Exploration of bacterial communities of economically important Rhipicephalus tick species in South Africa

Abstract

Tick infestation and tick-borne diseases are a major concern for domestic animal health. Accurate identification and characterization of ticks and their associated microbial communities may lead to the development of improved control strategies targeting known tick-bacterial interactions. Current technologies enable simultaneous detection of tick microbiota. These include microbial communities of ecological, commensal, symbiotic or parasitic importance in nature. The aim of this study was to characterize tick microbial populations from field derived Rhipicephalus tick species in terms of their symbiotic, medical, veterinary and environmental importance in comparison to colony reared controls. To achieve this, field-derived ticks collected from domestic animals including cattle, sheep, goats, horses, dogs and vegetation were morphologically identified as Rhipicephalus evertsi evertsi, R. appendiculatus and R. sanguineus using published taxonomic keys; whilst colony reared ticks were identified as R. e. evertsi, R. appendiculatus, R. decoloratus and R. microplus. To supplement morphological identification, the present study used mitochodrial DNA [cytochrome oxidase I (CO1) and 16S ribosomal ribonucleic acid (16S rRNA)] and nuclear DNA markers [(internal transcribed spacer 2 (ITS2) and 18S ribosomal RNA]. The performance of three DNA markers (CO1, 16S rRNA and ITS2) in identifying Rhipicephalus tick species was a success using PCR, sequencing, BLASTn and phylogenetic tree corroborating morphological identification. The phylogenetic analysis of the above-mentioned ticks indicated that the collected species were accurately identified as they clustered with their respective reference species from the GenBank database. There was no clear separation of species using the 18S rRNA marker, this marker is mostly significant in resolving relationships among higher taxa. The 16S metabarcoding of the whole tick was conducted on the Illumina MiSeq platform to characterize the microbiota harboured by Rhipicephalus ticks. A total of 221 tick DNA samples were extracted and sequenced from field derived and laboratory reared ticks. However, only 105 FASTQ sequences passed the quality check to generate a total of 689 ASV amplicon sequence variants (ASVs). These were composed of 27 bacterial phyla; 64 classes; 145 bacterial order, 234 bacterial family and 580 bacterial genera. The dominant phyla detected from ticks collected from the different domestic animals, vegetation and laboratory reared tick included Proteobacteria (30.8%) Firmicutes (18.3%), Actinobacteria (14.9%) and Bacteroidetes (13.8%). The remaining 23 phyla in this study contributed to 22.2% of the overall ASV sequences and each were in low abundance. Detected bacterial genera of veterinary, medical and environmental importance included Bacillus, Staphylococcus, Streptococcus, Neisseria, Campylobacter, Proteus, Serratia, Escherichia-Shigella, Providencia, Acinetobacter, Comamonas, Pseudomonas and Enterococcus, Achromobacter, Aerococcus, Caulobacter, Corynebacterium, Corynebacterium_1, Fusobacterium, Haemophilus, Lactobacillus, Lawsonella, Legionella, Micrococcus, Moraxella, Micobacterium, Paracoccus, Porphyromonas, Ralstonia, Rhodococcus, Streptomyces. Cloacibacterium, Clostridium sensu stricto 1,13,18, and 8. Potential genera of tick-borne zoonotic pathogens detected include Rickettsiae, Coxiella, Francisella, Ehrlichia and Anaplasma. Conventional PCR was conducted to detect bacterial zoonotic pathogens and endosymbionts from Rhipicephalus tick DNA samples which showed presence of their respective genera using metabarcoding. Conventional PCR and sequencing resolved the following species, Ehrlichia canis, Escherichia coli, Ricketssia aeschlimanni, Coxiella burnetii, Streptococcus dysgalactiae subspecies, Anaplasma ovis, and A. phagocytophilum. This study further identified symbionts such as Coxiella-like endosymbiont and Francisella- like endosymbiont. The livestock tick microbiota data generated in this study provides baseline knowledge of the microbiome of Rhipicephalus tick species harboured by domestic animals in South Africa.