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Item Pathophysiology and immunomodulation associated with Haemonchus contortus infection : a dissertation presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand(Massey University, 2015) Zia-ur-RehmanThe aim of this project was to investigate host-parasite interactions, which might lead to alternate strategies to control the sheep abomasal nematode Haemonchus contortus. The project focused on two aspects of host parasite interactions: the initiation of host pathology and suppression of host immune responses associated with the onset of infection. Adult H. contortus ES products increased the permeability of Caco-2 cell monolayers and this increase could be blocked by single chain antibodies against ES products displayed on phage. Recombinant H. contortus enolase may be one of the active components of ES as it mimicked the action of ES products on Caco-2 cells. This is the first study of immunomodulation by adult H. contortus ES products of the phenotypic and functional properties of human monocyte-derived dendritic cells (mdDCs). Incubation with ES products resulted in semi-maturation of mdDCs, with weak up-regulation of the co-stimulatory molecules CD40 and CD80 and increased surface expression of the tolerogenic markers CD32, CD305 and galectin-1. The highly variable responses of mdDCs of individual donors biased the group data, particularly in response to co-stimulation with ES products and LPS. This highlights genetic diversity in the immune system and possible difficulties in developing worm-based therapies. The blastogenic activities of cells from lymph nodes collected from two groups of infected and vaccinated sheep were measured by 3H-thymidine uptake after exposure to ConA or ES products. The Stimulation Index (SI) with ConA was 1 0-fold higher in cells from the older animals. Cells only from younger infected sheep had a reduced response to ConA and vaccinated groups with reduced parasite burdens had the highest Sl. There was little response to ES products in older sheep, but in younger animals there was a trend for lymphocyte Sl to be greater with 10% ES in sheep with the fewest parasites. These experiments show that H. contortus ES products may facilitate the initiation of host pathology and the potential to modulate responses of dendritic and lymph node cells during parasitism. Further identification of the specific ES components responsible may allow disruption of their actions, resulting in resilient and immune sheep.Item Bacteria associated with Haemonchus contortus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand(Massey University, 2012) Sinnathamby, GajenthiranInternal parasitism, a major cause of production losses in sheep, is routinely controlled by anthelmintic drenches, however, alternative control strategies are needed to combat the increasing resistance to these chemicals. [sic] A possible novel method of controlling abomasal nematodes, such as Haemonchus contortus, is manipulation of their essential resident bacteria, as is currently used to control filarial nematodes. For the first time, bacteria have been identified in the reproductive tract, as well as in the gut, of H contortus, using genetic fingerprinting, light and electron microscopy and fluorescence in situ hybridisation (FISH). PCR-DGGE analysis showed that adult worms had less complex bacterial profiles than did abomasal contents. L3, eggs and adult worms had similar bacterial profiles; 16S rRNA sequences obtained from seven major common DGGE bands were dominated by lactic acid bacterial and Proteobacterial sequences. PCR-DGGE short sequences and clone libraries of nearly full length sequences from all three life-cycle stages contained sequences belonging to Weissella, Lactococcus, Leuconostoc and Streptococcus. Clone library sequences were used to design group-, class- and species-specific FISH probes to locate bacteria in the parasites. The gut lumen of adult worms contained a mixed population of Grampositive and Gram-negative bacteria, which appeared to be multiple morphotypes in TEM images. The FISH probe (EUB338), which targets most bacteria, hybridised with the gut bacteria, but only some of these were targeted by Strc493, which targets most Streptococcus sp. and some Lactococcus sp. Neither the lactic acid bacterial group- nor the Weissella species-specific probes targeted any bacteria in the gut. A single morphotype of Gram-positive bacteria was seen in large numbers in the distal uterus of female H contortus in the TEM. They were close relatives of either Lactococcus sp. or Streptococcus sp., as they were targeted by the FISH probe Strc493. These bacteria seemed to be non-pathogenic to the nematodes, as adult female worms appeared to be healthy (nonnal in size and active) and carry normal eggs within them. Their roles in worm biology are unknown. A smaller number of bacteria were seen in the TEM in eggs within female wom1s. They were closely related to Weissella confusa, as all were targeted by lactic acid bacterial group- and Weissella species-specific probes, as well as by EUB338. These bacteria were dispersed throughout the eggs, as they could be seen at different focal panels in confocal microscopy. DNA fingerprinting and visualisation of these bacteria in eggs strongly suggest they are maternally transmitted endosymbionts. As this study was carried out on a parasite strain which has been maintained in the laboratory, practical applications of this research would depend on these bacteria being present in field strains of H. contortus.Item Nitrogen metabolism in Haemonchus contortus and Teladorsagia circumcincta : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand(Massey University, 2012) Umair, SallahThis is the first study to characterise proline, arginine and lysine metabolism in homogenates of L3 and adult Haemonchus contortus and Teladorsagia circumcincta. The properties of glutamate dehydrogenase (GDH), glutamate synthase and the GABA shunt were also compared in the two species. The kinetic properties of 26 enzymes were determined. The gene encoding T. circumcincta GDH was sequenced and recombinant TcGDH expressed and biochemically characterised. The ornithine-glutamate-proline pathway was fully functional. The mammalian α-AAA (saccharopine) and pipecolate pathways of lysine catabolism, but not the bacterial enzymes lysine dehydrogenase and decarboxylase, were present in adult worms. The pipecolate pathway was incomplete in L3 of both species, as Pip2CR activity was undetectable. Unusually, lysine ketoglutarate reductase and saccharopine dehydrogenase, Δ1-pyrroline-5-carboxylate synthase and reductase were able to use both cofactors. The glutamine synthetase-glutamate synthase pathway of ammonia incorporation into glutamate was present, except in L3 H. contortus. T. circumcincta GDH was cloned, purified and characterised and the predicted protein sequence was very similar to H. contortus GDH. T. circumcincta recombinant and H. contortus homogenate GDH were both dual co-factor specific, although the latter had 50% greater activity with NAD+/H as cofactor. GDH activity was inhibited by GTP and stimulated by ADP whereas ATP either inhibited or stimulated depending on the concentration and direction of the reaction. The GABA shunt enzymes glutamate decarboxylase and succinic semialdehyde dehydrogenase was not detected in homogenates of whole L3 or adult H. contortus or T. circumcincta. Neither parasite had a full functional ornithine urea cycle, nor appeared to use bacterial pathways to covert arginine to ornithine. NOS were demonstrated histochemically in nerves of adult H. contortus, but was undetectable in homogenates of both species. There was species variation in polyamine metabolism: T. circumcincta used arginase to form ornithine, followed by decarboxylation by ODC, while in H. contortus there was the additional pathway of first decarboxylation by ADC to form agmatine, then hydrolysis by agmatinase to putrescine. The present study helped in the better understanding of nitrogen metabolism and these enzymes can be useful targets if they differ antigenically from the host, provided the enzyme is accessible to blockage by immune effectors.Item Some aspects of the host-parasite relationship between goats and gastrointestinal nematodes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University(Massey University, 1994) Pomroy, William Ernest; Pomroy, William ErnestExperiments were conducted investigating the acquisition, by goats, of resistance to Haemonchus contortus and Trichostrongylus colubriformis. Neither 5.5 or 14 months old Saanen goats given a trickle infection with H. contortus, which was terminated with anthelmintic after 10 and 14 weeks respectively, showed significant resistance to a challenge infection. Serum pepsinogen levels rose significantly as a result of infection. Serum gastrin levels also rose as a result of infection but, following challenge, were generally higher for naive goats than previously infected goats. There were no significant correlations between worm counts, gastrin and pepsinogen levels at the time of slaughter. By contrast, a high level of resistance to T. colubriformis developed in 9 months old Saanen goats given trickle and two challenge infections. Goats exposed only to the two challenge infections developed worm burdens intermediate between, and significantly different from both trickle-infected and previously uninfected goats given just the second challenge. Both priming infections were removed with anthelmintic. Globule leukocyte (GL) counts in the proximal small intestine of trickle-infected goats were significantly higher than in goats given a single challenge infection, whilst counts for the group given two challenge infections were intermediate and not significantly different from other treatments. Nematode fecundity (eggs per female) and male:female ratios were also significantly decreased in the previously infected goats. Ninety four percent of all T. colubriformis were found in the proximal 50% of the small intestine in both young goats and older goats. Male:female ratios increased and eggs/female nematode decreased with distance down the small intestine. Only about 50% of adult T. colubriformis were recovered from recently killed goats by opening and massaging the small intestine under running water. Antiparasite activity of intestinal mucus from groups of Angora-cross goats killed 9, 18 or 27 days after infection with T. colubriformis, increased significantly in infected versus uninfected goats with no difference between infected groups. However, the establishment rate (57-67%) suggests little immunity was being expressed. Following infection, proximal small intestinal GL counts fell with time but there was still a clear negative relationship between GL counts and worm burdens. Mucosal mast cell and eosinophil counts showed no significant trends relative to duration of infection or worm burden. Differential cell counts were made using a monoclonal antibody to sheep mast cells developed with diaminobenzidine, combined with haematoxylin and Biebrich's scarlet. This method was shown to be superior to others tried. In Angora-cross goats killed sequentially after removal of an infection with T. colubriformis, proximal small intestinal GL counts increased with time. However, antiparasite activity of intestinal mucus was consistently low. GL counts in all these Angora-cross goats were generally higher further down the intestine beyond the location of a large proportion of the T. colubriformis burden. In Angora goats fitted with ileal cannulae and infected with T. colubriformis, antiparasite activity of ileal contents increased significantly with time after infection in all goats. However, the establishment rate (10.6-61.8%) indicated only a moderate degree of resistance was being expressed although a large proportion of established worms were inhibited L3s.Item Host-parasite interactions during abomasal parasitism and potential roles for ES products : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand(Massey University, 2009) Büring, WiebkeParasite excretory/secretory (ES) products are believed to play a role in the initiation of the host response to the abomasal parasites Haemonchus contortus and Teladorsagia circumcincta. Both parasites inhibit and cause loss of the acid-producing parietal cells. Three days after transplantation of adult T. circumcincta into parasite-naive sheep, a subpopulation of their parietal cells no longer expressed the proton pump β-subunit, but still stained for Transforming Growth Factor-α, suggesting loss of the proton pump preceded cell death. To investigate the ability of parasites to modify the function of mammalian cells in vitro, HeLa, AGS and CaCo-2 cells were exposed to ES products. ES products vacuolated all three cells, causing the development of large numbers of small vacuoles, which differed in appearance from those produced by Helicobacter pylori bacterial toxin VacA or ammonia. The vacuoles were unlike those which develop in parietal cells in the parasitised abomasum. Neither lipids nor prostaglandins appeared to play a role in vacuolation and the vacuolating factor in vitro is likely to be a protein because of its heat and acid lability. Vacuolation occurred within one hour and was partially reversible. ES products were also able to cause cytoskeletal rearrangement and detachment of HeLa cells, similar processes to those caused by bacterial pathogens, which also disrupt tight junctions in mammalian cells. H. contortus ES products also disrupted tight junctions of CaCo-2 cell monolayers, a model cell system used for these studies. The increased epithelial permeability was associated with structural rearrangements of the tight junction proteins occludin and ZO-1. This could explain protein loss and back-diffusion of pepsinogen into the blood, a marker of abomasal parasitism. Cell detachment and disruption of cell-cell adhesion in parasitised sheep may inhibit acid production by parietal cells, which cannot function when separated from adjacent cells. Increased permeability of the surface epithelium would allow parasite ES products to penetrate the mucosal barrier, causing further damage. This could also allow inhibition of parietal cells deeper in the abomasal glands and also allowing adult parasites living in the gastric lumen to modulate host immunity to enhance their survival.Item Studies on the occurrence of anthelmintic resistance in goat parasites in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Studies in Parasitology at Massey University, Palmerston North, New Zealand(Massey University, 2010) Kamaludeen, JuriahTwo studies were conducted to investigate anthelmintic resistance in goat parasites in New Zealand. In Study 1 parasites from goats on a farm with a long history of problems with anthelmintic efficacy were used to infect sheep for a controlled slaughter study. Nineteen lambs were acquired, effectively drenched and housed. Each was infected with a mixture of larvae comprising Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis and Oesophagostomum venulosum. After 28 days lambs were restrictively randomised into 3 groups based on faecal egg counts. Group 1 was left untreated (n=6), Group 2 (n=6) was given a single dose of abamectin (0.2mg/kg) + levamisole HCL (8mg/kg) + oxfendazole (4.5mg/kg) (“Matrix Oral Drench for Sheep”®, Ancare, New Zealand) and Group 3 (n=7) was treated at twice the dose rate of Group 2. Fourteen days after treatment all animals were killed for total worm counts. The mean burdens of T. circumcincta in Group 1 was 337, in Group 2 was 68 (efficacy 80%) and in Group 3 was 10 (efficacy 97%). The mean burdens of T. colubriformis in Group 1 was 375, in Group 2 was 220 (efficacy 41%) and in Group 3 was 81 (efficacy 78%). Although the worm burdens in these lambs were low, all animals were infected with each of these two species except for T. circumcincta in Group 3 where only 3 lambs were infected. Efficacy against other species was 100%. These results clearly indicate that a single dose of a combination drench was ineffective against two species and even when a double dose was used the efficacy against T. colubriformis was only 78%. In Study 2 a survey of drench efficacy was conducted on 17 goat farms using the DrenchRite® larval development assay. Evidence of concurrent resistance to benzimidazoles, levamisole and ivermectin was detected in T. colubriformis and T. circumcincta on 11/17 and 3/14 respectively. Only 5 of 14 farms had previously undertaken some form of testing for drench resistance prior to this survey. Evidence from these two studies suggests that severe anthelmintic resistance is common on goat farms in New Zealand