Absence of Tuberculosis-Causing Mycobacteria from Slaughtered Livestock Tissues and Environmental Samples, Gauteng Province, South Africa.

Mycobacterium tuberculosis complex (MTBC) is a group of bacteria responsible for causing tuberculosis in animals and humans. In South Africa (S.A), slaughterhouses are registered by the government and closely inspected and audited for hygienic slaughter practices. Meat inspection to detect lesions has been used for passive surveillance, monitoring, and diagnosis of the disease status. Information on the current status of bovine tuberculosis (bTB) in livestock in the country is limited. Hence, we investigated the occurrence of Mycobacterium spp. in the tissues of slaughtered livestock and environmental samples in abattoirs in Gauteng province of South Africa (S.A). The cross-sectional study employing random sampling from cattle, pigs, and sheep (with the collection of liver, lung, spleen, and different lymph nodes) irrespective of lesions was carried out in 19 red meat abattoirs. Five hundred animals were sampled, comprising cattle (n = 369), pigs (n = 90), and sheep (n = 41). Additionally, 19 environmental samples were collected from feedlots, or where animals drink water while awaiting slaughter, to identify mycobacterial species using culture, acid-fast bacteria staining, and polymerase chain reaction (PCR). The Chi-square and Fisher's Exact tests were used to detect statistically significant differences in the frequency of detection of Mycobacterium spp. according to the variables investigated (types of tissues, livestock, abattoirs, etc.). The PCR assays detected no MTBC complex species DNA in the bacterial isolates from cattle (n = 32). Sequence analysis (16S rDNA) of the isolates from eight cattle confirmed only two species, namely Mycobacterium colombiense (99.81% identity) and Mycobacterium simiae (99.42% identity). The remaining isolates were identified as members of the Actinomadura species. From the environmental samples, bacterial isolation was made from three samples, and two could only be identified up to the genus level (Mycobacterium species) while the remaining isolate was identified as Mycobacterium senuense (99.22% identity). The study revealed the absence of bovine tuberculosis-causing pathogens in red meat abattoirs of the Gauteng province. Although non-tuberculous Mycobacteria have been implicated as potentially causing tuberculosis-like diseases in livestock, their occurrence in the current study was found to be low, but the potential to cause disease cannot be ignored.

Application of the gamma-interferon assay to determine the prevalence of bovine tuberculosis in slaughter livestock at abattoirs in Gauteng, South Africa.

BACKGROUND: Bovine tuberculosis (bTB) is a zoonotic disease with great economic impact estimated at billions of dollars annually worldwide. Meat inspection represents a long-standing form of disease surveillance that serves both food safety and animal health. The objective of this study was to determine the prevalence of bTB in livestock at abattoirs using a cell-mediated immune (CMI) assay, the gamma interferon (IFN-γ) assay. This cross-sectional study was conducted at selected abattoirs (low-throughput, high-throughput and rural/informal) in Gauteng province, where animals were also subjected to routine meat inspection. RESULTS: A total of 410 fresh blood samples were collected from slaughter livestock (369 cattle and 41 sheep) from 15 abattoirs, and analysed using Bovigam® test kit with bovine, avian and Fortuitum purified protein derivatives (PPD) as blood stimulating antigens. The estimated prevalence of bTB in cattle was 4.4% (95% CI: 2.4%-7.3%). The prevalence of bTB in cattle varied between abattoirs (p = .005), ranging from 0% to 23%; however, there were no significant differences among genders, breeds, municipality, districts, origins of animals (feedlot, auction or farm) or throughput of abattoirs. The prevalence of avian reactors was 6.0% (95% CI: 3.6%-9.2%) in cattle, varying between abattoirs (p = .004) and ranging from 0% to 20.7%. None of the sheep with valid test results was positive for bTB and none was avian reactors (95% CI: 0%-15%). CONCLUSION: The detection of bTB reactor cattle in our study clearly shows the limitation of disease surveillance using a meat inspection approach, as all the 410 slaughter animals sampled had passed visual abattoir inspection and been classified as bTB-free. Our findings therefore emphasize the risk of zoonotic transmission of bTB to abattoir workers and potential food safety hazard to consumers. Furthermore, our study highlights the potential for the use of the IFN-γ assay to reduce this risk.

Expression of Rift Valley fever virus N-protein in Nicotiana benthamiana for use as a diagnostic antigen.

BACKGROUND: Rift Valley fever virus (RVFV), the causative agent of Rift Valley fever, is an enveloped single-stranded negative-sense RNA virus in the genus Phlebovirus, family Bunyaviridae. The virus is spread by infected mosquitoes and affects ruminants and humans, causing abortion storms in pregnant ruminants, high neonatal mortality in animals, and morbidity and occasional fatalities in humans. The disease is endemic in parts of Africa and the Arabian Peninsula, but is described as emerging due to the wide range of mosquitoes that could spread the disease into non-endemic regions. There are different tests for determining whether animals are infected with or have been exposed to RVFV. The most common serological test is antibody ELISA, which detects host immunoglobulins M or G produced specifically in response to infection with RVFV. The presence of antibodies to RVFV nucleocapsid protein (N-protein) is among the best indicators of RVFV exposure in animals. This work describes an investigation of the feasibility of producing a recombinant N-protein in Nicotiana benthamiana and using it in an ELISA. RESULTS: The human-codon optimised RVFV N-protein was successfully expressed in N. benthamiana via Agrobacterium-mediated infiltration of leaves. The recombinant protein was detected as monomers and dimers with maximum protein yields calculated to be 500-558 mg/kg of fresh plant leaves. The identity of the protein was confirmed by liquid chromatography-mass spectrometry (LC-MS) resulting in 87.35% coverage, with 264 unique peptides. Transmission electron microscopy revealed that the protein forms ring structures of ~ 10 nm in diameter. Preliminary data revealed that the protein could successfully differentiate between sera of RVFV-infected sheep and from sera of those not infected with the virus. CONCLUSIONS: To the best of our knowledge this is the first study demonstrating the successful production of RVFV N-protein as a diagnostic reagent by Agrobacterium-mediated transient heterologous expression in N. benthamiana. Preliminary testing of the antigen showed its ability to distinguish RVFV-positive animal sera from RVFV negative animal sera when used in an enzyme linked immunosorbent assay (ELISA). The cost-effective, scalable and simple production method has great potential for use in developing countries where rapid diagnosis of RVFV is necessary.

Validation of an ELISA for the concurrent detection of total antibodies (IgM and IgG) to Rift Valley fever virus.

Rift Valley fever virus (RVFV) infects humans and livestock, causing haemorrhaging and abortions in animals. Three major RVF epizootics have occurred in South Africa since the 1950s and the outbreak in 2010 had a mortality rate of 10.7% in humans. Accurate and early detection is therefore essential for management of this zoonotic disease. Enzyme-linked immunosorbent assays (ELISAs) have been developed for the detection of either IgM or IgG antibodies to RVFV in animal sera. In this study, data are presented on the validation of a double-antigen ELISA for the simultaneous detection of both classes of antibodies to RVFV ina single test. ELISA plates were coated with a recombinant nucleoprotein. The nucleoprotein,conjugated to horseradish peroxidase, was used as the detecting reagent. A total of 534 sera from sheep and cattle were used in the validation. The sheep sera were collected during a RVF pathogenesis study at the Agricultural Research Council (ARC) - Onderstepoort Veterinary Institute and the cattle sera were collected during an outbreak of RVF in 2008 at the ARC -Animal Production Institute in Irene, Pretoria. The ELISA had a diagnostic sensitivity of 98.4% and a specificity of 100% when compared to a commercial cELISA. This convenient and fast assay is suitable for use in serological surveys or monitoring immune responses in vaccinated animals.

Validation of an IgM antibody capture ELISA based on a recombinant nucleoprotein for identification of domestic ruminants infected with Rift Valley fever virus.

The presence of competent vectors in some countries currently free of Rift Valley fever (RVF) and global changes in climate, travel and trade have increased the risk of RVF spreading to new regions and have emphasised the need for accurate and reliable diagnostic tools for early diagnosis during RVF outbreaks. Highly sensitive viral detection systems like PCR have a limited use during outbreaks because of the short duration of viraemia, whereas antibodies like specific IgM which are serological indicators of acute infection, can be detected for up to 50 days after infection. Using the highly conserved and immunogenic recombinant nucleoprotein of RVF virus in an IgM capture ELISA, the risk of laboratory infection associated with traditional serological methods is avoided. The use of pre-coated/pre-blocked ELISA plates and the conjugation of the recombinant nucleoprotein with horseradish peroxidase simplified and shortened the assay procedure. Results showed the assay to be highly reproducible with a lower detection limit equal to that of a commercial competition ELISA. By receiver operating characteristic (ROC) curve analysis the area under curve (AUC) index was determined as 1.0 and the diagnostic sensitivity and specificity at a PP cut-off value of 4.1 as 100% and 99.78% respectively. The results of this study demonstrated that the IgM capture ELISA is a safe, reliable and highly accurate diagnostic tool which can be used on its own or in parallel with other methods for the early diagnosis of RVF virus infection and also for monitoring of immune responses in vaccinated domestic ruminants.