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. 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. 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.

Haemoparasites are responsible for some of the most prevalent and debilitating canine illnesses across the globe, whilst also posing a significant zoonotic risk to humankind. Nowhere are the effects of such parasites more pronounced than in developing countries in the tropics where the abundance and diversity of ectoparasites that transmit these pathogens reaches its zenith. Here we describe the use of a novel next-generation sequencing (NGS) metabarcoding based approach to screen for a range of blood-borne apicomplexan and kinetoplastid parasites from populations of temple dogs in Bangkok, Thailand. Our methodology elucidated high rates of Hepatozoon canis and Babesia vogeli infection, whilst also being able to characterise co-infections. In addition, our approach was confirmed to be more sensitive than conventional endpoint PCR diagnostic methods. Two kinetoplastid infections were also detected, including one by Trypanosoma evansi, a pathogen that is rarely screened for in dogs and another by Parabodo caudatus, a poorly documented organism that has been previously reported inhabiting the urinary tract of a dog with haematuria. Such results demonstrate the power of NGS methodologies to unearth rare and unusual pathogens, especially in regions of the world where limited information on canine vector-borne haemoparasites exist.

Blood- and vector-borne bacteria (VBB) can cause severe pathology and even be lethal for dogs in many regions across the globe. Accurate characterization of all the bacterial pathogens infecting a canine host is critical, as coinfections are common and emerging and novel pathogens that may go undetected by traditional diagnostics frequently arise.

Apicomplexan haemoparasites generate significant morbidity and mortality in humans and other animals, particularly in many low‐to‐middle income countries. Malaria caused by Plasmodium remains responsible for some of the highest numbers of annual deaths of any human pathogen, whilst piroplasmids, such as Babesia and Theileria can have immense negative economic effects through livestock loss. Diagnosing haemoparasites via traditional methods like microscopy is challenging due to low‐level and transient parasitaemia. PCR‐based diagnostics overcome these limitations by being both highly sensitive and specific, but they may be unable to accurately detect coinfections or identify novel species. In contrast, next‐generation sequencing (NGS)‐based methods can characterize all pathogens from a group of interest concurrently, although, the short‐read platforms previously used have been limited in the taxonomic resolution achievable. Here, we used Oxford Nanopore Technologies' (ONT) long‐read MinION™ sequencer to conduct apicomplexan haemoparasite metabarcoding via sequencing the near full‐length 18S ribosomal RNA gene, demonstrating its ability to detect Babesia, Hepatozoon, Neospora, Plasmodium, Theileria and Toxoplasma species. This method was tested on blood‐extracted DNA from 100 dogs and the results benchmarked against qPCR and Illumina‐based metabarcoding. For two common haemoparasites, nanopore sequencing performed as well as qPCR (kappa agreement statistics > 0.98), whilst also detecting one pathogen, Hepatozoon felis, missed by the other techniques. The long‐reads obtained by nanopore sequencing provide an improved species‐level taxonomic resolution whilst the method's broad applicability mean it can be used to explore apicomplexan communities from diverse mammalian hosts, on a portable sequencer that easily permits adaptation to field use.

Background: Filarial worms are important vector-borne pathogens of a large range of animal hosts, including humans, and are responsible for numerous debilitating neglected tropical diseases such as, lymphatic filariasis caused by Wuchereria bancrofti and Brugia spp., as well as loiasis caused by Loa loa. Moreover, some emerging or difficult-to-eliminate filarioid pathogens are zoonotic using animals like canines as reservoir hosts, for example Dirofilaria sp. ‘hongkongensis’. Diagnosis of filariasis through commonly available methods, like microscopy, can be challenging as microfilaremia may wane below the limit of detection. In contrast, conventional PCR methods are more sensitive and specific but may show limited ability to detect coinfections as well as emerging and/or novel pathogens. Use of deep-sequencing technologies obviate these challenges, providing sensitive detection of entire parasite communities, whilst also being better suited for the characterisation of rare or novel pathogens. Therefore, we developed a novel long-read metabarcoding assay for deep-sequencing the filarial nematode cytochrome c oxidase subunit I gene on Oxford Nanopore Technologies’ (ONT) MinION™ sequencer. We assessed the overall performance of our assay using kappa statistics to compare it to commonly used diagnostic methods for filarial worm detection, such as conventional PCR (cPCR) with Sanger sequencing and the microscopy-based modified Knott’s test (MKT). Results: We confirmed our metabarcoding assay can characterise filarial parasites from a diverse range of genera, including, Breinlia, Brugia, Cercopithifilaria, Dipetalonema, Dirofilaria, Onchocerca, Setaria, Stephanofilaria and Wuchereria. We demonstrated proof-of-concept for this assay by using blood samples from Sri Lankan dogs, whereby we identified infections with the filarioids Acanthocheilonema reconditum, Brugia sp. Sri Lanka genotype and zoonotic Dirofilaria sp. ‘hongkongensis’. When compared to traditionally used diagnostics, such as the MKT and cPCR with Sanger sequencing, we identified an additional filarioid species and over 15% more mono- and coinfections. Conclusions: Our developed metabarcoding assay may show broad applicability for the metabarcoding and diagnosis of the full spectrum of filarioids from a wide range of animal hosts, including mammals and vectors, whilst the utilisation of ONT’ small and portable MinION™ means that such methods could be deployed for field use.