Dissertations / Theses on the topic 'Parasitology – Research'

Studies on the biology of Schistosoma margrebowiei include, a simple means of culturing and infecting Bulinus natalensis snails; the morphology and ultrastructure of various stages in the life-cycle; pathology; cercarial longevity and infectivity; cross-reactivity with S. mansoni rabbit anti-sera and the possible use of S. margrebowiei egg homogenate in the serodiagnosis of S. haematobium patients. A simple method of maintenance and infection of B. natalensis snails en masse, was found to yield a rapid and continuous supply of material. The results indicate that the size of snails at the time of exposure is an important factor in successful infection. A wide range of morphological and ultrastructural similarities were found between S. margrebowiei larval stages and those of other species of the genus. Whereas the adult worms are among the largest, the eggs, miracidia and cercariae of S. margrebowiei, are among the smallest in the genus. The pathology associated with S. margrebowiei, is due to deposition of large numbers of eggs in various organs of the infected animal. Eggs were not only recovered from the liver and intestine but following 50 days post-infection, from the spleen. A large number (10-15%) of the total eggs recovered from mice 45 to 65 days post-infection were deposited in the spleen. The cercariae of S. margrebowiei by utilizing their glycogen reserves, can live for up to 70 hours in fresh water at temperatures of 26-28°C. This observed life-span can be prolonged when water temperatures were decreased to 8-12 °C. Cercariae kept in cold water although physically active and still infective, were found to be attenuated as measured by a reduced percentage of recovered worms compared with controls. The potential for immunizing mice with the hepatopancreas from infected and uninfected snails against schistosomiasis has been evaluated using S. mansoni. Although a reduction in the number of worms and eggs was observed in mice immunized with infected hepatopancreas when compared to the controls, this decrease was not significant. Sera from 53 patients infected with schistosomiasis were studied by ELISA using S. margrebowiei crude soluble egg antigen (SEA), S. mansoni SEA and cationic S. mansoni egg antigen (CEF6). It was found that S. margrebowiei SEA was more specific for the identification of S. haematobium infections.

Hawaii Island has the highest incidence of rat lungworm disease (RLWD) of all the Hawaiian Islands and the mainland United States. The relatively recent introduction of the semi-slug Parmarion martensi, an effective intermediate host, and the wide-spread use of rainwater catchment systems may play a role. Studies were designed to investigate the ability of drowned gastropods to shed larvae, the location in a water column where larvae would most likely be found, the potential for larval passage through a 20µm filter, and the ability of the larvae to survive outside the slug/snail host. Whole P. martensi shed many, viable A. cantonensis larvae with >90% of larvae found in samples taken from the bottom of the water column, suggesting they may settle near the bottom of a catchment tank. Larvae that were able to pass through a 20µm sieve could not survive acid, were active for at least 56 days outside the slug host, and tested positive for RLW by qPCR. Larvae that could not pass through a 20µm sieve were able to survive HCl-pepsin, were active for at least 21 days, and tested positive for RLW. First stage larvae can survive gut acid when swallowed after migration from the lungs but cannot withstand acid immersion again until they become third stage larvae.The study results merit further investigation into the potential link between poorly maintained rainwater catchment systems and the high incidence of RLWD on Hawaii Island, and the studies clearly demonstrate the need for control of hosts of Angiostrongylus cantonensis.

Hawaii’s remote location makes food security an important issue. State-wide efforts to promote the Grow Local, Eat Local movement are reflected in the growing number of residential gardens, small farms, farmers’ markets, school and youth garden projects, and the recent passage of the Farm to School Bill. However, efforts to educate farmers, food handlers, and consumers about rat lungworm disease and the need for disease prevention and host control has not been similarly supported. In collaboration with five partner schools on Hawaii Island, the University of Hawaii, Daniel K. Inouye College of Pharmacy’s Hawaii Island Rat Lungworm Working Group worked with students and teachers to develop an integrated pest management plan for school garden projects. Integrated pest management allows for the careful consideration of applications available to control a pest event and chooses those practices that are least harmful to human and environmental health. These best practices include preventative cultural practices, monitoring, mechanical control, biological control, and the responsible use of pesticides. Students were intensively educated about RLWD, the parasite’s life cycle, and prevention measures. Using best management practices, we set up traps and collected data on gastropod species abundance, and shelter-type capture rate. Integrating STEM curriculum makes the project attractive to schools as it supports student academic success. Adoption of this management project by the many school and youth garden projects in areas of RLWD can exponentially increase community awareness, encourage control efforts, and potentially map disease risk.

This thesis presents a descriptive and a functional analysis of the ecology of an island host-parasite system consisting of the Polynesian rat, Rattus exulans (Peale) and its gut helminths. The results, which include an historical perspective, are presented in the form of 7 papers or sections. Each of these examines a particular aspect of this host-parasite relationship. A review of the origin and an update of the theorised dispersal of this rat from Southeast Asia to New Zealand is given in the first section of chapter one. Previous theories have derived the New Zealand populations from a line which passed through Micronesia. In accordance with new information from the Lapita cultural assemblage, this rat is now theorised to have accompanied these "Lapita" peoples through the Bismarck Archipelago and Solomon Islands, arriving in the Tonga- Samoa region about 3600-3000 Before Present (B.P.). From here, the Proto- Polynesians then dispersed further east, taking with them the commensal R. exulans, pig, dog, and chicken. This rat is thus thought to have arrived in New Zealand, the most southern and last-settled landmass in Polynesia, in the canoes of the Maori about 1000 years ago. Information on the ecto- and endoparasites of the Polynesian rat from throughout its geographical range is collated and presented in section two. This includes the results of the two surveys (one being part of this thesis) done on the parasites of this rat in New Zealand. All populations of R. exulans sampled in these two surveys came from offshore islands, to which this rat is almost totally confined, and where, on many, it is the only rodent species present. In contrast, most of the populations sampled beyond New Zealand are now sympatric with other rodent species. For the New Zealand populations only, it was also possible to identify those parasites only accidentally associated with this host; these are listed as "transients". In section three, an attempt is made to determine the probable biogeographical origins of parasites recorded from populations of this rat on "exulans only" offshore islands of New Zealand. Such a study was possible only because of the archaeologically documented commensal relationship between rat and Polynesian man. This information, detailed in section one, together with the parasitological data base assembled in section two, provided the material for this analysis. Several "heirloom" species are identified, theorised to have been inherited by this rat during speciation somewhere in Island Southeast Asia. Parasites acquired during dispersal are divided into "old" and "new souvenirs"; the former are thought to have been acquired from sympatric rodent species in Near Oceania sometime prior to 3000 B.P., and the latter from R. rattus, R. norvegicus or Mus musculus introduced in the last 200 years during European settlement in New Zealand. The conclusions further suggest that some at least of the "new souvenir" species have been acquired by R. exulans on "exulans only" offshore islands of New Zealand by cross-transfer from other rodent species which have temporarily gained access to these islands. This theory is examined in more detail in the fourth section, and reports of such accidental colonizations of offshore islands are presented as supporting evidence. In Chapter two, the influence of habitat on the population demography of the host is investigated. Nearly 1000 rats were trapped and necropsied over a 17 month period in three different habitats on Tiritiri Matangi, an "exulans only" island at the entrance to the Auckland harbour. Rank grassland which covers most of this island formed one habitat; a second consisted of forest remnants confined to gullies, and the third consisted of the small, inhabitated, lighthouse station and farmed area at one end of the island. Between-habitat differences were observed in diet, adult longevity, mean weight of immatures, the time of onset of sexual maturation, and annual reproductive output. These results suggested several modifications to existing models of this host's demography in New Zealand. Shelter in particular appears to play an important role in the demography of this species in temperate latitudes. The effects of parasitism on potential fitness parameters e.g. reproduction, and adult mortality/survival, are examined in chapter three. Based on the results obtained in chapter two, a number of hypotheses were developed, and the predictions arising from these were tested. Few significant results were obtained; these revealed habitat and some sexual interactions with the level of infection, at certain times of the year. However, no causal relationship could be established between these effects and host reproduction or mortality. It is concluded that the helminth parasites of this rat on this island have little or no effect on these host parameters, and support the suggestion that these species constitute a depauperate and well-adapted rodent parasite fauna. The last chapter presents the results of an analysis of the effects of habitat, season, host age, and sex on the distribution and abundance of the helminths of this rat on this island. Together, the graphs and the statistical analyses demonstrate that habitat has the most important influence, significantly affecting all 7 species; this effect is of greater magnitude than the other 3 variables on 5/7 of these species. Season and age also have important effects, while sex had no apparent influence. Explanations for the observed patterns are sought in known aspects of the biology of the host in the three habitats described in chapter two, and in the life cycles of the parasites. In total, this thesis provides a comprehensive account of the ecology of the Polynesian rat and its helminth parasites on Tiritiri Matangi Island. It also identifies gaps in the existing data base, formulates certain hypotheses, and makes a number of predictions all of which will hopefully stimulate further interest in this rat and its parasites.

Site selection, and the means to access specific sites, is a keystone of parasitology. I evaluated migration and site selection behaviours of metacercariae of two congeneric species of strigeoid trematode throughout growth and encystment phases in the fathead minnow. Results showed that pre-encystment stages of Ornithodiplostomum ptychocheilus migrate along specific neural tracts to access sites in the optic lobes of the brain. Conversely, pre-encystment stages of Ornithodiplostomum sp. migrate via direct penetration, or via the vascular system to access visceral organs, especially the liver. Remarkably, both species have a bi-phasic pattern of development, with growth and encystment occurring in unique sites. Finally, I examined patterns of rodlet cell proliferation and maturation in response to growth and encystment phases of O. ptychocheilus. Cell densities were low (<11/mm2) in brain tissue adjacent to 1 and 2 week old metacercaria, but peaked to approximately 210/mm2 at 6 weeks. These results shed new light on the potential function of these enigmatic cells.
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Dendritic cells (DC) play a key role in immunity and represent a great target for modulation, because of their ability to prime T cells and direct their polarisation into effector subsets. Ticks release immunomodulatory compounds in their saliva, possibly in order to evade host immune responses during feeding. We have recently reported that Rhipicephalus appendiculatus ticks produce ‘Japanin’, a secretory lipocalin that arrests differentiation of monocytes into DC and reprogrammes maturation of DC in response to various stimuli towards a tolerogenic phenotype . Japanin was cloned and recombinantly expressed in a baculovirus system for subsequent immunological and biochemical analysis. This study was set out to further investigate the immunomodulatory activity of Japanin as well as the underlying mechanism of action. We have discovered that Japanin prevents DC-mediated proliferation and polarisation of allogeneic T cells. Experiments with labelled Japanin have demonstrated that it binds predominantly to ex vivo generated human monocyte-derived DC (moDC) and to a reduced degree to monocyte and DC populations in peripheral blood, yet to no other blood leucocytes. We have identified CD206, also known as the mannose receptor, as a Japanin-binding receptor on moDC. This identification has been achieved by crosslinking and subsequent pull-down of Japanin-receptor complexes from moDC. Affinity studies with recombinant CD206 constructs have confirmed the binding to Japanin. Moreover, the binding has been verified by specific siRNA knock-down of CD206 in moDC, which resulted in significantly decreased binding of Japanin. Unexpectedly, CD206 has appeared to be dispensable for at least most of the DC-modulatory activity of Japanin. Therefore, attempts were made to determine other factors in the mode of action of Japanin, through which we have found that IL-10 is not essentially involved. Further results have suggested that the activity of Japanin demands cell contact. Collectively, we have come to the conclusion that the mechanism of action of Japanin might require internalisation by DC, potentially enabling modulation of intracellular pathways involved in the regulation of DC maturation.

Mucins play important roles in host-pathogen interactions, influencing host resistance, establishment of infection, as pathogen recognition sites and a source of nutrients. They are highly glycosylated molecules and changes in monosaccharide composition during parasitism have been reported. Effects of parasites on monosaccharide component of fundic and duodenal mucins of sheep were investigated in 3 age ranges (i) 4-4.5, (ii) 6 and (iii) 8-9 months old: (1) noninfected; (2) infected with 10,000 Haemonchus contortus and euthanased 21 days post infection (p.i.); (3) infected with 50,000 Teladorsagia circumcincta and euthanased 28 days p.i. Three days-old lambs and 9 weeks-old lambs: (a) milk-fed, (b) solid-fed and (c) solid-fed, infected with T. circumcincta were also included. The effects of H. contortus and T. circumcincta infection in mucin changes were significantly different in the fundus, however, both of them shared some similarities. Infected sheep showed lower proportion of fucose and sialic acids in fundic and duodenal mucins compared with non-infected animals, the level of sulphation varied depending on the age of infected sheep: decrease in young sheep but increase in older animals. H. contortus infection also caused increased proportions of GlcNAc and Gal in fundic mucins and duodenal mucins respectively at all ages, however, in T. circumcincta infection, it was shown that the alterations of mucins were age-dependent. T. circumcincta infected sheep showed the significant changes at young ages (4-6 months-old) while 8-9 months-old animals showed less change in fundic mucins compared with non-infected animals. Effects of H. contortus and T. circumcincta infection differed in the fundic mucins but were similar in the duodenum. The study showed that parasitism caused the modifications of monosaccharide composition in gastrointestinal mucins of sheep. These alterations may result from parasite species differences, causing different effects from the host’s immune response. The changes in mucin profiles observed in the duodenum of sheep infected with abomasal nematodes suggested that the host may respond to parasitism. This would facilitate the use of mucins from accessible sources, without euthanasing the animals, to investigate the changes in mucin compositions which can be used to diagnose the susceptibility or resistance of sheep to parasites

Background: Neospora caninum is a parasite that causes disease, largely in cattle and dogs. It is a disease of significant interest within New Zealand due to its association with bovine abortion. The economic impact of bovine abortion justifies the development of a bovine vaccine against N. caninum. Aim: To develop and optimise diagnostic procedures for the detection of Neospora from a variety of blood and tissue samples and to isolate a New Zealand strain of Neospora caninum. Methods: A local strain of Toxoplasma gondii and an imported Neospora caninum strain, Nc-Liverpool, were used to optimise tachyzoite growing conditions in bovine endothelial (BE) cells and Vero host cell cultures. A serum study using 112 tissue culture flasks was performed to determine whether foetal bovine serum or horse serum supplemented media provided the optimal growing conditions for Nc-Liverpool tachyzoites. Nc-Liverpool tachyzoites were also used to determine the optimal growth period between passage, and harvest for cryopreservation and cryopreservation conditions. Percoll gradients were also tested using Nc-Liverpool tachyzoites. A known Neospora positive canine sample and murine tissues infected with Toxoplasma, were used during the development of the immunohistochemical diagnostic technique. Antibody concentrations and incubation temperatures were tested to reduce cross-reactivity and increase specific stain intensity. Immunohistochemistry was performed on sections of all tissue samples used for N. caninum isolation and experimentally infected murine tissue. Several PCR techniques were developed, the final PCR used being a combination of the different techniques, which produced a 250kb band. PCR-3 used the NF6/GA1 primer combination for Neospora detection and TF6/GA1 for Toxoplasma detection, additional Mg2+ and an annealing temperature of 55°C were required. Whole tissue was processed via DNA elution whereas cell culture and Percoll purified tachyzoites were used following crude lysis techniques. All bovine and canine tissues used for parasite isolation as well as all experimentally infected mouse tissues were tested for N. caninum using PCR. An immunoblot technique was developed for the detection of N. caninum antibodies in murine blood samples. Lysed Nc-Liverpool tachyzoites were used as antigen with varied results. The primary and secondary antibodies were commercially available and used at concentrations of 1:1,000 and 1:25,000 respectively. BALB/c and CF1 mice were experimentally infected with Toxoplasma gondii and Nc-Liverpool. Forty female BALB/c and 40 female CF1 mice were used in 2 studies to determine the optimal Nc-Liverpool inoculation dose and immunosuppression requirements. Mice were immunosuppressed with 2.5mg of methylprednisolone acetate (MPA) and Nc-Liverpool inoculation ranged from 1.3x106 to 5x103 tachyzoites. Upon death, the brain and blood was harvested from the mouse carcases. Attempts were made to isolate a New Zealand strain of N. caninum from bovine and canine central nervous system (CNS) tissue, and to maintain the parasites in cell culture and by small animal passage, in order to attenuate the parasite strain for use as a live large animal vaccine. Twenty one bovine tissue samples were used for N. caninum isolation attempts, 18 of which were positive for Neospora antibodies using a commercial IFAT. Isolation tissues were purified using a 30% Percoll gradient and inoculated onto 8 cell culture flasks and into 8 immunosuppressed mice (BALB/c and CF1). Results: Nc-Liverpool tachyzoites were found to be viable when grown at 37°C in antibiotic-MEM supplemented with either FBS or ES and grew optimally in FBS despite Neospora antibodies being detected using an IFAT. Passaging cultures at approx. 4 day intervals resulted in the greatest parasite growth. However, cryopreserved parasites should be harvested 2 days post inoculation (PI) for optimal viability. Viable parasites could be isolated using a 30% Percoll gradient and centrifuged at 2,700 x g (3,400 rpm) in a bucket centrifuge for 10 minutes. Tissue cysts could be detected using immunohistochemistry but some degree of cross reaction remained despite optimisation. Cysts were not found in tissues used for isolation attempts or in mouse brains following inoculation with Nc-Liverpool, however cysts were commonly found in mice experimentally infected with T. gondii tachyzoites. PCR-3 was successfully used to detect N. caninum and T. gondii infected tissue and tachyzoites from tissue culture. PCR-3 could detect N. caninum DNA in the brain tissue of 9/24 mice experimentally infected with Nc-Liverpool, even though most mice were culled within 1 week. Although production of N. caninum antigen was only moderately successful, N. caninum antibody detection in mouse blood using one specific antigen batch was reliable and specific. The immunoblot could only detect N. caninum antibody approximately 14 days PI, but was sensitive enough to detect 100% of mice experimentally infected with Nc-Liverpool tachyzoites. PCR-3 strongly correlated with the immunoblot results from 14 days PI. BALB/c mice were found to be far more sensitive to Nc-Liverpool than CF1 mice and developed severe disease at concentrations of approximately 1x106 Nc-Liverpool tachyzoites. Neither BALB/c nor CF1 mice developed peritoneal exudate, irrespective of the parasite inoculation concentration. Despite Neospora DNA being present in the brains of experimentally infected mice, re-isolation and continuous parasite passage from the brains could not be achieved. No mice experimentally infected with either Nc-Liverpool or isolation attempts were found to have brain cysts when tested using immunohistochemistry. Only 1 mouse inoculated with bovine isolation material was found to have a Neospora positive PCR. Through the detection of DNA, antigens and antibodies, parasites were determined to have been present in 10 of the 18 IFAT positive bovine isolation samples, indicating that 55% of calves born to seropositive dams were infected with N. caninum. However, despite numerous attempts to isolate Neospora parasites from naturally infected canine and bovine tissue and culturing using the optimised Nc-Liverpool technique, maintenance of a live culture of a New Zealand strain of N. caninum could not be established. Conclusions: Findings from this study could be used to assist in the maintenance of Neospora caninum and Toxoplasma gondii parasite strains and for detection or diagnosis of these parasites in host tissues.

Two indoor feeding experiments were conducted at the Animal Physiology Unit (APU) of Massey University, involving young sheep, to investigate the effect of feeding forage willow upon the death of established parasites and upon parasite fecundity, using chaffed lucerne as the control diet. Experiment 1: Twenty-four (24) parasite-free weaned hoggets weighing 29.9 ±1.2 kg (SD) were individually penned and fed chaffed lucerne ad libitum during a preexperimental adaption period of 5 weeks. They were then fed either lucerne chaff or chopped willow for a further 5 weeks (n = 12/group) and intakes were adjusted such that the DMI of the two groups was similar during weeks 9 & 10. All lambs were infected with L3 larvae parasites comprising 20,650 Teladorsagia, 1,320 Trichostrongylus and 330 Cooperia through oral drenching 12 days before willow feeding started. This was done after confirmation that the sheep were free of nematodes through FEC analysis. Total faeces were collected for 3 day periods towards the end of weeks 9 & 10, to measure diet digestibility and total faecal egg excretion. The sheep were slaughtered at the end of week 10. Voluntary feed intake (VFI), FEC and liveweight were measured weekly, whilst burdens of individual parasites and carcass characteristics were measured after slaughter. Duplicate samples of each feed offered and individual animal refusals were taken daily and pooled weekly per animal for chemical analysis. Female worm fecundity was calculated by two methods. Blood samples for immunological analysis were collected on days 20, 34, 51 and 70, and analysed for components of white blood cells (WBC) and for lymphocyte subsets. Experiment 2: A 2 x 2 changeover experiment was conducted, involving two time periods (Period 1 and Period 2 each of 14 days) with the same diets as used in Experiment 1, fed to 9 individually penned parasite-free young sheep randomly allocated to experimental diets. The parameters investigated were FEC and larvae hatching. Initially, a period of 7 days was allowed for acclimatisation in which both groups were fed on half willow and half lucerne chaff. This was followed by Period 1 with 4 lambs fed lucerne and 5 fed willow, after which the diets were changed over for Period 2. Total faeces produced were collected from all animals on the last day of each period using bagged sheep. A known number of Teladorsagia eggs (500 epg) was then added to faecal samples from these sheep and faeces-egg mixtures were made from which FEC was determined, to see if egg recovery was affected by these diets. Faecal samples for Period 2 with added eggs were also incubated for 10 days to measure hatchability. The recovery of added Teladorsagia eggs in Experiment 2 was 85% in lucerne-fed lambs and 53% willow-fed lambs (P<0.001); these were used as correction factors for Experiment 1 data. Larvae that hatched per gram of wet faeces in Experiment 2 tended to be lower for sheep fed willow than lucerne chaff (71% vs 83% of eggs added; P=0.08). Willow feed offered had lower DM (P<0.001) and CP (P<0.05) content, but had a significantly higher OM content (P<0.01) than lucerne chaff. Condensed tannin content of chopped willow was 27 g/kg DM, with only traces for lucerne. Apparent digestibility for DM (62.4% vs 59.5%; P≤0.05), OM (64.8% vs 59.9%; P≤0.001), DOMD (58.1% vs 55.0%; P≤0.01) and calculated ME (9.48 MJ/kg vs 8.96 MJ/kg; P≤0.01) were higher for the willow diet. VFI was similar for both groups during the adaption period (P>0.05) but declined with the introduction of willow in week 6 (P<0.001) and then progressively increased until it was similar to lucerne-fed sheep in weeks 9 & 10 (P>0.05). Calculated DM intake per head/day during the last two weeks of Experiment 1 was similar for the two groups (P>0.05); while the willow group had higher ME (P<0.01) and CP (P<0.001) intake per animal/day. Liveweight increased for the two groups during the adaption period (P>0.05), then declined for willow-fed lambs in week 6 (P<0.001) but later increased and by week 10 was similar to that of lucerne-fed lambs. The willow-fed lambs had lower carcass GR than the lucerne-fed lambs (P<0.01) when carcass weight was used as a covariate. Adjusted total daily egg production in Experiment 1 was lower in willow-fed sheep than lucerne-fed sheep, due to reductions for Haemonchus spp. (P<0.05) and Teladorsagia spp. (P<0.05). The per capita fecundity for Haemonchus worm spp. (P<0.05) and the in utero fecundity in both abomasal Teladorsagia spp. and small intestinal Trichostrongylus spp. (P<0.001) were lower for willow-fed sheep. There was reduced production of larvae for both Haemonchus spp. and Teladorsagia spp. (P<0.05) in willow-fed sheep. Feeding willow reduced the burden of Haemonchus adult worms in the abomasum (P<0.01) but reduced female worm burden only in Teladorsagia spp. (P<0.05) and reduced Cooperia spp. in the small intestines (P<0.01). Total WBC, total lymphocytes, subsets of lymphocytes and other white-cell groups were not affected by willow feeding (P>0.1). It was concluded that feeding chopped willow to young sheep reduced nematode worm burdens in the abomasum, especially both male and female Haemonchus spp., and reduced female worm burdens of Teladorsagia spp. Female worm fecundity of both species was also reduced by willow feeding. These reductions have been associated with CT content in the willow feed and the reduced worm burdens have been attributed to the death of the established worms by CT, since there was no evidence of immune priming in willow-fed sheep. Compounds present in the faeces of willow-fed sheep have been found to mask some of the nematode eggs, making them invisible by microscopic examination while keeping their viability. It is postulated that this could be due to binding of nematode eggs to insoluble CT associated with indigestible fibre in the faeces of willow-fed sheep. Conventional methods of measuring FEC therefore underestimated nematode eggs present in the faeces of willow-fed sheep and this needs to be checked for other CT-containing forages.

Two 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

Indiana University-Purdue University Indianapolis (IUPUI)
Histone acetylation has been linked to developmental changes in gene expression and is a validated drug target of apicomplexan parasites, but little is known about the roles of individual histone modifying enzymes and how they are recruited to target genes. The protozoan parasite Toxoplasma gondii (phylum Apicomplexa) is unusual among invertebrates in possessing two GCN5-family lysine acetyltransferases (KATs). While GCN5a is required for gene expression in response to alkaline stress, this KAT is dispensable for parasite proliferation in normal culture conditions. In contrast, GCN5b cannot be disrupted, suggesting it is essential for Toxoplasma viability. To further explore the function of GCN5b, we generated clonal parasites expressing an inducible HA-tagged form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative form of GCN5b was mediated through ligand-binding to a destabilization domain (dd) fused to the protein. Induced accumulation of the ddHAGCN5b(E703G) protein led to a rapid arrest in parasite replication. Growth arrest was accompanied by a decrease in histone H3 acetylation at specific lysine residues as well as reduced expression of GCN5b target genes in GCN5b(E703G) parasites, which were identified using chromatin immunoprecipitation coupled with microarray hybridization (ChIP-chip). We also demonstrate that GCN5b interacts with AP2-domain proteins, which are plant-like transcription factors in Apicomplexa. The interactions between GCN5b, AP2IX-7, and AP2X-8 were confirmed by reciprocal co-immunoprecipitation and revealed a “core complex” that includes the co-activator ADA2-A, TFIID subunits, LEO1 polymerase-associated factor (Paf1) subunit, and RRM proteins. The dominant-negative phenotype of ddHAGCN5b(E703G) parasites, considered with the proteomics and ChIP-chip data, indicate that GCN5b plays a central role in transcriptional and chromatin remodeling complexes. We conclude that GCN5b has a non-redundant and indispensable role in regulating gene expression required during the Toxoplasma lytic cycle.