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Item Use of adjusted cut-off values for Neospora caninum antibody ELISA in calves after colostrum intake: on-farm evaluation as part of a neosporosis eradication programme(Taylor and Francis Group on behalf of the New Zealand Veterinary Association, 2025-06-04) van Velsen CM; Laven LJ; Laven RA; Weston JFAims: To assess the effectiveness of testing young calves using an ELISA for antibodies to Neospora caninum with adjusted cut-off values to account for the presence of maternal antibodies, as an aid in decision-making during calf-rearing, with the purpose of eradicating neosporosis from endemically infected dairy herds. Methods: Replacement heifer calves on two dairy farms with endemic neosporosis were blood sampled at approximately 1–4 weeks of age. Sera were tested with an ELISA for antibodies to N. caninum, with the thresholds increased (based on unpublished data) to account for colostrum intake. The sample/positive (S/P) cut-off value for seronegative animals was increased from the manufacturer’s recommendation of S/P < 30 to < 70; the S/P value for seropositive was increased from ≥ 40 to ≥ 100; and S/P values 70–100 were considered inconclusive. Calves with inconclusive results were retested using standard thresholds at approximately 4 months of age (after colostral antibodies had waned). Seropositive calves were removed from the replacement herd. This protocol was first implemented in 2016. From 2018 onwards, parentage testing was carried out, and the calves’ results were extrapolated to their dams. Dams of seropositive calves were bred predominantly to beef semen. The proportion of seronegative calves in each cohort from 2016 to 2023 was calculated, and the reproductive performance of seronegative replacement calves (% producing a calf at approximately 24 months of age) was analysed. Results: The proportion of seropositive replacement calves peaked in 2017 (19.5%) and by 2023 had reduced to 1.2%. The odds of a heifer being seronegative in 2023 compared to 2016 were 14.0 (95% CI = 4.12–87.56) times higher. Compared to 2014/2015 when replacement heifers’ serostatus was unknown, after 2016 (the first year when replacement heifer serostatus was established) at least 12.9% more heifers produced a calf at approximately 24 months of age; compared to 2014 the odds were at least 2.88 (95% CI = 1.75–4.88) times higher. Conclusions and clinical relevance: Combining early testing of replacement heifers with the identification and breeding management of dams of seropositive replacement heifers reduced the proportion of seropositive heifer calves in subsequent years and improved the reproductive performance of heifer cohorts. Further research is required to establish optimal ELISA cut-off values, but this strategy is likely to be a useful tool to reduce the N. caninum seroprevalence in endemically infected dairy herds.Item Some aspects of the epidemiology of neosporosis in sheep in New Zealand : 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, 2014) Syed-Hussain, Sharifah SalmahRecent reports from New Zealand indicate Neospora caninum may have a role in causing reproductive problems in sheep. However, knowledge about the epidemiology of neosporosis in sheep in New Zealand is limited. Thus, the research presented in this thesis was undertaken to further understand the mode of transmission, seroprevalence, diagnosis and treatment of N. caninum in sheep in New Zealand. The initial study investigated venereal and vertical transmission. The results suggested that although N. caninum DNA can be found in the semen of experimentally infected rams (n=16), the transmission of N. caninum to ewes (n=16) via natural mating is unlikely. In a two year study, ewes inoculated prior to mating (n=25 in Year 1; n=7 in Year 2) did not have congenitally infected lambs that year (n=0/44) but did in Year 2 (n=7/11). Ewes re-inoculated on Day 120 of gestation in Year 2 (n=9) had congenitally infected lambs (n=12/12) with more severe lesions than those not re-inoculated (n=2/11) indicating that the initial inoculation did not induce protection. Ewes inoculated for the first time on Day 120 of gestation (n=12) gave birth to lambs (n=10) that were all congenitally infected. Treatment of these congenitally infected newborn lambs (n=11) with toltrazuril (20mg/kg) on Day 1, 7, 14 and 21 was not effective as determined by serology, histopathology and qPCR. An avidity ELISA assay was able to differentiate between recently and chronically infected sheep. A longitudinal study with serology on 3 farms where N. caninum infected sheep were previously identified, found an overall seroprevalence of 0.8% (n=7/880) for N. caninum antibodies. The low seroprevalence observed across selected farms did not allow a meaningful interpretation to be made about the role of neosporosis on these farms. A consistent observation was the value of using multiple diagnostic tests to detect the presence of Neospora rather than relying exclusively on any one of them. Observation of typical lesions was generally more rewarding then the detection of Neospora DNA. Overall, further work is required to fully determine if N. caninum is causing reproductive problems in sheep in New Zealand.Item Neospora and abortion in New Zealand dairy cattle : a thesis presented in partial fulfilment of the requirements for the degree of Master [of] Philosophy in Veterinary Science at Massey University(Massey University, 1994) Patitucci, Angel NazarenoNeospora caninum is a newly recognized Toxoplasma-like protozoan organism that infects dogs; Neospora also causes spontaneous abortion and neonatal disease in cattle and other animals although it is not clear if the organism concerned is N. caninum or another species. The present study aimed to improve the epidemiological knowledge of bovine Neospora abortion in New Zealand and describe the pathologic features of Neospora sp. infection in cattle and in dogs. In a retrospective study of preserved material, N. caninum was identified for the first time in New Zealand dogs in histologic sections of the CNS of 3/15 animals with a variety of CNS lesions and nervous signs. The diagnosis was confirmed by immunohistochemistry and, in one case, electron microscopy. Two cases of toxoplasmosis were confirmed but neither N. caninum or T. gondii could be demonstrated in ten cases with granulomatous meningoencephalomyelitis. In neosporosis the histopathological lesions were distributed more widely throughout the CNS and displayed a more marked inflammatory reaction than in toxoplasmosis cases. In an attempt to transmit the disease to dogs, puppies were inoculated with aborted bovine CNS material infected with Neospora organisms but this was unsuccessful. An epidemiological study of Neospora abortion in dairy cattle in the North Island revealed that the disease was diagnosed in 15% of abortion material submitted to Batchelar Animal Health Laboratory and Ruakura Animal Health Laboratory in 1992, thus making it the most frequently diagnosed cause of abortion. Descriptive epidemiologic information including age of aborted foetuses, age of aborting cows and seasonal distribution of the disease were obtained through a questionnaire survey of dairy farmers whose herds experienced Neospora abortion that year. Information on risk factors was sought but could not be related to Neospora infection because of the small scale of the survey. Nevertheless, some useful preliminary data which could be used in future investigations were obtained. An investigation of a herd with a recent history of neosporosis detected antibodies in cattle of different age groups using an indirect fluorescent antibody (IFA) test. A "cutoff" point of 1:400 was used in sera obtained one month after an abortion "storm". In all age groups on the farm at the time of the abortion there was a prevalence of approximately 29% (56/194) seropositive. However, the weaner heifers which were off the farm at that time, had a prevalence of 3% (1/32) (p<0.01) seropositive. This finding indicated that all cattle on the farm were exposed to a source of infection at the same time and no age-susceptibility was evident. The significance of these results and directions for future research are discussed.Item Investigations into the control of neosporosis in cattle : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Clinical Science, Massey University, Palmerston North, New Zealand(Massey University, 2011) Weston, Jennifer FaithThe research presented in this thesis was undertaken to further understanding of the control of neosporosis in cattle. A prospective cohort study of primiparous heifers on a farm with a history of Neospora-associated abortion found a 0.65 risk of abortion among seropositive heifers, suggesting that identification and culling of seropositive heifer replacements may be cost-effective. A clinical trial of a registered Neospora caninum vaccine utilising 2,246 cattle from five farms with endemic N. caninum infection was assessed for efficacy in preventing abortion and vertical transmission. Overall vaccine efficacy was 0.25 (p=0.12) and vaccination increased the risk of vertical transmission. Histopathological and serological results from 148 cases of abortion from this trial were compiled to establish aetiological diagnoses. Nine of 34 cases where the fetus was examined had histopathological evidence of N. caninum infection. Histopathology revealed dual infectious aetiologies in 2 cases and serology suggested that, in another 17 cases, there had been recent exposure to a second infectious agent capable of causing abortion in conjunction with N. caninum lesions in the fetus or fetal bacteraemia. As a prelude to cattle challenge trials, a challenge study conducted on pregnant sheep revealed a strong dose-response for abortion and that indirect fluorescent antibody test results did not correlate well with infection status or pregnancy outcome. A novel challenge method of applying tachyzoites to an abraded oral mucosa was undertaken in pregnant heifers to establish whether oral lesions could facilitate the direct horizontal transmission of N. caninum between cattle. One of eight heifers seroconverted, her calf and one other were seropositive when sampled within 12 hours of birth, and three other heifercalf pairs had at least one positive polymerase chain reaction result at parturition. This method of transmission between cattle may be responsible for only a small proportion of infections but is a major new finding in the epidemiology of N. caninum infection and warrants further investigation. Finally, inoculation with mouse-passaged N. caninum tachyzoites prior to mating did not prevent abortion when heifers were challenged again on Day 70 of gestation, suggesting that live inoculation may not be a suitable control option.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.