Massey Documents by Type
Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294
Browse
7 results
Search Results
Item The prevalence and production effects of liver fluke (Fasciola hepatica) in New Zealand cattle including evaluation of diagnostic tests : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand(Massey University, 2023) Dowling, AndrewThe liver fluke Fasciola hepatica, infects cattle worldwide and is considered a parasite of regional importance in New Zealand although the impact on milk production in that country have not been studied. The test characteristics of two antibody detection ELISAs; IDEXX ELISA (IDEXX Fasciolosis Verification) and an In-House assay using unrefined excretory secretory antigens, plus a coproantigen ELISA (Bio K 201–Monoscreen AgELISA) and faecal egg counts (FEC) were assessed against the gold standard of total fluke counts in naturally infected cattle (cows=29, steers=10). Vat milk of dairy herds on the West Coast of the South Island was assessed for liver fluke infection using the IDEXX ELISA in the autumn, near the end of one lactation (n=430), and spring, near the beginning of the subsequent lactation(n=403). A total of 156 questionnaires determining awareness of liver fluke infection and drenching practices were completed. A cross sectional study of 11 herds (n=1314 cows) in autumn and a longitudinal study of 4 herds (n=485 cows) in spring and autumn used the IDEXX ELISA (measured as SP%) on serum to analyse associations between liver fluke infection and milk production parameters in individual cows. A subset of cows was also faecal sampled for coproantigen and FEC analysis. Notably, a negative linear effect of the loge(total fluke count+1) on liveweight (p=0.02) was found and the coproantigen values showed a significant (p=0.01) quadratic effect for loge(total fluke count+1). The survey showed that infection of herds at a level likely to cause production losses on the West Coast is common, with regional clustering. Milk Fat % decreased 0.0004% points for every 1SP% increase (p=0.004), being 0.05 %points lower for cows with SP%150 than cows with SP%30, and 0.22 %points (p=0.014) lower in cows where SP% increased from ≤30 to ≥150 during lactation compared that those remained ≤30 with an economic cost of $55.19 per infected cow. Of the tests compared, the IDEXX ELISA was superior to the In-House ELISA for sensitivity (Se) and specificity (Sp) but the coproantigen ELISA had the highest Se (96%) and Sp (96%). Overall, liver fluke infection was common in dairy cows but the infection intensity was low, nevertheless a small effect on MF% was determined.Item A study of coccidial parasites in the hihi (Notiomystis cincta) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Science at Massey University(Massey University, 2001) Twentyman, Caroline MillicentA systemic protozoal disease resembling atoxoplasmosis has been found to be a serious problem in the captive hihi population at the National Wildlife Centre (N.W.C.), Mt Bruce, Masterton, causing high juvenile mortality. The literature on the Genus Atoxoplasma is reviewed, with attention focusing on the taxonomy, history, and life cycle of the organism, named and unnamed species, identification, epidemiology and clinical signs of infection. Atoxoplasma-like organisms have been recognized in birds since 1900 but difficulties in identification and in classification have meant that the genus is still inadequately defined and poorly understood. Monitoring of oocyst shedding from captive hihi at the N.W.C. during the 1997-1998 and 1998-1999 breeding seasons confirmed that the most consistent shedding was by the chicks/juveniles which had at least two periods of shedding: one in the nestling stage and one post-fledging. The earliest recorded excretion was at 9 days of age. Post-fledging, there was a period of high oocyst shedding between 6.5-8 weeks of age during both seasons. Some chicks had intermittent periods of excretion of high numbers of oocysts throughout the year although the months of December through to, and including, February were the times when high numbers of oocysts were shed by the chicks most consistently. The adult hihi at the N.W.C. passed oocysts only sporadically, with the exception of one hand-reared bird which had little exposure to conspecifics as a juvenile, and another bird that was in poor health at the time of shedding. Small numbers of coccidial oocysts were also present in faeces collected from hihi on Tiritiri Matangi and Mokoia Islands but, largely because of infrequent sampling, no shedding patterns were discernible. It is proposed that hihi normally develop immunity to this coccidial organism as they mature if they are reared naturally, but might shed oocysts if suffering from concurrent disease. Treatment with toltrazuril (Baycox solution 2.5%, Bayer) eliminated the shedding of oocysts in all birds. However, oocyst numbers sometimes rose again very quickly suggesting that toltrazuril is effective against the intestinal forms of this coccidia but not against the extra-intestinal forms. Difficulties were experienced in the in vitro sporulation of oocysts shed by birds from the N.W.C. although those recovered from the two islands sporulated relatively easily. The reasons for this were not established but it is suggested that the sporulation difficulties may have been due to management factors at the captive institution, such as the use of some medications. Preliminary morphological characteristics of sporulated oocysts of the Isospora-type are described. Two main types of coccidia were identified: Group A which comprised coccidia which had subspherical oocysts, and Group B which had ellipsoidal oocysts. Both types of coccidia were found in birds from all three locations. These preliminary epidemiological studies suggest that infection is maintained in chicks and juveniles with oocysts remaining viable in the environment for extended periods of time. Further work on oocyst shedding by adults during the breeding and oocysts viability in the environment is required in order to confirm this hypothesis. Transmission studies using starlings as recipient birds for both starling and hihi oocysts were not completed because of the unavailability of appropriate infective material at the required time. Another study using a single hihi as the recipient of sporulated hihi oocysts was also not completed because of the death of the hihi due to a fungal infection. A transmission study where sporulated hihi oocysts were inoculated into zebra finches, was completed and there was no evidence of infection, supporting the belief that these coccidia are species-specific. The gross and histological findings on necropsy of 12 cases of coccidial infection in hihi from the N.W.C. are described in detail including the locations of the various coccidial forms within the body. These findings are compared with cases of Atoxoplasma and Atoxoplasma-like infections in birds recorded in the literature. The most outstanding feature of the infection in hihi is the intestinal pathology which involves extreme thickening of the lamina propria with an overwhelming invasion by coccidial forms into the lamina propria and the intestinal epithelial cells. No atoxoplasmosis cases in other avian species exhibit similar intestinal pathology. Although there are some common aspects in the hepatic and splenic pathology, and in the tissue location of the different coccidial life cycle stages, there is currently insufficient consistent similarity to justify placing the hihi coccidia in the Genus Atoxoplasma. The taxonomic classification of this coccidia therefore remains uncertain.Item The development and evaluation of a village-based parasite control program for swamp buffalo and cattle in northeast Thailand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy at Massey University(Massey University, 1988) Meemark, NopadonInternal parasitism is a major problem in large ruminants in Thailand, especially nematodes in newborn calves and liver fluke in adults. Veterinary services are sparse, and can offer only very limited assistance at the village level. There are about 20,000 villages in the north-east of Thailand, where this study was conducted. To combat these major logistic problems a Basic Animal Health Service (BAHS) is being developed progressively within the region. The first component of the service to be developed was a "farmer self-help worm control program", commenced at a pilot level in 1983. Village farmers are selected on aptitude for the task, trained as BAHS "keymen" for one day, and then provide extension advice to farmers in up to 10 villages about disease control, with the initial emphasis being on internal parasites. This local effort is supported by wider promotional campaigns. Keymen are taught to dispense drugs for each type bi parasite, and receive part of the price paid by farmers for the drugs. purchase and distribution of drugs is supported out of a special revolving fund. Experience in the program since 1983 has shown that overall adoption of the program has been high, but that drug sales have varied greatly between keyman areas. A comparison was therefore made of "high adoption" and "low adoption" keyman areas, to determine levels of knowledge about parasites and the BAHS, and to assess which of a range of factors might be most closely associated with program success at the local level. Adoption rate was judged by sales of anthelmintics by each keyman. Results in four provinces which had participated in the program for either one or three years were compared with two provinces which had not yet begun the program. In total 420 farmers and 16 keymen were interviewed using a standardised questionnaire form. Farmers were classified into those showing high acceptance (understood the BAHS and had used the drugs within the last year), medium acceptance (understood the BAHS, but had not used the drugs for at least a year), and low acceptance (unfamiliar with the BAHS and its relevance to them, and had not used the drugs), Overall, 64% of farmers in the "high adoption" areas showed high acceptance of the program, compared with only 16% in the low adoption areas - producing a mean of 40% across the whole sample. Users of the control system were very satisfied that treatment provided economic benefits, and this view was supported by empiribal evidence from the study, which showed that owners who carried out treatment had lower calf mortality, higher market value of treated animals, and improved calving rates. The single most important determinant in the success of the program is the energy of the keyman in promoting the program and the sale of drugs, and acceptance of the program is almost entirely a function of this factor, rather than issues beyond the keyman's control. A number of quite simple and cheap modifications to details of the BAHS should further increase the already exceptionally high adoption rate. These include replacing ineffective keymen, increasing the density of keyman so that travel is not a limitation, and strengthening further the regional promotion effort to give maximum credibility to the keyman's local work. An economic analysis based on the data showed a return of US$143 to the typical farmer in the region for an investment of US$0.69, making very conservative assumptions about the nature and scale of the benefits. In contrast, the keymen make only a very small income from their efforts, estimated at US$0.70 per day worked on the program. The net benefit of the program across the six provinces studied was estimated at US$33.64 million. This can be increased by various improvements to the program, and costs and returns for such improvements were calculated. If 80% of farmers in the six provinces treated all of their animals, the net benefit to the region would be US$118 million for an investment of about $1 million, the costs being shared equally by Government and the farmers. Small scale farmers share more favourably in the benefiti than is the case for many improvements in village agricultural practices. The program has been very successful, primarily because it deals with a problem which farmers recognize as serious, and because everything the farmers need to carry out the program is available within the village. Various simple improvements identified in the study will further improve its acceptance and its benefit to the country.Item Epidemiology of coccidiosis in calves and control of coccidiosis using toltazuril at the time of weaning : this thesis is presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Veterinary Parasitology at Massey University, Palmerston North, New Zealand(Massey University, 2005) Gaddam, Mary JonesTwo separate studies were conducted to investigate the impact of coccidiosis in young calves. In one study calves were reared to weaning (100kg liveweight) by feeding meal with or without monensin added. The oocyst counts were low in both groups up to weaning and there was no statistically significant (p<0.05) improvement in terms of body weight or a decline in oocyst counts in the monensin-treated group At weaning a single dose of toltrazuril (20mg/kg) was given to half the calves in both groups. A similar treatment regime was given in a second study where calves had been raised to weaning by commercial calf rearers. Half of these were treated with toltrazuril (20mg/kg) and half not. In both studies there was a statistically significant (p<0.001) reduction in oocyst counts in treated calves which remained very low for 4-5 weeks post treatment. The treatment also significantly increased (p<0.001) weight gains in treated calves by 3-5kgs at 5-6 weeks post treatment. The coccidial status of other calves on a variety of farms were also monitored including a group of organic beef farms. High oocyst counts were noted on occasions where calves were not on anti-coccidial treatment. Low oocyst counts were noted in adult cows where they were examined. The two most prevalent species overall were Eimeria zuernii (95%) and E. bovis (87%) followed by E. auburnensis (62%), E. cylindrica (42%), E. canadensis (31%), E. wyomingensis (23%), E bukidnonensis (36%), E. ellipsoidalis (24%) E. alabamensis (12%), E. brasiliensis (12%), and E. subspherica (27%). The most predominant species, measured as the most numerous oocysts overall, were E. bovis (31%) followed by E. zuernii (27%), E. auburnensis (13%), E. bukidnonensis (7%), E. cylindrica (6%), E. wyomingensis (5.3%), E. canadensis (4.4%), E. ellipsoidalis (3.3%), E. brasiliensis (1.9%), E. subspherica (1.5%), and E. alabamensis (1%). The most prevalent species were also the most pathogenic species. On many occasions calves were infected with more than one species, sometimes as many as 5-6 Eimeria species. A redescription of the 11 species of Eimeria in cattle identified from New Zealand Farms was made.Item Sarcocystis gigantea : studies on sporocyst production, excystation and viability : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science(Massey University, 1984) McKenna, Philip BernardRecent advances in knowledge about the sporozoan genus Sacocystis (Protozoa: Apicomplexa) are reviewed and studies on the production, excystation and viability of sporocysts of Sarcocystis gigantea, undertaken. Investigation of a sedimentation/floatation procedure for the mass recovery of S. gigantea sporocysts from cat faeces showed that the greatest yields were obtained when a proportion of faeces to floatation medium of 5% and a centrifugal force of 6000 x g for at least 5 min were used. Ninety-six percent of the sporocysts recovered were obtained from the first centrifugation in aqueous NaCl solution, specific gravity 1.2. Although neither sieving nor additional washing of homogenised samples prior to floatation significantly affected sporocyst recovery both reduced the amount of debris present. A considerable reduction in the amount of debris resulted from feeding infected cats on tinned fish rather than tinned meat. The addition of CCl4 to the NaCl solution also improved sporocyst purity but with a marked reduction in the numbers recovered. A technique for determining the concentration of sporocysts in faeces, using a modification of the mass recovery procedure and a haemocytometer, was developed. This was shown to be more accurate and reliable than the McMaster method for performing faecal sporocyst counts. It resulted in a mean sporocyst recovery of 75.5% and was used to obtain information about patterns of sporocyst excretion and numbers of S. gigantea sporocysts shed by 28 experimentally infected cats. In all cats, sporocyst excretion commenced 10 or 11 days post-infection(PI). Peak production occurred between 13 and 22 days PI, in most instances on days 17 and 18. Peak numbers (rounded) ranged from 550 to 260,000 (mean = 53,000) sporocysts per gram of faeces or from 38,000 to 6.6. million (mean = 1.7 million) sporocysts per day. The number of days sporocysts were shed ranged from 26 to at least 60 days PI but in 26 of the 28 infections examined, more than 80% of the total sporocyst yield was produced within 30 days of infection. The total numbers of sporocysts produced by individual cats over the patent period ranged from 164,000 to 56.6 million (mean = 12.7 million). These numbers tended to increase with increasing infective dose and to be greater in those cats receiving multiple rather than equivalent single doses. Neither the sex of the cat, nor experience of one or two previous infections, had any significant effect on the numbers of sporocysts shed. Studies on the in vitro excystation of S.gigantea sporocysts revealed that pretreatment before exposure to trypsin and bile was an essential pre-requisite. However, in contrast to S. tenella and S. capracanis, incubation in cysteine hydrochloride under CO2 was largely unsuccessful for excysting S. gigantea: of the pretreatments tested only exposure to sodium hypochlorite proved effective. Excystation from sodium hypochlorite-pretreated S. gigantea sporocysts took place in trypsin and bile between temperatures of 30° and 43°C and occurred rapidly at 39°C. While the presence of bile or bile salts was essential for this process, that of trypsin was not although more sporocysts excysted in its presence than in its absence. Excystation occurred in the presence of all bile types tested but not when 'Tween 80' was substituted for bile. The highest levels of excystation were recorded when cattle or sheep bile or sodium taurocholate were used and the lowest when chicken or pig bile were employed. Neither the concentration of sheep bile above 2.5%, nor hydrogen ion concentration (pH range 5.0 to 10.0) appeared to have any marked effect on the level of excystation obtained. The excystation process for S. gigantea was similar to that described for other Sarcocystis species and for other coccidian genera that lack sporocyst Stieda bodies. Sporozoites escaped following the collapse of the sporocyst wall and its eventual separation into four elongated pieces. In vivo studies on excystation of S. gigantea indicated that this process was, as in vitro, diphasic involving pretreatment and treatment phases. They also tended to support in vitro observations that the requirements for the excystation of S. gigantea and S. tenella sporocysts were quite different. Although the results suggested that for neither species was the pretreatment stimulus likely to be provided by conditions in the rumen alone, exposure to abomasal conditions only, induced moderate levels of excystation in both when they were subsequently treated with trypsin and bile. For S. gigantea, 0.25 to 4 hr abomasal exposure was most effective,for S. tenella 24 hours. The stimuli necessary to complete the excystation process could, apparently, be provided by 1 hr placement in the duodenum for S. gigantea but not for S. tenella. Using in vitro excystation as a measure of viability, it was found that at 4°C, S. gigantea sporocysts survived considerably better in tap water (85% excystation after 174 days) than in either 2.5% potassium dichromate (15% excystation after 174 days) or 2% sulphuric acid (0% excystation after 5 days). Although they were able to resist 48 hr suspension at room temperature in most laboratory reagents, disinfectants and anti-coccidial drugs tested, six (sulphuric acid, ammonia, methanol, ethanol, potassium hydroxide, sodium hydroxide, 16% synergistic mixture of five chlorinated phenols.) had major sporocysticidal properties. Further investigation with three of these, showed that sporocyst excystation was reduced from 65% to less than 10% following contact with 2.5% sulphuric acid for 1 hr or with 2% ammonia or 4% Medol for 4 hours. Sporocysts were either killed, or their ability to excyst severely impaired, by heating to 60° and 55°C for 5 and 60 min, respectively, by exposure to ultraviolet radiation at a dose of 4000 ET, or by prolonged storage in water at 24°C. Sporocysts exposed to either constant or intermittent freezing at -18°C suffered a comparatively slow decline in excystation rate with time as did those subjected to desiccation. The duration of survival of desiccated sporocysts was inversely related to relative humidity and after 245 days at 33% RH and temperatures of 15 or 24°C, 60% of such sporocysts excysted. Studies on the survival of S. gigantea sporocysts in faeces outdoors showed that, viability declined most rapidly over the summer months and suggested that they were unlikely to remain infective for more than one year. Possible associations between the reported findings and both the epidemiology of S. gigantea infection and some of the previous unsuccessful or equivocal attempts to experimentally infect sheep with this species, are discussed.Item Abomasal secretion in parasitised sheep : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Physiology at Massey University(Massey University, 1995) Lawton, David Eric BenjaminThe effect of Ostertagia circumcincta on the secretory function of the ovine abomasum was studied in vivo and in vitro. In vivo, sheep were infected with larval or adult parasites and the changes in serum pepsinogen, serum gastrin and abomasal pH monitored. In vitro, the effect of worm extracts and incubates on the secretion of gastrin, somatostatin and pepsinogen were investigated using segments or dispersed cells of ovine abomasal mucosa. Using these, and a further perifusion technique, the pharmacology of gastrin and somatostatin secretion in the sheep was also investigated. The in vivo study revealed that adult worms transferred directly into the abomasum of parasite-naive sheep initiate immediate changes in serum pepsinogen, gastrin and abomasal pH, showing that larval stages are not essential for the pathophysiological changes. These changes also occurred following infection with larvae but not until about five days post-infection. The increase in abomasal pH and serum gastrin occurred at a similar time, regardless of the dose of larvae or the route of administration. Serum pepsinogen levels increased before gastrin and pH. The normal range for serum pepsinogen, serum gastrin and abomasal pH in the parasite-free sheep were defined (0-500 U tyrosine/litre, 12-64 pM and 2.34-3.26 respectively). When abomasal pH rose and was maintained above pH 5.5 in sheep infected with larvae, serum gastrin levels rapidly returned to normal. When pH subsequently declined below 5.5, gastrin rapidly returned to elevated levels. By three weeks after infection of parasite-naive sheep with larvae, pH had returned to the normal range despite the continued elevation of serum gastrin. Infection with adults and larvae significantly increased the wet weight of the abomasum and this occurred within 8 days of infection with adult worms. Tissue gastrin levels were decreased by infection. In vitro, solutions prepared with larvae and adult O. circumcincta had no effect on, or inhibited, gastrin release. These same solutions had no effect on, or stimulated, somatostatin secretion. Inhibition of gastrin secretion was always accompanied by increased somatostatin secretion although the converse was not true. Worm-derived solutions that inhibited gastrin release were possibly contaminated by microorganisms. Incubation of medium contaminated by an inoculum of abomasal content but without worms produced solutions that potently stimulated somatostatin and inhibited gastrin release. The pharmacological study revealed that mechanisms that have been identified in the regulation of gastrin secretion in other animals are present in the sheep. GRP, nicotine and carbachol but not adrenaline stimulated gastrin secretion from segments of antral mucosa in a concentration-dependent manner. Carbachol did not consistently inhibit somatostatin secretion and in most experiments somatostatin and carbachol release were both stimulated. Atropine inhibited basal gastrin release from segments of mucosa indicating a degree of tonic cholinergic discharge. Atropine partially or completely prevented the gastrin response to carbachol. VIP and GIP both stimulated somatostatin secretion but had no effect on gastrin, suggesting that somatostatin either does not restrain gastrin in the sheep or that this is maximal at basal levels. Somatostatin antiserum was not associated with increased gastrin secretion in most experiments.Item Neospora caninum : studies toward isolation in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Studies at Massey University, Palmerston North, New Zealand(Massey University, 2008) Walton, Julie K.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.