Adaptation of a surface antigen-based ELISA for the detection of antibodies against Neospora caninum in bovine milk

The aim of the present study was to determine whether an ELISA based on a 38-kDa surface antigen (NcSRS2) of Neospora caninum tachyzoites could be used to examine bovine milk for antibodies against N. caninum. A total of 797 undiluted milk samples from N. caninum-infected herds were examined in the p38-ELISA. Milk results of individual animals were compared with those obtained by the same ELISA for the corresponding serum samples. The linear correlation between milk and serum antibody results of individual animals was characterized by an adjusted R(2) of 0.644. The examination of the two-graph receiver-operating characteristics revealed an optimal cut-off of 0.150 to obtain similar results in the examination of milk and serum. With this cut-off, the test had a specificity and a sensitivity relative to the serum results of 93%. Using this cut-off, excellent agreement was observed between milk and serum results (Kappa 0.85; 95% CI, 0.781-0.920). A cut-off of 0.450 resulted in a specificity of 99% relative to the serum results. At this cut-off, the sensitivity of the test was 80% relative to the serum-ELISA and agreement was slightly lower (Kappa = 0.82; 95%CI, 0.749-0.887). An optimized linear regression model suggests that, in addition to the result in the p38-serum-ELISA, the variables abortion risk (ABRISK) (abortion between 100 and 269 days pregnant) and the age of the animal (AGE) affected the result of the p38-milk-ELISA. The optimized linear regression model had an adjusted R(2) of 0.8501.     

Prevalence of Neospora caninum infection in Australian (NSW) dairy cattle estimated by a newly validated ELISA for milk

AIM: To determine the performance characteristics of an Institut Pourquier (IP) enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Neospora caninum in bovine milk and subsequent determination of the prevalence of N. caninum infection in New South Wales (NSW) dairy cattle. METHODS: Matching serum and milk samples from 93 cattle were assayed in two commercially available ELISAs for the detection of anti-N. caninum antibodies. Serum test results of one ELISA (IDEXX) were used to determine the N. caninum infection status of the cattle. Optimised cut-off values for the IP ELISA using milk samples were determined by two-graph receiver operating characteristic (TG-ROC) analysis and then applied to a representative sample of 398 milk samples from dairy herds around NSW. RESULTS: When this ELISA was applied to a representative collection of 398 milk samples from dairy cattle across NSW it demonstrated a 21.1% prevalence of N. caninum infection in those cattle. From the TG-ROC analysis an IP ELISA protocol was derived which suggested a cut-off threshold that would allow milk testing with 97% sensitivity and specificity, respectively, relative to serum testing. CONCLUSIONS: The prevalence of N. caninum in NSW dairy cattle was higher than previously believed. When used on individual milk samples this ELISA demonstrated high sensitivity and specificity and so could be used to accurately identify N. caninum infection. TG-ROC analysis of the IP ELISA optimised the protocol and prescribed cut-off values enabling the ELISA to be used for the screening of N. caninum antibodies in the milk of dairy cattle.     

Adaptation of a Surface Antigen‐based ELISA for the Detection of Antibodies Against Neospora caninum in Bovine Milk

The aim of the present study was to determine whether an ELISA based on a 38‐kDa surface antigen (NcSRS2) of Neospora caninum tachyzoites could be used to examine bovine milk for antibodies against N. caninum. A total of 797 undiluted milk samples from N. caninum‐infected herds were examined in the p38‐ELISA. Milk results of individual animals were compared with those obtained by the same ELISA for the corresponding serum samples. The linear correlation between milk and serum antibody results of individual animals was characterized by an adjusted R² of 0.644. The examination of the two‐graph receiver‐operating characteristics revealed an optimal cut‐off of 0.150 to obtain similar results in the examination of milk and serum. With this cut‐off, the test had a specificity and a sensitivity relative to the serum results of 93%. Using this cut‐off, excellent agreement was observed between milk and serum results (Kappa 0.85; 95% CI, 0.781–0.920). A cut‐off of 0.450 resulted in a specificity of 99% relative to the serum results. At this cut‐off, the sensitivity of the test was 80% relative to the serum‐ELISA and agreement was slightly lower (Kappa = 0.82; 95%CI, 0.749–0.887). An optimized linear regression model suggests that, in addition to the result in the p38‐serum‐ELISA, the variables abortion risk (ABRISK) (abortion between 100 and 269 days pregnant) and the age of the animal (AGE) affected the result of the p38‐milk‐ELISA. The optimized linear regression model had an adjusted R² of 0.8501.     

Anti-Neospora caninum antibodies in milk in relation to production losses in dairy cattle

A comprehensive field study was carried out with the following objectives: (a) to assess the usefulness of individual and bulk tank milk analysis for determining Neospora caninum serostatus in individual cows and herds, and (b) to study the associations between N. caninum infection status (based on milk testing), and several productive and reproductive parameters in the animals. Antibodies were detected with a commercially available ELISA test (Bio K 192/5). Analysis of paired serum and milk samples from 1134 lactating cows on 38 farms revealed that 97.6% of the ELISA results were coincident, irrespective of whether serum or milk samples were used. Moreover, multiple linear regression analysis revealed that 86.0% of the variations in ELISA values in milk were due to variations in the serum. The measurement of antibodies in bulk tank milk was a good estimator of the herd level status of N. caninum infection, and enabled detection of infection in 94.7% herds with ≥10.0% seropositive cows and/or in all herds with >4% highly seropositive cows. The odds ratio for abortion in seropositive animals was 9.1 times higher than in seronegative animals. The infection serostatus was also a significant risk factor, as the odds ratio for abortion was even higher (12.0 times) in cows categorized as highly seropositive. ELISA values for the bulk milk from 387 randomly selected herds were negatively associated with average milk production. Moreover, milk production losses mainly occurred on farms categorized as highly positive (i.e. herds with ≥20.0% seropositive cows).     

Validation of a commercial ELISA for the detection of bluetongue virus (BTV)-specific antibodies in individual milk samples of Dutch dairy cows

A recently developed indirect ELISA for the detection of bluetongue virus (BTV)-specific antibodies in bovine milk samples was compared to that of the routinely used competitive ELISA on serum samples. During the bluetongue outbreak in the Netherlands in 2006, caused by BTV serotype 8, coupled serum and milk samples were obtained from 470 individual cows from 10 BTV-infected farms with an average seroprevalence of 57%. In addition, bulk milk samples of the same farms, and historically BT-negative samples were tested. Compared to the ELISA for sera, the relative specificity and sensitivity of the ELISA for milk samples is 96.5% and 98.9%, respectively when using a S/P% cut-off value of 50% as advised by the manufacturer. The optimal cut-off value was found at S/P% of 90% revealing an optimal specificity (99.0%) combined with an optimal sensitivity (98.1%). Titres in positive individual milk samples ranged from 1 to 2048 with a peak titre of 128. Bulk milk samples contained antibodies with titres ranging from 64 to 512. The ELISA for milk samples was found to be a reliable and robust test. This diagnostic tool is very useful, and may replace the ELISA for serum samples as first choice in order to get insight into the status of lactating individual animals and therewith of the entire herd with respect to BTV infection.     

Evaluation of three enzyme-linked immunosorbent assays for detection of antibodies to Neospora caninum in bulk milk

Three ELISAs for the detection of antibodies against Neospora caninum in bulk milk were evaluated in 162 Dutch dairy herds. The first ELISA was the Dutch Animal Health Service (AHS) in-house ELISA, developed from the routine in-house serum ELISA. The other two ELISAs were commercial milk ELISAs from IDEXX and LSI. Blood samples of all lactating cows in 162 dairy herds were tested using the AHS in-house serum ELISA. Based on previous studies in the Netherlands a within-herd N. caninum seroprevalence of 15% was associated with increased risk for reproductive losses. This percentage was therefore used as positive seroprevalence cut-off value. Repeatability of the ELISAs was evaluated by testing on three different days. The AHS in-house ELISA lacked specificity, probably due to use of a different batch of antigen on the second and third test-day. Cut-off values were determined using misclassification costs term calculations. At cut-off values 0.6 for the IDEXX and 0.2 for the LSI, a herd sensitivity of 61% (95% CI: 49--73%) and 47% (95% CI: 35--60%) was estimated. Herd specificity at these cut-off values was 92% (95% CI: 87--98%) for the IDEXX and 94% (95% CI: 90--99%) for the LSI ELISA. The positive and negative predictive values were 84% (95% CI: 68--100%) and 86% (95% CI: 79--94%) for the IDEXX ELISA, and 85% (95% CI: 67--100%) and 82% (95% CI: 74--90%) for the LSI ELISA. The agreement between all possible combinations of test-days was expressed by kappa values. These were found to be slightly higher for the IDEXX than for the LSI ELISA. It is concluded that both commercial ELISAs performed satisfactorily to detect a within-herd seroprevalence of N. caninum in lactating cows of at least 15%.     

Use of an enzyme-linked immunosorbent assay in bulk milk to estimate the prevalence of Neospora caninum on dairy farms in Prince Edward Island, Canada

This study evaluated the use of bulk milk as a diagnostic tool for estimation of herd-level Neospora caninum exposure in Atlantic Canada; it was used to estimate the prevalence of dairy farms with a within-herd N. caninum-seroprevalence > or = 15% in Prince Edward Island (PEI). The variation over time of N. caninum antibodies in bulk milk is also reported. Skimmed bulk milk and individual serum samples were analyzed for N. caninum antibodies by using an enzyme-linked immunosorbent assay (ELISA). Bulk milk samples were collected in May 2004 (n = 235), May 2005 (n = 189), and June 2005 (n = 235). The prevalence of dairy farms with a within-herd seroprevalence > or = 15% on PEI was 6.4% in May 2004. In May and June 2005, respectively, 10.1% and 10.2% of farms had a > or = 15% within-herd seroprevalence. In 11 farms that were considered positive based on bulk milk samples, blood samples were collected from all adult cows in September 2005, in conjunction with a 4th bulk milk sample on the same day. The correlation coefficient between serology and bulk milk ELISA was 0.87. The results of this study demonstrate that the prevalence of N. caninum in dairy farms can be estimated by using a bulk milk ELISA.     

Diagnostic performance of the Pourquier ELISA for detection of antibodies against Mycobacterium avium subspecies paratuberculosis in individual milk and bulk milk samples of dairy herds

The objective of the study was to determine the diagnostic performance of the Pourquier ELISA for detection of antibodies against Mycobacterium avium subsp. paratuberculosis (Map) in individual milk samples and in bulk milk samples. For individual milk samples the specificity of the Pourquier ELISA was estimated by testing a panel of individual milk samples from certified Map-free herds. The relative sensitivity of the assay in individual milk samples and agreement of the results with those of serum samples was estimated by testing panels of paired serum-milk samples from seropositive cattle, whole-herd investigations, and moderate or heavy shedders. The specificity of the ELISA for individual milk samples was still 99.8% at a cut-off of 20% sample to positive (S/P) value, clearly lower than the cut-off defined by the manufacturer (30% S/P). The relative sensitivity for individual milk samples as compared with positive serum samples was 87% for a cut-off of 20% S/P, and 80% for a cut-off of 30% S/P. The sensitivity of this ELISA for detection of high shedders was >90% both for individual milk and serum samples, also agreement was very good (kappa=0.91 for all paired samples). The specificity of the Pourquier ELISA in bulk milk samples was investigated by testing bulk milk samples from certified Map-free herds. Feasibility of bulk milk testing was investigated by titrating ELISA positive individual milk samples in negative milk. In addition, 383 bulk milk samples from herds with a known within-herd seroprevalence were tested. The specificity of the ELISA for bulk milk samples was 100% at a cut-off of 12.5% S/P. At the cut-off recommended by the manufacturer (30% S/P) performance of the bulk milk ELISA related to herd status (> or =2 seropositive cows) was rather poor, corresponding with a sensitivity of 24% and a specificity of 99% relative to serology. However, at the revised cut-off for bulk milk of 12.5% S/P and a within-herd seroprevalence of > or =3%, sensitivity and specificity relative to serology were 85% and 96%, respectively. Given the current herd-level seroprevalence in The Netherlands, these test characteristics corresponded with positive and negative predictive values for bulk milk of 67% and 94%, respectively. In conclusion, the diagnostic performance of the Pourquier ELISA for individual milk samples creates opportunities for a cheaper and more feasible testing scheme, while the diagnostic performance for bulk milk samples warrants further consideration.     

Variations of Neospora caninum antibody levels in milk during lactation in dairy cows

A longitudinal study was performed to investigate the variations of Neospora caninum antibody levels in individual milk during lactation as well as the association between antibody levels in serum and milk. Serum and milk samples of 15 milking cows were collected between February 2003 and September 2004 in three smallholder dairy farms in Khon Kaen province in northeast Thailand. All samples were analyzed for presence of antibodies by an N. caninum iscom ELISA test kit and the results were given as percent positivity (PP). The effects of time between calving and sampling, lactation number, and season on milk and serum PP were studied using Generalized Estimation Equations methods. All cows were antibody positive in either milk or serum at the first two consecutive samplings. Although serum and milk PP varied considerably, milk PP was consistently positive throughout the study. Cows of all lactation groups had a higher adjusted mean of milk PP at calving compared to later months after calving although the only significant difference was in first lactation. Serum and milk PP were always lower in first lactation than in second and later lactations. An adjusted mean of milk PP for cows classified as having serum PP> or =55 was significantly (P<0.05) higher than that of cows classified as having lower serum PP. Our results indicate that individual milk can be an alternative material to demonstrate presence of N. caninum antibodies in lactating cows.     

奶牛新孢子虫病血清流行病学调查

本研究用酶联免疫吸附试验（ELISA）对采集自北京地区的94份奶牛血清（随机采集）和河北地区的55份奶牛血清（有流产史奶牛），进行Neospora caninum血清抗体检测。结果发现，北京地区随机采集的奶牛血清N．caninum抗体阳性率为18．1％（17／94），河北地区有流产史的奶牛N．caninum血清抗体阳性率为23．6％（13／55）。采用牛奶记录体系（DHI）对北京地区17头N．caninum血清抗体阳性牛进行了日产奶量、乳中蛋白率和乳脂率的测定，并与同群牛中134头阴性牛比较。结果表明，N．caninum血清抗体阳性牛日产奶量比阴性牛降低9．7％，乳中蛋白率和乳脂率分别降低20％和15．4％。初步证明N．caninum血清抗体阳性奶牛产奶量降低及奶品质的下降。对不同N．caninum抗体滴度阳性牛的泌乳期主要生产性能比较发现，其生产性能的变化与抗体滴度无明显相关性。     

Performance characteristics and optimisation of cut-off values of two enzyme-linked immunosorbent assays for the detection of antibodies to Neospora caninum in the serum of cattle

AIM: To determine the performance characteristics of two enzyme-linked immunosorbent assays (ELISAs) manufactured by Institut Pourquier (IP) for the detection of antibodies against Neospora caninum in bovine sera. METHODS: Sera from 526 cattle were assayed in two ELISAs (IP) for the detection of anti-N. caninum antibodies. Results from a further ELISA (IDEXX) were used to provide the "gold standard"N. caninum infection status of the cattle and the ELISA results assessed by two-graph receiver operating characteristic (TG-ROC) analysis. RESULTS: TG-ROC analysis suggested changes to one of the IP ELISA protocols, arriving at a cut-off threshold that was different to the one recommended by the manufacturer. With that change, both of the ELISAs performed with high sensitivity and specificity (in excess of 98%) for bovine sera. CONCLUSIONS: The analysis of the two IP ELISAs when used on individual bovine sera demonstrated high sensitivity and specificity. TG-ROC analyses optimised the cut-off point suggested by the manufacturer for one of these commercial diagnostic assays and found agreement with the manufacturer's cut-off regarding the other assay. This will help with the accurate identification of infected animals and thereby contributing to the control of neosporosis.     

Trichinella spiralis infection induces β-actin co-localized with thymosin β4

A serological survey for Neospora caninum and Besnoitia besnoiti was carried out in beef and dairy cattle in South Australia. Serum samples of dairy cattle (n=133) from 9 properties and tank milk samples from a further 122 dairy herds were tested. An additional 810 sera from beef cattle from 51 properties were also tested. Testing at the individual animal level by IDEXX NEOSPORA X2 Ab test ELISA revealed a low prevalence of N. caninum antibodies of only 2.7% (95% CI; 1.6-3.7%) sera positive, as did the milk testing that showed 2.5% (95% CI; 1.4-3.6%) of tank milks being positive. At the herd level, 29.4% (95% CI; 16.9-41.9%) of beef, and 44.4% (95% CI; 12.0-76.9%) of dairy cattle herds showed serum antibodies. The highest within-herd prevalence in beef was 20% and 25%in dairy, which explains the low herd prevalence in dairy detected by bulk milk testing. Testing for B. besnoiti antibodies by PrioCHECK(?) Besnoitia Ab 2.0 ELISA initially identified 18.4% (95% CI: 15.8-21.0%) of 869 individual cattle sera as positive by ELISA at the manufacturer's suggested cut-off threshold (15 PP). Additional tests by immunoblot and IFAT, however, could not confirm any of the ELISA results. The use of a higher (40 PP) threshold in the ELISA is suggested to improve specificity. There is thus no evidence of B. besnoiti infection in South Australian cattle.     

Seroprevalence of Toxoplasma gondii and Neospora caninum in captive maned wolves (Chrysocyon brachyurus) from southeastern and midwestern regions of Brazil

The main purpose of the present study was to investigate the occurrence of antibodies against T. gondii and N. caninum in captive maned wolves from Brazil, considering that little information is available at the literature about infections by these parasites in this wild animal. Serum samples were obtained from 59 maned wolves originated from six zoos and from one ecological reserve of the southeastern and midwestern regions of Brazil. To detect IgG antibodies against T. gondii, an ELISA protocol was used and the results were expressed as ELISA reactivity indexes (EI). Serology for N. caninum was carried out by indirect fluorescent antibody test (IFAT) and cut-off titers were established at 1:25 dilution. From the total of the analyzed samples, 44 (74.6%) were seropositive for T. gondii and only 5 (8.5%) for N. caninum. Seropositivity for T. gondii ranged from 0 to 100% in the seven different origin locals, with rates over 50% among the six zoos, whereas no positivity was found in the samples from ecological reserve. For N. caninum, seroprevalence varied from 0 to 50% in the different locals, with the highest rates also detected in zoos. Seroprevalence for T. gondii was strongly related with age, with rates significantly higher among adult wolves (91.7%) when compared to newborn or young animals. Seropositive samples for N. caninum were found predominantly in adult wolves. For both parasites, seroprevalence did not show a significant distinction in relation to gender. Although seroprevalence for T. gondii was significantly higher when compared to N. caninum in the Brazilian captive maned wolves tested, these findings reflect the great exposure of this species to T. gondii and, in lower extension, to N. caninum. Also, the present study demonstrated for the first time the presence of antibodies to N. caninum in wild life from South America.     

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.     

Use of bulk milk for detection of Neospora caninum infection in dairy herds in Thailand

The relationship between the level of Neospora caninum antibodies in bulk milk and the seroprevalence in lactating cows was investigated. Bulk milk was also used to estimate the prevalence of N. caninum infection in dairy herds in the northeast and north Thailand. Bulk milk and individual serum from all lactating cows in 11 herds as well as 220 bulk milk samples from nine milk collection centres were analysed for presence of N. caninum antibodies using an iscom ELISA. In the 11 herds the bulk milk absorbances ranged between 0.04 and 0.89 and the seroprevalences varied between 0 and 46%. Five herds had milk absorbances below 0.20, among those were the two herds housing only seronegative lactating cows. In the remaining three herds with such low bulk milk absorbances one or two cows (5-14%) were seropositive. Six of the investigated herds had bulk milk absorbances above 0.20. In the two herds with the highest bulk milk absorbances more than 30% of the cows were seropositive. Using an absorbance of 0.20 to discriminate between negative and positive herds, 102 (46%) of 220 bulk milk samples were judged positive. There was no significant difference in mean bulk milk absorbance between the milk collection centres within each region. However, the proportion of herds with bulk milk absorbances > or =0.50 in the north was statistically (P < 0.01) higher than that in the northeast. It was concluded that bulk milk antibody testing can be used to identify N. caninum-infected herds and that N. caninum is a common infection in dairy herds in Thailand.     

Associations between Neospora caninum specific antibodies in serum and milk in two dairy herds in Scotland

The study evaluated the use of the Mastazyme ELISA for quantification of Neospora caninum (N. caninum) specific IgG in bovine milk and examined the relationship between serum and milk antibodies in two dairy herds. The serum and milk antibodies both had bimodal distributions in each herd. This was mainly due to between cow variation: in both herds, approximately two thirds of cows were either clearly and consistently seropositive or seronegative for N. caninum with one third consistently near the threshold. Milk and serum N. caninum IgG were strongly related. This relationship was modelled using a linear mixed model including a polynomial term for serum, the effect of herd, and between and within cow variance components. The latter gave a significantly better fit to the data than a model that allowed for a different relationship for the positive and negative (according to the serum test) groups of observations. The sensitivity and specificity (based on serum percentage positivity (pp)) of the milk antibody was determined for different milk pp thresholds. In spite of the differences between the relationship of milk to serum seen for the two herds, for those estimates with sufficient precision, sensitivity and specificity greater than 0.73 for both herds were obtained using single thresholds of 14 and 15.5 for milk pp in both herds based on, as our gold standard, serum antibody pp thresholds of 22.5 and 25, respectively. If milk antibody is to be used for detecting persistently infected cows, the higher threshold of 15.5 may be suitable while for epidemiological screening 14 would be preferable. Further validation in a greater number of herds is required, but our results suggest that this test may prove to be a useful adjunct to serum N. caninum IgG assays in the monitoring of N. caninum infection as part of herd health programmes and epidemiological studies.     

Considerations concerning diagnostic certainties and cut-off values for a bulk milk ELISA for Mycobacterium avium ssp. paratuberculosis

Serological tests for the examination of individual samples from single animals are evaluated based on their ability to detect true positives above a defined threshold value. If results are obtained not from an individual but from a bulk sample this concept usually is adopted such that the threshold is set to allow the detection of a single positive sample within the pool. In conjunction with the development of a diagnostic paratuberculosis ELISA for the examination of bulk milk samples it is discussed which interpolations of this concept are justified when defining the true status of a herd based on the test parameters and the seroprevalence within the herd. Here, bulk milk from up to 50 animals each and the corresponding individual samples of 4241 dairy cows from 28 herds in the state of Brandenburg are investigated, and results are subjected to different evaluation approaches. Based on epidemiological considerations and test parameters a "critical prevalence" is defined which then serves as basis for the deduction of a cut-off value to be used for bulk milk samples. Finally, the practical relevance of this approach is demonstrated by suggesting an initial scheme for paratuberculosis classification of dairy herds with respect to possible control measures.     

Seroprevalences of Neospora caninum and Toxoplasma gondii in nondomestic felids from southern Africa.

Sera from 68 nondomestic captive and free-ranging felids from southern Africa were tested for antibodies to Neospora caninum and Toxoplasma gondii by the indirect fluorescent antibody test. Four of the 68 (5.9%) serum samples were positive for antibodies to N. caninum, with titers ranging from 1:50 to 1:200. All other animals were negative for antibodies to N. caninum at a dilution of 1:50. Fifty of the 68 (74%) serum samples tested positive for antibodies to T. gondii, with titers ranging from 1:50 to 1:26,500. Four animals tested positive for antibodies to both N. caninum and T. gondii. None of these animals displayed clinical signs of disease. Results of this study indicate that nondomestic felids in southern Africa have been exposed to, and are likely infected with, N. caninum and T. gondii.     

Serum antibody profile and reproductive performance during two consecutive pregnancies of cows naturally infected with Neospora caninum.

The objective of this study was to record how the antibody levels change over time during pregnancy in dairy cows naturally infected with the protozoan parasite Neospora caninum, and relate this to the reproductive performance. Eighteen cows with antibodies to N. caninum were serum sampled monthly during their first pregnancy and 13 of them were also followed for a second pregnancy. In all, five pregnancies ended in abortion and two in stillbirth. Antibodies to N. caninum in serum were demonstrated by immune stimulating complex enzyme-linked immunosorbent assay (iscom ELISA). The N. caninum antibody titres remained well above the 1:100 cut-off limit for the test used during 2 years in all cows. In the non-aborting cows, mean values of antibody titres to N. caninum rose 1.5-2.5 dilution steps to reach a plateau 4-5 months before parturition, and thereafter decreased from 2 months before parturition. These changes were statistically significant (p < 0.001). The same pattern was seen in the aborting cows. The consistent pattern of rise in antibody titres observed during both pregnancies in all cows indicated a reactivation rather than a reinfection of the parasite at mid-gestation.     

Neospora caninum serostatus and milk production of Holstein cattle

OBJECTIVE: To determine whether Neospora caninum serostatus was associated with milk production among Holstein cattle in Ontario. DESIGN: Case-control study and cross-sectional observational study. ANIMALS: 3,702 Holstein cows in 83 herds (case-control study) and 3,162 Holstein cows in 57 herds. PROCEDURE: Herds in the case-control study were grouped on the basis of N. caninum abortion status. Herds in the observational study were considered representative of Ontario dairy herds. The N. caninum serostatus of individual cows was determined with a kinetic ELISA. Milk production was modeled to compare seropositive with seronegative animals while controlling for parity, days since parturition, and herd clustering. RESULTS: In the case-control study, 305-day milk production of seropositive cows was significantly less than milk production of seronegative cows in herds with abortions attributable to N. caninum infection and in herds with abortions attributable to pathogens other than N. caninum, but not in herds without abortion problems. In the observational study, 305-day milk production for seropositive cows was not significantly different from milk production of seronegative cows. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the association between N. caninum serostatus and milk production in Ontario Holstein dairy cattle may depend on abortion status of the herd. In herds with abortion problems, regardless of cause, N. caninum-seropositive cattle produced less milk, whereas in herds without abortion problems, N. caninum-seropositive cattle produced the same amount of milk as seronegative cattle.