First Portuguese isolate of Neospora caninum from an aborted fetus from a dairy herd with endemic neosporosis

Neospora caninum was isolated from the brain of an aborted 4-month-old fetus from a dairy cow herd with endemic neosporosis in Porto, Portugal. The fetal brain homogenate was inoculated interperitoneally first into outbred Swiss Webster mice given dexamethasone and then the peritoneal exudates from these mice was co-inoculated with mouse sarcoma cells in the peritoneal cavity of mice given dexamethasone. N. caninum tachyzoites were seen in peritoneal exudate of the second passage. Tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies and contained N. caninum specific DNA. This Portuguese isolate of N. caninum has been successfully maintained in cell culture. The dam of the aborted fetus had an antibody titer of 1:10240 in the Neospora agglutination test (NAT). Antibodies to N. caninum were found in 76 of 106 cows from this herd in titers of 1:40 in 31, 1:80 in 22, > or =1:160 or more in 23 in the Neospora agglutination test. This is the first isolation of a viable N. caninum-like parasite from any host in Portugal.     

The use of beef bull semen reduced the risk of abortion in Neospora-seropositive dairy cows

There is an evidence that the epidemiology of neosporosis differs in dairy and beef cattle, such that beef cattle carry a lower risk of abortion. The aim of the present study was to establish whether artificial insemination using semen from beef bulls could reduce the risk of abortion in dairy cows seropositive for the Neospora caninum parasite. Our study was based on yearly serological screening for neosporosis and on the confirmation of Neospora infection in aborted fetuses in two high-producing dairy herds with a mean 28% seroprevalence of N. caninum antibodies. The study population comprised of 273 pregnancies in seropositive animals: 156 pregnancies monitored after insemination using Holstein-Friesian semen and 117 after insemination using beef bull semen. Abortion rates for these animals were 28.2% (77 of 273), 34.6% (54 of 156) and 19.7% (23 of 117). Logistic regression analysis indicated no significant effects of lactation number and previous abortion on the abortion rate. Based on the odds ratio, a 1-unit increase in the Neospora antibody titre yielded a 1.01-fold increase in the abortion rate. The likelihood of abortion was two times higher for cows in one of the two herds and 2.8 times lower (one of 0.36) for pregnant cows inseminated with beef bull semen rather than Holstein-Friesian semen. Our results indicate that the use of beef bull semen can reduce the risk of abortion in dairy cows, and suggest that annual screening for neosporosis, specifically the antibody titre to the protozoan, could be an useful predictor of abortion risk in reproductive health programmes.     

Proportion of abortions due to neosporosis among dairy cattle in Japan

A retrospective cohort study was conducted to clarify the association between seropositive reactions to Neospora caninum and subsequent reproductive disorders among dairy cattle in Japan. A statistically significant association between Neospora seropositive reactions and abortions was observed (P=0.016), and seropositive cattle were 6.1 times more likely to abort compared to seronegative cows. No significant differences were observed between seropositive reactions and other reproductive disorders such as conception failure, perinatal death and calf mortality. As indicated by estimation of the attributable fraction, 83.6% of abortions in Neospora seropositive animals may be attributed to N. caninum. Considering seroprevalence of N. caninum in the cattle which aborted in Japan, 21.8% of abortions were estimated to be caused by neosporosis in Japan.     

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.     

Outbreak of abortion associated with Neospora caninum infection in a beef herd

Abortion outbreaks caused by infection with Neospora caninum in beef cattle have not been well documented. Neospora caninum infection was confirmed in 4 fetuses that were aborted by cattle in a 350-head beef herd; an additional 58 cattle aborted during the next 2.5 months. Overall, 44.4% (157/354) of the cattle in the herd did not become pregnant or experienced fetal loss during this period. Initially, 81.3% (282/347) of the herd was seropositive for antibodies to N caninum, and seropositive cows were 6.2 times as likely to not be pregnant as were seronegative cows. Other potential causes of abortion were not identified in this herd. Following the outbreak, few cattle in the herd or in a second exposed herd seroconverted, but high antibody titers persisted in most seropositive cattle through the end of the calving season. Evidence of cow-to-fetus transmission of the organism was detected in > 82% of seropositive cows.     

Serological evidence of Neospora caninum in dual-purpose cattle herds in Venezuela

Bovine neosporosis is a parasitic disease produced by Neospora caninum that induces abortion in cows, and consequently has a negative impact on the herd's reproductive efficiency. The main objective of this research was to determine the serological evidence of N. caninum in cattle herds from Venezuela using an indirect antibody capture ELISA test. Four hundred and fifty-nine (459) serum samples from crossbred adult cows were collected to be tested for Neospora antibodies. The sampled cows came from 15 large farms located in eight important cattle states that have predominant dual-purpose production systems (cattle from these farms are used for both milk and meat production). Fifty-two cows (11.3%) were seropositive to N. Caninum. Thirteen (86.7%) of 15 studied herds had cows seropositive to N. caninum. The average within-herd seroprevalence was 11.5% (range 3.8-36.7%). Cows that aborted in some of these farms had 2.71 (P: 0.009) greater odds to be seropositive when compared to cows that did not abort. Each one of the eight states represented in our study had seropositive animals. These results are the first evidence of exposure to N. caninum in Venezuelan cattle herds, indicating the possible circulation of this pathogen in the country. Further epidemiological studies should be granted to determine the spread of the disease in the Venezuelan cattle industry and its associated risk factors.     

Detection of antibodies to Neospora caninum in two species of wild canids, Lycalopex gymnocercus and Cerdocyon thous from Brazil

Domestic dog (Canis domesticus) and the coyote (Canis latrans) are the only known definitive hosts for the protozoan Neospora caninum that causes abortion in dairy cattle. In the present study, antibodies to N. caninum were sought in three species of wild canids, Cerdocyon thous, Lycalopex gymnocercus and Dusicyon vetulus from Brazil. Antibodies to N. caninum were assayed by the indirect fluorescent antibody test (IFAT) and the Neospora agglutination test (NAT). N. caninum antibodies were found in five of 12 L. gymnocercus with IFAT titers of 1:50 in three, 1:100 in one, and 1:1600 in one, and NAT titers of 1:40, 1:80, 1:160, 1:320, and 1:640 in five animals. Antibodies to N. caninum were found in four of 15 C. thous with IFAT titers of 1:50 in one, and 1:100 in three, and NAT titer of 1:40 in one animal. All 30 D. ventulus were seronegative by IFAT and NAT.     

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.     

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.     

Seroprevalence of Neospora caninum in dairy and beef cattle with reproductive disorders in Japan

Serum samples from 145 dairy and 65 beef cattle with reproductive disorders and 54 normally calving dairy cattle (controls) in Japan were tested for presence of Neospora caninum antibodies by use of an indirect fluorescent antibody test (IFAT, titer 1:200). Overall, seroprevalence of N. caninum was significantly higher (P < 0.001) in dairy cattle (20.0%, 29/145) than in beef cattle (1.5%, 1/65). In cattle which aborted, seroprevalence of N. caninum was significantly higher (P = 0.041) in dairy cattle (26.1%, 23/88, compared with controls (3.7%, 2/54)) than in beef cattle (5.0%, 1/20), indicating that neosporosis might be a more common problem in dairy cattle than in beef cattle in Japan. Seropositive cattle were 9.2 times more likely to abort compared to seronegative cows. Abortions associated with N. caninum seropositivity in this study were most frequently observed in the second trimester, and the mean gestational age of the fetuses aborted from seropositive dams was 5.7 months. In conclusions, N. caninum seems to be causing serious economic losses in the dairy industry in Japan. This is the first report on an objective comparison of seroprevalence of dairy and beef cattle with reproductive disorders in Asia.     

Artificial insemination of cows with semen in vitro contaminated with Neospora caninum tachyzoites failed to induce neosporosis

Neosporosis is a major cause of abortion in cattle all over the world. Congenital transmission as well as horizontal transmission by ingestion of oocysts has been described. The detection of Neospora caninum DNA in bull semen warrants the investigation of possible transmission through the use of contaminated semen. In this experiment four cows were artificially inseminated with frozen-thawed semen contaminated in vitro with viable N. caninum tachyzoites (group A) and four control cows were inseminated with tachyzoites-free frozen-thawed semen, from the same bull (group B). Serum samples were collected 15 days before the artificial insemination (AI) and at days 10, 14, 21, 28, 45, 60 and 75 post-insemination. All sera samples were tested for neosporosis by direct agglutination test (DAT). Three of the cows from group A had negative DAT titers (< or =1:20) in all of the samples, while the fourth cow from this group had a low titer of antibodies (1:80) at day 10, and became negative at day 45, suggesting a stimulation of the immune system by the tachyzoites placed in uterus, rather than the induction of an infection. All of the cows from group B had negative DAT titers (< or =1:20) in all of the samples. These results suggest that transmission of neosporosis by artificial insemination with frozen-thawed semen is an unlikely event.     

The Use of Beef Bull Semen Reduced the Risk of Abortion in Neospora‐seropositive Dairy Cows

There is an evidence that the epidemiology of neosporosis differs in dairy and beef cattle, such that beef cattle carry a lower risk of abortion. The aim of the present study was to establish whether artificial insemination using semen from beef bulls could reduce the risk of abortion in dairy cows seropositive for the Neospora caninum parasite. Our study was based on yearly serological screening for neosporosis and on the confirmation of Neospora infection in aborted fetuses in two high‐producing dairy herds with a mean 28% seroprevalence of N. caninum antibodies. The study population comprised of 273 pregnancies in seropositive animals: 156 pregnancies monitored after insemination using Holstein–Friesian semen and 117 after insemination using beef bull semen. Abortion rates for these animals were 28.2% (77 of 273), 34.6% (54 of 156) and 19.7% (23 of 117). Logistic regression analysis indicated no significant effects of lactation number and previous abortion on the abortion rate. Based on the odds ratio, a 1‐unit increase in the Neospora antibody titre yielded a 1.01‐fold increase in the abortion rate. The likelihood of abortion was two times higher for cows in one of the two herds and 2.8 times lower (one of 0.36) for pregnant cows inseminated with beef bull semen rather than Holstein–Friesian semen. Our results indicate that the use of beef bull semen can reduce the risk of abortion in dairy cows, and suggest that annual screening for neosporosis, specifically the antibody titre to the protozoan, could be an useful predictor of abortion risk in reproductive health programmes.     

Seroepidemiology of beef and dairy herds and fetal study of Neospora caninum in Argentina

The purpose of the present work was to study the epidemiology of Neospora caninum in beef and dairy herds in the Humid Pampas of Argentina. The seroprevalence of N. caninum was evaluated in 2414 serum samples of cows from beef and dairy farms. An indirect fluorescent antibody test (IFAT) was used to determine specific antibodies. The sera was screened at a dilution >or=1:200 and >or=1:600 in cows with reproductive disease antecedents and without them, respectively. Cows without history of reproductive diseases from nine beef and fifteen dairy farms were grouped according to the percentage (> or or 相似文献   

Repetitive abortion in Neospora-infected ewes.

Pregnant ewes inoculated with cultured Neospora caninum tachyzoites in 1995, 1996, or 1995 and 1996 aborted or delivered weak or clinically normal lambs in 1996. Nine of 11 ewes in the study had previously produced infected lambs or fetuses after being experimentally infected in 1995. Fetuses and lambs produced in 1996 showed histologic lesions and zoites indicative of Neospora. Serologic responses and production of infected fetuses/lambs indicated systemic neosporosis in the ewes during gestation, although tachyzoites could not be cultured from maternal tissues. The repetitive infection of fetuses, and resulting clinical and histopathologic findings in the present study are similar to those reported in naturally infected cattle, adding to the already established similarities of neosporosis between sheep and cattle.     

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.     

Pattern of recognition of Neospora caninum tachyzoite antigens by naturally infected pregnant cattle and aborted foetuses

Different aspects of Neospora tachyzoite antigen recognition by Neospora-infected heifers and cows and aborted foetuses were studied. The pattern of antigen recognition and the relationship between IFAT titres and number of Neospora antigens detected, were evaluated. In addition, the tachyzoite antigens involved in the humoral immune response developed against infection in normal cows and cows that aborted were also characterised throughout pregnancy. Comparison of tachyzoite antigen recognition was carried out in 13 thoracic and/or abdominal fluids from Neospora aborted foetuses and 33 sera from Neospora infected cows that had aborted. The kinetics of Neospora-antigen recognition was studied in Neospora-infected heifers and cows that had aborted foetuses (7) or not (14) during pregnancy.Based on the frequency and intensity of recognition, four IDAs-17-18, 34-35, 37 and 60-62kDa antigens-have been described. Moreover, a correlation was found between Western blot results and IFAT titres in both age groups. In relation to antigen recognition throughout pregnancy by seropositive cows that had aborted or not, the antibody fluctuations throughout pregnancy described in the literature could be due to differences in the intensity and frequency of recognition of particular antigens, especially the 17-18kDa antigen. We emphasize the important role that the 17-18kDa antigen could play in the serological diagnosis of Neospora infection in cattle as this was intensely detected in 100% of the animals.     

Detection of specific antibodies anti-Neospora caninum in the fallow deer (Dama dama)

Sera from 335 farmed fallow deer (Dama dama) at the breeding station in Kosewo Górne in the Mazurian Lake District, North-East Poland, were investigated for the presence of antibodies against Neospora caninum. The distribution of age groups was as follow: >4 years - 154 animals; 2 years - 76 animals; 1 year - 105 animals. Ten sera with the optical density exceeding 0.159 absorbance units (i.e., cut-off value) in ELISA test were also analyzed by Western blot. Western blot analysis revealed seroreactivity against immunodominant N. caninum antigens of 37, 25, and 16kDa; however, in some sera additional bands were also visible. This is the first screening studies for antibodies against N. caninum in farmed fallow deer in Poland, in the region where neosporosis was confirmed in cattle and in farmed and free-ranging European red deer (Cervus elaphus).     

Isolation of Neospora caninum from dairy zero grazing cattle in Israel

First Israeli Neospora caninum isolates were obtained from brain tissues of aborted fetuses (NcIs491 and NcIs580) from dairy farms endemic for neosporosis and maintaining cattle on zero grazing. Tissues from different parts of the fetus brains were used to infect Vero cells. Tachyzoites of N. caninum were first observed in cultures from days 30 and 32 after infection. To confirm the identity of the isolated parasites, DNA extracts from brains and cultures were tested by PCR with specific primers based on the Nc5 gene. Specific fragments were amplified by PCR from infected cultures of both fetuses on day 25. Susceptible seronegative gerbils (Meriones tristrami) were inoculated intraperitoneally with 10(3) to 10(5) tenfold dilutions of subculture tachyzoites. The inoculated gerbils developed specific antibodies to N. caninum, with end-point serum dilution of 1:4096 in the IFA assay, whereas no neurological signs or deaths were seen during 4 months of observation.     

Development of an indirect ELISA-NcSRS2 for detection of Neospora caninum antibodies in cattle

Neosporosis is of alarming economic concern in the cattle industry. The effectiveness of diagnostic tests for detecting specific antibodies against Neospora caninum is hampered by potential cross-reaction with other coccidia. Use of a single specific antigen might improve test specificity. An indirect enzyme-linked immunosorbent assay (ELISA) was developed using the truncated protein NcSRS2 expressed in Escherichia coli. The ELISA results were compared with those of the indirect fluorescence antibody test (IFAT). Receiver Operating Characteristic (ROC) and Tests in the Absence of a Gold Standard (TAGS) analysis revealed an assay having 96% specificity and 95% sensitivity when applied to 145 positive and 352 negative sera from two distinct cattle populations. Using OD ≤ 0.095 as the cut-off point, the assay's negative and positive predictive values ranged from 98.8% to 50.8% and from 58.8% to 99.1%, respectively, depending on neosporosis prevalence in a given area. The novel ELISA-NcSRS2 format described in the present report constitutes a specific and sensitive method for detecting N. caninum in cattle.     

Nationwide seroprevalence of Neospora caninum among dairy cattle in Japan

Serum samples from 2420 clinically healthy dairy cattle, randomly selected from stored sera in 18 districts of Japan, were tested for the presence of Neospora caninum antibodies using an indirect fluorescent antibody test (titer > or =1:200). Nationwide seroprevalence is estimated at 5.7% (139/2420). Seropositive cattle were detected in all surveyed districts despite the evidence of confirmed case reports of bovine neosporosis, showing that N. caninum is widely distributed throughout Japan. Age-specific seroprevalence did not increase with cattle age, suggesting that Neospora infection is likely to be transmitted vertically rather than horizontally in Japan. Considering that N. caninum seropositive cows are thought to be more likely to abort, substantial fetal losses may be induced by N. caninum infection in Japan. Devising strategies are needed to reduce the economic impact on the Japanese dairy industry. This is the first study to investigate the nationwide seroprevalence of N. caninum in cattle in Asia.