Novel High-Throughput Multiplex qPCRs for the Detection of Canine Vector-Borne Pathogens in the Asia-Pacific

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.

Keywords: Anaplasma platys; Asia-Pacific; Babesia; Ehrlichia canis; Hepatozoon canis; canine vector-borne disease; dogs; haemotropic Mycoplasma; molecular diagnostics; multiplex qPCR.

Figure 1

Standard curves generated from 12-fold serial dilutions of target gBlock Gene Fragments from the apicomplexans; B. vogeli (A), B. gibsoni (B) and H. canis (C).

Figure 2

Standard curves generated from 12-fold serial dilutions of target gBlock Gene Fragments for A. platys (A), E. canis (B), Mycoplasma spp. (C) and mammalian mtDNA (D).

Figure 3

Singleplex and multiplex qPCR efficiencies. Optimisation and comparison of the sensitivity and efficiency of each singleplex and multiplex qPCR using gBlock Gene Fragment controls for B. vogeli, B. gibsoni and H. canis.

Figure 4

Singleplex and multiplex qPCR efficiencies. Optimisation and comparison of the sensitivity and efficiency of each singleplex and multiplex qPCR using gBlock Gene Fragment controls for A. platys, E. canis, Mycoplasma spp. and mammalian mtDNA.