A Field-Deployable Insulated Isothermal PCR (iiPCR) for the Global Surveillance of Toxoplasma gondii Infection in Cetaceans

SIMPLE SUMMARY: Since high trophic levels marine mammal species share the coastal environments and diets with humans, cetaceans provide an indication of contaminant bioaccumulation in humans and may serve as sentinels for public health problems. Parasite monitoring in marine sentinels can assist in evaluating the quality of the aquatic ecosystem’s health. T. gondii infection in cetaceans is an indicator of land-to-sea coastal pollution. Although T. gondii infection cases in cetaceans have been reported in several countries, an information gap still exists in some areas. The present study employs a portable insulated isothermal PCR (iiPCR) with an automatic extraction device as a rapid, affordable, user-friendly, and field-deployable platform to rapidly detect nucleic acid of T. gondii in stranded cetaceans. The platform utilizes duplex iiPCR designed to simultaneously detect T. gondii and a housekeeping gene of cetacean on the samples, which can prevent the false-negative results of pathogen detection and improve the accuracy of surveillance. This study would contribute to improving the environment through the warning of the sentinel animals and building new strategies by detecting the occurrence of land-based biological pollution. ABSTRACT: Toxoplasmosis is a zoonotic disease with veterinary and public health importance worldwide. Toxoplasma gondii infection in cetaceans is an indicator of land-to-sea oocyst pollution. However, there is a critical knowledge gap within the distribution of the T. gondii infection in cetaceans. To facilitate the global surveillance of this important zoonotic pathogen, we developed a field-deployable duplex insulated isothermal PCR (iiPCR) with automated magnetic bead-based DNA extraction for the on-site detection of T. gondii in stranded cetaceans. It targets the B1 gene of T. gondii combined with β2-microglobulin (B2M) gene of cetaceans as an internal control. Compared with the conventional qPCR assay, B1/B2M duplex iiPCR assay showed comparable sensitivity (21~86 bradyzoites in 25 mg of tissue) to detect spike-in standard of T. gondii DNA in cerebrum, cerebellum, skeletal muscle and myocardium tissues. Moreover, the overall agreement between the duplex iiPCR and qPCR was in almost perfect agreement (92%; 95% CI: 0.78–0.90; κ = 0.84) in detecting a synthetic spike-in standards. The B1/B2M iiPCR assay coupled with a field-deployable system provides a prompt (~1.5 h), feasible, highly sensitive and specific on-site diagnostic tool for T. gondii in stranded cetaceans. This platform provides one approach to evaluating aquatic ecosystem health and developing early warnings about negative impacts on humans and marine animals.