IgG, IgM and IgA in the serum of cattle naturally infected with Mycobacterium paratuberculosis

Serum IgG, IgM and IgA antibody response in 20 cattle naturally infected with Mycobacterium paratuberculosis and in 15 non-infected cattle were measured by enzyme-linked immunosorbent assay. A strong IgG response was detected in 16 (80%) of the infected animals. Diagnostic levels of IgM were detectable in all of the infected animals as well as in 8 (53%) of the non-infected animals. Animals with paratuberculosis had a very weak specific serum IgA response and this appears to be of little value in detection of infection in these animals.     

Tonsillar lesions in cattle naturally infected with Mycobacterium bovis

The upper respiratory tract surfaces, the palatine and pharyngeal tonsils and associated lymph nodes of 32 tuberculin reactor cattle were examined pathologically and bacteriologically. Tuberculous lesions were observed histologically in the palatine tonsils of five animals and in both the palatine and pharyngeal tonsils of a sixth. Mycobacterium bovis was cultured from the tonsils of four of these animals and from the palatine or pharyngeal tonsils of a further eight cattle in which no lesions were observed. The upper respiratory tract surfaces of 10 animals were M bovis-positive.     

Cytokine gene expression in ileal tissues of cattle infected with Mycobacterium paratuberculosis

Cytokine gene expression in ileal tissues of cattle infected with Mycobacterium paratuberculosis was evaluated. The effects of infection with M. paratuberculosis on cytokine production may influence immune regulation at the site of colonization, resulting in the chronic inflammatory state associated with the latter stages of this disease. Ileal samples were obtained at necropsy from noninfected control cows (n=8) and clinically infected cows (n=7) and processed for immunohistochemistry and in situ hybridization. Cows infected with M. paratuberculosis were in the latter stages of disease with clinical signs such as weight loss, watery diarrhea, and inappetence. Among cytokines we studied, interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, and interferon-gamma (IFN-gamma) were expressed significantly more in infected animals than in noninfected control animals. The expression of tumor necrosis factor-alpha (TNF-alpha), however, was not different between the two groups of cattle. In addition, immunohistochemical staining demonstrated that the number of resident macrophages in the ileum of infected animals was three times greater than that of noninfected cows. In contrast to this, ileal tissues from noninfected control animals contained 1.5 times more neutrophils than the ileal tissues from cows infected with M. paratuberculosis. These data demonstrate that localized ileal cytokine production is different between cows chronically infected with M. paratuberculosis and noninfected control cows.     

Detection of Mycobacterium paratuberculosis antigen with colloidal immunogold in naturally infected sheep.

The anti-Mycobacterium paratuberculosis polyclonal serum is proved useful for labelling Mycobacterium paratuberculosis in glutaraldehyde-osmium-fixed and epon-embedded intestinal samples from sheep with clinical symptoms of paratuberculosis. M. paratuberculosis marked with antibody-coated colloidal gold stain was seen in macrophages, epithelioid cells, giant cells and neutrophils throughout intestinal mucosa. In large macrophages with a low lysosomal content, a great number of intact mycobacteria was seen within phagosomes. In macrophages with average lysosomal content, very few intact mycobacteria or mycobacterial debris were present and lysosome-phagosome fusions were observed. Mycobacteria within neutrophils were scanty. These results show the usefulness of colloidal immunogold techniques for studies of the pathogenesis of paratuberculosis.     

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.     

Polymerase chain reaction analysis of TNF-alpha and IL-6 mRNA levels in whole blood from cattle naturally or experimentally infected with Mycobacterium paratuberculosis.

Johne's disease is characterized by a chronic enteritis that results in granulomatous inflammation, cachexia, and eventual death of cattle infected with Mycobacterium paratuberculosis. The cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) have been associated with granuloma formation and wasting in other disease syndromes. The potential role of these cytokines in the development and progression of Johne's disease has not been investigated. Using the polymerase chain reaction (PCR) and specific bovine oligonucleotide cytokine primers and probes, we examined the expression of messenger RNA for these cytokines in whole blood from M. paratuberculosis infected and uninfected cattle. Cytokine mRNA levels were examined before and after in vitro incubation with E.coli lipopolysaccharide (LPS) and lipoarabinomannan (LAM) purified from M. paratuberculosis. Uninfected calves, experimentally infected calves, and naturally infected cattle all displayed similar cytokine mRNA expression patterns. However, individual animals demonstrated variability in the levels of IL-6 and TNF-alpha mRNA expression as determined by a semiquantitative PCR method using 32P-labelled oligonucleotide probes.     

Determination of adenosine deaminase activity in cattle naturally infected with Theileria annulata

The purpose of this study was to determine serum ADA activity in cattle naturally infected with Theileria annulata. In this study, a total of 37 cross-bred cattle which 27 of it showing clinical signs of theileriosis constituted infected group and 10 healthy cattle as control group were used as animal materials. Infected group divided into three groups according to their PCV values. Cattle with PCV > or = 25 were put on group I (n = 9), those with PCV 13-24 were put on group II (n = 11) and those with PCV < or = 12 were put on group III (n = 7). Microscopical diagnosis of the disease was also made. Hematological parameters, serum enzyme activities (ADA, AST, ALT and ALP) were determined in all cattle. Hematological results revealed that significant progressive decreases in HGB, PLT, PBML counts and ratios from group I onwards to group III, whereas the WBC, PBPL counts and ratios showed an increase from group I onwards to group III. The serum ADA, AST, ALT and ALP activity increased significantly in all infected groups compared to control group. However, these parameters were also observed to decrease progressively from group I to group III. Furthermore, the highest increase in enzyme activities observed in the infected group I. But, these enzyme's activities started to decrease in infected group II and III in parallel with PBML and PLT counts. Eventhough, this decrease did not reach to the values obtained from control group. On the contrary, PBPL counts and ratios increased in infected group II and III in contrast to decrease in PCV. As a result, increased serum ADA activity in tropical theileriosis may reflect the involvement of the cellular immune responses.     

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.     

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.     

Vaccination against paratuberculosis of lambs already infected experimentally with Mycobacterium avium subspecies paratuberculosis

OBJECTIVE: To assess the protective value of a live-attenuated vaccine in sheep already exposed to Mycobacterium avium subsp paratuberculosis and to investigate the progression of a systemic immune response in experimentally infected sheep. STUDY DESIGN: Twenty-eight lambs, aged 1 to 1.5 months, were dosed via stomach tube with approximately 4.4 x 10(8) M a paratuberculosis organisms. Two weeks later, 14 of these 28 animals received subcutaneous injections of 1 mL of a live-attenuated vaccine. Thirteen additional lambs were neither dosed nor vaccinated (negative controls). Antigen-induced production of IFN-gamma in blood, and antibody concentrations in serum were sequentially monitored in vaccinated, unvaccinated and control animals for 1 year. Each sheep was examined for infection by an IS900-based PCR test on samples of ileum and ileocaecal lymph node and histological examination at the time of necropsy. RESULTS: Seven of 14 unvaccinated and two of 14 vaccinated sheep developed clinical paratuberculosis that was later confirmed by histological examination and/or the IS900-based PCR test. The granulomatous inflammation in the jejunal and ileal mucosa was less severe in vaccinated than in unvaccinated sheep. Acid-fast organisms were detected only in the unvaccinated group. The PCR assay on ileal samples gave positive reactions in two vaccinated and eight unvaccinated sheep. Both the antibody response and IFN-gamma response were detected earlier and were more substantial in vaccinated than in unvaccinated sheep. Furthermore, in experimentally infected but unvaccinated sheep, the IFN-gamma concentrations were higher in those animals without acid-fast organisms than in those with them. CONCLUSIONS: Vaccination of lambs with live-attenuated vaccine 2 weeks after oral inoculation with M a paratuberculosis stimulated the host response against the organism and led to a reduced mycobacterial burden. The diminished IFN-gamma responses in experimentally infected sheep with acid-fast organisms suggest a positive relationship between the magnitude of the systemic cell-mediated immune response and an animal's ability to control infection.     

A technique for the purification of Mycobacterium paratuberculosis from the ileal mucosa of infected cattle

Mycobacterium paratuberculosis, the causative agent of paratuberculosis, produces considerable economic loss in the cattle industry in many countries. The slow growth of M. paratuberculosis has hindered investigations of the antigenic composition of the organism and the development of species-specific antigen for serological detection of this disease. This paper describes a simple method for the isolation of large quantities of viable M. paratuberculosis from the intestinal mucosa of infected cattle by a combination of trypsin digestion, deoxyribonuclease/lysozyme treatment and differential centrifugation. Purity was about 99% and yield between 10(5)-10(9) bacteria/g tissue.     

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

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

Osteopontin immunoreactivity in the ileum and ileocecal lymph node of dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Osteopontin (Opn), a highly acidic glycoprotein, promotes cellular adhesion and recruitment and has been shown to be upregulated in the granulomas of mycobacterial infections. Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is associated with granulomatous enteritis. The objective of this experiment was to identify Opn in the ileum and ileocecal lymph node (ICN) of dairy cows naturally infected with MAP and to compare the frequency and intensity of staining between noninfected healthy controls, subclinical and clinical cows. Sections from these three groups of animals were selected from a tissue archive. Immunohistochemical analysis was used to determine the location and expression of Opn. The frequency and intensity of staining was also reported. Confirmation of acid-fast bacilli in the tissue sections was achieved by the Ziehl-Neelsen method. Within the ileal tissue, macrophages, lymphocytes, and plasma cells stained positive for Opn. Clinical cows expressed Opn at a greater frequency in the lamina propria. Control and subclinical cows did not have areas of granulomatous inflammation but cells staining for Opn were equally intense for the three groups. The frequency of staining for Opn in the ICN was not affected by MAP infection. Results of this study confirm for the first time, the expression of Opn in the ileum and ICN of MAP-infected cattle.     

Repeat tuberculin skin testing leads to desensitisation in naturally infected tuberculous cattle which is associated with elevated interleukin-10 and decreased interleukin-1 beta responses

The principal surveillance tool used to control bovine tuberculosis in cattle is the removal of animals that provide a positive response to the tuberculin skin-test. In this study we performed a longitudinal investigation of the immunological and diagnostic consequences of repeated short-interval skin-tests in cattle naturally infected with Mycobacterium bovis. Tuberculin skin-test positive cattle were subjected to up to four further intradermal comparative cervical skin-tests at approximately 60-day intervals. A significant progressive reduction in the strength of the skin-test was observed after successive tests. In contrast, the magnitude of interferon-γ (IFN-γ) responses was not influenced by repeat skin-testing either transiently around the time of each skin-test or longitudinally following repeated tests. A significant boost in blood interleukin-10 (IL-10) production was observed within 3 days following each skin-test although the magnitude of this boosted response returned to lower levels by day 10 post-test. The application of a novel multiplex assay to simultaneously measure seven cytokines and chemokines also identified that skin-testing resulted in a significant and progressive reduction in antigen specific interleukin-1β (IL-1β) whilst confirming stable IFN-γ and elevated IL-10 responses in the blood. Therefore, we have demonstrated that in cattle naturally infected with M. bovis, repeat short-interval skin-testing can lead to a progressive reduction in skin-test responsiveness which has potential negative consequences for the detection of infected animals with marginal or inconclusive skin-test responses. The desensitising effect is associated with decreased IL-1β and elevated IL-10 responses, but importantly, does not influence antigen specific IFN-γ responses.     

Within-herd contact structure and transmission of Mycobacterium avium subspecies paratuberculosis in a persistently infected dairy cattle herd

Within-herd transmission of pathogens occurs either by direct or by indirect contact between susceptible and infected animals. In dairy herds that are structured into groups, the way in which animals encounter each other and share an environment can affect pathogen transmission. Dairy cattle are heterogeneous in terms of susceptibility and infectivity with respect to Mycobacterium avium subspecies paratuberculosis (Map) transmission. It is mainly young animals that are susceptible and adults that are infectious. Both vertical and horizontal transmission through the ingestion of Map shed into the environment by adults and transiently infected calves can occur. Our objective was to assess the effect of contact structure on Map transmission in persistently infected dairy herds and to examine the effect of isolating calves from other calves or from adults before weaning. We developed a stochastic compartmental model of Map transmission in a closed dairy herd. The model reflects the Map infection process and herd management characteristics. Indirect transmission via the environment was modelled explicitly. Six infection states (susceptible, resistant, transiently infectious, latently infected, subclinically infected, and clinically affected) and two contaminated farm area environments (whole farm and calf area) were modelled. Calves were housed in hutches, individual indoor pens, or group indoor pens. Two different levels of exposure of calves to a farm environment contaminated by adults were possible: no exposure and indirect exposure through fomites. Three herd sizes were studied. We found that contacts between calves before weaning did not influence Map transmission in a herd, whereas the level of exposure of calves to an environment contaminated by adults and the starting age of exposure of calves to adults were pivotal. Early culling of clinically affected adults led to a lower prevalence of infectious adults over time. The results were independent of herd size. Despite the many transmission routes that are known, the best control approach is to limit the exposure of calves to adult faeces through the systematic separation of adults and calves in combination with hygiene measures. Reducing contact between calves does not appear effective.     

Parturition invokes changes in peripheral blood mononuclear cell populations in Holstein dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Johne's disease (JD) is characterized by a protracted period of subclinical infection. Infected cows may remain in the subclinical state until stressors such as parturition and lactation invoke more clinical signs of disease. The objective of this study was to evaluate changes in the percentages of CD4(+), CD8(+), and gammadelta T-cells, B-cells, monocytes, as well as the expression of the activation marker, CD5, on these cell subpopulations in the peripheral blood of dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis (MAP) during the periparturient period. Peripheral blood mononuclear cells (PBMCs) were collected from 3 wk pre- to 4 wk post-calving and freshly isolated or cultured for 7d. Day 7 cultures were infected with live MAP at a 10:1 MOI (bacteria to adherent PBMC), and cultures were incubated for an additional 24h. Fluorescent antibody labeling of lymphocyte subsets and monocytes was conducted and analyzed with flow cytometry. Freshly isolated PBMCs from subclinical cows expressed a greater (P<0.05) percentage of CD8(+) and gammadelta T-cells compared with clinical cows. The percentage of CD4(+) T-cells increased (P<0.08) in clinical cows as parturition approached. During the postpartum period, clinical cows had greater (P<0.05) CD4:CD8 ratios compared with subclinical and control cows. After 8d, uninfected PBMCs from clinical cows had greater (P<0.05) percentages of CD14(+) cells compared with subclinical cows. When infected with live MAP, there was no effect of infection group or parturition on cell subpopulations. In fresh PBMCs, clinical cows expressed lower percentages of CD4(+)CD5(bright) and CD8(+)CD5(bright) compared with control cows, but greater percentages of CD5(dim) cells for all lymphocyte subsets. These results suggest changes in the percentages of lymphocyte subsets, monocytes, and CD5 markers are modulated by both infection status and the periparturient period.