Mycobacteria causing human cervical lymphadenitis in pastoral communities in the Karamoja region of Uganda.

Mycobacteria from lymph node biopsies of patients with cervical lymphadenitis reporting for tuberculosis treatment in Matany and Moroto Hospitals in the transhumant areas of Karamoja, Uganda were isolated and characterized. The AccuProbe culture identification kits for Mycobacterium tuberculosis complex (MTC), M. avium complex (MAC) and M. avium were used to identify the isolates. Spoligotyping, IS901 PCR and IS1311 and IS1245 restriction fragment length polymorphism (RFLP) were used to characterize the isolates. Of the 43 biopsies, ten M. avium, seven M. tuberculosis, three M. bovis, and two M. intracellulare were isolated. Two isolates could not be identified with AccuProbe and from 19 samples no mycobacteria could be isolated. Three isolates with the Beijing spoligotype were identified from the seven M. tuberculosis isolates. The spoligopatterns of the M. bovis isolates had previously been detected in cattle in Uganda. Isolation of members of the MAC group reflects the complex interaction between the transhumant communities, water sources and their cattle. None of the M. avium isolates harboured IS901, and all showed several bands on IS1311 and IS1245 RFLP, in accordance with M. avium subsp. hominissuis. Composite dendrograms of IS1311 and IS1245 RFLP showed that the isolates were similar and identical patterns were found. The isolation of M. bovis confirms the human infection with zoonotic mycobacteria in areas where consumption of raw milk and meat is routine. Isolation of environmental mycobacteria also confirms their increasing role in human disease and the occupational risk of infection in the transhumant ecosystem in the absence of safe drinking water and environmental contamination.

Characterisation of mycobacteria isolated from slaughter cattle in pastoral regions of Uganda.

BACKGROUND: Bovine tuberculosis is a zoonotic problem in pastoral cattle and communities in Uganda. Tuberculin tests in pastoral cattle had shown a high herd but low animal prevalence, with a high proportion of avian reactors. No work had been done to identify the mycobacterial species involved. The objective of the study was to isolate and characterise Mycobacterial species causing tuberculous lesions in slaughtered animals. Lesioned organs compatible with bovine tuberculosis in slaughtered cattle from pastoral areas in Uganda were collected and cultured to isolate mycobacteria. AccuProbe culture identification kits for the Mycobacterium tuberculosis complex, M. avium complex and M. avium were used to identify the isolates. Spoligotyping and Insertion Sequence (IS) 1311 and IS1245 Restriction Fragment Length Polymorphism analysis (RFLP) were used to further characterise the isolates. RESULTS: Of the 61 lesioned organs and tissues cultured, 19 isolates were identified as M. bovis, 3 as M. avium subsp.hominissuis, 1 as M. intracellulare, 1 as a mixed culture of M. bovis and M. avium sp. and 1 as M. avium sp. and unidentified mycobacteria. Eleven other mycobacteria outside the tuberculosis and avium complex groups were also isolated. Ten new spoligopatterns grouped into three clusters were identified from M. bovis isolates. Two of the three M. avium subsp.hominissuis isolates showed similar patterns on the IS1311 RFLP but all were different on the IS1245 RFLP. CONCLUSION: The isolation of M. bovis confirms the ongoing infection with spoligotypes unique to Uganda. Isolation of environmental mycobacteria could explain the high avian or non specific tuberculin reactor patterns commonly observed in pastoral cattle and suggests their pathogenic or opportunistic role in the infection of cattle with disseminated bovine tuberculous lesions.

Risk factors for herd-level bovine-tuberculosis seropositivity in transhumant cattle in Uganda.

We investigated the prevalence and risk factors to positive herd-level tuberculin reactivity between October 2003 to May 2004 to bovine tuberculosis (BTB) in the four transhumant districts of Uganda: three districts (Karamoja region) of nomadic transhumance cattle rearing (30 superherds and 1522 cattle), and one district (Nakasongola) of fixed-transhumance (7 herds and 342 cattle). We used the comparative intradermal skin-test, sampled 50 animals per superherd/herd, and considered herd positive if there was at least one reactor. Of the 30 superherds under nomadic transhumance, 60% (95% CI 41.4, 79) were tuberculin-test positive; of the 7 fixed herds, 14.3% (95% CI -20.7, 49.2) were tuberculin test positive. The true herd prevalence was estimated at 46.6%. Many risk factors were collinear. The final multivariable logistic-regression model included: recent introductions from market (OR=3.4; 95% CI 1.1, 10.3), drinking water form mud holes during dry season (OR=49; 95% CI 9.1, 262), and the presence of monkeys (OR=0.08; 95% CI 0.0, 0.6) or warthogs (OR=0.1; 95% CI 0.0, 0.3). No association was found between herd size or number of herd contacts with reactors; it was probably masked by the effect of high between-herd interactions. Provision of water from mud holes in dry river beds and introductions of new animals are risk factors that might be targeted to control BTB in transhumance areas.

Responses to tuberculin among Zebu cattle in the transhumance regions of Karamoja and Nakasongola district of Uganda.

Responses to tuberculin in Zebu cattle of the transhumant pastoral farming system in Karamoja region and Nakasongola district in the north-eastern and mid-central regions in Uganda, respectively, were investigated using a comparative intradermal tuberculin skin test. Of the 1864 cattle tested from 30 large units (superherds) in Karamoja and 7 herds in Nakasongola, a total of 28 animals from 19 herds (51.4%) tested positive. Inter-district tuberculin reactor prevalence variations seemed to be influenced by climate, with impact on both the management patterns and transmissibility of agent. High herd tuberculin reactor prevalence (51.4%) was attributed to widespread contacts and mixing of animals between herds. Low individual animal tuberculin test positivity (mean = 1.4%) was attributed to low transmissibility of the agent under the Karamoja climate, which is semi-arid, and to increased resistance due to non-specific response to environmental mycobacteria and natural selection, since there was no active control against bovine tuberculosis. Owing to similarities in management practices in Karamoja and widespread risk factors, it was difficult to identify which were more important, but variations in sources of drinking water pointed to provision of lake and borehole water during dry season as reducing the risk. Positive bovine tuberculin reactor prevalence and skin reactor status were related to age.

IS900 restriction fragment length polymorphism (RFLP) analysis of Mycobacterium avium subsp. paratuberculosis isolates from goats and cattle in Norway.

In Norway, paratuberculosis has been frequently diagnosed in goats, while cattle have been almost free of the infection. This difference in prevalence between goats and cattle has led to speculations about the existence of a Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) isolate that is non-pathogenic for cattle. There is little information available on genotypic variation of M. a. paratuberculosis isolated from animals in Norway. In the present study, genotypic information on 51 isolates from goats and four isolates from cattle in Norway was obtained by use of IS900 restriction fragment length polymorphism (RFLP) analysis. All isolates from cattle and 84% of the isolates from goats had the same RFLP pattern (B-C1). Five RFLP patterns not previously detected were found. No genotypic variation that could explain a difference in host origin was found between the isolates from cattle and the majority of the Norwegian goat isolates. This lack of difference indicates that the most common M. a. paratuberculosis isolates in Norway may infect both cattle and goats.

Description of the infection status in a Norwegian cattle herd naturally infected by Mycobacterium avium subsp. paratuberculosis.

The Norwegian surveillance and control programme for paratuberculosis revealed 8 seroreactors in a single dairy cattle herd that had no clinical signs of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) infection. Paratuberculosis had been a clinical problem in goats several years previously in this herd. All 45 cattle were culled and a thorough investigation of the infection status was conducted by the use of interferon-gamma (IFN-gamma) immunoassay, measurement of antibodies, and pathological and bacteriological examination. In the IFN-gamma immunoassay, 9 animals gave positive results, and 13 were weakly positive, while 19 animals were negative. In the serological test, 10 animals showed positive reactions, and 5 were doubtful, while 30 animals gave negative reactions. There appeared to be a weak trend toward younger animals having raised IFN-gamma and older animals having raised serological tests. Histopathological lesions compatible with paratuberculosis were diagnosed in 4 animals aged between 4 and 9 years. Three of these animals had positive serological reaction and one animal gave also positive results in the IFN-gamma immunoassay. Infection was confirmed by isolation of M. a. paratuberculosis from 2 of these 4 animals. One single bacterial isolate examined by restriction fragment length polymorphism (RFLP) had the same profile, B-C1, as a strain that had been isolated from a goat at the same farm several years previously. Despite many animals being positive in one or both of the immunological tests, indicative of a heavily infected herd, none of the animals showed clinical signs and only one cow was shown to be shedding bacteria. A cross-reaction with other mycobacteria might have caused some of the immunoreactions in these animals. It is also possible that the Norwegian red cattle breed is resistant to clinical infection with M. a. paratuberculosis.

Factors affecting the herd level of antibodies against Mycobacterium avium subspecies paratuberculosis in dairy cattle.

A case-control study was made of Norwegian dairy herds with high and low herd levels of antibodies against Mycobacterium avium subspecies paratuberculosis. A high proportion of the herds had a considerable number of seropositive cows, and environmental and management factors were examined for possible associations with the high serological levels of antibodies. The most important appeared to be: geographical location, red deer (Cervus elaphus) gaining access to the pastures for cattle, the observation of wild birds in the feed storage, and herds sharing common pasture with other herds of cattle. However, diagnostic tests showed that none of the animals in the case herds was infected with M a paratuberculosis.

Detection by immunomagnetic PCR of Mycobacterium avium subsp. paratuberculosis in milk from dairy goats in Norway.

Milk samples from 340 individual goats in 34 dairy herds throughout Norway were examined for Mycobacterium avium subsp. paratuberculosis (M.a. paratuberculosis) by culture and immunomagnetic separation combined with PCR (IMS-PCR). The samples included three categories; (A) vaccinated dairy goats in herds with paratuberculosis; (B) vaccinated dairy goats in herds with no history of paratuberculosis; (C) unvaccinated goats in herds with no history of paratuberculosis. Viable M.a. paratuberculosis were not detected by culture in any sample, but 24 samples (7.1%) tested positive by IMS-PCR when the PCR products were visualised by dot blot hybridisation. PCR products from five milk samples originating from five different herds were sequenced; all showed 99% homology with the IS900 sequence from M.a. paratuberculosis.M.a. paratuberculosis were detected in all sampled categories. The percentage of IMS-PCR positive samples from herds where paratuberculosis had previously been reported was significantly lower than from herds where the infection had never been diagnosed (3.3 and 9.1%, respectively, P=0.048). Similar proportions of milk samples from vaccinated and non-vaccinated goats tested positive for the presence of M.a. paratuberculosis. Vaccinated goats older than 4 years tested positive more often than vaccinated animals less than 2 years old. Samples collected in May tested significantly more often positive than milk sampled during February-March (13.8 and 2.9%, respectively, P=0.001). This study showed that raw goats' milk in Norway might be contaminated with M.a. paratuberculosis.

Paratuberculosis with special reference to cattle. A review.

Paratuberculosis is a chronic, granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis affecting domestic and wild ruminants. The symptoms of clinical paratuberculosis are chronic diarrhoea and progressive weight loss while subclinically infected animals mainly have decreased production. The infection is widespread throughout the world and causes substantial financial losses for the farming industry. One of the major obstacles in the control of this disease, is the difficulty of identifying subclinically infected animals. This review gives a summary of several aspects of paratuberculosis including clinical importance, pathology, immunology and properties of the infectious agent. Special emphasis will be on the available diagnostic methods, their use and limitations.

Subclinical paratuberculosis in goats following experimental infection. An immunological and microbiological study.

An experimental oral infection of goats with a caprine isolate of Mycobacterium a. subsp. paratuberculosis was used to investigate immunological and bacteriological events during the subclinical phase of infection. Seven goats at 5-8 weeks of age were given a bacterial suspension in milk-replacement three times weekly for 9 weeks. Six animals were kept as controls. Cellular recall responses against M. a. paratuberculosis were analysed by means of a lymphocyte proliferation test, an IFN-gamma assay and an IL-2 receptor assay. All inoculated animals had detectable CMI responses from 9 weeks post-inoculation and through the 2 years of study, although the responses were highest during the first year. Antibodies against M. a. paratuberculosis could be detected from weeks 15-20 in four of the seven animals, and one additional animal became antibody positive at week 35, while two inoculated animals did not produce significant antibody titres during the experiment. At about 1-year post-inoculation, two animals became faecal shedders, while two others started to excrete bacteria into faeces about 2 years post-inoculation. The appearance of M. a. paratuberculosis in faeces was not associated with a decline in cellular responses as far as could be assessed using the current methods for measuring CMI. Pathological lesions due to M. a. paratuberculosis infection and presence of bacteria were recorded in the intestine and/or mesenteric lymph nodes of five animals while lymph node changes suggestive of paratuberculosis were observed in one animal. Only the two animals with no signs of an active infection at necropsy showed a considerable decline in the cellular parameters during the last year of the study, particularly in the IFN-gamma assay. The two animals with the highest levels of M. a. paratuberculosis responsive CD8+ lymphocytes in the circulation about 1-year post-inoculation had no detectable lesions in the distal ileum and colon at necropsy, while high numbers of gammadelta T-cells responsive to M. a. paratuberculosis in the circulation were associated with disseminated lesions in the distal ileum and colon.

Disseminated Mycobacterium celatum (type 3) infection in a domestic ferret (Mustela putorius furo).

A 4-year-old male ferret (Mustela putoriusfuro) had a 6-month history of weight loss and gradual development of depression and coughing. Necropsy findings included pale gray tissue around the distal trachea, multiple nodules in the lungs, a single nodule in the stomach wall, gray foci in the liver, and enlarged lymph nodes. Histologic examination revealed multifocal to coalescing granulomatous inflammation in the trachea, lungs, stomach, liver, and lymph nodes, with acid-fast bacteria in epithelioid cells and macrophages. The acid-fast bacteria were identified as Mycobacterium celatum (type 3) using DNA sequence analysis of the 16S ribosomal DNA gene. M. celatum is a recently described mycobacterium isolated mainly from immunocompromised humans. This is the first report of M. celatum infection in an animal.

Nephrotoxicity of Narthecium ossifragum in cattle in Norway.

During the summer of 1992 renal failure was diagnosed in 232 grazing cattle in 85 herds on the west coast of Norway. The salient clinical signs were depression, anorexia and melaena or fresh blood in the faeces; diarrhoea was also commonly observed. The serum concentrations of creatinine, urea, magnesium and phosphorus, and the activities of glutamate dehydrogenase, aspartate aminotransferase and creatine kinase were above normal and the serum calcium concentration was below normal. Post mortem examinations consistently revealed renal tubular necrosis. In some cases there was liver necrosis and also erosions at the base of the tongue, in the oesophagus and in the jejunum and colon. The toxicity was probably caused by the plant Narthecium ossifragum (bog asphodel).