Abstract
Toxoplasma gondii is a ubiquitous protozoal parasite capable of causing clinical disease and death in all warm-blooded animals. Widespread and persistent in the environment, it has a complex lifecycle involving numerous intermediate hosts which may then reinfect cats, the only definitive host capable of shedding oocysts back into the environment. It is composed of several lineages each of which carries varying levels of pathogenicity to the intermediate hosts it infects.1 In New Zealand, T. gondii has increasingly been identified as the causative agent behind the deaths of several endangered native species including kaka, kiwi, and Hector’s dolphins.2,3 One genotype, variant type II or ToxoDB#3, has been responsible for all the investigated fatal NZ wildlife cases to date, but has not been identified from other sources.4 In fact, there is a limited understanding of the Toxoplasma genotypes present in NZ overall. The purpose of this study is to determine whether a cat prey species, the rat, can be used to investigate T. gondii genotypes in NZ. In the initial phase, 20 rats each, collected from 3 geographically distinct kill trapping sites, were dissected to collect brain, tongue, heart, and liver samples. DNA extraction and PCRs were performed on each sample, with any positives being submitted for genotyping.
Samples were either stored in RNA later at 4°C prior to being frozen at -20°C, or stored at 80°C prior to DNA extractions. DNA extractions were performed using a DNeasy Blood and Tissue Kit according to the manufacturer’s instructions and were frozen at -20°C prior to PCR analysis. A nPCR targeting a 530 bp fragment of the Toxoplasma B1 gene was conducted for each sample based on its increased sensitivity for detecting T. gondii tachyzoites in intermediate host tissues.5 The nPCR products were run on a 1.5% agarose gel stained with SYBR Safe prior to UV visualization on a GelDoc. All positive products, as identified by fluorescence at the ∼500 bp region when compared to a standard ladder, will be submitted for sequencing. An estimated prevalence of T. gondii in NZ rats will be calculated and tissue comparisons conducted if prevalence is high enough to determine whether a particular tissue type is more likely to yield a positive result on PCR. Any sequence-positive samples may also undergo genotyping to compare to the current understanding of Toxoplasma genotypes in New Zealand.
Despite Toxoplasma being widespread and known to cause production losses in livestock, a risk to public health, and a threat to wildlife, studies on its effects on NZ wildlife populations are very limited. Based on the current available evidence, toxoplasmosis may have a deleterious impact on NZ’s endangered and critically endangered Hector’s and Maui dolphins which seem to experience a greater incidence of fatal toxoplasmosis compared to other native wildlife.4 This research will further our understanding of the prevalence and genotypes of Toxoplasma in a common cat prey species which will help elucidate the overall picture of how this parasite has spread and may inform future control and management strategies to limit its impact.
Acknowledgements
The authors wish to thank the teams who provided rats for use in this study and the staff of the Hopkirk Research Institute, who provided endless support for the molecular biology work that was undertaken.
*Presenting author
+Student presenter
Literature Cited
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