Prevalence and characterization of Brucella spp. in slaughter animals in abattoirs in Gauteng Province, South Africa

Abstract

Brucellosis is a neglected zoonotic disease that affects humans, and domestic animals; and has huge food safety implication and economic significance. Globally, abattoirs are used for passive and active surveillance of diseases of both economic and public health significance. Surveys by serological assays of slaughtered animals can be used to detect newly introduced disease agents and monitor the effectiveness of disease control and eradication programmes. The research goal of the study was to determine the frequency of Brucella spp. detection in slaughtered livestock in abattoirs in Gauteng Province, and the risk posed to abattoir workers. Retrospective data from 2007 to 2015 were collated and analyzed for disease trends involving 376,757 animals. This objective was to review the reported frequency of brucellosis based on the diagnosis of the zoonosis in the Government brucellosis laboratory at the Onderstepoort Veterinary Research before conducting the current cross-sectional study in this thesis. It is evident from the review of the laboratory data over a nine year period (2007-2015), according to the species of animals tested, the individual animal seroprevalence in cattle (n=304,020 animals tested), sheep (n=39,672), goats (29,967) and pigs (3,098) was 5.23% (CI: 5.15- 5.31), 2.09% (CI: 1.95-2.23) 0.63% (CI: 0.54-0.72) and 0.13% (CI: 0.05-0.33) respectively. The diagnostic sensitivity and specificity for the RBT were assumed to be 100% and 75%, based on previous validation studies respectively (Nielsen et al., 2005, Stemshorn et al., 1985). The cut-off value for CFT test was 15 IU/ml or more as an indication of infection and compared to the positive and negative controls. We compared the seroprevalence of brucellosis within the four species and observed that the difference was statistically significant (P=0.00). In the study, the cattle population was over-represented because of the bias and focus of the brucellosis scheme on cattle in the country. The herd level seroprevalence varied from 1.01% (CI: 0.06-1.69) in goats to 3.64% (CI: 1.09-9.05) in pigs, 8.72% (CI: 7.770-9.87) in sheep and 23.3% (CI: 22.4-24.2) cattle. Importantly, the seroprevalence in pigs is most probably due to cross-reaction with Yersinia enterocolitica serotype O:9 as Brucella suis has not been reported in South Africa. However, we cannot exclude that some pigs may have been infected with B. abortus or B. melitensis spilling over form infected cattle and small ruminants, respectively. Current cross-sectional study sampled 14 abattoirs where un-clotted blood and lymph node samples were collected from 342 animals (200 cattle, 57 sheep and 85 pigs). Rose Bengal test (RBT), complement fixation test (CFT) and indirect enzyme linked immunosorbent assay (iELISA), were used to determine the seroprevalence of brucellosis in the slaughter animals. Animal tissue samples (lymph nodes, spleen and liver) were cultured for Brucella spp. isolation using standard methods. AMOS PCR (B. abortus, B. melitensis, B. ovis and B. suis) was used for molecular characterization of the Brucella isolates which were also biotyped using standard phenotypic methods. The RBT screening revealed a seroprevalence of 11.0% (22 of 200) in cattle, 0.0% in sheep and 0.0% in pigs. The CFT confirmed 18.2% (4 of 22) as seropositive and iELISA confirmed 5.5% (11 of 200) in cattle with a large amount of RBT cattle positives below the cut-off point of the iELISA. With the iELISA for B. ovis, 1.7% (1 of 57) was positive while the pig samples were seronegative. The genus specific 16-23 ribosomal DNA interspacer region (ITS) PCR assay on tissue samples detected Brucella DNA in 12.5% (25 of 200) of cattle, 93.0% (53 of 57) of sheep and 27.1% (23 of 85) Brucella-like DNA in pigs. Detection of Brucella DNA was from both seronegative sheep and pigs. AMOS PCR characterized Brucella DNA of 11 isolates from cattle as B. melitensis (n=6) and B. abortus (n=5). Of the 11 isolates (seven were from the seropositive cattle), five (all from seropositive animals) were biotyped consisting of two B. abortus biovar (bv) 1and three B. melitensis (n=3) isolates of which one was biovar 2 and two were biovar 3. In sheep, AMOS PCR characterized 25 (44.0%) isolates as 18 B. melitensis and 7 as B. ovis (which included the ELISA B. ovis seropositive sheep). The ITS PCR positive pig samples did not amplify using AMOS PCR and no culture was established. As such, brucellosis in the pigs was deemed negative. Brucellosis could not be detected in the pig samples using serology, culture and PCR. The ITS-PCR detected Brucella DNA in the tissue, but this PCR is not an OIE recommended and validated PCR. Furthermore, the ITS-PCR results could not be confirmed using qPCR and AMOS-PCR. This result showed that brucellosis is a much bigger problem in cattle as B. abortus and B. melitensis were isolated as well as B. melitensis were isolated from seronegative sheep slaughtered at abattoirs in Gauteng province. A thorough investigation needs to be established to investigate brucellosis in sheep and pigs in South Africa. A recommendation on the diagnostic strategy is to conduct a combination of serological tests, PCR and cultures to increase the chances of making positive diagnosis in animals. The seroprevalence of brucellosis for the abattoir workers were determined using RBT, BrucellaCap and IgG ELISA from 103 abattoir workers from six abattoirs with seropositive animals. Of the 103 abattoir workers’ serum samples tested with combined serological tests, the overall distribution and seroprevalence for Brucella spp. infection or exposure was 21 (20.4%, 95%CI=13.1-29.5). The distribution and seroprevalence of brucellosis were 13 (12.6%, 95%CI=6.89-20.6), 9 (8.74%, 95%CI=4.07-15.9) and 18 (17.5%, 95%CI=10.7-26.2) with RBT, BrucellaCap and IgG ELISA respectively. It was concluded that slaughtered livestock infected with Brucella spp. poses an exposure potential of zoonotic risk to abattoir workers and consumers of uncooked or undercooked meat and meat products. Differentiating acute febrile illnesses in human brucellosis infection using more specific diagnostic tools is also recommended.