A novel metabarcoding diagnostic tool to explore protozoan haemoparasite diversity in mammals: a proof-of-concept study using canines from the tropics

Lucas G Huggins,Rebecca J Traub,Anson V Koehler,Stephen Wilcox,Tawin Inpankaew,Dinh Ng-Nguyen,Bettina Schunack

doi:10.1038/s41598-019-49118-9

Abstract

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.

Highlights

Protozoan haemoparasites generate some of the highest rates of morbidity and mortality in canines worldwide, whilst some are zoonotic, capable of producing significant infections in humans as well[1,2,3,4]
Apicomplexan Babesia spp. parasites are transmitted by tick vectors which invade erythrocytes and cause a spectrum of anaemia-related pathology depending on the species, from the relatively benign Babesia vogeli to the more virulent Babesia canis and Babesia rossi species[1,10]
Two primer pairs were designed to exclusively amplify 18S rRNA sequences from the phylum Apicomplexa and the class Kinetoplastida using a diverse range of sequences from GenBank

Summary

Introduction

Protozoan haemoparasites generate some of the highest rates of morbidity and mortality in canines worldwide, whilst some are zoonotic, capable of producing significant infections in humans as well[1,2,3,4]. Www.nature.com/scientificreports immunocompromised humans and children, in regions of South America, the Middle East and the Mediterranean[12,13,14] Many of these haemoparasites are united by their ability to create enduring infections, that can last years, with periods of immunological control followed by remission[10,15,16]. Hepatozoon canis, typically generates a subclinical infection[15], when found to be coinfecting with Babesia spp. or bacterial VBDs a much more severe anaemia and overall disease outcome is generated[18] Taking this into consideration, canine VBD diagnostic methods must be able to characterise the entire haemoparasite microbiome and not just a dominant pathogen within a host. Amplified DNA from all the different species barcodes in a sample can be sequenced in massive parallelisation, generating a sample metabarcode of every species present from a taxonomic group of interest, thereby elucidating an entire microbiome from within a specific host environment[22,23]

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Results

Conclusion