Investigating Toxoplasma gondii in the marine environment in New Zealand : from cats to kai moana (shellfish) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, School of Veterinary Science, Massey University, Palmerston North, New Zealand

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Recent reports indicate that Toxoplasma gondii may be an important cause of mortality for the endangered Hector’s dolphin in New Zealand. Infections are thought to occur after dolphin exposure to freshwater runoff containing T. gondii oocysts, which are only shed by cats. This thesis investigated land-sea transmission of T. gondii in an effort to determine how widespread the parasite is in New Zealand coastal waters and to better understand whether representatives of variant Type II T. gondii are particularly pathogenic for Hector’s dolphins. Chapters 3 – 5 investigated feline infections in New Zealand, their effect on the environmental oocyst burden, and associated T. gondii genotypes. Chapter 3 used a novel Bayesian model to evaluate the performance of three serological assays in the absence of a gold standard, with true seroprevalence in companion cats estimated at 61 %. No significant differences were found between regions sampled, suggesting that T. gondii exposure is widespread, and relatively high compared to the worldwide estimate of 30 – 40 %. Chapter 4 found that 1.6 % of feral and stray cats sampled were shedding T. gondii oocysts. Although shedding prevalence was within expected global limits, between 51 and 62 trillion oocysts were determined to be shed into the environment annually, including in regions adjacent to Hector’s dolphin habitat. Genotyping of oocysts revealed the presence of two unique recombinant strains. Chapter 5 described feline cases of toxoplasmosis in New Zealand. A rare manifestation - toxoplasmic gastritis - was uncovered, only previously reported in one other naturally infected cat overseas. Partial genotyping was possible for two of five cases, revealing what appeared to be unique, atypical strains. Although the presence of variant Type II T. gondii could not be ruled out due to small sample sizes and PCR amplification difficulties, results from Chapters 4 and 5 support the hypothesis that this genotype is especially pathogenic for certain endemic wildlife species, notably the Hector’s dolphin. The large, estimated oocyst burden also suggests substantial exposure risks for nearshore marine mammals, especially Hector’s dolphins, which are known to forage in shallow, estuarine waters. Looking to the marine environment, Chapter 2 examined archived tissues from common, dusky, and striped dolphins for T. gondii presence using histological, immunohistochemical and molecular methods. None of the dolphins were found to be infected with T. gondii. Due to issues with sample preservation and sample size it was not possible to reach definitive conclusions, but results suggest that T. gondii infection and disease is more prevalent in Hector’s dolphins than other cetacean species in New Zealand. Whether this is due to differences in exposure or susceptibility to toxoplasmosis still remains to be determined. Chapters 6 – 8 focused on the use of green-lipped mussels as biosentinels, for T. gondii surveillance, as it has not yet been possible to molecularly confirm the presence of T. gondii oocysts in seawater directly. Chapter 6 provides the first report of sporulated T. gondii oocysts and Giardia duodenalis in commercial green-lipped mussels in New Zealand. Specifically, using optimised molecular methods, G. duodenalis assemblage B, known to be pathogenic in humans, was discovered in 1 % mussels tested. Moreover, 13 mussels (n = 104) were found to be positive for T. gondii DNA with a true prevalence of 16.4 % obtained via Bayesian statistics, which is relatively high compared to overseas estimates. As oocysts must sporulate outside the definitive host to become infective, the study also validated a reverse-transcriptase PCR, which confirmed the presence of a sporozoite-specific marker (SporoSAG) in four mussels. Importantly, this represented the first time that sporulated, potentially infectious, T. gondii oocysts were confirmed to be present in shellfish globally. Chapter 7 aimed to find a suitably rapid, cost-effective, and analytically sensitive PCR assay for testing large quantities of wild green-lipped mussel haemolymph, in order to assess the prevalence of T. gondii in Hector’s dolphin habitat (Chapter 8). Particularly, four different PCR assays were validated for T. gondii detection using oocyst spiking experiments. Results identified a real-time PCR targeting a 529-bp repetitive element (rep529) as being preferable for future mussel studies, having the lowest limit of detection (5 oocysts), good correlation between oocyst concentrations and Cq values, and acceptable efficiency. Definitive confirmation of T. gondii DNA via direct sequencing was shown to be required using this assay, however, as the rep529 primers cross-reacted with Sarcocystis spp. and N. caninum DNA. Chapter 8 aimed to investigate T. gondii in green-lipped mussels collected from field sites located in key Māui dolphin habitats. Haemolymph samples were collected between 2014 and 2017 and stored at -80°C for 5 – 36mths. A relatively high T. gondii prevalence was expected: in the initial stages of the study 32/166 (19.3 %) of mussel haemolymph samples tested fresh were positive for T. gondii DNA. Surprisingly, no haemolymph samples tested in 2017 were positive for T. gondii DNA, despite the use of the validated rep529 qPCR. Although not statistically significant, results of a subsequent storage study showed that fewer stored samples tested positive compared with samples processed within 72h, suggesting that long-term storage of haemolymph could negatively impact the quality and/or yield of extractable DNA. Nevertheless, the storage study showed that T. gondii was still present in wild green-lipped mussels, albeit at lower prevalence (2 %), supporting previous findings that T. gondii oocysts are reaching coastal waters in sufficient quantities to be detected in mussels grown in New Zealand. Moreover, the results also confirmed that T. gondii is still present in the habitat of the critically endangered Māui dolphin subspecies and therefore remains a pathogen of concern for the Hector’s dolphin.
Figures 1, 2, 3 & 16 were re-used with permission from the respective publishers.
Toxoplasma gondii, Marine ecology