Bacterial isolation, immunological response, and histopathological lesions during the early subclinical phase of experimental infection of goat kids with Mycobacterium avium subsp. paratuberculosis.

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.

Comparative use of DNA probes for Mycobacterium avium and Mycobacterium intracellulare and serotyping for identification and characterization of animal isolates of the M. avium complex.

Commercially available DNA probes for the Mycobacterium avium complex (MAC) were compared with conventional identification and serotyping of animal isolates of MAC. DNA hybridization of 44 strains of mycobacteria showed a test specificity of 100% and sensitivity of 100%. Hybridization of 12 serotype strains showed that serotypes 1-6 and 8-11 hybridized with the M. avium probe and serotypes 7 and 14 hybridized with the M. intracellulare probe. All of the 42 bovine and porcine isolates of MAC consisting of serotypes 1-6 and 8-10 hybridized only with the M. avium probe. Two strains originally identified as MAC by biochemical tests turned out to be negative in the hybridization test and were identified as rapid growers in gas chromatography analysis of fatty acids. This study furthermore indicates that MAC infections in animals in Norway are mainly caused by M. avium probe positive strains.

An outbreak of tuberculosis in pigs and cattle caused by Mycobacterium africanum.

An outbreak of tuberculosis in pigs and cattle caused by Mycobacterium africanum produced lesions in the pigs similar to those caused by M tuberculosis, M bovis and M avium, with caseation in the lymph nodes of the head and in the jejunal lymph nodes. Bacteriological examination of the dysgonic mycobacteria isolated showed that they were M africanum I. The intradermal tuberculin test was very reliable in pigs, differentiating between mammalian and avian reactions, and the results of the test were in accordance with the lesions found at meat inspection. No clinical signs were observed during the outbreak, and the infection was neither serious nor progressive. There were no lesions in the tuberculin-positive cattle. The source of the infection remains unknown.

Gen-Probe Rapid Diagnostic System for the Mycobacterium avium complex does not distinguish between Mycobacterium avium and Mycobacterium paratuberculosis.

Three reference and 16 field strains of Mycobacterium paratuberculosis were tested with the Gen-Probe Mycobacterium avium complex DNA probe (Gen-Probe Inc., San Diego, Calif.). All reference strains and 12 of 16 field strains gave positive hybridization results with the probe. This study shows that the M. avium complex probe does not distinguish between M. avium and M. paratuberculosis and indicates heterogeneity in the 16S rRNA gene of M. paratuberculosis.

Experimental infection of calves with an apparently specific goat-pathogenic strain of Mycobacterium paratuberculosis.

Four calves and four goat kids were inoculated perorally with a Norwegian goat-pathogenic strain of Mycobacterium paratuberculosis. None of the calves developed clinical disease, pathological lesions or humoral antibody response, but the organism was reisolated from the small intestine and mesenteric lymph nodes when the calves were slaughtered after 7, 12, 14 and 18 months, respectively. As one of the goats died of non-specific causes and one did not become infected, two remained as positive controls. One of these became subclinically and one clinically infected, but both showed distinct histopathological lesions at necropsy. Both were shown to be positive in the complement fixation test (CFT) and the enzyme-linked immunosorbent assay (ELISA) but only the clinically affected goat proved positive in the agar gel immunodiffusion (AGID) test. The AGID test was found to be of low diagnostic value, and the ELISA was as sensitive as the CFT in detecting infection at an early stage. However, when infection was finally established, the ELISA titres became far higher than the CFT titres. The results confirm previous experience in Norway, that the current Norwegian strain of M. paratuberculosis has little or no pathogenicity for cattle.

Mycobacterium avium infection of the knee in a child.

We report a case of Mycobacterium avium infection of the knee joint in a 5-year-old child. He was successfully treated by surgery and antituberculous drugs.

Characterization of clinical isolates of Mycobacterium paratuberculosis by DNA-DNA hybridization and cellular fatty acid analysis.

The DNA-DNA homologies obtained were more than 90 per cent for all strains examined, including the reference strains of Mycobacterium paratuberculosis and Mycobacterium avium. Consequently, in a genetic sense, M. paratuberculosis with its variants belong to a single species which should be considered a subspecies of M. avium. The same reference strains of M. paratuberculosis and M. avium showed small but distinct differences in the cellular fatty acid patterns. The Norwegian goat isolate proved to be M. paratuberculosis, while the three sheep isolates from Iceland and the Faroe Islands showed a typical M. avium pattern.

Relationship between Mycobacterium avium, Mycobacterium paratuberculosis and "wood pigeon mycobacteria". Determinations by DNA-DNA hybridization.

The DNA-DNA homology percentages obtained in this study indicate that M. avium and M. paratuberculosis belong to one species. Consequently, M. paratuberculosis ought to be considered a variant of M. avium, and the following designations are proposed: Mycobacterium avium, subsp. avium. Mycobacterium avium, subsp. paratuberculosis. Identification and classification of "wood pigeon mycobacteria" occurring in wild animals have been problematic due to their dysgonic and mycobactin-dependent growth. DNA-DNA homology percentages indicate that these bacteria are closely related to reference strains both of M. avium and of M. paratuberculosis. "Wood pigeon mycobacteria" should therefore be classified as atypical strains of M. avium, and the following designation is proposed: Mycobacterium avium subsp. columbae.

Isolation of Mycobacterium paratuberculosis from intestinal mucosa and mesenteric lymph nodes of goats by use of selective Dubos medium.

To isolate Mycobacterium paratuberculosis from contaminated material, a selective medium, selective Dubos medium (SDubos), was developed by supplementing conventional Dubos medium (CDubos) with carbenicillin, polymyxin, trimethoprim, and amphotericin B. The intestine and mesenteric lymph nodes of 1,501 goats were cultured in parallel on SDubos and CDubos after decontamination with oxalic acid. The contamination rate was reduced more than 150 times by the use of SDubos. The number of positive specimens (18, or 1.2%), i.e., those which revealed growth of M. paratuberculosis, was too small to evaluate the number of specimens which could have been missed due to contamination on CDubos. However, the number of specimens positive on SDubos (16, or 89%) showed that the antibiotics used were not harmful to M. paratuberculosis.

Control of paratuberculosis (Johne's disease) in goats by vaccination.

After several years of unsuccessful efforts to eradicate paratuberculosis in goats in Norway by conventional methods such as general hygienic precautions and the isolation and slaughtering of clinically affected and serologically positive animals, a vaccination programme was initiated in 1967. The vaccine used consists of two live attenuated strains of Mycobacterium paratuberculosis suspended in a mixture of liquid paraffin, olive oil and pumice powder. The vaccine may be stored at 4 degrees C for two weeks, the dose is 1 ml and the goat kids are vaccinated at the age of two to four weeks. The efficacy of the vaccine has been judged mainly by post mortem examination of vaccinated and unvaccinated goats in the period 1967-82. During this period about 131,000 goats were vaccinated and, based on the post mortem examination of 15,219 goats, the infection rate was reduced from 53 to 1 per cent. Moreover, infection occurred almost exclusively in goats which for some reason or other had not been vaccinated or which had been too old when vaccinated. The results of these examinations showed that the adjuvanted vaccine with live M paratuberculosis bacteria offers a high degree of protection against paratuberculosis in goats.

Fatty acid and polar lipid analysis as tools in the identification of Mycobacterium leprae and some related slow-growing mycobacterial species.

Some species of slow-growing Mycobacteria, including Mycobacterium leprae (1 strain), M. lepraemurium (2 strains), M. paratuberculosis (12 strains) and a group of 12 M. avium-like strains (isolates from wild animals) were examined by gas chromatography (GC) for cellular fatty acids and by thin-layer chromatography (TLC) for polar lipids. All the GC patterns, including that of M. leprae, contained high levels of tuberculostearic-, stearic-, octadecenoic- and palmitic acid. Tetradecanoic-, pentadecanoic-, hexadecenoic- and heptadecanoic acid were also generally present but in lower concentrations. In addition to these acids shared by all strains, each bacterial species or group was found to exhibit compounds which were not detected (or detected in considerably lower quantities) in the other taxa examined. Thus each bacterial species or group could be distinguished by their GC profiles. The corresponding TLC patterns were also rather complex. A total of 39 different spots were distinguished. A few of these were shared by all strains, some were characteristic of certain species or groups, whereas others were strain-specific. Both M. leprae and M. lepraemurium shared several features with the other strains but could be distinguished from each other and from the others by their patterns of slow-moving (polar) spots. The 12 M. avium-like strains were divided into two main groups, one with only a few slow-moving spots (rough stains), and one with several slow-moving spots (smooth strains) which included the M. avium reference strains.