No description available.

No description available.

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

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.

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 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

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.

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.