Intracellular fate of Mycobacterium avium subspecies paratuberculosis in monocytes from normal and infected, interferon-responsive cows as determined by a radiometric method.

The ability of Mycobacterium avium subsp. paratuberculosis to survive in bovine monocytes was studied using radiometric (BACTEC) culture, standard plate counting and microscopic counting of acid-fast stained monocyte monolayers. Results of microscopic counts sharply contrasted with results of viable counts determined both by plate counting and radiometric counting. We observed an early phase (the first 6 d after in vitro infection) of intracellular bacillary growth, followed by a later phase of mycobacteriostasis or killing (up to 12 d after in vitro infection) in monocytes from non-infected cows. The data suggest that multiplication and death of M. avium subsp. paratuberculosis occur simultaneously in bovine monocytes infected in vitro. Using the BACTEC method, we compared the ability of bovine monocytes from normal cows and cows infected with M. avium subsp. paratuberculosis and showing evidence of a strong Thl-like cellular immune response to ingest and inhibit the intracellular growth of M. avium subsp. paratuberculosis. There was a trend toward greater phagocytosis and faster killing of Mycobacterium avium subsp. paratuberculosis by monocytes from the infected, immune responder cows. However, the observed numbers of viable M. avium subsp. paratuberculosis at each time after monocyte infection were not significantly different between normal and infected cows.     

ATP release by infected bovine monocytes increases the intracellular survival of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease, a chronic intestinal infection in ruminants. Adenosine 5'-Triphosphate (ATP) has been reported to induce killing of several Mycobacterium species in human and murine macrophages. We investigated whether ATP secreted from M. avium subsp. paratuberculosis-infected bovine monocytes affects intracellular survival of the bacilli. Bovine monocytes constitutively secreted ATP during an 8-day incubation period in vitro; however, M. avium subsp. paratuberculosis infection did not enhance ATP release. Removal of extracellular ATP by the addition of apyrase increased the viability of infected monocytes, but surprisingly decreased the number of viable intracellular bacilli. In contrast to previous reports, addition of extracellular ATP (1mM) increased intracellular survival of M. avium subsp. paratuberculosis in bovine monocytes. Neither apyrase nor ATP altered production of reactive oxygen intermediates (ROI) or reactive nitrogen intermediates (RNI) by bovine monocytes. These results suggest that ATP release from infected bovine monocytes improves, rather than decreases, the intracellular survival of M. avium subsp. paratuberculosis.     

Comparison of two enzyme-linked immunosorbent assays for serologic diagnosis of paratuberculosis (Johne's disease) in cattle using different subspecies strains of Mycobacterium avium.

Serologic diagnosis of bovine paratuberculosis (Johne's disease) with currently available tests may give false-positive results due to cross-reactions with avian and bovine tuberculosis viruses and other infectious agents. Indirect enzyme-linked immunosorbent assays (ELISA) for detection of antibodies against paratuberculosis based on antigens from Mycobacterium avium subsp. avium (A-ELISA) and M. avium subsp. paratuberculosis (P-ELISA) were compared. Despite an expected higher specificity for M. a. paratuberculosis in the P-ELISA, the 2 antigens were equally suitable for demonstration of antibody to M. a. paratuberculosis in cattle. Receiver operating characteristic (ROC) curves was used to demonstrate the possible antigenic relationship. The area under the curve (AUC) was calculated for each of the 2 ROC curves. The AUC for the P-ELISA ROC curve was 0.9197, and the AUC for the A-ELISA ROC curve was 0.9149, demonstrating a negligible difference in efficiency of the 2 tests (z = 0.182).     

Development of ELISA to detect antibodies specific to Mycobacterium avium subsp. paratuberculosis with truncated 34 kDa proteins

To develop ELISA to detect antibodies specific to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), the carboxyl termini of the 34 kDa proteins of M. paratuberculosis and Mycobacterium avium subsp. avium (M. avium) were expressed in Escherichia coli expression system. Antibodies specific to M. paratuberculosis were detected with the truncated 34 kDa protein of M. paratuberculosis in ELISA after pre-absorption of serum samples with the truncated 34 kDa protein of M. avium. All the serum samples from cattle confirmed to be infected with M. paratuberculosis were positive and those from healthy cattle were negative in the present ELISA system. These results indicate that the established ELISA detects antibodies specific to M. paratuberculosis with high specificity and sensitivity and is an useful tool for the screening of Johne's disease.     

Development and application of quantitative polymerase chain reaction assay based on the ABI 7700 system (TaqMan) for detection and quantification of Mycobacterium avium subsp. paratuberculosis.

Numerous reports have described diagnostic methods based on the polymerase chain reaction (PCR) used to detect Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease. The result of conventional PCR tests has been only qualitative, either positive or negative; it does not present any quantitative information about the number of the agents in the specimen. A quantitative PCR method (IS900 TaqMan) was developed to measure the number of M. a. paratuberculosis organisms present in field and clinical samples. The sensitivity of IS900 TaqMan was 1 colony-forming unit (CFU) for M. a. paratuberculosis ATCC 19698. The specificity of the method was determined by testing 14 mycobacterial species (M. abscessus, M. asiaticum, M. avium subsp. avium, M. bovis, M. fortuitum subsp. fortuitum, M. intracellulare, M. kansasii, M. marinum, M. phlei, M. scrofulaceum, M. simiae, M. smegmatis, M. terrae, and M. ulcerans) and 9 nonmycobacterial species (Borrelia burgdorferi, Chlamydia psittaci, Ehrlichia canis, E. equi, E. risticii, Escherichia coli, E. coli O157:H7, Streptococcus equi, and S. zooepidemicus). Even at high cell numbers (10(5) CFU/reaction), most of the organisms tested negative for the IS900 insertion element except M. marinum and M. scrofulaceum. This finding for M. scrofulaceum was consistent with previous reports that several M. scrofulaceum-like isolates were positive for IS900. Those isolates had 71-79% homology with M. a. paratuberculosis in the region of IS900. When used in conjunction with the new liquid medium-based ESP culture system II for bovine clinical fecal samples, IS900 TaqMan confirmed that the ESP II-positive samples contained 10(5)-10(6) CFU/ml of M. a. paratuberculosis. All of the 222 ESP II-positive and acid-fast bacilli-positive samples tested in this study were positive by IS900 TaqMan. IS900 TaqMan was also useful in the study of growth characteristics of 3 groups of M. a. paratuberculosis strains in bovine fecal samples from 3 shedding levels (heavy, medium, and low) based on cell numbers measured by Herrold egg yolk (HEY) agar culture. When cultured in ESP medium, M. a. paratuberculosis reached 10(5)-10(6) CFU/ml within 2 weeks for heavy shedders, 3-4 weeks for medium, and 6-8 weeks for low shedders. No significant growth was observed after up to 5 weeks of incubation for some of low shedders. No or extremely slow growth characteristic of low shedders might be a possible explanation for frequent false-negative results by HEY. The detection time was dependent on the inoculum size and the growth rate of M. a. paratuberculosis. Generation times were inversely proportional to the shedding level: 1-2 days for medium and heavy shedders and >4 days for low shedders. IS900 TaqMan could be a useful tool for determining viable cell counts by measuring changes in cell numbers over the incubation period.     

Antigenic relationship between Mycobacterium paratuberculosis and Mycobacterium avium

Four prototype strains of Mycobacterium paratuberculosis contained the type-specific glycopeptidolipid antigen of serovar 8 of the M avium complex. This glycolipid was distinguished by a 4,6-(1'-carboxyethylidene)-3-O-methyl-beta-D-glucopyranosyl terminal unit. Of 59 low-passage, field isolates of M paratuberculosis, 2 contained this antigen, and these 2 isolates were indistinguishable from M avium serovar 8. However, most M paratuberculosis isolates had no characteristic surface glycopeptidolipid. Seemingly, M paratuberculosis, long regarded as a single species and the causative agent of bovine paratuberculosis, is not a homogeneous taxon. Most isolates obtained from infected ruminants may be antigenically defective, variants of M avium and, thereby, more successful pathogens.     

Analysis of antigens in Mycobacterium paratubercuolsis.

Analysis of antigens in Mycobacterium paratuberculosis. Acta vet. scand. 1979, 20, 200–215. — Using crossed immunoelectrophoresis (GIE) and crossed line immunoelectrophoresis (GLIE), antigens from different strains and variants of Mycobacterium paratuberculosis were compared, and cross-reactions between 1 of these strains and Mycobacterium avium and BGG studied. In each of 4 bovine laboratory strains of M. paratuberculosis examined, altogether 44 different antigens were demonstrated. This is the largest number of antigens in M. paratuberculosis which has been described so far. No important difference in the antigenic structure of the strains was found. The 4 laboratory strains are being used routinely in the production of vaccine against Johne’s disease in Norway and Iceland. One of the aims of the present work was to investigate the antigenic relationship between these strains and the goat-pathogenic Norwegian and the Icelandic variant of M. paratuberculosis. Out of 44 different antigens demonstrated in the laboratory strains, 39 and 31 gave cross-reactions against the Norwegian and the Icelandic variant, respectively. This is in accordance with practical experience, as the results of vaccination against Johne’s disease, performed in Norway for many years, are very good.Twenty-seven and 24 cross-reacting antigens between M. paratuberculosis and strains of M. avium and BGG, respectively, were observed. This finding agrees with clinical observations.Another aim of the investigation was to identify species-specific antigens as regards M. paratuberculosis. One antigen showed a marked cross-reaction between the strains of M. paratuberculosis examined, but did not react with antisera against M. avium and BGG. Some other antigens showed partial specificity.The results obtained stress the complicated antigenic situation in mycobacteria which is of decisive significance as regards the diagnosis and classification of mycobacterial infections.     

HPLC does not differentiate Mycobacterium paratuberculosis from Mycobacterium avium

HPLC, which is gaining its place as identification tool in mycobacteriology laboratories, has been proposed to distinguish Mycobacterium paratuberculosis from Mycobacterium avium. We had reported no significant difference between M. avium and M. paratuberculosis reference strain ATCC 19698. Because of the advantages offered by such a method, we enlarged our observations to include more isolates of M. paratuberculosis. Within the double cluster of peaks obtained by both M. avium and M. paratuberculosis, we could not find a consistent difference typical of M. paratuberculosis. Therefore, the present study confirmed that M. avium and M. paratuberculosis could not be distinguished by HPLC, raising doubts of a straightforward use of HPLC to identify M. paratuberculosis.     

Bacteriological investigations about paratuberculosis in dairy herds in Switzerland

In Switzerland clinical bovine paratuberculosis is registered sporadically with on average seven outbreaks per year. Our present studies are aimed to investigate the prevalence of Mycobacterium avium ssp. paratuberculosis (MAP)-infections in the Swiss cattle population and, therefore methods to culture MAP from bovine feces as well as a commercially available ELISA to detect MAP-specific antibodies are evaluated by using fecal samples and blood sera from herds with cases of clinical paratuberculosis. A series of molecular methods i.e. PCR-coupled RFLP analysis of the IS1311-insertion element of M. avium, PCR-coupled RFLP-analysis of the mycobacterial rpoB-gene, and DNA analysis of the mycobacterial 16S rRNA gene are used to identify mycobacterial isolates grown from bovine feces. Up to now, MAP was detected by culture in 12 of 155 (7.7%) animals from herds with paratuberculosis. A rather striking result is the finding of atypical mycobacteria in feces of 75 cattle (48.3%). Among these isolates, M. avium ssp. avium, M. thermoresistibile, and M. hassiacum/M. buckleii have been identified so far.     

Heat-shock protein-specific T-cell responses in various stages of bovine paratuberculosis.

Bovine paratuberculosis is characterized by a chronic inflammation of the small intestine, caused by infection with Mycobacterium avium ssp. paratuberculosis. Research regarding diagnostic as well as immunopathogenic aspects of paratuberculosis are hampered by the lack of specific antigens. The aim of the present study was to evaluate the potential of mycobacterial heat-shock proteins, as specific antigens, to measure cell-mediated immune responses during various stages of the disease. In a cross-sectional study, peripheral blood mononuclear cells of 179 cows in different stages of M. avium ssp. paratuberculosis infection, vaccinated against paratuberculosis or noninfected, were used to evaluate lymphoproliferative responses to mycobacterial heat-shock protein of 70 kD (HSP70) and 65 kD (HSP65). In addition, lymphoproliferative responses were measured using purified protein derivate (PPD) preparations from M. avium ssp. paratuberculosis, M. avium and M. bovis as antigens. Responses to HSP70 were higher in the vaccinated animals and in asymptomatic animals that shed the organism in their faeces. Compared with these animals, responses were lower in cows with clinical signs of paratuberculosis. Mycobacterial HSP65 induced less prominent responses compared with HSP70, but showed a similar pattern with regard to the stages of disease. Vaccinated and shedding animals also showed the highest responses to PPD derived from M. avium ssp. paratuberculosis (PPD-P). Observations with short-term cell lines raised to PPD-P and to HSP70 indicated that the similarity between those two antigens was not due to the presence of HSP70 in PPD-P. In conclusion, our study indicated that, as for PPD antigens the mycobacterial heat-shock protein-specific cell-mediated immune responses decrease when comparing the asymptomatic stage to the clinical stage in bovine paratuberculosis. Furthermore, this study shows that HSP70, being a well-defined antigen in comparison with PPD antigens, can be used to monitor cell-mediated immune responses in studies regarding the immunopathogenesis of bovine paratuberculosis.     

Antigenic profiles of recombinant proteins from Mycobacterium avium subsp. paratuberculosis in sheep with Johne's disease

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, antibody responses of infected and healthy sheep were evaluated using 18 purified recombinant proteins in an ELISA-based format for the serodiagnosis of ovine paratuberculosis. These selected recombinant proteins represent heat shock proteins, hypothetical proteins and cell surface proteins of M. avium subsp. paratuberculosis. Whereas, Map0862 (a gene uniquely present in M. avium subspecies paratuberculosis) and Map3786 encoded protein solicited the strongest antibody response in infected sheep. The protein encoded by Map2116c showed the weakest antibody response among the animals tested. Although none of the recombinant proteins detected all 11 infected sheep singly, antibodies to Map0862 were detected in 9 of 11 (81%) infected sheep. Furthermore, ovine responses to these selected antigens were assessed temporally over the course of 1 year during which we found a spiking effect rather than an incremental increase of antibody reactivity. This study evaluated multiple M. avium subsp. paratuberculosis recombinant proteins in an ELISA-based format for sheep.     

A critical role of interleukin-10 in the response of bovine macrophages to infection by Mycobacterium avium subsp paratuberculosis

OBJECTIVES: To evaluate the role of interleukin (IL)-10 in the inability of monocyte-derived bovine macrophages to kill Mycobacterium avium subsp paratuberculosis organisms in vitro. SAMPLE POPULATION: Monocytes were obtained from healthy adult Holstein dairy cows that had negative results when tested for infection with M avium subsp paratuberculosis. PROCEDURE: Monocyte-derived macrophages were incubated with M avium subsp paratuberculosis for 2, 6, 24, 72, or 96 hours with or without addition of saturating concentrations of a goat anti-human IL-10 that has been documented to neutralize bovine IL-10 activity. Variables assessed included ingestion and killing of M avium subsp paratuberculosis; expression of tumor necrosis factor (TNF)-alpha, IL-12, IL-8, major histocompatability (MHC) class II, vacuolar H+ ATPase, and B cell CLL/lymphoma 2 (BCL-2); production of nitric oxide; acidification of phagosomes; and apoptosis of macrophages. RESULTS: Neutralization of IL-10 enabled macrophages to kill 57% of M avium subsp paratuberculosis organisms within 96 hours. It also resulted in an increase in expression of TNF-alpha, IL-12, IL-8, MHC class II, and vacuolar H+ ATPase; decrease in expression of BCL-2; increase in acidification of phagosomes; apoptosis of macrophages; and production of nitric oxide. CONCLUSIONS AND CLINICAL RELEVANCE: The capacity of M avium subsp paratuberculosis to induce IL-10 expression may be a major determinant of virulence for this organism.     

Progress in molecular typing of Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (M. a. paratuberculosis) is responsible for paratuberculosis or Johne's disease, a chronic inflammation of the gastrointestinal tract in different animal species. Some studies have also established a link between this microorganism and Crohn's disease in humans. Although, M. a. paratuberculosis is a difficult microorganism to cultivate in the laboratory (occasionally is non-cultivable), a proper molecular characterization of M. a. paratuberculosis is necessary to better understand the epidemiology of the disease, and design strategies to eradicate it. In the present review, we compile and discuss the recent progress attained in the diagnostic and characterization of this pathogen.     

Carboxyl terminus of the 34 kDa protein of Mycobacterium paratuberculosis shares homologous B-cell epitopes with Mycobacterium avium and Mycobacterium intracellulare

Monoclonal antibodies (mAbs) against a recombinant carboxyl terminus of the 34 kDa protein of Mycobacterium paratuberculosis were produced in mice. Two of the mAbs cross-reacted with Mycobacterium avium and Mycobacterium intracellulare in both an elisa and immunoblot. The recombinant protein also reacted with polyclonal sera produced in rabbits against all three mycobacteria, indicating the presence of cross-reactive epitopes in the protein. To determine the reactivity of cattle sera against epitopes recognised by the mAbs, competition assays between bovine sera and the mAbs were carried out. Two mAbs were significantly inhibited by sera from cattle that were naturally infected with M paratuberculosis. The results indicate that epitopes on the carboxyl terminus of the 34 kDa protein common to M paratuberculosis, M avium and M intracellulare readily induce antibody production in naturally infected cattle. These epitopes reduce the diagnostic specificity of the carboxyl terminus of the 34 kDa protein, which was originally thought to contain only M paratuberculosis-specific epitopes.     

Mycobacterial diseases of deer

The most significant mycobacterial diseases of free-living, captive and farmed deer are bovine tuberculosis, caused by Mycobacterium bovis, Johne's disease (paratuberculosis), caused by Mycobacterium avium subsp paratuberculosis (basonym M. paratuberculosis), and avian tuberculosis, caused principally by M. avium subsp avium. The first case of M. bovis infection in farmed deer was identified in New Zealand in 1978. In 1983, a voluntary scheme was introduced in New Zealand to control tuberculosis in farmed deer, followed by a compulsory tuberculosis control scheme in 1990. The primary control measure is the slaughter of infected animals, detected by skin testing and blood testing, together with movement control and vector control. The number of infected deer herds peaked in the mid 1990s at over 160 herds, but by 30 June 2002 this had been reduced to 79 (1.45%), and to 67 (1.23%) by June 2003. Deer-to-deer transmission occurs, but the majority of herd breakdowns are believed to be from infected vectors. Factors likely to affect the susceptibility of deer include age, environment, population density, exposure and genetics. Avian tuberculosis occasionally causes clinical disease in wild, captive and farmed deer in New Zealand and overseas. Mycobacterium intracellulare, and subspecies of M. avium other than M. paratuberculosis, are widespread throughout New Zealand and are thought to be largely responsible for the high level of sensitisation to avian purified protein derivative (PPD), which is used for comparison purposes in tuberculosis skin testing of deer in this country. Infections with these organisms are usually subclinical in farmed deer, although M. avium subsp avium commonly causes lesions in retropharyngeal, mesenteric and ileocaecal lymph nodes. These lesions cause problems because of their gross and microscopic similarity to those due to M. bovis infection. Birds and domestic animals are most likely to become infected via environmental contamination of food, water, bedding litter or soil, while carnivores or scavengers may also become infected by ingesting infected carcasses. Johne's disease has been reported in deer in the wild and in zoos, especially in North America, the United Kingdom (UK) and Europe. Since first being confirmed in farmed deer in New Zealand in 1979, the incidence of Johne's disease has increased steadily. To date, M. paratuberculosis has been identified in >600 farmed deer on 300 properties. The majority of cases have been identified from suspected tuberculous lesions submitted from deer slaughter plants. Clinically, Johne's disease in deer is similar to the disease in sheep and cattle, with typical signs of loss of weight and condition, and diarrhoea. However, outbreaks of Johne's disease frequently occur in young red deer, 8-15 months of age, whereas the clinical disease in sheep and cattle is sporadic and usually affects adults 3-5 years of age. The disease is characterised by a chronic granulomatous enteritis and lymphadenitis, especially affecting the jejunum and ileum and the mesenteric lymph nodes. Deer affected subclinically may have lesions in these lymph nodes at slaughter, which are grossly indistinguishable from those due to bovine tuberculosis. Because of the antigenic similarity between M. intracellulare and all the subspecies of M. avium, including M. paratuberculosis, the diagnostic tests for Johne's disease lack sensitivity and specificity, making control difficult.     

The GroES antigens of Mycobacterium avium and Mycobacterium paratuberculosis.

The GroES antigen provokes a strong immune response in human beings with tuberculosis or leprosy. We cloned and sequenced the Mycobacterium avium and Mycobacterium paratuberculosis GroES genes. M. avium and M. paratuberculosis have identical GroES sequences which differ from other mycobacterial species. This supports the current formal designation of M. paratuberculosis as M. avium subsp. paratuberculosis. Immunodominant epitopes from Mycobacterium tuberculosis GroES are conserved in M. avium, but some Mycobacterium leprae epitopes are distinct. GroES is unlikely to be specific as a serologic or skin test reagent, but may be an appropriate component of a broad mycobacterial vaccine.     

Temporal patterns and quantification of excretion of Mycobacterium avium subsp paratuberculosis in sheep with Johne's disease

OBJECTIVES: To determine the frequency of excretion of Mycobacterium avium subsp paratuberculosis in Merino sheep with Johne's disease and to quantify excretion in a group of Merino sheep. DESIGN: A pen and laboratory experiment. PROCEDURE: Seven sheep selected from an affected flock on the basis of acid-fast bacilli in the sheep's faeces were housed and total daily faecal output was collected, weighed and subjected to culture for M avium subsp paratuberculosis. An end-point titration method was used to enumerate viable M avium subsp paratuberculosis in a 15 day pooled sample from five sheep that had acid-fast bacilli in their faeces while housed. RESULTS: Four sheep with subclinical multibacillary Johne's disease excreted M avium subsp paratuberculosis each day for 11 days of cultural observation. A further three sheep were intermittent excreters but lacked other evidence of infection with M avium subsp paratuberculosis. The average number of viable bacteria excreted was 1.09 x 10(8) per gram of faeces while total daily excretion was 8.36 x 10(10) viable M avium subsp paratuberculosis per sheep. Examination of faecal smears stained with Ziehl Neelsen was an unreliable means of assessing daily excretion in individual animals except in those with severe lesions. CONCLUSION: Excretion of M avium subsp paratuberculosis in Merino sheep with multibacillary Johne's disease occurred daily, proving that environmental contamination can be continuous on farms with endemic ovine Johne's disease. Faecal culture is a useful method for detecting infection as it does not appear to be affected by the timing of collection of a sample from sheep with multibacillary disease however, to maximise the sensitivity of disease surveillance using faecal culture, sampling rates should be adjusted to take account of the proportions of multibacillary and paucibacillary cases.     

Mucosal immune response in cattle with subclinical Johne's disease

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease, a chronic granulomatous enteritis of wild and domestic ruminants. During a long subclinical period, the organism persists in the intestine despite systemic cellular and humoral immune responses. To explore the mucosal immune response in Johne's disease, we isolated mononuclear leukocytes from the ileum of cows naturally infected with M. avium subsp. paratuberculosis and from cows that were not infected. We evaluated the immunophenotype of these cells and the proliferative responses after the addition of M. avium subsp. paratuberculosis sonicate or B-cell or T-cell mitogens. Although the percentage of T cells was increased in infected cows, these cells consisted mostly of memory (CD2+CD62L-) and regulatory (CD4+CD25+) T cells. Further evidence of immune hyporesponsiveness included a decrease in the percentage of T cells with an activated phenotype and a decrease in cells expressing major histocompatibility factor class II (MHC class II). Unlike the spleen, ileal lymphocytes from infected cows failed to proliferate in response to M. avium subsp. paratuberculosis sonicate. Additionally, ileal lymphocytes from infected cows proliferated poorly in response to concanavalin A and pokeweed mitogen, suggesting generalized T cell and B cell hyporesponsiveness. These results indicate that a state of tolerance may exist in the intestine of cows subclinically infected with M. avium subsp. paratuberculosis organisms in subclinically infected cows. This effect may be induced, at least in part, by proliferation of regulatory T cells that nonspecifically suppress mucosal immune responsiveness.