Assessment of a metabarcoding approach for the characterisation of vector-borne bacteria in canines from Bangkok, Thailand

Abstract

Background Globally, bacterial vector-borne disease (VBD) exerts a large toll on dogs in terms of morbidity and mortality but nowhere is this more pronounced than in the tropics. Tropical environments permit a burgeoning diversity and abundance of ectoparasites some of which can transmit an extensive range of infectious agents, including bacteria, amongst others. Although some of these vector-borne bacteria are responsible for both animal and human diseases in the tropics, there is a scarcity of epidemiological investigation into these pathogens’ prevalence. The situation is further exacerbated by frequent canine co-infection, complicating symptomatology that regular diagnostic techniques may miss or be unable to fully characterise. Such limitations draw attention to the need to develop screening tools capable of detecting a wide range of pathogens from a host simultaneously. Results Here, we detail the employment of a next-generation sequencing (NGS) metabarcoding methodology to screen for the spectrum of bacterial VBD that are infecting semi-domesticated dogs across temple communities in Bangkok, Thailand. Our NGS detection protocol was able to find high levels of Ehrlichia canis , Mycoplasma haemocanis and Anaplasma platys infection rates as well as less common pathogens, such as “ Candidatus Mycoplasma haematoparvum”, Mycoplasma turicensis and Bartonella spp. We also compared our high-throughput approach to conventional endpoint PCR methods, demonstrating an improved detection ability for some bacterial infections, such as A. platys but a reduced ability to detect Rickettsia . Conclusions Our methodology demonstrated great strength at detecting coinfections of vector-borne bacteria and rare pathogens that are seldom screened for in canines in the tropics, highlighting its advantages over traditional diagnostics to better characterise bacterial pathogens in environments where there is a dearth of research. Electronic supplementary material The online version of this article (10.1186/s13071-019-3651-0) contains supplementary material, which is available to authorized users.

A Host-Specific Blocking Primer Combined with Optimal DNA Extraction Improves the Detection Capability of a Metabarcoding Protocol for Canine Vector-Borne Bacteria

“…Pathogenic vector-borne bacterial species were detected from the species E. canis, A. platys, M. haemocanis, 'Candidatus Mycoplasma haematoparvum', Mycoplasma turicensis, Bartonella spp. as had been previously found by [22]. However, only the high prevalence of E. canis, A. platys and M. haemocanis, permitted statistically comparable results between the ability of the metabarcoding methodology to detect vector-borne bacterial DNA with and without the use of Canis-mito-blk.…”

“…Similar challenges have been tackled before by using blocking primers that selectively bind to and prevent the amplification of DNA sequences that would otherwise dominate amplicons in contexts where there is a stacked ratio of low copy number DNA of interest compared to overabundant DNA that is ideally excluded [26][27][28][29][30][31]. Considering this, we designed and tested a 3 -spacer C3 blocking primer that could prevent amplification of the canine 12S rRNA gene by our previously tested bacterial-universal primers [22]. We then compared our bacterial NGS metabarcoding pipeline with and without this blocking primer to assess its blocking efficacy and elucidate whether it improved bacterial detection capability.…”

“…16S rRNA metabarcoding is better able to elucidate diversity in explorative research by not relying on likely pathogen prevalence in a region nor on a priori knowledge of a pathogen's target genetic sequences, whilst also being better able to characterise coinfection [18,20]. Previous work has already developed a novel 16S and 18S rRNA metabarcoding methodology to characterise the range of blood-borne bacterial, apicomplexan and kinetoplastid organisms infecting canine hosts in Thailand, a country of substantial CVBD diversity [4,8,[21][22][23]. The current research takes this further, by tackling some of the inherent challenges of NGS microbiome research whilst also improving methods to be better at unearthing pathogen diversity in the context of the canine blood micro-environment [24].…”

“…Prior research employing NGS metabarcoding analysis of bacterial pathogens from canines suffered from issues of bacterial primer cross-reactivity with the canine mitochondrial 12S ribosomal RNA (12S rRNA) gene [22]. On average, 47% of total reads during previous experiments were from mitochondrial DNA (mtDNA) cross-reactivity despite poor complementarity between the bacterial primers and 12S rRNA gene sequences [22].…”

“…Prior research employing NGS metabarcoding analysis of bacterial pathogens from canines suffered from issues of bacterial primer cross-reactivity with the canine mitochondrial 12S ribosomal RNA (12S rRNA) gene [22]. On average, 47% of total reads during previous experiments were from mitochondrial DNA (mtDNA) cross-reactivity despite poor complementarity between the bacterial primers and 12S rRNA gene sequences [22]. This cross-reactivity is likely due to multiple causes, including a dominance of canine host DNA in the blood [22] in conjunction with the prokaryotic origin of mitochondria producing sequence similarities [25].…”