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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.

Ovine Johne's disease, or paratuberculosis, occurs in many countries. In Australia, surveillance using serology is used as part of a control program, but the testing regime is costly relative to its sensitivity. For this reason, culturing of Mycobacterium avium subsp. paratuberculosis in fecal samples pooled from a number of sheep was evaluated. Initially, the effect of pooling on the sensitivity of fecal culture was evaluated using samples from 20 sheep with multibacillary paratuberculosis and 20 sheep with paucibacillary paratuberculosis, each confirmed histologically. All multibacillary cases and 50% of paucibacillary cases were detected by culturing of feces at a pooling rate of 1 infected plus 49 uninfected sheep. In a pilot-scale study in 1997, M. avium subsp. paratuberculosis was detected by pooled fecal culture on 93% of 27 infected farms which were identified originally based on history, clinical signs, and one or more rounds of testing using serologic and histopathologic examinations. Pooled fecal culture was compared with serologic examination for submissions from 335 farms where both tests had been conducted on the same sheep and was significantly more sensitive (P<0.001). Computer simulation of random sampling indicated that the testing of 6 pools of 50 sheep would provide 95% confidence in detecting > or =2% prevalence of infection. The estimated laboratory cost of pooled fecal culture when applied as a flock test is approximately 30% that of serologic examination, and sample collection costs are lower. It is recommended that pooled fecal culture replace serologic examination for detection of M. avium subsp. paratuberculosis infection at the flock level.

Of 19 adult cows naturally infected with paratuberculosis, 13 were treated with monensin sodium and six remained untreated. At the beginning of the study, the severity of the histological lesions was assessed from biopsy samples of ileum, liver, mesenteric lymph node and rectal mucosa. From the data acquired it was possible to assign the animals so that the lesions in the two groups were similar (P=0. 8323). Monensin was administered in the feed, which contained 147.5 mg/kg, and each treated cow received 450 mg of monensin daily for 120 days. At the end of this period all cows were killed and histopathological findings in the ileum, liver, mesenteric lymph node and rectal mucosa were compared with the initial findings. A scoring system showed that monensin had a beneficial effect in the ileum (P=0.077), liver (P=0.0322) and rectal mucosa (P=0.0578), but under the conditions of the experiment no such effect could be demonstrated in mesenteric lymph node (P=0.3599). There appeared to be an overall effect on all tissues taken together (P=0.1335). The effect of monensin may have been due to both a halting and a reversal of the pathological process. In all but one of the six untreated cows, the lesions worsened during the course of the experiment., (Copyright 2000 Harcourt Publishers Ltd.)

Sixty-eight adult goats with clinical or sub-clinical paratuberculosis, from naturally infected flocks, were subjected post mortem to a pathological examination that focused on the intestinal lymphoid tissue. The lesions were divided into four categories. Focal lesions, found in 16 goats, consisted of small granulomata in the ileocaecal Peyer's patches or related lamina propria. Diffuse multibacillary lesions (34 goats) consisted of a granulomatous enteritis, affecting different intestinal sites. Numerous macrophages containing many mycobacteria were present, resulting in macroscopical changes in the normal gut morphology; in the ileum of nine of the 34 animals, however, these changes were confined to the apex of the villi and the intestinal wall was not thickened. In diffuse lymphocytic lesions (10 goats), the lymphocyte was the main inflammatory cell, with some macrophages (containing few if any mycobacteria). In diffuse mixed lesions (eight goats) the infiltrate consisted of numerous lymphocytes and macrophages, with small numbers of mycobacteria. The three types of diffuse lesion were often associated with necrosis in the lymph vessels of the mucosa, mesentery and lymph nodes, and with greater thickening of the jejunum than of the ileum. Mycobacterium avium subspecies paratuberculosis was cultured from 69% of goats with diffuse lesions and from 44.4% of those with focal lesions., (Copyright 2000 Harcourt Publishers Ltd.)

Molecular biological techniques have permitted the rapid and sensitive detection of the Mycobacterium paratuberculosis genome in infected tissues, most commonly by polymerase chain reaction amplification of sequences in the IS900 DNA insertion sequence. The aim of this work was the detection of M. paratuberculosis DNA in ovine tissues by in situ-polymerase chain reaction, which is sensitive and localises the signal within the tissue sample. Paraffin embedded tissues from three acid-fast positive ovine guts with classical lesions of paratuberculosis, and from negative control samples were tested. A 413-bp fragment of the IS900 sequence was amplified in-situ and hybridised to an internal PCR-synthesised digoxygenin-labelled probe. The samples from sheep affected by paratuberculosis clearly showed cell-specific cytoplasmic signals in mucosal and submucosal macrophages. This technique could be useful both in the diagnosis and study of the pathogenesis of infections in which involvement of M. paratuberculosis is suspected.

The diagnosis of Mycobacterium avium subsp. paratuberculosis infection is difficult, especially in the early stages of disease. This is due to the long incubation period, the variable lag phase associated with bacterial proliferation, and the multifocal distribution of slowly developing lesions. There are few previous studies of the early stages of experimental paratuberculosis in goats. In the present study, the ability of conventional diagnostic methods to detect M. a. paratuberculosis infection during the early stages of infection was assessed. Eight goat kids were experimentally infected with M. a. paratuberculosis and subjected to a series of immunological and bacteriological tests before being euthanatized at various times postinfection. At postmortem examination, the ages of the kids ranged from 1 1/2 to 12 months. Of the eight goats infected, three had histopathological evidence of paratuberculosis. Two of these goats were positive with bacteriology, but only one was also positive with all immunological tests. One animal had a positive immunological response, but infection could not be demonstrated by bacteriologic or histopathologic examination. Histopathologic lesions were found in the jejunum, in the ileum, and in one mesenteric lymph node, but only the mesenteric lymph nodes and one retropharyngeal lymph node gave positive results following bacteriologic culture. The disparity between the localization of histopathologic lesions and bacteriologic results emphasizes the need for exhaustive sampling to confirm a diagnosis during the early phase of an infection. It also highlights the need for a better understanding of the biology of M. a. paratuberculosis and its interaction with the immune system of the host.

Mycobacterium avium subspecies paratuberculosis is the etiologic agent of paratuberculosis (Johnes disease), a chronic enteritis in ruminants, which is one of the most widespread bacterial diseases of domestic animals, causing enormous economic losses worldwide. Though the disease was first described more than a century ago, the biology of the infecting organism and the mechanisms of its interactions with the host still remain a mystery. In this review, recent advances made on pathogenesis of paratuberculosis are summarized and future challenges are discussed.

The superficial similarity of Johne's disease to Crohn's disease led to the hypothesis that, like the former. Crohn's disease was caused by Mycobacterium paratuberculosis. Detailed pathologic comparisons, however, reveal little similarity between these two entities, including the lack of important extraintestinal manifestations. Attempts to recover M. paratuberculosis by culture have only rarely succeeded and the significance of spheroplasts that appear more frequently on culture is seriously in question. Five immunocytochemistry studies have failed to find mycobacterial antigens in diseased tissues and the five most recent polymerase chain reaction (PCR) attempts to find genomic evidence of M. paratuberculosis were uniformly negative. Numerous serologic studies failed to demonstrate antibody to M. paratuberculosis and attempts to show cell-mediated immunity were also unrewarding. Inoculation of numerous experimental animals with Crohn's disease tissue has failed to induce Johne's disease, and inoculation of various animal species with M. paratuberculosis has equally failed to result in Crohn's disease. Controlled studies of the treatment of Crohn's disease with antimycobacterial agents have generally resulted in no improvement, and most studies that have shown a positive response are either uncontrolled or include broad-spectrum antibiotics that may be acting on pathogens other than mycobacteria. Finally, although Johne's disease is common in farm animals, and infected animals shed M. paratuberculosis in large numbers, no record of zoonotic transmission has been recorded.

Objective: To investigate the relation between lesions and cellular immune response-based tests in sheep with naturally acquired paratuberculosis and to evaluate efficacy of an interferon-gamma (IFN-gamma) assay and the intradermal skin test., Animals: 110 and 88 adult sheep were used to evaluate the IFN-gamma assay and skin test, respectively., Procedure: Before euthanasia, heparinized blood was collected and incubated with avian purified protein derivative (PPD). Interferon-y production was assessed, using a commercial enzyme immunoassay. Skin testing in the caudal fold also was performed, using avian PPD. Sensitivity and specificity of these techniques were evaluated, using histopathologic findings as reference., Results: Of the sheep examined, 61 (55.4%) had lesions that were classified in 5 categories, according to location, extension, cellular types, and presence of acid-fast bacilli. Regarding cellular immunity test results, 55.4% of sheep with and 6.1 % without pathologic changes had positive results of the IFN-gamma assay. Skin test sensitivity (55.6%) and specificity (100%) were slightly greater., Conclusion: Close association between pathologic findings and cellular immune response was apparent. Most sheep with focal (tuberculoid) and some with diffuse lesions (borderline tuberculoid), all with scant or no acid-fast bacilli in the intestine, had positive results of the IFN-gamma assay and skin test. Most sheep with diffuse lesions and high numbers of bacilli (borderline lepromatous) were test negative., Clinical Relevance: Cellular immune response-based and serologic tests are useful tools for seeking evidence of paratuberculosis. Each technique detects different types of immune-reacting sheep, and their simultaneous use may detect most infected animals.

Nineteen adult sheep diagnosed as having clinical paratuberculosis (Johne's disease) and 16 unaffected controls were examined in this study. Animals were tested for the presence of circulating antibodies of Mycobacterium avium ssp. paratuberculosis (M. a. paratuberculosis) and lymphocytes derived from the blood, mesenteric lymph nodes and intestines were examined for cell-mediated immune (CMI) responses to Johnin pure protein derivative (Johnin-PPD: J-PPD). Bacteriological examinations were carried out on faeces and tissues and any mycobacterial isolates identified as M. a. paratuberculosis (IS900+) or M. avium ssp. silvaticum (M. a. silvaticum) (IS901+) by polymerase chain reaction (PCR). Full necropsy and histopathological studies were performed and diseased animals were categorised on the basis of having a lepromatous or tuberculoid form of intestinal pathology. Unaffected control sheep were generally antibody-negative and demonstrated varying CMI responses to J-PPD. Clinically-affected animals were almost always antibody-positive with variable CMI responses. A correlation was observed between the histological lesion type in the intestine and the cellular immune response. Tuberculoid-type lesions were associated with strong CMI responses in lymphocytes derived from the peripheral blood, mesenteric lymph node and intestine, whereas lepromatous-type lesions were associated with weak CMI responses in all tissues examined.

We have previously described the cloning and sequencing of a gene portion coding for the terminal part of a 34-kDa protein of Mycobacterium avium subsp. paratuberculosis, the etiological agent of Johne's disease (P. Gilot, M. De Kesel, L. Machtelinckx, M. Coene, and C. Cocito, J. Bacteriol. 175:4930-4935, 1993). The recombinant polypeptide (a362) carries species-specific B-cell epitopes which do not cross-react with other mycobacterial pathogens (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol. 31:947-954, 1993). The present work describes the preparation of polyclonal and monoclonal antibodies directed against a362 and the use of these immunoglobulins for histopathological diagnosis of Johne's disease. The new immunohistological procedures herewith detailed proved to be able to identify M. avium subsp. paratuberculosis antigens in the intestinal tissues and lymph nodes of cattle affected by either the paucibacillary or pluribacillary form of the disease. They yielded negative responses not only with healthy animals but also with those affected by tuberculosis (Mycobacterium bovis). Both immunohistological procedures proved to be as sensitive as or more sensitive than Ziehl-Neelsen staining and, in addition, to be endowed with species specificity.

An experimental oral infection of neonatal (< 2 weeks old) lambs with a cervine isolate of Mycobacterium avium subspecies paratuberculosis (M.a. paratuberculosis), the causal agent of ruminant paratuberculosis (Johne's disease) was used to investigate bacteriological, histopathological and immunological changes during the early (up to 8 weeks) post-infection phase. In vitro culture for mycobacteria was positive in one faecal and three mesenteric lymph node (MLN) samples from the eight infected lambs. All mycobacterial isolates from MLN were identified as M.a. paratuberculosis by polymerase chain reaction (PCR). Small-to-medium sized focal granulomata were observed in jejunal (JPP) and ileal Peyer's patches (IPP) from four of the eight infected lambs. Compared with controls, JPP from all infected lambs had significantly (p < 0.05) higher proportions of CD8+ and CD2+ lymphocytes, and there were significantly (p < 0.05) fewer cells expressing B lymphocyte-associated markers in IPP and MLN. The T/B cell ratio was significantly (p < 0.05) increased in both JPP and MLN from infected lambs. The expression of a range of genes for cytokines was examined using specific reverse transcriptase PCR (RT-PCR) amplification of messenger RNA (mRNA) template isolated from MLN, JPP and IPP from both groups of animals. Densitometric analyses indicated that, in infected animals, MLN expressed significantly (p < 0.05) more mRNA for TNF-alpha: JPP had significantly increased (p < 0.05) mRNA for GM-CSF and significantly decreased (p < 0.05) mRNA for IL-4 and IFN-gamma. Infected lambs had significantly (p < 0.05) decreased titres of both circulating IgG and gut mycobacteria-associated IgG antibody. Infection was not associated with any consistent changes in lymphocyte reactivity to specific mycobacterial antigens, IFN-gamma release into supernatants from in vitro intestinal lymphocyte cultures or gut IgA antibody levels.

The expression of macrophage surface markers that are likely to be important in antigen presentation and cell interactions was examined in normal sheep and those with clinical paratuberculosis. Immunohistological studies demonstrated that intestinal macrophages in diseased sheep expressed MHC class II, LFA-1 and CR4 antigens weakly compared with normal tissues. Reverse transcriptase-polymerase chain reaction analysis of MHC class II mRNA in intestinal whole tissue samples showed no significant difference between control and diseased groups. A reduction in molecules such as MHC class II and LFA-1 on the surface of infected macrophages could have implications for survival of the intracellular mycobacteria and the persistence of infection.

A local clinical Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) isolate was cultivated in large-scale culture. A procedure for efficient preparation of a lipoarabinomannan-containing antigen was developed and standardized; 25 mg of purified antigen were obtained per gram of bacterial wet weight. An ELISA based on this antigen was developed. Intra- and interassay variation were determined to be 20% and 27%, respectively. The ELISA was evaluated using the sera of groups of 39 non-randomly selected and 92 randomly selected animals from which ileocaecal lymph nodes were cultured to isolate viable M. paratuberculosis. Combining the results of both groups a positive predictive value of 74% and a negative predictive value of 99% were calculated. The ELISA was licensed by the German regulatory agencies and, in a direct comparison, was found to be superior to both other licensed assays.

A direct polymerase chain reaction (PCR) test was applied to DNA extracted from blood, liver, ileocecal lymph node and ileum from twelve ewes in poor condition with histologically confirmed paratuberculosis and ten clinically normal sheep which had no evidence of paratuberculosis. The assay was compared with four serological tests: complement fixation test (CFT), gel diffusion test (AGID) and two enzyme-linked immunosorbent assays (ELISA). The PCR detection rate of Mycobacterium avium subsp. paratuberculosis, when results of single tests were interpreted in duplicate, was 72% for ileocecal lymph node, 90% for liver, and 100% for ileum in sheep with confirmed paratuberculosis. A single PCR test detected the target DNA in 66% of 0.5 ml blood samples. Sensitivities of serological tests compared with histological diagnosis were: 33% for CFT, 66% for AGID, 75% for the Central Animal Health Laboratory (CAHL) ELISA, and 83% for the 'modified' Commonwealth Serum Laboratories (CSL) ELISA. The PCR assay gave no positive reaction in samples collected from 10 sheep considered to be free of paratuberculosis. Similarly, all four serological tests were also 100% specific. The results raise some hope for the development of a PCR-based test using liver biopsy specimens, or possibly blood, in the diagnosis of paratuberculosis in sheep.

Objective: To investigate the relation between lesions and serologic response in sheep with naturally acquired paratuberculosis and to evaluate the efficacy of agar gel immunodiffusion (AGID) and ELISA as diagnostic tests., Animals: 134 adult sheep, culled by the owner from flocks with history of severe chronic weight loss., Procedure: Before euthanasia, blood samples were collected and serum was obtained. AGID and ELISA were performed, using a protoplasmic antigen of Mycobacterium paratuberculosis. Sensitivity and specificity of these techniques were evaluated, using histopathologic findings as a reference. Histologic studies were carried out focusing on specimens containing intestinal organized lymphoid tissue., Results: Of the sheep examined, 46.3% had lesions that were classified in 5 categories, according to their location, extension, cellular types, and presence of acid-fast bacilli. Regarding serologic test results, 37.1% of sheep with lesions had a positive AGID result, and all sheep without lesions had negative results. However, ELISA positivity was slightly greater--48.4% of sheep with lesions; however, by use of ELISA, 11.1% of sheep without lesions had positive results., Conclusion: A close relation between pathologic findings and serologic response was determined, especially between presence of acid-fast bacilli (potentially heavily shedding sheep) in the intestinal lesions and positivity to serologic tests, especially AGID., Clinical Relevance: On the basis of these results, together with its high specificity, reliability, and the difficulty of fecal specimen culture in sheep, AGID is proposed as a useful tool for seeking evidence of paratuberculosis in live sheep, and therefore, is suggested as the basis for control programs in areas in which the disease is a serious threat to sheep production.

Developments in the diagnosis and treatment of paratuberculosis is constrained by the lack of an experimental animal model. To investigate this problem conventional chicks immunodepressed by a Cyclophosphamide injection and concurrent inoculation of Infectious Bursal Disease Virus (IBDV) were infected with Mycobacterium paratuberculosis and kept for 4 months. The immunodepressed chicks eliminated mycobacteria with their faces from the first month until the third month and developed typical intestinal lesions of mycobacterial infection characterized by aggregation of macrophages with monocytes and lymphocytes. Diarrhoea was absent. The number of lymphocytes decreased by about 80%. The serological tests carried out with Complement Fixation test were negative. For the positive bacteriology and typical granulomatous lesions, the conventionally reared chicks proved to be a useful laboratory model for reproduction of Johne's disease.

A survey of wild rabbits in Tayside, Scotland revealed that 67 per cent were infected with Mycobacterium avium subspecies paratuberculosis. In general, the infected rabbits had histopathological changes within the lymph nodes and intestines which were consistent with the changes due to paratuberculosis in ruminants. The survey raises the possibility that rabbits and other wildlife may be involved in the epidemiology of paratuberculosis, a possibility which has important implications for the control of the disease.

Characterisation of the T-cell subsets in intestinal lesions in sheep with paratuberculosis may contribute to our understanding of the pathogenesis of this disease. To determine the phenotype and distribution of lymphocytes in the normal sheep intestinal mucosa and in Mycobacterium avium subspecies paratuberculosis infected sheep, immunohistochemistry was performed on 12 normal sheep and 18 naturally infected, clinically diseased sheep of which 12 showed lepromatous and six tuberculoid forms of the disease. Immunoperoxidase staining was carried out on frozen sections of ileum using monoclonal antibodies against ovine CD4, CD8, and gamma delta T-cell receptor (TCR) markers. In all three sample groups, cells appeared to be non-randomly distributed throughout the lamina propria. Higher densities of lymphocytes were present in villus than in crypt areas. CD8+ cells were located principally around the epithelial basement membrane, whereas CD4+ cells were localised towards the central villus area of the lamina propria. Lymphocytes bearing the gamma delta T-cell receptor were more widely distributed, both in epithelial and lamina propria compartments. Ileum with tuberculoid lesions had higher densities of CD4 and gamma delta T-cell subsets while lepromatous lesions had lower densities of CD4 and CD8 cells compared with normal tissues. The median relative percentage of CD4+ cells was increased and that of CD8+ cells decreased in tuberculoid cases, with a corresponding increase in the CD4:CD8 ratio, while the relative percentage of gamma delta + cells was increased in lepromatous cases.

Polyclonal antisera were raised in rabbits against preparations of live and heat-killed Mycobacterium paratuberculosis and cell-wall proteins of M. paratuberculosis and were evaluated as diagnostic tools in immunohistochemical staining of bovine tissue. Live preparations of M. paratuberculosis (LMp) were inoculated intraperitoneally or intravenously at 10(9)/ml. Heat-killed M. paratuberculosis (HKMp) was prepared by treatment of bacteria at 85 C for 10 minutes. Cell-wall proteins were isolated from M. paratuberculosis and conjugated to keyhole limpet hemocyanin to improve antigenicity (KLH-CWPMp). The HKMp and KLH-CWPMp preparations were emulsified in incomplete Freund's adjuvant before subcutaneous inoculation of rabbits. Antibody titers in the terminal blood sample were higher for HKMp and KLH-CWPMp than for LMp rabbits (1:1,024 vs. 1:64). The KLH-CWPMp antibody did not cross-react with M. bovis-infected tissues. Sensitivity and specificity of immunohistochemical detection of Johne's disease (paratuberculosis) from bovine tissues was much higher for the KLH-CWPMp polyclonal antibody. Immunoreactivity of the antibody resulted in staining of bacteria in the cytoplasm of macrophages, mononuclear giant cells, and extracellular bacteria in both intestine and lymph node.

The term Johne's disease immediately brings to mind an image of an emaciated, debilitated ruminant with bottlejaw and fluid, pipestream diarrhea. However, the clinical case is merely the tip of the iceberg in terms of the total number of infected animals on the farm. If the clinically affected animal was born on the farm, a minimum of 25 other animals are probably infected and less than 30% of those will be detectable by currently available tests. Infected animals in the early stages of the disease progress slowly over a period of months or several years to stages that may be detected by antigen detection tests and antibody-based diagnostic tests. The duration of each stage of infection depends on age at the time of exposure and the dose of organisms ingested. The lack of tests sensitive enough to detect infected animals in the earliest stages of disease is a serious obstacle to eradication of Johne's disease. The animals with overt infection may be culled from the herd immediately on detection. However, the animals that are shedding organisms below the threshold of detectability and have not mounted an immune response remain behind to contaminate the environment and infect herdmates.

Paratuberculosis in small ruminants is widespread geographically. In some herds and flocks, clinical paratuberculosis, which primarily causes long-term weight loss, can be a significant cause of culling. The effects of subclinical disease are less defined, but they may include decreased milk production in milking sheep and decreased weight gain in deer raised for slaughter. Paratuberculosis also can cause economic losses due to reduced sales of breeding animals from purebred flocks and herds. Diagnosis in goats, deer, and SACs is based primarily on feces or tissue culture and histopathologic examination. Because of the difficulty in growing sheep strains of M. paratuberculosis, diagnosis is based on finding compatible histopathologic lesions with acid-fast organisms. The AGID is a highly specific serologic test for diagnosis of clinical paratuberculosis that correlates with fecal shedding of organisms and degree of severity of lesions; however, it is less sensitive than fecal culture at the herd level for detection of subclinical disease. The CF test detects clinically affected animals but yields poor specificity. The ELISA test is being evaluated for use as a herd screening test in sheep, goats, and SACs. The test seems to be as sensitive as the AGID test but shows a lack of specificity in herds and flocks infected with caseous lymphadenitis. Absorption of sera with Corynebacterium pseudotuberculosis or Mycobacterium phlei may improve specificity, but more studies are needed. Currently, the ELISA test is best used only as a herd or flock screening tool; its use for diagnosis of individual cases requires confirmation with a more specific serologic test or organism detection test such as fecal culture or PCR. Paratuberculosis can be managed and eliminated by combining stringent management with frequent testing and culling or by combining vaccination (where permitted) with management of fecal-oral transmission. Management of fecal-oral transmission is particularly important to prevent exposure of young stock to the infection. Control programs differ with the goals and economics of each farm. The frequency of testing and level of management intervention are determined by each farm's abilities, priorities, and finances.

In sheep clinically affected with paratuberculosis, two distinct forms of microscopical pathology were recognized, related to a high or a low degree of mycobacterial colonization ("multibacillary" or "paucibacillary" presence). These forms were characterized by different types of cellular infiltrate in the ileal mucosa and submucosa. Statistical analysis demonstrated strong correlations between the presence of large numbers of acid-fast organisms and macrophage infiltration, and between small numbers and lymphocyte infiltration. Correlations also existed between high numbers of acid-fast bacteria and a positive serum antibody test results; and between the presence of giant cells and lymphocytes in the gut. This study suggests that in ovine paratuberculosis the same clinical and gross pathological changes can result from different pathogenetic mechanisms.

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 lesions in naturally infected sheep were classified histologically. Adult sheep (n = 166) culled for various reasons, from four flocks in which clinical cases of the disease had occurred, were studied. Eight-two sheep (49.4%) showed lesions that could be divided into three main categories. Type 1 lesions, found in 24.1% of these animals, consisted of small granulomata formed by macrophages and were located exclusively in the ileocaecal Peyer's patch. In type 2 lesions, found in 4.8% of the sheep, granulomata were also observed in the mucosa associated with Peyer's patches. Type 3 lesions were characterized by granulomata in areas of the mucosa associated with, and also distinct from, the Peyer's patches. Three subtypes of type 3 lesions were recognized. In subtype 3a, found in 4.2% of the sheep, multifocal granulomata appeared in different areas of the lamina propria; they were not apparently associated with lymphoid tissue, and neither did they modify the morphology of the affected areas. Subtype 3b, found in 13.9% of animals, consisted of large numbers of macrophages, widespread in the lamina propria; in subtype 3c (2.4% of sheep) lymphocytes were the main inflammatory cell, with some macrophages scattered amongst them. In subtypes 3a and b, villi were distended and the mucosa appeared thickened. Mycobacteria could be demonstrated in tissue sections from all the samples with subtype 3b lesions and in almost all of those with type 2 and 3a lesions; these organisms were absent or sparse, however, in type 1 and 3c lesions. Macroscopical lesions were clearly visible only in sheep with type 3b and 3c lesions. Mycobacterium paratuberculosis was cultured from all the animals with type 3 lesions, from 87.5% of those with type 2 lesions, and from 47.5% of those with type 1. Type 3b lesions resembled the "borderline-lepromatous" form of mycobacterial lesions, whereas type 3c lesions resembled the "borderline-tuberculoid" form. The relationship between intestinal lymphoid tissue and paratuberculosis lesions is discussed, and the diagnostic importance of histological examination of the ileocaecal valve emphasized.

Macrophage activation, measured as increased acid phosphatase (AcPase)-positive areas by image analysis, and ultrastructural features were examined in granulomatous mycobacterial lesions of mice innately susceptible (BALB/c mice; Bcg) and innately resistant (C3H/HeJ mice; Bcg) to Mycobacterium paratuberculosis strain ATCC 19698. In the liver and spleen of BALB/c mice 3 weeks after intraperitoneal inoculation with M. paratuberculosis, AcPase activity detected in epithelioid cell nodules was high; it had decreased, however, in the liver and spleen after a further 3 and 6 weeks, respectively. In C3H/HeJ mice, the size of epithelioid cell nodules in the liver and spleen was smaller than in BALB/c mice, and infiltrating macrophages, which had increased by week 9 after inoculation, showed high AcPase activity. Ultrastructurally, by week 32 in BALB/c mice, small phagolysosomes (SPLs) had greatly increased in number in the epithelioid cells. These SPLs contained a few AcPase-positive areas and a small number of bacteria, most of which were surrounded by an electron-translucent space (or electron-transparent zone [ETZ]). In contrast, only a few SPLs were observed in C3H/HeJ mice at week 32; in the liver and spleen, large phagolysosomes (LPLs) showed high AcPase activity and contained many degenerated bacteria, which also had an ETZ. These results suggest that the enzymatic and ultrastructural differences in phagolysosomes between BALB/c mice and C3H/HeJ mice reflect the susceptibility of these mouse strains to M. paratuberculosis.

Mycobacterium avium subspecies paratuberculosis is an intracellular parasite of intestinal macrophages and causes a chronic granulomatous enteritis in sheep and other ruminants (paratuberculosis or Johne's disease). Macrophages can be produced a variety of immunoregulatory cytokines that may influence mycobacterial killing and produce disordered inflammation within the gut. In this study, messenger RNA (mRNA) was extracted from intestinal tissue from control and multibacillary diseased sheep and profiles for the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), IL-6, transforming growth factor-beta1 (TGF-beta1) and granulocyte-macrophage colony stimulating factor (GM-CSF) were semi-quantified using reverse transcriptase polymerase chain reactions (RT-PCR). Infected intestinal tissues had significantly increased mRNA for TNF-alpha, IL-1beta and IL-6 but TGF-beta1 and GM-CSF mRNA levels were significantly different from controls. Supernatants from in vitro intestinal cultures were assayed for TNF-alpha activity using the PK(15)-1512 cytotoxicity bioassay and levels were significantly raised in diseased samples. TNF-alpha was not detected in any serum samples. Further analysis on intestinal tissues from sheep with the different, paucibacillary, form of the disease showed significant elevation of TNF-alpha mRNA but not other cytokines tested. Increased pro-inflammatory cytokine expression in the intestine coincident with a failed or misdirected immune response may contribute to the pathogenesis of paratuberculosis and the persistence of a chronic inflammatory state.

Susceptibility of C57BL/6 (Bcgs) and C3H/HeN (Bcgr) mice to an intraperitoneal infection with Mycobacterium paratuberculosis strain 19698 was compared (by histopathology and the number of mycobacteria isolated from the spleen). Mycobacterial counts from the spleen of Bcgr mice progressively decreased over the course of infection but remained unchanged in Bcgs mice. Granulomatous lesions and acid-fast bacteria were consistently present in the liver and lymph nodes of Bcgs mice, whereas lesions were transient or absent in Bcgr mice. These results indicate that Bcgr mice are inherently resistant to M. paratuberculosis, whereas Bcgs mice are inherently susceptible. These differences may prove useful in elucidating the mechanisms of resistance and susceptibility to paratuberculosis and other mycobacterial infections.

Paratuberculosis (Johne's disease) was suspected in a flock of approximately 1000 sheep after weight losses and scouring had increased in adult animals despite repeated anthelmintic treatment. A total of 10 ewes showing clinical symptoms were submitted to laboratory examination. Gross pathological and histological examination of tissue samples rendered results compatible with Johne's disease in all 10 ewes. The diagnosis could only be confirmed by cultural isolation and identification of Mycobacterium paratuberculosis in the faeces of two ewes. This is considered the first documented isolation of Mycobacterium paratuberculosis in sheep in Morocco.

The susceptibility of C57BL/6 mice to oral inoculation with Myobacterium paratuberculosis was evaluated histopathologically. Granulomatous lesions containing acid-fast bacteria developed in the mesenteric lymph nodes in over 50% of the mice by 11 months after inoculation. The results suggest that C57BL/6 mice may be useful for studying infection, pathogenesis, and other aspects of paratuberculosis.

Johne's disease was diagnosed in 10 alpacas (Lama pacos) in Australia between February 1993 and May 1994. Eight of the animals were between 12 and 24 months of age, one was a 6-year-old female, and one was a 4-year-old male. Five, including the 6-year-old and the 4-year-old alpacas, showed weight loss and diarrhoea before death or slaughter. The other cases showed no clinical signs of Johne's disease but 4 gave a positive result on faecal culture and one gave a positive result on testing with the caprine AGID assay and had acid-fast organisms in its faeces. At necropsy, all cases had grossly enlarged mesenteric lymph nodes. Johne's disease was diagnosed after histological examination of the lymph nodes with conventional culture and polymerase chain reaction testing of tissue samples. This report outlines the clinical, epidemiological, and pathological findings in these cases.

Mycobacterium paratuberculosis was diagnosed in 2 llamas by bacteriologic culturing and DNA probe testing of fecal and intestinal specimens. Diarrhea and weight loss were observed in both animals. Lesions in the gastrointestinal tracts of the 2 llamas were similar to pathologic changes reported in cattle infected with M paratuberculosis.

Susceptibility to Mycobacterium paratuberculosis strain ATCC 19698 infection was compared histologically and morphometrically in BALB/c (Bcgs) and C3H/HeJ (Bcgr) mice. The granulomatous lesions, containing M. paratuberculosis, in the liver and spleen of BALB/c mice increased progressively until 32 weeks after intraperitoneal inoculation. In contrast, both the granulomatous lesions and number of bacteria decreased drastically in C3H/HeJ mice beginning at 6 weeks after inoculation. There were histological differences in granulomatous lesions between BALB/c and C3H/HeJ mice, indicating differing functional activity of cellular immunity. Intestinal lesions similar to those seen consistently in bovine paratuberculosis were observed first at 6 weeks after infection and later became disseminated widely, especially in BALB/c mice. Our results suggest that the innately susceptible BALB/c mice and the innately resistant C3H/HeJ mice may be useful and convenient models for further studies of immunoregulation and antimicrobial therapy of paratuberculosis.

Balb/C, C57/B10 and C57/B6 mice were examined for their susceptibility to disseminated Mycobacterium paratuberculosis infection after intraperitoneal inoculation with a suspension of organisms containing mineral oil. Animals were examined monthly by histopathology and bacterial tissue counts of liver and spleen over a 6-month period. Only Balb/c mice maintained a steady infection with an average of 4.1 x 10(5) +/- 7.8 x 10(3) and 8.1 x 10(5) +/- 2.6 x 10(4) colony forming units (cfu) per gram of liver and spleen, respectively, during the course of the study. In contrast, C57/B10 mice reduced the bacterial counts in the liver and spleen from 6.8 x 10(4) and 1.3 x 10(5) to 7.1 x 10(2) and 4.3 x 10(3), respectively during the first 120-150 days after infection. The reduction in cfu was associated with the development of caseous necrotic lesions. C57/B10 mice were of intermediate resistance, slowly reducing cfu in the liver, but not the spleen, during the 6-month period. Balb/c was found to be a suitable mouse strain for the study of chronic M. paratuberculosis infection.

Mycobacterium paratuberculosis was isolated from farmed deer in New Zealand on 21 occasions over a 7-year period. The number of cases increased during the last 3 years of the study. Clinical paratuberculosis was observed in 5 deer, 3 other cases came from grossly normal animals that reacted to a tuberculin skin test, and the remaining 13 animals had tuberculous lesions that were identified at meat inspection. Pathologic features of the lesions in these 13 cases included necrosis and mineralization in lymph nodes draining the gastrointestinal tract. The histologic changes in these lesions were very similar to those caused by Mycobacterium bovis and members of the M. avium complex. Characterization of 20 of the isolates of M. paratuberculosis by restriction endonuclease analysis and DNA hybridization revealed that 3 of these isolates were identical to New Zealand sheep isolates and 17 were the same as cattle isolates. The source of the cervine infections was not determined.

Paratuberculosis was diagnosed in one 18-month-old and two 30-month-old hinds in a herd of 70 red deer (Cervus elaphus) in Ireland. Loss of condition and intermittent diarrhoea were the main clinical findings. Clumps of acid-fast organisms were found in the faeces of the three deer. Post mortem examination of one deer showed a slight swelling and pallor of the intestinal tract and associated lymph nodes. Histopathology showed a severe, granulomatous enteritis and lymphadenitis, with extensive cellular infiltration, notably with epithelioid macrophages containing numerous acid-fast organisms. Mycobacterium paratuberculosis was isolated from intestinal and lymph node samples. Paratuberculosis was also confirmed in one of nine clinically normal, yearling stags, sampled at slaughter. Complement fixation tests and enzyme-linked immunosorbent assays gave higher readings for clinically affected deer than healthy ones. Acid soil on the farm was believed to be a contributory cause.

One hundred and seventy-six cattle, vaccinated against paratuberculosis in the first month of life with a whole-cell inactivated vaccine, containing Mycobacterium paratuberculosis suspended in a water/oil emulsion, were tested with intradermal Johnin between the ages of five and 14 months. Negative results in the skin test occurred more frequently in animals found positive in post mortem examinations (P = 0.008) using histological, microscopical and cultural techniques.

An agar gel immunodiffusion (AGID) test was used over a 3-year period to examine 1,871 serum samples from sheep representing 5 Mycobacterium paratuberculosis-infected flocks and 4 flocks presumed to be uninfected. Of 1,032 sheep, 31 had positive AGID test results (scoring 1 to 5), and 23 of these 31 were necropsied. Infection with M paratuberculosis was confirmed by 1 or more of the following findings: observation of typical lesions on histologic examination of sections of ileum or ileocecal lymph nodes, observation of clumps of acid-fast bacteria in mucosal smears of ileum, and isolation of the organism from feces or tissue. False-positive results on AGID testing were not found in sheep from flocks known to have exposure to Corynebacterium pseudotuberculosis. Diarrhea in infected sheep was observed infrequently; chronic, severe weight loss was the most common sign observed. On histologic examination of tissues from 20 infected sheep, 16 (80%) had diffuse lesions of the ileum and 13 (65%) had acid-fast bacteria in areas of ileal inflammation; 4 had discrete granulomas and peripheral lymphocytic infiltrates in the ileum. Sheep with diffuse lesions tended to have higher mean scores on AGID testing and examination for acid-fast bacteria, compared with those from sheep with more discrete lesions. Bacteriologic culture yielded M paratuberculosis from only 3 sheep with paratuberculosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Severe combined immunodeficient beige mice were inoculated orally and intraperitoneally with a bovine strain of Mycobacterium paratuberculosis to explore their potential as laboratory animal models in the study of paratuberculosis (Johne's disease). Control animals were similarly inoculated with heat-killed M. paratuberculosis. In the mice inoculated intraperitoneally, focal lesions and acid-fast bacilli were first detected in the livers (4 weeks postinfection) and later in the spleens and intestines of the test but not the control animals. No bacteria were seen in the hearts, kidneys, or lungs. At 12 weeks postinfection, all test mice had significant losses in body weight compared with those in controls (P less than 0.05), a characteristic sign of bovine paratuberculosis. Tumor necrosis factor alpha was not detected in the serum. Histologic lesions were seen in the intestines, livers, and spleens of the animals in the orally inoculated test group after 26 weeks of infection. Our results suggest that the severe combined immunodeficient beige mouse may be a useful model for the investigation of paratuberculosis and cachexia and the evaluation of antimycobacterial drugs.

Mycobacterium paratuberculosis was isolated in low numbers from the small intestine and associated mesenteric lymph nodes of a saiga antelope (Saiga tatarica) using routine culture techniques in spite of histologic evidence of high numbers of acid-fast bacteria in these tissues. Two newborn domestic sheep were fed the ground intestinal tissue containing acid-fast bacteria and the progression of the experimental disease was followed by fecal culture, immunodiffusion (AGID) and lymphocyte stimulation (LST) tests. One experimentally infected sheep developed progressive clinical illness 1 yr postinoculation. Few M. paratuberculosis were isolated from feces or tissues although an extensive granulomatous mycobacterial enteritis, lymphadenitis and lymphangitis were observed containing large numbers of typical acid-fast organisms. No clinical illness was observed in the second inoculated sheep after 18 mo of observation, although infection was demonstrated at necropsy. Both sheep developed AGID and LST reactions indicative of paratuberculosis. This study demonstrated that a difficult to culture isolate of M. paratuberculosis was responsible for paratuberculosis in captive wild ruminants and was transmissible to domestic sheep. Diagnosis of paratuberculosis in four of eight of the imported saiga antelope and in eleven of their 18 offspring indicates the importance of this disease in management of captive wild ruminants and the ease with which this organism can be transmitted.

The susceptibility of young chickens to infection with an isolate of Mycobacterium paratuberculosis which had been recovered from diseased tissues of a patient with Crohn's disease was determined. Two-week-old Leghorn-Cochin chicks were inoculated with strain Linda. Six birds received 10(7) organisms orally, six intraperitoneally, and five intracardially. Six uninoculated birds served as contact controls. Birds from each group were necropsied at two-week intervals. Focal granulomatous lesions occurred in two of six chickens which were inoculated orally, in six of six inoculated intraperitoneally, in three of five inoculated intracardially, but in none of six controls. Of the 11 birds with lesions, acid-fast bacilli were demonstrated in five. Immunoperoxidase reactivity paralleled the presence of acid-fast bacilli.

In this study, a paratuberculosis (Johne's disease) model was developed by intragastrically dosing gnotobiotic athymic nude mice with Mycobacterium paratuberculosis. The mice infrequently shed bacilli from their intestinal tracts during the first 4 months after inoculation. Following this time, increasing numbers of M. paratuberculosis (greater than 4.0 log10 bacilli per fecal pellet by 40 weeks) were recovered from the feces of the 12 mice that remained in the isolator. A similar pattern of recovery of M. paratuberculosis was obtained from the ileum, cecum, colon, and liver. Histopathologic lesions and acid-fast bacilli were rare during the first 4 months of infection and then, with time, increased in prevalence and severity. Mice maintained for 7 months or longer exhibited severe granulomatous inflammation and large numbers of acid-fast bacilli in the gastrointestinal tract and liver (up to 10(8) log10 colony forming units per gram wet weight). Five mice maintained for 7 months or more developed clinical signs consistent with those seen in paratuberculosis (weight loss, chronic diarrhea); three of these mice eventually died or became moribund and were euthanatized. M. paratuberculosis monoassociated mice released increased levels of tumor necrosis factor activity into their sera, as compared to uninfected control mice, when they were injected with bacterial lipopolysaccharide. The clinical signs, fecal shedding of M. paratuberculosis, granuloma formation, and progressive bacillary multiplication observed with these mice are consistent with naturally occurring M. paratuberculosis infection of ruminants (Johne's disease). This model will be useful for future studies of immunoregulation and antimicrobial therapy of paratuberculosis.

Using immunohistochemistry, the differential distribution of the alpha subunit (S100 alpha) and beta subunit (S100 beta) of S100 protein was studied in mesenteric lymph nodes from normal or Mycobacterium paratuberculosis-infected cattle. In epithelioid cell granulomas, S100 alpha-positive epithelioid cells and some giant cells were scattered among S100 alpha-negative cells, which were predominant. The S100 beta-positive and -negative cells contained acid-fast bacilli. The presence of S100 beta-positive cells was not demonstrated in the granulomas. In normal component cells in the lymph nodes, follicular dendritic cells in the germinal centres and endothelium of lymphatic sinus and lymph vessels were positive for S100 alpha. S100 beta was positive only in the endothelial cells of blood vessels. Results shown in the present paper are discussed in light of results obtained in other work on human tissues using the same sources of antibodies.

One of three feral rabbits from a colony on a hill grazing used to sustain farmed red deer in north-east Scotland, had chronic intestinal lesions resembling paratuberculosis (Johne's disease), but similar lesions were not found in 29 juvenile and adult rabbits killed subsequently on the same ground. Acid- and alcohol-fast bacteria were observed in fixed tissues, but material for culture was not obtained from the affected rabbit, thus the species of Mycobacterium involved was not identified. The farmed deer had experienced recently a severe outbreak of paratuberculosis.

To assess the rabbit as a model for the study of paratuberculosis infection, two groups of newborn rabbits were orally inoculated at one to two days of age with Mycobacterium paratuberculosis (ATCC 19698 or field strain 22206) and compared to uninoculated controls. Nine of thirteen rabbits (69%) inoculated with ATCC 19698, and all three rabbits inoculated with 22206, experienced episodes of intermittent diarrhea starting four months after inoculation. Multifocal granulomas containing acid-fast organisms were observed in the sacculus rotundus and vermiform appendix of the cecum in three of nine rabbits (all with diarrhea) that had been inoculated with ATCC 19698. Although M. paratuberculosis was not recovered from inoculated rabbits when fecal cultures were incubated three months in vitro, a slow-growing mycobactin-dependent form of Mycobacterium was recovered from feces and ileal tissue after incubation for 11-15 months. Reduced feed intake, body weight loss and reduced abdominal fat at necropsy, were not observed. Epithelial transport function across the distal ileum in vitro was not altered nine months subsequent to inoculation. Diarrhea and the histological lesions indicate that the rabbit may be a useful model for the study of paratuberculosis infection.