Characterisation of Brucella species in Zimbabwe

Abstract

Brucellosis is an important zoonotic disease of ruminants, suidae, canids, several wildlife species and humans caused by from the genus Brucella genus consisting of gram-negative bacteria that are facultative intracellular pathogens. Brucellosis is endemic in sub-Saharan African countries, which include Zimbabwe where Brucella abortus and B. melitensis have been reported. In order to control brucellosis, surveillance and identification of Brucella species is of paramount importance. The aim of the study was to carry out a survey of bovine brucellosis using serological tests, PCR assay and characterizing Brucella spp in Zimbabwe using molecular techniques. Serological tests and PCR based assay were used to detect brucellosis in cattle and wildlife in Chiredzi district of Zimbabwe. Blood and serum samples from cattle (n=700) from Chiredzi district (Malipati and Pesvi communal areas), as well as, African buffalo (n=10) and impalas (n=14) (from Gonarezhou National Park (GNP)) were tested for bovine brucellosis using rose bengal test (RBT), serum agglutination methods (SAT) and indirect enzyme linked immunoassays (iELISA). The seroprevalence of bovine brucellosis in cattle was found to be 8.3% using RBT and iELISA and 6.0% using RBT, SAT and iELISA. No antibodies for Brucella spp were detected in the African Buffaloes and impalas. Brucella strains were cultured from milk and blood sample from cows in Chiredzi district. DNA from the two Brucella strains was amplified using the ITS66 and ITS279 primers specific for Brucella 16- 23S rDNA intergenic spacer (ITS) region. This Brucella specific PCR assay was also used to detect Brucella DNA in blood and buffy coat samples from cattle that were seropositive using RBT, SAT and iELISA. Despite the fact that the blood and buffy coat samples were from animals that were bacteriemia since Brucella was isolated from blood and milk and all samples had SAT titres above 148, no Brucella DNA were detected using the specific PCR assay. Furthermore Brucella strains (23) isolated at the Central Veterinary Laboratory (CVL) in Zimbabwe were identified using molecular techniques like MLVA, AMOS-PCR and Bruceladder PCR. Only a few isolates were classified up to species level using bacteriology method (biotyping). MLVA-16 was able to identify B. abortus, B. melitensis, B. ovis and B. suis strains amongst Zimbabwean isolates. The latter two species is the first report of these species in Zimbabwe. The MLVA assay furthermore indicated that some Zimbabwean isolates significantly differ from isolates of other origin in specific species clusters. These Zimbabwean isolates could not be speciated and future research using bacteriology and molecular characterization are necessary to characterize these isolates. A few strains identified using bacteriological methods and MLVA were also verified using AMOS-PCR and Bruceladder. For these results it is clear that MLVA, AMOS-PCR and Bruceladder can be used to identify Brucella species and biovars and can therefore contribute towards the control of brucellosis in African countries.