Molecular epidemiology of Mycobacterium bovis isolates from free-ranging wildlife in South African game reserves

Bovine tuberculosis is endemic in African buffalo and a number of other wildlife species in the Kruger National Park (KNP) and Hluhluwe-iMfolozi Park (HiP) in South Africa. It was thought that the infection had been introduced into the KNP ecosystem through direct contact between cattle and buffalo, a hypothesis which was confirmed in this study by IS6110 and PGRS restriction fragment length polymorphism (RFLP) typing. The molecular characterisation of 189 Mycobacterium bovis isolates from nine wildlife species in the HiP, including three smaller associated parks, and the Kruger National Park with adjacent areas showed that the respective epidemics were each caused by an infiltration of a single M. bovis genotype. The two M. bovis strains had different genetic profiles, as demonstrated by hybridisation with the IS6110 and PGRS RFLP probes, as well as with regard to evidence of evolutionary changes to the IS profile. While the M. bovis type in HiP was transmitted between buffaloes and to at least baboon, bushpig and lion without obvious genetic changes in the RFLP patterns, in the KNP a dominant strain was represented in 73% of the M. bovis isolates, whilst the remaining 27% were variants of this strain. No species-specific variants were observed, except for one IS6110 type which was found only in a group of five epidemiologically related greater kudu. This finding was attributed to species-specific behaviour patterns rather than an advanced host-pathogen interaction.     

A lymphocyte transformation assay for the detection of Mycobacterium bovis infection in the Eurasian badger (Meles meles).

The Eurasian badger (Meles meles) is a significant wildlife reservoir of Mycobacterium bovis in Great Britain. Improved control strategies against the disease in badgers require the development of diagnostic tests and vaccines. Here, we report the development of a comparative lymphocyte transformation assay (LTA) using bovine and avian tuberculin as antigen to detect cell-mediated responses in M. bovis-infected badgers. In a pilot study, the performance of this assay was compared with the existing indirect ELISA assay for the detection of tuberculous badgers. The sensitivity of the Comparative LTA was 87.5% compared with 62.5% for the indirect ELISA whereas the ELISA test gave a greater specificity (100% compared with 84.6% for the comparative LTA). Preliminary evidence suggests that for the comparative LTA, the blood may be stored overnight prior to testing and that this procedure might improve the specificity of the assay without compromising the sensitivity.     

A systematic review on the distribution of Mycobacterium bovis infection among wildlife in the Americas

Tropical Animal Health and Production - The occurrence of Mycobacterium bovis infection in wildlife places at risk livestock, public health, and ecosystems that house endangered species. However,...     

Development of a loop-mediated isothermal amplification assay for the detection of Mycobacterium bovis

Bovine tuberculosis caused by Mycobacterium bovis is an important zoonosis. In this study, a simple, rapid method for detecting this organism was developed based on loop-mediated isothermal amplification of the mpt83 gene. The technique will be of value in the clinical and field-based diagnosis of M. bovis and can differentiate it from other bacteria such as Corynebacterium diphtheriae, Streptococcus pneumoniae, β-haemolytic streptococci, Pseudomonas aeruginosa, Yersinia pseudotuberculosis, Staphylococcus aureus, and Klebsiella pneumoniae. The limit of detection was 10 copies/μL and the results were corroborated by PCR. The method was highly specific and more sensitive than PCR.     

Fluorescence polarization assay for the detection of antibodies to Mycobacterium bovis in bovine sera

The performance of a fluorescence polarization assay (FPA) that detects antibodies to Mycobacterium bovis in bovine sera is described. The FPA reported here is a direct binding primary screening assay using a small polypeptide derived from the M. bovis MPB70 protein. A secondary inhibition assay confirms suspect or presumed positive samples. Specificity studies involved five different veterinary laboratories testing 4461 presumed negative bovine samples. FPA specificity was 99.9%. The FPA was used to identify herd status as either M. bovis infected or non-infected. Herd surveillance studies (nine herds) were performed in Mexico and South Africa. The FPA had a specificity of 100% (two negative herds), and correctly identified six of seven infected herds. Finally, sera from 105 slaughter animals that had gross lesions in lymph nodes similar to those seen with bovine tuberculosis were tested by the FPA. Thin sections from the associated formalin-fixed paraffin-embedded samples of lymph nodes were stained using hematoxylin and eosin (H&E) for morphologic examination and using the Ziehl-Neelsen (ZN) method for detection of acid-fast bacilli. Of the 105 animals, 78 were classified as TB suspect based on lesion morphology, 21 were positive by ZN, 9 were positive by FPA and 13 were positive by PCR for the tuberculosis group of Mycobacterium. Among the 21 ZN positives, 11 (52.4%) were PCR positive. Among the 9 FPA positives, 8 (88.9%) were PCR positive. For the 13 PCR positives, 8 (61.5%) were FPA positive and 11 (84.6%) were ZN positives. These results show that use of the FPA for detection of M. bovis infection of cattle has value for bovine disease surveillance programs.     

Use of a macrophage cell line for rapid detection of Mycobacterium bovis in diagnostic samples

Mycobacterium bovis isolation on bacteriological media from suspected cases of bovine tuberculosis (TB) demands laborious and time-consuming procedures. Even polymerase chain reaction (PCR) and radiometric analyses are secondary procedures and not alternatives to bacteriological procedures. Therefore, there is a need to develop new techniques aimed at rapid M. bovis detection in diagnostic samples. The human macrophage cell line THP-1 was thus investigated in experiments of M. bovis propagation and isolation from reference lymph node suspensions. THP-1 cells were shown to support a high-titered propagation within 48h of minute amounts of both M. bovis BCG and fully pathogenic M. bovis strain 503. A semi-nested PCR for TB-complex-specific insertion sequence IS6110 revealed M. bovis infection in THP-1 cells. The same was true of a flow cytometry (FC) assay for expression of M. bovis chaperonin 10 in infected cells. The reduced time for isolation and identification of M. bovis (48-72h) and the consistency of the test results make the use of macrophage cell cultures attractive and cost-effective for veterinary laboratories involved in TB surveillance.     

A fluorescence polarization assay for the detection of antibodies to Mycobacterium bovis in cattle sera

A fluorescence polarization assay (FPA) utilizing fluorescein-labelled MPB70 protein as the antigen was developed and evaluated for its ability to detect antibodies to Mycobacterium bovis in cattle sera. Three panels of sera were examined in this study. These included: (A) sera (n=28) obtained from cattle from which M. bovis was cultured; (B) sera (n=5666) from Canadian field cattle which were presumed to be free from M. bovis; (C) sera (n=10) from cattle infected with Mycobacterium paratuberculosis and known to contain antibodies to this organism. Receiver operating characteristic (ROC) curve analysis of the results of panels A and B yielded an area under the curve value of 0.975 (95% confidence interval=0.971-0.979), which indicated that this FPA is an accurate indicator of M. bovis infection. At the cut-off point recommended by the ROC curve analysis, the FPA sensitivity and specificity estimates were 92.9% (95% confidence interval=76.5-98.9%) and 98.3% (95% confidence interval=97.9-98.6%) respectively. The FPA results were compared to the results of the single intradermal (SID) test for the 28 infected cattle. Fifteen of these animals were scored positive with the SID test (sensitivity=53.6%). The FPA detected 15/15 (100%) of the SID test-positive animals and 11/13 (84.6%) of the SID test-negative animals. Two of the culture-positive cattle were not detected by either test. None of the sera that were obtained from the M. paratuberculosis-infected animals cross-reacted in this assay.     

Evaluation of historical factors influencing the occurrence and distribution of Mycobacterium bovis infection among wildlife in Michigan

OBJECTIVES: To determine historical events leading to establishment of bovine tuberculosis in the white-tailed deer population in the northeastern corner of the lower peninsula (NELP) of Michigan and describe factors relevant to the present outbreak of bovine tuberculosis in Michigan. SAMPLE POPULATION: Cattle and white-tailed deer in Michigan from 1920 to 1990. PROCEDURES: A search of extant historical documents (eg, scientific journals, books, public reports, and correspondence and internal reports from governmental agencies) was conducted. Factors investigated included the number of cattle and prevalence of tuberculosis, deer population and density levels, and changes in regional environments affecting the population and management of cattle and wild deer. RESULTS: High deer numbers and severe winter feed shortages resulting from habitat destruction in the NELP in 1930 contributed to the transmission of tuberculosis from cattle to deer. Starvation increased the susceptibility of deer to infection and modified behavior such that exposure to infected cattle was increased. Relocation of deer resulted in spread of infection to other sites, including locations at which spatial clusters of tuberculosis presently exist. Ribotyping of Mycobacterium bovis from a human patient suggests that the strain of M. bovis presently infecting white-tailed deer in the region is the same strain that affected cattle farms at that time. CONCLUSIONS AND CLINICAL RELEVANCE: Feeding deer to maintain numbers above the normal carrying capacity of the NELP led to deer depending on consumption of livestock feed for survival during winter and increased contact with domestic cattle. This practice should be avoided.     

The development and evaluation of an enzyme-linked immunosorbent assay for the detection of Mycobacterium bovis

A double antibody sandwich layer enzyme-linked immunosorbent assay (ELISA) was used to detect Mycobacterium bovis. The ELISA detected M. bovis is pure culture at concentrations of 1 x 10(5) colony-forming units (CFU) ml-1 and greater, compared to a minimum detection level of 1 x 10(6) CFU ml-1 for isolation techniques. Neither technique detected M. bovis at 1 x 10(4) CFU ml-1. The ELISA did not cross-react with common mycobacterial contaminants such as Mycobacterium avium intracellulare-scrofulaceum complex serotypes 18 and 42, M. terrae, M. fortuitum, M. flavescens and with Escherichia coli or Rhodococcus equi. Further work is needed to evaluate this assay in detecting M. bovis in tissues and the environment.     

Surveillance of wildlife for Mycobacterium bovis infection using culture of pooled tissue samples from ferrets (Mustela furo)

AIM: To compare culture results of homogenates of pooled lymph nodes from individual ferrets with and without macroscopic lesions of bovine tuberculosis for the presence of Mycobacterium bovis, and to determine whether homogenates from 10-30 ferrets could be combined and cultured without loss of sensitivity as a possible method for improving cost-effectiveness of surveillance for M. bovis infection in wildlife populations. METHODS: Numbers of colony forming units (cfu) of M. bovis present in cultures of homogenates of pooled lymph nodes from individual ferrets known to be infected and having no visible lesions (NVL) or macroscopic lesions consistent with bovine tuberculosis were determined. Prevalences of M. bovis infection in populations of ferrets in the Marlborough region of the South Island of New Zealand were determined by culturing homogenates of pooled lymph nodes from individual animals. Samples from homogenates from North Canterbury were combined to form pools representing 10, 20 and 30 animals and also cultured for M. bovis. RESULTS: Fewer M. bovis cfu were isolated from ferrets with NVL (mean=0.77 log10) compared with ferrets with macroscopic lesions (mean=3.22 log10; p<0.05). The mean prevalence of infection in eight different surveys involving 427 ferrets from the Marlborough region was 18% (range 8-44%), which included a small number of animals with macroscopic lesions of tuberculosis. Pooling of samples from up to 30 different ferrets with NVL did not reduce the sensitivity of detecting M. bovis infected populations. CONCLUSION: Culturing of pools of lymph node samples detected a significant proportion of M. bovis-infected ferrets that would otherwise have gone unnoticed based on samples that had only macroscopic lesions. Culturing of samples pooled from up to 30 different ferrets could provide significant cost savings in surveys of wildlife for the presence of M. bovis infection without any apparent loss of sensitivity.     

Accidental Mycobacterium bovis infection in a veterinarian

CASE: A veterinarian developed tenosynovitis and secondary carpal tunnel syndrome following accidental inoculation of Mycobacterium bovis during the necropsy of a tuberculous possum from Westland, New Zealand.

CLINICAL RELEVANCE: M. bovis infection is a zoonotic disease, and occupational exposure to tuberculous animals places people at risk of contracting the disease.

CONCLUSIONS: Adhering to safe work practices reviewed in this article is important to minimise the risk of infection to people handling tuberculous animals.     

Pulmonary Mycobacterium bovis infection in a dog

AIM: To describe the clinical course of a dog infected with Mycobacterium bovis causing a granulomatous pneumonia. CLINICAL FINDINGS: The dog initially presented with a persistent cough, inappetence and weight loss. Clinical findings included a fever, dyspnoea and tachypnoea, with haematological evidence of a mild neutrophilia and hypoalbuminaemia. Radiographs of the chest demonstrated a concomitant pneumothorax, pleural effusion, and a consolidated area within the left caudal lung lobe. An exploratory thoracotomy revealed this to be a ruptured granulomatous lesion. Subsequent histopathological, microbiological and genetic studies identified M. bovis as the causal agent. CLINICAL SIGNIFICANCE: Mycobacterium bovis infections should be included in the differential diagnosis of pulmonary disease and pleural effusions in dogs living in regions of New Zealand known to have a high incidence of mycobacterial infection in wildlife and farm animals.     

Assessment of an enzyme linked immunosorbent assay for the detection of cattle infected with Mycobacterium bovis

An indirect enzyme linked immunosorbent assay (ELISA) using an unpurified antigen was assessed for its accuracy in detecting Mycobacterium bovis infected cattle. The ELISA test recorded sensitivities of 88.7% and 63%, respectively, for infected cattle tuberculin tested positive and for infected cattle never tuberculin tested. Specificity was determined at 52.6% for cattle from confirmed free herds which had never been tuberculin tested. Significant differences in the mean ELISA values were recorded between the 3 groups. No evidence was found for long term effects of tuberculin testing upon the titre of antibodies detected by the ELISA in unaffected cattle. The indirect ELISA using the unpurified antigen of this assay was considered to be unsuitable as an alternative to tuberculin testing for the detection of M. bovis infected animals.     

Mycobacterium bovis infection and tuberculosis in cattle

This review considers the possible events that can occur when cattle are exposed to Mycobacterium bovis and, where appropriate, draws on principles accepted for tuberculosis infection in humans and laboratory animal models. Consideration is given to the many complex factors which influence the outcome of challenge with tubercle bacilli. These include features inherent to the mycobacterium, the host and the environment. It is apparent that clinical disease probably occurs only in a relatively small, but undetermined, proportion of cattle that are exposed to Al. bovis. The majority of animals may clear infection or control the bacilli, possibly in a condition of latency. It is concluded that a better understanding of the dynamics of the events following M. bovis exposure and subsequent infection in cattle would be of significant benefit in developing new tools appropriate for disease control and to designing optimal approaches for their application.     

Pulmonary Mycobacterium bovis infection in a dog

dAim: To describe the clinical course of a dog infected with Mycobacterium bovis causing a granulomatous pneumonia.

dClinical findings: The dog initially presented with a persistent cough, inappetence and weight loss. Clinical findings included a fever, dyspnoea and tachypnoea, with haematological evidence of a mild neutrophilia and hypoalbuminaemia. Radiographs of the chest demonstrated a concomitant pneumothorax, pleural effusion, and a consolidated area within the left caudal lung lobe. An exploratory thoracotomy revealed this to be a ruptured granulomatous lesion. Subsequent histopathological, microbiological and genetic studies identified M. bovis as the causal agent.

dClinical significance: Mycobacterium bovis infections should be included in the differential diagnosis of pulmonary disease and pleural effusions in dogs living in regions of New Zealand known to have a high incidence of mycobacterial infection in wildlife and farm animals.     

Accidental Mycobacterium bovis infection in a veterinarian

CASE: A veterinarian developed tenosynovitis and secondary carpal tunnel syndrome following accidental inoculation of Mycobacterium bovis during the necropsy of a tuberculous possum from Westland, New Zealand. CLINICAL RELEVANCE: M. bovis infection is a zoonotic disease, and occupational exposure to tuberculous animals places people at risk of contracting the disease. CONCLUSIONS: Adhering to safe work practices reviewed in this article is important to minimise the risk of infection to people handling tuberculous animals.     

Histopathogenesis of experimental Mycobacterium bovis infection in mice

In-bred strains of mice are commonly used to model pathogenic infections due to their cost and utility. In order to understand better the nature of experimental tuberculosis in mice, we infected BALB/c mice with a virulent field isolate of Mycobacterium bovis. Mice were sacrificed at intervals in order to visualise the pathological lesions in major internal organs. Pathological lesions in tissues increased in number and severity over time and replicated many of the salient features observed in badgers and cattle infected with M. bovis. These similarities are discussed. Examination of pathological lesions at terminal stages of infection enabled us to suggest the lethal effects of M. bovis mediated through the host response. We conclude that the mouse is a relevant surrogate species in which to study the virulence of M. bovis, as well as the influence of vaccination on its pathogenicity.     

Use of DNA amplification for the rapid identification of Mycobacterium bovis

DNA amplification using the polymerase chain reaction technique was evaluated for rapid identification of Mycobacterium bovis. Two oligonucleotide primers of 20 bases in length were constructed to target a region of the gene encoding the M. bovis secretory protein, MPB70. The amplification reaction produced a single product 372 bp in size which was readily detected by agarose gel electrophoresis. All 84 strains of M. bovis tested produced a positive signal in the amplification reaction. In addition all isolates fro the M. tuberculosis complex tested, with the exception of M. microti, gave a single band at 372 bp. No amplified product was detected when 24 other species of mycobacteria and species from four other genera were tested. The sensitivity of the test was such that a single viable cell could be detected in the reaction. This technique provides a simple and extremely sensitive method of identifying isolates of M. bovis and other pathogenic M. tuberculosis complex organisms.