Altered patterns of toll-like receptor gene expression in cull cows infected with Mycobacterium avium subsp. paratuberculosis

Johne's disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteric disease of cattle. The mechanism how MAP can co-exist in the gastro-intestinal tract despite a massive infiltration of immune cells is not known. Toll-like receptors (TLRs) are known to play an important role in both innate and acquired immune responses but it is unclear what role different TLRs play in response to MAP. In this study, 38 cull cows from herds infected with MAP were classified into four groups, based on MAP culture from gut tissues and histopathological lesion scores. The expression of TLR1, 2 and 4 mRNA from MAP antigen-stimulated mesenteric lymph node (MLN) cultures and peripheral blood mononuclear cells (PBMCs) and in the MLN and ileum tissues of these animals was determined. MAP antigen-specific expression of TLR1 in MLN and PBMC was significantly lower in the MAP-infected groups than the non-infected control group, suggesting that in MAP-infected animals there is impairment in the up-regulation of TLR1 in response to MAP antigen. TLR4 expression in MLN tissues was significantly higher in the severely infected group than the control group suggesting up-regulation of endogenous TLR4 expression at a site of MAP infection in animals severely affected with Johne's disease. A preliminary screening of TLR1, 2 and 4 in the cull cows revealed the presence of polymorphisms in TLR1 and TLR2. In summary, one mechanism how MAP may subvert the immune system is that there is an apparent lack of recognition of MAP antigens as foreign by TLR1 in MAP-infected cows.     

Differential expression of CD5 on B lymphocytes in cattle infected with Mycobacterium avium subsp. paratuberculosis

CD5 is a cell surface molecule involved in antigen recognition and is present on all T lymphocytes and a subset of B lymphocytes. The purpose of this study was to examine CD5+ expression on peripheral blood B cells from healthy, noninfected cattle and cattle with subclinical and clinical paratuberculosis. Peripheral blood mononuclear cells (PBMC) were freshly isolated or cultured for 7 days in the presence or absence of live Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis), and then analyzed by flow cytometry for CD5 expression within the B cell subpopulation. Analysis demonstrated a significant increase (P<0.01) in B cells in clinical animals as compared to healthy control cows and subclinically infected cows. In addition, three subpopulations within the CD5+ B cell population were identified: CD5dim, CD5bright, and a minor population that was characterized as CD5extra bright. A decrease in the CD5dim B cell population along with a concomitant increase in CD5bright B cells was observed in infected cows, an effect that was highly significant (P<0.01) for subclinically infected cows in cultured PBMC. In vitro infection with live M. avium subsp. paratuberculosis did not affect CD5+ expression patterns on B cells, regardless of animal infection status. Addition of exogenous IL-10 to PBMC cultures resulted in decreased numbers of CD5(bright) B cells for healthy control cows, whereas, a synergistic effect of IL-10 and infection with live M. avium subsp. paratuberculosis resulted in increased CD5bright B cells for subclinically infected cows. These results suggest that differential expression of CD5bright and CD5dim subpopulations on B cells in animals with paratuberculosis may reflect a shift in host immunity during the disease process.     

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

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 were positive by the 6-sample environmental testing method, resulting in a herd sensitivity of 0.40 (95% CI: 0.26-0.54). None of the 3 FC-negative sampling dates produced positive environmental samples. Although environmental sampling can be used as a tool in understanding the level of MAP infection in a herd or pen, it did not appear to be a sensitive diagnostic method for herd positivity in these low prevalence herds, and its use may require caution.     

The use of interleukin-2 receptor expression as a marker of cell-mediated immunity in goats experimentally infected with Mycobacterium avium ssp. paratuberculosis

The purpose of the present work was to demonstrate cell-mediated immune response to paratuberculosis in experimentally infected animals, using quantification of interleukin-2 receptor (IL-2R) expression on activated lymphocytes by means of in vitro stimulation with Mycobacterium avium ssp. paratuberculosis-derived purified protein derivative (PPDp). A whole-blood technique was developed, and optimal conditions for quantification of IL-2R expression on caprine lymphocytes, using monoclonal antibodies (anti-bovine IL-2R-alpha) and low cytometrical analysis, were determined. Different PPDp-antigen concentrations and incubation times were compared. The whole-blood method was also compared to the more traditional IL-2R assay using peripheral blood mononuclear cultures (Hesketh et al., 1993). Cross-reactivity to Mycobacterium avium was studied at different mycobacteria-PPD concentrations. An immune response could be demonstrated in animals infected with Mycobacterium avium ssp. paratuberculosis. We found that a PPDp concentration of 10microgml(-1) together with an incubation time of 72h, gave the best results using the whole-blood method. The whole-blood method eliminates many laborious steps involved in lymphocyte separation, and the effects of all the constituents of blood are expressed in a way which corresponds more to in vivo conditions. The risk of selecting subpopulations of lymphocytes during cell separation is avoided.     

Immunogenicity of eight Mycobacterium avium subsp. paratuberculosis specific antigens in DNA vaccinated and Map infected mice

Mycobacterium avium subsp. paratuberculosis (Map), the etiological agent of chronic enteritis of the small intestine in domestic and wild ruminants, causes substantial losses to livestock industry. Control of this disease is seriously hampered by the lack of adequate diagnostic tools and vaccines. Here we report on the immunogenicity of eight Map specific antigens, i.e. MAP1693c, Ag3, MAP2677c (identified by post-genomic and immunoproteomic analysis of Map secretome) and Ag5, Ag6, MAP1637c, MAP0388 and MAP3743 (identified by bioinformatic in silico screening of the Map genome). Strong, antigen-specific IFN-γ responses were induced in mice vaccinated with plasmid DNA encoding MAP1693c, MAP1637c, MAP0388 and MAP3743. In contrast, T cell responses in Map infected mice were directed preferentially against Ag5 and to a lesser extent against MAP3743. None of the tested DNA vaccines conferred protection against subsequent challenge with Map.     

Use of Mycobacterium avium subsp. paratuberculosis specific coding sequences for serodiagnosis of bovine paratuberculosis

In this study, the finished complete genome of Mycobacterium avium subsp. paratuberculosis (Map) was screened for specific coding sequences that could be very valuable in the design of a sensitive and specific Map detection serological assay. Eighty-seven Map-specific sequences were retained. Among these, three candidate antigens have been analysed for their serodiagnostic potential. These antigens were selected on the basis of their putative immunogenicity as predicted by in silico analysis. The antigens were cloned in Escherichia coli, expressed, and purified before testing in an antibody detection ELISA test, using a well characterized panel of 18 and 48 sera from Map infected and uninfected cattle, respectively. Two of these antigens, antigen 6 and MAP1637c, yielded in our conditions a sensitivity of 72% and 82%, respectively, for a specificity of 98%. It is particularly noticeable that, when probed with the same serum panel, the most widely used European paratuberculosis commercial seroassay (Pourquier test) yielded a sensitivity of 72% for a specificity of only 92%.     

Detection by immunomagnetic PCR of Mycobacterium avium subsp. paratuberculosis in milk from dairy goats in Norway

Milk samples from 340 individual goats in 34 dairy herds throughout Norway were examined for Mycobacterium avium subsp. paratuberculosis (M.a. paratuberculosis) by culture and immunomagnetic separation combined with PCR (IMS-PCR). The samples included three categories; (A) vaccinated dairy goats in herds with paratuberculosis; (B) vaccinated dairy goats in herds with no history of paratuberculosis; (C) unvaccinated goats in herds with no history of paratuberculosis.Viable M.a. paratuberculosis were not detected by culture in any sample, but 24 samples (7.1%) tested positive by IMS-PCR when the PCR products were visualised by dot blot hybridisation. PCR products from five milk samples originating from five different herds were sequenced; all showed 99% homology with the IS900 sequence from M.a. paratuberculosis.M.a. paratuberculosis were detected in all sampled categories. The percentage of IMS-PCR positive samples from herds where paratuberculosis had previously been reported was significantly lower than from herds where the infection had never been diagnosed (3.3 and 9.1%, respectively, P=0.048). Similar proportions of milk samples from vaccinated and non-vaccinated goats tested positive for the presence of M.a. paratuberculosis. Vaccinated goats older than 4 years tested positive more often than vaccinated animals less than 2 years old. Samples collected in May tested significantly more often positive than milk sampled during February-March (13.8 and 2.9%, respectively, P=0.001). This study showed that raw goats' milk in Norway might be contaminated with M.a. paratuberculosis.     

Pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in goats

OBJECTIVE: To evaluate pooled faecal culture for herd diagnosis of caprine Johne's disease and relate these findings to faecal shedding rates of Mycobacterium avium subsp paratuberculosis (Map). DESIGN: Radiometric broth culture was applied to several pooling dilutions, and shedding rates were estimated from a regression equation based on bacterial growth rates and known processing losses during radiometric culture. PROCEDURE: Sixteen faecal samples from goats naturally infected with sheep (n = 3) or cattle (n = 13) strains of Map, were diluted in normal goat faeces from 1 in 5 to 1 in 50. Cultures were confirmed by IS900 polymerase chain reaction and restriction endonuclease analysis, and mycobactin dependency. The numbers of viable Map in the culture inocula were determined by endpoint titration (most probable number) of nine samples and related to a cumulative growth index. RESULTS: A pooling dilution of 1 in 25 with an incubation period of 10 weeks detected 13 of 16 culture positive goats, all shedding > or = 2 x 10(4) Map per gram of faeces. Two samples containing very low numbers of Map (< 2 x 10(3)/g) were only culture positive from undiluted faeces. Thirteen of 16 goats were considered to be shedding low to moderate concentrations of Map (< 2 x 10(5)/g faeces). CONCLUSIONS: These data support a pooling dilution of 1 in 25 for application of pooled faecal culture as a diagnostic tool in caprine Johne's disease control. A test based on this dilution would reduce laboratory costs of whole herd testing in goats by approximately 40% relative to serology and 75 to 90% relative to individual faecal culture.     

Strain-specific sensitivity estimates of Mycobacterium avium subsp. paratuberculosis culture in Greek sheep and goats

The requirements for the isolation of Mycobacterium avium subsp. paratuberculosis (Map) may be related to the strain-type [sheep (S)- or cattle (C)-type] and not to the host. The objective of this paper was to estimate and compare strain- and biological sample (faeces or pooled-tissue)--specific sensitivities (Ses) of two solid culture media, Herrold's egg yolk medium (HEYM) and Lowenstein-Jensen (LJ) medium, for the isolation of Map from Greek dairy sheep and goats. From 400 faecal samples collected from sub-clinically infected sheep and goats of four flocks and from 214 pooled-tissue samples (142 from sheep and 72 from goats) collected, at the abattoir, from >1-year-old routinely slaughtered animals, with gross pathology suggestive of paratuberculosis, we isolated 34 Map strains. Of those, by the IS1311 PCR, 18 were categorized into the C-type and nine into the S-type; seven were not typed. We used a Bayesian approach to estimate the strain-specific Ses. SeHEYM-C-faecal=17% (95% credible interval: 7, 40) was higher than SeHEYM-S-faecal=2% (0.3, 11). Also, SeHEYM-C-faecal was higher than SeLJ-C-faecal=4% (1, 12). In pooled-tissue samples, the strain-specific Ses did not differ between the two media.     

Detection of Mycobacterium avium subsp. paratuberculosis from the testicles of a clinically infected breeding animal

During a post mortem of a six year old simmental bull with severe paratuberculosis infection the testicles were further examined by pathological, histological and microbiological methods. No gross or histological lesions could be observed. Single acid fast organisms were detected in smears taken from sterile testicle tissue. Tissue material was additionally cultured in mycostatin culture media and after 8 weeks of incubation acid fast colonies were demonstrated. Polymerase chain reaction with DNA extracted from cultured bacteria and testicle tissue material resulted in M. avium subsp. paratuberculosis-specific amplicons. The detection of M. avium subsp. paratuberculosis in the testicle of the bull demonstrates the possibility of bacteriemia in the final stage of clinical paratuberculosis infection. The evidence of transmitting paratuberculosis through contaminated semen and its relevance for artificial insemination is being discussed in the presented paper.     

Cell-mediated immune response in swine infected with Mycobacterium avium subsp. avium

The zoonotic characteristic of Mycobacterium avium subsp. avium (MAA) represents a veterinary and economic problem in infected pigs. In this study, we analysed cell-mediated immunity six months after experimental infection by measuring interferon-γ (IFN-γ) production and by performing lymphocyte transformation tests after in vitro re-stimulation with the MAA-derived antigen. At the same time, IFN-γ-producing cells were characterised by flow cytometry. In MAA-infected animals, the production of IFN-γ increased in response to the MAA antigen in the blood, spleen and mesenteric lymph nodes. Similarly, a positive antigen-driven response was detected by the proliferation assay. In contrast, IFN-γ production and proliferation was undetectable after stimulation with the MAA antigen in uninfected control animals. These results indicate that both methods can be used for the identification of individual MAA-infected pigs. Using flow cytometry, we found that double-positive CD4(+)CD8(+) lymphocytes were the major T lymphocyte subset producing IFN-γ after in vitro re-stimulation.     

Isolation of Mycobacterium avium subsp paratuberculosis from environmental samples collected from farms before and after destocking sheep with paratuberculosis

OBJECTIVE: To determine whether Mycobacterium avium subsp paratuberculosis could be isolated from soil-pasture, faecal, water and sediment samples on farms before and after removal of sheep with paratuberculosis. A feasibility study and subsequent field survey. PROCEDURE: First the analytical sensitivity of radiometric culture of the organism from two types of soil was determined relative to faeces. Then soil-pasture, faecal, water and sediment samples were collected for culture from a range of sites from 6 farms with paratuberculosis affected sheep and goats. Similar samples were collected from 20 farms at least 9 months after removal of infected stock. RESULTS: The analytical sensitivity of culture of M a paratuberculosis from soil samples was 2 orders of magnitude less than that from faeces, and environmental samples required longer incubation periods to yield significant growth in radiometric culture (BACTEC) medium. However, the organism was recovered from approximately 20% of 163 soil-pasture, water and sediment samples from 6 properties with clinically-affected animals with paratuberculosis. The positive samples were from a range of topographic sites, including open exposed and dry areas, however, low lying areas tended to have larger numbers of organisms. When the same sites were sampled again about 5 months later, only 1 was culture positive, and none were culture positive > 12 months later. Of 17 water and dam sediment samples collected from farm 6, which had long-standing high prevalence OJD infection, only one water sample and one sediment from the same dam were culture positive. None of the 5 water samples from the other farms were culture positive. Of 96 water samples, 90 sediment samples and 93 soil samples from farms that had been destocked of infected sheep/goats for 9 to 24 months, one sediment sample from a farm in Victoria (destocked for 12 months) and two sediment samples from a farm in New South Wales (10, 19 months) were culture positive. Recontamination from cattle or water could not be excluded as a cause of the positive cultures from the second farm. CONCLUSION: M a paratuberculosis can be detected by radiometric culture in environmental samples from farms grazed by sheep or goats with paratuberculosis. There is a relatively low likelihood of recovery of the organism from water samples from such farms, and at 5 or more months after removing stock with paratuberculosis the likelihood of positive cultures from environmental samples is very low. Although the analytical sensitivity of culture from environmental samples is less than that from faeces, surveys of environmental sites are nevertheless feasible. However, improved culture methods are needed for critical surveys and to study the movement and fate of the organism in the environment.     

Immunological response to Mycobacterium avium subsp. paratuberculosis in chickens

Enzyme-linked immunosorbent assay (ELISA) and culture are 2 common diagnostic tests for detecting Mycobacterium avium subsp. paratuberculosis (Map) in Johne's disease, but they are not as sensitive as polymerase chain reaction (PCR). However, inhibitors can coextract with the target DNA and cause interference in PCR. Development of an immune capture assay followed by PCR amplification can alleviate this problem. In this study, we were able to induce an immune response in chickens using heat or formalin inactivated Map. The purified immunoglobulin (Ig)Y has a molecular weight of 160 kDa. The titers were at 1:6400 and 1:12 800 at weeks 5 to 6 and 8 to 9, respectively, as determined by the IDEXX modified ELISA kit for Johne's disease. The IgY produced from inactivated bacterial cells had no effect on its ability to recognize live Map cells as illustrated by immunofluorescence assay and immune capture PCR results.     

A long-term study in Angora goats experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies

Two longitudinal experiments involving Angora goats challenged with either bovine or ovine strains of Mycobacterium avium subspecies paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the interferon-gamma (IFN-gamma) test and the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Persistent shedding, IFN-gamma production, seroconversion and clinical disease occurred earlier with the bovine Map gut mucosal tissue challenge inoculum than with cultured bacteria. The IFN-gamma responses of the gut mucosal tissue and bacterial challenge groups were substantially and consistently higher than those of the control group. The in vivo and cultured cattle strains were much more pathogenic for goats than the sheep strains with persistent faecal shedding, seroconversion and clinical disease occurring in the majority of bovine Map challenged goats. With the ovine Map, 3 goats developed persistent antibody responses but only one of these goats developed persistent faecal shedding and clinical disease. However, there was no significant difference between the IFN-gamma responses of the tissue challenged, bacterial challenged and control groups. Compared with sheep, the ELISA appeared to have higher sensitivity and the IFN-gamma test lower specificity.     

Efficacy of spheroplastic and cell-wall competent vaccines for Mycobacterium avium subsp. paratuberculosis in experimentally-challenged baby goats

A Mycobacterium avium subspecies paratuberculosis (MAP) vaccine that reduced the incidence of clinical disease or reduced fecal shedding of MAP would aid control of Johne's disease (JD). The objective of the present study was to evaluate the efficacy of four MAP vaccine combinations, including cell-wall competent (CWC) alum adjuvant, CWC-QS21 adjuvant, cell-wall deficient (CWD) alum adjuvant and CWD-QS21 adjuvant vaccines. Eighty baby goats were vaccinated at 1 and 4 weeks of age with one of these vaccines or a sham control vaccine consisting of alum adjuvant. Kids were challenged orally with approximately 6.0 × 10⁹ organisms in four divided doses of 1.5 × 10⁹ organisms using a goat isolate of MAP. Vaccinated challenged and challenged control groups had 10 and 6 kids per group, respectively. Half of the kids within each group were necropsied at either 6 or 9 months post-challenge. Gross and microscopic lesions and relative number of acid-fast bacilli were evaluated and scored at necropsy. Results indicated all challenged kids had some lesions compatible with JD suggesting none of the vaccines prevented infection. Three vaccines (CWC-alum, CWC-QS21 and CWD-QS21) reduced lesion scores by 46–51% at 9 months. CWD-alum vaccine resulted in a more severe (+33.5%) lesion score than sham-vaccinated challenged control. Lesion scores were greater at 9 months than at 6 months post-challenge in the sham-vaccinated challenged group and CWD-alum vaccinated group, while lesion scores were generally stable with remaining vaccines. Mean fecal CFU/g were significantly different across time from challenge to 9-month necropsy (p = 0.043) and the CWC-QS21 vaccine group had a marked reduction in fecal CFU/g at all time points post-challenge. A reduction in MAP CFU/g was also detected in necropsy tissues from kids given the CWC-alum, CWC-QS21 and CWD-QS21 vaccines, and increased CFU/g were detected in tissues from kids given the CWD-alum vaccine. Immunological tests evaluated included, humoral response evaluation by AGID, ELISA and Western blot, and cell mediated response by comparative PPD skin testing (M. avium, Old Johnin, M. bovis and Lot 2 Johnin PPD's), and production of MAP induced γ-interferon. Vaccination also resulted in false-positive PPD skin test reactions for M. avium PPD, Old Johnin PPD and γ-interferon tests. When a 2-mm cutoff above normal skin thickness was used to define positive skin test reactions, false-positive reactions for M. bovis were detected in only 2 of 32 kids given a vaccine with QS21 adjuvant.     

Distribution of Mycobacterium avium subsp. paratuberculosis in organs of naturally infected bull-calves and breeding bulls

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, has particular importance in cattle due to the resulting chronic diarrhoea, weight loss, decreased production, infertility and eventual death. While faecal oral route of infection is generally recognised, reports about semen-derived infection are rare. The objective of this work was to assess whether M.a. paratuberculosis may disseminate from the gastrointestinal tract to reproductive organs, and compare this event between naturally infected bull-calves and breeding bulls. Ten bull-calves, aged 6-28 weeks and four breeding bulls were tested by serology, faecal and tissue culture, IS900 PCR and RFLP. In seven bull-calves M.a. paratuberculosis was isolated predominantly from mesenteric lymph nodes (75%); isolates from mucosa of the intestine constituted 25%. In three breeding bulls, M.a. paratuberculosis was isolated both from intestinal mucosa and mesenteric lymph nodes. Head and mediastinal lymph nodes, liver, spleen and semen of bull no. 1 (Holstein-Friesian); testes and epididymis of bull no. 2 (Piemonte); testes, epididymides and seminal vesicle of bull No. 3 (Hereford); and seminal vesicle of bull No. 4 (Simmental) tested positive by culture. Hot-start PCR revealed M.a. paratuberculosis in semen, seminal vesicle and intestinal tissue where culture isolation was difficult. Isolates from bull-calves and breeding bulls were of RFLP types B-C9 and B-C1, respectively. Bull-calves born in infected herd can be sources of infection when later used for natural mating or artificial insemination. Sub-clinically infected bulls release M.a. paratuberculosis into semen, consequently infecting the uterine environment of cows.     

Review of Mycobacterium avium subsp. paratuberculosis antigen candidates with diagnostic potential

Mycobacterium avium subsp. paratuberculosis (MAP) is a slow growing bacterium that can infect ruminants and remain latent for years without development of any clinical signs or disease. Diagnosis is often based on detection of MAP antibodies in milk or serum samples or culture of bacteria from faeces; however, these diagnostic tools are often not applicable until years after infection. Detection of MAP specific cell-mediated immune (CMI) responses can serve as an alternative and be implemented in a diagnostic tool. CMI responses can be measured at an early stage of infection, prior to development of antibodies and shedding of detectable amounts of MAP. At present, available diagnostic assays are limited by the lack of MAP specific antigens included in these assays resulting in poor specificity. The objective of this review is to provide a systematic overview of diagnostic MAP antigen candidates described to date with special emphasis on antigen candidates tested for CMI responses. Relevant information on 115 different MAP antigens was systematically extracted from literature and summarized in 6 tables of CMI antigens, secreted antigens, cell wall and membrane antigens, lipoprotein antigens, heat shock antigens and hypothetical antigens. Strategies for evaluation of novel antigen candidates are discussed critically. Relatively few of the described antigens were evaluated for their use in CMI based diagnostic assays and so far, no obvious candidate has been identified for this application. Most of the novel diagnostic candidates were evaluated in few animals and it is recommended that an appropriate sample size is included for evaluation of antigen candidates in future studies.     

Comparison between two in situ methods for Mycobacterium avium subsp. paratuberculosis detection in tissue samples from infected cattle

Paratuberculosis or Johne's disease is a chronic infectious disorder caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease produces diarrhea and weight loss in cattle and other animal species, and it is characterized by granulomatous enteritis and lymphadenitis. Histopathology and in situ techniques can be used as a diagnostic test, but the performance of these methods was not previously compared. The aim of this paper was to evaluate the ability of immunohistochemistry and in situ hybridization to detect Map in formalin-fixed tissue samples from infected cattle. Samples (ileum or ileocecal lymph node) from four animals that had positive Map culturing, lesions and detectable acid fast bacilli, as well as from two control animals, were tested by immunohistochemistry and in situ hybridization. Immunostaining and positive hybridization were observed in areas with lesions from infected animal samples, inside the cytoplasm of macrophages, epithelioid and giant cells. Immunostaining was intense in three samples and weak in one, while hybridization was weak in all cases. In situ hybridization was positive in negative areas of tissues analyzed by immunohistochemistry, which could be related to spheroplast detection as it was previously described for this method. Control samples resulted negative by these two methods. Both techniques were able to detect Map in formalin fixed and paraffin embedded tissues, however immunohistochemistry produced higher intensity staining and was easier to perform. Therefore, we believe that immunohistochemistry and in situ hybridization to be useful for the post-mortem diagnosis and research of Paratuberculosis.     

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.