Abstract
Bacterial canine vector-borne diseases are responsible for some of the most life-threatening conditions of dogs in the tropics and are typically poorly researched with some presenting a zoonotic risk to cohabiting people. Next-generation sequencing based methodologies have been demonstrated to accurately characterise a diverse range of vector-borne bacteria in dogs, whilst also proving to be more sensitive than conventional PCR techniques. We report two improvements to a previously developed metabarcoding tool that increased the sensitivity and diversity of vector-borne bacteria detected from canine blood. Firstly, we developed and tested a canine-specific blocking primer that prevents cross-reactivity of bacterial primer amplification on abundant canine mitochondrial sequences. Use of our blocking primer increased the number of canine vector-borne infections detected (five more Ehrlichia canis and three more Anaplasma platys infections) and increased the diversity of bacterial sequences found. Secondly, the DNA extraction kit employed can have a significant effect on the bacterial community characterised. Therefore, we compared four different DNA extraction kits finding the Qiagen DNeasy Blood and Tissue Kit to be superior for detection of blood-borne bacteria, identifying nine more A. platys, two more E. canis, one more Mycoplasma haemocanis infection and more putative bacterial pathogens than the lowest performing kit.
Highlights
Despite recent developments in veterinary care and medicine, canine vector-borne diseases (CVBD) continue to inflict a large burden with regard to morbidity and mortality on dogs across the globe [1,2,3]
Initial experimentation using conventional PCR (cPCR) analysis with gel electrophoresis assessed the ability of our blocking primer: Canis-mito-blk, to bind to canine 12S mitochondrial sequences when it did not have the 30 -spacer C3 modification, which was observable via production of a 137 bp product
Bacterial primers were not prevented from amplifying bacterial sequences as an approximately 250 bp product was still produced by these primers when used on canine blood samples positive for the pathogens E. canis, A. platys and R. felis
Summary
Despite recent developments in veterinary care and medicine, canine vector-borne diseases (CVBD) continue to inflict a large burden with regard to morbidity and mortality on dogs across the globe [1,2,3]. This is especially true in low-socioeconomic countries, that may have little available resources to invest into disease prevention programs [1,2,3]. Lower but significant levels of infection by haemotropic mycoplasmas (3.7–12.8%) and A. platys (3.7–4.4%) have been detected in these same countries [6,8]
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