Metabarcoding using nanopore sequencing enables identification of diverse and zoonotic vector-borne pathogens from neglected regions: A case study investigating dogs from Bhutan.

The diversity and prevalence of canine vector-borne pathogens (VBPs) in Bhutan have to date remained unexplored, whilst recent epidemiological surveys in other South Asian nations have found diseases caused by VBPs to be rife in local dog populations. Importantly, many of such VBPs can infect people as well, with a building body of evidence identifying potentially zoonotic rickettsial organisms infecting humans in Bhutan. Given the lack of data on canine pathogens in Bhutan we employed a suite of deep-sequencing metabarcoding methods using Oxford Nanopore Technologies' MinION™ device to holistically characterise the bacterial, apicomplexan and filarial worm blood-borne pathogens of dogs in the country's south. Of the 95 stray, owned and community dogs sampled 78% (95% CI = 69%-85%) were infected with at least one VBP. Pathogen species detected were highly diverse including the bacteria Mycoplasma haemocanis in 16% (95% CI: 10-24%), Ehrlichia canis in 4% (95% CI: 2-10%), Anaplasma platys in 2% (95% CI: 0.5-7%) of dogs as well as the zoonotic species Bartonella clarridgeiae in 1% (95% CI: 0.1-6%), a potentially novel Bartonella spp. and an Ehrlichia chaffeensis-like bacterium, both in 1% (95% CI: 0.1-6%) of dogs. The apicomplexan haemoparasites Hepatozoon canis in 62% (95% CI: 52-71%), Babesia gibsoni in 45% (95% CI: 36-55%) and Babesia vogeli in 3% (95% CI: 1-9%) of dogs were also detected. Finally, 5% (95% CI: 2-12%) of dogs were found to be infected with the filarioid Acanthocheilonema reconditum and 1% (95% CI: 0.1-6%) with zoonotic Dirofilaria sp. hongkongensis. One canine was found positive to the filarioid Setaria tundra, a species normally found infecting cervids. The elucidated diversity of VBP communities highlights the strength of assumption-free diagnostics, such as metabarcoding, in detecting rare, novel, and unexpected pathogens. This approach to identifying pathogen diversity is of critical importance when investigating regions and populations that have thus far been neglected, with the findings aiding the development of future One Health informed strategies for disease control.

Development and validation of a long-read metabarcoding platform for the detection of filarial worm pathogens of animals and humans.

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.

Dogs are reservoir hosts of the zoonotic Dirofilaria sp. 'hongkongensis' and potentially of Brugia sp. Sri Lanka genotype in Sri Lanka.

In 2016, the World Health Organization declared Sri Lanka as having successfully eliminated lymphatic filariasis as a public health concern. However, in recent decades, several infections with subperiodic filarial species suggestive of zoonotic infections have been recorded across the country. The arthropod-borne filarioids Dirofilaria repens, Brugia malayi, Brugia ceylonensis, and Acanthocheilonema reconditum are historically known to be endemic in dogs in Sri Lanka. Despite this, limited information on the prevalence, diversity, and predictors of filarial infections in dogs in the country has resulted in suboptimal control and prevention of these parasites, some of which are known to be zoonotic. To address this, whole blood and metadata were collected and analysed from 423 pet dogs across three geo-climatic zones within Sri Lanka. Blood samples were screened using the Modified Knott's Test (MKT) and PCR followed by Sanger sequencing. Multivariable logistic regression models were used to assess predictors for canine filarial infections. Dirofilaria sp. 'hongkongensis' (Dirofilaria sp. HK) and Brugia sp. Sri Lanka (SL) genotype were identified infecting dogs. The overall prevalence of filarial infection in pet dogs by PCR was 36.9% (95% CI 32.3-41.7%, n = 156), compared to 18.8% (95% CI 15.2-22.9%, n = 79) detected using the MKT. >80% of filarial-positive dogs were infected by Dirofilaria sp. HK, while the remaining dogs were infected by Brugia sp. SL genotype. Increasing age (p < 0.001) and residing in the low-country wet zone (p < 0.001), which includes regions that were endemic for human filariasis in Sri Lanka, were associated with filarial infections in dogs. No clear pathognomonic signs for filarial infection were identified, indicating that dogs act as reservoirs for these potentially zoonotic pathogens. Given the morphological similarity of Dirofilaria HK and Brugia sp. SL microfilariae with those of D. repens and B. malayi, respectively, it is likely that these species have been misidentified in the past. Prevention and control measures of these potentially zoonotic canine filarial infections are highly advocated to safeguard both canine and human health.

Nanopore Sequencing Using the Full-Length 16S rRNA Gene for Detection of Blood-Borne Bacteria in Dogs Reveals a Novel Species of Hemotropic Mycoplasma.

Dogs across the globe are afflicted by diverse blood- and vector-borne bacteria (VBB), many of which cause severe disease and can be fatal. Diagnosis of VBB infections can be challenging due to the low concentration of bacteria in the blood, the frequent occurrence of coinfections, and the wide range of known, emerging, and potentially novel VBB species encounterable. Therefore, there is a need for diagnostics that address these challenges by being both sensitive and capable of detecting all VBB simultaneously. We detail the first employment of a nanopore-based sequencing methodology conducted on the Oxford Nanopore Technologies (ONT) MinION device to accurately elucidate the "hemobacteriome" from canine blood through sequencing of the full-length 16S rRNA gene. We detected a diverse range of important canine VBB, including Ehrlichia canis, Anaplasma platys, Mycoplasma haemocanis, Bartonella clarridgeiae, "Candidatus Mycoplasma haematoparvum", a novel species of hemotropic mycoplasma, and Wolbachia endosymbionts of filarial worms, indicative of filariasis. Our nanopore-based protocol was equivalent in sensitivity to both quantitative PCR (qPCR) and Illumina sequencing when benchmarked against these methods, achieving high agreement as defined by the kappa statistics (k > 0.81) for three key VBB. Utilizing the ability of the ONT' MinION device to sequence long read lengths provides an excellent alternative diagnostic method by which the hemobacteriome can be accurately characterized to the species level in a way previously unachievable using short reads. We envision our method to be translatable to multiple contexts, such as the detection of VBB in other vertebrate hosts, including humans, while the small size of the MinION device is highly amenable to field use. IMPORTANCE 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. Deep sequencing using devices from Oxford Nanopore Technologies (ONT) provides a solution, as the long read lengths achievable provide species-level taxonomic identification of pathogens that previous short-read technologies could not accomplish. We developed a protocol using ONT' MinION sequencer to accurately detect and classify a wide spectrum of VBB from canine blood at a sensitivity comparable to that of regularly used diagnostics, such as qPCR. This protocol demonstrates great potential for use in biosurveillance and biosecurity operations for the detection of VBB in a range of vertebrate hosts, while the MinION sequencer's portability allows this method to be used easily in the field.

Genomic Variability of Canine Parvoviruses from a Selected Population of Dogs and Cats in Sri Lanka.

The aim of the study was to identify canine parvovirus type 2 (CPV-2) subtypes circulating among a selected population of domestic dogs and cats in Sri Lanka and to investigate the evolutionary patterns among Sri Lankan viruses in the context of contemporary global CPV-2 sequences. Altogether, 40/61 (65.6%) samples tested were positive for CPV-2 DNA, including 31/48 (64.6%) dogs and 9/13 (69%) cats. All three subtypes (CPV-2a, CPV-2b and CPV-2c) were detected, with CPV-2a being most common. International median joining haplotype network of 291 CPV-2 sequences suggested that there was little barrier for CPV-2 moving between different geographical regions worldwide, including Sri Lanka, and that there was no correlation between the genetic structure within the molecular network and the decade of sample collection. By contrast, there was correlation between CPV-2 subtype and genetic structure, both within the international network and within the network built from 31 Sri Lankan CPV-2 sequences only. The structure within the latter was not correlated with the location of the veterinary clinic where the samples were submitted, the age or species of the host. Altogether, we have shown that there is considerable variability of CPV-2 genotypes circulating in Sri Lanka, which is likely driven by both local evolution and introduction from other countries. The similarity of CPV-2 obtained from cats and dogs suggests that cats may play a role in the epidemiology of CPV-2 in Sri Lanka.