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ELISA serodiagnosis of Johne's disease in cattle.

An ELISA test for the serodiagnosis of M. paratuberculosis in cattle has been developed. The test is based on antigen 60 from BCG. It is specific for myocobacteria and detects M. bovis and M. paratuberculosis antibodies. The correlation between sero-units and complement fixation is not an absolute one. On the basis of these preliminary results, a cut-off established at an optical density (OD) of 0.42-0.45 when the sera are analysed at a 1:100 dilution, corresponding to 200 serounits, seems to be adequate. The greatest usefulness of the test appears to consist in the exclusion of a presumed mycobacterial infection.

Animals↗

Antibacterial therapy for Crohn's disease: a review emphasizing therapy directed against mycobacteria.

The most commonly used antibiotics in Crohn's disease are nitroimidazoles and macrolides often combined with corticosteroids or sulfasalazine. There has been interest in a mycobacterial involvement in Crohn's disease since its earliest description. It is not recognized that Mycobacterium avium subspecies paratuberculosis, a proven but uncommon cause of human disease, is widespread in the human food chain especially in dairy products and beef. M. paratuberculosis has been identified in tissues from a higher proportion of Crohn's disease patients than controls, suggesting that it may be one of the causes of Crohn's disease. We review the large number of antibiotic trials in Crohn's disease. Although studies have been performed with many different protocols and variations in the definition of success, preliminary reports of multiple drug therapies are encouraging. Nevertheless, large-well designed preferably placebo-controlled studies are needed before one could recommend such therapy.

Anti-Bacterial Agents↗

Mycobacteria of Runyon groups I, II and IV do not play an aetiological role in Crohn's disease.

OBJECTIVE: In addition to Mycobacterium paratuberculosis, other slow-growing as well as rapid-growing mycobacteria were isolated from mucosa or full thickness samples of intestine from patients with Crohn's disease. The meaning of these data remained unclear. To investigate the possible aetiological role of these rapid- and slow-growing mycobacteria, serosa and mesenteric lymph nodes were also cultivated in the present study. DESIGN AND METHODS: Mucosa, lymph nodes and serosa of 23 patients with Crohn's disease and 23 patients with other intestinal afflictions were incubated at 37 degrees C on Löwenstein-Jensen medium and Herrold egg yolk medium. These methods allow the cultivation of most atypical mycobacteria, including Mycobacterium paratuberculosis. In addition, full thickness samples of some of these patients were analysed for mycobacterial DNA using polymerase chain reaction. RESULTS: Despite lack of decontamination of 60% of lymph nodes and serosa, no mycobacterial growth was observed over a long incubation period (an average of 718 days in Crohn's disease and 552 days in controls) on Löwenstein-Jensen medium. Polymerase chain reaction was based on the amplification of 16S ribosomal DNA sequences specific for mycobacteria of tissues derived from four patients with Crohn's disease and one control was negative. CONCLUSIONS: These findings suggest that mycobacteria, such as M. fortuitum and M. chelonei, which are widespread in the environment, are not involved in the aetiology of Crohn's disease but, rather, should be considered as environmental opportunists.

Adolescent↗

Studies on Johne's disease in Egyptian cattle.

Johne's disease (JD) or paratuberculosis is a serious problem of cattle industry worldwide. For a long period of time, Egypt was considered to be free of JD. In the present study, 2150 Egyptian cattle were examined clinically for JD. Among these, samples from 160 cows were investigated for the presence of Mycobacterium avium ssp. paratuberculosis using various laboratory methods including direct microscopic examination, faecal culture and polymerase chain reaction (PCR). According to the data obtained by the culture method, positive results could be observed for 75 cows from three of five investigated districts in Egypt. Comparably investigated samples from 40 cows of one known positive flock from Hesse, Germany yielded positive reactions for 20 cows. The present study is the first description of JD in Egypt.

Animals↗

Metastatic Crohn's disease: a case report.

Metastatic Crohn's disease (MCD) is a rare extraintestinal manifestation of Crohn's disease characterized by the histologic finding of granulomatous dermatitis at a site noncontiguous to the gastrointestinal tract. An adolescent had MCD of the face that was initially mistaken for severe, treatment-resistant acne. Histopathologic and microbiologic evaluation, combined with clinical examination and response to therapy, ultimately led to the correct diagnosis. Mycobacterium paratuberculosis is a suspected cause of Crohn's disease. Polymerase chain reaction was used to detect genomic DNA specific for the organism in biopsy specimens from the patient's cutaneous lesions. This study failed to demonstrate M. paratuberculosis in the specimens.

Acne Vulgaris↗

A preliminary study of possible genetic influences on the susceptibility of sheep to Johne's disease.

OBJECTIVE: To investigate possible genetic influences on susceptibility or resistance of sheep to Johne's disease. DESIGN: A field and laboratory study of two fine-wool Merino flocks with a high prevalence of disease due to Mycobacterium avium subsp paratuberculosis infection. PROCEDURE: Adult sheep were phenotypically classified as having severe, mild or no disease on the basis of clinical, pathological and cultural tests for paratuberculosis, and as positive or negative in tests for humoral immunity (agar gel immunodiffusion test) or cell mediated immunity (skin test for delayed type hypersensitivity). Correlations with phenotype were sought for polymorphisms at loci within selected immune function genes (NRAMP, MHC complex, IFN-gamma, lysozyme, leukaemia inhibiting factor). RESULTS: Possible associations of particular NRAMP and MHC alleles with susceptibility or resistance to Johne's disease were detected. CONCLUSION: If the results of this preliminary study are confirmed in further work, then the use of rams with "resistant" genotypes may assist in the control of Johne's disease in infected flocks.

Animals↗

Structure and relevance of the oligosaccharide hapten of Mycobacterium avium serotype 2.

The type-specific antigen of Mycobacterium avium serotype 2, the most ubiquitous of serotypes within the M. avium complex and a major cause of disseminated and localized infections, was isolated and purified. It is of the glycopeptidolipid (mycoside C) class with a characteristic oligosaccharide hapten. This was released as the oligosaccharide alditol by base-catalyzed reductive beta-elimination, and the structure was established by a combination of gas chromatography-mass spectrometry, methylation analysis, fast atom bombardment mass spectrometry, and 13C and 1H nuclear magnetic resonance as 2,3-di-O-methyl-L-fucopyranosyl-(alpha 1----3)-L-rhamnopyranosyl-(alpha 1----2)- 6-deoxytalitol. A feature of the work was the elucidation of the absolute (enantiomeric) configuration of the sugars. This same structure, in much less detail, was previously reported as the species-specific hapten of strains of Mycobacterium paratuberculosis. Thus, the work raises the intriguing possibility that some M. avium serotypes are synonymous, at least in outer cell wall anatomy, with the agent(s) of paratuberculosis (Johne's disease), an insidious disease of vast proportions in ruminants. In addition, recognition of a specific determinant will allow a precise study of the epidemiology of M. avium infections in humans and animals.

Antigens, Bacterial↗

Extensive genomic polymorphism within Mycobacterium avium.

We have initiated comparative genomic analysis of Mycobacterium avium subspecies by DNA microarray, uncovering 14 large sequence polymorphisms (LSPs) comprising over 700 kb that distinguish M. avium subsp. avium from M. avium subsp. paratuberculosis. Genes predicted to encode metabolic pathways were overrepresented in the LSPs, and analysis revealed a polymorphism within the mycobactin biosynthesis operon that potentially explains the in vitro mycobactin dependence of M. avium subsp. paratuberculosis.

Chromosomes, Bacterial↗

Determination of the etiology of presumptive feline leprosy by 16S rRNA gene analysis.

PCR-amplified 16S rRNA gene sequences were obtained directly from tissue specimens from eight cats with presumptive feline leprosy. Acid-fast bacilli were observed in sections from all eight specimens, but culture for mycobacteria was successful for one specimen only. Analysis of the V2 variable region of each 16S rRNA PCR product identified a sequence with 100% nucleotide identity to the sequences of Mycobacterium lepraemurium, Mycobacterium avium, and Mycobacterium paratuberculosis in four of the specimens from cats with feline leprosy. Separate M. paratuberculosis- and M. avium-specific PCR amplifications of the four specimens were negative, thus substantiating the identification of M. lepraemurium in these specimens from cats with feline leprosy. Further sequence analysis of the V3 variable region of one of the four specimens provided conclusive evidence of the presence of M. lepraemurium. This is the first report of the definitive identification of M. lepraemurium in cats with feline leprosy by molecular biology-based analyses. M. avium, which is rarely reported in cats, and Mycobacterium chitae, a reported nonpathogenic, rapidly growing mycobacterial species found in the environment, were identified in the specimen from which acid-fast bacilli were cultured. Two of the specimens from cats were infected with a potentially novel species of mycobacteria which had a 16S rRNA gene sequence sharing the closest nucleotide sequence identity with that of Mycobacterium malmoense. Molecular biology-based analyses provided for the accurate and rapid diagnosis of mycobacterial infections in cats and circumvented the problems of culture and misdiagnosis of feline leprosy associated with traditional methods.

Animals↗

Control of Johne's disease in four commercial dairy herds in Iowa.

A 6-year study was conducted in 4 dairy herds in Iowa in which Johne's disease was diagnosed previously. Fecal specimens were collected at 6-month intervals from animals 2 years of age and over for mycobacteriologic examination. Serum samples were obtained at 3-month intervals and tested by enzyme-linked immunosorbent assay (ELISA). The antigen used in the ELISA was a potassium chloride extract of a field strain of Mycobacterium paratuberculosis. The ELISA reactions were observed in 87% of the cows from which M. paratuberculosis was isolated. Dairy producers that participated in the Johne's control program reported reduced economic losses. Increased income was attributed to improved milk production, increased value of vaccinated animals sold as replacements to other dairy herds in which Johne's disease had been diagnosed, and the increased market value of slaughter animals removed from the herd.

Animals↗

Estimating receiver operating characteristic curves with covariates when there is no perfect reference test for diagnosis of Johne's disease.

Paratuberculosis (Johne's disease) is a significant animal health problem. Evaluation of diagnostic tests for Johne's disease has been difficult due to lack of a gold standard test. In recent years, there has been interest in receiver operating characteristic (ROC) curve estimation without any gold standard test. Typically, either Bayesian or maximum likelihood methods are proposed. Although these methods overcome the lack of a gold standard test in ROC curve estimation, little work has been done to incorporate covariates in the analysis. In this paper, we propose a method for estimation of ROC curves based on statistical models to adjust for covariate effects when the true disease states of test animals are unknown. The covariates may be correlated with the disease process or with the diagnostic testing procedure, or both. We propose a 2-part Bayesian model: first, a logistic regression model for disease prevalence is used to fit the covariates; second, a linear model is used to fit the covariates to the distribution of test scores. We used Markov chain Monte Carlo methods to compute the posterior estimates of the sensitivities and specificities that provide the groundwork for inference concerning the diagnostic procedure's accuracy. We applied the methodology to milk ELISA scores from several dairy-cow herds for the diagnostic testing of paratuberculosis. We found that both milk yield and its interaction with age had significant effects on the disease process whereas only milk yield was significant on the testing procedure.

Animals↗

The occurrence of infectious diseases in mixed farming of domesticated wild herbivores and livestock in Kenya. II. Bacterial diseases.

The prevalence of antibodies to Brucella spp., Mycobacterium paratuberculosis and the Mycoplasma spp. causing contagious bovine pleuropneumonia and contagious caprine pleuropneumonia was determined in various species of ruminants on a ranch in the semi-arid zone of southeastern Kenya. Antibody titers to Brucella spp. were found in eland (Taurotragus oryx), oryx (Oryx beisa) and camels (Camelus dromedarius). Reactors were not found in buffalo (Syncerus caffer), sheep (Ovis aries) and goats (Capra hircus). Brucella sp. was not isolated from eland and camels. Antibody titers to M. paratuberculosis were found only in camels and goats. Mycobacteria were not detected in feces of two serologically positive camels. Significant serum antibody titers to Mycoplasma mycoides mycoides were found only in camels. Antibody titers to Mycoplasma sp. (strain F38), which causes contagious caprine pleuropneumonia, were found in buffalo, cattle and camels but not in the other species. Attempts to isolate the Mycoplasma sp. from nasal secretion of the buffalo and camels failed. The possible occurrence of tuberculosis in camels is discussed. Under the conditions at the ranch, contagious bacterial diseases appear to be of minor importance in the domesticated wild herbivores. The introduced camels, however, might be a source of various infections such as brucellosis, mycoplasmosis and possibly tuberculosis for the other susceptible species.

Animals↗

[Diagnosis of Johne's disease].

The authors carried out clinical observations and allergical, bacteriological and serological investigations of 73 dairy cows in a herd affected with paratuberculosis. Durings 18 months, clinical signs of the disease were noted in 2 animals only. It was found that the allergic test with the use of johnin had a limited diagnostic value and made it possible only to suspect the disease in the herd. Avian tuberculin had higher potency than johnin and gave, in most cases, stronger allergic reactions in animals infected with M. johnei that the homologous preparation. Bacterioscopic and cultural examination of the faeces with the use of Stuart's serum-agar medium had a significant diagnostic value because they made it possible to detect the excretion of Johne's bacilli before the clinical signs could be observed. The complement fixation test gave positive results in animals with the clinical symptoms of the disease, or when pathological lesions in the intestines were pronounced. In most of the cows investigated, the pathological changes of the ileocecal valve, characteristic for paratuberculosis, appeared earlier than the positive results of the complement fixation test.

Animals↗

The use of antimycobacterial agents in Crohn's disease.

Seventy-five years ago, a Scottish surgeon, Dalziel, clearly described Crohn's disease (CD) and suggested that it might be caused by a mycobacteria. However, mycobacteria were not isolated from CD tissue until 1978 and 1984. Since then several investigators, using sophisticated polymerase chain reaction (PCR) techniques, found Mycobacterium paratuberculosis sequences in cultures of Crohn's disease tissue in approximately two thirds of patients. Because of a possible mycobacterial etiology, Rutgeerts et al. (J Clin Gastroenterol 1992;15:24-8) treated resected ileocolonic CD patients who had early evidence of recurrence with ethambutol and rifabutin. Patients were followed by colonoscopy to observe changes in the intestinal lesions, but no improvement was noted. Rutgeerts et al. concluded that antimycobacterial therapy did not affect the course of CD. Failure of these antibiotics does not necessarily negate the mycobacterial theory of CD, however. The study population of Rutgeerts et al. was too small, and the treatment period too short. Furthermore, ethambutol shows little or no activity against the proposed Crohn's organism, Mycobacterium paratuberculosis; use of only one active agent (rifabutin) can lead to drug resistance and failure of antibiotic therapy. We hope that Rutgeerts et al. will continue these important studies, but with a more active combination of antimycobacterial drugs for a longer time.

Antitubercular Agents↗

Isolation of mycobacteria inducing cross-reactions in the complement fixation test for Johne's disease.

Eight bulls which gave positive reactions in the complement fixation test for Johne's disease were identified in a group of 29 bulls which had previously given only negative reactions at repeated annual tests. One reacting bull was confirmed to be infected with Mycobacterium paratuberculosis at autopsy, but another reactor which was slaughtered had no visible lesions and mycobacteria were not recovered in culture. Faecal samples from seven further bulls (six reactors and one non-reactor) yielded mycobacteria which were not dependent on mycobactin for growth. When one of these strains was injected into two calves they developed positive reactions in the complement fixation test for Johne's disease and to intradermal skin testing with purified protein derivatives of M avium, M paratuberculosis and M bovis. The strains did not satisfy the exact requirements for classification into any recognised mycobacterial species.

Animals↗

Immune response to other agents of calves persistently infected with bovine virus diarrhoea virus (BVDV).

The ability of calves persistently infected (PI) with bovine virus diarrhoea virus BVDV to respond immunologically to defined antigens other than BVDV was studied. Five clinically healthy PI calves were studied together with 5 non-PI calves serving as controls. The humoral immune response was tested by measuring the serum antitoxin titre following immunization against tetanus. The cellular immune response was tested by the ability to develop a positive reaction in a cutaneous tuberculin test performed 1 month after immunization against Johne's disease (paratuberculosis). Finally, a skin-sensitizing agent, dinitrochlorobenzene (DNCB), was employed to study whether PI calves would react by hypersensitization following skin exposure to DNCB for 7 consecutive days followed by application of DNCB to a new skin area remote from the area that had first been exposed. The response of PI calves to the various types of antigenic stimuli applied was not significantly different from that of the control calves. Thus, PI calves developed a potent antitoxin response after tetanus immunization, they showed a positive reaction to tuberculin skin test after immunization against paratuberculosis, and were skin sensitized with DNCB.

Animals↗

Immunohistochemical examination for mycobacteria in intestinal tissues from patients with Crohn's disease.

We conducted an immunohistochemical search for mycobacteria in the intestinal tissues of patients with Crohn's disease. Tissues obtained by biopsy or surgical resection and fixed by a variety of methods (formalin, periodate-lysine-paraformaldehyde, fresh-frozen) were reacted by an immunoperoxidase method with antibodies to (a) Mycobacterium paratuberculosis strain linda, (b) M. tuberculosis, and (c) the common mycobacterial antigen, lipoarabinomannan. Each of the antibody preparations was shown capable of detecting a variety of typical and atypical mycobacteria (M. tuberculosis, M. kansasii, M. fortuitum, M. chelonei, M. paratuberculosis, and cell wall-defective as well as cell wall-intact forms of M. avium intracellulare) under conditions identical to those used for staining the patients' tissues. We did not detect mycobacteria in any of the 67 specimens from 30 patients examined. These results, in conjunction with those of our previous serologic studies, do not support the hypothesis that infection with a Mycobacterium causes Crohn's disease.

Antibodies, Monoclonal↗

Complete sequence of the gene encoding the bacterioferritin subunit of Mycobacterium avium subspecies silvaticum.

A gene encoding the bacterioferritin subunit (Bfr) of Mycobacterium avium (Ma) subspecies silvaticum has been cloned, sequenced and expressed. The 477-bp open reading frame codes for 159 amino acids, which were shown to share up to 92% identity with the Bfr of five bacterial genera. The recombinant Bfr exhibits serological cross-reactivity with Ma paratuberculosis antigen D, a protein of approx. 20 kDa in cell lysates of Ma paratuberculosis and Ma silvaticum and a protein of 20-22 kDa in sonicates of M. leprae.

Amino Acid Sequence↗