Advanced approaches for the diagnosis and chemoprevention of canine vector-borne pathogens and parasites—Implications for the Asia-Pacific region and beyond

Metabarcoding using nanopore long‐read sequencing for the unbiased characterization of apicomplexan haemoparasites

“…In the case of coinfections, if assays like cPCR and Sanger sequencing are used to target conserved genes then often only the dominant pathogen will be detected, whilst rarer or novel pathogens may be obscured (Zepeda Mendoza et al, 2015). This is in stark contrast to metabarcoding methods which can holistically characterize the entire pathogen community simultaneously, identifying both common and rare sequences from a sample (Huggins et al, 2023;Zepeda Mendoza et al, 2015).…”

“…The detection of unusual parasites in non‐typical hosts is one of the primary advantages of employing NGS‐based metabarcoding assays for pathogen surveillance, particularly across study sites with little‐to‐no prior epidemiological data. Conventional molecular diagnostics would likely miss such rare or unexpected findings as they rely on careful selection of assays to detect just one or a few pathogens that are expected to be found in the region under investigation (Huggins et al, 2023; Ondrejicka et al, 2014). In the case of coinfections, if assays like cPCR and Sanger sequencing are used to target conserved genes then often only the dominant pathogen will be detected, whilst rarer or novel pathogens may be obscured (Zepeda Mendoza et al, 2015).…”

“…Among animals that live in close association with humans, dogs act as an ideal species in which to investigate tools for exploring apicomplexan haemoparasite diversity as they act as hosts for a diverse array of species, including tick transmitted Babesia that cause varying degrees of haemolytic anaemia and can prove fatal. The ‘large’ ( Babesia canis , Babesia vogeli , Babesia rossi ) and ‘small’ ( Babesia vulpes , Babesia gibsoni ) dog infecting Babesia not only vary in geographical distribution and pathogenicity, but also respond variably to specific treatments, making their accurate detection and diagnosis imperative (Davitkov et al, 2015; Huggins et al, 2023; Karasová et al, 2022; Penzhorn, 2020). Haemoparasites of the genus Hepatozoon are also important canine pathogens, for example, Hepatozoon canis , which is found throughout the tropics and can generate clinical signs that overlap with those caused by Babesia (Baneth et al, 2003; Baneth & Allen, 2022).…”

“…Diagnostic tools that use next‐generation sequencing (NGS) bypass many of the issues that classical molecular methods face, as they can deep‐sequence conserved genetic barcodes from a target group of organisms simultaneously, generating a ‘metabarcode’ of all the pathogens present (Huggins et al, 2019a, 2019b; Wahab et al, 2020). Common targets, such as the 16S ribosomal RNA (16S rRNA) gene for bacteria or 18S ribosomal RNA (18S rRNA) gene for haemoparasites have been used before to characterize entire pathogen communities and detect rare and/or novel species (Ghafar et al, 2021; Huggins et al, 2023; Kumar et al, 2022). Nevertheless, to date, most studies have utilized short‐read NGS platforms, limiting the size of amplicons that can be sequenced and, therefore, the taxonomic resolution achievable (Ghafar et al, 2021; Huggins, Colella, et al, 2021; Mohamed et al, 2021; Squarre et al, 2020).…”

“…Common targets, such as the 16S ribosomal RNA (16S rRNA) gene for bacteria or 18S ribosomal RNA (18S rRNA) gene for haemoparasites have been used before to characterize entire pathogen communities and detect rare and/or novel species (Ghafar et al, 2021;Huggins et al, 2023;Kumar et al, 2022). Nevertheless, to date, most studies have utilized short-read NGS platforms, limiting the size of amplicons that can be sequenced and, therefore, the taxonomic resolution achievable (Ghafar et al, 2021;Mohamed et al, 2021;Squarre et al, 2020).…”