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Item Discovery of novel plant based compounds to address the drug resistance problem in nematode infested ruminants : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University, Manawatū, New Zealand(Massey University, 2022) Gupta, ArkaIn this thesis, the discovery of novel compounds from plant extracts towards addressing the drug resistance issue in nematode infested ruminants is presented. The nematocidal efficacy of the plant extracts was tested against the L3 stage nematode larvae through bioassay-guided fractionation and chromatographic separation. Quebracho crude powder (QCP) is the only commercially available source of polyphenols. The efficacy of a separated fraction of the QCP was found to be higher (P<0.05) than the crude extract against batches of L3 larvae. However, the research with QCP encountered many challenges and attention was shifted towards medicinal plants found in New Zealand. From a screening study, the Māori plant Piper excelsum (Kawakawa) was found to be the most effective. Of its different components, the leaf component was found to have the highest nematocidal efficacy. Kawakawa (KK) leaf samples were collected over three seasons and the nematocidal efficacy of the leaf samples was found to be independent. From the bioassay-guided fractionation study, the Water and MeOH solvent fractions were found to be most effective, and they were subjected to further reverse-phase chromatographic separation. It was found that the separated fractions had better anthelmintic efficacy than the parent crude solvent fractions (P<0.05). An improvised separation technique named ‘Hand Controlled Countercurrent Separation’ (HCCCS) based on the principle of CCS was developed. It was found that the fractions obtained from the HCCCS study had better anthelmintic efficacy than the parent MeOH-Fraction-4 and the anthelmintic ivermectin (IVM) (P<0.05). A series of combination formulations were made with the separated fractions and IVM. It was observed that these formulations had better efficacy than IVM and the individual HCCCS Fractions (P<0.05). The HCCCS Fractions were subjected to LC-MS/MS investigation and 34 compounds were identified. Of which, 8 were reported in the literature from published KK research. However, the anthelmintic properties of these compounds were not previously reported. The cytotoxicity evaluation of a series of fractions did not reveal any toxic effect to mammalian epithelial cells. Therefore, there is further potential towards isolating leaf fractions into pure isolates which may possess higher nematocidal efficacy compared to the fractions presented in this thesis.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 Genome-wide copy number variation in sheep : detection and utility as a genetic marker for quantitative traits, with reference to gastrointestinal nematodiasis : 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, 2018) Yan, JuncongGastrointestinal nematodes are perhaps the most important parasites of domestic sheep world-wide. Genetic selection for nematode resistance in domestic sheep is being promoted in many countries including New Zealand. There are several strategies to identify genetic markers associated with quantitative traits. Single nucleotide polymorphism (SNP)-based strategies have been widely used in animal breeding. However, SNP cannot explain all the genetic variation for a particular trait. A new kind of variation, copy number variation (CNV) has been identified as contributing to genetic variation in production and disease traits. Compared with other domestic animals, CNV in sheep is poorly investigated. The primary objective of this thesis was to explore the utility of genome-wide CNV as a genetic marker for the analysis of quantitative traits in sheep. Five different studies were undertaken to fulfill the objective. The first two studies used 50 K SNP BeadChip genotype data and next generation sequencing (NGS) data to detect CNV. Extensive CNV differences were evident between breeds as well as detection algorithms. NGS-based detection resulted in better CNV resolution than that by SNP. Subsequently, a genome-wide association study (with a small sample size) using CNV detected from a high density (HD) SNP genotype data identified four CNV regions to be significantly associated with a couple of traits pertaining to gastrointestinal nematodiasis in Romney sheep, while no significant SNP associations were found. Somatic mosaicism of CNV, influenced by age (high in foetuses, compared to adults), individuals, detection algorithm and type of tissue analysed, was also evident in separate study. The final study detected CNV differences and SNP based selection signatures in two Romney lines selected for gastrointestinal nematode resistance or resilience. Several significant SNPs and line-specific CNV regions were identified. However, only one SNP overlapped to a CNV region, indicating that SNP-based selection signatures and CNV could represent different aspects of sheep immunogenetics. Overall, CNV could be a potential genetic marker, albeit with methods for detection and validation needing to be refined. The conclusions from this thesis expand our understanding of CNV in sheep and its potential application prospects for genetic breeding of sheep in the future.Item Development of a low-cost automated sample presentation and analysis system for counting and classifying nematode eggs : a thesis presented in partial fulfilment of the requirements for the degree of Master of Engineering in Mechatronics at Massey University, Manawatu, New Zealand(Massey University, 2017) Pedersen, BenjaminThis thesis discusses the concept development and design of a low-cost, automated, sample presentation system for faecal egg counting, and classification. The system developed uses microfluidics to present nematode eggs for digital imaging to produce images suitable for image analysis and classification. The system costs are kept low by using simple manufacturing methods and commonly available equipment to produce microfluidic counting chambers, which can be interfaced with conventional microscopes. This thesis includes details of the design and implementation of the software developed to allow capture and processing of images from the presentation system. This thesis also includes details on the measures taken to correct for the optical aberrations introduced by the sample presentation system.Item An ecological study of some nematodes associated with apple trees in a grassed orchard : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science at Massey University(Massey University, 1967) Egunjobi, Olufunke AgbebePlant nematology appears to have been studied in New Zealand for the past 68 years, during which period a number of publications have accumulated on this subject (Kirk, 1899; Reid & Cottier, 1935; Jacks, 1944; Cottier, 1956; Stanton, 1956; Atkinson, Brien, Chamberlain, Cottier, Jacks, Reid & Taylor, 1949; Blair & Morrison, 1949; Morrison, 1957; Clark, 1964). However until 1961 when Clark returned to New Zealand from the United Kingdom, there was no trained nematologist in this field and most of the earlier identifications were done either through the disease symptoms or by overseas authorities. Hence, there is no record of specific ecological studies for this country. The first attempt to study the New Zealand soil nematode ecology was made by Clark at the commencement of his Ph.D. studies, but because of the high endemism encountered, which presented an absorbing taxonomic problem, he ended up merely opening the gate to this field by describing a vast number of the hitherto undescribed species. This work is the second attempt made, and it aims, not only to venture into the yet untouched ecological realm, but also to investigate some of the possible relationships of nematodes and apple trees in this primarily agricultural country.Item Aspects of anthelmintic resistance in nematodes in sheep : this thesis is presented in partial fulfillment of the requirements of the degree of Master of Veterinary Science at Massey University(Massey University, 1989) Butler, Andrew RoyThe increasing prevalence of anthelmintic resistance in nematodes of sheep is causing concern among animal scientists and farmers. In Australia anthelmintic resistance has become widespread since the first case was reported in 1968, and in some districts up to 68% of farms are affected. Benzimidazole resistance is most common, but levamisole and morantel resistance also occurs, and some farms have nematodes resistant to both major anthelmintic groups. Strains of the following species have shown resistance: Haemonchus contortus. Ostertagia circumcincta. Trichostrongylus spp. and, least commonly, Nematodirus spp. In New Zealand, anthelmintic resistance was first reported in 1980, and subsequent surveys found that its prevalence was generally low except on farms with above average anthelmintic usage. The same anthelmintics and nematodes as in Australia were implicated, although resistant Nematodirus spp. were reported more frequently in New Zealand. The most common test used to identify anthelmintic resistance - the faecal egg count reduction (or depression) test - was used in the survey of 15 Manawatu sheep farms which is the subject of this report. On each farm the anthelmintics oxfendazole, ivermectin and levamisole were given by mouth to groups of 12 young sheep, at dose rates of 5.0, 0.2 and 8.0 mg/kg, respectively. Twelve additional sheep were designated as untreated controls. Faecal samples for egg counting were taken from all sheep on the day of treatment and 7 days later. Composite faecal samples from each farm were cultured for identification of larvae present before treatment and, when anthelmintic treatments were deemed unsatisfactory, post-treatment faeces were also cultured for larval identification. Using the simplest method of calculation of faecal egg count reduction, and a cut-off point of 90% or below, there were (respectively) 4,2 and 0 farms with oxfendazole, ivermectin and levamisole resistant strongylate nematodes (excluding Nematodirus spp.). However, supporting evidence for the existence of ivermectin resistant nematodes was weak, and no claim is made that true ivermectin resistance has been detected. On the 4 farms with oxfendazole resistant nematodes, resistant Trichostrongylus spp. were the most common (4 farms), followed by H. contortus (3 farms), 0. circumcincta (2 farms) and Oesophagostomum spp. (2 farms). Oxfendazole resistant Nematodirus spp. were very common, but it was not possible to reach any conclusion about the susceptibility of the Nematodirus spp. present on each farm because of the low numbers of sheep passing Nematodirus spp. eggs and the small numbers of those eggs. Resistant Nematodirus spp. have probably been overlooked in the past and methods to reduce the likelihood of this are suggested. The faecal egg count reduction test is a useful field screening test, but it is difficult to interpret when only a slight degree of anthelmintic resistance is encountered. This is partly due to uncertainty in where the cut-off point should be, and partly to variation in calculation methods. The acceptance of recently formulated standard test protocols for Australia and New Zealand should reduce this problem, but supplementary tests will still be needed in many cases of anthelmintic resistance testing.Item Studies on the epidemiology of Nematodirus spathiger and Nematodirus filicollis in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Animal Science at Massey University, Manawatu, New Zealand(Massey University, 2015) Oliver, Anne-Maree BridgetThis thesis studies aspects of the epidemiology of Nematodirus spathiger and Nematodirus filicollis, both are common and potentially pathogenic parasites of lambs in New Zealand. Three studies were undertaken; the first a presence/absence survey to determine the distribution of the two species on farms, the second examined the prevalence of benzimidazole (BZ) resistance in the two species and the third experiment investigated the requirements for chilling for N. filicollis eggs to hatch. The first two studies utilised faecal samples from routine faecal egg counts and/or routine faecal egg count reduction testing on farms. To identify the Nematodirus species involved, eggs from these samples were chilled and then incubated to facilitate hatching of N. filicollis, before larvae were recovered. The identity of the larvae was determined using PCR of the ITS-2 region of rDNA. The third experiment used a bulk collection of N. filicollis from naturally infected lambs. Extracted eggs were incubated at temperatures between 2.7°C - 9.9°C, for up to 224 days. The proportion of eggs hatching was assessed against chill units. Chill units (degree-day) were calculated by subtracting the culture temperature from a constant threshold of 11°C and multiplying by the number of days for which the sample was cultured. In Study 1, N. spathiger was present on all farms tested, while N. filicollis was found on 76% of farms. Both species were distributed throughout New Zealand, with no regional differences. In Study 2, the BZ-resistance study, efficacies below 95% were recorded for N. spathiger and N. filicollis on 95% (20/21) and 40% (4/10) respectively of farms tested. In Study 3, the chilling experiment, the overall hatching of N. filicollis eggs was low, but increased with chill accumulation to plateau at about 11%, with 800-1000 chill units required for maximum hatching. In conclusion the two species of Nematodirus were commonly found on most New Zealand farms. The prevalence of BZ-resistance in N. filicollis was lower than that in N. spathiger. N. filicollis required a considerable period of chilling to enable hatching to occur and this will influence their epidemiology.Item Nematodiasis and larval migrans in kiwi (Apteryx spp.) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Zoology at Massey University, Palmerston North, New Zealand(Massey University, 2014) van Zyl, NickiThe aim of this thesis is to investigate the inter-relationships between host and parasite specificity and aberrant larval migration, using the kiwi family (Apteryx spp) and their nematodes as the study system. Kiwi are endemic to New Zealand, and have been estimated to have declined by 90% since human settlement in New Zealand. The effect of disease, mainly parasitism on kiwi populations have been a minor issue compared to predation, however with intensive conservation management the effect of parasites on kiwi are likely to increase. Very little is known about parasites in kiwi and how they impact the birds, especially the aberrant larval migrans syndromes. I determined the prevalence of nematodes in all kiwi species from 1991-2012 (n=642) that were necropsied at Massey University, using the National Wildlife Pathology Database (Huia), with a specific focus on larval migrans. Brown kiwi (Apteryx mantelli) were found to have the highest prevalence of nematodiasis, and were also the only species to show evidence of neural larval migrans. Visceral larval migrans were found in brown, rowi (A. rowi) and little spotted kiwi (A. owenii). With the brown kiwi showing the highest incidence of larval migrans, I focused on this species for further investigation. It has previously been proposed that the larval migrans could be caused by nematodes from the Toxocara spp, since wild kiwi can share habitats with wild cats and dogs, which are the normal host of this nematode). However, with specific PCR analysis conducted on archived tissue, I concluded that neither Toxocara cati or T. canis was present in the tissues. I also investigated the cause of cutaneous larval migrans (CLM) in rowi. Uniquely, CLM has not been previously recorded in any avian host. A generic nematode PCR analysis followed by DNA sequencing were used in this study to identify the nematode involved in CLM as being closely related to a nematode from the Trichostrongylus species. ii My results suggest that introduced animal hosts are having an indirect effect on our native bird populations, but to what extent is still unknown. The parasites transmitted between the introduced species and our native species are causing adverse effects to our native populations’ health, in the case of larval migrans; but how often are these introduced parasites infecting our native population and could our native species cope with this competition? From my study it can be presumed that our native species are struggling to adapt to new parasites, and competition between introduced and native species could be contributing to the decline in our species.Item Some aspects of the population dynamics of Cooperia oncophora : 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, 2014) Sauermann, Christian WilhelmTo develop better control strategies for Cooperia oncophora a detailed understanding of the population dynamics is desirable. To achieve this, aspects of the life cycle have been studied in a series of experiments, including the free-living and the parasitic phases. To investigate the development from egg to third stage larvae, faeces containing C. oncophora eggs gathered from different donor animals were incubated at different constant and variable temperatures as well as under natural conditions in the field. To examine the survival of C. oncophora third stage larvae they were kept under similar conditions as for the development experiments. Based on the results at constant temperatures, parameters were calculated for a model to simulate the effect of temperature during the free-living phase of this nematode. A further experiment was conducted over an 11 month period to investigate the effect of host age and previous exposure on the establishment rate of third stage larvae in 3 groups of young calves. Two groups received a high or low dose of trickle-infection and the third remained as an uninfected control group. At lower temperatures the development rate and success from egg-third stage larvae were both low but increased with higher temperatures. At 8°C 50% development was reached in 56 days with a success rate of 5.5% compared to 5 days and 26.4% respectively at 32°C. The highest development success rate of 37.4% was observed at 28°C. For larval survival, the median survival was 512.2 days at 8°C and decreased to 6.4 days at 37°C. Both development and survival were significantly (p<0.05) influenced by the host animal from which the faeces were sourced. Utilising these parameters in a model provides a useful tool to further understand the effect of temperature on the free-living stages. The establishment rate of C. oncophora in the trickle-infected groups declined rapidly compared to the control group but was not significantly different (p>0.05) to the control group if the existing worm burden was removed before challenge. A decline in establishment rate over the 11 month period of the experiment in the control animals was due to the age of the larvae.