Immunohistochemical expression of interferon-γ in different types of granulomatous lesions associated with bovine paratuberculosis

Animals infected with Mycobacterium avium subsp. paratuberculosis (Map) show a variety of lesions, from focal forms, seen in subclinical stages to diffuse lesions in clinical cases. The purpose of this study was to evaluate the local expression of IFN-γ by immunohistochemistry in relation with the type of lesion in naturally Map-infected cows. The number of immunolabelled cells, −the majority morphologically consistent with lymphocytes-, was higher in focal and diffuse paucibacillary forms than in diffuse multibacillary lesions, where they appeared closely related to epithelioid cells. Diffuse multibacillary lesions had the lowest numbers, but higher than controls, and positive cells were intermingled among the macrophages. The peripheral IFN-γ production was higher in all Map infected cows and a positive correlation was found with the number of immunolabelled cells in the intestine. The findings of this study show that IFN-γ would play a role in the development of the different types of lesions in paratuberculosis, and also points out the importance of adequate sampling of lymphoid tissue containing samples when studying the local immune response in which IFN-γ expression may be involved, especially in cases where focal lesions are present.     

Failure of antigen-stimulated gammadelta T cells and CD4+ T cells from sensitized cattle to upregulate nitric oxide and mycobactericidal activity of autologous Mycobacterium avium subsp. paratuberculosis-infected macrophages

The function of gammadelta T cells during ruminant paratuberculosis (Johne's disease) is presently unknown. An ex vivo system was used to test the hypothesis that gammadelta T cells are capable of activating Mycobacterium avium subsp. paratuberculosis-(M. paratuberculosis)-infected macrophages. Peripheral blood-derived macrophages were infected in vitro with live M. paratuberculosis, and autologous LN-derived gammadelta T cells or CD4+ T cells were co-cultured with infected macrophages for 48h, at which time bacterial survival as well as production of nitrites and IFN-gamma was evaluated. Incubation of M. paratuberculosis-infected macrophages with autologous gammadelta T cells did not result in reduced intracellular bacterial viability compared to infected macrophage cultures without added T cells. IFN-gamma production by-infected cultures containing added gammadelta T cells was not enhanced compared to that of infected macrophages alone. Although infection of macrophage cultures caused increased production of nitrites at both post-infection day (PID) 0 and PID 60, the addition of gammadelta T cells did not further increase nitrite production. In contrast, addition of PPD-stimulated CD4+ T cells obtained at PID 60 to M. paratuberculosis-infected macrophages resulted in significantly increased IFN-gamma production compared to cultures without added T cells or cultures containing unstimulated CD4+ T cells or unstimulated or antigen-stimulated gammadelta T cells. However, the increased production of IFN-gamma by co-cultures containing PPD-stimulated CD4+ T cells did not result in increased bacterial killing or increased production of nitrites compared to cultures without added T cells. In additional in vitro experiments, M. paratuberculosis-infected macrophages, but not uninfected macrophages, were unable to increase nitrite production when stimulated with recombinant IFN-gamma. Taken together, the data suggest that (1) gammadelta T cells do not produce significant IFN-gamma and do not significantly increase NO production from M. paratuberculosis-infected macrophages in vitro, (2) the production of significant IFN-gamma by antigen-stimulated CD4+ T cells from infected calves is insufficient to enhance mycobacterial killing or nitrite production by infected macrophages, and (3) macrophages may have an impaired NO response following intracellular M. paratuberculosis infection, even in the presence of significant concentrations of IFN-gamma.     

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.     

Local assessment of WC1+ γδ T lymphocyte subset in the different types of lesions associated with bovine paratuberculosis

The local expression of WC1⁺ γδ T lymphocytes subset has been evaluated by immunohistochemical methods at the different types of lesions present in cows naturally infected with Mycobacterium avium subsp. paratuberculosis (Map) and in non-infected control animals. Infected cattle were either in the latent/subclinical (focal lesions) or clinical (diffuse paucibacillary and multibacillary forms) stage of paratuberculosis. To assess the cell distribution, a differential cell count was carried out at the lamina propria, gut-associated lymphoid tissue and submucosa. A significant increase in the number of WC1⁺ γδ T cells was observed in all the infected animals, regardless of the type of lesion. Cows with focal lesions showed higher number of labeled cells than those with diffuse forms, where no differences were found between the two types. This increase in the number of positively immunolabelled lymphocytes in infected animals was seen in the lamina propria, with higher values in those with focal lesions. While in the lymphoid tissue no differences in the numbers were observed, in animals with focal lesions, WC1⁺ γδ T cells tended to be located at the periphery of the granulomas. These findings suggest a proinflammatory action of WC1⁺ γδ T lymphocytes in bovine paratuberculosis, which might play an important role in the containment of the Map-infection in the focal granulomas located in the lymphoid tissue, helping to prevent the progression toward diffuse forms responsible for the clinical signs.     

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.     

Immunoperoxidase studies of cell mediated immune effector cell populations in early Mycobacterium avium subsp. paratuberculosis infection in sheep

Immunoperoxidase (IPX) labelling for CD4, CD8, TCR-gammadelta, WC1, CD1b, IFN-gamma, CD45R, CD56 and lysozyme was used to investigate changes in cell mediated immune effector cell populations in the intestinal Peyer's patches (PP) and mesenteric lymph nodes of lambs, 2 and 4 months after experimental infection with low doses of sheep strain Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis). The organism was cultured from the tissues of each infected lamb, but histological lesions were not present. This infection model was considered to be more representative of natural M. a. paratuberculosis infection than previous studies. Infected sheep had significantly more CD4+ cells in the mucosa, domes and interfollicular areas of the terminal ileum, and in the interfollicular areas of the jejunal Peyer's patch. Infected sheep also had significantly increased numbers of TCR-gammadelta+ cells in the mucosa and interfollicular areas of the jejunal Peyer's patch, and increased numbers of WC1+ cells in the ileal Peyer's patch. These findings are consistent with previous findings in sheep given higher doses of cattle strain M. a. paratuberculosis. Significantly fewer CD1b+ cells were present in the paracortical areas of the mesenteric lymph nodes of infected sheep, and the reduction was greater in sheep infected for 4 months compared to sheep infected for only 2 months. Down-regulation of CD1b expression may be important for the continued survival and multiplication of M. a. paratuberculosis as specific adaptive immunity develops. Across all sheep, jejunal Peyer's patches had higher numbers of CD4+, CD8+, TCR-gammadelta+, WC1+ and CD45R+ cells, and lower numbers of CD56+ fibres compared to ileal Peyer's patches. These findings confirm and extend the peculiarities of the terminal ileal Peyer's patch in the young ruminant, with possible implications for the early establishment of M. a. paratuberculosis infection.     

Quantitative analysis of inflammatory and immune responses in dogs with gastritis and their relationship to Helicobacter spp. infection

The present study sought to quantitatively examine mucosal inflammatory and immune responses in dogs with gastritis and the relationship of these responses to infection with Helicobacter. Gastric biopsies from 30 dogs were evaluated for B- and T-lymphocytes, neutrophils, eosinophils, macrophages, and mast cells. Mucosal atrophy, fibrosis, cellularity, and severity of gastritis were graded qualitatively. Messenger-RNA (mRNA) for actin, interleukin-1beta (IL-1beta), IL-4, IL-8, and IL-10, transforming growth factor beta (TGF-beta), and interferon gamma (IFN-gamma) was quantified by polymerase chain reaction (PCR). The presence of Helicobacter spp. was determined by urease activity, histology, PCR, and enzyme-linked immunosorbent assay. mRNA for IL-1beta, IL-8, IL-10, TGF-beta, and IFN-gamma was detected in most dogs. IL-4 mRNA was detected in only 1 dog. Correlations were observed for IL-1beta versus IL-8 and IL-10; IL-8 versus IL-10, IFN-gamma, and TGF-beta; and IL-10 versus IFN-y. Mucosal pathology was related to cytokine mRNA expression (neutrophils to IL-8 and IFN-gamma, macrophages and lymphocytes to IFN-gamma, and fibrosis to IL-1beta). Gastritis was categorized as lymphoplasmacytic in all dogs, and its histologic severity correlated with atrophy, infiltration with lymphocytes and macrophages, and expression of IL-10 and IFN-gamma. Of the dogs examined, 76.7% were infected with Helicobacter spp. Infection was associated with increased expression of TGF-beta and fibrosis. Circulating anti-Helicobacter immunoglobulin G titers were higher in uninfected than infected dogs. We conclude that lymphoplasmacytic gastritis in dogs is characterized by concurrent activation of proinflammatory and immunomodulatory cytokines, with increased mRNA expression related to mucosal pathology. No significant associations between Helicobacter infection and proinflammatory cytokine expression, severity of gastritis, or differences in the pathogenicity of different Helicobacter spp. were found.     

Analysis of repeated tests for interferon-gamma (IFN-gamma) response and faecal excretion for diagnosis of subclinical paratuberculosis in Danish cattle

A total of 315 cattle were tested for infection with Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) at three consecutive samplings, using the interferon-gamma (IFN-gamma) test on whole blood and bacteriological culture of faecal samples. Of 205 cattle from 10 infected herds 99 (48%) were positive in the IFN-gamma test on at least one sampling using "IDEXX-criteria" for interpretation, and of 110 cattle from five non-infected herds three (3%) were positive. Forty-four animals from infected and one from non-infected herds tested positive at all three samplings. Although support for the specificity of the IFN-gamma test was provided by these results, they also indicate problems with false positives. Approximately half of the positive animals did not give the same result at all three samplings, indicating that repeated testing increases the chance of detecting reactors. Changing, or fluctuating, IFN-gamma test results occurred most frequently in animals younger than 1 year, indicating that the IFN-gamma test should be applied only to animals 1 year and older. M. paratuberculosis was isolated from 16 (4%) of 371 cattle, all from infected herds. Fifteen culture-positive cattle tested positive at least once in the IFN-gamma test. It was not possible to predict from the IFN-gamma test result the number of animals that would eventually develop disease. However, the test may be useful to detect animals that have been exposed to M. paratuberculosis earlier in their lives, and the testing of young cattle could be included in a control program to check for the effectiveness of preventing transmission of infection to calves and to identify animals at risk of developing disease later in their lives.     

Effects of positive results for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA on results of caudal fold tuberculin test and interferon-gamma assay for tuberculosis in cattle

OBJECTIVE: To determine whether cattle testing positive for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA were more likely to have false-positive responses on the caudal fold tuberculin (CFT) test or interferon-gamma (IFN-gamma) assay for Mycobacterium bovis than cattle testing negative for M paratuberculosis. ANIMALS: 1043 cattle from 10 herds in Michigan. PROCEDURE: Feces and blood samples for plasma were collected from cattle > or =24 months old on the day the CFT test was read. Fecal samples were submitted for microbial culture for M paratuberculosis. Plasma samples were tested for antibody against M paratuberculosis, and IFN-gamma after stimulation with purified protein derivative tuberculin from M bovis or M avium. RESULTS: Of 1043 cattle, 180 (17.3%) had positive CFT test results (suspects) and 8 (0.8%) had positive IFN-gamma assay results after stimulation with purified protein derivative tuberculin from M bovis. Forty-five (4.3%) and 115 (11.0%) cattle tested positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA, respectively. Cattle with positive responses for M paratuberculosis appeared to have an increased likelihood of false-positive results on the CFT test, although this association was not significant. CONCLUSIONS AND CLINICAL RELEVANCE: No significant association was detected among cattle testing positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA and positive CFT test and IFN-gamma assay results for M bovis.     

Description of the infection status in a Norwegian cattle herd naturally infected by Mycobacterium avium subsp. paratuberculosis

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

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.     

Immunohistochemical staining of IFN-gamma positive cells in porcine reproductive and respiratory syndrome virus-infected lungs

Paraffin-embedded lungs were obtained from a previous porcine reproductive and respiratory syndrome virus (PRRSV)-challenged experiment involving three groups: an uninfected control group, a low virulence (LV, Resp PRRSV/Repro)-infected group, and a high virulence (HV, VR-2385)-infected group. Tissues were collected at 3, 7, 10, 14 or 28 days post-inoculation (DPI) (n=5). Lungs were examined to detect IFN-gamma positive cells by immunohistochemical staining using polyclonal antibodies to IFN-gamma. The microscopic lung lesions induced by the HV group were more severe than those in the LV group. A significant increase in number of lymphocytes in the HV group was observed at 10 DPI (24.90+/-9.79%), 14 DPI (22.00+/-11.47%) and 28 DPI (28.95+/-15.11%) (P<0.05). A relative decrease in macrophage numbers was observed and correlated well with the increase in lymphocyte numbers when the disease progressed. IFN-gamma positive cells were demonstrated in both lymphocytes and macrophages, particularly pulmonary alveolar macrophages. A significant increase in IFN-gamma positive cells was found at 7 DPI (15.90+/-13.65%), 10 DPI (46.95+/-13.79%), 14 DPI (10.90+/-5.13%) and 28 DPI (13.40+/-4.89%) in the HV group (P<0.05). The results suggested that the increase in IFN-gamma positive cells in the HV group correlated well with the severity of the lung lesions, which may be because of the presence of PRRSV in the lung.     

An evaluation of a modified interferon-gamma assay for the detection of paratuberculosis in dairy herds

Whole blood samples were obtained from multiple dairy herds in Pennsylvannia and in Wisconsin which were previously determined to be infected with Mycobacterium paratuberculosis (MpS) (Johne's disease) by fecal culture. Blood samples were shipped overnight to the National Animal Disease Center (NADC) in Ames, IA for processing and interferon-gamma (IFN-gamma) analysis. Blood samples were incubated alone (non-stimulated) or with concanavalin A (ConA), a T-cell mitogen used as a positive control in the assay, for 18h. In addition, samples were incubated with M. avium purified protein derivative (AvPPD), M. bovis purified protein derivative (BoPPD), or a whole cell sonicate of M. paratuberculosis for 18h to elicit antigen-specific IFN-gamma production. After incubation, plasma was harvested and analyzed for IFN-gamma by ELISA. Values for IFN-gamma for non-stimulated blood samples (background) were consistently low for animals in all herds evaluated. In contrast, ConA stimulation of blood samples evoked a significant secretion of IFN-gamma regardless of infection status or fecal culture results for individual cows, indicating that immune cells were still viable after overnight shipment and capable of responding to stimulation. Antigen-specific IFN-gamma results were positively correlated with infection status as determined by previous fecal shedding and/or current fecal shedding of M. paratuberculosis. Accuracy of the IFN-gamma assay for correctly predicting infection status of individual cows in the herds with low levels of infection ranged from 50 to 75% when used as a single test. Combined use of the IFN-gamma test and a commercial ELISA antibody test accurately predicted infection status of 73% of cows from a dairy herd with a high level of M. paratuberculosis infection and 90% from a well-characterized group of dairy cows at the NADC. These results indicate that the antigen-specific IFN-gamma assay is a very sensitive diagnostic tool for detection of subclinical paratuberculosis in cattle and may be useful on an individual animal basis to remove infected animals from the herd.     

In vitro responses to purified protein derivate of caprine T lymphocytes following vaccination with live strains of Mycobacterium avium subsp paratuberculosis

Live attenuated vaccines provide protection against intestinal lesions in goats infected with Mycobacterium avium subsp. paratuberculosis. To examine the role of different T lymphocyte subsets in the development of this protective immunity, CD4(+), CD8(+) and gamma delta T cell receptor (TCR)(+) cells from peripheral blood of goat kids vaccinated with live attenuated strains of M. a. paratuberculosis were studied. After in vitro stimulation with purified protein derivate, the expression of gamma-interferon (IFN-gamma) and the activation marker interleukin-2 receptor (IL-2R) was analysed by flow cytometry. A depletion experiment was performed, where the phenotypes and IL-2R expression was studied after stimulation of cultures depleted of a T lymphocyte subpopulation. Close to all of the IFN-gamma producing cells were of the CD4(+) subset, while only a small number were CD8(+) cells. The gamma delta TCR(+) cells were highly activated, but did not produce IFN-gamma after in vitro stimulation. Depletion of CD4(+) cells lead to a decrease in the percentage of total gamma delta TCR(+) cells and gamma delta TCR(+)IL2-R(+) cells. Removing the gamma delta TCR(+) cells increased the relative numbers of CD4(+), but not the CD4(+)IL-2R(+) cells. Insight into the in vitro recall responses of T cell subsets from animals vaccinated with live paratuberculosis vaccines is essential in the development of more efficient vaccines.     

The presence of CD25 on bovine WC1+ gammadelta T cells is positively correlated with their production of IL-10 and TGF-beta, but not IFN-gamma

WC1+ cells in cattle exhibit both regulatory and effector activities. However, it has not been elucidated whether they are so plastic that both activities co-exist in one cell or there are separate subpopulations of effector and regulatory cells. Since the production of IFN-gamma and IL-10 seems to be related to WC1+ cells' effector and regulatory function, respectively, the main aim of this study was to determine whether those cytokines are produced by separate subpopulations of WC1+, or are co-produced by the same cells. Due to increasingly frequent emphasised role of consumption of IL-2 in the mechanism of suppressor action of mouse CD25+CD4+ T regulatory cells, expression of the receptor's alpha chain for interleukin 2 (CD25) on WC1+ lymphocytes has been evaluated. An average of 5.21% of WC1+ cells obtained from PBMCs of 12-month-old heifers show constitutive expression of the CD25 molecule, with CD25(high)WC1+ and CD25(low)WC1+ cells accounting for 1.05% and 4.10% of WC1+ lymphocytes, respectively. For detection of intracellular cytokine production, PBMCs were stimulated with concanavalin A. Both IFN-gamma(-) and IL-10-producing cells within the CD25(-)WC1+ and CD25+WC1+ subpopulations were mainly separate subpopulations. The average percentage of IFN-gamma(+)IL-10(-), IFN-gamma(-)IL-10+ and IFN-gamma(+)IL-10+ cells among CD25(-)WC1+ lymphocytes was 4.03%, 2.67% and 0.51%, respectively. A positive correlation was observed between the presence of the CD25 molecule on WC1+ lymphocytes and production of IL-10 and TGF-beta, because the average percentage of IFN-gamma(-)IL-10+ and IFN-gamma(+)IL-10+ among CD25+WC1+ lymphocytes was 3 and 4.5 times higher as compared to the corresponding cells in the CD25(-)WC1+ subpopulation, whereas the percentage of IFN-gamma(+)IL-10(-) cells in both the subpopulations was not significantly different. The percentage of TGF-beta+ cells within the CD25+WC1+ subpopulation was 2.72 times as high as that of CD25(-)WC1+ lymphocytes. Therefore, with respect to the production of IFN-gamma, IL-10 and TGF-beta, CD25+WC1+ lymphocytes turn out to have a more suppressor profile than CD25(-)WC1+.     

Interferon-gamma and interleukin-2 release by lymphocytes derived from the blood, mesenteric lymph nodes and intestines of normal sheep and those affected with paratuberculosis (Johne's disease).

This study sought to determine if T-cell cytokine responses to mycobacterial infections in sheep were similar to those in other species and if such responses correlated with prevailing gut pathology. Lymphocytes were isolated from the blood (PBL), mesenteric lymph nodes (MLN) and ileal lamina propria (LPL) of control sheep and of sheep with clinical Johne's disease due to infection with Mycobacterium avium ssp. paratuberculosis (M.a. paratuberculosis). These animals had previously been categorised into two groups exhibiting either the 'tuberculoid' (paucibacillary) form of lesion or the 'lepromatous' (multibacillary) form. Lymphocytes were examined for their capacity, following stimulation with johnin-PPD, to release interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) characteristic of the Th1 subset of MHC Class II-restricted CD4+ (helper) T-cells in other species. The expression of the two cytokines appeared related to the type of histological lesion observed. Antigen-stimulated lymphocytes from the tuberculoid group exhibited greater release of IFN-gamma and IL-2 than lymphocytes from the lepromatous group suggesting a Th1-type of response in the former in which expression of IFN-gamma by PBL showed a significant positive correlation with that expressed by MLN and LPL. Lymphocytes from animals with lepromatous lesions released lesser mycobacterium-induced IFN-gamma and IL-2 indicating a diminished role for a Th1 subset in this group of sheep. Differences in cytokine expression were much more apparent with lymphocytes which were derived from MLN.     

Paratuberculosis in sheep: its possible role in the epidemiology of paratuberculosis in cattle

A total of 50 sheep originating from 15 Dutch farms with a known paratuberculosis infection in their cattle herd, but with no history of paratuberculosis infection in their sheep flock, were examined for infection with Mycobacterium avium subsp. paratuberculosis (Map). The sheep had been grazing on the same pastures as the cattle or on pastures fertilised with manure from these cows. The sheep were screened for paratuberculosis by serum biochemistry, serology and intradermal skin tests. At necropsy they were examined macroscopically, microscopically and bacteriologically for paratuberculosis.From 10 sheep, originating from eight flocks, Map could be isolated from various tissues but not from the intestinal contents, after an incubation period of 2.5-4 months. Six of these culture-positive sheep had no macroscopic signs of paratuberculosis at necropsy. Seven sheep were Map culture negative but showed macroscopic and microscopic lesions consistent with a paratuberculosis infection. Results of serology and skin tests did not correlate with the results of bacteriological culture. Serum concentrations of calcium, albumin and total protein of the infected, suspected and negative sheep were not different. These results indicate that a substantial number of the sheep examined were infected with Map. Even though this bacterium was not isolated from their faeces, the possibility that these sheep could have been shedding Map with their faeces below detection level or at a later stage of the disease cannot be eliminated. Map infected sheep should, therefore, be considered as a possible factor in the epidemiology of with Map infected cattle herds in The Netherlands. At necropsy bacteriological culture of Map should be performed on a routine basis to improve the diagnosis of paratuberculosis in sheep.     

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.     

Antigen-induced production of interferon-gamma in samples of peripheral lymph nodes from sheep experimentally inoculated with Mycobacterium avium subsp. paratuberculosis.

The production of interferon-gamma (IFN-gamma) in response to Johnin purified protein derivate was measured in samples of the prescapular lymph node (PLN) from 10 sheep, aged 2 years, and nine sheep, aged 1 year that had been inoculated orally with Mycobacterium avium subsp. paratuberculosis within their first month of life. Ten non-inoculated sheep, aged 1 year, constituted the negative control group. The results obtained in the PLN IFN-gamma assay were compared with those derived from serological tests: a complement fixation test (CFT), agar gel diffusion test (AGID) and enzyme-linked immunosorbent assay (ELISA), as well as an IFN-gamma test on samples of blood. Among the 19 inoculated sheep, 16 gave positive reactions in the PLN IFN-gamma assay on samples incubated overnight, and 18 tested positive when the assay was applied to PLN samples incubated for 48h. In comparison, three, four and seven inoculated sheep gave positive reactions in the ELISA, CFT and in the blood IFN-gamma assay on samples incubated overnight, respectively. The AGID and IFN-gamma assay on blood samples incubated for 48h detected eight inoculated animals. Twelve inoculated sheep, that tested positive in the PLN IFN-gamma assay were clinically normal, gave negative results in an IS900-based polymerase chain reaction (PCR) assay on samples of ileum and ileocaecal lymph node and had no histological evidence of paratuberculosis, but tested positive on more than two occasions in sequential serological testing before necropsy. None of the 10 non-inoculated sheep tested positive in the AGID, CFT, ELISA, blood IFN-gamma assay on samples incubated overnight and for 48h or the PLN IFN-gamma assay on samples incubated overnight, but one gave a positive result in the PLN IFN-gamma assay on samples stimulated for 48h. It is likely that the positive reactions obtained by the PLN IFN-gamma assay in the 12 inoculated sheep that tested negative in the PCR assay and histopathological examination represents immunological evidence of latent infection or previous exposure to M. paratuberculosis rather than active infection.