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Item The prevalence and production effects of liver fluke (Fasciola hepatica) in New Zealand cattle including evaluation of diagnostic tests : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand(Massey University, 2023) Dowling, AndrewThe liver fluke Fasciola hepatica, infects cattle worldwide and is considered a parasite of regional importance in New Zealand although the impact on milk production in that country have not been studied. The test characteristics of two antibody detection ELISAs; IDEXX ELISA (IDEXX Fasciolosis Verification) and an In-House assay using unrefined excretory secretory antigens, plus a coproantigen ELISA (Bio K 201–Monoscreen AgELISA) and faecal egg counts (FEC) were assessed against the gold standard of total fluke counts in naturally infected cattle (cows=29, steers=10). Vat milk of dairy herds on the West Coast of the South Island was assessed for liver fluke infection using the IDEXX ELISA in the autumn, near the end of one lactation (n=430), and spring, near the beginning of the subsequent lactation(n=403). A total of 156 questionnaires determining awareness of liver fluke infection and drenching practices were completed. A cross sectional study of 11 herds (n=1314 cows) in autumn and a longitudinal study of 4 herds (n=485 cows) in spring and autumn used the IDEXX ELISA (measured as SP%) on serum to analyse associations between liver fluke infection and milk production parameters in individual cows. A subset of cows was also faecal sampled for coproantigen and FEC analysis. Notably, a negative linear effect of the loge(total fluke count+1) on liveweight (p=0.02) was found and the coproantigen values showed a significant (p=0.01) quadratic effect for loge(total fluke count+1). The survey showed that infection of herds at a level likely to cause production losses on the West Coast is common, with regional clustering. Milk Fat % decreased 0.0004% points for every 1SP% increase (p=0.004), being 0.05 %points lower for cows with SP%150 than cows with SP%30, and 0.22 %points (p=0.014) lower in cows where SP% increased from ≤30 to ≥150 during lactation compared that those remained ≤30 with an economic cost of $55.19 per infected cow. Of the tests compared, the IDEXX ELISA was superior to the In-House ELISA for sensitivity (Se) and specificity (Sp) but the coproantigen ELISA had the highest Se (96%) and Sp (96%). Overall, liver fluke infection was common in dairy cows but the infection intensity was low, nevertheless a small effect on MF% was determined.Item Developing a risk prediction model for the seasonality of Lucilia spp. in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Manawatū, New Zealand(Massey University, 2023) Brett, Paul Thomas JamesFlystrike of sheep in New Zealand is principally caused by Lucilia cuprina and Lucilia sericata. A series of studies were conducted to develop models to describe and predict the seasonal occurrence of these Lucilia spp. in New Zealand. Dipterans were collected on a weekly basis on eight farms across New Zealand over three fly seasons (2018/2019, 2019/2020, 2020/2021) using the LuciTrap® with a Stickytrap attachment (Lucilia spp. n = 10,559). Covid 19 travel restrictions restricted the collection of samples during the 2019/2020 and 2020/2021 seasons. Dipterans were initially identified using morphological characteristics, with further validation using the nuclear 28s rRNA and the mitochondrial ND4 gene. The morphological identification had an accuracy of 71 % for L. cuprina and 55 % for L. sericata, compared to the molecular method (p < 0.05). Consequently, the counts of both species were combined for modelling purposes. The seasonality of Lucilia spp. adult flies span from early October until late May with variability of weeks duration between farms and three weeks between seasons for individual farms. A hurdle model was used to describe the occurrence of Lucilia spp. from the 2018/2019 season (p < 0.05). Significant variables include soil temperature, rainfall, maximum temperature and photoperiod with lag times of one to seven weeks. A second model used the 2018/2019 seasonal data to predict the start of the 2019/2020 season using a mixed-effects logistic regression model using weather data from the closest Virtual Climate Station. 10 cm soil temperature and Soil Moisture Deficit Index predicted the start of the season within two weeks of the observed season (p < 0.05). Four trap and bait combinations were compared to help choose a supplementary on-farm technique to confirm model predictions. A negative binomial model fitted for Lucilia spp. catch data found no difference between LuciTrap® combined with LuciLures and the Western Australian Trap combined with sheep liver preserved in 30 % sodium sulphide (p > 0.05). While the other two trap and bait combinations were significantly worse for catching Lucilia spp. (p < 0.05). These models should provide information to allow farmers to make more informed decisions for flystrike control.Item Diagnosis and prevalence of ventricular nematodiasis in kiwi (Apteryx spp.) : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Veterinary Science at Massey University, Manawatū, New Zealand(Massey University, 2021) Chidambaram, BhargaviOnly one species of nematode has been formally described from the ventriculus of kiwi (Apteryx spp.); a spirurid nematode of the Cyrnea genus. However, apart from its morphology, little is known about this parasite. This thesis describes investigations into its pathogenicity, prevalence and diagnosis. A case series examining four cases of ventricular nematodiasis in kiwi (VNK) highlighted that repeated faecal examinations did not detect the eggs of the ventricular nematodes. Diagnoses were achieved via gastroscopy or post-mortem histological examination of the ventricular wall. These cases illustrated how the lack of an accurate ante-mortem diagnostic technique limits what is known about the parasite responsible for VNK including its pathogenesis, transmission and management. Four-hundred and fifty samples were collected opportunistically from captive kiwi housing facilities over the course of eight months with the dual aims of refining diagnostic methodology and establishing the prevalence of the Cyrnea sp. nematode in captive kiwi faecal samples. These samples were obtained from 167 individual kiwi of four different subspecies across a wide age-range from nine different submission locations. An ante-mortem test to diagnose VNK was formulated and refined using coprological testing. The most sensitive method of detecting Cyrnea ova was centrifugal flotation in 80% zinc sulphate solution but examination of the centrifugal sediment from a 33% zinc sulphate solution was more accurate in quantifying the number of ova present. Centrifugal sedimentation reduces the time and effort required for sample processing, making it the efficient choice. The prevalence of ventricular nematode ova in faecal samples from captive kiwi was found to be 16.77% (95% CI) based on faecal flotation in 80% zinc sulphate solution. Using this method, kiwi aged 4 months or younger and those of the North Island brown (A. mantelli) species were the most likely to have detectable parasitic infection with Cyrnea spp. This research highlighted the importance of developing an accurate ante-mortem faecal test for the diagnosis of VNK. A viable preliminary test for laboratory use was developed and the prevalence of infection was examined in captive kiwi through faecal testing.Item Host-parasite dynamics in the endangered yellow-eyed penguin (Megadyptes antipodes) : investigations of Plasmodium and Eimeria in geographically distinct populations : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Science in Wildlife Health at Massey University, Palmerston North, Manawatu, New Zealand(Massey University, 2021) Kay, Emily ElizabethThe yellow-eyed penguin (Megadyptes antipodes) is an endangered species endemic to New Zealand, facing ongoing threats to the persistence of the species. The yellow-eyed penguin occupies a restricted range including mainland New Zealand and adjacent islands, and the subantarctic Auckland Islands and Campbell Island. There has been no effective migration between the birds occupying mainland New Zealand, and the subantarctic islands, creating two population centres that are both geographically and genetically distinct. The two population centres of the yellow-eyed penguin, defined by the geographical range, occupy vastly different habitat ranges and face ongoing site-specific threats. The mainland population has declined by up to 65% in the last 20 years, while the sub-Antarctic population appears stable through limited monitoring. Examining host-pathogen dynamics of geographically distinct wildlife populations can inform predictions of a population’s response to major climate shifts or translocations. This study therefore explored possible differences in the host-pathogen dynamics between the two population centres of the yellow-eyed penguin. The study sampled wild penguins from the two major population centres, the mainland and sub-Antarctic population centres, to examine the prevalence and pathogen load differences of two known parasites Eimeria and Plasmodium. In addition, the study documented differences in the prevalence of Plasmodium infection between penguins in rehabilitation and those in the wild. Eimeria is a host-adapted parasite that principally causes disease in wild birds when the host is immunosuppressed through stress or intercurrent disease. Therefore, this study used Eimeria as an ecological biomarker to assess the distinct wild yellow-eyed penguin populations. The first morphological description of a novel species of Eimeria protozoa from a yellow-eyed penguin host is provided in this study. A high prevalence and pathogen load of Eimeria oocysts in faecal samples from wild yellow-eyed penguins was identified from the wild sub-Antarctic (apparent prevalence 76.6%, 95% CI 62.78-86.40% and mean pathogen load 9723 +/- 5831 oocysts/gram) and mainland populations surveyed (apparent prevalence 58.5%, 95% CI 43.37-72.24% and mean pathogen load 1050 +/- 398 oocysts/gram), with only weak evidence for a difference in the apparent prevalence and pathogen load between the two population centres. There was, however, a significant difference in body condition scores of infected penguins between the two populations, suggesting possible differences in the host-pathogen dynamics between the two distinct population centres. These results confirm the hypothesis that these coccidia are characteristic of a highly host-adapted endemic parasite in the yellow-eyed penguins. Penguins have a known susceptibility to Plasmodium sp., with infection capable of causing significant morbidity and mortality. This study provides the first report of a positive result for Plasmodium sp. from wild yellow-eyed penguins using LSU-RNA qPCR, and documents the estimated pathogen load (mean pathogen load 122 +/-29 Plasmodium DNA copies / 10,000 avian cells) of infected wild birds. There was good evidence for a difference in apparent prevalence between the two population centres, with a higher apparent prevalence of avian malaria in the wild mainland population (6.8%, 95% CI 2.96-15.05%), and an apparent absence of infection in the sub-Antarctic population surveyed (0%, 95% CI 0-5.58%). The study also documented a high apparent prevalence (65.9%, 95% CI 51.14-78.12%) of Plasmodium sp. infection in yellow-eyed penguins in rehabilitation, identifying three separate strains of avian malaria (Plasmodium sp. Lineage LINN1, Plasmodium relictum lineage SGS1 and Plasmodium elongatum lineage GRW06) via molecular sequencing. These results confirm the hypothesis that avian malaria is characteristic of a vector-borne parasite in the yellow-eyed penguins, and despite the presence of competent vectors in both habitats, only the northernmost population of yellow eyed penguins is currently infected. However, this disease is likely to emerge in the sub-Antarctic population in response to predicted climate shifts. The results of this work provide a platform for further research into the host-pathogen dynamics of the novel Eimeria species identified, and the potential host effects of this parasite during environmental stressors. In addition, the findings of this study suggest future monitoring of avian malaria prevalence and mortality rates in the yellow-eyed penguin are critical to understand the emerging risk of this pathogen in the context of ongoing climate shifts. Finally, the high prevalence of Plasmodium infection documented in yellow-eyed penguins in rehabilitation demonstrates the threat of this parasite to the success of ongoing rehabilitation efforts, and the need to investigate measures to mitigate infection risk for this species in the future.Item Nematode infections of kiwi (Apteryx spp.) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand(Massey University, 2021) French, AdrienneOverall, little is known about the nematodes of kiwi (Apteryx spp.) and there has, thus far, been little indication that such infections are associated with significant clinical disease in these species. However, over the past 15 to 20 years there has been increasing recognition of cases of nematode larva migrans identified in kiwi at necropsy, caused by the aberrant migration of nematode larvae within visceral organs and brain. The initial study of this research re-examines archived cases of larva migrans through DNA extraction and PCR using formalin-fixed, paraffin-embedded tissue sections. Sequencing and BLAST analysis of positive results showed 100% alignment to database sequences from Toxocara cati, a nematode parasite whose definitive host is the domestic cat, indicating an infection acquired from an invasive mammalian species. Following the success of this methodology, similar techniques were applied to archived biopsies from an outbreak of cutaneous nematodiasis in a geographically isolated population of juvenile rowi kiwi (Apteryx rowi). The resultant sequences aligned among members of the capillarid genus Eucoleus although without an exact match present in the database, and so the precise, species-level identification and original source of this unusual presentation of cutaneous capillariasis remains unknown. Concurrent with these investigations into aberrant nematode infections, an opportunistic, prospective survey that included 50 kiwi presenting for necropsy examination was performed, with the objective of examining as far as practicable the prevalence, diversity, and potential pathogenicity of gastrointestinal nematodes, including any potential association with aberrant migratory lesions. The results confirmed that gastrointestinal nematodiasis was common, with 94% of the kiwi examined infected to some degree, and at least five morphologically distinct nematode types were found. Species-level identification of the nematodes was not reached based on limited morphological and molecular evaluations. However, while such gastrointestinal nematode infections appeared on the whole to be well-tolerated by the kiwi hosts, occasional cases of significant ventriculitis were identified in association with gizzard infections, particularly in juvenile, captive-bred kiwi. Furthermore, histological lesions consistent with nematode larva migrans were identified in around 43% of the survey kiwi in which visceral sections were also examined, indicating that this disease may be routinely under-diagnosed.Item Towards developing support tools for sustainable control of gastrointestinal nematodes in sheep : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, Manawatū, New Zealand(Massey University, 2021) Ikurior, SeerGastrointestinal nematode (GIN) parasitism is a major animal health challenge for sheep. Parasitized animals typically display a number of clinical signs, including a reduction in voluntary feed intake, altered grazing behaviour and lethargy. The aim of this thesis was to use remote sensing technologies to advance the development of a methodology where early changes in animal behaviour can be used to help identify sheep suffering ill effects of GIN parasitism, especially in a pre-clinical situation. It was hypothesised that lambs with even modest worm burdens will be less active, graze for less time and spend more time resting than those herd mates that were less heavily parasitized. The movement and behavioural activity of young and mature, infected and uninfected sheep were monitored in a series of studies using global positioning system (GPS) and tri-axial accelerometer sensors. Key behaviours were identified using machine learning techniques. Also assessed was the influence of host genotype on movement activity. Accelerometry data accurately identified grazing, resting and walking activities of sheep. The sensors were able to identify the effects of GIN parasitism on movement and behaviour in sheep. Clear evidence was found that GIN were associated with reduced movement and overall activity in growing lambs, with reductions in time spent ‘grazing’ and ‘walking’ occurring concomitantly with increases in ‘resting’ activity, and before effects were recorded on growth rates. Host genotype also had an effect on movement activity of lambs in untreated sheep, but not in treated individuals. Adult sheep, however, showed no consistent changes in movement and behaviour associated with parasitism, as measured by faecal egg counts. Overall, the findings in this thesis have demonstrated the potential value in remote monitoring of sheep as a diagnostic marker to detect the generally subtle behavioural changes associated with changing GIN infection status. Such monitoring could therefore be used as the basis for deciding whether animals need to be treated with anthelmintic on the basis of individual need, and such decisions could be taken early, i.e. before animals have failed to grow adequately or started to manifest more overt signs of clinical illness such as weight loss.Item 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(Massey University, 2021) Coupe, AliciaRecent 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.Item Morphological and molecular characterisation of coccidia (Eimeria spp.) in kiwi (Apteryx spp.) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand(Massey University, 2021) Coker, SarahThe coccidia of kiwi were first reported in the 1970s; however, in-depth, morphological descriptions of oocysts and endogenous stages were not described until 2013. The development and success of Operation Nest Egg (ONE) between 1994 to the present provided the ideal environment for the proliferation of coccidia, i.e., increased density of immunologically naïve hosts. These conditions led to an increase in morbidity and mortality of kiwi chicks due to high coccidial burdens. Current methods of detection and treatment rely heavily on husbandry practices and, potentially, over medication with coccidiocides. Treatment-resistant species of coccidia may exacerbate these problems causing increased cost of frequent hospitalisation and additional supportive care required by kiwi with high coccidial burdens. This thesis advances the ability to quantify oocyst loads in kiwi droppings by determining that a modified Mini-FLOTAC protocol is more accurate than the current method used in New Zealand diagnostic labs. This alternative protocol has the added benefit of not requiring large centrifuges, potentially enabling practitioners to determine coccidial burdens on site. The morphological descriptions of oocysts are continued in brown kiwi and established in Haast tokoeka. The four morphotypes previously described were represented in the brown kiwi dataset; however, E. paraurii was not described in Haast tokoeka. A new morphotype similar to E. kiwii was described in both brown kiwi and Haast tokoeka. This thesis provides the first genetic data at the mitochondrial cytochrome c oxidase I gene, which was initially amplified from the morphologically described species using Sanger sequencing. A larger dataset that included brown kiwi, Haast tokoeka, rowi, and a great spotted kiwi was targeted and sequenced using Illumina amplicon technology. This in-depth analysis allowed for the detection of the full variation of sequences within the samples, informing the development of diagnostic testing, pathogenicity studies, and treatment efficacy monitoring in the future. Further, extensive optimisation of extraction protocols provides key guidelines for breaking open unsporulated oocyst walls to ensure all the oocysts in a sample are represented in the results.