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.     

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.     

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.     

Immunohistochemical localization of galectin-3 in the granulomatous lesions of paratuberculosis-infected bovine intestine

The presence of galectin-3 was immunohistochemically quantified in bovine intestines infected with paratuberculosis (Johne''s disease) to determine whether galectin-3 was involved in the formation of granulation tissue associated with the disease. Mycobacterium avium subsp. paratuberculosis infection was histochemically confirmed using Ziehl-Neelsen staining and molecularly diagnosed through rpoB DNA sequencing. Galectin-3 was detected in the majority of inflammatory cells, possibly macrophages, in the granulomatous lesions within affected tissues, including the ileum. These findings suggest that galectin-3 is associated with the formation of chronic granulation tissues in bovine paratuberculosis, probably through cell adhesion and anti-apoptosis mechanisms.     

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.     

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.     

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.     

Comparison of diagnostic detection methods for Mycobacterium avium subsp. paratuberculosis in North American bison

Tissues and fecal material were collected from 14 North American bison (Bison bison) that were suspected of having Johne's disease and analyzed for the presence of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis). Sections of ileum, ileal-cecal lymph node, and three sequential sections of jejunum with their associated mesenteric lymph nodes were taken from each animal. Fecal culture indicated that 5 of 14 (35.7%) animals were infected, whereas cultures from tissues detected 12 of 14 (85.7%) animals as infected and 59 of 111 (53.2%) of the tissues as positive for M. paratuberculosis. Polymerase chain reaction analysis identified infection in 14 of 14 (100%) animals and in 91 of 112 (81.2%) tissues. In addition, tissues were processed for Ziehl-Neelsen acid-fast staining, auramine O/acridine orange fluorescent staining, and immunohistochemical staining. Ziehl-Neelsen and auramine O staining identified 7 of 14 (50%) and 5 of 14 (35.7%) animals as infected and 24 of 112 (21.4%) and 28 of 112 (25%) tissues as positive, respectively. Immunohistochemical analyses of bison tissues, using antisera collected from rabbits immunized with four different preparations of M. paratuberculosis, identified a greater percentage of infected animals (ranging from 57 to 93%) and positive tissues (ranging from 28 to 46%). Collectively, these data indicate that DNA-based detection of M. paratuberculosis was more sensitive than bacterial culture or staining, identified infection in all the bison, and detected the greatest number of positive tissues within each animal.     

Immunohistochemical methods for the visualization of Mycobacterium paratuberculosis in bovine tissues

The peroxidase-antiperoxidase (PAP), streptavidin-biotin (SB), and avidin-biotin-complex (ABC) techniques have been evaluated for the visualization of Mycobacterium paratuberculosis (Mp) in formalin-fixed, paraffin-embedded bovine tissues. The used immunoperoxidase techniques were comparatively better than the Ziehl-Neelsen stain, specially for the demonstration of small number of mycobacteria in tissue sections.     

Disseminated Mycobacterium paratuberculosis infection in a cow

A cow with chronic diarrhea and weight loss caused by localization of Mycobacterium paratuberculosis in the intestinal tract (Johne's disease) had gross and microscopic changes indicative of a disseminated infection. A direct association between the remote lesions and the intestinal infection was shown by isolation of M paratuberculosis from renal tissue, detection of intracellular M paratuberculosis antigen(s), using an indirect immunoperoxidase method, and by the characteristic granulomatous nature of the lesions. This case illustrates the potential for extra-intestinal lesions in M paratuberculosis infection of cattle and should cause veterinarians to consider mycobacterial disease when confronted with multinodular lesions of the bovine kidney. The immunoperoxidase method was useful in determining the cause of the inflammatory lesion in which intact organisms were not evident.     

Histopathology of grossly normal mesenteric lymph nodes of New Zealand farmed red deer (Cervus elaphus) including identification of lipopigment

This article describes the histopathology of grossly normal mesenteric lymph nodes (MLNs) of New Zealand farmed red deer (Cervus elaphus). Eighty MLNs were sourced from 10 deer from 5 North Island herds and 5 South Island herds classified as low risk and high risk of Mycobacterium avium subspecies paratuberculosis (MAP) infection, respectively. Fixed sections were stained with hematoxylin and eosin; Ziehl-Neelsen; and, selectively, periodic acid-Schiff, Perl's, and Sudan black. Positive Ziehl-Neelsen stain, follicular hyperplasia, capsular eosinophil infiltration, focal granulomas, foci of macrophages containing lipopigment, parasitic granulomas, and calcified foci are described and severity graded where appropriate. Animal age, sex, and herd of origin are variably associated with the presence of one or more features. Trabecular fibrosis and dilated edema-filled sinusoids are described. These observations allow differentiation between likely nonpathologic histologic features in deer MLNs and features possibly attributable to infection with a pathogen such as MAP.     

Negative staining method with nigrosin for the detection of cryptosporidial oocysts: a comparative study

A comparison was made between two methods for the detection of cryptosporidial oocysts in human faecal sediments of formalin-ether concentrates: a cover-slipped, wet method (containing Gram's iodine) and an air-dried, negative staining method using nigrosin solution. A modified Ziehl-Neelsen technique was used as a reference method. The negative staining method with nigrosin gave a positive diagnosis more often than the cover-slipped wet method. A comparison of the nigrosin method with the modified Ziehl-Neelsen method gave almost identical results. Restaining the nigrosin slides with the modified Ziehl-Neelsen technique showed that the round, refractile bodies were cryptosporidial occysts.     

Effects of altered dietary iron intake in Mycobacterium paratuberculosis-infected dairy cattle: sequential observations on growth, iron and copper metabolism and development of paratuberculosis

Twenty calves were orally inoculated with Mycobacterium paratuberculosis at six weeks old. At six months old, 10 of these, plus four uninfected controls were maintained on limited dietary copper and supplemented iron intake for a further 27 months. During this time all these animals, together with a further four untreated controls, were bred before being killed and examined for evidence of paratuberculosis. Despite significant reduction in weight gain, attributable to both iron supplementation and infection, no significant difference was found in the numbers of iron-supplemented and unsupplemented animals that developed clinical signs nor in the extent and severity of intestinal lesions between groups. Accumulation of iron in paratuberculosis lesions was not affected by iron supplementation but was positively correlated with the frequency of shedding of M paratuberculosis in faeces (P less than 0.05). Dietary iron supplementation alone resulted in serum hyperferraemia, hepatic siderosis and slight hypocuprosis, whereas, in infected animals, this resulted in marked hypocuprosis and anaemia within groups (P less than 0.05). Infection alone resulted in serum hypoferraemia and intestinal and hepatic siderosis which was positively correlated with the severity of infection within groups (P less than 0.05). Susceptibility to paratuberculosis may result from failure ultimately to limit monokine-mediated iron sequestration in intestinal tissue.     

Detection of Mycobacterium avium subsp. paratuberculosis in intestinal and lymph node tissues of water buffaloes (Bubalus bubalis) by PCR and bacterial culture

The efficacy of bacterial culture and IS900-specific polymerase chain reaction (PCR) was compared for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) from the intestinal and mesenteric lymph node tissues of water buffaloes (Bubalus bubalis) showing lesions of paratuberculosis (Johne's disease). Out of 20 (4.9%) animals showing histological lesions suggestive of paratuberculosis, 14 (70%) and 6 (30%) were positive in the PCR and bacterial culture, respectively. The results of this study suggested that PCR was more sensitive than bacterial culture in detection of subclinical paratuberculosis in water buffaloes. The bacterial concentration from large amount of tissues by differential and density gradient centrifugation method was found to facilitate the diagnosis by smear examination and PCR. The specificity of the PCR was confirmed by the product size and restriction digestion pattern of the amplicons. The sequence analysis of the amplified products (626bp of IS900 gene) from buffalo strain showed more than 97% homology with the published sequences.     

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.     

A simple, rapid, and effective method for the extraction of Mycobacterium paratuberculosis DNA from fecal samples for polymerase chain reaction.

Diagnosis of paratuberculosis (Johne's disease) is stymied by the lack of 1 diagnostic tool that can be used to detect both subclinically and clinically infected animals. At present, fecal culture remains the single diagnostic test that can detect infection in both disease states provided the animals actively shed Mycobacterium paratuberculosis in their feces. Yet, fecal culture has a disadvantage associated with the protracted incubation period of 8-16 weeks before results are available. Detection of nucleic acids specific to M. paratuberculosis in fecal samples is a technique that can circumvent the culture method. This study describes a rapid, simple, and effective method to extract DNA from fecal samples and modification of a polymerase chain reaction assay for optimal sensitivity of detection. An evaluation of 1,000 well-characterized fecal samples was performed by the Colorado Department of Agriculture (Denver, CO) and the National Animal Disease Center (Ames, IA) to determine the sensitivity, specificity, and reproducibility of the new method. Results from this study show that the sensitivity of detection was highly dependent on the load of bacteria in the fecal sample with 81% detection of samples containing >70 colony-forming units (cfu)/g of feces and a 45% detection rate for samples containing less than 1 cfu/g. Similarly, reproducibility of the technique between the 2 laboratories (n = 250 samples) was much higher (75%) for the fecal samples containing high levels of M. paratuberculosis and reduced to 25% for samples with less than 1 cfu/g. An overall specificity of 83% was obtained for known negative samples. The method described here is rapid, simple, and inexpensive compared with other techniques. In addition, this method can detect animals that are shedding less than 1 cfu/g.     

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.     

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.