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

Abstract

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.

Keywords: Bartonella; Dirofilaria sp. hongkongensis; Ehrlichia; MinION; Next-generation sequencing; One Health; Setaria tundra; Zoonosis.

Fig. 1

Map of districts and field sites in Bhutan where canine samples were collected. The size of the site circle is proportional to the number of samples taken. Map created in QGIS 3.4 via QGIS.org, 2021. QGIS Geographic Information System. QGIS Association.

Fig. 2

Phylogenetic relationship of the uncharacterised Ehrlichia taxon (bold) from the blood of one Bhutanese dog alongside representative sequences from across the family Ehrlichiaceae. Phylogenetic inference was made using a Bayesian and neighbour-joining distance method for a 1357 bp segment of the 16S rRNA gene. Posterior probability values and bootstrap support (where available) for tree branches are indicated, with Neorickettsia risticii used as an outgroup. The taxonomic label in bold indicates the Ehrlichia sequence (accession # PP158804) generated in this study.

Fig. 3

Phylogenetic relationship of Dirofilaria sp. hongkongensis (bold) from the blood of a Bhutanese dog alongside representative sequences from across the families Onchocercidae and Setariidae. Phylogenetic inference was made using a Bayesian and neighbour-joining distance method for a 465 bp segment of the filarial worm COI gene. Posterior probability values and bootstrap support (where available) for tree branches are indicated, with Brugia malayi used as an outgroup. The taxonomic label in bold indicates the Dirofilaria sp. hongkongensis sequence (accession # PP158772) generated in this study.

Fig. 4

Phylogenetic relationship of Setaria tundra (bold) from the blood of one Bhutanese dog alongside representative sequences from across the families Onchocercidae, Setariidae and Thelaziidae. Phylogenetic inference was made using a Bayesian and neighbour-joining distance method for a 577 bp segment of the filarial worm COI gene. Posterior probability values and bootstrap support (where available) for tree branches are indicated, with Thelazia callipaeda used as an outgroup. The taxonomic label in bold indicates the Setaria tundra sequence (accession # PP158773) generated in this study.