Mycobacterium avium infection in a farmed deer herd

Tuberculous lesions were identified over a 2-year period in 36 clinically normal red deer from a single herd. The lesions were only present in the retropharyngeal lymph nodes and lymph nodes draining the intestinal tract, indicating infection by the oral route. Mycobacterium avium was isolated from 27 of 29 lesions examined by bacterial culture. Grossly and histologically, the lesions were indistinguishable from those caused by Mycobacterium bovis. DNA restriction endonuclease analysis revealed that all the 26 M. avium isolates available for examination had identical cleavage patterns. These patterns were identical to a New Zealand M. avium serotype 2 isolate from a pig and were very similar to a reference strain of M. avium serotype 2. The DNA examinations indicated that the deer were infected from a common source that was not identified.     

Diagnostic detection methods for Mycobacterium avium subsp. paratuberculosis in white-tailed deer

This study compares the results and suitability of serological testing, microscopic examination, deoxyribonucleic acid (DNA) detection, and bacterial culture for detecting Mycobacterium avium subsp. paratuberculosis (Map) infection in asymptomatic farmed white-tailed deer (WTD) (Odocoileus virginianus). Deer were classified as infected if culture slants from their feces, lymph nodes, or ileum were positive, or if a polymerase chain reaction (PCR) assay detected Map DNA in any of its tissues. Deer identified as positive by agar gel immunodiffusion (AGID) testing or enzyme-linked immunosorbent assay (ELISA) but not by bacterial culture, Ziehl-Neelsen staining, or PCR assay were classified as suspect. Culture of tissues classified 10/16 (62.5%), histopathologic examination 1/16 (6.3%), tissue smears 4/16 (25%), culture slant (CS)-PCR on feces 12/15 (80%), CS-PCR on tissue 13/16 (81.3%), and direct PCR on uncultured tissues 5/16 (31.3%) deer as infected. The ELISA classified 2/15 (13.3%) deer as positive and therefore suspect. The AGID test was negative for all deer. Fifteen of 16 deer were positive by 1 or more tests; only 1 deer was negative on all 11 assays. The CS-PCR gave superior results on antemortem fecal testing as well as postmortem tissue testing and can be recommended for improving the detection of Map in WTD at every stage of infection.     

Intra-uterine transmission of Mycobacterium avium subsp paratuberculosis in subclinically affected red deer (Cervus elaphus)

AIM: To determine the rate of transmission of Mycobacterium avium subsp paratuberculosis (M. ptb) from hind to fetus in utero, and the risk of transmission from dam to fawn via infected colostrum and milk in subclinically affected red deer hinds.

METHODS: Hinds were sourced from farms in Otago or Southland and selected for the study if they were positive to the immunoglobulin G1 (IgG1) modified enzyme-linked immunosorbent assay (ELISA) (Paralisa) and exhibited no clinical signs of Johne's disease. The hinds (n=35) were sent to a deer slaughter premises (DSP; n=31) or were killed on-farm (n=4). All post-mortem samples were collected from the fetus first and then from the dam, taking care to avoid cross contamination between samples. Fresh samples (n=185) were collected for culture, and tissue samples (n=72) were collected from 24 hinds and their fetuses for histopathological examination.

RESULTS: A total of 24/35 hinds selected were suitable for inclusion in the study. Eighteen of these pregnant hinds were culture-positive for M. ptb, and 14 of these had culture-positive fetuses, representing a transmission rate of 78% (95% confidence interval (CI) =0.58–0.98) from dam to fetus. Of the 16 mammary glands sampled, 11 (69%) were culture-positive for M. ptb while 12/15 (80%) mammary lymph nodes sampled were also culture-positive.

CONCLUSIONS: This study demonstrated a high rate of transmission of M. ptb from dam to fetus in red deer, and a potential risk of transmission to fawns suckling from mothers that are subclinically affected with Johne's disease.     

Intra-uterine transmission of Mycobacterium avium subsp paratuberculosis in subclinically affected red deer (Cervus elaphus)

AIM: To determine the rate of transmission of Mycobacterium avium subsp paratuberculosis (M. ptb) from hind to fetus in utero, and the risk of transmission from dam to fawn via infected colostrum and milk in subclinically affected red deer hinds. METHODS: Hinds were sourced from farms in Otago or Southland and selected for the study if they were positive to the immunoglobulin G1 (IgG1) modified enzyme-linked immunosorbent assay (ELISA) (Paralisa) and exhibited no clinical signs of Johne's disease. The hinds (n=35) were sent to a deer slaughter premises (DSP; n=31) or were killed on-farm (n=4). All post-mortem samples were collected from the fetus first and then from the dam, taking care to avoid cross contamination between samples. Fresh samples (n=185) were collected for culture, and tissue samples (n=72) were collected from 24 hinds and their fetuses for histopathological examination. RESULTS: A total of 24/35 hinds selected were suitable for inclusion in the study. Eighteen of these pregnant hinds were culture-positive for M. ptb, and 14 of these had culture-positive fetuses, representing a transmission rate of 78% (95% confidence interval (CI) =0.58-0.98) from dam to fetus. Of the 16 mammary glands sampled, 11 (69%) were culture-positive for M. ptb while 12/15 (80%) mammary lymph nodes sampled were also culture-positive. CONCLUSIONS: This study demonstrated a high rate of transmission of M. ptb from dam to fetus in red deer, and a potential risk of transmission to fawns suckling from mothers that are subclinically affected with Johne's disease.     

Mycobacterium avium subsp. paratuberculosis infection in wildlife on three deer farms with a history of Johne's disease

Abstract

AIM: To determine the prevalence of Mycobacterium avium subsp. paratuberculosis (Map) infection in wildlife, in pastoral landscapes with a recent history of clinical Johne's disease in livestock.

METHODS: A total of 449 wild mammals and birds from three farms in the South Island of New Zealand with recent histories of clinical Johne's disease in their deer herds were trapped and examined for gross pathological changes in the gastrointestinal tract. Additionally, individual mesenteric lymph nodes from 380 mammals, and segments of gastrointestinal tract from 32 birds were excised, homogenised and cultured for viable Map bacilli. The prevalence of Map infection was then calculated for the various species. Faecal samples from those mammals which had culture-positive tissues were further cultured for the presence of Map.

RESULTS: Gross pathological changes were identified in the gastrointestinal tract of four brushtail possums, one cat, six ferrets, 12 hares, six hedgehogs, three rabbits, one stoat, and one paradise shelduck. Infection with Map in the gastrointestinal tract was confirmed in only three of these cases, one each of brushtail possums, hares and hedgehogs. In contrast, Map infection in the absence of gross pathological changes was frequently recorded in enteric tract tissues of mammals and birds. Among mammals, Map infection was recorded in 18/73 (25%) brushtail possums, 4/23 (17%) cats, 15/42 (36%) hedgehogs and 29/113 (26%) rabbits. Among birds, intestinal tract tissue Map infection was recorded in 3/17 (18%) paradise shelducks. Among 64 of the 74 mammals which had Map culture-positive tissues, 38% (n=5) of hedgehogs and 11% (n=3) of rabbits also had culture-positive faecal samples.

CONCLUSIONS: This study is the first to identify that Map infection can be prevalent in wildlife in New Zealand. There was a high prevalence of Map infection among both scavenging and grazing wild animals. Both mammals and birds are capable of harbouring viable Map organisms in their gastrointestinal tract; further, viable Map was excreted into the environment via faeces by hedgehogs and rabbits.

CLINICAL RELEVANCE: Previous studies overseas have postulated a role of wildlife as reservoirs of Map infection and possible vectors of Johne's disease to livestock. Here, brushtail possums, hedgehogs and rabbits and in particular were identified as potential wildlife hosts for Map infection in NewZealand. This suggests that several wildlife species could contribute to the persistence of Map infection within a wildlife/livestock complex, and potentially, perhaps more importantly, to the spread of infection between farms.     

Mycobacterium avium subsp. paratuberculosis from free-ranging deer and rabbits surrounding Minnesota dairy herds

The objectives of this study were to estimate the prevalence of Mycobacterium avium subsp. paratuberculosis (MAP) among deer and rabbits surrounding infected and noninfected Minnesota dairy farms using fecal culture, and to describe the frequency that farm management practices were used that could potentially lead to transmission of infection between these species. Fecal samples from cows and the cow environment were collected from 108 Minnesota dairy herds, and fecal pellets from free-ranging white-tailed deer and eastern cottontail rabbits were collected from locations surrounding 114 farms; all samples were tested using bacterial culture. In addition, a questionnaire was administered to 114 herd owners. Sixty-two percent of the dairy herds had at least 1 positive fecal pool or environmental sample. A total of 218 rabbit samples were collected from 90% of the herds, and 309 deer samples were collected from 47% of the herds. On 2 (4%) of the farms sampled, 1 deer fecal sample was MAP positive. Both farms had samples from the cow fecal pool and cow environment that were positive by culture. On 2 (2%) other farms, 1 rabbit fecal sample was positive by culture to MAP, with one of these farms having positive cow fecal pools and cow environmental samples. Pasture was used on 79% of the study farms as a grazing area for cattle, mainly for dry cows (75%) and bred or prebred heifers (87%). Of the 114 farms, 88 (77%) provided access to drylot for their cattle, mainly for milking cows (77/88; 88%) and bred heifers (87%). Of all study farms, 90 (79%) used some solid manure broadcasting on their crop fields. Of all 114 farms, the estimated probability of daily physical contact between cattle manure and deer or rabbits was 20% and 25%, respectively. Possible contact between cattle manure and deer or rabbits was estimated to occur primarily from March through December. The frequency of pasture or drylot use and manure spreading on crop fields may be important risk factors for transmission of MAP among dairy cattle, deer, and rabbits. Although the MAP prevalence among rabbits and deer is low, their role as MAP reservoirs should be considered.     

Mycobacterium avium subsp. paratuberculosis infection in an addax (Addax nasomaculatus).

Mycobacterium avium subsp. paratuberculosis was cultured from a single fecal sample collected from a 10-yr-old, captive-bred male addax (Addax nasomaculatus). Attempts to confirm infection with additional fecal cultures, serology, semen culture, and tissue biopsy were unsuccessful. There were no gross lesions on necropsy. On histopathology there were neither acid-fast organisms nor microscopic changes suggestive of active or clinical Johne's disease. Mycobacterium avium subsp. paratuberculosis was isolated from four organ tissues: ileum, jejunum, colon, and mesenteric lymph node.     

Experimental infection of weaner sheep with S strain Mycobacterium avium subsp. paratuberculosis

We sought to determine whether infection of recently weaned 12-16-week-old Merino lambs with an Australian S strain M. a. paratuberculosis, at doses consistent with natural exposure, could be detected in the first few months post-inoculation. Such detection would facilitate the use of weaner sheep as sentinel animals for the presence of infectious doses of M. a. paratuberculosis on pastures. In controlled pen trials, oral doses of approximately 10(7)-10(8) viable organisms were demonstrated to be infective, whereas doses below 10(4) organisms failed to produce detectable infection. Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) was isolated from intestinal and/or lymphoid tissues collected at necropsy 7 or 14 weeks after first infection, but there were no associated gross or microscopic lesions. Skin testing with intradermal Johnin detected all three infected lambs at 13 weeks post-infection, and one of the three infected lambs at 6 weeks post-infection, with 100% specificity. Results for whole blood IFN-gamma assay showed some correlation with infection status but lacked specificity. One infected lamb gave a positive result in an ELISA for antibodies to M. a. paratuberculosis, 14 weeks post-infection and 1 week after skin testing. This was the first demonstration of experimental infection with S strain M. a. paratuberculosis in Australian Merino sheep at doses likely to be representative of natural infection. Culture from tissues in the first few months post-exposure could facilitate the use of naive weaner sheep as tracer animals to detect heavy contamination of pastures with M. a. paratuberculosis, but low-level contamination may not be detected in such a system.     

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.     

Immunological and pathological responses of red deer resistant or susceptible genotypes, to experimental challenge with Mycobacterium avium subsp. paratuberculosis

This study aimed to monitor the clinical, immunological and pathological changes in red deer for 49 weeks after experimental oral challenge with Mycobacterium avium subsp. paratuberculosis (MAP) and to assess the heritability of resistance in the offspring of two red stags. Eighteen young deer, which were bred from unselected hinds and sired by two stags resistant (R) or susceptible (S) to paratuberculosis, were challenged with MAP and monitored for 49 weeks. Biopsy samples of the jejunal lymph node were collected at Weeks 4 and 13 and at necropsy after euthanasia of clinically affected animals or when electively killed at Week 49. Three animals (two S and one R) developed clinical disease and were euthanised. The nine S offspring had significantly more severe lesions than the nine R offspring (Mantel-Haenszel Chi-square P=0.017). The average Lesion Severity Score (LSS) of R offspring was 5.9 (mild), and 7/9 had no or very mild lesions. In contrast, the LSS of S offspring averaged 11.7 (severe), and 7/9 had severe lesions. Most of the resistant, but not the susceptible, animals showed evidence of resolving lesions and a reduction in the number of MAP between 13 and 49 weeks after challenge. One R offspring appeared to completely cure itself, and progressed from mild culture-positive paratuberculosis lesions at Week 13 to having no signs of disease or infection 36 weeks later. This study showed significant heritable resistance/susceptibility to paratuberculosis and key differences in immunological responses in the first 3 months after challenge, indicating different paths to relative success or failure to control MAP. In general, R deer had higher IFN-γ levels, low antibody titres and fewer MAP, while S deer had lower IFN-γ levels, higher antibody and more MAP.     

应用TaqMan荧光定量PCR快速检测鹿血中副结核分枝杆菌

为建立快速检测鹿血中副结核分枝杆菌(Mycobacterium avium subsp. paratuberculosis,MAP)的荧光定量PCR方法,本研究根据GenBank中登录的MAPf57基因序列设计并合成引物及探针,并检测该方法的特异性和敏感性。试验结果显示,该方法具有良好的特异性,对MAP的检测灵敏度可以达到单个菌细胞。对长春地区采集的549份血清样品进行检测,结果显示阳性血清101份,阳性率达到18.4%。本研究结果表明,荧光定量PCR用于动物性产品MAP的检测具有快速、准确的特点。     

Modulation of gammadelta T cells and CD1 in Mycobacterium avium subsp. paratuberculosis infection

M.a. paratuberculosis is the causal agent of paratuberculosis (Johne's disease). Recent work has suggested that gammadelta T cells may play an important role in the early immunological response to mycobacterial diseases, and that CD1 may act as a non-classical MHC molecule in antigen presentation to these gammadelta T cells. Experimental infection of neonatal lambs with M.a. paratuberculosis was used to investigate the changes in gammadelta T cells and CD1 molecules in the gut associated lymphoid tissue 4 weeks after inoculation. Immunohistochemistry was used to label the gammadelta lymphocytes and CD1 molecules. An increase in the number of gammadelta T cells was noted in both the jejunal and ileal Peyer's patches in the gut of infected lambs, but no statistically significant change was found in the mesenteric lymph nodes. There were no obvious changes in the CD1 molecules in any tissue. This work suggests that gammadelta T cells may play a role in the initial immunological events of paratuberculosis infection.     

鹿副结核病抗体间接ELISA检测方法的建立

本研究以副结核杆菌亲和层析抗原为检测抗原,检测以草分枝杆菌抗原吸收的待检鹿血清,建立检测鹿副结核病血清抗体的间接酶联免疫吸附试验,确定其抗原最佳包被浓度为40μg/mL,血清样品稀释度为1:80,兔抗鹿IgG辣根过氧化物酶标记抗体稀释度为1:8000。经特异性试验和重复性试验证明该方法特异性高、重复性好。对不同地区4个鹿场的760头份鹿血清进行副结核病抗体检测,其中阳性61头份,阳性率为8%,获得副结核病在我国鹿群中的血清流行病学资料,从而为防制鹿副结核病提供一定的依据。     

Lewis rats are not susceptible to oral infection with Mycobacterium avium subsp. paratuberculosis

Pathogenesis studies of Mycobacterium avium subsp. paratuberculosis infection in ruminants are hampered by the long incubation time of the disease. A laboratory animal model with a shorter incubation time would facilitate research in this field. Although small rodents are usually considered to be resistant to M.a. paratuberculosis infection, several susceptible murine strains have been found. To our knowledge, there are no detailed reports with regard to susceptibility in rats. The Lewis rat is a valuable model for inflammatory bowel disease studies as well as autoimmune diseases involving mycobacteria as inducing agents. In this study Lewis rats were used to investigate their potential as a small laboratory animal model for paratuberculosis.In total 28 female Lewis rats were orally inoculated with M.a. paratuberculosis. The rats were first inoculated at 3 weeks of age, and 12 more inoculations followed in increasing intervals during the 3 months to follow. Eight control rats received a sham inoculation. Over 9 months, two rats from each group were sacrificed at regular intervals and immunological and histopathological examinations were performed on the gastrointestinal tract, the liver and the spleen.None of the rats developed lesions which were indicative of mycobacterial infection as determined by histology with HE and Ziehl-Neelsen staining. The bacteria could not be recultured from samples taken from the gut, the liver or the spleen. The immunological tests however, showed that bacteria had entered via the intestinal tract. From this study it appears that Lewis rats are resistant to oral inoculation with M. a. paratuberculosis, and not suitable as a model to study the immunopathogenesis of paratuberculosis as it occurs in ruminants.     

Different immune response of pigs to Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis infection

Mycobacterium avium subsp. avium (MAA) and Mycobacterium avium subsp. hominissuis (MAH) are the most common mycobacterial species isolated from granulomatous lesions in swine in countries with controlled bovine tuberculosis. This study is focused on the immunological aspect of MAA and MAH infection in pigs. We detected induction of humoral and cell-mediated immunity in experimentally infected pigs. Specific antibodies were analyzed in serum by ELISA and the IFN-γ release assay was used for evaluation of cell-mediated immunity. While MAA induced a significant increase of both types of immune responses, MAH-infected pigs had an unvarying level of specific antibodies and showed low cell-mediated immunity with high individual variability. The subsequent in vitro experiment confirmed the lower immunogenicity of the MAH strain in comparison to MAA. MAH-infected porcine monocyte-derived macrophages showed a weaker induction of pro-inflammatory mediators in comparison to MAA, which included mRNA for IL-1β, TNF-α, IL-23p19, IL-18 and chemokines CCL-3, CCL-5, CXCL-8 and CXCL-10. Additionally, qualitative proteomic analysis revealed 28 proteins exclusively in MAA and 7 proteins unique to MAH. In conclusion, closely related M. avium subspecies MAA and MAH showed different capacities to stimulate the porcine immune system. From a diagnostic point of view, the IFN-γ release assay showed higher sensitivity than the detection of specific antibodies by ELISA and seems to be an effective tool for discrimination of MAA-infected pigs. In the case of MAH infection, the IFN-γ release assay could fail because of the low immunogenic capacity of the MAH strain.     

Experimental infection of a bovine model with human isolates of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (Map), the etiologic agent of Johne's disease (JD) in ruminants, has been implicated in the pathogenesis of Crohn's disease (CD) in humans. We developed a bovine ileal cannulation model to facilitate comparison of the immune response to Map and the mechanisms of pathogenesis in cattle and humans. Initial studies showed a T cannula could be maintained for up to a year in calves without inducing inflammation or adversely affecting intestinal function. Map introduced through the cannula established a persistent low level of infection without inflammation. Infection elicited an immune response to Map antigens detectable by flow cytometry. Further studies now show the cannulation model can be used with cows during the later stage of infection, affording access to the target tissue at all stages of infection. The studies also revealed no difference in infectivity or immunogenicity of isolates of Map obtained from cattle or humans with CD. Comparison of the immune response to Map during the early and late stages of infection using PCR, flow cytometry and QRT-PCR, showed the immune response early in the disease process is dominated by CD4 T cells. A CD8 response is delayed but comparable at later stages of infection. Genes for pro-inflammatory cytokines IFN-γ and the recently identified genes encoding IL-17 and IL-22 are up regulated in infected animals. These findings reveal that both human and bovine isolates of Map can establish infection and induce similar immune responses in a bovine model. They also reveal the cytokine responses elicited in cattle are similar to those implicated in CD pathogenesis.     

Herd-level prevalence of Mycobacterium avium subsp. paratuberculosis infection in United States dairy herds in 2007

Testing of composite fecal (environmental) samples from high traffic areas in dairy herds has been shown to be a cost-effective and sensitive method for classification of herd status for Mycobacterium avium subsp. paratuberculosis (MAP). In the National Animal Health Monitoring System's (NAHMS) Dairy 2007 study, the apparent herd-level prevalence of MAP was 70.4% (369/524 had ≥1 culture-positive composite fecal samples out of 6 tested). Based on these data, the true herd-level prevalence (HP) of MAP infection was estimated using Bayesian methods adjusting for the herd sensitivity (HSe) and herd specificity (HSp) of the test method. The Bayesian prior for HSe of composite fecal cultures was based on data from the NAHMS Dairy 2002 study and the prior for HSp was based on expert opinion. The posterior median HP (base model) was 91.1% (95% probability interval, 81.6 to 99.3%) and estimates were most sensitive to the prior for HSe. The HP was higher than estimated from the NAHMS Dairy 1996 and 2002 studies but estimates are not directly comparable with those of prior NAHMS studies because of the different testing methods and criteria used for herd classification.     

Fecal shedding of Mycobacterium avium subsp. paratuberculosis by dairy cows

Between 1982 and 2000, fecal samples were obtained from 786 cows that were shedding Mycobacterium avium subsp. paratuberculosis (Map). These cows were resident on 93 Pennsylvania dairies (mean herd size, 64 milk cows) that had no or minimal previous testing for Map. Feces were cultured on four tubes of Herrold's egg yolk medium and the distribution of mean Map colony forming units (CFU) was evaluated. Most cows were light (< 10 CFU/tube, 51.4%) or high (> 50 CFU/tube, 30.8%) fecal shedders with fewer cows in the moderate category (10-50 CFU/tube). Of the 786 cows, 192 (24.4%) had colonies in only one of four tubes. In the multivariable negative binomial model, there were significant associations between mean CFU/tube and prevalence, herd size, and season and an interaction between herd size and season. The linear mixed model of continuous tube counts with a random herd effect yielded similar findings with associations with herd size as a continuous variable, season, and an interaction between categorized prevalence and continuous herd size. Variability in CFU/tube was greatest among cows in the same herd, intermediate for replicate tubes from the same cow, and smallest among cows in different herds. Reduction in the number of replicate tubes from four would have reduced the sensitivity of fecal culture for Map by approximately 6% (for three tubes) to 12% (for two tubes).