Detection and verification of Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn's disease

Mycobacterium avium subsp. paratuberculosis is a robust and phenotypically versatile pathogen which causes chronic inflammation of the intestine in many species, including primates. M. avium subsp. paratuberculosis infection is widespread in domestic livestock and is present in retail pasteurized cows' milk in the United Kingdom and, potentially, elsewhere. Water supplies are also at risk. The involvement of M. avium subsp. paratuberculosis in Crohn's disease (CD) in humans has been uncertain because of the substantial difficulties in detecting this pathogen. In its Ziehl-Neelsen staining-negative form, M. avium subsp. paratuberculosis is highly resistant to chemical and enzymatic lysis. The present study describes the development of optimized sample processing and DNA extraction procedures with fresh human intestinal mucosal biopsy specimens which ensure access to M. avium subsp. paratuberculosis DNA and maximize detection of these low-abundance pathogens. Also described are two nested PCR methodologies targeted at IS900, designated IS900[L/AV] and IS900[TJ1-4], which are uniquely specific for IS900. Detection of M. avium subsp. paratuberculosis in mucosal biopsy specimens was also evaluated by using mycobacterial growth indicator tube (MGIT) cultures (Becton Dickinson). IS900[L/AV] PCR detected M. avium subsp. paratuberculosis in 34 of 37 (92%) patients with CD and in 9 of 34 (26%) controls without CD (noninflammatory bowel disease [nIBD] controls) (P = 0.0002; odds ratio = 3.47). M. avium subsp. paratuberculosis was detected by IS900[L/AV] PCR in MGIT cultures after 14 to 88 weeks of incubation in 14 of 33 (42%) CD patients and 3 of 33 (9%) nIBD controls (P = 0.0019; odds ratio = 4.66). Nine of 15 (60%) MGIT cultures of specimens from CD patients incubated for more than 38 weeks were positive for M. avium subsp. paratuberculosis. In each case the identity of IS900 from M. avium subsp. paratuberculosis was verified by amplicon sequencing. The rate of detection of M. avium subsp. paratuberculosis in individuals with CD is highly significant and implicates this chronic enteric pathogen in disease causation.     

Insertion sequence IS900 revisited

Many studies investigating Mycobacterium avium subsp. paratuberculosis in Crohn's disease have used molecular detection of IS900 in clinical samples, but some have described polymorphisms in IS900 as variants of this organism. Analysis of 23 M. avium subsp. paratuberculosis isolates revealed that IS900 is highly conserved, with only two sequevars distinguishing sheep and cattle lineages. Amplification of IS900-like sequences is not sufficient as a proxy for M. avium subsp. paratuberculosis.     

Results of multiple diagnostic tests for Mycobacterium avium subsp. paratuberculosis in patients with inflammatory bowel disease and in controls

Mycobacterium avium subsp. paratuberculosis has been incriminated as a cause of Crohn's disease (CD); however, studies to date have been relatively small and generally only used a single diagnostic assay. The objective of the study was to reexamine the association of M. avium subsp. paratuberculosis and CD using multiple diagnostic tests. Five methods were used to detect M. avium subsp. paratuberculosis infections in 439 inflammatory bowel disease (IBD) patients and 324 control subjects in the United States and Denmark. Most assays were adaptations of diagnostic tests for this infection performed routinely on animals. PCR for IS900, a genetic element unique to M. avium subsp. paratuberculosis, was positive significantly more often on resected bowel and lymph node tissues from CD patients (19.0%) and ulcerative colitis (UC) patients (26.2%) than from controls (6. 3%) (P < 0.05). Positive IS900 PCR results occurred more often in U. S. than in Danish IBD patients, 32.0 versus 13.3% (P = 0.025). The majority of Danish patients were bacillus Calmette-Guérin (Mycobacterium bovis BCG) vaccinated (CD, 77.5%; UC, 86.6%; controls, 83.0%) whereas none of the U.S. patients with IBD and only 2% of U. S. controls were vaccinated. Among Danish IBD patients, positive PCR findings were four times more common among subjects who were not BCG vaccinated (33.3%) than among BCG vaccinates (8.8%, P = 0.02). Culture of the same tissues tested by PCR using modified BACTEC 12B medium failed to grow M. avium subsp. paratuberculosis from patients or controls. U.S. CD patients had the highest serological evidence (enzyme-linked immunosorbent assay [ELISA] for serum antibodies) of M. avium subsp. paratuberculosis infection (20.7% of patients positive) which was higher than for all UC patients studied (6.1%) or healthy controls (3.8%, P < 0.005). Among Danish patients alone, however, no significant differences in rates of ELISA-positive results among CD, UC, or control patients were found. For 181 study subjects, both IS900 PCR and ELISA were performed. Although 11 were ELISA positive and 36 were PCR positive, in no instance was a patient positive by both tests, suggesting that these states are mutually exclusive. Evaluation of cytokine-mediated immune responses of IBD patients was complicated by the influence of immunosuppressive therapy given most IBD patients. Gamma interferon (IFN-gamma) release by peripheral blood leukocytes after M. avium purified protein derivative PPD antigen stimulation showed significantly lower responses in CD patients than in UC patients or controls in both U.S. (by ex vivo assay) and Danish (by in vitro assay) populations (P < 0.05). Interleukin-5 responses were not different among CD, UC, or control groups. Collectively, the PCR, ELISA, and IFN-gamma tests for M. avium subsp. paratuberculosis together with the unexpected observation that BCG vaccination influenced M. avium subsp. paratuberculosis detection, lead us to conclude that M. avium subsp. paratuberculosis, or some similarly fastidious mycobacterial species, infects at least a subset of IBD patients. Whether the infection is primary (causal) or secondary, it may contribute to the etiopathogenesis of IBD.     

Use of multilocus variable-number tandem-repeat analysis for typing Mycobacterium avium subsp. paratuberculosis

The etiology of Crohn's disease in humans is largely unknown. Clinical signs of Crohn's disease partly resemble the clinical picture of Johne's disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis. Because of the high prevalence of these bacteria in (products of) ruminants and their remarkable thermostability, concern has been raised about the possible role of these bacteria in the pathogenesis of Crohn's disease. In an attempt to develop a molecular typing method to facilitate meaningful comparative DNA fingerprinting of M. avium subsp. paratuberculosis isolates from the human and animal reservoirs, multilocus variable-number tandem-repeat analysis (MLVA) was explored and compared to IS900 restriction fragment length polymorphism (RFLP) typing. MLVA typing subdivided the most predominant RFLP type, R01, into six subtypes and thus provides a promising molecular subtyping approach to study the diversity of M. avium subsp. paratuberculosis.     

Highly specific and quick detection of Mycobacterium avium subsp. paratuberculosis in feces and gut tissue of cattle and humans by multiple real-time PCR assays

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease (JD) in cattle and may be associated with Crohn's disease (CD) in humans. It is the slowest growing of the cultivable mycobacteria, and culture from clinical, veterinary, food, or environmental specimens can take 4 months or even longer. Currently, the insertion element IS900 is used to detect M. avium subsp. paratuberculosis DNA. However, closely related IS900 elements are also present in other mycobacteria, thus limiting its specificity as a target. Here we describe the use of novel primer sets derived from the sequences of two highly specific single copy genes, MAP2765c and MAP0865, for the quantitative detection of M. avium subsp. paratuberculosis within 6 h by using real-time PCR. Specificity of the target was established using 40 M. avium subsp. paratuberculosis isolates, 67 different bacterial species, and two intestinal parasites. Using the probes and methods described, we detected 27 (2.09%) M. avium subsp. paratuberculosis-positive stool specimens from 1,293 individual stool samples by the use of either IS900 or probes deriving from the MAP2765c and MAP0865 genes described here. In general, bacterial load due to M. avium subsp. paratuberculosis was uniformly low in these samples and we estimated 500 to 5,000 M. avium subsp. paratuberculosis bacteria per gram of stool in assay-positive samples. Thus, the methods described here are useful for rapid and specific detection of M. avium subsp. paratuberculosis in clinical samples.     

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: IS900 restriction fragment length polymorphism and IS1311 polymorphism analyses of isolates from animals and a human in Australia

The distribution and prevalence of strains of Mycobacterium avium subsp. paratuberculosis were determined among sheep, cattle, and other species with Johne's disease in Australia. A total of 328 isolates were evaluated from numerous farms in New South Wales, Victoria, Tasmania, and South Australia, Australia. Restriction fragment length polymorphism (RFLP) analysis of genomic DNA using BstEII and an IS900 probe and IS1311 polymorphism analysis using PCR and restriction endonuclease analysis (PCR-REA) was used to classify isolates as cattle (C) or sheep (S) strains. IS1311 PCR-REA provided similar information to IS900 RFLP analysis but was more useful than RFLP analysis where DNA was degraded or scarce. Twelve IS900 RFLP types were found. Johne's disease in sheep was always due to S strains, while cattle were infected only with C strains. RFLP type S1 was the dominant strain in sheep in New South Wales (97% of isolates) and was the only strain found in sheep from Victoria. Seven RFLP types were present in cattle. RFLP types C3 and C1 were most common (collectively, 85% of isolates), but C1 was not found in New South Wales and C3 was present in dairy cattle but not in beef cattle in Victoria. These differences may be explained by restricted livestock trading patterns between different segments of the cattle industry. Up to five RFLP types were present in some geographic regions in Victoria, while up to three RFLP types were found among cattle on some farms. Individual cattle usually were infected with only one RFLP type, but one animal was infected with both C5 and CU4. Two isolates from goats were C type as were three from alpacas, one from a rhinoceros, and two from a human with Crohn's disease. The prevalences of specific RFLP types in Australia differ from those reported in Europe and elsewhere. Given the existence of geographical and farm enterprise differences in IS900 RFLP type, this technique may be applied selectively to trace the spread of Johne's disease, at least in the cattle industries. As these observations reflect past exposure of livestock to M. avium subsp. paratuberculosis, the monitoring of strains present in animals in Australia is continuing.     

Absence of Mycobacterium avium subsp. paratuberculosis in the microdissected granulomas of Crohn's disease.

The etiology of Crohn's disease remains unknown with inflammatory, infectious, and/or genetic causes suspected. Granulomatous inflammation is a characteristic feature of the disorder, resembling the tissue response to mycobacterium. Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent in Johne's disease, a chronic ulcerative intestinal condition in cattle, and has been implicated as a likely candidate. We carefully microdissected the granulomas from the paraffin-embedded resection specimens of 18 patients with well-established Crohn's disease. The DNA obtained was PCR amplified for the IS900 and IS1311 repeat elements of MAP, PCR product size maintained at 101 and 124 base pairs, respectively. Archival tissue from bovine Johne's disease was used as a positive control. MAP-specific DNA, confirmed by sequencing and comparison with prototype strain sequence, was appropriately amplified from the positive control. None of the Crohn's disease cases yielded a positive amplification product, failing to support a role for the organism in the pathogenesis of this illness.     

Experimental infection model for Johne's disease in sheep

Johne's disease in ruminants results in chronic enteritis caused by the pathogenic bacterium Mycobacterium avium subsp. paratuberculosis. This study examined two M. avium subsp. paratuberculosis strains (JD3 and W), using different doses and routes of infection, to establish the optimal time postchallenge when predictable levels of infection, gut lesions, and clinical disease occur in a large proportion of sheep. While a small proportion (25%) of sheep challenged with a low-passage-number laboratory culture of M. avium subsp. paratuberculosis (strain W) became infected, no infection was found in animals exposed to a high-passage-number culture isolate of strain W. In contrast, a primary tissue homogenate of M. avium subsp. paratuberculosis (JD3) resulted in high (90%) infection rates and gut histopathology following oral or intratonsillar challenge. The optimal conditions necessary to produce Johne's disease involve oral inoculation of 3-month-old lambs with four doses of 5 x 10(8) CFU of M. avium subsp. paratuberculosis isolated directly from the gut lymphatic tissues of clinically affected sheep. This resulted in consistent gut histopathology at 9 months and the onset of clinical disease by 11 months postchallenge.     

Study of animal-borne infections in the mucosas of patients with inflammatory bowel disease and population-based controls

Crohn's disease may be triggered by an infection, and it is plausible to consider that such an infection may be animal borne and ingested with our food. There has been considerable interest in the past in determining whether Mycobacterium avium subsp. paratuberculosis (M. avium) might be the etiologic agent in Crohn's disease since it causes a disease in cattle that is similar to Crohn's disease in humans. We aimed to determine if there was an association between Crohn's disease and infection with M. avium or other zoonotic agents and compared the findings with those for patients with ulcerative colitis, unaffected siblings of Crohn's disease patients, or population-based controls without inflammatory bowel disease. Patients under age 50 years with Crohn's disease or ulcerative colitis, unaffected siblings of patients, or healthy controls drawn from a population-based age- and gender-matched registry were enrolled in a study in which subjects submitted to a questionnaire survey and venipuncture. A nested cohort underwent colonoscopy plus biopsy. Samples were batched and submitted to PCR for the detection of M. avium and other zoonotic agents known to cause predominately intestinal disease in cattle, sheep, or swine. Only one patient with ulcerative colitis, no patients with Crohn's disease, and none of the sibling controls were positive for M. avium, whereas 6 of 19 healthy controls were positive for M. avium. Since the control subjects were significantly older than the case patients, we studied another 11 patients with inflammatory bowel disease who were older than age 50 years, and another single subject with ulcerative colitis was positive for M. avium. One other subject older than age 50 years with ulcerative colitis was positive for circovirus, a swine-borne agent of infection. In conclusion, by performing PCR with mucosal samples from patients with Crohn's disease and controls, no association between Crohn's disease and infection with M. avium or any of the other six zoonotic agents studied could be found.     

Evaluation of in situ methods used to detect Mycobacterium avium subsp. paratuberculosis in samples from patients with Crohn's disease

In common with other diagnostic tests, detection of mycobacteria in tissue by microscopic examination is susceptible to spectrum bias. Since Crohn's disease is defined by the absence of detectable pathogenic organisms, the use of in situ techniques to search for Mycobacterium avium subsp. paratuberculosis in Crohn's disease samples requires validation of methods in a paucibacillary setting. To generate paucibacillary infection, C57BL/6 mice were artificially infected with Mycobacterium avium subsp. paratuberculosis strain K10 and M. tuberculosis H37Rv, yielding tissues harboring fewer than one bacillus per oil immersion field. Serial sections of organs were then studied by cell wall-based staining techniques (Ziehl-Neelsen and auramine rhodamine) and nucleic acid-based staining techniques (in situ hybridization [ISH] and indirect in situ PCR [IS PCR]). Microscopic examination and measurement of morphometric parameters of bacilli revealed that for all methods, Mycobacterium avium subsp. paratuberculosis bacilli were observed to be shorter, smaller, and less rod shaped than M. tuberculosis bacilli. Ziehl-Neelsen, auramine rhodamine stains, ISH targeting rRNA, and IS-PCR targeting the IS900 element afforded comparable sensitivities, but for all methods, visualization of individual bacterial forms required magnification x1,000. Auramine rhodamine staining and IS-PCR generated positive signals in negative controls, indicating the nonspecificity of these assays. Together, our results indicate that detection of Mycobacterium avium subsp. paratuberculosis bacilli in tissue requires oil immersion microscopy, that rRNA-ISH provides sensitivity and specificity comparable to those of Ziehl-Neelsen staining, and that the microscopic detection limit for Mycobacterium avium subsp. paratuberculosis in tissue is governed more by bacterial burden than by staining method.     

Current perspectives on Mycobacterium avium subsp. paratuberculosis, Johne's disease, and Crohn's disease: a review

Mycobacterium avium subsp. paratuberculosis (MAP) causes the disease of cattle, Johne's. The economic impact of this disease includes early culling of infected cattle, reduced milk yield, and weight loss of cattle sold for slaughter. There is a possible link between MAP and Crohn's disease, a human inflammatory bowel disease. MAP is also a potential human food borne pathogen because it survives current pasteurization treatments. We review the current knowledge of MAP, Johne's disease and Crohn's disease and note directions for future work with this organism including rapid and economical detection, effective management plans and preventative measures.     

Identification of Mycobacterium avium subsp. paratuberculosis in Biopsy Specimens from Patients with Crohn's Disease Identified by In Situ Hybridization

Crohn's disease is a chronic inflammatory disease of the gastrointestinal tract of unknown etiology. We report on the presence of cell wall-deficient Mycobacterium avium subsp. paratuberculosis in 35 of 48 paraffin-embedded tissue specimens from 33 patients with Crohn's disease by in situ hybridization with IS900 as a probe.     

Mycobacterium avium subsp. paratuberculosis strains isolated from Crohn's disease patients and animal species exhibit similar polymorphic locus patterns

Analysis of short sequence repeats of Mycobacterium avium subsp. paratuberculosis isolated from Crohn's disease patients identified two alleles, both of which clustered with strains derived from animals with Johne's disease. Identification of a limited number of genotypes among human strains implies the existence of human disease-associated genotypes and strain sharing with animals.     

Invasion and persistence of Mycobacterium avium subsp. paratuberculosis during early stages of Johne's disease in calves

Infection with Mycobacterium avium subsp. paratuberculosis causes Johne's disease in cattle and is a serious problem for the dairy industry worldwide. Development of models to mimic aspects of Johne's disease remains an elusive goal because of the chronic nature of the disease. In this report, we describe a surgical approach employed to characterize the very early stages of infection of calves with M. avium subsp. paratuberculosis. To our surprise, strains of M. avium subsp. paratuberculosis were able to traverse the intestinal tissues within 1 h of infection in order to colonize distant organs, such as the liver and lymph nodes. Both the ileum and the mesenteric lymph nodes were persistently infected for months following intestinal deposition of M. avium subsp. paratuberculosis despite a lack of fecal shedding of mycobacteria. During the first 9 months of infection, humoral immune responses were not detected. Nonetheless, using flow cytometric analysis, we detected a significant change in the cells participating in the inflammatory responses of infected calves compared to cells in a control animal. Additionally, the levels of cytokines detected in both the ileum and the lymph nodes indicated that there were TH1-type-associated cellular responses but not TH2-type-associated humoral responses. Finally, surgical inoculation of a wild-type strain and a mutant M. avium subsp. paratuberculosis strain (with an inactivated gcpE gene) demonstrated the ability of the model which we developed to differentiate between the wild-type strain and a mutant strain of M. avium subsp. paratuberculosis deficient in tissue colonization and invasion. Overall, novel insights into the early stages of Johne's disease were obtained, and a practical model of mycobacterial invasiveness was developed. A similar approach can be used for other enteric bacteria.     

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: evidence for limited strain diversity,strain sharing,and identification of unique targets for diagnosis

The objectives of this study were to understand the molecular diversity of animal and human strains of Mycobacterium avium subsp. paratuberculosis isolated in the United States and to identify M. avium subsp. paratuberculosis-specific diagnostic molecular markers to aid in disease detection, prevention, and control. Multiplex PCR of IS900 integration loci (MPIL) and amplified fragment length polymorphism (AFLP) analyses were used to fingerprint M. avium subsp. paratuberculosis isolates recovered from animals (n = 203) and patients with Crohn's disease (n = 7) from diverse geographic localities. Six hundred bacterial cultures, including M. avium subsp. paratuberculosis (n = 303), non-M. avium subsp. paratuberculosis mycobacteria (n = 129), and other nonmycobacterial species (n = 168), were analyzed to evaluate the specificity of two IS900 integration loci and a newly described M. avium subsp. paratuberculosis-specific sequence (locus 251) as potential targets for the diagnosis of M. avium subsp. paratuberculosis. MPIL fingerprint analysis revealed that 78% of bovine origin M. avium subsp. paratuberculosis isolates clustered together into a major node, whereas isolates from human and ovine sources showed greater genetic diversity. MPIL analysis also showed that the M. avium subsp. paratuberculosis isolates from ovine and bovine sources from the same state were more closely associated than were isolates from different geographic regions, suggesting that some of the strains are shared between these ruminant species. AFLP fingerprinting revealed a similar pattern, with most isolates from bovine sources clustering into two major nodes, while those recovered from sheep or humans were clustered on distinct branches. Overall, this study identified a high degree of genetic similarity between M. avium subsp. paratuberculosis strains recovered from cows regardless of geographic origin. Further, the results of our analyses reveal a relatively higher degree of genetic heterogeneity among M. avium subsp. paratuberculosis isolates recovered from human and ovine sources.     

Isolation of high-affinity single-chain antibodies against Mycobacterium avium subsp. paratuberculosis surface proteins from sheep with Johne's disease

Johne's disease, caused by infection with Mycobacterium avium subsp. paratuberculosis, causes significant economic losses to the livestock farming industry. Improved investigative and diagnostic tools-necessary to understand disease processes and to identify subclinical infection-are much sought after. Here, we describe the production of single-chain antibodies with defined specificity for M. avium subsp. paratuberculosis surface proteins. Single-chain antibodies (scFv) were generated from sheep with Johne's disease by cloning heavy-chain and lambda light-chain variable regions and expressing these in fusion with gene III of filamentous phages. Two scFv clones (designated SurfS1.2 and SurfS2.2) were shown to be immunoreactive against M. avium subsp. paratuberculosis surface targets by flow cytometry, and immunoblotting identified specificity for a 34-kDa proteinase-susceptible determinant. Both antibodies were cross-reactive against Mycobacterium avium subsp. avium but nonreactive against Mycobacterium bovis or Mycobacterium phlei cells and were shown to be capable of enriching M. avium subsp. paratuberculosis cells by a factor of approximately 10(6)-fold when employed in magnetic bead separation of mixed Mycobacterium sp. cultures. Further, magnetic bead separation using SurfS1.2 and SurfS2.2 was capable of isolating as few as 10(3) M. avium subsp. paratuberculosis cells from ovine fecal samples, indicating the diagnostic potential of these reagents. Finally, inclusion of SurfS1.2 or SurfS2.2 in in vitro broth culture with M. avium subsp. paratuberculosis indicated that surface binding activity did not impede bacterial growth, although colony clumping was prevented. These results are discussed in terms of the potential use of single-chain phage display monoclonal antibodies as novel diagnostic reagents.     

Rapid and sensitive detection of Mycobacterium avium subsp. paratuberculosis in bovine milk and feces by a combination of immunomagnetic bead separation-conventional PCR and real-time PCR

Immunomagnetic bead separation coupled with bead beating and real-time PCR was found to be a very effective procedure for the isolation, separation, and detection of Mycobacterium avium subsp. paratuberculosis from milk and/or fecal samples from cattle and American bison. Samples were spiked with M. avium subsp. paratuberculosis organisms, which bound to immunomagnetic beads and were subsequently lysed by bead beating; then protein and cellular contaminants were removed by phenol-chloroform-isopropanol extraction prior to DNA precipitation. DNA purified by this sequence of procedures was then analyzed by conventional and real-time IS900-based PCR in order to detect M. avium subsp. paratuberculosis in feces and milk. By use of this simple and rapid technique, 10 or fewer M. avium subsp. paratuberculosis organisms were consistently detected in milk (2-ml) and fecal (200-mg) samples, making this sensitive procedure very useful and cost-effective for the diagnosis of clinical and subclinical Johne's disease (paratuberculosis) compared to bacteriological culture, which is constrained by time, labor, and expense under diagnostic laboratory conditions.     

Immunological and molecular characterization of susceptibility in relationship to bacterial strain differences in Mycobacterium avium subsp. paratuberculosis infection in the red deer (Cervus elaphus)

Johne's disease (JD) infection, caused by Mycobacterium avium subsp. paratuberculosis, represents a major disease problem in farmed ruminants. Although JD has been well characterized in cattle and sheep, little is known of the infection dynamics or immunological response in deer. In this study, typing of M. avium subsp. paratuberculosis isolates from intestinal lymphatic tissues from 74 JD-infected animals showed that clinical isolates of M. avium subsp. paratuberculosis from New Zealand farmed red deer were exclusively of the bovine strain genotype. The susceptibility of deer to M. avium subsp. paratuberculosis was further investigated by experimental oral-route infection studies using defined isolates of virulent bovine and ovine M. avium subsp. paratuberculosis strains. Oral inoculation with high (10(9) CFU/animal) or medium (10(7) CFU/animal) doses of the bovine strain of M. avium subsp. paratuberculosis established 100% infection rates, compared to 69% infection following inoculation with a medium dose of the ovine strain. The high susceptibility of deer to the bovine strain of M. avium subsp. paratuberculosis was confirmed by a 50% infection rate following experimental inoculation with a low dose of bacteria (10(3) CFU/animal). This study is the first to report experimental M. avium subsp. paratuberculosis infection in red deer, and it outlines the strong infectivity of bovine-strain M. avium subsp. paratuberculosis isolates for cervines.     

Evaluation of modified BACTEC 12B radiometric medium and solid media for culture of Mycobacterium avium subsp. paratuberculosis from sheep

Definitive diagnosis of Johne's disease in ruminants depends on confirming the presence of the causative bacterium, Mycobacterium avium subsp. paratuberculosis, in tissues of the host. This is readily achieved in most ruminant species by culture. However, culture of clinical specimens from sheep in many countries has been unrewarding. Such a culture from sheep was achieved recently in Australia by using a radiometric culture medium. The aims of the present study were to evaluate the culture of M. avium subsp. paratuberculosis from sheep by using modified BACTEC 12B radiometric medium, to determine the sensitivity of culture in relation to histopathology, and to evaluate a range of solid media. Culture of M. avium subsp. paratuberculosis from sheep with Johne's disease is a sensitive method of diagnosis: intestinal tissues from all 43 animals with multibacillary disease and all 22 animals with paucibacillary disease were culture positive, while 98% of feces from 53 animals with multibacillary disease and 48% of feces from 31 animals with paucibacillary disease were culture positive. Of sheep without histological evidence of Johne's disease from infected flocks, intestinal tissue from 32% of 41 were culture positive, while feces from 17% of 41 were culture positive. Consequently, culture is recommended as the "gold standard" test for detection of ovine Johne's disease. Of the wide range of solid media that were evaluated, only modified Middlebrook 7H10 and 7H11 agars, which were very similar in composition to modified BACTEC 12B medium, yielded growth of ovine strains of M. avium subsp. paratuberculosis. The sensitivity of detection of M. avium subsp. paratuberculosis on solid media was slightly lower than that in modified BACTEC 12B radiometric medium. Both egg yolk and mycobactin J were essential additives for growth of ovine strains of M. avium subsp. paratuberculosis in both liquid and solid media.     

Rapid detection of Mycobacterium avium subsp. paratuberculosis from cattle and zoo animals by nested PCR

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, a suspect causative agent of Crohn's disease in man, is an emerging disease of international proportions affecting all ruminants. Early stage detection of Mycobacterium avium subsp. paratuberculosis infection would accelerate progress in control programmes. Despite new molecular approaches the standard diagnostic test for this disease is at present still the time consuming classic isolation procedure. Therefore, alternative diagnostic tests such as PCR, are needed for quick detection of infected animals. In this study, the conventional enrichment and isolation procedure and two IS900-based PCR methods for detection of Mycobacterium avium subsp. paratuberculosis in clinical samples from zoo animals and cattle were compared. A total number of 48 different clinical specimens obtained from animals suspected of having paratuberculosis were examined. The samples included faeces (n = 15) and organ tissues (n = 33). Of the faecal specimens two were identified as positive by nested PCR, whereas none was positive by single PCR or by culture. 28 organ specimens were found positive by culture. Mycobacterium avium subsp. paratuberculosis DNA was detected by nested PCR in 82% of the organ specimens identified positive by culture (23 samples) as opposed to 57% by single PCR (16 samples). Nested PCR also identified two positive samples that were not detected by either culture or single PCR. These findings show the great potential of nested PCR as a useful tool for the rapid diagnosis of paratuberculosis in animals.