Bovine brucellosis in South Africa is caused mainly by Brucella abortus biovar (bv.) 1 and less frequently by B. abortus bv. 2. Bacterial isolation is regarded as the gold standard for diagnosis of Brucella species; however, it is not very sensitive. The aim of this study was to determine the selective medium with optimum antibiotic composition that will allow the growth of Brucella species (spp.) while inhibiting moulds, yeast and most, if not all, Gram-negative contaminants in South Africa. In the controlled experiment, modified Agrifood Research and Technology Center of Aragon (CITA) medium (mCITA) seemed to be the optimum selective medium for isolation of Brucella spp. as compared with Farrell’s medium (FM) and modified Thayer Martin (mTM), while FM inhibited the growth of most fungal and bacterial contaminants. Mean comparison between the three media used to culture B. abortus resulted in lower mean difference ranging from 0 to 2.33. In case of Brucella ovis, high mean difference was obtained when comparing FM with mCITA (10.33) and mTM (12). However, the mean differences of 0.67 and 1.67 were obtained when comparing mCITA and mTM media used to, respectively, culture pasteurised and raw milk spiked with B. ovis. Further optimisation at the Agricultural Research Council – Onderstepoort Veterinary Research Institute resulted in a comparable performance between FM and mCITA; however, mCITA allowed optimal growth of the fastidious B. ovis, which is generally inhibited on FM. Generally, mCITA seemed to be the optimum selective medium for isolation of Brucella spp., while FM inhibits the growth of most fungal and bacterial contaminants. Thus, veterinary laboratories can use mCITA and/or FM but should take into consideration the detection of factious Brucella isolated in the country or region.

Brucella abortus and B . melitensis have been reported in several studies in animals in Zimbabwe but the extent of the disease remains poorly known. Thus, characterizing the circulating strains is a critical first step in understanding brucellosis in the country. In this study we used an array of molecular assays including AMOS-PCR, Bruce-ladder, multiple locus variable number tandem repeats analysis (MLVA) and single nucleotide polymorphisms from whole genome sequencing (WGS-SNP) to characterize Brucella isolates to the species, biovar, and individual strain level. Sixteen Brucella strains isolated in Zimbabwe at the Central Veterinary laboratory from various hosts were characterized using all or some of these assays. The strains were identified as B . ovis , B . abortus , B . canis and B . suis , with B . canis being the first report of this species in Zimbabwe. Zimbabwean strains identified as B . suis and B . abortus were further characterized with whole genome sequencing and were closely related to reference strains 1330 and 86/8/59, respectively. We demonstrate the range of different tests that can be performed from simple assays that can be run in laboratories lacking sophisticated instrumentation to whole genome analyses that currently require substantial expertise and infrastructure often not available in the developing world.

The availability of whole genome sequences in public databases permits genome-wide comparative studies of various bacterial species. Whole genome sequence-single nucleotide polymorphisms (WGS-SNP) analysis has been used in recent studies and allows the discrimination of various Brucella species and strains. In the present study, 13 Brucella spp. strains from cattle of various locations in provinces of South Africa were typed and discriminated. WGS-SNP analysis indicated a maximum pairwise distance ranging from 4 to 77 single nucleotide polymorphisms (SNPs) between the South African Brucella abortus virulent field strains. Moreover, it was shown that the South African B. abortus strains grouped closely to B. abortus strains from Mozambique and Zimbabwe, as well as other Eurasian countries, such as Portugal and India. WGS-SNP analysis of South African B. abortus strains demonstrated that the same genotype circulated in one farm (Farm 1), whereas another farm (Farm 2) in the same province had two different genotypes. This indicated that brucellosis in South Africa spreads within the herd on some farms, whereas the introduction of infected animals is the mode of transmission on other farms. Three B. abortus vaccine S19 strains isolated from tissue and aborted material were identical, even though they originated from different herds and regions of South Africa. This might be due to the incorrect vaccination of animals older than the recommended age of 4–8 months or might be a problem associated with vaccine production.