Molecular detection of bovine pathogens and microbiota harboured by Stomoxys calcitrans occurring in South African feedlots

Abstract

Stomoxys calcitrans are biting flies commonly known as stable flies. They belong to the family Muscidae which is composed of 18 described species under the genus Stomoxys with a cosmopolitan distribution. Stomoxys spp. are of economic importance worldwide due to their ability to mechanically transmit various pathogens including bacteria, viruses, and protozoa. Vector borne lumpy skin disease virus, rickettsiosis, anaplasmosis and ehrlichiosis are major diseases that threaten and affect livestock production in tropical and sub-tropical regions of Africa. Some bacteria are essential for the larval survival and development for stable flies, but little is known about the innate microbial communities of stable flies. The aim of this study was to characterize stable flies inhabiting selected feedlots in South Africa and detect disease-causing pathogens they are harbouring using PCR. Furthermore, this study utilized Illumina MiSeq next generation sequencing of 16S amplicons to characterize stable fly microbiome. A total of 10195 stable flies were collected from three feedlots, with 9993 from Van der Leeuw Boerdery in Potchefstroom, North West province, 175 from GysbertHoek in Sasolburg, Free State province, and 27 from Doornbult in Limpopo province. Morphological identification of stable flies was further supported by amplification of CO1 and 16S rRNA genes whereby their sequences matched with respective stable fly genes on NCBI database. Furthermore, phylogenetic analysis of CO1 gene also showed that Stomoxys calcitrans characterized in this study clustered with other Stomoxys spp. from around the world. PCR detected Anaplasma marginale infections from S. calcitrans with infection rates of 10% and 16% in flies from Free State and North West respectively, whilst none of the flies from Limpopo were positive for the presence of A. marginale. This study is the first to report on the detection of A. marginale infections in stable flies by PCR in South Africa. The current study has detected 27% LSDV by PCR from S. calcitrans collected from North West alone. None of Rickettsia and Ehrlichia spp. were detected from all the sampled provinces. This study also attempted to determine the best washing method for the removal of microbes from the fly’s surface by washing in 70 % ethanol, 10% bleach and 10% tween 20. In the current study, 70% EtOH was one of the less effective disinfecting methods tested, while 10% bleach and 10% tween20 solutions appeared to be the most effective methods of sterilizing the external surface of stable fly without interfering with the analysis of the mitochondrial DNA of fly internal contents. Analysis of gut microbes from 50 South African Stomoxys flies produced a total of 462 operational taxonomic units (OTUs). The most abundant genera at Van der Leeuw Boerdery, Potchefstroom was Sphingomonas at 12.1%, followed by Wolbachia at 11.7%. At GysbertHoek, Sasolburg, the most abundant genera were Sphingomonas at 13.4 %, followed by Agrobacterium at 3.2%. Bacterial genera of medical, veterinary and ecological importance detected in the current study include Clostridium, Bacillus, Anaplasma, Rickettsia, Wolbachia, and Rhizobium.