Exploration of tick-borne pathogens and microbiota of dog ticks collected at Potchefstroom Animal Welfare Society

Abstract

Ticks are blood-feeding pests of domestic and wild animals. Next to mosquitoes ticks are notorious for the transmission of pathogenic organisms to their mammalian hosts. Companion animals also suffer from tick infestations and this is problematic due to their close association with humans. In South Africa, there is a need for constant supply of up to date information of ticks infesting companion animals and their associated tick-borne pathogens using modern diagnostic techniques. Furthermore, some of the pathogens infecting companion animals are zoonotic resulting in diseases which are also of importance to human health. The current study was aimed at identifying tick species infesting urban dogs admitted at the Potchefstroom Animal Welfare Society (PAWS) and known pathogens of veterinary importance using microscopy and PCR. Moreover, the 16S metabarcoding of the tick gut, salivary gland, and egg microbiota was conducted using Illumina next-generation sequencing (NGS) platform. Different tick sterilization methods were also tested to determine their effectiveness to eliminate surface bacteria on the ticks. During the current study, a total of 592 ticks were collected from 61 dogs admitted to PAWS, mainly originating from the JB Marks Local Municipality and a few dogs originating from Merafong City and Emfuleni Local Municipalities. Tick species were identified as Haemaphysalis elliptica (39%) and Rhipicephalus sanguineus (61%) by both morphological and molecular analysis. Phylogenetic analysis of the Heamaphysalis and Rhipicephalus genera, using the CO1 and ITS2 genes, supported the respective monophyly of these ticks compared to other ticks of the same genera on the NCBI database. Results of this study indicated ticks were most abundant the PAWS kennels, where companion animals are co-housed than in stray dogs from residential areas where grooming is rare. Morphological detection of pathogens infecting H. elliptica indicated respective infestation rates of 29% (32/112) and 2% (2/112) with Rickettsia sp. and Anaplasma sp. Whilst in R. sanguineus there was respective infestation rates of 14% (13/92) and 1% (1/92) with Rickettsia sp. and Babesia sp. Molecular analysis detected respective infestation rates of 2% (1/49), 8.2% (4/49), 14.3% (7/49), 22.4% (11/49) of Anaplasma phagocytophilum, Ehrlichia canis, Rickettsia conorii, and Coxiella burnetii from H. elliptica DNA. Whilst 3.6% (2/55), 5.5% (3/55), and 5.5% (3/55) of E. canis, R. conorii, and C. burnetii was PCR positive from R. sanguineus DNA. In addition, H. elliptica eggs had an infestation rate of 10.5% (2/19) for C. burnetii. This is an indication that C. burnetii is possibly being transmitted both transstadially and transovarially in this tick species. The metagenomic analysis of data generated by NGS from DNA of H. elliptica and R. sanguineus whole ticks, midguts, salivary glands, and eggs produced 1111515 assembled sequences and 404 Operational Taxonomic Units (OTUs) up to genus level. There was a total of 25 detected bacterial phyla. Dominating bacterial phyla detected were Proteobacteria (57.2%), Actinobacteria (17.4%), and Bacteroidetes (12%). This was followed by Firmicutes (7.8%) and Cyanobacteria (2.7%). Bacterial phyla that were unassigned had a total relative abundance of 14.2%. Bacterial genera of veterinary, ecological, and medical importance were also detected in lower relative abundances including Bacillus, Staphylococcus, Streptococcus, Clostridium, Ehrlichia, Rickettsia, Wolbachia, Neisseria, Campylobacter, Proteus, Coxiella, Borrelia, and Pseudomonas. Several bacterial phyla detected during metagenomic diagnosis in this study were previously reportedly isolated from agricultural environments. This is in accordance with Ixodid ticks spending most of their life cycle off hosts. Due to this, the current study also evaluated several tick sterilization methods in order to determine the most effective method for removing external bacteria on tick surface. After washing ticks with 70% ethanol, PBS, 10% Bleach and 10% Tween 20, observations of this study revealed that a combination of washing first with 10% Tween 20 and then followed by 70% ethanol is effective for removal of external bacteria on tick surface without causing damage to the tick tissue and DNA. This current study has documented that H. elliptica and R. sanguineus ticks are infesting urban dogs admitted at PAWS. Furthermore, this study has showed that these tick harbour several pathogens which are known for causing tick-borne disease, some of which are zoonotic. This data will be useful to veterinarians in municipalities where sampled dogs originated, as it will enable formulation of proper tick and tick-borne disease control approaches. Additional studies are required to determine the effect of these ticks, their associated tick-borne pathogens and their microbiota on other companion animals and humans