Serology of a Neospora abortion outbreak on a dairy farm in New Zealand: a case study

AIM: To describe the kinetics of serological titres after an abortion outbreak in April-May 1995 due to Neospora caninum affected 17 dairy cows in a herd of 320. METHODS: Thirty-five cows, that had either aborted, carried mummified calves, were not pregnant or calved normally were: bled several times at regular intervals and the sera tested for Neospora antibodies in the indirect fluorescence antibody test (IFAT). RESULTS: Maximal IFAT titres of up to 1:4000 occurred within 6 weeks of the abortion outbreak, decreased over the next 2 months to < or = 1:200 and remained at this level until the next scheduled bleed a further 2 months later. A rise in titres was subsequently observed in the cows that had aborted or were not pregnant (at the time of the abortions) or had carried mummified foetuses. Seroconversion was also observed in some of the control cows, which had, up until then, remained seronegative. A dog and cat in contact with the cows in the herd investigated were, however, negative in the IFAT. CONCLUSIONS: Maximal serological titres in Neospora abortions are observed within weeks of the abortion event and then quickly return to very low levels. Subsequently, a recrudescence of titres can be observed in infected cows during the next pregnancy, without it being associated with repeat abortions.     

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.     

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 ELISApositive 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 ELISAnegative.

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 (≥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.     

Seroepidemiology of neosporosis in dairy cattle in Uruguay

A seroepidemiological survey of Neospora caninum was done in a dairy farm in Uruguay employing indirect fluorescent antibody test (IFAT). Sera from 217 cattle (155 cows, 31 heifers and 31 calves) were tested for N. caninum antibodies in July 2000 and it was found that abortions were significantly associated with the seropositivity of the cows by chi(2)-test (P = 0.025). The cows with an IFAT titre of 1:3200 were at a significantly higher risk of abortion than seronegative cows (P = 0.0013). Neospora organisms were detected in the brains of two aborted foetuses by immunohistochemical examination. The infection of N. caninum was considered postnatal because of the even distribution of seropositive cows (60%), low seroprevalence (20%) in calves and no significant correlation in the serostatus between calves and their dams (P = 0.43). A fitted binomial generalised linear model revealed that there was an increasing risk of abortion with increasing IFAT titre and increasing age between 2 and 6 years.     

Toxoplasma gondii infection in adult llamas (Lama glama) and vicunas (Vicugnavicugna) in the Peruvian Andean region

The present study was designed to investigate Toxoplasma gondii infection in adult llamas (Lama glama) and vicunas (Vicugna vicugna) in the Peruvian Andean region, for which to date no information has been available. Serum samples from 43 llamas (L. glama) and 200 vicunas were tested by IFAT detecting titres of 1:50 or higher in 55.8% (33.9-70.9%) and 5.5% (2.8-9.6%), respectively. IFAT titres ranged from 1:50 to 1:6400. In order to avoid cross reactions with closely related coccidian parasites and to confirm the existence of T. gondii specific antibodies, IFAT positive sera from both ruminant species were also analysed by western blot. T. gondii specific antigens were recognised by IFAT positive sera, although different IFAT cut-off points could be selected for llamas (1:200) and vicunas (1:50) meaning seroprevalence of 44.2% (29.1-60.1%) and 5.5% (2.8-9.6%), respectively. Based on the frequency and intensity of tachyzoite antigen recognition, at least three immunodominant antigens with apparent molecular weights of 22-24, 30, and 38-40 kDa were detected, together with other minor protein fractions located in the 18-73 kDa range. This study documents for the first time the presence of T. gondii infection and reports the target T. gondii antigens in adult llamas and vicunas in Peru.     

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.     

Influence of age and purpose for testing on the cut-off selection of serological methods in bovine neosporosis

The aim of this study was to investigate the need for different cut-off points, according to animal age and the purpose of testing, for two of the most widely used serological techniques in bovine neosporosis, IFAT and a crude antigen ELISA (Civtest, HIPRA). Therefore, the population reference sera used were defined using a combination of multiple criteria such as epidemiological/clinical and histopathological parameters and an immunoblot test. Firstly, foetuses and breeding cattle (heifers and cows) were considered as separate subpopulations for serological evaluation. Secondly, cut-off points for each serological technique (IFAT and ELISA) according to age group (foetuses and breeding cattle) and the different practical applications (detection of infection and abortion) were calculated following the receiver operating characteristics (ROC) analysis. Cut-off points were defined, for IFAT and ELISA for aborted breeding cattle and for IFAT alone in the case of the foetuses, assuming an equivalent cost of false positive and negative results. In infected breeding cattle, for IFAT and ELISA and in foetuses for ELISA, two possible cut-off values were obtained, one for a maximum sensitivity and one for a maximum specificity and the intervals of unclear results were defined. In this case, a cut-off value for equal sensitivity and specificity was also estimated. When cut-off points for infected breeding cattle, 1:100-1:250 for IFAT and 0.306-0.451 for ELISA were applied to a target population, optimal and similar negative and positive predictive values together with similar apparent and true prevalence results were observed suggesting the possibility of using both tests interchangeably.     

Immune response to Neospora caninum in naturally infected heifers and heifers vaccinated with inactivated antigen during the second trimester of gestation

The objective of this study was to compare the immune response to Neospora caninum in naturally infected heifers and heifers inoculated with a killed whole N. caninum tachyzoite preparation during the second trimester of gestation. Nine Holstein heifers were used in this study; three naturally infected heifers were born from seropositive dams, and six seronegative heifers were born from seronegative dams. Four seronegative heifers were subcutaneously vaccinated with a killed whole N. caninum tachyzoite preparation at weeks 13, 15 and 17 of gestation. A killed whole N. caninum tachyzoite preparation containing 45 mg of protein/5 ml dose was formulated with 70% of mineral oil adjuvant (13% consisting of Arlacel C, 85% Marcol 52 and 2% Tween-80). Similarly, two seronegative heifers (negative controls) were inoculated with mock-infected bovine monocytes in oil adjuvant. Humoral immune responses were tested by using an indirect fluorescent antibody test (IFAT) and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting isotype specific antibodies. Cellular immune responses were assessed by lymphocyte proliferation test (LPT) and IFN-gamma production. N. caninum-specific antibody responses increased in immunized cattle by week 15 of gestation (mean reciprocal antibody titers 450+/-252), peaked at week 23 (mean 16,000+/-6400). Maximum antibody response in naturally infected heifers was observed at week 19 of gestation (mean: 3467+/-2810). Mean serum IFAT titers were significantly higher in immunized heifers compared with those in naturally infected heifers from weeks 17 to 25 (P < 0.05). Analysis of isotype specific antibodies in naturally infected heifers revealed a predominant IgG1 response in one heifer and a predominant IgG2 response in the other two. Similar titers of IgG1 and IgG2 occurred in immunized heifers. Control heifers remained seronegative throughout the study by IFAT and ELISA. Significant antigen-specific proliferation responses were only detected in naturally infected heifers in week 19 of gestation. Peripheral mononuclear blood cells (PMBC) from immunized animals produced IFN-gamma in similar concentrations to those of infected animals (P > 0.05). No abortion was seen in any experimental group; however, one calf from a vaccinated heifer died due to dystocia. All calves from vaccinated and control heifers were seronegative by IFAT at 6 months of age; in contrast, calves born from naturally infected heifers remained seropositive with titers > or = 200. Killed vaccine induced similar immune responses to those found in chronically, naturally infected cattle which did not abort; however, different immune pathways may be followed in vaccinated and natural infected heifers with differences in degree of protective immunity.     

Serological study of a dairy herd with a recent history of Neospora abortion

HERD HISTORY: A cluster of 19 abortions occurred in April and May 1994 on a seasonal dairy farm of 277 cows in Taranaki. Lesions consistent with neosporosis were seen in several foetuses. The age of the aborting cows ranged from 2 to 12 years. Pregnant rising 2-year-old heifers had been grazed off the main farm on a run-off' until returning in May, when they were replaced on the run-off by the rising l-year-old calves. None of these pregnant heifers aborted. METHODS: Sera were obtained from all calves (n = 32), 32 of 33 pregnant heifers, 59 of 60 3-4 year-old cows and 101 of 150 older cows, plus all cows that had aborted. Sera were tested using an indirect fluorescent antibody assay. Non-aborting cattle sera were screened at 1:400, and further dilutions were tested from 15 aborted cows. RESULTS: The percentages of seropositive cattle were: 32% of the calves, 3% of the heifers, 31% of the younger cows and 27% of the older cows. The proportion of seropositive heifers was significantly lower (p< 0.01) than in the other age classes. There was no significant difference in the proportion of seropositive animals between these other age classes (p >0.05). Of the 15 cows that aborted, and for which further dilutions were tested, nine had titres of 1: 12,800, four had titres of 1:6400, one had a titre of 1: 1600 and one had a titre of 1:400. Twenty cows were the dams of pregnant heifers. Six of these cows were seropositive but all their offspring were seronegative. Fifteen aborting cows were held over to the following year when 13 became pregnant and calved normally. There were only two to three abortions in the whole herd in this following year. CONCLUSION: These results suggest that either a point-infection occurred sometime in early 1994 with an infective period short enough so that the heifers did not get infected when they returned to the main farm, or some factor precipitated a recrudescence of latent infection with a subsequent rise in titres in about 29% of the animals on. the main farm only. Although neither hypothesis can be proven or disproven in this observational study, the former hypothesis seems more likely.     

Serological status and abortion rate in progeny obtained by natural service or embryo transfer from Neospora caninum-seropositive cows

Bovine neosporosis is an important disease in dairy herds in Argentina. Embryo transfer (ET) technology offers the opportunity to minimize vertical transmission of neosporosis. The objective of this work was to compare the serological status and the incidence of abortion between heifers obtained via natural service and heifers obtained by ET from Neospora-infected cows. Progeny from eight Neospora-infected cows belonging to a commercial dairy farm where ET was routinely performed were studied. Between 1996 and 2001, 16 and 15 heifers were obtained by natural service and ET, respectively. Serostatus and reproductive performance were recorded in both progeny. Serostatus was determined by indirect fluorescent antibody test (IFAT). All heifers obtained by natural service were seropositive and six of 16 pregnancies ended in abortion. All heifers obtained by ET were seronegative and only two abortions of 15 pregnancies were recorded.     

Cloprostenol for termination of pregnancy in cattle

One hundred and eight cows and 65 maiden heifers which were pregnant from 32 to 250 days were treated with 500 meg cloprostenol. Abortions occurred in 53 (49%) cows and 47 (72%) heifers, with 62% of aborted foetuses being expelled within 5 days of treatment. Of 96 pregnancies treated before 150 days, 86 (89.5%) were terminated. Luteolysis, followed by oestrus and ovulation occurred in both those which aborted and those which did not, although serum progesterone levels and ovarian palpations suggest that luteolysis was not complete in those animals which maintained pregnancy. Abortions occurred in only 2 of 51 cows at 151 – 250 days pregnancy, while 12 of 26 similar pregnancies were terminated in heifers. Reasons for this difference are discussed, as are possible methods for improving the success rate in aborting pregnancies between 151 – 250 days.     

Monoclonal antibodies preventing invasion of Neospora caninum tachyzoites into host cells

Twelve monoclonal antibodies (MAbs) against Neospora caninum tachyzoites were produced to specify the antigens related to the invasion of tachyzoites into host cells. In the assay to evaluate the inhibition activity, all these MAbs prevented the cultured Vero cells from the invading by the tachyzoites. These MAbs recognized approximately a 73 kDa antigen in Western blot analysis. Immunofluorescence assay and immune electron microscopy revealed that this 73 kDa antigen is a part of the surface antigens of N. caninum tachyzoite, and that the tachyzoite antigen identified plays an important role for invasion of host cells.     

Evaluation by different diagnostic techniques of bovine abortion associated with Neospora caninum in Spain

Eighty foetuses from some of the main cattle-producing regions in Spain were analysed to investigate the participation of Neospora caninum in cases of bovine abortion. Diagnosis of the infection was determined by histopathological analysis complemented with immunohistochemistry, serology (IFAT and ELISA) and PCR tests. A total of 38.8% of the bovine foetuses analysed were considered to be infected by at least one of the diagnostic techniques used. Microscopic lesions consistent with Neospora infection in brain were identified in 31.3% of the samples, whereas only 10.7 and 15.3% were positive using serological and PCR analysis, respectively. Perfect agreement was shown between IFAT and ELISA, although there was little agreement among results of the other diagnostic techniques. Gestational age of aborted foetuses checked ranged from <3 to 9 months, with a mean of 5.9 months, and no difference in age was found between infected and non-infected foetuses (P>0.05). This study confirms the importance of N. caninum as a cause of abortion in Spain and underlines the need to use different diagnostic techniques to increase the chance to detect the infection in aborted foetuses.     

Dynamics of anti-Neospora caninum antibodies during gestation in chronically infected dairy cows

The dynamics of antibody production against Neospora caninum during the gestation period was examined in chronically infected dairy cows. Data were obtained from 86 pregnant parous dairy cows, 21 of which had suffered abortion. The cows belonged to two herds in which a diagnosis of N. caninum infection had been previously confirmed in aborted foetuses. Pregnancy diagnosis and blood collection were performed on post-insemination Days 40, 90, 120, 150, 180, 210, and at parturition or until the time of abortion detection. Blood plasma was tested for antibodies against N. caninum using ELISA. The non-aborting cows were divided into two groups according to whether their antibody values in the second half of gestation had increased or not, while aborting cows were classified as those showing an antibody peak before abortion or those not showing a pre-abortion peak. Differences in antibody values throughout pregnancy in each group of non-aborting and aborting cows were analysed by GLM repeated measures of analysis of variance. While 32 non-aborting cows (49%) showed a significant and consistent increase in anti-Neospora antibody values during the second half of gestation, antibody values in the remaining 33 non-aborting cows were practically constant throughout gestation. An antibody peak around abortion was observed in 11 aborting cows (52%), while antibody values in the remaining 10 aborting cows were similar before and at abortion. Seroprevalence fluctuations, defined as seronegative blood samples at some point during the gestation period, were, furthermore, observed in 2 aborting and 11 non-aborting cows. Our results indicate two clearly distinguishable types of humoral immune dynamics throughout gestation: an increased or flat production of antibodies during the second half of gestation in non-aborting animals and before abortion in aborting cows. The observation that some Neospora-infected dams can exhibit negative antibody values at any time during gestation, particularly at parturition or abortion, prompts future studies designed to explore the use of new ELISA strategies at the farm level.     

The specificity of antibody responses in cattle naturally exposed to Fasciola hepatica

Fasciola hepatica causes significant morbidity and mortality in dairy cattle in the Andean region of Cajamarca, Peru, where prevalence of infection of up to 78% has been reported. ELISA and Western blot analyses were used to characterise antibody responses in dairy cattle to adult F. hepatica to excretory-secretory (E/S), somatic (SO) and surface (SU) antigens. Three groups of dairy cattle - calves, heifers and adult cows - naturally exposed to F. hepatica in this region, were monitored every 2 months over a 2-year period. Calves, heifers and adult cows all had antibodies which recognised a 28kDa protein in the SO preparation, whereas only adult cows had antibodies that recognised a 28kDa protein in E/S products. All three groups of cattle responded to a 60-66kDa group of proteins in E/S and SU preparations and a 17kDa antigen in SO products was recognised by antibodies from cows and heifers but not calves. The total antibody response to E/S antigens measured by ELISA, increased over time in calves and remained constantly high over the 2-year period in all three groups of cattle. Slight fluctuations in the antibody response occurred in the group of heifers and cows coinciding with seasonal changes in the level of challenge.     

Neospora caninum antigens recognized by mouse IgG at different stages of infection including recrudescence

Western blotting was performed to analyze Neospora caninum tachyzoite antigens recognized by mouse IgG at different stages of infection including recrudescence. At the early stage of infection, a 36-38 kDa antigen was clearly recognized by the mouse antisera. After day 48 postinoculation, the signal of the 36-38 kDa antigen gradually weakened. Meanwhile, a 43 kDa antigen was intensely and continuously recognized from 48 to 125 days postinoculation. This 43 kDa antigen was clearly detectable with the antisera from the mice under immunosuppression. Sera from naturally infected cattle strongly reacted with the 43 kDa antigen. Therefore, the 43 kDa antigen may be useful for immunological reactions to detect infected animals except in the early stage of the infection.     

Efficacy of a Neospora caninum killed tachyzoite vaccine in preventing abortion and vertical transmission in dairy cattle

A clinical trial was undertaken to assess the efficacy of Bovilis(?) Neoguard, a killed Neospora caninum tachyzoite vaccine on 5 commercial dairy farms in New Zealand with a history of Neospora-associated abortion. Cattle were enrolled in the trial at 30-60 days of gestation and randomly allocated to treatment or control groups. Treatment consisted of 5 mL doses of Bovilis Neoguard administered subcutaneously at enrolment then 4 weeks later. Isotonic saline was administered to the control group. Of 2246 cattle enrolled in the trial, 10.7% of cows and 12.6% of heifers were seropositive to N. caninum. Sampling of a randomly selected proportion of enrolled animals 6 weeks after the second treatment showed that 188/232 (81.0%) vaccinated with Bovilis(?) Neoguard had seroconverted, while 11/130 (8.5%) cows and 10/36 (27.8%) heifers in the control group had seroconverted. Forty-eight vaccinated and 63 control animals aborted. On one farm 12.5% of control animals and 6.1% of vaccinated animals aborted (vaccine efficacy 0.61; p=0.03). On another farm with a high level of abortion 8.4% of control animals and 8.7% of vaccinates aborted. On the remaining 3 farms fewer abortions occurred than expected. A modified Poisson regression approach was used to calculate relative risks for abortion and vertical transmission. Overall vaccine efficacy was 0.25 (p=0.12). Heifer replacement calves from the animals enrolled in the trial were sampled for antibodies to N. caninum at 6-9 months of age. Fourteen of 17 calves from vaccinated, seropositive cows were seropositive as were 13/23 calves from seropositive cows in the control group. The interaction between dam serostatus and treatment group was significant (p=0.05) with vaccination increasing the risk of vertical transmission. It was concluded that vaccination after conception prevented 61% abortions in one of five herds and that vaccination may have increased the risk of early embryonic death.     

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.     

First identification of Neospora caninum infection in aborted bovine foetuses in China

The first identification of Neospora caninum infection in the tissues of aborted bovine foetuses in China is reported. Aborted foetuses were collected from 16 dams, and 12 of the dams had high serum antibody titres to N. caninum determined using an ELISA test kit. The Nc-5 gene of N. caninum was amplified from DNA samples extracted from brains of four aborted foetuses using a Neospora-specific PCR assay, confirming N. caninum infection in the aborted foetuses. Histology and immunohistochemistry showed thick-walled (3 microm) tissue cyst in 25 microm diameter in the brain of one foetus. Non-suppurative encephalomyelitis, focal haemorrhage, hepatic lesions consisted of lymphocyte infiltration and haemorrhage were also found in the heart and lung of the foetus. Thus, we have confirmed for the first time the infection of N. caninum in aborted foetuses of cattle in the People's Republic of China.