Contribution to the diagnosis of Johne's disease in cattle. Comparative studies on the validity of Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test in detecting Mycobacterium paratuberculosis in faeces from cattle

In the present study, 132 selected faecal samples from clinically affected and subclinically infected cattle from dairy herds known to be affected by Johne's disease were investigated for the presence of Mycobacterium paratuberculosis using Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test. The sensitivity was 36.4% for Ziehl-Neelsen staining, 85.6% for faecal culture and 47.7% for the DNA-Probe test. Proving the presence of acid-fast bacteria in 49.3% of the samples from clinically affected cattle and 19.3% of those from subclinically infected cattle, Ziehl-Neelsen staining had the lowest detection rate of the three tests under investigation. Faecal culture showed the highest detection rate of M. paratuberculosis in samples from both clinically affected (84.0%) and subclinically infected (87.7%) animals. The DNA-Probe test showed a positive result in 68.0% of the samples from clinically affected cattle and 21.1% of those from subclinically infected cattle. Ziehl-Neelsen staining proved unreliable in diagnosing Johne's disease. Faecal culture was the most sensitive method for detecting M. paratuberculosis both in clinically affected and subclinically infected cattle. The sensitivity of a commercially available DNA-Probe test has to be enhanced to enable a quick and reliable diagnosis of Johne's disease.     

Contribution to the Diagnosis of Johne’s Disease in Cattle.Comparative Studies on the Validity of Ziehl-Neelsen Staining,Faecal Culture and a Commercially Available DNA-Probe® Testb in Detecting Mycobacterium paratuberculosis in Faeces from Cattle

In the present study, 132 selected faecal samples from clinically affected and subclinically infectedcattle from dairy herds known to be affected by Johne’s disease were investigated for the presence of Mycobacterium paratuberculosis using Ziehl-Neelsen staining, faecal culture and a commercially available DNAProbe ® test. The sensitivity was 36.4% for Ziehl-Neelsen staining, 85.6% for faecal culture and 47.7% for the DNA-Probe® test. Proving the presence of acid-fast bacteria in 49.3% of the samples from clinically affected cattle and 19.3% of those from subclinically infected cattle, Ziehl-Neelsen staining had the lowest detection rate of the three tests under investigation. Faecal culture showed the highest detection rate of M. paratuberculosis in samples from both clinically affected (84.0%) and subclinically infected (87.7%) animals. The DNA-Probe® test showed a positive result in 68.0% of the samples from clinically affected cattle and 21.1% of those from subclinically infected cattle. Ziehl-Neelsen staining proved unreliable in diagnosing Johne’s disease. Faecal culture was the most sensitive method for detecting M. paratuberculosis both in clinically affected and subclinically infected cattle. The sensitivity of a commercially available DNAProbe® test has to be enhanced to enable a quick and reliable diagnosis of Johne’s disease.     

Detection of Mycobacterium paratuberculosis in formalin-fixed paraffin-embedded tissues by the polymerase chain reaction.

The polymerase chain reaction (PCR) utilizing primers specific for the IS900 sequence of Mycobacterium paratuberculosis was applied to tissue sections of formalin-fixed, paraffin-embedded ileum from cattle with Johne's disease and the results compared to those obtained with acid-fast (Ziehl-Neelsen) and immunohistochemical staining. The PCR was positive in 19/21 tissues (90%) while Ziehl-Neelsen staining was positive in 18/21 (86%) and immunohistochemical staining in 21/21 (100%). The Ziehl-Neelsen and immunohistochemical stains are not, however, specific for M. paratuberculosis. The PCR for detection of M. paratuberculosis using the IS900 sequence is a specific and relatively sensitive method for confirmation of Johne's disease and its application to formalin-fixed, paraffin-embedded tissues may be useful for confirmation of dubious cases, for retrospective studies and for epidemiological analyses.     

Paratuberculosis in red deer (Cervus elaphus): an immunohistochemical study

In the present study, we compared the utility of immunohistochemistry with serological and histological results for the characterization of Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) in tissues of affected red deer. Bacterial isolation was considered the standard reference. Samples were taken from seven clinically affected animals with typical macroscopic lesions. The enzyme linked immunosorbent assay (ELISA) and the gel diffusion tests (GD) were used for serological determinations. Samples from intestine and mesenteric lymph nodes were processed for bacterial isolation and histology. M. paratuberculosis was isolated from all the animals. Histologically, lymph nodes displayed necrosis and mineralization at the cortical and medullar areas. Ziehl-Neelsen stained bacteria were numerous inside macrophages and Langhans-type giant cells. Giant and epithelioid cells and lymphocytes were prominent at the ileal mucous membrane. The immunostaining of M. paratuberculosis was very clear inside epithelioid and giant cells. Image analysis was carried out to determine the immunostained area. There was total agreement among the methods employed. Immunohistochemistry can be very useful when the microorganism cannot be recovered from tissues or faeces.     

Skin testing, fecal culture, and lymphocyte immunostimulation in cattle inoculated with Mycobacterium paratuberculosis.

Fourteen calves at 21 days of age were experimentally inoculated with 100 mg (wet weight) of Mycobacterium paratuberculosis. Three calves were inoculated orally, 4 intravenously, and 7 subcutaneously. Lymphocyte immunostimulation, fecal culture, and intradermal tuberculin skin testing were done between 112 to 150 days following exposure. Lymphocyte immunostimulation test results, conducted at 112 days after inoculation, showed all animals positive to Mycobacterium avium purified protein derivative. Fecal culture results, taken at 120 days after inoculation, showed that 2 of 3 animals inoculated intravenously were positive, whereas only 2 of 7 inoculated subcutaneously were positive (8 of 14 total were positive). Intradermal skin testing results at 150 days with M avium purified protein derivative showed 13 of the 14 calves were positive. Calves were examined at necropsy 153 days after inoculation, and M paratuberculosis was isolated from tissues of each of the 14 calves.     

The pathology of spontaneous paratuberculosis in the North American bison (Bison bison)

Gross and histopathologic examinations were performed on 70 North American bison (Bison bison) from a Mycobacterium avium paratuberculosis culture-positive herd. The bison examined were part of a breeding herd totaling 2,800 animals. Eight of 70 (11%) animals had gross findings of intestinal mucosal thickening, and 16 of 70 (23%) of the animals had enlarged mesenteric lymph nodes. Histologic lesions compatible with Johne's disease were diagnosed in 30 of 70 (43%) bison on the basis of the demonstration of noncaseating granulomatous inflammatory infiltrates and of one or more acid-fast bacilli characteristic of Mycobacterium avium paratuberculosis. A suspicious diagnosis of Johne's disease was obtained in 11 of 70 (16%) bison on the basis of the observation of noncaseating granulomatous inflammatory infiltrates without demonstrable acid-fast bacteria. Twenty-nine of 70 (41%) animals were assessed as histologically paratuberculosis free. Histologic results were compared to Johne's disease tests such as culture, serology, and polymerase chain reaction, which were performed on some of the cohort animals.     

The emergence of Mycobacterium paratuberculosis in farmed deer in New Zealand - a review of 619 cases

AIM: To review cases in which Mycobacterium paratuberculosis was identified in farmed deer in New Zealand. METHODS: Case histories were reviewed where M. paratuberculosis was identified in deer by either culture or a polymerase chain reaction (PCR) test using primers from IS900. RESULTS: Between 1986 and 2000, M. paratuberculosis was identified by bacterial culture and/or PCR in 619 farmed deer from 299 herds, representing approximately 6% of deer herds in New Zealand. Over 85% of cases were identified during the last 6 years. In 60% of the infected herds, only one infected animal was identified. The maximum number of cases identified in a single deer herd was 47, and these were identified over a period of 8 years. Only 36 (5.8%) cases came from clinically affected animals identified on farms by veterinarians. The majority (89.7%) of the 619 cases were identified from lesions in mesenteric lymph nodes, including the ileocaecal lymph nodes, identified at meat inspection as being macroscopically either typical or equivocal of bovine tuberculosis (M. bovis). While the overwhelming majority of lesions were identified in mesenteric lymph nodes, M. paratuberculosis was also identified in 27 lesions in lymph nodes of the head, especially the retropharyngeal lymph node. CONCLUSIONS: The figures presented underestimate the true prevalence of infection with M. paratuberculosis, especially since not all suspect cases were submitted for culture or PCR. However, they do show that M. paratuberculosis appears to be spreading in farmed deer in New Zealand and highlight the possibility that Johne's disease is emerging as a potential major problem affecting this species. Identification of the organism by bacterial culture or PCR is required in many cases to distinguish lesions in mesenteric lymph nodes and lymph nodes of the head caused by M. paratuberculosis from those caused by M. bovis and M. avium.     

Bovine fetal infection with Mycobacterium paratuberculosis

In utero transmission of Mycobacterium paratuberculosis, the causative agent of paratuberculosis in cattle, has been suggested. Tissue specimens were obtained at a packing plant from pregnant dairy cows and their fetuses and from cows with clinical signs of paratuberculosis and from their fetuses. Specimens were processed according to methods described for isolating M paratuberculosis from bovine tissues and were incubated on Herrold egg yolk medium for 16 weeks. Presumed positive specimens were confirmed to be M paratuberculosis, using acid-fast staining and subculturing. Of 407 lymph nodes from cows, 34 (8.4%) were culture positive for M paratuberculosis; 9 of 34 (26.4%) of these culture-positive cows had fetuses from which specimens were also culture positive. The results estimated the risk of fetal infection with M paratuberculosis to be 26.4% (95% confidence interval between 11.3 and 40.7%).     

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.     

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.     

The detection of Mycobacterium paratuberculosis in bovine faeces by isolation and the comparison of isolation with the examination of stained smears by light microscopy

The detection of Mycobacterium paratuberculosis organisms in bovine faeces by isolation was compared with that by the microscopical examination of Ziehl-Neelsen stained faecal smears for the presence of clumps of acid-fast M. paratuberculosis organisms. Faeces were obtained from cattle naturally or experimentally infected with M. paratuberculosis as well as from uninfected cattle. Microscopical examination was an unreliable method for the detection of M. paratuberculosis organisms, since the organisms were only detected in 99 (=55.9%) of 177 culturally positive faecal samples. 1111 addition, clumps of acid-fast organisms indistinguishable from M. paratuberculosis were also observed iin three of 18 samples from cattle free from Johne's disease and in 18 of 37 culturally negative samples from paratuberculous cattle. When M. paratuberculosis organisms were added to faeces from an uninfected cow, results showed that isolation attempts should be positive when 15 or more M. paratuberculosis organisms per gram of faeces are present.     

Parallel faecal and organ Mycobacterium avium subsp. paratuberculosis culture of different productivity types of cattle

Faecal (at least 3 months before slaughtering) and organ examinations were carried out in 611 animals (497 dairy, 69 dual-purpose and 44 beef cattle) originating from eight paratuberculosis infected cattle herds. The diagnosis in cattle was established by routine intestinal culture (ileum and the adjacent lymph nodes) after slaughter. In selected 132 animals, post-mortem intensive culture was performed on tissue samples collected from the gastrointestinal tract (duodenum, jejunum, ileum, ileocecal valve, caecum, rectum) and the corresponding lymph nodes, submandibular, retropharyngeal, tracheobronchial, liver and supramammary lymph nodes, kidney, liver and spleen. In 251 (41.1%) of all 611 animals, Mycobacterium avium subspecies paratuberculosis could be isolated from the faeces; in 164 (65.7%) out of 251 shedding animals the infection was detected in the ileum and adjacent lymph nodes. The detection of M. paratuberculosis by routine intestinal culture of faecal culture positive animals varied from 46.0% in animals shedding 1 CFU (colony forming unit), to 94.7% in massive shedders. On the contrary, M. paratuberculosis was detected by routine intestinal culture in 92 (25.5%) of the 360 faecal culture negative animals. Shedding animals had significantly higher (P<0.01) number of organisms in their organs than non-shedding animals. During the intensive tissue cultivation from selected 132 animals, 72 (54.5%) of them were positive. For the negative animals, no significant difference was found between the detection rate in organs examined after slaughter with routine and intensive method. However, in the subgroup of tissue culture positive animals a highly significant difference (P<0.01) was found by intensive examination (83.0%) compared with the routine examination (60.4%). Out of 72 tissue culture positive animals 73.6% of them harboured M. paratuberculosis in the gastrointestinal tract, 16.7% in the gastrointestinal tract and the parenchymatous organs, tracheobronchial and mandibular lymph nodes. The rest of the 9.7% of the infection was detected in the lymph nodes of head and lungs. Our study concerning the distribution of M. paratuberculosis by intensive examinations revealed a minimum effect of breed and production type on localisation of the agent. Thus, the results suggest that in case of an active infection, M. paratuberculosis can be localised in different organs of animals irrespective of their breed or production type.     

Accuracy assessment and screening of a dairy herd with paratuberculosis by three different ELISAs

Although the culture of Mycobacterium avium subsp. paratuberculosisis is the gold standard for the diagnosis of paratuberculosis, this bacterium is difficult to grow. In contrast, serological tests like ELISAs are inexpensive, rapid, and easy to perform. The aims of this study were to evaluate the accuracy of three different ELISAs: one with the commercial antigen PPA-3, another one with L5P (a recently described lipopentapeptide), and a third one with an in-house antigen whole cell lysates (WCL) of M. avium (MAA) strain D4ER (Study 1), and to compare them with other tests for paratuberculosis (PTB) diagnosis (Study 2). In Study 1, the sensitivities of the three ELISAs tested were 74.1%, 37% and 74.1%, respectively, whereas their specificities were 98.9%, 100% and 100%, respectively. In Study 2, we compared the three above-mentioned ELISAs with the intradermal reaction test using Avian PPD (PPDa) and fecal culture associated with Ziehl-Neelsen stain and PCR tests, in a dairy herd with 4.6% of cows with clinical signs of PTB. The results showed that fecal samples from 14 cows (16%) were culture-positive and that fecal samples from nine cows (10%) were PPDa-positive. Most of these animals (culture-positive and PPDa-positive) were detected as positive with any of the three ELISAs tested. Serological results showed that 31% of the animals were positive to ELISA-PPA-3, 17% to ELISA-L5P and 42.5% to ELISA-WCL. The combination of these three ELISAs identified 50.6% of the animals as positive in the infected herd. In particular, the results show that the locally developed ELISA seems to be useful for identifying many infected animals in a herd.     

The pathology of goat paratuberculosis: gross and histopathological lesions in the intestines and mesenteric lymph nodes

From a pathological examination of the intestinal tracts of 1590 goats killed at slaughterhouses in the Fars Province of Iran, 59 cases (3.71%) were suspected, on gross examination, of having paratuberculosis. The diagnosis was confirmed by histopathological study and Ziehl-Neelsen staining of direct smears of rectal faeces. On the basis of severity of involvement of the terminal ileum and mesenteric lymph nodes, the microscopic lesions were classified to mild, moderate and severe forms. Caseous necrosis and calcification were observed only in the mesenteric lymph nodes. High numbers of acid-fast organisms were present in the epithelioid macrophages of the intestine but were inapparent or sparse in the mesenteric lymph nodes. On microscopic examination, 13.5% of the suspected animals were found to have paratuberculosis, in comparison with 3.38% by direct faecal smears. In addition, 30.5% and 15.3% of the animals were diagnosed as having eosinophilic enteritis and linguatulosis, respectively. These findings stress the importance of a careful histopathological examination of the intestines and mesenteric lymph nodes for the diagnosis of paratuberculosis in goats.     

The prevalence of paratuberculosis in culled New England cattle

The prevalence of bovine paratuberculosis in New England was determined to be 18% based on a random survey of abattoir cattle. Six tissues from each of 100 animals were examined by histologic and bacteriologic methods. The cecal lymph node and the ileocecal valve yielded positive cultures most frequently, but isolations were also made from liver, tonsil, colon, and ileum. On the basis of the prevalence data, the economic significance of paratuberculosis to the New England dairy industry was estimated in excess of +15.4 million annually. Field studies identified 25 infected herds in New England. One of the infected herds from Connecticut was sold at public auction, an act which resulted in the dissemination of 95 potentially infected cattle to 28 farms in 8 different states.     

Evaluation of the agar gel immunodiffusion test for diagnosis of subclinical paratuberculosis in cattle

Concurrent bacteriologic culture of feces and agar gel immunodiffusion (AGID) testing was performed on all cows and bred heifers over 14 months old in 10 dairy herds during a 32-month period to determine the effectiveness of the AGID test for the detection of subclinical paratuberculosis. Herds were sampled 5 times and, when possible, culled animals were tested again at slaughter. During 5 herd-wide samplings, Mycobacterium paratuberculosis was isolated from 139 fecal specimens obtained from 109 cattle. Results of the AGID test were simultaneously positive 40 of 139 times (28.8%). Thirty-six of the 109 cattle (33.0%) determined to be infected had a positive AGID test result at some point during the 5 herd-wide samplings. When results of tests performed at time of slaughter were included, 117 cattle were identified as infected by culture methods; 55 of these (47.0%) were AGID test-positive at some point during the study. The upper limit of the maximal false-positive rate for the AGID test was 2.1%. On the basis of colony counts from cultures, subclinically infected cows shedding higher numbers of M paratuberculosis in their feces were more likely to have positive AGID test results (P less than 0.0001). In known infected cattle, neither the culture nor AGID test results were consistently positive on repeated testing. Of 48 official calfhood paratuberculosis vaccinates tested as adults, 3 had positive AGID test results and in 1 of these, M paratuberculosis was also isolated from the feces, indicating that the rate of false-positive AGID test results in calfhood vaccinates is low.     

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.     

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.     

Polymerase chain reaction identification of Mycobacterium avium in formalin-fixed, paraffin-embedded animal tissues.

A PCR procedure previously developed for identification of Mycobacterium bovis in formalin-fixed tissues was used to identify mycobacteria of the M. avium complex. Tissues were examined from 100 culture-positive cases of M. avium complex infection, including 86 in which the subspecies was not identified and 14 that had been identified as M. avium subsp. paratuberculosis. Each sample was tested with 5 primer sets, 16S ribosomal RNA (rRNA), IS900, IS901, IS1245, and a heat shock protein (hspX), that detect 1 or both M. avium subspecies. The success rate of PCR detection varied with the primers used and the animal species tested. Among the 86 cases with no M. avium subspecies designation, primers for the 16S rRNA gene were clearly the most efficient because they produced amplicons from all samples that reacted with any other primer set. The overall detection rate in this group of samples was 71%: highest in avian tissues (89%) followed by swine (72%) and ruminants (57%) None of the avian or swine tissues reacted with primers for IS900 or hspX, which identify M. a. paratuberculosis. In contrast, 7 of the 12 ruminant samples that were 16S rRNA positive reacted with 1 or both of these primers. All of the 14 cases shown by culture to be M. a. paratuberculosis infections were positive with IS900 primers, whereas only 11 were positive for 16S rRNA. These results indicate that 16S rRNA primers are the most useful for PCR identification of M. avium in formalin-fixed tissues of nonruminant species. However, IS900 primers should also be used when ruminant tissues are examined because these primers provide the greatest sensitivity for detection of M. a. paratuberculosis infections.