Serological investigation of aborted sheep and pigs for infection by Neospora caninum

Serology for Neospora caninum was undertaken using direct ELISAs on sera from 660 aborted sheep and 454 breeding sows, which had aborted or were considered infertile. All ovine sera were further tested by indirect fluorescent antibody test (IFAT) for N. caninum, and a latex agglutination test (LAT) for Toxoplasma gondii was performed on 423 of the samples, including all those positive by ELISA. ELISA-positive porcine sera were tested by IFAT and an inhibition ELISA for antibodies to N. caninum and by LAT for T. gondii. Only 3 (0.45%) of the ovine sera were seropositive for N. caninum by both ELISA and IFAT whereas although 40 porcine sera were seropositive by ELISA all were negative by IFAT. The results suggest that environmental exposure to N. caninum occurs rarely in sheep and pigs.     

Potential involvement of Neospora caninum in naturally occurring ovine abortions in New Zealand

Neospora caninum is an obligate intracellular parasite and is recognised as the leading cause of bovine abortion worldwide. Natural infection with N. caninum has been described in sheep but it has generally not been regarded as a significant cause of abortion. Recently, there have been several New Zealand cases of foetal abortions where N. caninum was detected which strongly suggested the involvement of Neospora in these abortions. However, there is minimal information about the prevalence of N. caninum infection naturally occurring in New Zealand sheep flocks and particularly its impact on reproduction success. Thus, this present study provides preliminary data on the role that Neospora is playing in ovine reproductive failure by establishing the prevalence of N. caninum antibodies and DNA in ewe blood and foetal material present in 21 New Zealand sheep farms with ongoing unexplained abortion problems and 10 farms with consistently high fertility levels. The results of this study demonstrated an overall seroprevalence of 1.4% which varied between Aborting/non-pregnant (1.8%), age-matched pregnant controls (0.6%) and high fertility (2.1%) ewes. However, despite the variation observed, there was no statistical difference between the three groups. In addition, Neospora DNA was detected by PCR in 13% of submitted foetal brains and in ewe blood from aborting/non-pregnant (6.9%), age-matched pregnant controls (3.6%) and high fertility pregnant (2.1%) ewes. When the PCR results were considered with the IFAT and IDEXX ELISA results, there was no correlation between serology positive and PCR positive blood samples. Taken together, these results reveal that reliance on ELISA-based serology or PCR alone may underestimate the involvement of Neospora. Furthermore, determining the involvement of Neospora appears to require a multi-facetted approach where diagnostic methods and serological cut-off values may need to be adjusted as further information about the effect of natural infections with N. caninum in the ovine host is elucidated.     

Evaluation of serological tests for the diagnosis of Neospora caninum infection in dogs: optimization of cut off titers and inhibition studies of cross-reactivity with Toxoplasma gondii

Diagnosis of Neospora caninum infection in dogs is based on serological assays such as the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assays (ELISA). This study evaluated two serological tests (IFAT and ELISA) for the detection of IgG antibodies to N. caninum in 300 serum samples of dogs through the optimization of cut off titers by using the two-graph receiver-operating characteristic (TG-ROC) curve. In addition, the identification of major cross-reactive antigens with Toxoplasma gondii was investigated by inhibition ELISA and immunoblotting (IB) assays. IFAT and ELISA results showed 74% agreement, with a good negative concordance (P(neg)=0.83), but a poor positive concordance (P(pos)=0.42). The great majority (86%) of sera with positive concordant results (IFAT+/ELISA+) recognized at least two out of three N. caninum immunodominant antigens, particularly the 29-32 and 35-37 kDa bands. Optimization of cut off titers in IFAT and ELISA was performed considering the reactivity to at least two out of three N. caninum immunodominant antigens as infection markers, obtaining a titer of 50 for IFAT and 200 for ELISA. Seropositivity to N. caninum was significantly associated with T. gondii-seropositive samples, particularly in ELISA (55.4%). Inhibition ELISA curves for N. caninum showed a partial heterologous inhibition, indicating some degree of cross-reactivity between N. caninum and T. gondii antigens. Inhibition IB assays showed a moderate heterologous inhibition for N. caninum antigens above 45-50 kDa. These results indicate that ELISA should be used critically when crude tachyzoite antigen preparations are employed, due to possible cross-reactivity with other related parasites as T. gondii. Also, the cut off dilution of 1:50 in IFAT showed to be the most appropriated for N. caninum serology in dogs. Therefore, we suggest that N. caninum immunodominant antigens, specially the 17 and 29-32 kDa proteins, should be selected markers in serological assays for canine neosporosis.     

Serological investigation of an outbreak of Neospora caninum-associated abortion in a dairy herd in southeastern United States

An outbreak of Neospora caninum-associated abortion occurred in a South Carolina dairy wherein greater than 10% of the herd aborted over a 4-month period. Of the total number of cows at mid-late gestation, nearly 40% (28/71) aborted while the remaining 60% (43/71) gave birth to normal calves. Immunohistochemical examination of brain tissue from a subset of aborted fetuses confirmed N. caninum as the causative agent of abortion in these animals. A variety of serological assays, including indirect fluorescence antibody test (IFAT), recombinant enzyme-linked immunosorbent assay (rELISA), ISCOM-ELISA, avidity ELISA, and Neospora agglutination test (NAT), were used to evaluate sera collected during the outbreak from 240 cows for antibodies to N. caninum. IFAT and ISCOM-ELISA testing showed that nearly 80% of the dairy cows had antibodies to N. caninum. NAT and rELISA had similar levels of seropositivity relative to IFAT and ISCOM-ELISA, but the percentage of positive sera was dependent on the cut-off value chosen. As indicated by kappa coefficient statistical analysis, ISCOM-ELISA and IFAT exhibited the highest level of agreement in identifying N. caninum-positive and -negative cows. A decrease in the percentage of seropositive cows as determined by ISCOM-ELISA and IFAT with increasing cow age was noted. However, no significant difference was observed between cow age and abortion status. In addition to these tests, an avidity ELISA was performed on all sera with high (> or =0.4) ISCOM-ELISA readings. Avidity index (AI) increased with time post-abortion suggesting that most abortions were due to recent N. caninum infection. Of the cows at risk for abortion, the mean serological AI of aborting cows was significantly lower (P<0.05) than mean serological AI of non-aborting cows.     

Evaluation of four serological techniques to determine the seroprevalence of Neospora caninum in foxes (Vulpes vulpes) and coyotes (Canis latrans) on Prince Edward Island, Canada

The objectives of this study were (1) to evaluate the performance and agreement of serological assays (ELISA, IFAT, Neospora caninum agglutination test and immunoblot) using reference sera and field sera from foxes and coyotes and (2) to estimate the N. caninum seroprevalence in foxes and coyotes on Prince Edward Island, Canada. With fox and coyote reference sera the test performance of the ELISA, IFAT and IB was excellent (100% sensitivity and specificity). NAT showed a low sensitivity (50%). Serum was collected from 201 coyotes and 271 foxes. The seroprevalence observed in the different assays ranged from 0.5 to 14.0% in coyotes and 1.1 to 34.8% in foxes. The seroprevalence, when taking more than one test positive as cut-off value was 3.3 and 1.1% for coyotes and foxes, respectively. From the N. caninum-positive group, all coyotes were older than 3 years. Agreement among assays (measured as prevalence-adjusted bias-adjusted kappa) using the field sera ranged from 0.17 to 0.97. Best agreement was observed between ELISA and IFAT, poor agreement was observed between NAT and the other assays. Positive agreement was moderate to poor among all assays utilized in this study. Although the seroprevalence observed was low, N. caninum antibodies are present in foxes and coyotes on Prince Edward Island (PEI) and their role in the N. caninum epidemiology needs further study.     

Seroprevalence of Neospora caninum in aborting dairy cattle in the Czech Republic

A serological survey for antibodies against Neospora caninum in aborting cattle was carried out in the Czech Republic. Serum samples from 463 aborting dairy cows originated from 137 farms from different parts of the Czech Republic were tested for presence of N. caninum antibodies by use of an enzyme-linked immunosorbent assay (ELISA) and an indirect fluorescent antibody test (IFAT). Antibodies (> or = 1:640) to N. caninum were found in 18 (3.9%) of 463 aborting cows. Farm prevalence in aborting cows was 12.4% (17/137). The antibody titres of cows were 1:200 (9 cows), 1:640 (7 cows), 1:1280 (3 cows), 1:2560 (3 cows), 1:5120 (3 cows), 1:10,240 (2 cows) and 1:20,480 (0 cow). A case-control study was conducted to estimate the association of N. caninum infection and abortion. For this 407 serum samples were collected from cows on five dairy farms with repeated occurrence of endemic and sporadic abortion of unidentified etiology. These samples were obtained from aborting cattle (n=44) and normally calving cattle (control group; n=363) and tested for N. caninum antibodies by an immunofluorescent antibody test (IFAT). Overall, 3.19% (13/407) of cows sampled had positive N. caninum fluorescence with a cut-off titre of 1:200. The prevalence of N. caninum was significantly higher (P<0.05) in the aborting group (13.64%; 95% confidence interval (CI): 5.2, 27.4) than in the control group (1.93%; 95% CI: 0.8, 3.9). A strong association between seropositivity and abortion was found, with seropositive cows being eight times more likely to abort than seronegative cows (odds ratio=8; 95% CI: 2.6, 25.1). This first report on the serological prevalence of N. caninum in cows in the Czech Republic verified a strong association between N. caninum infection and abortions in five dairy farms. Thus, the neosporosis should be considered in differential diagnosis of bovine abortion.     

Prevalence of antibodies to Neospora caninum in dogs of rural or urban origin in central New Zealand

AIM: To investigate the seroprevalence of Neospora caninum infection in populations of dogs from dairy farms, sheep/beef farms and urban areas in the central part of New Zealand. It was postulated seroprevalence would be higher for farm dogs than urban dogs if the life-cycle of this parasite involves transmission between dogs and cattle. METHODS: Serum samples were obtained from dogs that lived on dairy farms (n=161), sheep/beef farms (n=154) and in urban situations (n=150). The relative risk of detecting antibodies to N. caninum using an immunofluorescent antibody test (IFAT) was compared between farm and urban dogs. RESULTS: The relative risk of having a titre of > or = 1:200 to N. caninum was 2.43 (95% CI=1.88-3.14) for dairy-farm dogs and 3.16 (95% CI=2.48-4.02) for sheep/beef-farm dogs, compared with urban dogs. At this titre, which is currently used in New Zealand to indicate seropositivity, seroprevalence of N. caninum infection was 30.7% in urban dogs, 74.5% in dairy-farm dogs and 96.8% in sheep/beef-farm dogs. CONCLUSION: This observation is consistent with a cycling of this disease between cattle and dogs on farms in New Zealand and with higher exposure of dogs to N. caninum on farms than occurs in urban environments. The prevalence of antibodies in all three groups of dogs tested in this study (dairy-farm dogs, sheep/beef-farm dogs and urban dogs) is higher than has generally been reported elsewhere. New Zealand farm dogs have a higher serological prevalence of N. caninum infection than urban dogs. CLINICAL RELEVANCE: Management and disease control practices that break the life-cycle of transmission between cattle and dogs should assist in controlling cattle abortion due to N. caninum.     

Evaluation of Toxoplasma gondii and Neospora caninum infections in sheep from Uberlândia, Minas Gerais State, Brazil, by different serological methods

Toxoplasmosis and neosporosis have been recognized as economically important diseases with considerable impact on the livestock industry. Considering the scarce information on the occurrence of Toxoplasma gondii and Neospora caninum infections in sheep from Uberlandia, Minas Gerais State, Brazil, this study aimed to investigate the frequency of antibodies against these parasites in sheep sera from this region by using different serological methods. A total of 155 sheep serum samples were analyzed by the indirect fluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) for the detection of IgG against T. gondii and N. caninum. Seroreactivity by IFAT showed 80% of samples with titers between 512 and 2048 for T. gondii (cutoff ≥ 64) and 78% presenting titers between 50 and 200 for N. caninum (cutoff ≥ 50). Seroreactivity by ELISA showed 75% of samples with ELISA index (EI) between 2.0 and 3.0 for T. gondii (cutoff ≥ 1.3) and 54% presenting EI between 1.3 and 2.0 for N. caninum (cut off ≥ 1.3). Discordant results by both tests were analyzed by immunoblot, resulting in a total seropositivity of 61% for T. gondii and 23% for N. caninum, with 41% to T. gondii only, 3% to N. caninum only, and 20% to both parasites. There was a significant positive association between seropositivity to T. gondii and age over one year (P<0.001), but such association was not found for N. caninum infection. In conclusion, as T. gondii and N. caninum infections are simultaneously present in sheep flocks of this region, it should be emphasized the importance to carry out a regular monitoring of Toxoplasma infection due to its high prevalence, its zoonotic potential and induction of reproductive disorders leading to economic losses. For neosporosis, sheep farmers should be instructed about the presence of the parasite in the flock, its risk factors and potential abortifacient role in sheep. Differential flock management could be valuable tool to establish the association of serological positivity and reproductive disease induced by N. caninum in sheep.     

Elaboration of a crude antigen ELISA for serodiagnosis of caprine neosporosis: validation of the test by detection of Neospora caninum-specific antibodies in goats from Sri Lanka

The protozoan parasite N. caninum is a major pathogen in cattle and dogs. However, clinical symptoms are occasionally described for other potential hosts. Natural abortion in goats due to N. caninum has been rarely reported and only little data is available on the seroprevalence of N. caninum in this species. In the present study, 486 goats from Sri Lanka were tested in a crude antigen ELISA for the presence of serum antibodies against N. caninum. Additionally, the sera were analysed by N. caninum-Western blot and indirect fluorescence antibody test (IFAT). In all three tests applied, only three sera (0.7%) were scored clearly positive for anti-N. caninum antibodies. The optimal correlation between ELISA, IFAT, and Western blot confirms the suitability of the ELISA for large-scale seroepidemiologic studies, not only in cattle but also in goats.     

Serodiagnosis of Neospora caninum infection in cattle by enzyme-linked immunosorbent assay with recombinant truncated NcSAG1

Neospora caninum is a veterinary medically important pathogen capable of causing abortion in cattle and neuromuscular paralysis in dogs. The surface antigen 1 of N. caninum (NcSAG1) is an important candidate for the development of a diagnostic reagent for neosporosis. In order to establish an effective diagnostic method, the gene encoding truncated NcSAG1 (NcSAG1t) lacking a signal peptide and C-terminal hydrophobic regions was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The purified GST-NcSAG1t was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of N. caninum antibodies in cattle. The ELISA with GST-NcSAG1t clearly differentiated between immunofluorescent antibody test (IFAT)-positive and -negative sera from cattle. In addition, the ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Toxoplasma gondii. Field serum samples collected from cattle in Brazil were examined for the diagnosis of neosporosis by using the ELISA. Of the 197 samples analyzed, 66 (33.5%) samples were positive for antibodies to N. caninum. Of the 66 ELISA-positive samples, 60 (90%) samples were confirmed as positive by Western blot analysis with whole parasite antigens. These results suggest that the recombinant NcSAG1t could be a reliable reagent for use as an antigen in ELISA for the serodiagnosis of N. caninum infection in cattle.     

Comparison of serological methods for the diagnosis of Neospora caninum infection in cattle

The aims of this study were to evaluate the performance and agreement of various commercial and in-house Neospora caninum antibody assays used in dairy cattle in North America, and to investigate reproducibility of two assays performed in different laboratories. From 1998 to 2005, three enzyme linked immunosorbent assays (ELISAs, a competitive ELISA-VMRD Inc., an indirect ELISA-Biovet Inc., and another indirect ELISA-Herdchek IDEXX Corp.), two indirect fluorescent antibody tests (IFATs, VMRD Inc., and in-house USDA) and one N. caninum agglutination test (NAT, in-house USDA) were utilized to test 397 randomly selected dairy cattle serum samples from 34 herds in eastern Canada for antibodies to N. caninum. The manufacturers' recommended cut-off values were used to evaluate test performance and agreement between tests. One IFAT (VMRD Inc.) performed well (sensitivity and specificity: 0.97 and 0.97, respectively) using reference sera (n = 452), therefore, results from this IFAT on the 397 samples could subsequently be used as the reference standard to calculate test characteristics for the other assays. Only 11% of the 397 sera were found to be N. caninum-positive with the IFAT. Prevalence-adjusted bias-adjusted kappa (PABAK) ranged from 0.06 to 0.99. Positive agreement was moderate to very good (P(pos) = 0.25-0.96). Negative agreement was very good for all assays (P(neg) > 0.94) except NAT (P(neg) = 0.66). Sensitivity was > or =0.89 for all assays except the NAT, which had a significantly lower sensitivity (0.66). Specificity was high (>0.94) for all assays except for one indirect ELISA (specificity = 0.52). This indirect ELISA did not perform satisfactorily when used in 1998, but an improved version of the ELISA performed as one of the best assays in 2004. Reproducibility of the competitive ELISA was excellent, but the reproducibility of the indirect ELISA that was improved was low (concordance correlation coefficient = 0.90 and 0.36, respectively). The performance characteristics observed for most assays in this study make them useful for screening antibodies to N. caninum in cattle.     

Seroprevalence of Neospora caninum in non-carnivorous wildlife from Spain

Serum samples from 1034 non-carnivorous wildlife from Spain were tested for antibodies to Neospora caninum by competitive screening enzyme linked immunosorbent assay (ELISA) and confirmed by an indirect fluorescent antibody test (IFAT). High agreement was observed between results in both techniques (kappa value higher than 0.9). Prevalences of N. caninum antibodies positive by both techniques were 11.8% of 237 red deer (Cervus elaphus), 7.7% of 13 barbary sheep (Ammotragus lervia), 6.1% of 33 roe deer (Capreolus capreolus) and 0.3% of 298 wild boar (Sus scrofa). In one of 53 hares (Lepus granatensis), antibodies were found in the ELISA but could not be confirmed by IFAT due to lack of sample. Antibodies to N. caninum were not found in any of 251 wild rabbits (Oryctolagus cuniculus), 79 fallow deer (Dama dama), 27 mouflon (Ovis ammon), 40 chamois (Rupicapra pyrenaica) and three Spanish ibex (Capra pyrenaica). Statistically significant differences were observed between N. caninum seroprevalence in red deer and management of hunting estates (open versus fenced) with higher prevalence in fenced estates, and among sampling sites. Seroprevalence was particularly high in some areas (MO estate in South-Central Spain or some estates of Catalonia, North-East Spain), while no contact with N. caninum was observed in others. Results indicate that in certain areas of Spain, N. caninum is present in wildlife, especially in red deer. These results have important implications in both sylvatic cycles and may influence the prevalence of infection in cattle farms in those areas. To our knowledge, this is the first report of antibodies to N. caninum in wildlife from Spain and the first report of N. caninum antibodies in barbary sheep and wild boar.     

Postnatal neosporosis in dairy cattle in northeast Thailand

A total of 83 dairy cows in Loei Province (Muang) and Nong Bua Lamphu (NBL) Province, northeast Thailand were sampled three times within 6 months in 1998 and their sera were examined for antibodies to Neospora caninum at a dilution of 1:100 in the indirect fluorescent antibody test (IFAT). In Muang the seroprevalence of N. caninum was 37.5% (June), 60% (August), and 62.5% (November). In NBL the prevalence was 50% (August) and 70% (November). In both areas abortions were observed between 1 and 3 months after the introduction of these cattle from another area. Nine of 14 and seven of 17 calves were descendants of seropositive dams, of which only two calves from Muang and two calves from NBL were positive for N. caninum antibodies. These findings suggest postnatal N. caninum transmission.     

Seroprevalence of Neospora caninum infection following an abortion outbreak in a dairy cattle herd

OBJECTIVE: To investigate the seroprevalence of Neospora caninum infection in a commercial dairy cattle herd, 15 months after detection of an abortion outbreak. PROCEDURE: Sera from the whole herd (n = 266) were examined for N caninum antibodies by indirect fluorescent antibody test (IFAT) and immunoblot analysis. Herd records were reviewed to collate serological results with abortion history, proximity to calving, and pedigree data. RESULTS: The seroprevalence of N caninum infection was 24% (63/266) for IFAT titre > or = 160, 29% (78/266) for immunoblot positive (+ve), and 31% (82/266) for IFAT > or = 160 and/or immunoblot +ve; 94% (59/63) of animals with IFAT > or = 160 were immunoblot +ve. The association between seropositivity (IFAT > or = 160 and/or immunoblot +ve) and history of abortion was highly significant (P < 0.001); the seroprevalence was 86% (18/21) in aborting cows, compared with 30% (50/164) in non-aborting animals. The abortion rate for seropositive cows was 26% (18/68) compared with 3% (3/117) for seronegative animals. IFAT titres of infected cows were higher within 2 months of calving than at other times (P < 0.001). The association between seropositivity in dams and daughters was highly significant (P = 0.009). CONCLUSIONS: The abortions were associated with N caninum infection and there was evidence of reactivation of latent infection close to calving and congenital transmission of infection. Immunodominant antigens identified by immunoblots may prove useful for improved diagnostic tests.     

Evaluation of a commercial ELISA for detecting serum antibody to Neospora caninum in cattle

A commercially available serum antibody detection enzyme-linked immunosorbent assay for Neospora caninum in cattle was evaluated against an immunofluorescent antibody test (IFAT) by applying it to 397 sera from normal adult cattle, 352 sera from cattle which had recently aborted, and 422 sera from two herds which had a history of N caninum-associated abortions. It was evaluated in two laboratories and showed high reproducibility, repeatability and almost perfect or substantial agreement with the IFAT.     

Prevalence of Toxoplasma gondii and Neospora caninum antibodies in wild boars in the Czech Republic

Sera collected from hunter-killed wild boars (Sus scrofa) during 1999-2005 from seven different regions of the Czech Republic were assayed for antibodies to Toxoplasma gondii by indirect fluorescence antibody test and to Neospora caninum by competitive-inhibition enzyme-linked immunosorbent assay and by indirect fluorescence antibody test. Antibodies to T. gondii were detected in 148 (26.2%) of 565 wild boars with serum dilutions of 1:40 in 40, 1:80 in 40, 1:160 in 27, 1:320 in 19, 1:640 in 18 and 1:1280 in 4 wild boars. Antibodies to N. caninum were detected in 102 (18.1%) of 565 wild boars with 30.1-94.6% inhibition in ELISA; statistical significant differences were observed between sampling regions, ranging from 0% to 31.8%. Sera, positive in ELISA, were examined in IFAT; 58 of 102 (56.9%) were positive with titres 1:40-1:160. Mixed infection (concurrent presence of both T. gondii and N. caninum antibodies) was found in 38 wild boars. It is the first report of antibodies to N. caninum in wild boar. Serological results indicate a common exposure to T. gondii and to N. caninum among wild boars in the Czech Republic.     

Seroprevalence of Toxoplasma gondii and Neospora caninum infection of cats in Hungary

Blood samples were collected from 330 cats in Hungary in order to evaluate their seroconversion to Toxoplasma gondii and Neospora caninum using the indirect fluorescent antibody test (IFAT). The overall prevalence of toxoplasmosis was 47.6%, the prevalence being 22.4% among urban, 50% among suburban and 61.3% among rural animals. Significantly more cats had high IFAT titres (1:640 to 1:5120) in the countryside. Female cats were more frequently infected with T. gondii than males (53.3% vs. 39.3%), and seropositivity increased with the age of animals. The prevalence (0.6%) and titre (1:40) of antibodies to N. caninum was low. Sixty-two cats were also screened for seroconversion to feline infectious peritonitis (FIP) virus. Higher titres to T. gondii were more frequently detected among FIP-positive cats, but this difference was non-significant due to the small number of cats with concurrent infection.     

Associations between pregnancy outcome and serological response to Neospora caninum among a group of dairy heifers

AIM: To monitor pregnancy in a group of rising 2-year-old dairy heifers on a farm on which abortion due to Neospora caninum was known to occur in previous years. METHODS: A prospective cohort study group of 164 rising 2-year-old heifers was pregnancy-tested and blood-sampled at 4-5-week intervals throughout gestation. Sera were tested for antibodies to N. caninum at 3-4-month intervals, using an enzyme-linked immunosorbent assay (ELISA). When loss of pregnancy was detected, an N. caninum indirect fluorescent antibody test (IFAT) was conducted retrospectively on stored sera collected the month before abortion, the month abortion was detected, and for the following 2 months, from heifers that aborted. All fetal and placental material detected following abortion was subjected to gross post-mortem and histopathological examination. RESULTS: Eleven of 18 (61%) heifers that were seropositive and 4/146 (3%) heifers that were seronegative to N. caninum by ELISA, aborted. The relative risk for abortion among ELISA-positive heifers was 23.6. Abortion occurred predominantly between Days 120 and 152 gestation among the ELISA-positive heifers and throughout gestation among the ELISA-negative heifers. IFAT titres rose around the time of abortion in most of the heifers that were previously seropositive by ELISA, but dropped rapidly again in post-abortion samples. IFAT titres among 4/6 ELISA-positive heifers that did not abort increased, but later in gestation than the time other heifers aborted. IFAT titres remained negative in heifers that aborted that were ELISA negative. CONCLUSIONS: Heifers that were seropositive to N. caninum by ELISA had a much greater risk of abortion than seronegative heifers. Most seropositive heifers showed evidence of a reactivation of infection during pregnancy. High (> or =1:2,000) N. caninum IFAT titres also occurred in non-aborting heifers. CLINICAL RELEVANCE: Culling of replacement heifers seropositive to N. caninum may be a cost-effective strategy for minimising risk of abortion. Pregnancy testing heifers before 5 months gestation may overestimate the number that calve in N. caninum-infected herds, but would assist in documenting the occurrence of abortion. Reliance on a high (>1:2,000) IFAT titre to rule-in N. caninum as a cause of abortion is likely to produce false-positive results.     

新孢子虫dNcSRS2重组蛋白间接ELISA的建立及其应用

利用新孢子虫体外重组表面蛋白dNcSRS2蛋白作为包被抗原，对各项条件进行优化，确定判定标准，建立了检测新孢子虫血清抗体的间接ELISA方法。经对多例血清检测表明，所建立的诊断试剂盒重复性好、特异性强、灵敏度高，与进口的IFAT及两种商品化ELISA试剂盒的检测结果相比较，符合率均达到92％以上。应用建立的ELISA方法对236份奶牛血清的新孢子虫抗体进行检测，阳性率为22％。这是国内首次利用重组蛋白建立的诊断试剂盒，该方法的建立将为牛新孢子虫病的诊断与流行病学调查提供有效的技术手段。     

Validation of a Neospora caninum iscom ELISA without a gold standard

Neospora caninum is an intracellular parasite which causes abortion in cattle worldwide. One problem in the validation of the different methods for demonstration of this parasite is the lack of an appropriate gold standard. To validate an immunostimulating complex (iscom) enzyme-linked immunoassay (ELISA) used to detect antibodies to N. caninum, sera from 244 cattle in five Swedish dairy herds infected with N. caninum were analysed. The sera also were analysed by a standard indirect-fluorescent antibody test (IFAT). The results obtained by the two tests were compared using the Gibbs sampler. Gibbs sampling is a latent-class approach based on Bayesian statistics; neither test is assumed to be more correct in stating the true status of infection. The Gibbs sampler was run using both informative and non-informative prior probabilities. We also simulated different cut-offs in the iscom ELISA (providing data to inform selection of optimal cut-off values for different applications).The ELISA produced fewest incorrect test results over all at a cut-off value of 0.200. The sensitivity and specificity at this cut-off were 99 and 96%, respectively. The IFAT had a high specificity (99%) but a lower sensitivity (78%) than expected-confirming that the IFAT cannot be treated as a true gold standard. Sensitivity and specificity of the ELISA were presented in a two-graph receiver operating characteristic (TG-ROC) plot. Any cut-off between 0.150 and 0.300 will have both sensitivity and specificity > or =95%. Optical densities of < or =0.150 and > or =0.550 (or > or =0.350) were suggested as limits to rule out and rule in infection, respectively.