Bovine neosporosis: immunoblot improves foetal serology.

The purpose of the study is the comparative evaluation of the immunofluorescent antibody test (IFAT) and an immunoblot (IB) test for the examination of foetal fluids for specific antibodies against Neospora caninum. Peritoneal and pleural fluids as well as abomasal contents were analysed. The results of the serological examinations were compared to those obtained by histological, immunohistochemical, and PCR analysis of foetal tissues as well as to the results of maternal serological examinations. Fluids were used undiluted in the IB and reactions against six immunodominant antigens were recorded. When the recognition of at least two immunodominant antigens was regarded as positive, the agreement of the IB with other diagnostic methods was good to moderate as characterised by kappa-values of 0.76 (histology/immunohistochemistry), 0.69 (maternal serology) and 0.54 (PCR on foetal tissues). The IB results agreed better with the results of the other diagnostic methods than those of the IFAT. The higher relative sensitivity of the IB was regarded as the main reason for the better agreement. However, also the specificity of the IB was superior to that of the IFAT in relation to histology/immunohistochemistry, maternal serology and PCR.     

Abortions caused by protozoa in agricultural animals]

A review is given on abortions in livestock caused by Toxoplasma gondii, Neospora caninum and Sarcocystis spp. Special emphasis is put on diagnostic procedures. T. gondii is considered to be a major cause of abortions in sheep and goats. Diagnosis is based on the characteristic lesions in the cotyledons, histologic and immunohistochemical examination, and on the detection of specific antibodies in body fluids or serum of aborted fetuses. Whether abortions in swine due to T. gondii are of practical importance is still unclear. The most important causative protozoal agent of abortions in cattle is Neospora caninum. This protozoon was found in 19 per cent of cattle fetuses submitted for examination in USA. Occasionally it was found also in aborted lambs, kids and foals. Diagnosis is based on the histologic or immunohistochemical detection of the parasite. Abortions can be regularly induced experimentally in large and small ruminants and in pigs by the oral inoculation of sporocysts of certain Sarcocystis species. However, under natural conditions abortion due to Sarcocystis was diagnosed only occasionally and only in cattle and sheep. The histologic examination of the fetus and fetal membranes is the only way to diagnose Sarcocystis abortion or neonatal infection at present.     

Investigation of Neospora sp. antibodies in aborted mares from Normandy, France

Neospora caninum, an apicomplexan protozoan parasite, is recognized as a major cause of abortion in cattle while limited information is presently available on association between equine Neospora infections and abortions. The aim of the present study was to document prevalence of antibodies against Neospora sp. in aborted mares as a clue to the role of N. caninum in mare reproductive failure in Normandy, France. Using an agglutination test, the number of animals with elevated (>80) anti-Neospora sp. antibody titer was higher in a group of 54 aborted mares than in randomly chosen groups of 45 mares and 76 horses sampled for equine arteritis virus and Fasciola hepatica antibodies, respectively (P<0.001). N. caninum DNA was found in 3/91 fetal brains, 2/77 fetal hearts, and 1/1 placenta, and present in both brains and hearts of two fetuses. In 13 cases for which both mare serum and fetus were available, no fetal N. caninum amplification product was present while a large variation of maternal antibody titers was found. Data prompt at additional surveys of association between equine reproductive failure and Neospora sp. infection.     

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.     

Neospora abortion in New Zealand cattle

Protozoal abortion in cattle was retrospectively studied by examining material submitted to the Batchelar Animal Health Laboratory in the years 1987-90. Only cases in which foetal brain had been submitted were examined. Histologically, protozoal lesions were seen in the brains of 28% of 320 aborted foetuses. Lesions were also seen in 10% of the hearts and 34% of the placentas examined, but these were not as characteristic as those in the brains. Protozoa, confirmed immunohistochemically as being Neospora caninum-like in two brains, were seen in 5% of aborted foetuses with lesions. No protozoa or associated lesions were seen in 57 late term foetuses or neonatal controls. A farm survey supported the hypothesis that Neospora is an important cause of multiple abortions in cattle. Laboratory and farm data suggested that protozoal abortion was more common in early gestation. No predisposing causes of abortion were found and there was no evidence of point infection of affected herds.     

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.     

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.     

First Confirmed Case of Neospora caninum-associated Abortion Outbreak in Portugal

In a dairy herd experiencing an abortion outbreak, 49% of the 119 cows were positive for Neospora caninum antibodies and the remaining 51% were seronegative. Using immunohistochemical analysis of foetal brain tissue, Neospora cysts and bradyzoite clusters were identified in two of the 15 aborted foetuses submitted for testing. Other agents that commonly cause abortions were not found. These results, coupled with neurological clinical symptoms in new-born calves, implicated N. caninum as the major cause of abortions in this dairy herd. This is the first report that associates N. caninum infection with bovine abortions in Portugal.     

Neosporosis in bovine dairy herds from the west of France: detection of Neospora caninum DNA in aborted fetuses, seroepidemiology of N. caninum in cattle and dogs.

Neospora caninum is considered one of the major causes of abortion in cattle in most parts of the world. In this study, the role of N. caninum was investigated in groups of aborted cattle and dairy herds from the west of France. Good correlation was found between parasite DNA detection in fetuses and serologic statuses of dams. In groups with documented abortion status and no antibodies to other pathogens, 17-45% of aborted animals were seropositive for N. caninum, and significant relationship between prevalence of Neospora antibodies and frequency of abortions was found. Neospora-associated abortions were observed all the year round, with a peak in summer. Higher ratios of seropositive abortions were found before the 6th month of gestation. In 12 herds studied in the field, serologic prevalence ranged 6-47%. No difference in age was found between seropositive and seronegative cows. Results indicate that N. caninum is an important and stable cause of abortion in cattle in France.     

Bovine abortion associated with Leptospira interrogans serovar hardjo infection

During 1981, 265 bovine abortions were investigated by serological and histological methods for evidence of leptospiral infection. Leptospires were demonstrated in the tissues of 10 foetuses by a Levaditi silver impregnation technique. Serological testing of maternal sera indicated that Leptospira interrogans serovar hardjo was associated with 5 of the abortions while the remaining 5 were due to L. interrogans serovar pomona infection. In cases of abortion associated with L. interrogans serovar hardjo leptospires were readily demonstrated in foetal liver, kidney, intestine and heart. They were demonstrated less often in lung and placenta and could not be found in foetal brain. Autolysis did not appear to interfere with the demonstration of leptospires by silver impregnation. No lesions attributable to leptospiral infection were seen in placentas but mild interstitial nephritis was found in some of the foetuses. Fourteen other cows had serological evidence of recent leptospiral infection but leptospires were not detected in foetal tissues. Histological examination of silver impregnated foetal tissues in combination with the microscopic agglutination test was shown to be an effective method for diagnosing abortion associated with L. interrogans serovar hardjo in cattle.     

Determination of an optimized cut-off value for the Neospora agglutination test for serodiagnosis in cattle

A definitive diagnosis of neosporosis in cattle implies the examination of the aborted fetus. However, in many instances fetal material is not available. Therefore, most diagnosis are based on serological tests. At the moment, there are no consensuses about the cut-off for serodiagnosis of neosporosis in cattle, for any test. The objective of the present study was to estimate the best cut-off for the Neospora agglutination test (NAT) for serodiagnosis in cattle. For that purpose, 246 serum samples from 4 groups of dairy cows (aborted Neospora positive; not aborted healthy; aborted other diseases and herds endemic neosporosis) were collected and antibodies anti-N. caninum were determined by NAT. Additionally, immunoblot (IB) was performed with sera from all cows of the endemic neosporosis group and the patterns of seroreactivity were contrasted with the NAT titers for this group of cows. Evaluation of the optimized sensitivity and specificity was calculated using Youden's J-statistics. The best Youden's J-statistic was obtained at 1:40 titer, presenting 100% of sensitivity and 90.4% of specificity with negative and positive predictive values of 100 and 75.0%, respectively. The comparison between NAT titers and the IB banding pattern support the results of the statistical analysis, i.e. titers of 1:40 and higher showed a complex pattern of bands, while titers lower than 1:40 did not precipitate any bands. These results indicate that 1:40 was an optimized NAT cut-off for serodiagnosis in cattle.     

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.     

A Swiss case-control study to assess Neospora caninum-associated bovine abortions by PCR, histopathology and serology.

Neospora caninum is one of the most frequent infectious organisms causing abortion in cattle worldwide. The present case-control study was designed to assess the importance of bovine neosporosis for causing abortion in Swiss cattle and to identify selected risk factors. Infection was primarily diagnosed by a N. caninum-specific PCR and serology, complemented with histopathology and immunohistochemistry. A total of 113 case and 113 corresponding control-farms were studied for 1.5 year. During this time period, 242 abortions were reported and referred for bacteriological, virological, parasitological and pathohistological examinations. N. caninum was detected by PCR in the brains of 21% of all aborted fetuses. Microscopic lesions indicative for cerebral protozoa infection were detected in 84% of PCR-positive fetal brains. Bovine viral diarrhea virus (BVDV) was demonstrated in 7% of the cases, and bacterial infections were detected in 4% of the abortions. One or more N. caninum-abortions occurred in 20% of the herds (41 case-farms and 3 control-farms). Serological examination of aborting mother cows revealed a significantly higher percentage of N. caninum-seropositive animals (44%) in comparison to the prevalence in a randomly selected population (12%). However, in eight cases (4% of all investigated abortions) seronegative cows aborted N. caninum PCR-positive fetuses, and in 50 cases the fetus remained negative although the respective mother cow was N. caninum-seropositive. Repetitive serological investigations (at a 3-12 months interval) of 3551 cows from case- and control-farms showed a decrease of the overall N. caninum-seroprevalence from 17 to 12%. Ninety out of 3008 seronegative animals were converted to N. caninum-seropositivity. Conversely, 212 out of 543 initially seropositive animals became seronegative for their second serum sample. The obtained data underlined the importance of N. caninum as a causative agent for abortion in Swiss cattle. Furthermore, PCR was confirmed to be a valuable diagnostic tool for the primary diagnosis of N. caninum in aborted fetuses. On the other hand, the value of serology appears to be hampered by the temporal instability of N. caninum antibody concentrations in adult cattle, including especially seronegativity of some individual animals. Thus, seronegativity in a mother cow or heifer does not exclude N. caninum-associated abortions.     

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.     

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.     

Experimental Leptospira borgpetersenii serovar hardjo infection of pregnant cattle

Objective To observe the effect upon the foetus of experimental infection of pregnant cattle with Leptospira borg-petersenii serovar hardjo .
Design A disease transmission study using pregnant cattle.
Procedure Fourteen heifers serologically negative to L hardjo were artificially inseminated and later challenged with a north-Queensland isolate of L hardjo by conjunctival inoculation. The heifers were serologically monitored and their urine examined for the presence of leptospires using culture and fluorescent-antibody tests at appropriate intervals. Elective caesarean sections were performed on pregnant heifers at 6.5 weeks after the challenge. Foetuses were examined using serological, histopathological, microbiological and fluorescent-antibody tests.
Results Ten of the heifers became pregnant, but three subsequently aborted before challenge. After challenge, all 14 heifers seroconverted and L hardjo was isolated from the urine of 6 of the 7 pregnant heifers. No evidence of foetal L hardjo infection was detected. Two of the foetuses had histopatho-logical lesions consistent with Neospora s p infection.
Conclusion It is likely that the isolate of L hardjo used in this study does not normally infect the foetus. Neospora s p may be a more significant cause of bovine reproductive wastage.     

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.     

Seroprevalence of Neospora caninum infection in dairy and beef cattle in Paraguay

Seroprevalence of Neospora caninum in 879 beef and dairy cattle in different locations of Paraguay was determined by an ELISA. In the survey, 262 (29.8%) cattle were positive to N. caninum, and animals with anti-Neospora antibody titre were observed in all the locations tested in the country. Serum samples taken from a herd that exhibited persistent abortion had the highest percentage of animals being positive to the parasite (17/30, 56.7%). In the same herd, abortion was significantly more likely in animals with high anti-Neospora antibody titre. Immunoblot analysis demonstrated that the banding pattern from positive Paraguayan cattle was similar to that seen with the positive control sample. In conclusion, N. caninum infection is present among Paraguayan beef and dairy cattle, and it may be an important cause of bovine abortion in Paraguay.     

Neosporosis in a pup

CASE: A 13-week-old female boxer pup was found to be suffering from rigidity of the left hindleg. Antibiotic and anti-inflammatory treatment over a 3-week period failed to improve the condition and the pup was humanely killed. METHODS: Serological examination for Neospora antibodies was carried out by the indirect fluorescent antibody test and for Toxoplasma gondii antibodies with a latex agglutination test. A variety of tissues were examined histologically, and the central nervous system by immunohistochemistry and the polymerase chain reaction. RESULTS: The IFAT for anti-Neospora antibodies showed a titre of 1:51 200 in the clinically affected pup while the latex agglutination test for Toxoplasma antibodies was negative. The dam and one of two tested litter-mates had anti-Neospora IFAT titres of 1:1600, the other litter mate was negative. All three were not clinically affected. Histological, immunohistochemical and polymerase chain reaction examinations of the affected pup confirmed the diagnosis of Neospora infection. CONCLUSION: In the live animal, serological examination is thought to be the most useful specific test. Post-mortem examination by traditional histology, immunohistochemistry and the polymerase chain reaction confirmed the diagnosis. The case is discussed in the context of present knowledge about Neospora infection in New Zealand.