Genetic characterization of ticks and tick borne prokaryotic pathogens of urban stray dogs in Potchefstroom, South Africa

Abstract

Ticks are well documented for their ability to transmit pathogenic organisms of medical, veterinary and economical importance mostly to a variety of animal hosts and humans. Ticks harbour various members of bacteria, archaea, and eubacteria and several eukaryotic organisms, including fungi, nematodes, and protozoa, collectively referred to as the microbiome. Associations between the ticks and the microbiota affect several aspects of the ticks such as their vectorial competence, survival, and reproductive fitness. The bacterial composition of ticks varies due to a lot of factors, including but not limited to the tick’s life cycle, host ecology, and the absence or presence of bacterial pathogens. Therefore, this study aimed at conducting a systematic review focusing on the global prevalence of R. sanguineus and H. elliptica, as well as the tick-borne pathogens they harbour. This study also sought to identify the tick species and tick-borne pathogens infesting dogs collected from the Potchefstroom Animal Welfare Society (PAWS). Lastly this study aimed to identify the bacterial communities of dog blood and ticks from the PAWS in comparison to colony ticks from gnotobiotic hosts. For the systematic review, the Scopus, ScienceDirect, PubMed, and Web of Science databases were searched for English journal articles which were assessed by following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. This systematic review was registered on PROSPERO under the following ID: CRD42022327372. Of the studies included in the systematic review, 247 and 19 articles collected and identified R. sanguineus and H. elliptica respectively, whereas 15 articles managed to collect and identify both tick species. In total 121 articles were included in the systematic review that reported the presence of haemoparasite infections within R. sanguineus or H. elliptica ticks. Respectively, 119 and 2 of the aforementioned articles reported the presence of tick-borne pathogens within R. sanguineus and H. elliptica ticks respectively. In addition, only 1 article reported the presence of tick-borne pathogens within both R. sanguineus and H. elliptica ticks. The prevalence of the haemopathogens was similar to the prevalence of their tick hosts, thus indicating the potential of tick-borne pathogens to spread and inhabit new niches. Articles included in the systematic review reported the presence of R. sanguineus and H. elliptica ticks collected from companion animals, vegetation, livestock, humans, as well as several wildlife species. The tick species identification method in the included articles of the systematic review was done employing morphological identification (n=222), as well as a combination of morphological identification and conventional PCR (n=29). Articles that detected tick-borne pathogens within R. sanguineus used molecular techniques (n=139) as well as morphological techniques (n=5). Articles that detected tick-borne pathogens within H. elliptica used molecular techniques (n=2). There were 64 countries that reported the presence of R. sanguineus, whereas H. elliptica was only reported in 6 countries. It is considered that R. sanguineus has a global prevalence, mainly in regions that are tropical or subtropical. H. elliptica was reported in South Africa and countries with close proximity to South Africa. The close proximity between ticks and travelling hosts or migratory hosts (such as birds), is one of the main contributors to the global prevalence of R. sanguineus and the prevalence of H. elliptica in the neighbouring countries of South Africa. In this study tick specimens (n=300) and blood (n=50) were collected from naturally infected dog hosts (n=50) residing at the PAWS located in Potchefstroom of the North West Province, South Africa. In addition, eggs were also collected (n=6 egg batches) from ticks that laid eggs after collection. Tick specimens were morphologically identified and genetically using PCR CO1 and 18S rRNA assays, whilst genus or species specific PCR assays were performed for detection of various bacterial pathogens of veterinary importance from tick pools (n=81), egg batches (n=6), and dog blood (n=50). The PCR amplicons were Sanger sequenced and aligned using the nucleotide Basic Local Alignment Search Tool (BLASTn) for confirmation of detected pathogen identity. In this study, two tick species were identified namely R. sanguineus with an overall abundance of 70.7% (212/300) and H. elliptica with an overall abundance of 29.3% (88/300) based on morphological key features and DNA analysis. For the CO1 gene all of the tick specimens morphologically identified as H. elliptica were 96.89 to 97.74% similar to H. elliptica gene sequences on the NCBI database. On the other hand, the tick species morphologically identified as R. sanguineus were 97.20 to 99.84% similar to R. sanguineus gene sequences on the NCBI database. For the 18S rRNA gene all of the tick specimens morphologically identified as H. elliptica were 99.26 to 99.63% similar to H. elliptica gene sequences on the NCBI database. On the other hand, the tick species morphologically identified as R. sanguineus were 98.53 to 99.90% similar to R. sanguineus gene sequences on the NCBI database. None of E. canis or A. phagocytophylum were detected by PCR from DNA of ticks, egg batches, or dog blood samples from this study. The overall occurrence of C. burnetii within pooled tick DNAs was 9.88% (8/81). The C. burnetii were harboured by 8.82% (3/34) of H. elliptica ticks and 10.64% (5/47) of R. sanguineus ticks. The overall occurrence of C. burnetii within egg batches was 16.67% (1/6). The C. burnetii was present in 25% (1/4) H. elliptica egg batches. One of the PCR products was successfully sequenced and BLASTn search results revealed that the aforementioned gene sequence was 96.89 to 100% similar to C. burnetii sequences on the NCBI database. The overall occurrence of Rickettsia spp. within tick pools was 6.17% (5/81). Rickettsia spp. were harboured by 11.76% (4/34) of H. elliptica ticks and 2.13% (1/47) of R. sanguineus ticks. The overall occurrence of Rickettsia spp. within egg batches was 16.67% (1/6). Rickettsia spp. were present in 25% (1/4) H. elliptica egg batches. The BLASTn results revealed that two of the Rickettsia spp. were 98.75 to 100% similar to R. conorii gene sequences on the NCBI database. Another two Rickettsia spp. sequences. BLASTn search results revealed that they were 97.18 to 99.70% similar to R. massiliae gene sequences on the NCBI database. In addition, one R. sanguineus tick pool was co-infected with both C. burnetii and Rickettsia spp. Lastly, this study used Illumina next-generation sequencing targeting the V3 and V4 regions of the 16S rRNA gene to determine bacterial microbiota from R. sanguineus (n=1) and H. elliptica (n=1), dog blood samples from which ticks were collected. The microbiota of these ticks and dog blood was compared with that of colony reared ticks. Dominant bacterial phyla were identified as Proteobacteria (54.02%), Bacteroidetes (18.03%), and Actinobacteria (11.76%). The dominant bacterial classes were identified as Gammaproteobacteria (41.61%), Bacteroidia (17.93%), Alphaproteobacteria (11.20%), and Actinobacteria (11.11%). Dominant orders were identified as Betaproteobacteriales (14.78%) and Coxiellales (14.40%). Dominant bacterial families were identified as Coxiellaceae (14.40%) and Burkholderiaceae (12.12%). The dominating bacterial genera were identified as Coxiella (14.40%). Additionally, at class and genus levels the relative abundance between the R. sanguineus and H. elliptica of the colony ticks from gnotobiotic hosts were similar, whereas the relative abundances of the R. sanguineus and H. elliptica ticks and their natural dog hosts were similar indicating similarities between the species richness, whereas the relative abundance between the colony ticks from gnotobiotic hosts and the ticks collected from naturally infected dog hosts was different indicating differentiation between the species richness. The results of the study indicate the presence of several bacterial communities harboured by and shared between R. sanguineus and H. elliptica ticks from naturally infected dog hosts, dog blood, and R. sanguineus and H. elliptica ticks from gnotobiotic hosts. The aforementioned bacterial communities are taxonomically similar among all the sample types but the relative abundances of the bacterial communities of both the dog blood and ticks from natural dog hosts differ compared to the ticks from gnotobiotic hosts, specifically among the Rickettsia spp. and Coxiella spp. that were very high in the tick specimens collected from gnotobiotic hosts. Additionally, several bacterial genera detected in the sample types were of medical, veterinary, and ecological importance. The systematic review has provided consolidated insight on the prevalence of R. sanguineus, H. elliptica, and their tick-borne pathogens, as well as other associated subjects, such as the expansion of ticks and their tick-borne pathogens to non-endemic areas, the effects of coinfections, and tick identification and parasite detection methods. The results of this study indicate that dogs admitted to PAWS are infected by R. sanguineus and H. elliptica ticks which are harbouring tick-borne pathogens responsible for causing zoonotic diseases.