified in the in vitro models of stress were also expressed in vivo in MAP-infected cattle at a relatively early stage after infection and therefore stimulate the host's immune system. It has been assumed that the sensitivity of antibody ELISA tests is dependent on the stage of infection and the age of the animals. However, we have provided some evidence that humoral immunity occurs at an early stage of paratuberculosis and can be detected using appropriate antigens such as MAP stress-associated proteins.

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

    PubMed Central

    Lingle, Cari K.; Adam, Philip R.; Ramyar, Kasra X.; McWhorter, William J.; Stabel, Judith R.; Picking, William D.

    2016-01-01

    Abstract 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

  12. 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.

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

    PubMed

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

    2013-12-27

    Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis, or Johne's disease, in animals. Diagnosis of MAP infection is challenging because of the pathogen's fastidious in vitro growth requirements and low-level intermittent shedding in feces during the preclinical phase of the infection. Detection of these "low-shedders" is important for effective control of paratuberculosis as these animals serve as sources of infection for susceptible calves. Magnetic separation technology, used in combination with culture or molecular methods for the isolation and detection of pathogenic bacteria, enhances the analytical sensitivity and specificity of detection methods. The aim of the present study was to evaluate peptide-mediated magnetic separation (PMS) capture technology coupled with IS900 PCR using the Roche real-time PCR system (PMS-PCR), in comparison with fecal culture using BACTEC-MGIT 960 system, for detection of MAP in bovine fecal samples. Among the 351 fecal samples 74.9% (263/351) were PMS-PCR positive while only 12.3% (43/351) were MGIT culture-positive (p=0.0001). All 43 MGIT culture-positive samples were also positive by PMS-PCR. Mean PMS-PCR crossing-point (Cp) values for the 13 fecal samples with the highest number of MAP, based on time to detection, (26.3) were significantly lower than for the 17 fecal samples with <100 MAP per 2g feces (30.06) (p<0.05). PMS-PCR technology provided results in a shorter time and yielded a higher number of positive results than MGIT culture. Earlier and faster detection of animals shedding MAP by PMS-PCR should significantly strengthen control efforts for MAP-infected cattle herds by helping to limit infection transmission at earlier stages of the infection.

  14. 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.

  15. Use of enoyl coenzyme A hydratase of Mycobacterium avium subsp. paratuberculosis for the serological diagnosis of Johne's disease.

    PubMed

    Nagata, Reiko; Kawaji, Satoko; Mori, Yasuyuki

    2013-10-01

    Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), remains difficult to control because of the lack of specific and sensitive diagnostic tests. In order to improve the specificity of sero-diagnosis for JD, the phage display library derived from genomic DNA of MAP was immunoscreened to identify novel antigenic targets. We selected a clone using antibodies from MAP experimentally infected cattle, and annotated its coding sequence as MAP1197 in the MAP genome, which encoded "echA12_2" in the MAP protein (Map-echA) belonging to Enoyl-CoA hydratase, known as a crotonase enzyme. The Map-echA was expressed in Esherichia coli and purified as a histidine-tag recombinant protein (rMap-echA), and the diagnostic potential of the protein was further evaluated by enzyme-linked immunosorbent assays (ELISA). Antibody responses to rMap-echA were higher in MAP-infected cattle than in uninfected cattle. The specificity of the Map-echA ELISA was also confirmed by evaluation with hyper-immune sera against various kinds of Mycobacterium species. Furthermore, in all experimentally infected cattle the antibody against rMap-echA was detected 2-7months earlier than by a commercially available ELISA kit. These results suggested that Map-echA can be used as a specific and sensitive serological diagnostic antigen for the detection of MAP infection.

  16. Characterization of total deoxyribonucleic acid of Mycobacterium paratuberculosis (ATCC 19698) and of M. avium complex (ATCC 25291) using restriction enzymes.

    PubMed

    Labidi, A

    1988-01-01

    Total DNA was extracted from M. paratuberculosis (ATCC 19698) and from M. avium complex (ATCC 25291) cultivated on RVB-10 enriched liquid media. Restriction endonuclease analysis was conducted of Total DNA using 34 enzymes and DNA digestion profiles were compared. Fifteen enzymes revealed important differences between the two species. Two pairs of enzymes (EcoRII, BstNI) and (MboI, Sau3AI) provide evidence for the presence of dcmI and dam methylation in DNA of M. avium complex and M. paratuberculosis. The differences in DNA fragments of these two species could be of potential value in differentiating these clinically significant mycobacteria.

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

    PubMed Central

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

    2015-01-01

    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 GreenTM 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. 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.

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

    PubMed

    Fernández, Miguel; Benavides, Julio; Sevilla, Iker A; Fuertes, Miguel; Castaño, Pablo; Delgado, Laetitia; García Marín, J Francisco; Garrido, Joseba M; Ferreras, M Carmen; Pérez, Valentín

    2014-01-16

    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.

  20. 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

  1. Differences in the peripheral immune response between lambs and adult ewes experimentally infected with Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Delgado, Laetitia; Juste, Ramón A; Muñoz, María; Morales, Silvia; Benavides, Julio; Ferreras, M Carmen; Marín, J Francisco García; Pérez, Valentín

    2012-01-15

    The peripheral immune response, and its relationship with the outcome of the infection according to the age of the animal, has been investigated in young lambs and adult ewes experimentally infected with two different doses of Mycobacterium avium subspecies paratuberculosis (Map). Sixteen 1.5-month-old lambs out of 24 and 23 adult ewes out of 30 were orally challenged with an ovine Map field isolate. Animals were divided into two groups: HD, infected with a higher dose of Map and LD, with a lower dose. The remaining animals were used as uninfected control groups. Animals were euthanized at 110-120 and 210-220 days post-infection (dpi). Along the experiment, the humoral response and the specific and non-specific IFN-γ production were assessed. An intradermal skin test (IDT), using avian PPD, was also performed at 90 and 195 dpi. Samples of intestine and related lymphoid tissue were taken for histological, bacteriological and PCR studies. The Ab and IFN-γ production as well as the IDT response appeared earlier and with more intensity in the adult ewes compared to the lambs. The basal non-specific IFN-γ levels increased only in the adult ewes from the HD group. Animals from the LD and HD groups were positive to PCR; however, lesions consistent with paratuberculosis were exclusively observed in the HD group, both in lambs and in adult sheep, but they only progressed to more advanced stages in the former. These results suggest that the peripheral immune response induced by Map infection in the adult ewes is more efficient to control the progression of the infection than in lambs. This could likely be due to the existence of previous contacts with Map or other mycobacteria in the adult sheep compared to the young lambs.

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

    PubMed

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

    2006-01-01

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

  3. Directed evolution of a quorum-quenching lactonase from Mycobacterium avium subsp. paratuberculosis K-10 in the amidohydrolase superfamily.

    PubMed

    Chow, Jeng Yeong; Wu, Long; Yew, Wen Shan

    2009-05-26

    The PLL(PTE-like lactonase)-group of enzymes within the amidohydrolase superfamily hydrolyze N-acyl-homoserine lactones (AHLs) that are involved in bacterial quorum-sensing pathways. These enzymes possess the (beta/alpha)(8)-barrel fold and serve as attractive templates for in vitro evolution and engineering of quorum-quenching biological molecules that can serve as antivirulence therapeutic agents. Using a quorum-quenching lactonase from Mycobacterium avium subsp. paratuberculosis K-10 (GI: 41409766) as the initial template for in vitro evolution experiments, we enhanced the catalytic efficiency and increased the substrate range of the wild-type enzyme through a single point mutation on the loop at the C-terminal end of the eighth beta-strand. This N266Y mutant had an increased value of k(cat)/K(M) of 30- and 32-fold toward 3-oxo-N-octanoyl-l-homoserine lactone and N-hexanoyl-l-homoserine lactone, respectively; the evolved mutant also exhibited lactonase activity toward 3-oxo-N-hexanoyl-l-homoserine lactone and N-butyryl-l-homoserine lactone, AHLs that were previously not hydrolyzed by the wild-type enzyme. This article reinforces the evolutionary potential of the (beta/alpha)(8)-barrel fold and highlights the possibility of using quorum-quenching lactonases in the amidohydrolase superfamily as templates for engineering biomolecules of therapeutic use.

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

    PubMed

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

    2010-07-31

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease; moreover, it seems to be implicated in the development of Crohn's disease in humans. In the present study, fluorescence ratio imaging microscopy (FRIM) was used to assess changes in intracellular pH (pH(i)) of one strain of MAP after exposure to nisin and neutralized cell-free supernatants (NCSs) from five bacteriocin-producing lactic acid bacteria (LAB) with known probiotic properties. The evaluation of pH(i) by FRIM provides information about the physiological state of bacterial cells, bypassing the long and problematic incubations needed for methods relying upon growth of MAP such as determination of colony forming units. The FRIM results showed that both nisin and the cell-free supernatant from Lactobacillus plantarum PCA 236 affected the pH(i) of MAP within a few hours. However, monitoring the population for 24h revealed the presence of a subpopulation of cells probably resistant to the antimicrobial compounds tested. Use of nisin and bacteriocin-producing LAB strains could lead to new intervention strategies for the control of MAP based on in vivo application of probiotic cultures as feed additives at farm level.

  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. 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

  7. Comparison of fecal pooling methods and DNA extraction kits for the detection of Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Mita, Akiko; Mori, Yasuyuki; Nakagawa, Tetsuo; Tasaki, Tomoko; Utiyama, Katsuo; Mori, Hitomi

    2016-02-01

    The aim of the study was to develop a sensitive method using quantitative real-time polymerase chain reaction (qPCR) with pooled fecal samples for the screening of Johne's disease (JD). Manufacturer-specified and our new pooling method in combination with five commercial kits for DNA extraction and purification were compared. Different volumes of pooled fecal suspensions were tested, and the results were compared for individual samples and three pool sizes (5, 10, and 50 samples); each of the fecal suspensions, which were prepared from healthy dairy and beef cattle was spiked with 0, 10, 100, or 1000 cultured Mycobacterium avium subspecies paratuberculosis (MAP) organisms or was mixed with fecal suspensions from experimentally infected cattle. The MAP DNA detection proportion with our pooling method in combination with Johne-Spin kit (Fasmac, Japan) was 100% for all models and all pool sizes, except for the low shedder model with a pool size of 50. There was no loss of sensitivity in pools of 10 subjects or less by using the new method. These results suggest that new method is a sensitive, practical, and cost-effective screening test for the detection of MAP-infected cattle and the monitoring of JD-free herds.

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

    PubMed

    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

    2015-06-01

    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.

  9. A systematic review of risk factors associated with the introduction of Mycobacterium avium spp. paratuberculosis (MAP) into dairy herds

    PubMed Central

    Rangel, Saray J.; Paré, Julie; Doré, Elizabeth; Arango, Juan C.; Côté, Geneviève; Buczinski, Sebastien; Labrecque, Olivia; Fairbrother, Julie H.; Roy, Jean P.; Wellemans, Vincent; Fecteau, Gilles

    2015-01-01

    The objective of this study was to systematically collect and appraise the scientific evidence related to risk factors associated with the introduction of Mycobacterium avium spp. paratuberculosis (MAP) into a herd of cattle. An electronic search was conducted to collect relevant references addressing 2 specific questions: are i) purchasing/introduction of cattle into a herd, and ii) presence of wildlife or domestic animals, risk factors for the introduction of MAP into a herd? The screening was based on titles and abstracts and selected studies were fully analyzed. Seventeen manuscripts published between 1996 and 2011 were ultimately analyzed. Unit of interest was mainly the herd (n = 17). The specific description of the risk factors studied varied between studies. The principal study design was cross-sectional (n = 15). The review indicated that purchase/introduction of animals was an important risk factor and that the importance of wildlife or other domestic species as a mechanism for transmission into a cattle herd was not measurable. PMID:25694667

  10. Mycobacterium avium ssp. paratuberculosis recombinant heat shock protein 70 interaction with different bovine antigen-presenting cells.

    PubMed

    Langelaar, M F M; Hope, J C; Rutten, V P M G; Noordhuizen, J P T M; van Eden, W; Koets, A P

    2005-03-01

    Abstract Heat shock proteins (Hsp) can deliver antigen into the major histocompatibility complex class I presentation pathway of antigen-presenting cells (APC), a process called cross priming, thus stimulating antigen-specific CD8+ T-cell reactions. Hsp were shown to elicit proinflammatory responses in APC. Both processes require interaction of Hsp with APC via specific receptors. This study describes the interaction of recombinant Hsp70 (rHsp70) of Mycobacterium avium subspecies paratuberculosis with bovine peripheral blood mononuclear cells that was restricted to CD14+ cells. Characterized monocyte-derived macrophages, monocyte-derived dendritic cells (DC) and BoMac, an immortalized bovine macrophage cell line, were used to investigate the interaction of rHsp70 with different bovine APC. Saturation of immature DC with high concentrations of rHsp70 is demonstrated, and it was found that interaction of rHsp70 with DC was related to the maturation stage of the DC. Involvement of CD91 as a cellular receptor for rHsp70 was demonstrated; however, competition studies with immature DC demonstrated that other receptors exist on bovine APC. These data suggest that rHsp70-based vaccines may be useful for the successful immunization of cattle.

  11. 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

  12. Proteome-scale identification of outer membrane proteins in Mycobacterium avium subspecies paratuberculosis using a structure based combined hierarchical approach.

    PubMed

    Rana, Aarti; Rub, Abdur; Akhter, Yusuf

    2014-07-29

    Outer membrane proteins (OMPs) in eubacteria have several important roles, which range from membrane transport to the host-pathogen interactions. These are directly involved in pathogen attachment, entry and activation of several pathogen-induced signaling cascades in the cell. The cardinal structural features of OMPs include the presence of a β-barrel, a signal peptide and the absence of the transmembrane helix. This is the first report on proteome-wide identification of OMPs of ruminant pathogen, Mycobacterium avium subsp. paratuberculosis (MAP). The complete proteome of MAP was analyzed using a pipeline of algorithms, which screens the amino acid sequences and structural features shared by OMPs in other bacteria. Secondary structure of these proteins is also analyzed and scores are calculated for amphiphilic β-strands. From the set of 588 exported proteins, 264 proteins are predicted to be inner membrane proteins while 83 proteins are identified as potential OMPs in MAP. Finally, this study identified 57 proteins as top candidates, on the basis of computed isoelectric points, as the core set of OMPs. Significantly, the resulting data for OMPs are not only useful in designing novel vaccines but may also open avenues for the development of early serodiagnostic tools for MAP.

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

    PubMed

    Bannantine, John P; Everman, Jamie L; Rose, Sasha J; Babrak, Lmar; Katani, Robab; Barletta, Raúl G; Talaat, Adel M; Gröhn, Yrjö T; Chang, Yung-Fu; Kapur, Vivek; Bermudez, Luiz E

    2014-01-01

    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 vaccine for Johne's disease, we evaluated eight attenuated mutant strains of MAP using a C57BL/6 mouse model. The persistence of the vaccine candidates was measured at 6, 12, and 18 weeks post vaccination. Only strains 320, 321, and 329 colonized both the liver and spleens up until the 12-week time point. The remaining five mutants showed no survival in those tissues, indicating their complete attenuation in the mouse model. The candidate vaccine strains demonstrated different levels of protection based on colonization of the challenge strain in liver and spleen tissues at 12 and 18 weeks post vaccination. Based on total MAP burden in both tissues at both time points, strain 315 (MAP1566::Tn5370) was the most protective whereas strain 318 (intergenic Tn5367 insertion between MAP0282c and MAP0283c) had the most colonization. Mice vaccinated with an undiluted commercial vaccine preparation displayed the highest bacterial burden as well as enlarged spleens indicative of a strong infection. Selected vaccine strains that showed promise in the mouse model were moved forward into a goat challenge model. The results suggest that the mouse trial, as conducted, may have a relatively poor predictive value for protection in a ruminant host such as goats.

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

    PubMed

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

    2016-03-09

    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.

  15. 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

    2016-07-07

    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.

  16. 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

  17. 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

  18. Concurrent Resolution of Chronic Diarrhea Likely Due to Crohn’s Disease and Infection with Mycobacterium avium paratuberculosis

    PubMed Central

    Singh, Shoor V.; Kuenstner, J. Todd; Davis, William C.; Agarwal, Prabhat; Kumar, Naveen; Singh, Devendra; Gupta, Saurabh; Chaubey, Kundan K.; Kumar, Ashok; Misri, Jyoti; Jayaraman, Sujatha; Sohal, Jagdip S.; Dhama, Kuldeep

    2016-01-01

    Examination of samples of stool from a 61-year-old male patient, presenting with the clinical symptoms of Crohn’s disease (CD), revealed massive shedding of acid fast bacilli with the morphology of Mycobacterium avium paratuberculosis (MAP), the causative agent of Johne’s disease in cattle. MAP was cultured from the stool. Biotyping of the bacterium isolated from cultures of stool demonstrated, it was the Indian Bison biotype of MAP, the dominant biotype infecting livestock and humans in India. Based on this finding and because the patient was unresponsive to standard therapy used in India to treat patients with gastrointestinal inflammatory disorders, the patient was placed on a regimen of multi-antibiotic therapy, currently used to treat tuberculosis and CD. After 1 year of treatment, the patient’s health was restored, concurrent with cessation of shedding of MAP in his stool. This patient is the first case shown to shed MAP from the stool who was cured of infection with antibiotics and who was concurrently cured of clinical signs of CD. PMID:27833911

  19. The fibroblast growth factor-2 arrests Mycobacterium avium sp. paratuberculosis growth and immunomodulates host response in macrophages.

    PubMed

    Wang, Jianjun; Wang, Zeyou; Yao, Yongliang; Wu, Jianhong; Tang, Xin; Gu, Tao; Li, Guangxin

    2015-07-01

    Mycobacterium tuberculosisis (M. tb) epidemic is one of the most severe health problem worldwide, while mechanisms underlying its pathogenesis and host immune responses remain unclear. Mycobacterium avium (M. avium), a mycobacterial species related to M. tb, shares similarities with M. tb in many ways. In this study, using M. avium infection of macrophages as a model, we systematically studied the effect of fibroblast growth factor-2 (FGF-2) on M. avium infection of macrophages. Our results showed that M. avium infection could increase FGF-2 expression on both mRNA and protein levels. M. avium infection elevated TNF-α and IFN-γ production while the addition of FGF-2 could further increase TNF-α but not IFN-γ level. M. avium infection could increase the expression of oxygen/nitrogen metabolism proteins iNOS and SOD-1, and FGF-2 had additive effect on the expression of these two proteins. M. avium infection had inhibitive effect on actin expression while FGF-2 could partly counteract such inhibition. Moreover, FGF-2 could inhibit M. avium proliferation in macrophages. Our results together indicate that macrophage-secreted FGF-2 upon M. avium infection could suppress M. avium proliferation through various ways including cytokine production, enhancement of phagocytosis as well as oxygen/nitrogen metabolism.

  20. 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.

  1. Gene expression profiles of putative biomarker candidates in Mycobacterium avium subsp. paratuberculosis-infected cattle.

    PubMed

    Park, Hyun-Eui; Shin, Min-Kyoung; Park, Hong-Tae; Jung, Myunghwan; Cho, Yong Il; Yoo, Han Sang

    2016-06-01

    This study was conducted to analyze the gene expression of prognostic potential biomarker candidates using the whole blood of cattle naturally infected with ITALIC! Mycobacterium aviumsubsp. ITALIC! paratuberculosis(MAP). We conducted real-time PCR to evaluate 23 potential biomarker candidates. Experimental animals were divided into four groups based on fecal MAP PCR and serum ELISA. Seven ( ITALIC! KLRB1, ITALIC! HGF, ITALIC! MPO, ITALIC! LTF, ITALIC! SERPINE1, ITALIC! S100A8and ITALIC! S100A9) genes were up-regulated in fecal MAP-positive cattle and three ( ITALIC! KLRB1, ITALIC! MPOand ITALIC! S100A9) were up-regulated in MAP-seropositive cattle relative to uninfected cattle. In subclinically infected animals, 17 genes ( ITALIC! TFRC, ITALIC! S100A8, ITALIC! S100A9, ITALIC! MPO, ITALIC! GBP6, ITALIC! LTF, ITALIC! KLRB1, ITALIC! SERPINE1, ITALIC! PIGR, ITALIC! IL-10, ITALIC! CXCR3, ITALIC! CD14, ITALIC! MMP9, ITALIC! ELANE, ITALIC! CHI3L1, ITALIC! HPand ITALIC! HGF) were up-regulated compared with the control group. Moreover, six genes ( ITALIC! CXCR3, ITALIC! HP, ITALIC! HGF, ITALIC! LTF, ITALIC! TFRCand ITALIC! GBP6) showed significant differences between experimental groups. Taken together, our data suggest that six genes ( ITALIC! LTF, ITALIC! HGF, ITALIC! HP, ITALIC! CXCR3, ITALIC! GBP6and ITALIC! TFRC) played essential roles in the immune response to MAP during the subclinical stage and therefore might be useful as prognostic biomarkers.

  2. 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

  3. 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

  4. Analysis of Mycobacterium avium subsp. paratuberculosis Mutant Libraries Reveals Loci-dependent Transposition Biases and Strategies to Novel Mutant Discovery.

    PubMed

    Rathnaiah, Govardhan; Bannantine, John P; Bayles, Darrell O; Zinniel, Denise K; Stabel, Judith R; Gröhn, Yrjö T; Barletta, Raúl G

    2016-02-16

    Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of Johne's disease, is one of the most important bacterial pathogens in ruminants. A thorough understanding of MAP pathogenesis is needed to develop new vaccines and diagnostic tests. The generation of comprehensive random transposon mutant libraries is a fundamental genetic technology to determine the role of genes in physiology and pathogenesis. In this study, whole MAP genome analysis compared the insertion sites for the mycobacterial transposon Tn5367 derived from the Mycobacterium smegmatis insertion sequence IS1096 and the mariner transposon MycoMarT7 carrying the Himar1 transposase. We determined that only MycoMarT7 provides a random representation of insertions in 99% of all MAP genes. Analysis of the MAP K-10 genome indicated that 710 of all open reading frames do not possess IS1096 recognition sites, while only 37 do not have the recognition site for MycoMarT7. Thus, a significant number of MAP genes remain underrepresented in insertion libraries from IS1096 derived transposons. Analysis of MycomarT7 and Tn5367 mutants showed that Tn5367 has a predilection to insert within intergenic regions, suggesting that MycoMarT7 is more adequate to generate a comprehensive library. However, we uncovered the novel finding that both transposons have loci-dependent biases with Tn5367 being the most skewed. These loci-dependent transposition biases lead to an underestimation of the number of independent mutants required to generate a comprehensive mutant library, leading to an overestimation of essential genes. Herein, we also demonstrated a useful platform for gene discovery and analysis by isolating three novel mutants for each transposon.

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

    PubMed Central

    2013-01-01

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

  6. Antigenicity in sheep of synthetic peptides derived from stress-regulated Mycobacterium avium subsp. paratuberculosis proteins and comparison with recombinant protein and complex native antigens.

    PubMed

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

    2014-03-15

    Serum antibody enzyme-linked immunosorbent assay is the most commonly used test for diagnosis of Mycobacterium avium subsp. paratuberculosis infection in ruminants. However, the assay requires serum preabsorption with Mycobacterium phlei proteins to reduce cross reactions potentially contributed by the exposure of livestock to environmental mycobacteria. To trial the discovery of novel antigens which do not require serum absorption, synthetic MAP-specific peptides were selected based on in silico research to identify putative B cell epitopes. Four peptides from previously identified stress-regulated proteins were synthesized and evaluated using enzyme linked immunosorbent assay to detect Mycobacterium avium subsp. paratuberculosis specific antibodies in sheep. Two peptides were from hypothetical MAP proteins (MAP3567 and MAP1168c) and two were from proteins with known function (MAP2698c, an acyl-acyl carrier protein desaturase-DesA2 and MAP2487c a carbonic anhydrase). The ability of each peptide to discriminate between unexposed and MAP exposed (infected and vaccinated) animals was similar to that of the parent recombinant MAP antigen, with area under receiver operating curve values of 0.86-0.93. Assays run with a combination of two peptides showed slightly higher reactivity than those of individual peptides. Peptides evaluated in this study had diagnostic potential similar to corresponding recombinant proteins but not superior to a complex native MAP antigen or a commercial assay. Further study is required to investigate other peptides for their diagnostic potential, and this may be simpler and cheaper than subunit protein-based research.

  7. Mycobacterium avium ssp. paratuberculosis detection in animals, food, water and other sources or vehicles of human exposure: A scoping review of the existing evidence.

    PubMed

    Waddell, Lisa; Rajić, Andrijana; Stärk, Katharina; McEwen, Scott A

    2016-09-15

    Mycobacterium avium ssp. paratuberculosis is the etiologic agent of Johne's disease in ruminants and is hypothesized to be an infectious cause of Crohn's disease, as well as some other human diseases. Due to key knowledge gaps, the potential public health impact of M. paratuberculosis is unknown. This scoping review aims to identify and characterised the evidence on potential sources and vehicles of M. paratuberculosis exposure for humans to better understand how exposure is likely to occur. Evidence from 255 primary research papers is summarized; most examined the prevalence or concentration of M. paratuberculosis in animals (farmed domestic, pets and wildlife) (n=148), food for human consumption (62) (milk, dairy, meat, infant formula) or water (drinking and recreational) and the environment (farm, pasture and areas affected by runoff water) (20). The majority of this research has been published since 2000 (Figure- abstract). Nine case-control studies examining risk factors for Crohn's disease highlighted significant associations with the consumption of processed meats and cheese, while direct contact with ruminants, high risk occupations (farmer, veterinarian), milk consumption and water source were factors not associated with the disease and/or M. paratuberculosis exposure status. Molecular epidemiology studies demonstrated strain-sharing between species. Produce and seafood were the only previously suggested sources of human exposure for which there was no supporting evidence identified in this scoping review. The results of this review indicate that ruminant populations from around the globe are infected with M. paratuberculosis and many non-ruminant species have also been found to carry or be infected with M. paratuberculosis. Several potential sources for human exposure to M. paratuberculosis were identified; however there remain important gaps in quantitative information on the prevalence and concentration of M. paratuberculosis in contaminated sources of

  8. 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 ...

  9. 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...

  10. 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...

  11. 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...

  12. 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.

  13. Evaluation of the single cervical skin test and interferon gamma responses to detect Mycobacterium bovis infected cattle in a herd co-infected with Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Seva, Juan; Sanes, Jose M; Ramis, Guillermo; Mas, Alberto; Quereda, Juan J; Villarreal-Ramos, Bernardo; Villar, David; Pallares, Francisco J

    2014-06-25

    This study reports the performance of the single intradermal tuberculin (SIT) test and the interferon-gamma (IFN-γ) assay for Mycobacterium bovis in a cattle herd with high prevalence of paratuberculosis (PTB). A total of 58/350 animals were selected for necropsy based on one or more of the following criteria: positive to SIT, IFN-γ, a breeding cow that seroconverted to PTB and showed signs compatible with a wasting disease. Infection status was determined by post mortem diagnostic tests that included histopathology examination, mycobacterial cultures and PCR identification for M. bovis and Mycobacterium avium subsp. paratuberculosis (MAP). In 7/58 animals primary tuberculosis (TB) lesions, affecting only the retropharyngeal and/or mediastinal lymph nodes, were found; 3/7 animals were found SIT positive. PTB was confirmed in 35/58 animals, of which 30 had seroconverted and 14 had typical clinical signs. 45/58 animals were IFN-γ(+) using the most stringent criterion (cut-off point ≥ 0.05); however, IFN-γ test was only positive in 33 animals when using a higher threshold (cut-off point ≥ 0.1). Three animals co-infected also showed extensive TB and diffuse PTB lesions. These results show that the combined use of SIT and IFN-γ, as interpreted using official guidelines, detected all confirmed cases of TB. Individually, the sensitivity of the SIT was inadequate to diagnose TB-positive animals with an advanced stage of PTB. The large number of IFN-γ(+) animals with no visible TB lesion could be due, in part, to some protection conferred by prior infection with MAP.

  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

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

    2005-04-01

    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.

  15. Genome-wide association analysis and genomic prediction of Mycobacterium avium subspecies paratuberculosis infection in US Jersey cattle.

    PubMed

    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).

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

    PubMed

    Richardson, Ekb; Mee, Jf; Sánchez-Miguel, C; Crilly, J; More, Sj

    2009-06-01

    The demography of bovine infections caused by Mycobacterium avium subspecies paratuberculosis (MAP) in Ireland is poorly defined. The objective of this study was to describe the demographics of cattle positive to MAP on faecal culture, based on submissions to the Cork Regional Veterinary Laboratory (Cork RVL) from 1994 to 2006. The study focused on all available faecal samples from adult cattle with non-responsive chronic diarrhoea that were submitted by private veterinary practitioners to Cork RVL for MAP culture. For each MAP-positive by faecal culture animal, data were collated from Cork RVL and Cattle Movement Monitoring Scheme (CMMS) records. Johne's disease (JD) was confirmed in 110 animals from 86 herds by the Cork RVL between 1994 and 2006, with a rate of positive cases between 15% and 18% over last four years of the study. Two breeds (Holstein/Friesian or Limousin) made up 78% of submissions. Movements were assessed for the 57 study animals with available movement information, 90% died within one year of the test and 26% tested positive in the herd they were born into. The study provides preliminary information about movement trends and demographics of animals with MAP positive submissions. Although the study area is restricted, it includes the most intensive (and economically-important) dairy region in Ireland. The demographics of JD infection from the study area are in agreement with international reports. Further work is required to determine demographic trends, incidence and prevalence of JD throughout Ireland. It is hoped this work may contribute to the development of a surveillance strategy for MAP by regional veterinary laboratories.

  17. 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

  18. 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

  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.

  20. Deletion of relA abrogates the capacity of Mycobacterium avium paratuberculosis to establish an infection in calves.

    PubMed

    Park, Kun Taek; Allen, Andrew J; Barrington, George M; Davis, William C

    2014-01-01

    Previous comparative studies in goats revealed deletion of relA but not pknG abrogates the capacity of Mycobacterium avium subsp. paratuberculosis (Map) to establish a persistent infection. The immune response elicited by the mutant cleared infection. The objective of the present study was to extend the studies in calves and compare the proliferative response elicited by the relA deletion mutant (ΔrelA) and Map using flow cytometry and quantitative reverse transcription real-time PCR (qRT-PCR). Six 3-day-old calves were divided into two groups. Three were vaccinated with ΔrelA and 3 inoculated with wild type Map. The calves were challenged with Map 1 month later and necropsied 3 months post challenge. Three untreated calves were used as uninfected controls. Examination of tissues revealed the ΔrelA mutant was immune eliminated. Bacterial load of Map was significantly reduced in the calves vaccinated with ΔrelA and challenged with Map in comparison with calves inoculated and challenged with Map. A vigorous CD4 memory T cell response was detected at necropsy in PBMC from both infected groups. CD8 positive NK cells proliferated in the presence and absence of antigen stimulation in both treated groups but not in the uninfected group. IFN-γ, IL17, and IL22 gene expression were up-regulated with an associated increase in their transcription factors, Tbet and RORC, in both treated groups. TGF-β, IL-10, and FoxP3 were not up-regulated, indicating no activation of regulatory T cells. The findings show that the immune response to ΔrelA is clearly different than the response to Map. Understanding the immunological basis for this difference should facilitate development of a vaccine that elicits sterile immunity.

  1. 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.

  2. Analysis of the Bovine Monocyte-Derived Macrophage Response to Mycobacterium avium Subspecies Paratuberculosis Infection Using RNA-seq

    PubMed Central

    Casey, Maura E.; Meade, Kieran G.; Nalpas, Nicolas C.; Taraktsoglou, Maria; Browne, John A.; Killick, Kate E.; Park, Stephen D. E.; Gormley, Eamonn; Hokamp, Karsten; Magee, David A.; MacHugh, David E.

    2015-01-01

    Johne’s disease, caused by infection with Mycobacterium avium subsp. paratuberculosis, (MAP), is a chronic intestinal disease of ruminants with serious economic consequences for cattle production in the United States and elsewhere. During infection, MAP bacilli are phagocytosed and subvert host macrophage processes, resulting in subclinical infections that can lead to immunopathology and dissemination of disease. Analysis of the host macrophage transcriptome during infection can therefore shed light on the molecular mechanisms and host-pathogen interplay associated with Johne’s disease. Here, we describe results of an in vitro study of the bovine monocyte-derived macrophage (MDM) transcriptome response during MAP infection using RNA-seq. MDM were obtained from seven age- and sex-matched Holstein-Friesian cattle and were infected with MAP across a 6-h infection time course with non-infected controls. We observed 245 and 574 differentially expressed (DE) genes in MAP-infected versus non-infected control samples (adjusted P value ≤0.05) at 2 and 6 h post-infection, respectively. Functional analyses of these DE genes, including biological pathway enrichment, highlighted potential functional roles for genes that have not been previously described in the host response to infection with MAP bacilli. In addition, differential expression of pro- and anti-inflammatory cytokine genes, such as those associated with the IL-10 signaling pathway, and other immune-related genes that encode proteins involved in the bovine macrophage response to MAP infection emphasize the balance between protective host immunity and bacilli survival and proliferation. Systematic comparisons of RNA-seq gene expression results with Affymetrix® microarray data generated from the same experimental samples also demonstrated that RNA-seq represents a superior technology for studying host transcriptional responses to intracellular infection. PMID:25699042

  3. High herd-level prevalence of Mycobacterium avium subspecies paratuberculosis in Western Canadian dairy farms, based on environmental sampling.

    PubMed

    Wolf, R; Barkema, H W; De Buck, J; Slomp, M; Flaig, J; Haupstein, D; Pickel, C; Orsel, K

    2014-10-01

    Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive enteritis in ruminants. The pathogen is present in most countries with modern dairy production, causing substantial economic losses for the industry. The objectives of this study were to estimate dairy herd prevalence of MAP in the Western Canadian provinces of Alberta and Saskatchewan, and to determine whether herd size and housing system (tie-stall versus freestall or loose housing) affected the risk of a herd testing positive for MAP. Six environmental samples were collected on 360 Alberta farms (60% of registered producers) and on 166 Saskatchewan dairy farms (99%). In total, 47% of the sampled farms in Alberta and 53% of the sampled farms in Saskatchewan had at least one environmental sample that was MAP culture positive and were, therefore, defined as infected. Sensitivity of environmental sampling was estimated using 3 subsequent annual tests performed on 82 farms. Because laboratory protocols were continuously improved throughout the project, the sensitivity increased over time. Therefore, a mean of the sensitivity estimates weighted on sampling year was constructed; this resulted in sensitivities of 68 and 69% for Alberta and Saskatchewan, respectively. Implementing those estimates in an approximate Bayesian computation model resulted in a true herd prevalence of 68% (95% probability interval: 60-80%) for Alberta and 76% (95% probability interval: 70-85%) for Saskatchewan. Herds with >200 cows had 3.54 times higher odds of being environmental sample positive and had more positive samples than herds with <50 cows (neither province nor housing system affected those results). In conclusion, the majority of Alberta and Saskatchewan dairy farms were infected with MAP and larger herds were more often MAP positive than smaller herds.

  4. Use of conventional and real-time polymerase chain reaction for confirmation of Mycobacterium avium subsp. paratuberculosis in a broth-based culture system ESP II.

    PubMed

    Kim, Sung G; Kim, Eun H; Lafferty, Caroline J; Miller, Loretta J; Koo, Hye J; Stehman, Susan M; Shin, Sang J

    2004-09-01

    The ESP II Culture System (ESP II), a broth-based culture system, has been modified and optimized for culturing Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in animal feces since 2000. Conventional and real-time polymerase chain reaction (PCR) assays based on the IS900 sequence were performed as confirmatory tests for M. paratuberculosis in ESP II liquid culture medium. There were no differences between test results of conventional and real-time PCR assays. During the 5-week incubation period, if acid-fast bacilli (AFB) were detected in ESP culture-positive samples, IS900 PCR assays were performed to confirm whether those AFB were M. paratuberculosis. At the end of the 5-week incubation, AF staining was performed on all ESP II-negative cultures to screen any false-negative cultures; IS900 PCR assays were performed on AFB-positive cultures. During a period of 1 year, of a total of 18,499 ESP II cultures, 2,814 (15.2%) PCR confirmation assays were performed. Of those, 2,259 (80%) were both ESP and PCR positive; 104 (4%) were ESP positive and PCR negative; 423 (15%) were ESP negative and PCR positive; 28 (1%) were both ESP and PCR negative. The AF-staining step after the 5-week incubation produced 423 (15%) more PCR-positive cultures. Of a total of 2,814 AFB-positive cultures, 132 (5%) were not confirmed as M. paratuberculosis. Further studies are needed for speciation of non-M. paratuberculosis isolates.

  5. 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

  6. Multiple strain infections and high genotypic diversity among Mycobacterium avium subsp. paratuberculosis field isolates from diseased wild and domestic ruminant species in the eastern Alpine region of Austria.

    PubMed

    Gerritsmann, H; Stalder, G L; Spergser, J; Hoelzl, F; Deutz, A; Kuebber-Heiss, A; Walzer, C; Smith, S

    2014-01-01

    Johne's disease, or paratuberculosis, is a chronic fatal ruminant gastroenteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP) whose foodborne zoonotic potential and association with Crohn's disease are still under debate. The disease is widespread but its epidemiology and epizootiology remains elusive. Wildlife is suspected to play a major role. After a surge in MAP seroprevalence in Austrian cattle, paratuberculosis was declared a notifiable disease in Austria in 2006. At the same time a rise in MAP cases in wild ruminant populations in the Austrian province of Styria was reported. All five autochthonous ruminants were affected. Genetic analysis of isolates, yielded numerous genotypes (>15) and several multiple strain infections (15%) across host species. Identical MIRU-VNTR profiles were identified in different species and sampling locations. On the other hand varying MIRU-VNTR profiles were revealed at the same location and in conspecifics. Our data, taken together with earlier epidemiological studies on MAP and other mycobacteria, raised concerns about the organisms' ecology. Constraints regarding in vitro culture of this highly fastidious organism potentially bias our current understanding of its epidemiology. We suggest that MAP infections could be polyclonal and question the informative value of genotyping a single MAP colony derived from a single specimen for epidemiological analysis of MAP.

  7. Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection

    PubMed Central

    Alonso-Hearn, Marta; Abendaño, Naiara; Ruvira, Maria A.; Aznar, Rosa; Landin, Mariana; Juste, Ramon A.

    2017-01-01

    Johne's disease is a chronic granulomatous enteritis of ruminants caused by the intracellular bacterium Mycobacterium avium subsp. paratuberculosis (Map). We previously demonstrated that Map isolates from sheep persisted within host macrophages in lower CFUs than cattle isolates after 7 days of infection. In the current study, we hypothesize that these phenotypic differences between Map isolates may be driven be the fatty acids (FAs) present on the phosphadidyl-1-myo-inositol mannosides of the Map cell wall that mediate recognition by the mannose receptors of host macrophages. FAs modifications may influence Map's envelope fluidity ultimately affecting pathogenicity. To test this hypothesis, we investigated the responses of two Map isolates from cattle (K10 isolate) and sheep (2349/06-1) to the bovine and ovine macrophage environment by measuring the FAs content of extracellular and intracellular bacteria. For this purpose, macrophages cell lines of bovine (BOMAC) and ovine (MOCL-4) origin were infected with the two isolates of Map for 4 days at 37°C. The relative FAs composition of the two isolates recovered from infected BOMAC and MOCL-4 cells was determined by gas chromatography and compared with that of extracellular bacteria and that of bacteria grown in Middlebrook 7H9 medium. Using this approach, we demonstrated that the FAs composition of extracellular and 7H9-grown bacteria was highly conserved within each Map isolate, and statistically different from that of intracellular bacteria. Analysis of FAs composition from extracellular bacteria enabled the distinction of the two Map strains based on the presence of the tuberculostearic acid (18:0 10Me) exclusively in the K10 strain of Map. In addition, significant differences in the content of Palmitic acid and cis-7 Palmitoleic acid between both isolates harvested from the extracellular environment were observed. Once the infection established itself in BOMAC and MOCL-4 cells, the FAs profiles of both Map

  8. Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolated from sheep, cattle and deer on New Zealand pastoral farms.

    PubMed

    Verdugo, Cristobal; Pleydell, Eve; Price-Carter, Marian; Prattley, Deborah; Collins, Desmond; de Lisle, Geoffrey; Vogue, Hinrich; Wilson, Peter; Heuer, Cord

    2014-12-01

    The present study aimed to describe the molecular diversity of Mycobacterium avium subsp. paratuberculosis (MAP) isolates obtained from sheep, cattle (beef and dairy) and deer farms in New Zealand. A total of 206 independent MAP isolates (15 beef cattle, 89 dairy cattle, 35 deer, 67 sheep) were sourced from 172 species-mobs (15 beef cattle, 66 dairy cattle, 31 deer, 60 sheep). Seventeen subtypes were identified, using a combination of variable number of tandem repeats (VNTR) and short sequence repeat (SSR) methods. Rarefaction analysis, analysis of molecular variance (AMOVA), Fst pairwise comparisons and proportional similarity index (PSI) were used to describe subtype population richness, genetic structure and potential associations between livestock sectors and New Zealand two main islands (North and South). The rarefaction analysis suggests a significantly higher subtype richness in dairy cattle herds when compared to the other livestock sectors. AMOVA results indicate that the main source of subtype variation is attributable to the livestock sector from which samples were sourced suggesting that subtypes are generally sector-specific. The pairwise Fst results were similar, with low Fst values for island differences within a livestock sector when compared to between sector analyses, representing a low subtype differentiation between islands. However, for a given island, potential associations were seen between dominant subtypes and specific livestock sectors. Three subtypes accounted for 76% of the isolates. The most common of these was isolated from sheep and beef cattle in the North Island, the second most frequent subtype was mainly isolated from dairy cattle (either island), while the third most common subtype was associated with deer farmed in the South Island. The PSI analysis suggests similarities in subtypes sourced from sheep and beef cattle. This contrasted with the isolates sourced from other livestock sectors, which tended to present sector

  9. Implications of PCR and ELISA results on the routes of bulk-tank contamination with Mycobacterium avium ssp. paratuberculosis.

    PubMed

    Beaver, A; Cazer, C L; Ruegg, P L; Gröhn, Y T; Schukken, Y H

    2016-02-01

    Mycobacterium avium ssp. paratuberculosis (MAP), the etiologic agent of Johne's disease in dairy cattle, may enter the bulk tank via environmental contamination or direct excretion into milk. Traditionally, diagnostics to identify MAP in milk target either MAP antibodies (by ELISA) or the organism itself (by culture or PCR). High ELISA titers may be directly associated with excretion of MAP into milk but only indirectly linked to environmental contamination of the bulk tank. Patterns of bulk-milk ELISA and bulk-milk PCR results could therefore provide insight into the routes of contamination and level of infection or environmental burden. Coupled with questionnaire responses pertaining to management, the results of these diagnostic tests could reveal correlations with herd characteristics or on-farm practices that distinguish herds with high and low environmental bulk-tank MAP contamination. A questionnaire on hygiene, management, and Johne's specific parameters was administered to 292 dairy farms in New York, Oregon, and Wisconsin. Bulk-tank samples were collected from each farm for evaluation by real-time PCR and ELISA. Before DNA extraction and testing of the unknown samples, bulk-milk template preparation was optimized with respect to parameters such as MAP fractionation patterns and lysis. Two regression models were developed to explore the relationships among bulk-tank PCR, ELISA, environmental predictors, and herd characteristics. First, ELISA optical density (OD) was designated as the outcome in a linear regression model. Second, the log odds of being PCR positive in the bulk tank were modeled using binary logistic regression with penalized maximum likelihood. The proportion of PCR-positive bulk tanks was highest for New York and for organic farms, providing a clue as to the geographical patterns of MAP-positive bulk-tank samples and relationship to production type. Bulk-milk PCR positivity was also higher for large relative to small herds. The models

  10. 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

  11. Detection of Mycobacterium avium subsp. paratuberculosis in a sheep flock in Tuscany.

    PubMed

    Galiero, Alessia; Fratini, Filippo; Turchi, Barbara; Colombani, Giuseppe; Nuvoloni, Roberta; Cerri, Domenico

    2015-12-01

    Paratuberculosis is an infectious disease which affects ruminants. In this work, a sheep flock in Garfagnana district (Tuscany, Italy) was examined by agar gel immune-diffusion (AGID) tests, culture, and PCR from feces, milk, tissue samples, and cheeses. At the first AGID test, 7/280 (2.5 %) animals were positive. From these animals, feces and milk samples were collected: 4/7 feces (57.14 %) and 2/7 milk samples (28.57 %) were positive to culture and 7/7 (100 %) feces samples and 1/7 milk samples (14.28 %) were positive to PCR; 2/4 (50 %) cheeses ripened for 14 days and 1/3 (33.33 %) cheese ripened for 20 days were positive to PCR, from which no viable microorganisms were isolated. Then, the AGID-positive animals were slaughtered and tissue samples were taken from one sheep with PCR-positive feces and milk: the liver, intestine, mesenteric lymph nodes, but not the spleen and mammary lymph nodes were positive to culture; all these samples were instead PCR-positive. After 1 year, a second AGID survey was performed on the remaining animals: 6/244 (2.45 %) subjects were positive to this test. Data obtained revealed the presence of paratuberculosis in a sheep population in Garfagnana.

  12. Seroprevalence of and agroecological risk factors for Mycobacterium avium subspecies paratuberculosis and Neospora caninum infection among adult beef cattle in cow-calf herds in Alberta, Canada

    PubMed Central

    Scott, H. Morgan; Sorensen, Ole; Wu, John T.Y.; Chow, Eva Y.W.; Manninen, Ken

    2007-01-01

    A province-wide cross-sectional seroprevalence and agroecological risk factor study of Mycobacterium avium subspecies paratuberculosis (MAP) and Neospora caninum (NC) infection among cattle in 100 cow-calf herds in Alberta was conducted. The seroprevalence of MAP in adult cattle was 1.5% across all herds. Using a widely accepted herd test cutpoint of 2 or more seropositive cows out of 30 animals tested, 7.9% of herds were estimated to be infected (95% confidence interval (CI): 2.3–23.4%). Seroprevalence of MAP differed by agroecological region; specifically, cattle and herds in areas with high soil pH (> 7.0), southern latitudes, and arid climates had a moderately reduced risk of infection (P < 0.10). Seroprevalence of NC infection was 9.7% among adult beef cattle province-wide — these levels also varied by agroecological region — with 91.0% of herds infected overall. PMID:17494367

  13. 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.

  14. Virulence attenuation of a Mycobacterium avium subspecies paratuberculosis S-type strain prepared from intestinal mucosa after bacterial culture. Evaluation in an experimental ovine model.

    PubMed

    Fernández, Miguel; Delgado, Laetitia; Sevilla, Iker A; Fuertes, Miguel; Castaño, Pablo; Royo, Marcos; Ferreras, M Carmen; Benavides, Julio; Pérez, Valentín

    2015-04-01

    The differences in pathogenicity between an inoculum derived directly from an intestinal tissue homogenate from a paratuberculosis affected sheep and the S-type Mycobacterium avium subsp. partuberculosis (Map) strain isolated in laboratory media from the mentioned homogenate were assessed in two experiments in lambs. Specific peripheral immune responses were significantly lower in animals inoculated with the cultured organisms that showed only granulomatous lesions in the intestinal lymphoid tissue. However, in the homogenate group, more abundant granulomata also occurred in the lamina propria. Map was isolated only in lambs infected with the culture strain. Map DNA was demonstrated by nested-PCR in all the lambs but in a lower proportion (57.1% vs 100%) in those from the culture group. Under these particular experimental conditions, the results suggest that an attenuation of Map virulence has occurred in the cultured strain compared to the initial tissue homogenate, even after a low number of passages.

  15. Epstein-Barr virus and Mycobacterium avium subsp. paratuberculosis peptides are cross recognized by anti-myelin basic protein antibodies in multiple sclerosis patients.

    PubMed

    Mameli, Giuseppe; Cossu, Davide; Cocco, Eleonora; Masala, Speranza; Frau, Jessica; Marrosu, Maria Giovanna; Sechi, Leonardo A

    2014-05-15

    Epstein-Barr virus and Mycobacterium avium subsp. paratuberculosis (MAP) have been associated to multiple sclerosis (MS). We searched for antibodies against the homologous peptides Epstein-Barr virus nuclear antigen 1 (EBNA1)400-413, MAP_0106c protein (MAP)121-132, and myelin basic protein (MBP)85-98 on a MS Sardinian cohort, showing that these antibodies are highly prevalent among MS patients compared to healthy controls. Competitive assay demonstrated that antibodies recognizing EBNA1400-413 and MAP121-132 cross-react with MBP85-98, possibly through a molecular mimicry mechanism. Indeed, the fact that peptides from different pathogens can be cross-recognized by antibodies targeting self-epitopes supports the hypothesis that EBV and MAP might trigger autoimmunity through a common target.

  16. 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.

  17. 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...

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

    PubMed

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

    2009-12-15

    Johne's disease or paratuberculosis is widespread in almost all countries and remains difficult to eradicate. Nowadays, diagnosis of Mycobacterium avium subsp. paratuberculosis (MPTB) infection is one of the main concerns. In this work, we evaluated the expression, biochemical properties and antigenicity of the Apa antigen, encoded by the gene annotated as MAP1569, in the MPTB genome. We confirmed its expression in MPTB and its glycosylation by the ConA binding assay. Although the MPTB-Apa is not an immunodominant antigen, MPTB-infected cattle showed a strong humoral response to recombinant Apa by Western blot and ELISA. Milk was also a suitable sample to be tested by ELISA. We comparatively analysed the humoral cross-reactivity to the Apa from MPTB (MPTB-Apa) and the orthologue from Mycobacterium tuberculosis (MT-Apa, identical to that from Mycobacterium bovis) in both infected and control cows. Response of M. bovis- and MPTB-infected animals against MT-Apa was similar (P=0.6985) but the response of the M. bovis-infected ones to MPTB-Apa was differential, being significantly diminished (P<0.0001). Although 6 out 45 animals from MPTB-infected herds responded to MPTB-Apa stimulation in the IFNgamma release assay, we found no significant differences when compared infected herds with non-infected ones (P=0.34). This antigen, in contrast to bovine Purified Protein Derivative (PPDb), was strongly represented in avian PPD (PPDa), as shown by the recognition of BALB/c mice hyperimmune sera against MPTB-Apa by Dot-blot immunoassay. We therefore demonstrated the antigenicity of Apa in MPTB-infected animals and a differential response to the recombinant antigen when compared to M. bovis-infected animals. These traits herein described, added to the usefulness of milk samples to detect IgG anti-Apa, could be important for routine screening in dairy cattle, considering a multiantigenic approach to overcome the lack of immunodominance.

  19. Potential role of real-time PCR for detecting Mycobacterium avium subsp. paratuberculosis in chronically diseased milking cows: a case control study.

    PubMed

    Alajmi, Ahmad; Klein, Günter; Greiner, Matthias; Grabowski, Nils Th; Fohler, Svenja; Campe, Amely; Scheu, Theresa; Hoedemaker, Martina; Abdulmawjood, Amir

    2016-01-01

    Paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (MAP) is economically important to dairy operations. In the present study, a real-time PCR kit (certified for use in Germany) was used to detect MAP in bovine fecal and milk samples within a case control study examining different factors for their association with chronic disease in Northern Germany.The aim of this study was to describe the suitability of the MAP real-time PCR kit to detect MAP in feces and milk of chronically diseased dairy cows. For MAP detection a total of 928 fecal, 922 composite foremilk, and 92 bulk milk samples obtained from 58 cases and 35 control dairy herds were investigated. The real-time PCR showed MAP positive results for 11 (18.96%) and 6 (17.14%) of the case and control herds, respectively. All bulk milk samples were MAP negative. The results of fecal and milk samples were moderately correlated (kappa = 0.27). These data indicate that real-time PCR results have diagnostic value for diagnosing MAP positive animals, and that fecal samples are more suitable than milk samples for assessing the reasons for chronic disease on dairy farms.

  20. Detection of Mycobacterium avium subspecies paratuberculosis by IS900-based PCR assays from an alpaca (Vicugna pacos) kept in a German zoological garden.

    PubMed

    Münster, Pia; Völkel, Inger; von Buchholz, Aloys; Czerny, Claus-Peter

    2013-03-01

    An alpaca (Vicugna pacos) bred and kept in a German zoological garden exhibited clinical signs consistent with paratuberculosis. The presence of Mycobacterium avium ssp. paratuberculosis (MAP) was confirmed in feces and in the ileocecal lymph node (ILN) by IS900-based polymerase chain reaction (PCR) assays and culture. A bacterial burden of 7.6 x 10(6) MAP/g in feces and 4.4 x 10(7) MAP/g in lymph node tissue was determined by real-time PCR. For further characterization, a conventional PCR was developed. After sequencing of the 864-bp PCR amplicon covering nucleotide positions 13 to 876 within the IS900, the alpaca isolate shared 100% nucleotide homology with the bovine MAP-K10 IS900 reference sequence (GenBank: AE16958), indicating a cattle strain. This report supports the present occurrence of MAP in German camelid populations and highlights the need to expand routine MAP surveillance to South American camelids held in European zoos.

  1. Expression and purification of a gene encoding a 9.7 kDa PE protein of Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Sekar, S Chandra; Goswami, P P; Deb, R

    2016-12-01

    Mycobacterium avium subsp. paratuberculosis (Map) contains PE family antigens which are Proline and glutamic acid rich and may play important role as T-cell antigens. In the present study, the Map 1507 ORF encoding 9.7 kDa PE protein was amplified by polymerase chain reaction and cloned into E. coli vector pQE30 UA. The recombinant plasmid designated as pQ PE was transformed into E. coli M15 cells and induced with IPTG revealed the high level expression of 11.9 kDa His-fusion protein as estimated by migration in 15 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Recombinant PE protein was purified by Ni-NTA agarose chromatography. Polyclonal antibodies raised against purified recombinant PE protein reacted with expressed PE protein as well as with Map sonicate. The recombinant PE protein was also recognized by serum from goat with clinical paratuberculosis. The protein elicited significant delayed type hypersensitivity (DTH) skin reaction in mice sensitized with Map. The results indicated that the recombinant PE protein of Map was associated with T-cell response.

  2. Expression of genes associated with cholesterol and lipid metabolism identified as a novel pathway in the early pathogenesis of Mycobacterium avium subspecies paratuberculosis-infection in cattle.

    PubMed

    Thirunavukkarasu, Shyamala; Plain, Karren M; de Silva, Kumudika; Begg, Douglas; Whittington, Richard J; Purdie, Auriol C

    2014-08-15

    Johne's disease (JD) is a chronic disease affecting ruminants and other species caused by the pathogenic mycobacterium, Mycobacterium avium subsp. paratuberculosis (MAP). MAP has developed a multitude of mechanisms to persist within the host, and these in turn are counteracted by the host through various immune pathways. Identifying and characterising the different strategies employed by MAP to alter the host immune system in its favour, and thereby persist intracellularly, could hold the key to developing strategies to fight this disease. In this study we analysed a subset of bovine microarray data derived from early time points after experimental infection with MAP. A specifically developed integrated approach was used to identify and validate host genes involved in cholesterol homeostasis (24DHCR, LDLR, SCD-1), calcium homeostasis and anti-bacterial defence mechanisms, (CD38, GIMAP6) which were downregulated in response to MAP exposure. A trend for upregulation of granulysin gene expression in MAP-exposed cattle in comparison to unexposed cattle was also observed. From these analyses, a model of potential pathogen-host interactions involving these novel pathways was developed which indicates an important role for host lipids in mycobacterial survival and persistence.

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

    PubMed

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

    2015-08-01

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

  4. 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.

  5. Early infection dynamics after experimental challenge with Mycobacterium avium subspecies paratuberculosis in calves reveal limited calf-to-calf transmission and no impact of Hsp70 vaccination.

    PubMed

    Santema, Wiebren J; Poot, Jacqueline; Segers, Ruud P A M; Van den Hoff, Daniëlle J P; Rutten, Victor P M G; Koets, Ad P

    2012-11-19

    Efficient control of bovine paratuberculosis is hampered by lack of a vaccine. The purpose of this study was to evaluate efficacy of a candidate vaccine, consisting of recombinant Mycobacterium avium subspecies paratuberculosis (MAP) Hsp70 with DDA adjuvant, in calves experimentally infected with MAP. Four groups of 14 animals each were used. Animals in group 1 and 2 were all vaccinated with Hsp70/DDA at day 0, 84, 168 and 357, and those in group 3 and 4 were non-vaccinated controls. In each group half (n=7) of the animals were challenged and the remaining half served as contacts. Blood and fecal samples were collected at three week intervals until day 588, and subsequently all animals were subjected to necropsy. The primary outcomes assessed were fecal culture (FC) of MAP, tissue colonization of MAP, and transmission of infection to contact animals. The kinetics of MAP shedding in feces of challenged animals showed a peak around 130 days post-challenge, irrespective of vaccination status. At necropsy no differences in the level of tissue colonization between vaccinated animals and controls were observed in the challenged groups. Only one contact animal (non-vaccinated) was positive at necropsy, indicating limited to no transmission within groups. These findings indicate that Hsp70/DDA vaccination does not influence early infection dynamics after experimental infection. However, early shedding of MAP in calves did not result in efficient transmission of infection to contact animals. The latter implies that introduction of an infected calf in a cohort of susceptibles has limited consequences for spread of infection.

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

    PubMed

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

    2010-01-01

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

  7. Bayesian estimation of the sensitivity and specificity of individual fecal culture and Paralisa to detect Mycobacterium avium subspecies paratuberculosis infection in young farmed deer.

    PubMed

    Stringer, Lesley A; Jones, Geoff; Jewell, Chris P; Noble, Alasdair D; Heuer, Cord; Wilson, Peter R; Johnson, Wesley O

    2013-11-01

    A Bayesian latent class model was used to estimate the sensitivity and specificity of an immunoglobulin G1 serum enzyme-linked immunosorbent assay (Paralisa) and individual fecal culture to detect young deer infected with Mycobacterium avium subsp. paratuberculosis. Paired fecal and serum samples were collected, between July 2009 and April 2010, from 20 individual yearling (12-24-month-old) deer in each of 20 South Island and 18 North Island herds in New Zealand and subjected to culture and Paralisa, respectively. Two fecal samples and 16 serum samples from 356 North Island deer, and 55 fecal and 37 serum samples from 401 South Island deer, were positive. The estimate of individual fecal culture sensitivity was 77% (95% credible interval [CI] = 61-92%) with specificity of 99% (95% CI = 98-99.7%). The Paralisa sensitivity estimate was 19% (95% CI = 10-30%), with specificity of 94% (95% CI = 93-96%). All estimates were robust to variation of priors and assumptions tested in a sensitivity analysis. The data informs the use of the tests in determining infection status at the individual and herd level.

  8. Sensitivity of solid culture, broth culture, and real-time PCR assays for milk and colostrum samples from Mycobacterium avium ssp. paratuberculosis-infectious dairy cows.

    PubMed

    Laurin, Emilie; McKenna, Shawn; Chaffer, Marcelo; Keefe, Greg

    2015-12-01

    Mycobacterium avium ssp. paratuberculosis (MAP) can be shed in feces, milk, and colostrum. The goal of this study was to assess assays that detect MAP in these sample types, including effects of lactation stage or season. Understanding the performance of these assays could improve how they are used, limiting the risk of infection to calves. Forty-six previously confirmed MAP-positive cows from 7 Atlantic Canadian dairy farms were identified for colostrum sampling and monthly sampling of milk and feces over a 12-mo period. Samples were assayed for MAP using solid culture, broth culture, and direct real-time PCR (qPCR). Across assay types, test sensitivity when applied to milk samples averaged 25% of that when applied to fecal samples. For colostrum samples, sensitivity depended on assay type, with sensitivity of qPCR being approximately 46% of that in feces. Across sample types, sensitivity of qPCR was higher than that of the other assays. Sensitivity of qPCR, when applied to milk samples, was significantly higher in summer than in other seasons. Summer was also the season with highest agreement between milk and fecal samples collected within the same month. Our results suggest that qPCR would detect more cows shedding MAP in their milk and colostrum than solid or broth culture assays, particularly during the summer, thus providing better management information to limit exposure of calves to this infectious organism.

  9. Occurrence, diagnosis, and strain typing of Mycobacterium avium subspecies paratuberculosis infection in Rocky Mountain bighorn sheep (Ovis canadensis canadensis) in southwestern Alberta.

    PubMed

    Forde, Taya; Kutz, Susan; De Buck, Jeroen; Warren, Amy; Ruckstuhl, Kathreen; Pybus, Margo; Orsel, Karin

    2012-01-01

    The role that wildlife may play in the transmission of Mycobacterium avium subspecies paratuberculosis (Map), the causative agent of Johne's disease (JD), and the potential consequences of infection in these populations are being given increasing consideration. A yearling male Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from southwestern Alberta, Canada, was found infected with Map in August 2009. Clinical signs of emaciation and diarrhea and histologic findings of diffuse granulomatous enteritis of the distal ileum, lymphadenitis of the mesenteric lymph nodes, and lymphangitis of the ileum were similar to previously described cases of JD in bighorn sheep. Infection with Map was confirmed by bacterial isolation through fecal culture, acid-fast staining, and polymerase chain reaction (PCR) of IS900. The Map1506 gene was sequenced, and the isolate was identified as a Cattle (Type II) strain. In a follow-up herd-level survey, three of 44 fecal samples (7%) from individual bighorn sheep from the same herd as the index case were PCR-positive and identified as Type II Map strains. Twenty-five samples from a distant bighorn population were negative. Additional strain typing of the isolates from the index case and the positive fecal samples was done by sequencing three discriminatory short sequence repeat (SSR) regions. All four SSR profiles differed from one another, suggesting multiple introductions or a long-existing circulation of Map within this bighorn population. Detailed molecular analyses are essential for understanding and managing diseases at the wildlife-livestock interface.

  10. Evaluation of environmental fecal culture for Mycobacterium avium subspecies paratuberculosis detection in dairy herds and association with apparent within-herd prevalence

    PubMed Central

    Lavers, Carrie J.; McKenna, Shawn L.B.; Dohoo, Ian R.; Barkema, Herman W.; Keefe, Greg P.

    2013-01-01

    This study evaluated test characteristics of environmental culture (EC) for the detection of Mycobacterium avium subspecies paratuberculosis (MAP) in 32 herds over a 2-year period. Individual fecal samples were collected every 6 mo and environmental samples every 3 mo. Individual fecal culture was performed on samples from positive pools. Samples were cultured in broth, with confirmatory polymerase chain reaction performed on positive fecal samples. Repeated measures were accounted for using GEE logistic models. Relative to a MAP herd-status based on all pooled fecal culture results collected during the study, sensitivity of a set of 6 EC-samples collected from prescribed locations within the herd environment (EC-6) was 71% [95% confidence interval (CI): 49% to 86%] and specificity was 99% (95% CI: 95% to 100%). Sensitivity of EC increased as apparent within-herd fecal culture prevalence (aWHP) increased. The estimated aWHP increased as the proportion of positive EC-samples within an EC-6 set increased. Environmental culture is an acceptable tool for herd diagnosis of MAP in low-prevalence herds. PMID:24179240

  11. 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.

  12. Efficacy of 'indigenous vaccine' using native 'Indian bison type' genotype of Mycobacterium avium subspecies paratuberculosis for the control of clinical Johne's disease in an organized goat herd.

    PubMed

    Singh, K; Chandel, B S; Chauhan, H C; Dadawala, A; Singh, S V; Singh, P K

    2013-06-01

    Therapeutic efficacy of a new 'Indigenous vaccine' prepared from native highly pathogenic 'Indian Bison Type' genotype of Mycobacterium avium subspecies paratuberculosis (MAP) of goat origin has been evaluated with respect to control of clinical Johne's disease in naturally infected Mehsana breed of goat in North Gujarat. Fifty goats from Sheep and Goats Research Station, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar, were randomly divided into 2 groups viz.,'Vaccinated'(n = 35) and 'Control'(n = 15). After vaccination, goats were monitored for physical condition, morbidity, mortality, body weights, shedding of MAP in feces, internal condition, gross lesions and humoral immune responses up to 120 days (at each interval of 30 days). At the end of 120 days trial, there was marked overall improvement in physical condition and body weights of vaccinated goats as compared to 'Control' goats. Vaccinated goats gained significantly (P < 0.05) higher body weights, hardly exhibited any lesions characteristic of JD, had significantly higher (P < 0.01) antibody titers and shedding of MAP was significantly (P < 0.01) reduced. Few of the vaccinated goats were positive for MAP DNA in faecal PCR and blood PCR before vaccination. However, all were found as negative at 120 days post vaccination (DPV). Overall vaccine exhibited effective in restriction of MAP infection and significant improvement in production parameters and reduction in mortality and morbidity due to JD. The trial in the herd will be continued.

  13. Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h☆

    PubMed Central

    Swift, Benjamin M.C.; Denton, Emily J.; Mahendran, Sophie A.; Huxley, Jonathan N.; Rees, Catherine E.D.

    2013-01-01

    The aim of this study was to develop a methodology to rapidly detect viable Mycobacterium avium subsp. paratuberculosis (MAP) in clinical blood samples. MAP cells spiked into commercially available blood were recovered using optimised peptide-mediated magnetic separation (PMMS) and detected using a phage-based method, and the identity of the cells detected confirmed using nested-PCR amplification of MAP signature sequences (IS900). The limit of detection was determined to be 10 MAP cells per ml of blood and was used to detect MAP present in clinical bovine blood samples. Using the PMMS-phage method there was no difference when detecting MAP from whole blood or from isolated buffy coat. MAP was detected in animals that were milk-ELISA positive (15 animals) by PMMS-phage and no MAP was detected in blood samples from an accredited Johne's disease free herd (5 animals). In a set of samples from one herd (10 animals) that came from animals with variable milk ELISA status, the PMMS-phage results agreed with the positive milk-ELISA results in all but one case. These results show that the PMMS-phage method can detect MAP present in naturally infected blood. Total assay time is 48 h and, unlike PCR-based detection tests, only viable cells are detected. A rapid method for detecting MAP in blood could further the understanding of disseminated infection in animals with Johne's disease. PMID:23811207

  14. Within-herd prevalence thresholds for the detection of Mycobacterium avium subspecies paratuberculosis-positive dairy herds using boot swabs and liquid manure samples.

    PubMed

    Donat, K; Hahn, N; Eisenberg, T; Schlez, K; Köhler, H; Wolter, W; Rohde, M; Pützschel, R; Rösler, U; Failing, K; Zschöck, P M

    2016-01-01

    The control of Johne's disease requires the identification of Mycobacterium avium ssp. paratuberculosis (MAP)-positive herds. Boot swabs and liquid manure samples have been suggested as an easy-to-use alternative to sampling individual animals in order to diagnose subclinical Johne's disease at the herd level, but there is a need to evaluate performance of this approach in the field. Using a logistic regression model, this study aimed to calculate the threshold level of the apparent within-herd prevalence as determined by individual faecal culture, thus allowing the detection of whether a herd is MAP positive. A total of 77 boot swabs and 75 liquid manure samples were taken from 19 certified negative and 58 positive dairy herds. Faecal culture, three different polymerase chain reaction (PCR) methods and the combination of faecal culture with PCR were applied in order to detect MAP. For 50% probability of detection, a within-herd prevalence threshold of 1·5% was calculated for testing both matrices simultaneously by faecal culture and PCR, with the threshold increased to 4·0% for 90% probability of detection. The results encourage the use of boot swabs or liquid manure samples, or a combination both, for identifying MAP-positive herds and, to a certain extent, for monitoring certified Johne's disease-negative cattle herds.

  15. 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

  16. 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

  17. 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

  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.

  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 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

  20. Gamma-delta T-cell responses during subcutaneous Mycobacterium avium subspecies paratuberculosis challenge in sensitized or naive calves using matrix biopolymers.

    PubMed

    Plattner, B L; Huffman, E L; Hostetter, J M

    2013-07-01

    We have developed a model to explore the early immune response against Mycobacterium avium subspecies paratuberculosis (Map) infection in the bovine calf using subcutaneously placed liquid gel matrix biopolymer (matrigel) containing live Map. Matrigel rapidly polymerizes in vivo, retains recruited cellular infiltrates and soluble immune mediators, and can be rapidly removed 48 hours later and depolymerized for analysis. In this study, we examined early host immune events at matrigel/Map sites; recruited cells were evaluated by histopathology and flow cytometry, and cytokines were measured by flow cytometry, enzyme-linked immunosorbent assay, and Luminex bead immunoassay. Our results demonstrate earlier recruitment of gamma-delta (γδ) T cells to matrigel/Map challenge sites compared to CD4+ T cells. We also show that significantly more γδ T cells were recruited to matrigel/Map sites postinfection day 7 compared to postinfection day 30 and that these cells produced significant amounts of the cytokine interferon gamma. We also provide evidence that peripheral blood-derived γδ T-cell subsets in cattle differentially generate interferon gamma, suggesting distinct roles for these cells. These data provide unique insight into initial antimycobacterial host cellular immune responses following Map infection in calves.

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

    PubMed

    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.

  2. 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

    2016-04-22

    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).

  3. 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.

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

    PubMed

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

    2017-01-01

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

  5. 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.

  6. 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.

  7. Examination of Mycobacterium avium subspecies paratuberculosis mixed genotype infections in dairy animals using a whole genome sequencing approach

    PubMed Central

    Davidson, Fraser W.; Ahlstrom, Christina; De Buck, Jeroen; Whitney, Hugh G.

    2016-01-01

    Many pathogenic mycobacteria are known to cause severe disease in humans and animals. M. avium subspecies paratuberculosis (Map) is the causative agent of Johne’s disease—a chronic wasting disease affecting ruminants such as cattle and sheep, responsible for significant economic losses in the dairy and beef industries. Due to the lack of treatment options or effective vaccines, mitigating losses can be difficult. In addition, the early stages of Map infection may occur in asymptomatic hosts that continue to shed viable bacteria in their faeces, leading to the infection of other healthy animals. Using multi-locus short sequence repeat (ML-SSR) analysis we previously reported that individual Johne’s positive dairy cattle from farms across the island of Newfoundland were infected by Map with multiple SSR-types simultaneously. The occurrence of multiple mixed genotype infections has the potential to change pathogen and disease dynamics as well as reduce the efficacy of treatments and vaccines. Therefore, we conducted whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis on a subset of these isolates for a more in-depth examination. We also implemented a PCR assay using two discriminatory SNPs and demonstrated the incidence of a mixed infection by three genotypically diverse Map isolates in a single animal. In addition, results show that WGS and SNP analysis can provide a better understanding of the relationship between Map isolates from individual and different animals. In the future such studies on the occurrence of mixed genotype infections could potentially lead to the identification of variable pathogenicity of different genotypes and allow for better tracking of Map isolates for epidemiological studies. PMID:27994984

  8. Examination of Mycobacterium avium subspecies paratuberculosis mixed genotype infections in dairy animals using a whole genome sequencing approach.

    PubMed

    Davidson, Fraser W; Ahlstrom, Christina; De Buck, Jeroen; Whitney, Hugh G; Tahlan, Kapil

    2016-01-01

    Many pathogenic mycobacteria are known to cause severe disease in humans and animals. M. avium subspecies paratuberculosis (Map) is the causative agent of Johne's disease-a chronic wasting disease affecting ruminants such as cattle and sheep, responsible for significant economic losses in the dairy and beef industries. Due to the lack of treatment options or effective vaccines, mitigating losses can be difficult. In addition, the early stages of Map infection may occur in asymptomatic hosts that continue to shed viable bacteria in their faeces, leading to the infection of other healthy animals. Using multi-locus short sequence repeat (ML-SSR) analysis we previously reported that individual Johne's positive dairy cattle from farms across the island of Newfoundland were infected by Map with multiple SSR-types simultaneously. The occurrence of multiple mixed genotype infections has the potential to change pathogen and disease dynamics as well as reduce the efficacy of treatments and vaccines. Therefore, we conducted whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis on a subset of these isolates for a more in-depth examination. We also implemented a PCR assay using two discriminatory SNPs and demonstrated the incidence of a mixed infection by three genotypically diverse Map isolates in a single animal. In addition, results show that WGS and SNP analysis can provide a better understanding of the relationship between Map isolates from individual and different animals. In the future such studies on the occurrence of mixed genotype infections could potentially lead to the identification of variable pathogenicity of different genotypes and allow for better tracking of Map isolates for epidemiological studies.

  9. Identification and Characterization of a Spore-Like Morphotype in Chronically Starved Mycobacterium avium Subsp. Paratuberculosis Cultures

    PubMed Central

    Lamont, Elise A.; Bannantine, John P.; Armién, Aníbal; Ariyakumar, Don Sanjiv; Sreevatsan, Srinand

    2012-01-01

    Mycobacteria are able to enter into a state of non-replication or dormancy, which may result in their chronic persistence in soil, aquatic environments, and permissive hosts. Stresses such as nutrient deprivation and hypoxia provide environmental cues to enter a persistent state; however, a clear definition of the mechanism that mycobacteria employ to achieve this remains elusive. While the concept of sporulation in mycobacteria is not novel, it continues to spark controversy and challenges our perceptions of a non-replication. We investigated the potential role of sporulation in one-year old broth cultures of Mycobacterium subsp. paratuberculosis (MAP). We show that dormant cultures of MAP contain a mix of vegetative cells and a previously unknown morphotype resembling a spore. These spore-like structures can be enriched for using sporulating media. Furthermore, purified MAP spore forms survive exposure to heat, lysozyme and proteinase K. Heat-treated spores are positive for MAP 16SrRNA and IS900. MAP spores display enhanced infectivity as well as maintain acid-fast characteristics upon germination in a well-established bovine macrophage model. This is the first study to demonstrate a new MAP morphotype possessing spore-like qualities. Data suggest that sporulation may be a viable mechanism by which MAP accomplishes persistence in the host and/or environment. Thus, our current understanding of mycobacterial persistence, pathogenesis, epidemiology and rational drug and vaccine design may need to be reevaluated. PMID:22292005

  10. Identification and characterization of a spore-like morphotype in chronically starved Mycobacterium avium subsp. paratuberculosis cultures.

    PubMed

    Lamont, Elise A; Bannantine, John P; Armién, Aníbal; Ariyakumar, Don Sanjiv; Sreevatsan, Srinand

    2012-01-01

    Mycobacteria are able to enter into a state of non-replication or dormancy, which may result in their chronic persistence in soil, aquatic environments, and permissive hosts. Stresses such as nutrient deprivation and hypoxia provide environmental cues to enter a persistent state; however, a clear definition of the mechanism that mycobacteria employ to achieve this remains elusive. While the concept of sporulation in mycobacteria is not novel, it continues to spark controversy and challenges our perceptions of a non-replication. We investigated the potential role of sporulation in one-year old broth cultures of Mycobacterium subsp. paratuberculosis (MAP). We show that dormant cultures of MAP contain a mix of vegetative cells and a previously unknown morphotype resembling a spore. These spore-like structures can be enriched for using sporulating media. Furthermore, purified MAP spore forms survive exposure to heat, lysozyme and proteinase K. Heat-treated spores are positive for MAP 16SrRNA and IS900. MAP spores display enhanced infectivity as well as maintain acid-fast characteristics upon germination in a well-established bovine macrophage model. This is the first study to demonstrate a new MAP morphotype possessing spore-like qualities. Data suggest that sporulation may be a viable mechanism by which MAP accomplishes persistence in the host and/or environment. Thus, our current understanding of mycobacterial persistence, pathogenesis, epidemiology and rational drug and vaccine design may need to be reevaluated.

  11. 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

  12. 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

  13. Association between caudal fold tuberculin test responses and results of an ELISA for Mycobacterium avium subsp paratuberculosis and mycobacterial culture of feces in tuberculosis-free dairy herds.

    PubMed

    Brito, Barbara P; Aly, Sharif S; Anderson, Randall J; Fossler, Charles P; Garry, Franklyn B; Gardner, Ian A

    2014-03-01

    OBJECTIVE--To evaluate associations between Mycobacterium avium subsp paratuberculosis (MAP) and caudal fold tuberculin (CFT) test results in cattle. DESIGN--Longitudinal and cross-sectional evaluations. ANIMALS--1 California (approx 3,600 cows) and 3 Colorado (approx 640, 1,190, and 1,480 cows) dairy herds considered free of Mycobacterium bovis infection. PROCEDURES--In the California herd, the association between CFT response and MAP status was determined with ELISA and mycobacterial culture of feces within 1 year before and after CFT testing. The association between CFT and MAP status in all herds was modeled with mixed-effects logistic regression. RESULTS--In the California herd, significantly higher odds of being classified as suspect by CFT were found for cows with results of MAP ELISA negative before and positive after CFT testing (OR, 5.6) and cows positive before and after CFT testing (OR, 8.1). Higher odds were found for cows positive for mycobacterial culture of feces before and negative for culture after CFT testing (OR, 4.6) and cows negative for mycobacterial culture of feces before and positive for culture after CFT testing (OR, 13.2). All herds had higher odds of being classified as suspect by CFT testing for cows with positive results for ELISA (OR, 2.9) or mycobacterial culture of feces (OR, 5.0), compared with cows with negative results of the same tests. CONCLUSIONS AND CLINICAL RELEVANCE--A strong association was found between positive MAP test results and being classified as a suspect by CFT testing. Within-herd MAP prevalence may affect specificity of CFT testing for tuberculosis in cattle.

  14. 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

  15. Genotyping of Mycobacterium avium complex organisms using multispacer sequence typing.

    PubMed

    Cayrou, Caroline; Turenne, Christine; Behr, Marcel A; Drancourt, Michel

    2010-03-01

    Mycobacterium avium complex (MAC) currently comprises eight species of environmental and animal-associated, slowly-growing mycobacteria: Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium colombiense, Mycobacterium arosiense , Mycobacterium bouchedurhonense, Mycobacterium marseillense and Mycobacterium timonense. In humans, MAC organisms are responsible for opportunistic infections whose unique epidemiology remains poorly understood, in part due to the lack of a genotyping method applicable to all eight MAC species. In this study we developed multispacer sequence typing (MST), a sequencing-based method, for the genotyping of MAC organisms. An alignment of the genome sequence of M. avium subsp. hominissuis strain 104 and M. avium subsp. paratuberculosis strain K-10 revealed 621 intergenic spacers <1000 bp. From these, 16 spacers were selected that ranged from 300 to 800 bp and contained a number of variable bases, <50 within each of the 16 spacers. Four spacers were successfully PCR-amplified and sequenced in 11 reference strains. Combining the sequence of these four spacers in 106 MAC organisms, including 83 M. avium, 11 M. intracellulare , six M. chimaera, two M. colombiense and one each of M. arosiense, M. bouchedurhonense, M. marseillense and M. timonense, yielded a total of 45 spacer types, with an index of discrimination of 0.94. Each spacer type was specific for a species and certain spacer types were specific for subspecies of M. avium. MST is a new method for genotyping of organisms belonging to any one of the eight MAC species tested in this study.

  16. A novel real-time PCR assay for specific detection and quantification of Mycobacterium avium subsp. paratuberculosis in milk with the inherent possibility of differentiation between viable and dead cells

    PubMed Central

    2010-01-01

    Background Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of paratuberculosis (Johne's disease) in ruminants and is suggested to be one of the etiologic factors in Crohn's disease in humans. Contaminated milk might expose humans to that pathogen. The aim of the present study was to develop a novel real-time PCR assay providing the additional possibility to detect viable Mycobacterium avium subsp. paratuberculosis (MAP) based on the MAP-specific Mptb52.16 target. The design included an internal amplification control to identify false negative results. Findings Inclusivity and exclusivity tested on 10 MAP strains, 22 non-MAP mycobacteria, and 16 raw milk microflora strains achieved 100%. The detection limit in artificially contaminated raw milk was 2.42 × 101 MAP cells/ml milk. In a survey of naturally contaminated samples obtained from dairy herds with a known history of paratuberculosis, 47.8% pre-milk and 51.9% main milk samples tested positive. Real-time PCR-derived MAP-specific bacterial cell equivalents (bce) ranged from 1 × 100 to 5.1 × 102 bce/51 ml; the majority of samples had less than one bce per ml milk. Expression of the chosen target was detected in artificially contaminated raw milk as well as inoculated Dubos broth, thus confirming the real-time PCR assay's potential to detect viable MAP cells. Conclusions Concentrating the DNA of a large sample volume in combination with the newly developed real-time PCR assay permitted quantification of low levels of MAP cells in raw milk and pasteurized milk. The selected target - Mptb52.16 - is promising with regard to the detection of viable MAP. Future studies integrating quantitative DNA- and RNA-based data might provide important information for risk assessment concerning the presence of MAP in raw milk and pasteurized milk. PMID:20925922

  17. SOLiD SAGE sequencing shows differential gene expression in jejunal lymph node samples of resistant and susceptible red deer (Cervus elaphus) challenged with Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Mackintosh, C G; Griffin, J F T; Scott, I C; O'Brien, R; Stanton, J L; MacLean, P; Brauning, R

    2016-01-01

    This study compared in vivo lymph node gene expression levels between six young red deer that were either relatively resistant (R) or susceptible (S) to paratuberculosis following experimental challenge with Mycobacterium avium subsp. paratuberculosis. Intestinal lymph nodes were biopsied at 4, 12 and 50 weeks post challenge (pc) and parallel changes in histopathology, immunology and bacterial load monitored. SOLiD SAGE (serial analysis of gene expression) next generation sequencing of biopsied lymph node samples generated a total of 373 million transcript tags 26-28bp in length after filtering. A total of 36,632 unique transcripts were identified and 14,325 of these were able to be annotated. The copy number of each transcript was counted, averaged and compared for R and S animals (R-S). P values and False Discovery Rates (FDR) were calculated for each transcript. Genes differentially upregulated ≥2 fold (FDR<0.5) totalled 9, 40 and 32 in R animals (+ values) and 23, 164 and 47 in S animals (- values) at weeks 4, 12, and 50pc, respectively. Transcripts displaying greatest differential expression between R and S animals at each time point were IFIT2 (189 fold) and S100A8 (-32.7 fold) at week 4, LRR1 (52.7 fold), SERPINF2 (-214.6 fold) at week 12 and CEACAM8 (84.6 fold), and STK31 (-129.5 fold) at week 50, respectively. All 9 genes significantly upregulated at week 4 in R animals relate specifically to host defence and all involve Type I interferon stimulated genes. By contrast genes upregulated in S animals at week 4, relate predominantly to inflammation, but also involve adaptive immune responses, mitochondrial function and apoptosis regulation. At week 12, the genes differentially upregulated in R animals are linked predominantly to regulation of adaptive immunity and mucosal immunity, while many of the genes in S animals are associated with pro-inflammatory interleukins involved with innate and adaptive immunity. These correlated with greater lesion severity

  18. 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

    2015-06-19

    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

  19. 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

  20. Mycobacterium avium subspecies paratuberculosis isolates from sheep and goats show reduced persistence in bovine macrophages than cattle, bison, deer and wild boar strains regardless of genotype.

    PubMed

    Abendaño, Naiara; Sevilla, Iker A; Prieto, José Miguel; Garrido, Joseba M; Juste, Ramon A; Alonso-Hearn, Marta

    2013-05-03

    Assessment of the virulence of isolates of Mycobacterium avium subsp. paratuberculosis (Map) exhibiting distinct genotypes and isolated from different hosts may help to clarify the degree to which clinical manifestations of the disease in cattle can be attributed to bacterial or to host factors. The objective of this study was to test the ability of 10 isolates of Map representing distinct genotypes and isolated from domestic (cattle, sheep, and goat), and wildlife animal species (fallow deer, deer, wild boar, and bison) to enter and grow in bovine macrophages. The isolates were previously typed using IS1311 PCR followed by restriction endonuclease analysis into types C, S or B. Intracellular growth of the isolates in a bovine macrophage-like cell line (BoMac) and in primary bovine monocyte-derived macrophages (MDM) was evaluated by quantification of CFU numbers in the initial inoculum and inside of the host cells at 2h and 7 d p.i. using an automatic liquid culture system (Bactec MGIT 960). Individual data illustrated that growth was less variable in BoMac than in MDM cells. All the isolates from goat and sheep hosts persisted within BoMac cells in lower CFU numbers than the other tested isolates after 7 days of infection regardless of genotype. In addition, BoMac cells exhibited differential inflammatory, apoptotic and destructive responses when infected with a bovine or an ovine isolate; which correlated with the differential survival of these strains within BoMac cells. Our results indicated that the survival of the tested Map isolates within bovine macrophages is associated with the specific host from which the isolates were initially isolated.

  1. Within- and between-herd prevalence variation of Mycobacterium avium subsp. paratuberculosis infection among control programme herds in Denmark (2011-2013).

    PubMed

    Verdugo, Cristobal; Toft, Nils; Nielsen, Søren Saxmose

    2015-10-01

    This study aimed to estimate the between- (HTP) and within- (TP) herd true prevalence distribution of Mycobacterium avium subsp. paratuberculosis (MAP) infection in dairy cattle herds participating in the Danish MAP control programme. All herds enrolled in the programme between 2011 and 2013 were included in the analysis, and one annual milk-ELISA test of all lactating cows present in such herds was considered. A Bayesian latent class model was used to obtain HTP and TP posterior distributions for each year. The model adjusts for uncertainty in age-specific test sensitivity and prior prevalence estimates. Bayesian posterior probabilities were computed in order to compare prevalence between the years. A total of 665,700 samples were included in the study, from 221,914, 224,040, and 220,466 cows sourced from 1138, 1112, and 1059 herds in years 2011, 2012, and 2013, respectively. In that period, HTP estimates of 0.92 (95% posterior probability interval (PPI), 0.87-0.96), 0.78 (95% PPI, 0.74-0.83), and 0.75 (95% PPI, 0.71-0.78) were recorded, respectively. Low TP were observed, with population mean estimates of 0.08 (95% PPI, 0.07-0.08), 0.07 (95% PPI, 0.07-0.08), and 0.07 (95% PPI, 0.06-0.07) for the three consecutive years. Statistically-important differences were recorded for HTP and population mean TP estimates between years, indicating a trend for a decreasing level of MAP infection at both herd and animal level. Model results showed that MAP infection was widespread among the Dairy cattle herds participating in the Danish control programme, though in general it was kept at very low levels.

  2. Detection of Mycobacterium avium subspecies paratuberculosis in tie-stall dairy herds using a standardized environmental sampling technique and targeted pooled samples

    PubMed Central

    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

    2016-01-01

    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

  3. 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.

  4. Bovine WC1(+) γδ T lymphocytes modify monocyte-derived macrophage responses during early Mycobacterium avium subspecies paratuberculosis infection.

    PubMed

    Baquero, Monica M; Plattner, Brandon L

    2016-02-01

    Following Mycobacterium avium subspecies paratuberculosis (Map) infection, some calves are apparently able to successfully clear the pathogen whereas others become persistently infected; however the reasons for this remain unknown. The importance of innate immunity, and in particular the role of γδ T lymphocytes, during early anti-mycobacterial immune response is recognized but specific mechanisms remain incompletely characterized. The objective of this study was to investigate how bovine WC1(+) γδ T lymphocytes mediate macrophage function during early Map infection. To achieve this objective, Map-infected monocyte-derived macrophages (MDMs) were co-cultured either in direct contact with, or separated by a semi-permeable membrane from, autologous WC1(+) γδ T lymphocytes. Nitrites, IL-17A, IFN-γ, IL-4 and IL-10 from cell culture supernatants were measured. Expression of CD25 on WC1(+) γδ T lymphocytes, expression of MHC-I and MHC-II on MDMs and the viability of Map recovered from MDM cultures 72h after Map infection were also assessed. Map viability was significantly reduced when WC1(+) γδ T lymphocytes were co-cultured in direct contact with Map-infected MDMs. Both MDMs and WC1(+) γδ T lymphocytes generated increased concentrations of IFN-γ and IL-4 in our system, and MDM/WC1(+) γδ T lymphocyte synergism was identified for IFN-γ production. MDMs but not WC1(+) γδ T lymphocytes were a significant source of IL-17A. The presence of WC1(+) γδ T lymphocytes was associated with higher expression of MHC-I on MDMs and increased concentration of nitrites in supernatants 72h after Map infection. In conclusion, this study showed that WC1(+) γδ lymphocytes had differential effects on Map-infected macrophages in vitro.

  5. PCR Inhibition of a Quantitative PCR for Detection of Mycobacterium avium Subspecies Paratuberculosis DNA in Feces: Diagnostic Implications and Potential Solutions

    PubMed Central

    Acharya, Kamal R.; Dhand, Navneet K.; Whittington, Richard J.; Plain, Karren M.

    2017-01-01

    Molecular tests such as polymerase chain reaction (PCR) are increasingly being applied for the diagnosis of Johne’s disease, a chronic intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Feces, as the primary test sample, presents challenges in terms of effective DNA isolation, with potential for PCR inhibition and ultimately for reduced analytical and diagnostic sensitivity. However, limited evidence is available regarding the magnitude and diagnostic implications of PCR inhibition for the detection of MAP in feces. This study aimed to investigate the presence and diagnostic implications of PCR inhibition in a quantitative PCR assay for MAP (High-throughput Johne’s test) to investigate the characteristics of samples prone to inhibition and to identify measures that can be taken to overcome this. In a study of fecal samples derived from a high prevalence, endemically infected cattle herd, 19.94% of fecal DNA extracts showed some evidence of inhibition. Relief of inhibition by a five-fold dilution of the DNA extract led to an average increase in quantification of DNA by 3.3-fold that consequently increased test sensitivity of the qPCR from 55 to 80% compared to fecal culture. DNA extracts with higher DNA and protein content had 19.33 and 10.94 times higher odds of showing inhibition, respectively. The results suggest that the current test protocol is sensitive for herd level diagnosis of Johne’s disease but that test sensitivity and individual level diagnosis could be enhanced by relief of PCR inhibition, achieved by five-fold dilution of the DNA extract. Furthermore, qualitative and quantitative parameters derived from absorbance measures of DNA extracts could be useful for prediction of inhibitory fecal samples. PMID:28210245

  6. Dam's infection progress and within-herd prevalence as predictors of Mycobacterium avium subsp. paratuberculosis ELISA response in Danish Holstein cattle.

    PubMed

    Nielsen, Søren Saxmose; Hansen, Kira Frello; Kvist, Louise; Kostoulas, Polychronis

    2016-03-01

    Understanding the primary routes of transmission of Mycobacterium avium subsp. paratuberculosis (MAP) is pivotal to manage the pathogen in cattle herds. MAP is transmitted both vertically and horizontally, and both the dam's stage of infection and the prevalence in the population are therefore potentially important for MAP transmission control. The objective of this study was therefore to assess the dam's infection progress and the within-herd test-prevalence as predictors of MAP infection in Danish dairy cattle. MAP specific antibody ELISA records from 95,025 dam-offspring pairs were combined with test-prevalence estimates from 939 Danish Holstein herds. The odds of testing ELISA-positive given the within-herd test-prevalence and the time-period a dam had had MAP specific antibodies were estimated for the offspring. Both dams and offspring were tested as adults, and parity-group was used to correct for the effect of age. The results showed that both the within-herd test-prevalence and the dam's infection progress were significant predictors, while the dams that had tested positive when giving birth and up to 0.7 years after were more likely to have offspring that would test positive. The odds of testing positive were about 1.5 to 2.5 times higher for these offspring, compared to offspring of dams that never tested positive. Furthermore, offspring born in high (>5% ELISA-positive) and medium (2.5 to 5% ELISA-positive) prevalence herds had 9 and 3, respectively, times higher odds of testing positive, compared to animals born in a low prevalence herd. The variance heterogeneity reduced 81% through the included predictors. The results of this study suggest that irrespective of the prevalence, offspring of dams with MAP specific antibodies should be considered as high-risk animals when managing the infection in cattle herds, but both the prevalence and the dam's infection status are important in MAP control.

  7. 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

  8. 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

  9. PCR Inhibition of a Quantitative PCR for Detection of Mycobacterium avium Subspecies Paratuberculosis DNA in Feces: Diagnostic Implications and Potential Solutions.

    PubMed

    Acharya, Kamal R; Dhand, Navneet K; Whittington, Richard J; Plain, Karren M

    2017-01-01

    Molecular tests such as polymerase chain reaction (PCR) are increasingly being applied for the diagnosis of Johne's disease, a chronic intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Feces, as the primary test sample, presents challenges in terms of effective DNA isolation, with potential for PCR inhibition and ultimately for reduced analytical and diagnostic sensitivity. However, limited evidence is available regarding the magnitude and diagnostic implications of PCR inhibition for the detection of MAP in feces. This study aimed to investigate the presence and diagnostic implications of PCR inhibition in a quantitative PCR assay for MAP (High-throughput Johne's test) to investigate the characteristics of samples prone to inhibition and to identify measures that can be taken to overcome this. In a study of fecal samples derived from a high prevalence, endemically infected cattle herd, 19.94% of fecal DNA extracts showed some evidence of inhibition. Relief of inhibition by a five-fold dilution of the DNA extract led to an average increase in quantification of DNA by 3.3-fold that consequently increased test sensitivity of the qPCR from 55 to 80% compared to fecal culture. DNA extracts with higher DNA and protein content had 19.33 and 10.94 times higher odds of showing inhibition, respectively. The results suggest that the current test protocol is sensitive for herd level diagnosis of Johne's disease but that test sensitivity and individual level diagnosis could be enhanced by relief of PCR inhibition, achieved by five-fold dilution of the DNA extract. Furthermore, qualitative and quantitative parameters derived from absorbance measures of DNA extracts could be useful for prediction of inhibitory fecal samples.

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

    PubMed

    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.

  11. 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

  12. 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

  13. Immunity, safety and protection of an Adenovirus 5 prime--Modified Vaccinia virus Ankara boost subunit vaccine against Mycobacterium avium subspecies paratuberculosis infection in calves.

    PubMed

    Bull, Tim J; Vrettou, Christina; Linedale, Richard; McGuinnes, Catherine; Strain, Sam; McNair, Jim; Gilbert, Sarah C; Hope, Jayne C

    2014-10-29

    Vaccination is the most cost effective control measure for Johne's disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) but currently available whole cell killed formulations have limited efficacy and are incompatible with the diagnosis of bovine tuberculosis by tuberculin skin test. We have evaluated the utility of a viral delivery regimen of non-replicative human Adenovirus 5 and Modified Vaccinia virus Ankara recombinant for early entry MAP specific antigens (HAV) to show protection against challenge in a calf model and extensively screened for differential immunological markers associated with protection. We have shown that HAV vaccination was well tolerated, could be detected using a differentiation of infected and vaccinated animals (DIVA) test, showed no cross-reactivity with tuberculin and provided a degree of protection against challenge evidenced by a lack of faecal shedding in vaccinated animals that persisted throughout the 7 month infection period. Calves given HAV vaccination had significant priming and boosting of MAP derived antigen (PPD-J) specific CD4+, CD8+ IFN-γ producing T-cell populations and, upon challenge, developed early specific Th17 related immune responses, enhanced IFN-γ responses and retained a high MAP killing capacity in blood. During later phases post MAP challenge, PPD-J antigen specific IFN-γ and Th17 responses in HAV vaccinated animals corresponded with improvements in peripheral bacteraemia. By contrast a lack of IFN-γ, induction of FoxP3+ T cells and increased IL-1β and IL-10 secretion were indicative of progressive infection in Sham vaccinated animals. We conclude that HAV vaccination shows excellent promise as a new tool for improving control of MAP infection in cattle.

  14. 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.

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

    PubMed

    Nielsen, Søren Saxmose; Toft, Nils

    2014-01-01

    Detection of bulk tank milk (BTM) antibodies using ELISA (BTM-ELISA) may constitute an inexpensive test for surveillance of Mycobacterium avium subsp. paratuberculosis (MAP) infection in dairy cattle herds provided that the test is accurate and consistent. The objectives of this study were to determine: (a) the correlation between repeated BTM reactions; and (b) the association between the BTM antibody ELISA-level and the within-herd prevalence of antibody-positive cows. Eight BTM samples per herd and approximately four milk samples per lactating cow per herd were collected from each of 108 Danish Holstein herds over a period of one year. All samples were tested using a commercial indirect ELISA for detection of MAP specific antibodies. The individual cow's results were dichotomised and used to estimate the within-herd antibody prevalence at each test-date. These prevalences were then combined with the ELISA reading on the BTM test-date closest to the cow-level test-date. A mixed-effect analysis of covariance with autoregressive type 1 correlation structure was carried out using the log-transformed BTM-ELISA results as outcome. This model was used to assess the correlation between repeated tests with and without correction for within-herd antibody prevalence. The repeated BTM-recordings were highly correlated with a correlation of 0.80 between samples collected 1.5 months apart. The within-herd antibody prevalence significantly influenced this estimate (p<0.0001), which dropped to 0.60 when corrected for the within-herd antibody prevalence. Although the test-results were relatively consistent and correlated with the within-herd prevalence, the magnitude of the test-values makes it difficult to use the BTM-ELISA for surveillance of MAP infections in practice.

  16. Effect of Mycobacterium avium subsp. paratuberculosis (MAP) infection on the diagnostic accuracy for Mycobacterium bovis (M. bovis) infection under field conditions in cattle belonging to low M. bovis prevalence herds.

    PubMed

    Raffo, E; Steuer, P; Monti, G; Salgado, M

    2017-03-10

    Currently, the Chilean authority has implemented a National Eradication Program for bovine tuberculosis (bTB), aimed at controlling and eradicating the disease in Chile. The area under study has a low within-herd prevalence, has a relatively low number of infected herds, and is one of the major milk and beef producing areas in the country. However, so far, no attempts at eradicating the disease have been successful. It has been suggested that the diagnostic tests used were either not sensitive or specific enough. In addition, previous studies have shown that a great number of herds are infected with Mycobacterium avium subsp. paratuberculosis (MAP). The present study estimates the effect of MAP infection under field conditions, on the diagnostic sensitivity and specificity of routine tests in live animals for Mycobacterium bovis infection diagnosis in cattle. In general, the estimations of test accuracy observed an increase in the sensitivity and specificity on MAP-infected animals for tuberculin test but observed a decrease in the sensitivity of gamma interferon tests for MAP-infected cattle. These results are different from those of previous studies considering the role of MAP infection as an interfering infection. More research is needed in order to understand the complex interactions of the different mycobacteria that can be found infecting production cattle.

  17. Novel secreted antigens of Mycobacterium paratuberculosis as serodiagnostic biomarkers for Johne's disease in cattle.

    PubMed

    Facciuolo, Antonio; Kelton, David F; Mutharia, Lucy M

    2013-12-01

    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.

  18. 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...

  19. Successful treatment of asymptomatic or clinically terminal bovine Mycobacterium avium subspecies paratuberculosis infection (Johne disease) with the bacterium Dietzia used as a probiotic alone or in combination with dexamethasone

    PubMed Central

    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

  20. 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

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

    PubMed

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

    2015-04-01

    The influence of milk yield and milk composition on the diagnosis of Mycobacterium avium ssp. paratuberculosis (MAP) by milk ELISA in the context of the total IgG secretion patterns in milk throughout lactation and serum concentrations were investigated. A 2-yr trial was performed in which 1,410 dairy cows were sampled monthly and MAP milk ELISA status and milk yield and composition were determined. Data were analyzed by mixed model analysis. Milk yield was found to significantly influence ELISA results expressed as sample-to-positive (S/P) ratios. For each 5-kg increase in milk, the S/P ratio has to be multiplied by 0.89; therefore, high milk yield can change the MAP milk ELISA outcome of a cow in early infection from positive to negative. Parity influenced ELISA outcome significantly, indicating that cows with a parity >1 are more likely to be identified by milk testing. Also, herd was an important predictor, showing that herd prevalence influences the milk ELISA strongly. Other factors influencing the S/P ratios were protein concentration, somatic cell count, and days in milk. The IgG concentration and mass excreted per day were determined longitudinally in a subset of 41 cows of which samples and data of a complete lactation were available. Again, the IgG concentration in milk was mainly influenced by milk yield. The total IgG mass secreted per day in milk was found to be relatively constant, with a mean of 8.70 ± 5.38 g despite an increasing IgG concentration in serum at the same time. The variation of IgG concentration in milk can be mainly attributed to dilution through changes in milk yield. This supports the assumption that concentrations of MAP-specific antibodies are influenced by changes in milk yield similarly. In conclusion, we confirmed that antibody concentrations, and therefore MAP ELISA outcome, were influenced by milk yield, herd, and parity. To enhance performance, milk ELISA tests should either be performed in early or late lactation, when

  2. The investigation of the truncated mbtA gene within the mycobactin cluster of Mycobacterium avium subspecies paratuberculosis as a novel diagnostic marker for real-time PCR.

    PubMed

    de Kruijf, Marcel; Coffey, Aidan; O'Mahony, Jim

    2017-05-01

    The inability of Mycobacterium avium subspecies paratuberculosis (MAP) to produce endogenous mycobactin in-vitro is most likely due to the presence of a truncated mbtA gene within the mycobactin cluster of MAP. The main goal of this study was to investigate this unique mbtA truncation as a potential novel PCR diagnostic marker for MAP. Novel primers were designed that were located within the truncated region and the contiguous MAP2179 gene. Primers were evaluated against non-MAP isolates and no amplicons were generated. The detection limit of this mbtA-MAP2179 target was evaluated using a range of MAP DNA concentrations, MAP inoculated faecal material and 20 MAP isolates. The performance of mbtA-MAP2179 was compared to the established f57 target. The detection limits recorded for MAP K-10 DNA and from MAP K-10 inoculated faecal samples were 0.34pg and 10(4)CFU/g respectively for both f57 and mbtA-MAP2179. A detection limit of 10(3)CFU/g was recorded for both targets, but not achieved consistently. The detection limit of MAP from inoculated faecal material was successful at 10(3)CFU/g for mbtA-MAP2179 when FAM probe real-time PCR was used. A MAP cell concentration of 10(2)CFU/g was detected successfully, but again not consistently achieved. All 20 mycobacterial isolates were successfully identified as MAP by f57 and mbtA-MAP2179. Interestingly, the mbtA-MAP2179 real-time PCR assay resulted in the formation of a unique melting curve profile that contained two melting curve peaks rather than one single peak. This melting curve phenomenon was attributed towards the asymmetrical GC% distribution within the mbtA-MAP2179 amplicon. This study investigated the implementation of the mbtA-MAP2179 target as a novel diagnostic marker and the detection limits obtained with mbtA-MAP2179 were comparable to the established f57 target, making the mbtA-MAP2179 an adequate confirmatory target. Moreover, the mbtA-MAP2179 target could be implemented in multiplex real-time PCR assays and

  3. Designing a risk-based surveillance program for Mycobacterium avium ssp. paratuberculosis in Norwegian dairy herds using multivariate statistical process control analysis.

    PubMed

    Whist, A C; Liland, K H; Jonsson, M E; Sæbø, S; Sviland, S; Østerås, O; Norström, M; Hopp, P

    2014-11-01

    Surveillance programs for animal diseases are critical to early disease detection and risk estimation and to documenting a population's disease status at a given time. The aim of this study was to describe a risk-based surveillance program for detecting Mycobacterium avium ssp. paratuberculosis (MAP) infection in Norwegian dairy cattle. The included risk factors for detecting MAP were purchase of cattle, combined cattle and goat farming, and location of the cattle farm in counties containing goats with MAP. The risk indicators included production data [culling of animals >3 yr of age, carcass conformation of animals >3 yr of age, milk production decrease in older lactating cows (lactations 3, 4, and 5)], and clinical data (diarrhea, enteritis, or both, in animals >3 yr of age). Except for combined cattle and goat farming and cattle farm location, all data were collected at the cow level and summarized at the herd level. Predefined risk factors and risk indicators were extracted from different national databases and combined in a multivariate statistical process control to obtain a risk assessment for each herd. The ordinary Hotelling's T(2) statistic was applied as a multivariate, standardized measure of difference between the current observed state and the average state of the risk factors for a given herd. To make the analysis more robust and adapt it to the slowly developing nature of MAP, monthly risk calculations were based on data accumulated during a 24-mo period. Monitoring of these variables was performed to identify outliers that may indicate deviance in one or more of the underlying processes. The highest-ranked herds were scattered all over Norway and clustered in high-density dairy cattle farm areas. The resulting rankings of herds are being used in the national surveillance program for MAP in 2014 to increase the sensitivity of the ongoing surveillance program in which 5 fecal samples for bacteriological examination are collected from 25 dairy herds

  4. Responses of Bovine Innate Immunity to Mycobacterium avium subsp. paratuberculosis Infection Revealed by Changes in Gene Expression and Levels of MicroRNA

    PubMed Central

    Malvisi, Michela; Morandi, Nicola

    2016-01-01

    Paratuberculosis in cattle is a chronic granulomatous gastroenteritis caused by Mycobacterium avium subsp. paratubercolosis (MAP) which is endemic worldwide. In dairy herds, it is responsible for huge economic losses. However, current diagnostic methods do not detect subclinical infection making control of the disease difficult. The identification of MAP infected animals during the sub-clinical phase of infection would play a key role in preventing the dissemination of the pathogen and in reducing transmission. Gene expression and circulating microRNA (miRNA) signatures have been proposed as biomarkers of disease both in the human and veterinary medicine. In this paper, gene expression and related miRNA levels were investigated in cows positive for MAP, by ELISA and culture, in order to identify potential biomarkers to improve diagnosis of MAP infection. Three groups, each of 5 animals, were used to compare the results of gene expression from positive, exposed and negative cows. Overall 258 differentially expressed genes were identified between unexposed, exposed, but ELISA negative and positive groups which were involved in biological functions related to inflammatory response, lipid metabolism and small molecule biochemistry. Differentially expressed miRNA was also found among the three groups: 7 miRNAs were at a lower level and 2 at a higher level in positive animals vs unexposed animals, while 5 and 3 miRNAs were respectively reduced and increased in the exposed group compared to the unexposed group. Among the differentially expressed miRNAs 6 have been previously described as immune-response related and two were novel miRNAs. Analysis of the miRNA levels showed correlation with expression of their target genes, known to be involved in the immune process. This study suggests that miRNA expression is affected by MAP infection and play a key role in tuning the host response to infection. The miRNA and gene expression profiles may be biomarkers of infection and

  5. Comparative risk assessment for new cow-level Mycobacterium avium ssp. paratuberculosis infections between 3 dairy production types: Organic, conventional, and conventional-grazing systems.

    PubMed

    Beaver, A; Ruegg, P L; Gröhn, Y T; Schukken, Y H

    2016-12-01

    Johne's disease, a granulomatous enteritis of ruminant animals, is a hidden threat on dairy farms, adversely affecting animal welfare as well as herd productivity. Control programs in the United States advocate for specific management practices to temper the spread of the causal organism (Mycobacterium avium ssp. paratuberculosis, MAP), such as improving calving area hygiene and limiting introduction of replacement stock with unknown infection status. A need remains for direct exploration of Johne's disease prevention strategies in the United States with respect to production type. Alongside the growing demand for organic products, the safety of organic dairy practices with respect to MAP control is warranted. Further, conventional herds for which organic practices such as pasture grazing are used should be situated within the risk spectrum. We developed a risk assessment model using the US Voluntary Bovine Johne's Disease Control Program as a framework, with the goal of evaluating the risk of new cow-level MAP infections. A total of 292 organic and conventional farms in 3 states were surveyed on management practices, and an overall analysis was conducted in which each farm was first scored on individual practices using a range of "no risk" to "high risk," according to the literature. The sum of all risk factors was then analyzed to quantify and compare the risk burden for each production type. Organic herds received higher overall risk scores compared with both conventional grazing and nongrazing subtypes. To identify which factors contributed to the overall increased risk for organic herds, the management practices were categorized and evaluated by logistic regression. We determined that the increased risk incurred by organic herds was predominantly due to decisions made in the calving area and preweaned calf group. However, although certain individual risk factors related to calf management are commonly involved in prevention strategies (e.g., cow

  6. Estimation of flock/herd-level true Mycobacterium avium subspecies paratuberculosis prevalence on sheep, beef cattle and deer farms in New Zealand using a novel Bayesian model.

    PubMed

    Verdugo, Cristobal; Jones, Geoff; Johnson, Wes; Wilson, Peter; Stringer, Lesley; Heuer, Cord

    2014-12-01

    The study aimed to estimate the national- and island-level flock/herd true prevalence (HTP) of Mycobacterium avium subsp. paratuberculosis (MAP) infection in pastoral farmed sheep, beef cattle and deer in New Zealand. A random sample of 238 single- or multi-species farms was selected from a postal surveyed population of 1940 farms. The sample included 162 sheep flocks, 116 beef cattle and 99 deer herds from seven of 16 geographical regions. Twenty animals from each species present on farm were randomly selected for blood and faecal sampling. Pooled faecal culture testing was conducted using a single pool (sheep flocks) or two pools (beef cattle/deer herds) of 20 and 10 samples per pool, respectively. To increase flock/herd-level sensitivity, sera from all 20 animals from culture negative flocks/herds were individually tested by Pourquier(®) ELISA (sheep and cattle) or Paralisa™ (deer). Results were adjusted for sensitivity and specificity of diagnostic tests using a novel Bayesian latent class model. Outcomes were adjusted by their sampling fractions to obtain HTP estimates at national level. For each species, the posterior probability (POPR) of HTP differences between New Zealand North (NI) and South (SI) Islands was obtained. Across all species, 69% of farms had at least one species test positive. Sheep flocks had the highest HTP estimate (76%, posterior probability interval (PPI) 70-81%), followed by deer (46%, PPI 38-55%) and beef herds (42%, PPI 35-50%). Differences were observed between the two main islands of New Zealand, with higher HTP in sheep and beef cattle flocks/herds in the NI. Sheep flock HTP was 80% in the NI compared with 70% (POPR=0.96) in the SI, while the HTP for beef cattle was 44% in the NI and 38% in the SI (POPR=0.80). Conversely, deer HTP was higher in the SI (54%) than the NI (33%, POPR=0.99). Infection with MAP is endemic at high prevalence in sheep, beef cattle and deer flocks/herds across New Zealand.

  7. 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

  8. 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

  9. Risk factors for herds to test positive for Mycobacterium avium ssp. paratuberculosis-antibodies with a commercial milk enzyme-linked immunosorbent assay (ELISA) in Ontario and western Canada.

    PubMed

    Sorge, Ulrike S; Lissemore, Kerry; Godkin, Ann; Jansen, Jocelyn; Hendrick, Steven; Wells, Scott; Kelton, David F

    2012-09-01

    The objectives of this study were to identify risk factors associated with i) a Mycobacterium avium subsp. paratuberculosis (MAP)-antibody milk enzyme-linked immunosorbent assay (MAP milk ELISA)-positive herd status, and ii) the within-herd MAP milk ELISA-positive prevalence in Canadian dairy herds. This prospective cohort study was conducted between 2005 and 2009 on 226 herds in Ontario and western Canada, which participated in a voluntary risk assessment (RA)-based Johne's disease control program. Two MAP milk ELISA and risk assessments and a previsit survey were available per herd. The overall farm RA scores alone could not be used to predict whether a herd would test positive for MAP antibodies. However, the results of this study indicated that increasing the likelihood of exposing calves to MAP through certain management practices, as assessed with the RA, increased the likelihood of a herd being test-positive for MAP antibodies.

  10. 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

  11. 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

  12. 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...

  13. Bayesian estimation of sensitivity and specificity of a commercial serum/milk ELISA against the Mycobacterium avium subsp. Paratuberculosis (MAP) antibody response for each lactation stage in Greek dairy sheep.

    PubMed

    Angelidou, Elisavet; Kostoulas, Polychronis; Leontides, Leonidas

    2016-02-01

    A total of 854 paired milk and blood samples were collected from ewes of a Greek flock and used to estimate the sensitivity and specificity of a commercial ELISA for detection of Mycobacterium avium subsp. paratuberculosis (MAP) specific antibodies in each stage of lactation. We implemented Bayesian mixture models to derive the distributions of the test response for the healthy and the infected ewes. In the colostrum period, early, mid and late lactation stage the median values of the area under the curves (AUC) were 0.61 (95% credible interval: 0.50; 0.84), 0.61 (0.51;0.84), 0.65 (0.51;0.91), 0.65(0.51;0.89) for the serum ELISA and and 0.60 (0.50; 0.84), 0.61 (0.50; 0.84), 0.67(0.51; 0.91), 0.66(0.50; 0.90) for the milk ELISA, respectively. Both serum and milk ELISA had low to average overall discriminatory ability as measured by the area under the curves and comparable sensitivities and specificities at the recommended cutoffs.

  14. Depletion of CD4 T Lymphocytes at the time of infection with M. avium subsp. paratuberculosis does not accelerate disease progression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A calf model was used to determine if the depletion of CD4 T cells prior to inoculation of Mycobacterium avium subsp. paratuberculosis (Map) would delay development of an immune response to Map and accelerate disease progression. Ileal cannulas were surgically implanted in 5 bull calves at two month...

  15. 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.

  16. 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.

  17. The diagnostic performance of an antibody enzyme-linked immunosorbent assay using serum and colostrum to determine the disease status of a Jersey dairy herd infected with Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Jenvey, Caitlin J; Reichel, Michael P; Cockcroft, Peter D

    2016-01-01

    Colostrum may have the ability to improve the diagnostic accuracy of some tests when compared to serum for important livestock diseases because of the high concentrations of immunoglobulins present within this sample type. The ELISA for Johne's disease is one such test, as it suffers from low sensitivity when testing serum samples collected during the subclinical stage of infection. Blood and colostrum samples were collected from 34 Jersey dairy cows and tested for antibodies against Mycobacterium avium subspecies paratuberculosis (MAP) by ELISA. Fecal samples were also collected and tested by a high-throughput Johne's polymerase chain reaction (HT-J PCR) assay and fecal culture (FC), with the latter being used as the reference test. A receiver operating characteristic (ROC) analysis was performed, and the area under the curve (AUC) was calculated. The HT-J PCR and FC results were also compared. Of the 34 cows in this study, 4 had FC results consistent with MAP infection. The HT-J PCR did not identify any FC-positive cows. Using a 1:20 dilution and sample-to-positive (S/P) ratio cutoff threshold of 0.15, the relative sensitivity values of both serum (AUC 0. 56) and colostrum (AUC 0.63) were 0%. With lower sample dilutions, the relative sensitivity values of serum were 0% (1:2, AUC 0.62; 1:5, AUC 0.55); however, the relative sensitivity value of colostrum was 75% (95% confidence interval [CI]: 19-99%) at a dilution of 1:5, S/P ratio cutoff threshold of 0.15, and AUC of 0.73 (95% CI: 0.55-0.87). The testing of colostrum samples for MAP-specific antibodies by ELISA may provide improved identification of animals in the early stages of infection with MAP when compared with serum samples.

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

    PubMed Central

    Bull, Tim J.; Munshi, Tulika; Mikkelsen, Heidi; Hartmann, Sofie B.; Sørensen, Maria R.; Garcia, Joanna S.; Lopez-Perez, Paula M.; Hofmann, Sven; Hilpert, Kai; Jungersen, Gregers

    2017-01-01

    The quantitative detection of viable pathogen load is an important tool in determining the degree of infection in animals and contamination of foodstuffs. Current conventional culture methods are limited in their ability to determine these levels in Mycobacterium avium subspecies paratuberculosis (MAP) due to slow growth, clumping and low recoverability issues. The principle goal of this study was to evaluate a novel culturing process (TiKa) with unique ability to stimulate MAP growth from low sample loads and dilutions. We demonstrate it was able to stimulate a mean 29-fold increase in recoverability and an improved sensitivity of up to three logs when compared with conventional culture. Using TiKa culture, MAP clumping was minimal and produced visible colonies in half the time required by standard culture methods. Parallel quantitative evaluation of the TiKa culture approach and qPCR on MAP loads in tissue and gut mucosal samples from a MAP vaccine-challenge study, showed good correlations between colony counts (cfu) and qPCR derived genome equivalents (Geq) over a large range of loads with a 30% greater sensitivity for TiKa culture approach at low loads (two logs). Furthermore, the relative fold changes in Geq and cfu from the TiKa culture approach suggests that non-mucosal tissue loads from MAP infected animals contained a reduced proportion of non-viable MAP (mean 19-fold) which was reduced significantly further (mean 190-fold) in vaccinated “reactor” calves. This study shows TiKa culture equates well with qPCR and provides important evidence that accuracy in estimating viable MAP load using DNA tests alone may vary significantly between samples of mucosal and lymphatic origin. PMID:28101082

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

    PubMed

    Bull, Tim J; Munshi, Tulika; Mikkelsen, Heidi; Hartmann, Sofie B; Sørensen, Maria R; Garcia, Joanna S; Lopez-Perez, Paula M; Hofmann, Sven; Hilpert, Kai; Jungersen, Gregers

    2016-01-01

    The quantitative detection of viable pathogen load is an important tool in determining the degree of infection in animals and contamination of foodstuffs. Current conventional culture methods are limited in their ability to determine these levels in Mycobacterium avium subspecies paratuberculosis (MAP) due to slow growth, clumping and low recoverability issues. The principle goal of this study was to evaluate a novel culturing process (TiKa) with unique ability to stimulate MAP growth from low sample loads and dilutions. We demonstrate it was able to stimulate a mean 29-fold increase in recoverability and an improved sensitivity of up to three logs when compared with conventional culture. Using TiKa culture, MAP clumping was minimal and produced visible colonies in half the time required by standard culture methods. Parallel quantitative evaluation of the TiKa culture approach and qPCR on MAP loads in tissue and gut mucosal samples from a MAP vaccine-challenge study, showed good correlations between colony counts (cfu) and qPCR derived genome equivalents (Geq) over a large range of loads with a 30% greater sensitivity for TiKa culture approach at low loads (two logs). Furthermore, the relative fold changes in Geq and cfu from the TiKa culture approach suggests that non-mucosal tissue loads from MAP infected animals contained a reduced proportion of non-viable MAP (mean 19-fold) which was reduced significantly further (mean 190-fold) in vaccinated "reactor" calves. This study shows TiKa culture equates well with qPCR and provides important evidence that accuracy in estimating viable MAP load using DNA tests alone may vary significantly between samples of mucosal and lymphatic origin.

  20. 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.

  1. Identification of single nucleotide polymorphisms in the bovine solute carrier family 11 member 1 (SLC11A1) gene and their association with infection by Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Ruiz-Larrañaga, O; Garrido, J M; Manzano, C; Iriondo, M; Molina, E; Gil, A; Koets, A P; Rutten, V P M G; Juste, R A; Estonba, A

    2010-04-01

    Johne's disease is a chronic enteritis caused by Mycobacterium avium ssp. paratuberculosis (MAP) that causes substantial financial losses for the cattle industry. Susceptibility to MAP infection is reported to be determined in part by genetic factors, so marker-assisted selection could help to obtain bovine populations that are increasingly resistant to MAP infection. Solute carrier family 11 member 1 (SLC11A1) was adjudged to be a potential candidate gene because of its role in innate immunity, its involvement in susceptibility to numerous intracellular infections, and its previous association with bovine MAP infection. The objectives of this study were to carry out an exhaustive process of discovery and compilation of polymorphisms in SLC11A1 gene, and to perform a population-based genetic association study to test its implication in susceptibility to MAP infection in cattle. In all, 57 single nucleotide polymorphisms (SNP) were detected, 25 of which are newly described in Bos taurus. Twenty-four SNP and two 3'-untranslated region polymorphisms, previously analyzed, were selected for a subsequent association study in 558 European Holstein-Friesian animals. The SNP c.1067C>G and c.1157-91A>T and a haplotype formed by these 2 SNP yielded significant association with susceptibility to MAP infection. The c.1067C>G is a nonsynonymous SNP that causes an amino acid change in codon 356 from proline to alanine (P356A) that could alter SLC11A1 protein function. This association study supports the involvement of SLC11A1 gene in susceptibility to MAP infection in cattle. Our results suggest that SNP c.1067C>G may be a potential causal variant, although functional studies are needed to assure this point.

  2. Zoonotic aspects of Mycobacterium bovis and Mycobacterium avium-intracellulare complex (MAC).

    PubMed

    Biet, Franck; Boschiroli, Maria Laura; Thorel, Marie Françoise; Guilloteau, Laurence A

    2005-01-01

    Pathogens that are transmitted between the environment, wildlife, livestock and humans represent major challenges for the protection of human and domestic animal health, the economic sustainability of agriculture, and the conservation of wildlife. Among such pathogens, the genus Mycobacterium is well represented by M. bovis, the etiological agent of bovine tuberculosis, M. avium ssp. paratuberculosis (Map) the etiological agent of Johne disease, M. avium ssp. avium (Maa) and in a few common cases by other emergent environmental mycobacteria. Epidemiologic surveys performed in Europe, North America and New Zealand have demonstrated the existence and importance of environmental and wildlife reservoirs of mycobacterial infections that limit the attempts of disease control programmes. The aim of this review is to examine the zoonotic aspects of mycobacteria transmitted from the environment and wildlife. This work is focused on the species of two main groups of mycobacteria classified as important pathogens for humans and animals: first, M. bovis, the causative agent of bovine tuberculosis, which belongs to the M. tuberculosis complex and has a broad host range including wildlife, captive wildlife, domestic livestock, non-human primates and humans; the second group examined, is the M. avium-intracellulare complex (MAC) which includes M. avium ssp. avium causing major health problems in AIDS patients and M. avium ssp. paratuberculosis the etiological agent of Johne disease in cattle and identified in patients with Crohn disease. MAC agents, in addition to a broad host range, are environmental mycobacteria found in numerous biotopes including the soil, water, aerosols, protozoa, deep litter and fresh tropical vegetation. This review examines the possible reservoirs of these pathogens in the environment and in wildlife, their role as sources of infection in humans and animals and their health impact on humans. The possibilities of control and management programmes for

  3. Multilocus enzyme electrophoresis analysis of the Mycobacterium avium complex and other mycobacteria.

    PubMed Central

    Wasem, C F; McCarthy, C M; Murray, L W

    1991-01-01

    Multilocus enzyme electrophoresis analysis was used to evaluate the Mycobacterium avium complex (MAC), M. paratuberculosis, and nine other mycobacterial species. The average number of alleles per locus was 2.8 for the 35 MAC and 2 M. paratuberculosis strains which represented 24 electrophoretic types (ETs) and two distinct groups. The M. avium group was resolved into 17 ETs and contained the M. paratuberculosis ET. The M. intracellulare group consisted of six ETs. There was complete agreement between Gen-Probe identification and group placement by multilocus enzyme electrophoresis. The mean genetic diversity per locus for the 24 MAC ETs was 0.38. This procedure subdivided some serovars and, if implemented, should prove to be a powerful epidemiologic tool for the MAC. Eleven additional ETs were formed after the data for the other mycobacterial species were pooled with those for the MAC. PMID:2007633

  4. Mycobacterium paratuberculosis as a cause of Crohn's disease.

    PubMed

    McNees, Adrienne L; Markesich, Diane; Zayyani, Najah R; Graham, David Y

    2015-01-01

    Crohn's disease is a chronic inflammatory bowel disease of unknown cause, affecting approximately 1.4 million North American people. Due to the similarities between Crohn's disease and Johne's disease, a chronic enteritis in ruminant animals caused by Mycobacterium avium paratuberculosis (MAP) infection, MAP has long been considered to be a potential cause of Crohn's disease. MAP is an obligate intracellular pathogen that cannot replicate outside of animal hosts. MAP is widespread in dairy cattle and because of environmental contamination and resistance to pasteurization and chlorination, humans are frequently exposed through contamination of food and water. MAP can be cultured from the peripheral mononuclear cells from 50-100% of patients with Crohn's disease, and less frequently from healthy individuals. Association does not prove causation. We discuss the current data regarding MAP as a potential cause of Crohn's disease and outline what data will be required to firmly prove or disprove the hypothesis.

  5. Mycobacterium paratuberculosis as a cause of Crohn's disease

    PubMed Central

    McNees, Adrienne L.; Markesich, Diane; Zayyani, Najah R.; Graham, David Y.

    2016-01-01

    SUMMARY Crohn's disease is a chronic inflammatory bowel disease of unknown cause, affecting approximately 1.4 million North American people. Due to the similarities between Crohn's disease and Johne’s disease, a chronic enteritis in ruminant animals caused by Mycobacterium avium paratuberculosis (MAP) infection, MAP has long been considered to be a potential cause of Crohn's disease. MAP is an obligate intracellular pathogen that cannot replicate outside of animal hosts. MAP is widespread in dairy cattle and because of environmental contamination and resistance to pasteurization and chlorination, humans are frequently exposed through contamination of food and water. MAP can be cultured from the peripheral mononuclear cells from 50 to 100% of patients with Crohn's disease, and less frequently from healthy individuals. Association does not prove causation. We discuss the current data regarding MAP as a potential cause of Crohn's disease and outline what data will be required to firmly prove or disprove the hypothesis. PMID:26474349

  6. Disseminated Mycobacterium avium infection in a cat

    PubMed Central

    Barry, Maureen; Taylor, Judith; Woods, Paul

    2002-01-01

    A domestic shorthair cat was presented for lethargy and ataxia. Clinical findings included an abdominal mass, lumbosacral pain, ataxia. Aspirates from the liver and lymph nodes revealed intracellular, negative-staining rods. Treatment for presumptive mycobacterium infection was unsuccessful and the cat was euthanized. Disseminated Mycobacterium avium was confirmed on culture. PMID:12001504

  7. 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...

  8. Improvement of sensitivity for Mycobacterium avium subsp. paratuberculosis (MAP) detection in bovine fecal samples by specific duplex F57/IC real-time and conventional IS900 PCRs after solid culture enrichment.

    PubMed

    Fawzy, Ahmad; Eisenberg, Tobias; El-Sayed, Amr; Zschöck, Michael

    2015-04-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease in ruminants and a probable pathogen of Crohn's disease in humans. Accurate, cost-effective, and time-relevant diagnostics are the basis for efficient control programs. This study was conducted as an attempt to re-evaluate MAP detection improvement by coupling solid media enrichment to a more specific IS900 conventional PCR and a very specific F57/IC real-time PCR. In a spiking experiment, we investigated the improvement of molecular-based MAP detection in feces after a culture-based enrichment step into Herrold's egg yolk media with mycobactin J (HEYM-MJ) for different time intervals, when compared to traditional culture. Detection limit of culture was 0.33 × 10(4) bacteria × g(-1) (33 CFU g(-1)), while that of IS900 PCR when coupled with an enrichment step for 2, 4, and 6 weeks was 0.33 × 10(5) (0.33 × 10(3) CFU g(-1)), 0.33 × 10(4) (33 CFU g(-1)), and 33 (>3.3 CFU g(-1)) bacteria × g(-1), respectively. Whereas the detection limits of F57/IC real-time PCR after the enrichment step for the same time intervals were 0.33 × 10(5) (0.33 × 10(3) CFU g(-1)), 0.33 × 10(3) (3.3 CFU g(-1)), and 33 (>3.3 CFU g(-1)) bacteria × g(-1), respectively. Altogether, enrichment of bovine fecal samples into solid media increased the sensitivity of specific molecular detection of MAP using IS900 conventional PCR and duplex F57/IC real-time PCR and offers an expedited and accurate alternative for MAP detection in bovine feces. Validation of these results is further recommended using field bovine fecal samples.

  9. 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

    Shaughnessy, Ronan G; Farrell, Damien; 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

  10. 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

  11. 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...

  12. 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 ...

  13. Iron-chelating compound from Mycobacterium avium.

    PubMed Central

    McCullough, W G; Merkal, R S

    1976-01-01

    A iron-chelating monohydroxamate was isolated from cultures of Mycobacterium avium grown on an iron-limiting medium. The hydroxyamate metabolite was characterized by chemical degradation and spectral measurements as L-alpha-asparaginyl-L-alpha-(N-hydroxy)-asparagine. PMID:185194

  14. Comparison of two DNA extractions and nested PCR, real-time PCR, a new commercial PCR assay, and bacterial culture for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces.

    PubMed

    Christopher-Hennings, Jane; Dammen, Matthew A; Weeks, Shelleen R; Epperson, William B; Singh, Shri N; Steinlicht, Gina L; Fang, Ying; Skaare, Jessica L; Larsen, Jill L; Payeur, Janet B; Nelson, Eric A

    2003-03-01

    In this study, 5 combinations of 2 DNA extractions and 3 polymerase chain reaction (PCR) techniques were compared with culture for the detection of Mycobacterium paratuberculosis directly from bovine feces. These combinations included a new commercial extraction technique combined with a commercial PCR/Southern blot technique, nested PCR (nPCR), or real-time PCR, and a university-developed extraction combined with nPCR or real-time PCR. Four of the 5 combinations had statistically similar sensitivities between 93% and 100% and specificity between 95% and 100%, when compared with culture results from 63 bovine fecal samples. These results indicated that using a commercial extraction with a commercial PCR/Southern blot, nPCR, or real-time PCR, or a university-developed extraction with real-time PCR would result in similar sensitivities to culture for the identification of M. paratuberculosis from bovine feces and are valid alternatives to culture.

  15. Efficient Differentiation of Mycobacterium avium Complex Species and Subspecies by Use of Five-Target Multiplex PCR▿

    PubMed Central

    Shin, Sung Jae; Lee, Byung Soo; Koh, Won-Jung; Manning, Elizabeth J. B.; Anklam, Kelly; Sreevatsan, Srinand; Lambrecht, Randall S.; Collins, Michael T.

    2010-01-01

    Infections caused by the Mycobacterium avium complex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all Mycobacterium species, a chromosomal target called DT1 that is unique to M. avium subsp. avium serotypes 2 and 3, to M. avium subsp. silvaticum, and to M. intracellulare, and three insertion sequences, IS900, IS901, and IS1311. The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. hominissuis. Analytical sensitivity was 10 fg of M. avium subsp. paratuberculosis genomic DNA, 5 to 10 fg of M. avium subsp. avium genomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media. PMID:20810779

  16. Mycobacterium paratuberculosis heat shock protein 70 as a tool in control of paratuberculosis.

    PubMed

    Langelaar, M; Koets, A; Müller, K; van Eden, W; Noordhuizen, J; Howard, C; Hope, J; Rutten, V

    2002-09-10

    Paratuberculosis in cattle is a chronic intestinal disease in which a distinctive cellular reactivity of a Th1-type preceeds the phase in which antibody titers are easily detectable and the animal becomes clinically ill. During infection with Mycobacterium avium ssp. paratuberculosis (M.a.p), a decrease in CD4 T-helper cells has been observed in the clinical phase. Our ultimate aim is to elicit a cytotoxic reaction against infected macrophages, using recombinant Hsp70 (rHsp70) of M.a.p. as a tool to shuttle antigen into the MHC class I antigen presentation pathway. To investigate the mechanism of rHsp70 as a carrier for antigen into the cell, we studied the interaction between APC and Fitc-labelled rHsp70, using FACS analysis and confocal microscopy. Interaction of rHsp70 with the cell surface of bovine APC, presumably via a receptor, was shown on monocytes, monocyte derived macrophages and dendritic cell (DC). The interaction is detectable on the complete population of freshly derived monocytes, although peak intensity of fluorescence is lower on these cells than on macrophages and DCs. DCs show interaction on a high percentage of the cells, with high intensity, while in the case of macrophages only a subpopulation interacts with rHsp70. Efficient uptake of rHsp70 as compared to OVA is shown. Preincubation of DC with unlabelled rHsp70 leads to a decreased interaction with rHsp70-FITC. DC interacting with rHsp70 in addition showed high expression of MHC I, MHC II, Myd-1 (CD172a) and CD40. Further research will focus on loading of the rHsp70 with M.a.p. antigen for presentation in MHC class I.

  17. 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...

  18. Comparative genome analyses of Mycobacterium avium reveal genomic features of its subspecies and strains that cause progression of pulmonary disease

    PubMed Central

    Uchiya, Kei-ichi; Tomida, Shuta; Nakagawa, Taku; Asahi, Shoki; Nikai, Toshiaki; Ogawa, Kenji

    2017-01-01

    Pulmonary disease caused by nontuberculous mycobacteria (NTM) is increasing worldwide. Mycobacterium avium is the most clinically significant NTM species in humans and animals, and comprises four subspecies: M. avium subsp. avium (MAA), M. avium subsp. silvaticum (MAS), M. avium subsp. paratuberculosis (MAP), and M. avium subsp. hominissuis (MAH). To improve our understanding of the genetic landscape and diversity of M. avium and its role in disease, we performed a comparative genome analysis of 79 M. avium strains. Our analysis demonstrated that MAH is an open pan-genome species. Phylogenetic analysis based on single nucleotide variants showed that MAH had the highest degree of sequence variability among the subspecies, and MAH strains isolated in Japan and those isolated abroad possessed distinct phylogenetic features. Furthermore, MAP strains, MAS and MAA strains isolated from birds, and many MAH strains that cause the progression of pulmonary disease were grouped in each specific cluster. Comparative genome analysis revealed the presence of genetic elements specific to each lineage, which are thought to be acquired via horizontal gene transfer during the evolutionary process, and identified potential genetic determinants accounting for the pathogenic and host range characteristics of M. avium. PMID:28045086

  19. Isolation of Mycobacterium paratuberculosis from Milk by Immunomagnetic Separation

    PubMed Central

    Grant, Irene R.; Ball, Hywel J.; Rowe, Michael T.

    1998-01-01

    An immunomagnetic separation (IMS) technique was developed to facilitate selective isolation of Mycobacterium paratuberculosis cells from milk. Rabbit polyclonal antibodies against radiation-killed intact M. paratuberculosis cells were produced and used to coat sheep anti-rabbit immunoglobulin G (IgG) type M-280 Dynabeads. The rabbit anti-M. paratuberculosis IgG-coated beads (IMB) reacted strongly with laboratory strains of M. paratuberculosis as determined by slide agglutination, and microscopic examination confirmed that M. paratuberculosis cells attached to the IMB. The IMB were found to have a maximum binding capacity of 104 to 105 CFU of M. paratuberculosis. Studies showed that IMS selectively recovered M. paratuberculosis from inoculated milk containing as few as 10 CFU of M. paratuberculosis per ml when 10 μl of IMB (ca. 106 beads) was added to 1 ml of milk and the preparation was incubated for 30 min at room temperature with gentle agitation. Larger volumes of milk (10 and 50 ml) were centrifuged and resuspended in 1 ml of phosphate-buffered saline–0.05% Tween 20 prior to IMS in order to increase the sensitivity of the method. Currently, primary isolation of M. paratuberculosis from a milk sample relies on chemical decontamination, followed by culturing on Herrold’s egg yolk medium, which must be incubated at 37°C for up to 18 weeks. The potential value of our IMS method is as an aid for rapid detection of M. paratuberculosis in milk when it is used in conjunction with end point detection methods, such as IS900 PCR or an enzyme-linked immunosorbent assay. PMID:9726853

  20. 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

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

    SciTech Connect

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

    1987-12-01

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

  2. Mycobacterium Avium subsp. paratuberculosis isolates induce in vitro granuloma formation and show successful survival phenotype, common anti-inflammatory and antiapoptotic responses within ovine macrophages regardless of genotype or host of origin.

    PubMed

    Abendaño, Naiara; Tyukalova, Lyudmila; Barandika, Jesse F; Balseiro, Ana; Sevilla, Iker A; Garrido, Joseba M; Juste, Ramon A; Alonso-Hearn, Marta

    2014-01-01

    The analysis of the early macrophage responses, including bacterial growth within macrophages, represents a powerful tool to characterize the virulence of clinical isolates of Mycobcaterium avium susbp. paratuberculosis (Map). The present study represents the first assessment of the intracellular behaviour in ovine monocyte-derived macrophages (MDMs) of Map isolates representing distinct genotypes (C, S and B), and isolated from cattle, sheep, goat, fallow deer, deer, and wild boar. Intracellular growth and survival of the selected isolates in ovine MDMs was assessed by quantification of CFUs inside of the host cells at 2 h p.i. (day 0) and 7 d p. i. using an automatic liquid culture system (Bactec MGIT 960). Variations in bacterial counts over 7 days from the baseline were small, in a range between 1.63 to 1.05-fold. After 7 d of infection, variations in the estimated log10 CFUs between all the tested isolates were not statistically significant. In addition, ovine MDMs exhibited enhanced anti-inflammatory, antiapoptotic and antidestructive responses when infected with two ovine isolates of distinct genotype (C and S) or with two C-type isolates from distinct hosts (cattle and sheep); which correlated with the successful survival of these isolates within ovine MDMs. A second objective was to study, based on an in vitro granuloma model, latter stages of the infection by investigating the capacity of two Map isolates from cattle and sheep to trigger formation of microgranulomas. Upon 10 d p.i., both Map isolates were able to induce the formation of granulomas comparable to the granulomas observed in clinical specimens with respect to the cellular components involved. In summary, our results demonstrated that Map isolates from cattle, sheep, goats, deer, fallow-deer and wild boar were able not only to initiate but also to establish a successful infection in ovine macrophages regardless of genotype.

  3. Inactivation of Mycobacterium avium with monochloramine.

    PubMed

    Luh, Jeanne; Tong, Ning; Raskin, Lutgarde; Mariñas, Benito J

    2008-11-01

    Batch experiments were performed to study the inactivation kinetics of Mycobacterium avium in the presence of monochloramine at 5-30 degrees C, pH 6-10, and 0.30-42.3 mg Cl2/ L. For each temperature and pH investigated, limiting high and low inactivation rates were observed for high and low disinfectant concentrations, respectively, within the range investigated. The rate of inactivation transitioned from high to low over a relatively narrow range of intermediate monochloramine concentrations. The observed temperature dependence of inactivation was consistent with an Arrhenius expression with activation energies of 58.0 and 71.7 kJ/mol for the high and low concentration ranges, respectively. The rate of inactivation increased with decreasing pH, consistent with trends reported for the reaction of monochloramine with protein thiols. Experiments performed at pH approximately 3.5 showed that dichloramine was a weaker disinfectant than monochloramine, and that its contribution to the overall inactivation of M. avium with combined chlorine was negligible at pH 6-10. A kinetic model incorporating disinfectant concentration, temperature, and pH effects was used to illustrate that monochloramine efficiency to inactivate M. avium in water could vary broadly from adequate (e.g., 99.9% inactivation efficiency in 32 min at 5 mg Cl2/L, pH 6, 30 degrees C) to impractical (e.g., 99.9% inactivation efficiency in 9 d at 1 mg Cl2/L, pH 9, 5 degrees C).

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

    PubMed Central

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

    2012-01-01

    The aim of the present study was to determine whether lipoarabinomannan (LAM), in combination with Freund's incomplete adjuvant (FIA), was able to improve cell-mediated and antibody-mediated immune responses against ovalbumin (OVA) in cattle. Twenty-three calves were assigned to four treatment groups, which were subcutaneously immunized with either OVA plus FIA, OVA plus FIA and LAM from Mycobacterium avium subsp avium, FIA plus LAM, or FIA alone. Lymphoproliferation, IFN-γ production and cell subpopulations on peripheral blood mononuclear cells before and 15 days after treatment were evaluated. Delayed hypersensitivity was evaluated on day 57. Specific humoral immune response was measured by ELISA. Inoculation with LAM induced higher levels of lymphoproliferation and IFN-γ production in response to ConA and OVA (P < 0.05). Specific antibody titers were similar in both OVA-immunized groups. Interestingly, our results showed that the use of LAM in vaccine preparations improved specific cell immune response evaluated by lymphoproliferation and IFN-γ production by at least 50 and 25%, respectively, in cattle without interfering with tuberculosis and paratuberculosis diagnosis. PMID:22286534

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

    PubMed

    Thakur, Aneesh; Riber, Ulla; Davis, William C; Jungersen, Gregers

    2013-10-01

    T cells, which encounter specific antigen (Ag), require additional signals to mount a functional immune response. Here, we demonstrate activation of signal 2, by anti-CD28 mAb (aCD28) and other costimulatory molecules (aCD49d, aCD5), and signal 3, by recombinant IL-12, enhance Ag-specific IFN-γ secretion by CD4, CD8, γδ T cells and NK cells. Age matched male jersey calves, experimentally infected with Mycobacterium avium subsp. paratuberculosis (MAP), were vaccinated with a cocktail of recombinant MAP proteins or left unvaccinated. Vaccine induced ex vivo recall responses were measured through Ag-specific IFN-γ production by ELISA and flow cytometry. There was a significant increase in production of IFN-γ by T cell subsets or NKp46+ cells cultured in the presence of Ag and aCD28/aCD49d. The increase was accompanied by an increase in the integrated median fluorescence intensity (iMFI) of activated T cells. Addition of rIL-12 induced a significant additive effect leading to a maximum increase in responder frequency of Ag-specific T cell subsets or NKp46+ cells with a heavy bias toward IFN-γ production by CD4 T cells. We provide the first description of using aCD28/aCD49d costimulation to potentiate an Ag-specific increase in the production of IFN-γ in bovine immunology. The study also shows the degree of signaling in T cells is regulated by the costimulatory environment.

  6. 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.

  7. 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

  8. 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).

  9. Mycobacterium avium Complex Cervical Lymphadenitis in an Immunocompetent Adult▿

    PubMed Central

    Christensen, Joshua B.; Koeppe, John

    2010-01-01

    Nontuberculosis mycobacterial cervical lymphadenitis is a relatively common disease in immunocompetent children but a rare disease in immunocompetent adults. We report the diagnosis and treatment of Mycobacterium avium complex cervical lymphadenitis in an adult female. Our evaluation of immune competence, including gamma interferon (IFN-γ) and interleukin-12 (IL-12) signaling, found no evidence of deficiency. PMID:20668140

  10. 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...

  11. Structure of a New Glycolipid from the Mycobacterium avium-Mycobacterium intracellulare Complex

    PubMed Central

    Watanabe, Motoko; Ohta, Akihiro; Sasaki, Shun-ichi; Minnikin, David E.

    1999-01-01

    From the lipid fraction of a freeze-dried cell mass of a strain of the Mycobacterium avium-Mycobacterium intracellulare complex, a new glycolipid was isolated and was characterized as 5-mycoloyl-α-arabinofuranosyl (1→1′)-glycerol, mainly on the basis of nuclear magnetic resonance spectroscopy studies. PMID:10094713

  12. Evaluation of Vitek MS for rapid classification of clinical isolates belonging to Mycobacterium avium complex.

    PubMed

    Boyle, Daniel P; Zembower, Teresa R; Qi, Chao

    2015-01-01

    We evaluated the ability of the Vitek MS system to classify clinical pulmonary Mycobacterium avium complex isolates compared to multilocus sequence analysis. Vitek MS accurately identified 55% of the isolates as M. avium and 18% as M. intracellulare, but misidentified 24 (27%) Mycobacterium chimaera isolates as Mycobacterium intracellulare.

  13. Whole chromosomal DNA probes for rapid identification of Mycobacterium tuberculosis and Mycobacterium avium complex.

    PubMed Central

    Roberts, M C; McMillan, C; Coyle, M B

    1987-01-01

    Whole chromosomal DNA probes were used to identify clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium complex, and Mycobacterium gordonae. The probe for M. tuberculosis was prepared from Mycobacterium bovis BCG, which has been shown to be closely related to M. tuberculosis. A probe for the M. avium complex was prepared from three strains representing each of the three DNA homology groups in the M. avium complex. The probes were used in dot blot assays to identify clinical isolates of mycobacteria. The dot blot test correctly identified 57 of the 61 (93%) cultures grown on solid media, and 100% of antibiotic-treated broth-grown cells were correctly identified. Identification by dot blot required a maximum of 48 h. When the probes were tested against 63 positive BACTEC (Johnston Laboratories, Inc., Towson, Md.) cultures of clinical specimens, 59% were correctly identified. However, of the 14 BACTEC cultures that had been treated with antibiotics before being lysed, 13 (93%) were correctly identified. PMID:3112180

  14. 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.

  15. 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.

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

    PubMed Central

    Viale, Mariana Noelia; Imperiale, Belén; Gioffre, Andrea Karina; Colombatti Olivieri, María Alejandra; Moyano, Roberto Damián; Morcillo, Nora; Santangelo, María de la Paz; Davis, William; Romano, María Isabel

    2014-01-01

    The lprG-p55 operon of Mycobacterium tuberculosis and Mycobacterium bovis is involved in the transport of toxic compounds. P55 is an efflux pump that provides resistance to several drugs, while LprG is a lipoprotein that modulates the host's immune response against mycobacteria. The knockout mutation of this operon severely reduces the replication of both mycobacterial species during infection in mice and increases susceptibility to toxic compounds. In order to gain insight into the function of LprG in the Mycobacterium avium complex, in this study, we assayed the effect of the deletion of lprG gene in the D4ER strain of Mycobacterium avium subsp. avium. The replacement of lprG gene with a hygromycin cassette caused a polar effect on the expression of p55. Also, a twofold decrease in ethidium bromide susceptibility was observed and the resistance to the antibiotics rifampicin, amikacin, linezolid, and rifabutin was impaired in the mutant strain. In addition, the mutation decreased the virulence of the bacteria in macrophages in vitro and in a mice model in vivo. These findings clearly indicate that functional LprG and P55 are necessary for the correct transport of toxic compounds and for the survival of MAA in vitro and in vivo. PMID:24967408

  17. Chlorine, Chloramine, Chlorine Dioxide, and Ozone Susceptibility of Mycobacterium avium

    PubMed Central

    Taylor, Robert H.; Falkinham, Joseph O.; Norton, Cheryl D.; LeChevallier, Mark W.

    2000-01-01

    Environmental and patient isolates of Mycobacterium avium were resistant to chlorine, monochloramine, chlorine dioxide, and ozone. For chlorine, the product of the disinfectant concentration (in parts per million) and the time (in minutes) to 99.9% inactivation for five M. avium strains ranged from 51 to 204. Chlorine susceptibility of cells was the same in washed cultures containing aggregates and in reduced aggregate fractions lacking aggregates. Cells of the more slowly growing strains were more resistant to chlorine than were cells of the more rapidly growing strains. Water-grown cells were 10-fold more resistant than medium-grown cells. Disinfectant resistance may be one factor promoting the persistence of M. avium in drinking water. PMID:10742264

  18. Mechanisms involved in the intrinsic isoniazid resistance of Mycobacterium avium.

    PubMed

    Mdluli, K; Swanson, J; Fischer, E; Lee, R E; Barry, C E

    1998-03-01

    Isoniazid (INH), which acts by inhibiting mycolic acid biosynthesis, is very potent against the tuberculous mycobacteria. It is about 100-fold less effective against Mycobacterium avium. This difference has often been attributed to a decreased permeability of the cell wall. We measured the rate of conversion of radiolabelled INH to 4-pyridylmethanol by whole cells and cell-free extracts and estimated the permeability barrier imposed by the cell wall to INH influx in Mycobacterium tuberculosis and M. avium. There was no significant difference in the relative permeability to INH between these two species. However, the total conversion rate in M. tuberculosis was found to be four times greater. Examination of in vitro-generated mutants revealed that the major resistance mechanism for both species is loss of the catalase-peroxidase KatG. Analysis of lipid and protein biosynthetic profiles demonstrated that the molecular target of activated INH was identical for both species. M. avium, however, formed colonies at INH concentrations inhibitory for mycolic acid biosynthesis. These mycolate-deficient M. avium exhibited altered colony morphologies, modified cell wall ultrastructure and were 10-fold more sensitive to treatment with hydrophobic antibiotics, such as rifampin. These findings may significantly impact the design of new therapeutic regimens for the treatment of infections with atypical mycobacteria.

  19. Mycobacterium avium-intracellulare: a rare cause of subacromial bursitis.

    PubMed

    Sinha, Raj; Tuckett, John; Hide, Geoff; Dildey, Petra; Karsandas, Alvin

    2015-01-01

    Septic subacromial bursitis is an uncommon disorder with only a few reported cases in the literature. The most common causative organism is Staphylococcus aureus. We report the case of a 61-year-old female with a septic subacromial bursitis where the causative organism was found to be Mycobacterium avium-intracellulare (MAI). The diagnosis was only made following a biopsy, and we use this case to highlight the importance of recognising the need to consider a biopsy and aspiration in atypical situations.

  20. 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.

  1. 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...

  2. 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...

  3. 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.

  4. 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...

  5. Intramacrophage growth of Mycobacterium avium during infection of mice.

    PubMed Central

    Frehel, C; de Chastellier, C; Offredo, C; Berche, P

    1991-01-01

    Growth of the virulent Mycobacterium avium strain TMC 724 in host tissues during persistent infection of mice was studied. Following intravenous infection of C57BL/6 mice, the kinetics of bacterial growth was biphasic in the spleen and liver, with a significant reduction of the multiplication rate after day 21 to 28 of infection. An electron-microscopic study of the liver and spleen of infected mice showed that the bacteria were strictly intracellular. They were observed within inflammatory macrophages populating granulomas disseminated in host tissues. The bacteria were confined to the phagosome compartment, and they were encapsulated. Phagosome-lysosome fusions were encountered, but the bacteria showed no visible signs of degradation and continued to multiply. These results are the first in vivo evidence that virulent M. avium multiplies exclusively intracellularly and that encapsulated bacteria resist the microbicidal mechanisms of macrophages inside the phagosomal compartment. Images PMID:2037382

  6. Identification of members of Mycobacterium avium species by Accu-Probes, serotyping, and single IS900, IS901, IS1245 and IS901-flanking region PCR with internal standards.

    PubMed

    Bartos, Milan; Hlozek, Pavel; Svastova, Petra; Dvorska, Lenka; Bull, Tim; Matlova, Ludmila; Parmova, Ilona; Kuhn, Isolde; Stubbs, Janine; Moravkova, Monika; Kintr, Jaromir; Beran, Vladimir; Melicharek, Ivan; Ocepek, Matjaz; Pavlik, Ivo

    2006-03-01

    From Mycobacterium avium species Mycobacterium avium subsp. paratuberculosis (n=961), Mycobacterium a. avium (n=677), Mycobacterium a. silvaticum (n=5), and Mycobacterium a. hominissuis (n=1566) were examined, and from Mycobacterium tuberculosis complex M. tuberculosis (n=2), Mycobacterium bovis (n=13), M. bovis BCG (n=4), and Mycobacterium caprae (n=10) were examined. From other mycobacterial species Mycobacterium intracellulare (n=60) and atypical mycobacteria (n=256) including Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium scrofulaceum, Mycobacterium gastri and other species of conditionally pathogenic mycobacteria were analysed. The internal standard molecules corresponding to insertion sequences IS900, IS901, IS1245, and flanking region (FR300) of IS901 were produced by PCR of alfalfa genome segment and inserted into plasmid vector. The resulting recombinant plasmid molecules were used as internal standards in coamplification with a total of 4729 mycobacterial collection strains and field isolates between 1996 and 2003. The size differences between amplicons obtained from IS900 (258 bp), IS901 (1108 bp), IS1245 (427 bp), and FR300 (300 bp) and from corresponding internal standard molecules ISIS900 (591 bp), ISIS901 (1 336 bp), ISIS1245 (583 bp), and IS901 flanking region of 300 bp ISFR300 (488 bp), respectively, allowed easy discrimination. The internal amplicons were visible by naked aye on agarose gel when 10(1), 10(3), 10(2), and 10(2) molecules for ISIS900, ISIS901, ISIS1245, and ISFR300 were used in the PCR, respectively, when no bacterial DNA was added to the reaction. The system was tested to define the amount of internal standards that could be used in the PCR without affecting the amplification of the specific segment. Non-specific amplifications were observed in M. fortuitum with IS1245 PCR and mixed infections with M. a. avium and M. a. hominissuis from pigs and cattle were found. PCR results of typing were compared with serotyping

  7. In Vitro Susceptibility Testing of Bedaquiline against Mycobacterium avium Complex.

    PubMed

    Brown-Elliott, Barbara A; Philley, Julie V; Griffith, David E; Thakkar, Foram; Wallace, Richard J

    2017-02-01

    We performed bedaquiline broth microdilution susceptibility testing using Clinical and Laboratory Standards Institute (CLSI) guidelines on 103 respiratory isolates of Mycobacterium avium complex (MAC), including multidrug-resistant isolates. Approximately 90% of isolates had bedaquiline MICs of ≤0.008 μg/ml, and 102/103 isolates had MICs of ≤0.015 μg/ml. Bedaquiline has excellent potential for use in patients with MAC infections, although for reasons of its metabolism by the cytochrome P450 system, it should not be given with rifampin.

  8. Mycobacteriosis in ostriches (Struthio camelus) due to infection with Mycobacterium bovis and Mycobacterium avium complex.

    PubMed

    Kelly, Pamela; Jahns, Hanne; Power, Eugene; Bainbridge, John; Kenny, Kevin; Corpa, Juan M; Cassidy, Joseph P; Callanan, John J

    2013-12-01

    Avian tuberculosis rarely affects ratites compared to other bird species and is typically caused by Mycobacterium avium species. This study describes the pathological and microbiological findings in three adult ostriches with mycobacteriosis, in one of which Mycobacterium bovis was isolated from the lesions. Post mortem examinations on ostriches from two different zoological collections in Ireland revealed multifocal caseous granulomas affecting the spleen and liver in all cases, with additional involvement of intestines in two cases. In one case, granulomas were present within the pharynx, at the thoracic inlet and multifocally on the pleural surface. Acid-fast bacilli were observed in all lesions. Mycobacterium sp. of the M. avium complex was isolated from the intestinal lesions in the two cases with intestinal involvement, and M. bovis sp. oligotype SB0140 was cultured from the liver of the third ostrich. This represents the first reported case of M. bovis infection in an ostrich. Avian tuberculosis due to M. bovis is rare and to date has been reported in only parrots and experimentally inoculated birds. Mycobacterium bovis needs to be considered as a possible cause of tuberculosis in ostriches because the lesions are similar to those observed with M. avium complex infection.

  9. Clarithromycin-ciprofloxacin-amikacin for therapy of Mycobacterium avium-Mycobacterium intracellulare bacteremia in patients with AIDS.

    PubMed Central

    de Lalla, F; Maserati, R; Scarpellini, P; Marone, P; Nicolin, R; Caccamo, F; Rigoli, R

    1992-01-01

    A combination of clarithromycin, ciprofloxacin, and amikacin for the treatment of Mycobacterium avium-Mycobacterium intracellulare bacteremia was evaluated in 12 AIDS patients. Mycobacteremia cleared in all patients by 2 to 8 weeks of treatment, and symptoms resolved. Four patients died; all had negative blood cultures until death, and disseminated M. avium-M. intracellulare complex infection was not considered the primary cause of death. PMID:1387303

  10. 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...

  11. 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...

  12. 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...

  13. REAL-TIME QUANTITATIVE PCR DETECTION OF MYCOBACTERIUM AVIUM COMPLEX ORGANISMS IN DRINKING WATER

    EPA Science Inventory

    The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and others. MAC are listed on the U.S. Environmental Protection Agency's Contaminant Candidate List (CCL) due to their association with human disease and occurrence in public drinkin...

  14. Detection of quantification of Mycobacterium avium complex organisms in drinking water

    EPA Science Inventory

    The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and others. MAC are listed on the U. S. Environmental Protection Agency’s Contaminant Candidate List 2 (CCL2) due to their association with human disease and occurrence in public dr...

  15. 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...

  16. Infection by Mycobacterium avium intracellulare in AIDS: endoscopic duodenal appearance mimicking Whipple's disease.

    PubMed

    Vázquez-Iglesias, J L; Yañez, J; Durana, J; Arnal, F

    1988-09-01

    We report the case of a 24-year-old woman who presented with diarrhea, weight loss and abdominal lymph node enlargement. A diagnosis of infection by Mycobacterium avium intracellulare with a clinical picture similar to Whipple's disease was established. The endoscopic study of the duodenum revealed multiple yellow nodules that became confluent in the second portion, entirely replacing the normal mucosa. These endoscopic findings have not been described previously in intestinal infection by Mycobacterium avium intracellulare.

  17. 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.

  18. 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.

  19. Diversity of Mycobacterium avium subsp. hominissuis mycobacteria causing lymphadenitis, France.

    PubMed

    Despierres, L; Cohen-Bacrie, S; Richet, H; Drancourt, M

    2012-07-01

    The knowledge of Mycobacterium avium complex (MAC) genotypes responsible for lymphadenitis is limited. We retrospectively characterized all of the MAC isolates made in our laboratory in the last 18 years by sequence-based identification and genotyping, and compared the clinical and laboratory data for lymphadenitis-associated and non-lymphadenitis-associated MAC isolates. Of 67 MAC-infected patients, 25 lymphadenitis patients were significantly younger than 42 non-lymphadenitis patients, while the male/female ratio did not significantly differ between the two groups. Cervical topography found in 76.5% of lymphadenitis patients was significantly more frequent in non-immunocompromised patients (p=0.04). M. avium subsp. hominissuis was identified in 53 patients (24 lymphadenitis, 29 non-lymphadenitis), M. colombiense in six patients (five non-lymphadenitis, one lymphadenitis), M. intracellulare in four non-lymphadenitis patients, and M. chimaera in three non-lymphadenitis patients, while negative controls remained negative. M. hominissuis was significantly associated with lymphadenitis (p=0.03). M. hominissuis isolates yielded 15 genotypes in 29 non-lymphadenitis isolates (molecular diversity, 0.622) versus 11 genotypes in 24 lymphadenitis isolates (molecular diversity, 0.578), demonstrating a non-significant lower diversity of M. hominissuis isolates cultured from lymphadenitis. The genotypes did not correlate with the clinical features. These data suggest the presence of several environmental reservoirs for M. hominissuis causing lymphadenitis in France.

  20. Preservation of monocyte effector functions against Mycobacterium avium-M. intracellulare in patients with AIDS.

    PubMed Central

    Johnson, J L; Shiratsuchi, H; Toba, H; Ellner, J J

    1991-01-01

    Mycobacterium avium-M. intracellulare is a frequent cause of late disseminated infection in patients with AIDS. The ability of human peripheral blood monocytes to phagocytose and kill M. avium was examined in an in vitro model. Monocytes were obtained from 13 healthy volunteers and 11 patients with AIDS, three of whom had documented disseminated M. avium infection. Monocytes were precultured for 2 days before infection with two AIDS-associated and two non-AIDS-associated strains of M. avium. Uptake of M. avium as measured by counting intracellular acid-fast bacilli did not differ among healthy subjects, patients with AIDS, or patients with AIDS and previously documented disseminated M. avium infection. Intracellular growth of M. avium was examined by a CFU assay of cell lysates from M. avium-infected monocytes after 0, 4, and 7 days of culture. Intracellular growth inhibition of M. avium at 7 days after infection was comparable between patients with AIDS and healthy donors for all M. avium strains tested. The effects of the addition of recombinant gamma interferon on M. avium uptake and intracellular growth in monocytes also were studied. Pretreatment of monocytes with gamma interferon prior to infection suppressed monocyte phagocytosis of M. avium. Continuously coculturing of monocytes with gamma interferon after infection augmented killing of M. avium among both patients with AIDS and healthy controls for three of the four strains of M. avium tested. The magnitude of this effect, however, was variable from donor to donor and strain to strain. No significant differences were noted between the growth-inhibiting abilities of gamma-interferon-treated monocytes obtained from healthy volunteers and those obtained from patients with AIDS. PMID:1910011