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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 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 Epidemiological studies of parasitism in sheep and reproduction in horses : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Studies (Epidemiology) at Massey University(Massey University, 1997) Macchi, ChiaraAnthelmintic resistance is a well-recognised problem for control of nemotodes in sheep in most countries of the world. The climatic conditions in New Zealand are particularly favourable to the survival and development of gastrointestinal nemo=atodes. As a consequence, gastrointestinal parasitism is a major impediment to profitable sheep raising in New Zealand. A random postal survey of 300 sheep farmers in the southern North Island region of Manawatu was conducted with the purpose of examining current farming and drenching practices and investigating possible risk factors in the development of anthelmintic resistance. The results of this study, reported in Chapter 2, revealed a high degree of awareness and concern about the problem of resistance, but also a lack of understanding on how grazing management strategies should be combined in order to achieve integrated control over gastrointestinal nematodes while minimising the use of anthelmintic drugs. Only 31% of respondents had performed at least one drench test on their property. Amond testing farms, prevalence of resistance approached 70% and involved benzimidazole products in all but one case. Subsequently, a trial was undertaken to investigate the economic consequences of anthelmintic resistance in growing lambs on commercial farms (Chapters 3 and 4). Five farms with a history of resistance to benzimidazole drenches were selected. The effects of three treatment strategis on nematode egg counts, bodyweight gains and susceptibility to diarrhoea were compared between groups of ewe lambs. Suppressively treated lambs performed significantly better than effectively treated lambs, which in turn performed better than ineffectively treated lambs. However, a partial budgeting analysis carried out by means of a stochastic simulation model (Chapter 4) indicated that effective treatment yielded the highest net returns. The model also showed that the range of possible outcomes oscillated substantially around the mean, reflecting the degree of uncertainty about the outcome on any single farm due to variation between farms. Chapter 5 describes a study which was carried out with the objective of evaluating two management strategies for breeding mares after foaling. Mares were examined on day 7-9 postpartum by palpation and ultrasound. Pregnancy rates, pregnancy loss rates and time from foaling to conception in two groups were compared. The statistical power of the study as determined by power analysis, was insufficient for the observed differences to reach statistical significance.Item A survey of anthelmintic resistance and parasite management practices on sheep farms in New Zealand : a dissertation presented in partial fulfilment of the requirements for the degree of Master of Veterinary Studies (Veterinary Parasitology) at Massey University(Massey University, 2004) Sharma, Shyam SundarThe occurrence of anthelmintic resistance in sheep nematodes in New Zealand was conducted by using DrenchRite and Inhouse larval development assays (LDA) on 25 randomly selected farms. Samples from 6 farms were put onto both DrenchRite and Inhouse LDA plates and were available for comparison. Both showed a similar LC50 and LC50 well for benzimidazoles but not for levamisole. Including results from both assay systems it is concluded that anthelmintic resistance in Trichostrongylus (either suspected or high level) was demonstrated in 60% of the farms (9/15) to benzimidazole (BZ), resistance to levamisole (LEV) in 66% of farms (10/15), combination drench (BZ+LEV) on 43% of farms (3/7) and avermectin on 1 of 8 farms. For those farms where Trichostrongylus was the predominant genus there was resistance to at least one anthelmintic on all tested farms (n=12). A survey of parasite control procedures over 2002/2003 was conducted on 38 farms. The principle findings were: about 58% farmers (n=37) performed quarantine drenching of brought-in sheep for which a majority of farmers (52%) used macrocyclic lactones alone or in combination with other anthelmintics; about 78% (n=37) of farmers followed a 5-6 drench programme to lambs/hoggets; the annual frequency of anthelmintic treatments (n=38) were 6.1 drenches to lambs/hoggets; 1.4 to two-tooths and 1.8 to mature ewes; about 71% farmers used macrocyclic lactones for lambs/hoggets on at least one occasion either alone or in combination with other anthelmintics; a majority (61%) of farmers followed a planned annual drench family rotation; about 76.5% (29/38) farmers had never carried out a test for drench resistance and about 57%(4/7) of those farmers who had tested did identify resistance on their farms to either the benzimidazole or combination (benzimidazole + levamisole) drench. Worms were considered "very important" by 55% farmers (n=38) for the cause of economic losses in their farm, whereas 42% farmer considered it "important". Drench resistance was considered as a "serious problem" today for the industry by 47% farmers (n=38), whereas, 34% farmers believe it as a problem but "not serious one today" for the industry.Item Molecular epidemiology of Salmonella typhimurium DT160 in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Studies in Public Health at Massey University, Palmerston North, New Zealand(Massey University, 2010) Omar, Sharina; Omar, SharinaSalmonellosis is a zoonotic bacterial disease of national and international importance. In New Zealand (NZ), the most common foodborne notifiable disease is campylobacteriosis, which is followed by salmonellosis. In 1998, Salmonella enterica subsp. enterica serotype Typhimurium Definitive Type 160 (DT160) was identified in NZ. Since first reported, S. Typhimurium DT160 has caused several epidemics in the country but has not produced significant illness worldwide. Therefore, the objectives of the project were to investigate the molecular epidemiology of S. Typhimurium DT160 and the association between isolates from human and animal origin. Ninety Salmonella isolates obtained in the period between 1999 and 2009 from the Institute of Environmental Science and Research, NZ were assessed for colony morphology, serotype, susceptibility to 11 antimicrobials, virulotyped using Polymerase Chain Reaction (PCR) and the Pulsed Field Gel Electrophoresis (PFGE) patterns were also determined. In addition, 4 isolates were further assessed with Triple Sugar Agar, API20E biochemical and motility tests. All 90 isolates were confirmed as Salmonella spp. with no indications for resistance to multiple antimicrobials. All isolates were susceptible to the antimicrobials used in this study with the exception of 26 and 8 isolates that had intermediate susceptibility against tetracycline and oxytetracycline, respectively. In an attempt to discriminate between potentially pathogenic and pathogenic Salmonella isolates, PCR-based virulotyping was performed based on 12 potential virulence genes. Results revealed that all isolates were positive for at least 10 of the 12 virulence genes. Two of the six isolates negative for one of the virulence genes (invA, iroN, pefA or sifA) were of human origin and the remaining four were sparrow isolates. The PFGE patterns determined with restriction enzymes XbaI and SpeI demonstrated that the genotype profile AA1 accounted for 78/90 (86.7%) of the isolates, whilst the second most common profile, AA2, was found in only three isolates (3.3%), comprising two isolates from sparrows and one from a human. The remaining nine profiles were found in single isolates. All isolates of AA2 profile were PCR negative for sifA. In conclusion, no obvious correlation was observed between the phenol- and geno-type and the isolates, year and month of isolation, and source of the samples. There was no obvious evidence for multidrug resistance among DT160 isolates. The PFGE and virulotyping profiles suggest close relation among majority of isolates with predominant and epidemiologically important genotype persistent in multiple hosts. Finally, the few genotypes with low prevalence in multiple hosts may indicate emergence of sporadic genomic variants in the population.Item Some aspects of ivermectin resistance in gastrointestinal nematodes of goats and sheep : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University(Massey University, 2000) Gopal, Rajesh MohanExperiments were conducted to determine whether sheep are at risk from grazing pasture previously grazed by goats harbouring ivermectin-resistant Ostertagia spp. by monitoring the level of resistance with faecal egg count reduction tests and larval development assays. Ivermectin resistance emerged rapidly in goats grazed on the paddocks previously grazed by goats or sheep. In contrast, resistance was never consistently identified in sheep grazed on paddocks previously grazed by sheep although ivermectin resistance emerged after about 5 months in sheep grazed on paddocks previously grazed by goats. Ivermectin resistance was suspected in Trichostrongylus colubriformis and Ostertagia species on a goat farm in Northland, New Zealand. A controlled efficacy study was conducted in lambs (n=12) and young Angora goats (n=10) with this isolate. The establishment rate of T. colubriformis and Ostertagia spp. was similar (p>0.05) in the sheep and goats. Following ivermectin treatment (0.2 mg/kg b.wt. per oral) to half of the lambs and goats, the burdens of T. colubriformis were reduced by 39% and 13% in lambs and goats respectively whereas Ostertagia spp. burdens were reduced by 33% and 0% in lambs and goats respectively. In series of larval development assays with ivermectin aglycone, ivermectin and avermectin B2, this isolate of T. colubriformis had a resistance ratio of 37, 4-5 and 3-4 respectively indicating ivermectin aglycone is the drug of choice for detecting ivermectin resistance in T colubriformis. However, both ivermectin and avermectin B2 were still able to discriminate between resistant and susceptible T. colubriformis under controlled experimental conditions. The LC50 values of ivermectin were found to be influenced by the age of the infection of T. colubriformis in sheep. These LC50 values were relatively constant at 23 to 37 days after infection, then rose about four fold to peak between 51 to 58 days post infection, followed by a decline close to the original starting values by 72 days post infection. The resistance ratios recorded with thiabendazole were also high (14 to 15) suggesting that the ivermectin-resistant strain of T. colubriformis was also resistant to benzimidazoles. The LC50 values of ivermectin for T. colubriformis in a larval development assay for the two reciprocal first generation (F1) crosses of ivermectin-resistant and -susceptible parents were slightly lower than the LC50 values for ivermectin-resistant parents but greater than the ivermectin-susceptibie parents suggesting that ivermectin resistance in T colubriformis is inherited as an incompletely dominant trait. The fitness of the ivermectin-resistant strain of T. colubriformis was investigated and there was no significant differences (p>0.05) in infectivity, longevity of infection, fecundity and development of eggs to larvae under natural or laboratory conditions between the ivermectin-resistant strain and two susceptible field isolates. The survival of ivermectin-resistant strain larvae was intermediate between the two susceptible field isolates at 10°C, 20°C, 25°C and 30°C under laboratory conditions. An efficacy study in sheep indicated that the moxidectin oral formulation (0.2 mg/kg b.wt.), moxidectin injectable formulation (0.2 mg/kg b.wt.) and ivermectin oral formulation were 98%, 4% and 62% effective against this ivermectin-resistant strain of T. colubriformis respectively. These findings indicate that formulation of an anthelmintic plays an important role in the efficacy against resistant nematodes.Item Differentially regulated proteins in breast cancer chemotherapy : a thesis presented to Massey University in partial fulfilment of the requirement for the degree of Doctor of Philosophy in Biochemistry(Massey University, 2005) Koehn, HenningIntrinsic or acquired drug resistance of tumours is a major problem for successful therapy of breast cancer patients. The efficacy of doxorubicin, one of the most important and commonly used drugs in chemotherapy, can be severely compromised by a variety of unspecific mechanisms rendering tumours drug resistant. Little is known however, about the specific events taking place in response to doxorubicin treatment, which may repair doxorubicin-induced damage, leading to drug resistance. Doxorubicin is a topoisomerase II poison, which interferes with topoisomerase II enzymes during DNA replication, resulting in DNA double-strand breaks. Topoisomerase II enzymes mediate the passage of DNA strands by introducing transient DNA breaks, and are essential for changes in DNA topology during replication. The DNA lesions induced by the combination of topoisomerase II and doxorubicin can be repaired by either non homologous end-joining or homologous recombination repair, as both pathways are specifically responsible for the repair of DNA double-strand breaks. The DNA-dependent protein kinase catalytic subunit in non homologous end-joining and Rad51 in homologous recombination repair are essential for each of these pathways. If it was possible to specifically target these proteins or other antagonistic mechanisms of doxorubicin-induced cell death, which may be activated in response to doxorubicin treatment, chemosensitivity of tumours could be restored and chemotherapy made more effective. Hence it was the purpose of this study to investigate proteome-wide changes in protein expression in response to drug treatment, as well as specifically analysing alterations in the protein levels of the DNA-dependent protein kinase catalytic subunit and Rad51. Global changes in protein regulation of breast and breast cancer cells were investigated using mass spectrometric and electrophoretic analysis techniques. These experiments however, could not reproducibly identify any genuine drug-induced changes in protein levels, as only proteins of relatively high abundance could be analysed. Immunoblotting results however, showed that Rad51 was differentially regulated in a cell line- and drug dosage-dependent manner, while levels of the DNA-dependent protein kinase catalytic subunit remained largely unchanged. Furthermore, increased levels of topoisomerase II alpha protein were also detected. In addition, immunohistochemical analysis demonstrated that both Rad51 and the DNA-dependent protein kinase catalytic subunit could be independently overexpressed in breast tumours and therefore may represent potential targets for selectively enhancing chemosensitivity of breast cancers.