Abstract
The Asia-Pacific hosts a large diversity of canine vector-borne pathogens (VBPs) with some of the most common and most pathogenic, generating significant mortality as well as a spectrum of health impacts on local dog populations. The VBPs Anaplasma platys, Babesia gibsoni, Babesia vogeli, Ehrlichia canis, Hepatozoon canis and haemotropic Mycoplasma spp. are all endemic throughout the region, with many exhibiting shifting geographical distributions that warrant urgent attention. Moreover, many of these species cause similar clinical signs when parasitising canine hosts, whilst knowledge of the exact pathogen is critical to ensure treatment is effective. This is complicated by frequent coinfection that can exacerbate pathology. Here, we describe the development, optimisation and validation of two novel quadruplex Taq-Man based real-time PCRs (qPCRs) for the specific and sensitive detection of the aforementioned VBPs. To ensure accurate evaluation of diagnostic performance, results of our qPCRs were evaluated on field samples from Thai dogs and compared with both conventional PCR (cPCR) results and next-generation sequencing (NGS) metabarcoding. Our qPCRs were found to be more sensitive at detecting canine VBP than cPCR and generated results similar to those achieved by NGS. These qPCRs will provide a valuable high-throughput diagnostic tool available to epidemiologists, researchers and clinicians for the diagnosis of key canine VBPs in the Asia-Pacific and further afield.
Highlights
A plethora of pathogenic agents ranging in size from minute arboviruses to bacteria, protozoa, and multicellular parasites, such as filarial nematodes, can all be transmitted by blood-feeding arthropods and are collectively known as vector-borne pathogens (VBPs) [1,2]
We developed two multiplex Taq-Man quadruplex Taq-Man based real-time PCRs (qPCRs) assays to simultaneously detect the common canine VBPs circulating in the Asia-Pacific, i.e., A. platys, B. gibsoni, B. vogeli, E. canis, H. canis and haemotropic Mycoplasma spp. [11,12,18,39,40,41]
We have demonstrated the development, optimisation and validation of two novel quadruplex qPCR assays for the simultaneous detection of six different canine VBPs
Summary
A plethora of pathogenic agents ranging in size from minute arboviruses to bacteria, protozoa, and multicellular parasites, such as filarial nematodes, can all be transmitted by blood-feeding arthropods and are collectively known as vector-borne pathogens (VBPs) [1,2]. VBPs due to their vectors’ ubiquitous nature are found globally and affect a vast range of animals including humans. This has been highlighted through outbreaks of diseases like Zika [3], Dengue Fever [4], leishmaniasis [5], Chagas disease [6] and tick-borne diseases, for example Lyme disease [7]. One key phylum from which many canine VBPs belong to is the Apicomplexa, which includes
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