Mark C. Thurmond

Detection of Serum Antibody Responses in Cattle with Natural or Experimental Neospora Infections

Parasite-specific antibody responses were detected using an indirect fluorescent antibody (IFA) test in cattle that were naturally or experimentally infected with Neospora parasites. The test was developed using Neospora tachyzoites isolated from an aborted bovine fetus and grown in bovine cell cultures (isolate BPA1). In all cases, infections were confirmed by the identification of Neospora tachyzoites and/or bradyzoite cysts in fetal or calf tissues using an immunoperoxidase test procedure. Fifty-five naturally infected cows that aborted Neospora-infected fetuses had titers of 320-5,120 at the time of abortion. The titer of 6 cows that were serologically monitored over a prolonged period decreased to 160–640 within 150 days after they aborted infected fetuses. Two of the cows showed an increase in their Neospora titers during their subsequent pregnancy, and they gave birth to congenitally infected calves that had precolostral titers of 10,240-20,480. Postcolostral titers of these calves and of 4 other calves with congenital Neospora infections were all 25,120, whereas calves with no detectable parasites had titers ≤ 160. Two pregnant heifers that were experimentally infected with the BPA1 isolate at approximately 120 days gestation seroconverted to Neospora antigens within 9 days and developed peak titers of 5,120 and 20,480 within 32 days of infection. The fetus taken by caesarian section 32 days postinfection from 1 heifer and the full-term calf born to the other had Neospora titers of 640 and 10,240, respectively. Nine cows that aborted uninfected fetuses and 61 adult cattle maintained under pasture or feedlot conditions, where risk of exposure to Neospora was considered to be low, had titers ≤ 320. Some of the feedlot cattle tested had serologic reactivity that was restricted to antigens at the apical end of both Neospora and Toxoplasma gondii tachyzoites. This type of reactivity, which may result from serologic cross-reactivity between conserved apical complex antigens of closely related sporozoan parasites, differed from the whole parasite fluorescence that was observed with sera from Neospora-infected animals. The significance of these results and the potential application of the IFA test for the diagnosis of Neospora infections in cattle are discussed.

NEOSPORA CANINUM ANTIBODIES IN COWS DURING PREGNANCY AS A PREDICTOR OF CONGENITAL INFECTION AND ABORTION

A cohort study was undertaken on a dairy experiencing endemic Neospora caninum abortions, to characterize dam serologic variations during pregnancy, and to determine if dam N. caninum antibody levels during gestation predicted congenital infection or abortion. Blood samples were collected monthly during pregnancies of 254 cows and precolostrally from 87 of their calves. Antibody levels, as measured by an enzyme-linked immunosorbent assay, indicated 60.6% of cows were seropositive at some time during pregnancy and 87.4% of seropositive cows were seropositive throughout pregnancy. The rate of seroconversion was 8.5/100 cows/yr. The risk of abortion for seropositive cows at the time of pregnancy diagnosis and during gestation was twice that for seronegative cows (P = 0.025, P = 0.006). Calves born to seropositive cows were more likely to be seropositive at birth if the dam had high antibody levels at 240 days of gestation (P = 0.04) and an increase in antibody levels between 90 and 240 days (P = 0.08) than if the respective values of the dam were low or decreasing. Seropositive cows with high antibody levels at 180 and 210 days of gestation were less likely to abort than cows with low antibody levels at those times (P = 0.05, P = 0.03). Results support a causal effect between exposure to N. caninum and abortion, indicate that acquisition of infection during pregnancy is not necessary for congenital infection or abortion to occur, and suggest that maternal immune response influences congenitial infection and abortion.

An Enzyme-Linked Immunosorbent Assay (ELISA) for Serological Diagnosis of Neospora Sp. Infection in Cattle

A kinetic enzyme-linked immunosorbent assay (ELISA) was developed and optimized for detection of antibodies to Neospora sp. in cattle. Sonicated tachyzoites of Neospora sp. isolated from an aborted bovine fetus were used as antigen. Variability in immunoblot patterns among positive sera, and the fact that all life stages of the parasites are unknown, justified use of a multiple-antigen ELISA to allow for maximum sensitivity. Immunoblot analysis revealed negligible cross-reactions between Toxoplasma gondii antigen and Neospora sp. antisera and between Neospora sp. antigen and antisera from various apicomplexan parasites. The maximum positive-to-negative Vmax (average maximum slope of the optical density over time) ratio was obtained using 200 ng/well of sonicated tachyzoite antigen and a 1:200 serum dilution. Using logistic regression to determine the optimal cutoff point between known infected and noninfected cattle, a sample-to-positive control Vmax ratio of 0.45 was found to maximize the percent correct classification, with an estimated sensitivity of 88.6% and specificity of 96.5%. Use of Neospora caninum antigen following the same protocol demonstrated no difference in ELISA interpretation. Comparison with an existing indirect immunofluorescent antibody (IFA) test showed the ELISA to be the more sensitive and specific test for serodiagnosis of Neospora infection in cattle.

Interpretation of an Indirect Fluorescent Antibody Test for Diagnosis of Neospora sp. Infection in Cattle

Neospora is a recently discovered protozoan that may cause bovine protozoal abortion (BPA) in cows and encephalomyelitis in congenitally infected calves. An indirect fluorescent antibody (IFA) test has been described for diagnosing Neospora sp. infection in cattle. We report here results using the IFA test on peritoneal and pleural fluids from aborted fetuses, precolostral calf sera, and selected adult cattle. The IFA test employed was the same as described elsewhere, with the following modifications. Teflon-coated slides were precleaned in absolute ethanol and fixed in an-

Seroprevalence of Neospora caninum and abortion in dairy cows in northern Spain

The seroprevalence of Neospora caninum infection was estimated from a sample of 889 cattle from 43 dairy herds in three counties in the Asturias region of Spain. The true prevalence of infection was estimated to be 30-6 per cent (95 per cent confidence interval (ca) 27.6 to 33.6). Seropositivity was associated with abortion during the previous year (odds ratio (OR)=3.31, P<0.001) and was slightly higher among purchased cattle (37.6 per cent), than among cattle raised on the farm (29.1 per cent) (P=0.078). Seropositive cows were more likely than seronegative cows to have had a seropositive dam (OR=2.3, P=0.011), suggesting that congenital transmission contributed to about 56 per cent of the infections. Herds with a true seroprevalence above 10 per cent had more dogs on the farm, than herds with a lower prevalence (P=0.032). The ORs relating abortion to seropositivity in individual herds ranged from 0.7 to 19, indicating that some herds experienced few abortions caused by N caninum, while others experienced more abortions due to the organism. Overall, 38.7 per cent of the abortions were estimated to have been attributable to N caninum.

Pooled-Sample Testing as a Herd-Screening Tool for Detection of Bovine Viral Diarrhea Virus Persistently Infected Cattle

The study was conducted to develop methodology for least-cost strategies for using polymerase chain reaction (PCR)/probe testing of pooled blood samples to identify animals in a herd persistently infected with bovine viral diarrhea virus (BVDV). Cost was estimated for 5 protocols using Monte Carlo simulations for herd prevalences of BVDV persistent infection (BVDV-PI) ranging from 0.5% to 3%, assuming a cost for a PCR/probe test of

Conceptual Foundations for Infectious Disease Surveillance

20. The protocol associated with the least cost per cow involved an initial testing of pools followed by repooling and testing of positive pools. For a herd prevalence of 1%, the least cost per cow was

Automatic online news monitoring and classification for syndromic surveillance

2.64 (95% prediction interval =

A Simulation Model of Intraherd Transmission of Foot and Mouth Disease with Reference to Disease Spread before and after Clinical Diagnosis

1.72,

Relationships between Maternal and Fetal Liver Copper, Iron, Manganese, and Zinc Concentrations and Fetal Development in California Holstein Dairy Cows

3.68), where pool sizes for the initial and repooled testing were 20 and 5 blood samples per pool, respectively. Optimization of the least cost for pooled-sample testing depended on how well a presumed prevalence of BVDV-PI approximated the true prevalence of BVDV infection in the herd. As prevalence increased beyond 3%, the least cost increased, thereby diminishing the competitive benefit of pooled testing. The protocols presented for sample pooling have general application to screening or surveillance using a sensitive diagnostic test to detect very low prevalence diseases or pathogens in flocks or herds.