Zoonotic tuberculosis and brucellosis in Africa: neglected zoonoses or minor public-health issues? The outcomes of a multi-disciplinary workshop.

Late in 2007, veterinary, medical and anthropological professionals from Europe and Africa met in a 2-day workshop in Pretoria, South Africa, to evaluate the burden, surveillance and control of zoonotic tuberculosis and brucellosis in sub-Saharan Africa. Keynote presentations reviewed the burden of these diseases on human and livestock health, the existing diagnostic tools, and the available control methods. These presentations were followed by group discussions and the formulation of recommendations. The presence of Mycobacterium bovis and Brucella spp. in livestock was considered to be a serious threat to public health, since livestock and animal products are the only source of such infections in human beings. The impact of these pathogens on human health appears to be relatively marginal, however, when compared with Mycobacterium tuberculosis infections and drug resistance, HIV and malaria. Appropriate diagnostic tools are needed to improve the detection of M. bovis and Brucella spp. in humans. In livestock, the 'test-and-slaughter' approach and the pasteurization of milk, which have been used successfully in industrialized countries, might not be the optimal control tools in Africa. Control strategies should fit the needs and perceptions of local communities. Improved intersectoral and international collaboration in surveillance, diagnosis and control, and in the education of medical and veterinary personnel, are advocated.

Cloning, sequencing and expression of white rhinoceros (Ceratotherium simum) interferon-gamma (IFN-gamma) and the production of rhinoceros IFN-gamma specific antibodies.

Bovine tuberculosis (BTB) is endemic in African buffalo (Syncerus caffer) in the Kruger National Park (KNP). In addition to buffalo, Mycobacterium bovis has been found in at least 14 other mammalian species in South Africa, including kudu (Tragelaphus strepsiceros), Chacma baboon (Papio ursinus) and lion (Panthera leo). This has raised concern about the spillover into other potentially susceptible species like rhinoceros, thus jeopardising breeding and relocation projects aiming at the conservation of biodiversity. Hence, procedures to screen for and diagnose BTB in black rhinoceros (Diceros bicornis) and white rhinoceros (Ceratotherium simum) need to be in place. The Interferon-gamma (IFN-gamma) assay is used as a routine diagnostic tool to determine infection of cattle and recently African buffalo, with M. bovis and other mycobacteria. The aim of the present work was to develop reagents to set up a rhinoceros IFN-gamma (RhIFN-gamma) assay. The white rhinoceros IFN-gamma gene was cloned, sequenced and expressed as a mature protein. Amino acid (aa) sequence analysis revealed that RhIFN-gamma shares a homology of 90% with equine IFN-gamma. Monoclonal antibodies, as well as polyclonal chicken antibodies (Yolk Immunoglobulin-IgY) with specificity for recombinant RhIFN-gamma were produced. Using the monoclonals as capture antibodies and the polyclonal IgY for detection, it was shown that recombinant as well as native white rhinoceros IFN-gamma was recognised. This preliminary IFN-gamma enzyme-linked immunosorbent assay (ELISA), has the potential to be developed into a diagnostic assay for M. bovis infection in rhinoceros.

A sensitive seminested PCR method for the detection of Shigella in spiked environmental water samples.

A rapid seminested polymerase chain reaction (PCR) method for the specific, sensitive detection of virulent Shigella spp. in spiked environmental water samples was developed. A set of primers specific for the invasion plasmid antigen gene (ipaH) of virulent Shigella spp. and enteroinvasive Escherichia coli produced a 620-bp fragment that was used as template for the seminested primer pair delineating a 401-bp fragment. By using agarose gel electrophoresis for detection of the seminested PCR-amplified products, a detection limit of 1.6 x 10(3) cfu S. flexneri was obtained with amplification reactions from crude bacterial lysates. The PCR procedure coupled with an enrichment culture incubated for 6 h detected as few as 1.6 S. flexneri organisms in pure culture. Treated sewage, ground, surface and drinking water samples collected from various sources were seeded with S. flexneri and incubated in GN broth for 6 h before detection by seminested PCR. A detection limit lower than 14 cfu/ml was achieved in some water samples. The results indicate that the described seminested PCR has the advantage of a rapid turnaround time and it fulfills the requirements of sensitivity and specificity for use in an environmental laboratory.

Towards establishing a rhinoceros-specific interferon-gamma (IFN-γ) assay for diagnosis of tuberculosis.

Mycobacterium bovis is the causal agent of bovine tuberculosis (BTB), with a diverse host range, extending from livestock to domestic and captive wild animals as well as free-ranging wildlife species. In South Africa, BTB is endemic in the Kruger National Park (KNP) and the Hluluwe iMfolozi National Park (HiP), where the high prevalence of M. bovis infections in buffalo herds has led to infection of a number of wildlife species. This has raised concerns about the spillover into the rhinoceros population, a species known to be susceptible to both M. bovis and Mycobacterium tuberculosis, jeopardizing breeding and relocation projects that serve to conserve and protect this species. In view of the advantages of the interferon-gamma (IFN-γ) assay in the diagnosis of BTB in a variety of species worldwide, such an assay has been developed for rhinoceroses by Morar and co-workers in 2007. In this study, this assay was optimized using recombinant eukaryotic rhinoceros IFN-γ and the lower detection limit was calculated to be 0.5 ng/ml. Subsequently, assessing the detection of native rhinoceros IFN-γ protein in whole-blood samples revealed stimulation with each of the mitogens: pokeweed (PWM), phytohaemagglutinin (PHA) & phorbol 12-myristate 13-acetate and calcium ionophore (PMA/CaI), though most prominently with the latter two. In addition, samples collected from 52 clinically healthy rhinoceroses, of presumed negative BTB status, from two different areas in South Africa were used to determine the cut-off value for a negative test result. This was calculated to be 0.10 (OD490 nm ) and as determined in this study is a preliminary recommendation based on IFN-γ responses observed in samples from BTB-free rhinoceroses only.

The elephant interferon gamma assay: a contribution to diagnosis of tuberculosis in elephants.

Mycobacterium tuberculosis (M. tb) has been shown to be the main causative agent of tuberculosis in elephants worldwide. M. tb may be transmitted from infected humans to other species including elephants and vice versa, in case of prolonged intensive contact. An accurate diagnostic approach covering all phases of the infection in elephants is required. As M. tb is an intracellular pathogen and cell-mediated immune (CMI) responses are elicited early after infection, the skin test is the CMI assay of choice in humans and cattle. However, this test is not applicable in elephants. The interferon gamma (IFN-γ) assay is considered a good alternative for the skin test in general, validated for use in cattle and humans. This study was aimed at development of an IFN-γ assay applicable for diagnosis of tuberculosis in elephants. Recombinant elephant IFN-γ (rEpIFN-γ) produced in eukaryotic cells was used to immunize mice and generate the monoclonal antibodies. Hybridomas were screened for IFN-γ-specific monoclonal antibody production and subcloned, and antibodies were isotyped and affinity purified. Western blot confirmed recognition of the rEpIFN-γ. The optimal combination of capture and detection antibodies selected was able to detect rEpIFN-γ in concentrations as low as 1 pg/ml. The assay was shown to be able to detect the native elephant IFN-γ, elicited in positive-control cultures (pokeweed mitogen (PWM), phorbol myristate acetate plus ionomycin (PMA/I)) of both Asian and African elephant whole-blood cultures (WBC). Preliminary data were generated using WBC from non-infected elephants, a M. tb infection-suspected elephant and a culture-confirmed M. tb-infected elephant. The latter showed measurable production of IFN-γ after stimulation with ESAT6/CFP10 PPDB and PPDA in concentration ranges as elicited in WBC by Mycobacterium tuberculosis complex (MTBC)-specific antigens in other species. Hence, the IFN-γ assay presented potential as a diagnostic tool for the detection of elephant tuberculosis. Validation of the assay will require its application in large populations of non-infected and infected elephants.

Development of a lion-specific interferon-gamma assay.

The ongoing spread of bovine tuberculosis (BTB) in African free-ranging lion populations, for example in the Kruger National Park, raises the need for diagnostic assays for BTB in lions. These, in addition, would be highly relevant for zoological gardens worldwide that want to determine the BTB status of their lions, e.g. for translocations. The present study concerns the development of a lion-specific IFN-γ assay, following the production and characterization of monoclonal antibodies specific for lion interferon-gamma (IFN-γ). Recombinant lion IFN-γ (rLIFN-γ) was produced in mammalian cells and used to immunize mice to establish hybridoma cell lines producing monoclonal antibodies. These were used to develop a sensitive, lion IFN-γ-specific capture ELISA, able to detect rLIFN-γ to the level of 160 pg/ml. Recognition of native lion IFN-γ was shown in an initial assessment of supernatants of mitogen stimulated whole blood cultures of 11 known BTB-negative lions. In conclusion, the capture ELISA shows potential as a diagnostic assay for bovine tuberculosis in lions. Preliminary results also indicate the possible use of the test for other (feline) species.