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PubMed1996

An immunohistochemical method for detecting bradyzoite antigen (BAG5) in Toxoplasma gondii-infected tissues cross-reacts with a Neospora caninum bradyzoite antigen.

The previously cloned gene of a bradyzoite-specific antigen (BAG5) of Toxoplasma gondii was used to express a fusion protein for subsequent antiserum production in rabbits. The BAG5 antiserum was used in an immunohistochemical procedure to look for reactive epitopes in bradyzoites and tachyzoites of T. gondii within animal tissues. Encysted bradyzoites in brain were stained deeply and diffusely. Although most unencysted organisms in brain were not stained, occasional free organisms had mild to deep staining. There was no staining of tachyzoites in liver where cysts were not observed. Neospora caninum organisms within animal tissues were also examined using the BAG5 immunohistochemical procedure. The BAG5 antiserum cross-reacted with N. caninum bradyzoites but had no affinity for tachyzoites.

Animals↗
PubMed1996

Serologic responses of cattle and other animals infected with Neospora caninum.

OBJECTIVE: To examine cross-reactivity among Neospora caninum and closely-related apicomplexans. DESIGN: Sera from animals were examined for antibody production to N caninum and cross-reactivity to Toxoplasma gondii. ANIMALS: Cattle were experimentally infected with 3 tissue cyst-forming protozoan parasites N caninum, T gondii, and Sarcocystis sp, and calves were monospecifically inoculated with the intestinal coccidia, Eimeria bovis and Cryptosporidium parvum. Similar studies were done in laboratory rabbits inoculated with N caninum, T gondii, Hammondia hammondi, and Sarcocystis sp. Additionally, sera were obtained from ewes, lambs, goats, sows, cats, rats, and mice inoculated with N caninum tachyzoites. PROCEDURE: The indirect fluorescent antibody (IFA) and ELISA antibody tests (cattle only) were used to examine reactivity to N caninum; the modified direct agglutination, Sabin-Feldman dye, and IFA tests were used to evaluate reactivity to T gondii. RESULTS: Serologic cross-reactivity among N caninum, T gondii, and Sarcocystis sp was none or minimal by the IFA test. There was some reactivity to N caninum by the use of ELISA in cattle inoculated with Sarcocystis sp. CONCLUSIONS: The IFA test for N caninum was specific for the diagnosis of neosporosis in animals.

Animals↗
PubMed1996

Fetal loss in BALB/C mice infected with Neospora caninum.

The suitability of mice as a model for reproductive loss due to Neospora caninum infection was investigated. Groups of mice were infected with 2 x 10(6) N. caninum before pregnancy (10 days) and during pregnancy (days 5 and 10 of gestation). In mice infected before and during early pregnancy, fetal loss was evaluated throughout gestation, and pregnancy loss was evaluated by enumeration of fetal resorptions and total fetuses. In mice infected before pregnancy, no difference was present in resorptions between infected and control mice, although litter size was decreased in the infected mice (P < 0.05). In mice infected during early pregnancy (day 5 gestation) and examined temporally throughout gestation, resorptions were increased in the infected mice compared to the control mice (P < 0.05). In mice infected at 5 days gestation and examined at one time point (day 14 of gestation), the resorption rate for infected mice was 33% and 12% for controls (P < 0.05). Routine histopathologic examination and immunohistochemistry using N. caninum-specific antisera did not identify tachyzoites in placental and fetal tissues during the pre- and early pregnancy infections. In mice infected late midgestation (day 10), N. caninum tachyzoites were identified in placenta and fetal muscle and neural tissue. In the placenta, there was multifocal necrosis and hemorrhage with intralesional tachyzoites. Tachyzoites in fetal tissues were not associated with pathologic changes. No reproductive loss was associated with mice infected late in gestation. These data demonstrate that mice can be used as a model for the study of fetal resorption and congenital infection associated with N. caninum.

Animals↗
PubMed1996

Demonstration of synergistic effects of sulfonamides and dihydrofolate reductase/thymidylate synthase inhibitors against Neospora caninum tachyzoites in cultured cells, and characterization of mutants resistant to pyrimethamine.

OBJECTIVE: To examine the efficacies of combinations of 7 sulfonamides and 5 dihydrofolate reductase/thymidylate synthase (DHFR/TS) inhibitors against tachyzoites of Neospora caninum in cultured cells. Mutant tachyzoites that were resistant to pyrimethamine were produced and examined for resistance to other DHFR/TS inhibitors. DESIGN AND PROCEDURES: After 5 days of treatment, a cell culture flask lesion-based assay was used to determine efficacies of combinations of sulfonamides and DHFR/TS inhibitors against N caninum tachyzoites and to evaluate the sensitivity of pyrimethamine-resistant mutants of N caninum to test agents. Cultured cells that were infected with the appropriate strains of N caninum and treated or not treated (controls) with test agents were examined. Mutations were induced by chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine or by selection for growth in permissive concentration of pyrimethamine. RESULTS: Synergism was detected for combinations of pyrimethamine, ormetoprim, trimethoprim, or diaveridine with the sulfonamides. Methotrexate did not have improved efficacy when combined with sulfonamides. Two mutants were produced that were resistant to pyrimethamine. Both mutants were resistant to other DHFR/TS inhibitors. Both mutants remained resistant to pyrimethamine in the absence of continuous exposure to the agent, indicating that the induced resistance was stable. Synergism was detected for combinations of DHFR/TS inhibitors and sulfonamides against these pyrimethamine-resistant mutants. CONCLUSIONS: Combinations of suboptimal concentrations of sulfonamides with suboptimal concentrations of DHFR/TS inhibitors results in improved efficacy of the agents in a cell culture assay. Stable resistance to pyrimethamine can be induced in N caninum tachyzoites by use of chemical mutagenesis or by selection. CLINICAL RELEVANCE: In vitro evidence indicated that combination treatment, using sulfonamides and DHFR/TS inhibitors, may be effective in treating neosporosis.

Animals↗
PubMed1996

Congenital Neospora caninum infection in dairy cattle and associated calfhood mortality.

A prospective cohort study was undertaken on two central California dairies, A and B, to estimate prevalence of congenital infection with Neospora caninum, to characterize temporal variation in prevalence, to determine if occurrence of congenital infection was associated with specific dam and calf attributes, and to estimate the effect of congenital infection on calfhood mortality. Of the 405 calves enrolled over a period of 2 1/2 y on dairy A and dairy B, 30.6% (85/278) and 53.5% (68/127), respectively, were seropositive precolostrally to N. caninum, as determined by an ELISA test. Adult cow seroprevalence at calving was 36.0% (82/228) for dairy A, and 57.9% (33/57) for dairy B. No evidence was found for a significant increasing or decreasing trend in adult and precolostral seroprevalence through the study period (P > or = 0.26). For both herds combined, 81% of seropositive cows (93/115) and 5% of seronegative cows (8/170) had congenitally infected calves. Seroprevalence did not increase with cow age on either dairy (P > or = 0.47). The probability of a calf being congenitally infected was not associated with dam age, dam lactation number, dam history of abortion, calf gender, or length of gestation (P > or = 0.11). High dam ELISA values at calving were significantly associated (P < or = 0.001) with an increased probability of congenital infection in her calf. Results of survival analyses of female calves available for follow-up indicated a consistently greater survivorship to 90 d in congenitally infected calves than in noninfected calves on both dairies, which was significant for dairy A (P = 0.07, n = 186) but not for dairy B (P = 0.69, n = 72), thus indicating that congenital infection does not necessarily have a detrimental effect on calf health. The findings of a similar magnitude in congenital infection rate and adult cow prevalence, the lack of increasing seroprevalence with cow age, the lack of an effect of dam age on precolostral seropositivity, and the constant seroprevalences during the study period, suggest that, in the two dairies studied, congenital transmission constituted a substantial amount of infection and was likely the major mode of transmission of N. caninum.

Abortion, Veterinary↗
PubMed1996

Culling associated with Neospora caninum infection in dairy cows.

OBJECTIVES: To estimate the extent to which cows infected with Neospora caninum were culled, compared with noninfected cows, and to identify differences in reasons for culling between infected and noninfected cows. ANIMALS: 442 Holstein cows on a commercial dairy with 36% seroprevalence for N caninum. PROCEDURE: Culling of cows was done after first calving without knowledge of N caninum serologic status. RESULTS: Risk of a seropositive cow dying was not different from that of a seronegative cow (P = 0.50). Seropositive cows were culled 6.3 months earlier than seronegative cows, and had a 1.6 times greater risk of being culled, compared with seronegative cows (P = 0.004), after adjusting for culling risk associated with abortion. For cows culled for low milk production, culling risk for a seropositive cow was twice that for a seronegative cow (P = 0.007). CONCLUSIONS: The economic impact of N caninum infection in dairy cattle can be expected to extend beyond that for abortion alone. Costs of the disease also may include premature culling and diminished milk production. CLINICAL RELEVANCE: Plans to control N caninum infection on dairies should include consideration that benefits may include reduction in premature culling and increase in milk production.

Abortion, Veterinary↗
PubMed1997

Effect of Neospora caninum infection on milk production in first-lactation dairy cows.

OBJECTIVE: To determine whether cows seropositive to Neospora caninum produced less milk during their first lactation than seronegative cows. DESIGN: Repeated-measures, prospective study. ANIMALS: 372 Holstein cows in their first lactation. PROCEDURE: Cows were tested repeatedly before and during their first lactation for antibodies to N caninum. One-way and repeated-measures ANOVA were used to determine whether mean daily milk weights and milk weights from Dairy Herd Improvement Association testing were less for seropositive cows than for seronegative cows. RESULTS: Weekly mean daily milk weights for the 118 seropositive cows were significantly less than those for the 254 seronegative cows, and milk production for seropositive cows (mean, 55.2 lb/cow/d) was 2.5 lb/cow/d less than that for seronegative cows (mean, 57.7 lb/cow/d). Analysis of results from Dairy Herd Improvement Association testing revealed that production of seropositive cows was less for milk (3.1 lb/cow/d), fat-corrected milk (3.6 lb/cow/d), and fat (0.14 lb/cow/d) than production of seronegative cows. CLINICAL IMPLICATIONS: The economic impact of N caninum infection in dairy cows can include reduced revenues from decreased milk production, which may warrant culling of young, seropositive replacement stock.

Analysis of Variance↗
PubMed1997

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.

Abortion, Veterinary↗
PubMed1997

Expression of Toxoplasma gondii genes in the closely-related apicomplexan parasite Neospora caninum.

We have established the feasibility of using Neospora caninum as a heterologous system for the expression of genes from the closely-related parasite Toxoplasma gondii. Plasmid construct containing the lacZ gene from Escherichia coli driven by T. gondii promoters were electroporated into N. caninum parasites, and expression of beta-galactosidase (beta-Gal) activity was assayed enzymatically. In transient assays, expression of beta-Gal driven by the GRA1 promoter was approximately 10-fold higher than the expression obtained with the SAG1 promoter. Enzyme activity was not detected when N. caninum parasites were transfected with a promoterless lacZ construct. Transfection of N. caninum with complete genomic clones of SAG1 or GRA2 from T. gondii yielded parasites that transiently expressed the respective gene products, as detected by immunofluorescence and Western blot. Additionally, these transiently expressed T. gondii proteins appeared by immunofluorescence localization and Triton X-114 partitioning to be correctly targeted in both extracellular and intracellular N. caninum parasites. Heterologous gene expression should be useful for studying the function of specific gene products and may facilitate the identification of genes responsible for the phenotypic differences observed between these two closely-related apicomplexan parasites.

Animals↗
PubMed1997

Immunohistochemical diagnosis of Toxoplasma gondii: potential for cross-reactivity with Neospora caninum.

A monoclonal antibody (MAB 3F5) was compared with a commercial polyclonal antibody for specificity for Toxoplasma gondii and cross-reactivity with Neospora caninum in paraffin-embedded tissue sections. Both antibody preparations reliably recognized T. gondii tachyzoites (RH isolate) in animal tissues but did not always label tissue cysts (ME-49 isolate). The commercial antibody strongly cross-reacted with N. caninum tachyzoites, but MAB 3F5 did not when the immunoperoxidase, immunofluorescence, or immunogold procedures were employed, nor did it cross-react with other apicomplexans, e.g., Isospora suis, Eimeria bovis. Sarcocystis cruzi, Hammondia hammondi, or Caryospora bigenetica. Immumoelectron microscopy revealed that MAB 3F5 bound to dense granules in extracellular T. gondii tachyzoites. In western blots of T. gondii tachyzoites, a major band at 38 kDa and a minor band at 32 kDa were labeled by MAB 3F5, but no labeling of the proteins of N. caninum tachyzoites or uninfected host cells occurred at the ultrastructural level or in immunoblots.

Animals↗
PubMed1997

A protocol for the production of Neospora caninum tissue cysts in mice.

Identification of a definitive host for Neospora caninum has been inhibited by lack of an efficient method for producing bradyzoites, needed for oral infectivity trials. An improved protocol for producing bradyzoite-containing tissue cysts in mouse brains is described. Six variables, including mouse strain (Balb/C, CBA/Ca, and ICR), sex, N. caninum isolate (NC-2 and NC-Liverpool), tachyzoite inoculum dose, immunosuppression with methylprednisolone acetate (MPA), and sulfadiazine treatment were tested. Tissue cyst numbers were estimated using an immunohistologic staining procedure specific for bradyzoites. Male ICR mice (> or = 30 g) that were immunosuppressed with 2 mg MPA 7 days prior to and 2.5 mg MPA at the time of subcutaneous inoculation with 400,000 N. caninum tachyzoites produced the highest numbers of tissue cysts. Significant numbers were produced by methods using the NC-2 strain of N. caninum; however, protocols using NC-Liverpool produced greater numbers of tissue cysts. Sulfadiazine treatment did not appear to contribute to tissue cyst production. The procedure described is superior to previously described methods with regard to numbers of tissue cysts produced, protocol reproducibility, and survival of mice until tissue cyst formation.

Animals↗
PubMed1997

Effect of congenitally acquired Neospora caninum infection on risk of abortion and subsequent abortions in dairy cattle.

OBJECTIVES: To estimate the extent to which abortion risk in dairy cattle during subsequent pregnancies was associated with congenitally-acquired Neospora caninum infection and previous abortions. ANIMALS: 468 Holstein cattle. PROCEDURE: Newborn heifer calves were tested for evidence of congenital infection attributable to N caninum and examined repeatedly until the completion of their second lactation for serologic status and evidence of abortion. RESULTS: Compared with noninfected cows, congenitally infected cows had a 7.4-fold higher risk of abortion during their initial pregnancy and a 1.7-fold higher risk of aborting the first pregnancy during their first lactation. During the first pregnancy of their second lactation, congenitally infected cows that had aborted previously had a 5.6-fold higher risk of abortion, compared with cows that had not previously aborted and that were seronegative. The fetal risk period for N caninum-associated death began sooner and extended later during the initial pregnancy compared with subsequent pregnancies. CONCLUSION: Congenitally acquired N caninum infection can cause a substantial number of abortions during the initial pregnancy of heifers, with abortion risk attributable to N caninum decreasing in subsequent pregnancies, possibly because of selective culling. Subsequent abortions can be expected in congenitally infected cows that have aborted previously.

Abortion, Veterinary↗
PubMed1997

Prevalence of antibodies to Neospora caninum in different canid populations.

A total of 1,554 dogs from 5 countries on 3 continents were tested for antibodies to Neospora caninum using an indirect fluorescent antibody test. In Australia, overall, 42/451 (9%, 95% confidence interval [CI] 6-12%) dogs were seropositive (Melbourne 11/207 [5%, 95% CI 2-9%]; Sydney 18/150 [12%, 95% CI 7-18%]; Perth 13/94 [14%, 95% CI 8-22%]). Antibodies to N. caninum were also detected in dogs in South America (Uruguay [20%, 95% CI 16-24%, n = 414]) and sub-Saharan Africa (Tanzania [22%, 95% CI 12-36%, n = 49]). In contrast, only 1 of 500 dogs tested from the Falkland Islands and none of 140 dogs from Kenya was seropositive. Of wild canids, 1/54 (2%, 95% CI 0-10%) British foxes and 15/169 (9%, 95% CI 5-14%) Australian dingoes had antibodies to N. caninum.

Africa, Eastern↗
PubMed1998

Oral inoculation of cats with tissue cysts of Neospora caninum.

OBJECTIVE: To test the hypothesis that cats are definitive hosts of Neospora caninum. ANIMALS: 6 weaned male kittens obtained from 2 sources, and several dozen outbred mice. PROCEDURE: Cats were fed large numbers of 3 strains of N caninum: tissue cysts in buffered saline solution, mouse brain homogenates, and whole carcass homogenates from seropositive mice. Fecal specimens were examined for 4 weeks by use of flotation tests, and bioassays were performed in mice. One cat was inoculated parenterally with tachyzoites, to determine whether cats could respond serologically to N caninum. Tissue cysts from portions of oral inocula were cultured to verify viability. Indirect fluorescent antibody serologic testing, histologic and immunohistologic examinations, cell culture, and polymerase chain reaction procedures were performed 4 to 8 weeks after oral exposure, to seek evidence of infection of cats and mice. RESULTS: None of the cats or mice seroconverted to N caninum, with the exception of the single cat inoculated parenterally. Fecal shedding of oocysts was not observed, except for Isospora felis oocysts that were shed by 2 cats beginning prior to oral challenge exposure. Evidence of infection was not detected in tissues of cats or mice, with the exception of the parenterally inoculated cat. CONCLUSIONS: The hypothesis that cats are definitive hosts of N caninum is not supported. CLINICAL RELEVANCE: Extermination of cats in efforts to control bovine neosporosis is not warranted.

Animals↗
PubMed1998

Vaccination of mice with Neospora caninum: response to oral challenge with Toxoplasma gondii oocysts.

Neospora caninum is a protozoan parasite that can cause severe disease in mammals. Two experiments were conducted to examine the effects of subcutaneous (s.c.) vaccination with Hank's balanced salt solution (HBSS), 1 x 10(5) N. caninum NC-1 strain tachyzoites or 1 x 10(5) Toxoplasma gondii TS-4 strain tachyzoites on challenge oral infections in mice with sporulated VEG strain T. gondii oocysts (1 X 10(3) oocysts exp. 1 and 5 x 10(3) oocysts exp. 2). An additional study, experiment 3, evaluated s.c. challenge with 2.5 X 10(3) tachyzoites of the highly virulent RH strain of T. gondii after vaccination with HBSS, NC-1 tachyzoites, or TS-4 tachyzoites. Mice vaccinated with NC-1 strain tachyzoites survived significantly (P < 0.05) longer than mice given HBSS in experiment 1, but not in experiments 2 and 3. Mice vaccinated with TS-4 strain tachyzoites survived significantly longer than HBSS-vaccinated mice in experiments 1, 2, and 3 and significantly longer than mice vaccinated with the NC-1 strain in experiments 2 and 3. Toxoplasma gondii tissue cyst numbers were significantly lower for mice vaccinated with TS-4 strain tachyzoites than mice vaccinated with HBSS or the NC-1 strain tachyzoites in experiment 1. No difference was observed in tissue cyst numbers in mice vaccinated with HBSS or NC-1 strain tachyzoites in experiment 1. No HBSS-vaccinated mice survived experiment 2, and the numbers of T. gondii tissue cysts were significantly lower for mice vaccinated with the TS-4 strain tachyzoites compared to NC-1 strain tachyzoites. No HBSS- or NC-1-vaccinated mice survived RH strain challenge in experiment 3. Results of these experiments indicate that infection with N. caninum provides some protection against fatal oral infection with T gondii oocysts of a moderately pathogenic strain but not tachyzoites of a highly pathogenic strain. The protection provided by N. caninum is much less than that provided by previous exposure to T. gondii, and the numbers of tissue cysts in the brains of mice are not significantly (P > 00.5) lowered.

Animals↗
PubMed1998

A chicken anti-conoid monoclonal antibody identifies a common epitope which is present on motile stages of Eimeria, Neospora, and Toxoplasma.

The chicken monoclonal antibody (mAb) 6D12-G10, raised against Eimeria acervulina sporozoites, has previously been shown to recognize the conoid of E. acervulina sporozoites and inhibit sporozoite invasion of lymphocytes in vitro. In indirect immunofluorescent assay, the mAb 6D12-G10 also reacted with merozoites from E. acervulina and identified a 21-kDa merozoite protein on western blots. By confocal laser scanning microscopy, the conoid of sporozoites from 6 different avian Eimeria species (E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox, and E. tenella) were reactive with 6D12-G10 mAb. Furthermore, the 6D12-G10 mAb also showed cross-reactivity with motile stages of 2 closely related apicomplexans, Neospora, and Toxoplasma. These results indicate that the mAb 6D12-G10 identifies a conserved epitope on the conoid that is important in host cell invasion by the apicomplexan parasites.

Animals↗
PubMed1998

Detection of antibodies to Neospora caninum in bovine postabortion blood samples from Hungary.

In order to obtain data on the occurrence of the protozoan parasite Neospora caninum as a potential factor in the aetiology of reproduction problems in cattle, 97 postabortion sera were examined by ISCOM ELISA and IFAT for the presence of antibodies in N. caninum. The results showed 10% and 9% seropositivity by the ELISA and IFAT, respectively. In 2 of the 10 seropositive cases no other fetopathogenic agents (such as Chlamydia sp., Leptospira spp. or bovine viral diarrhoea virus) were detected. These data confirm the presence of N. caninum in cattle in Hungary.

Abortion, Veterinary↗
PubMed1998

Differential expression of cell surface- and dense granule-associated Neospora caninum proteins in tachyzoites and bradyzoites.

Morphologically, the tachyzoites and the tissue cysts of Neospora caninum are difficult to distinguish from those of other cyst-forming apicomplexan parasites such as Toxoplasma gondii. Several stage-specific antigens have been identified in T. gondii tachyzoites and bradyzoites, and respective antibodies are useful tools for discriminating between the 2 stages during tachyzoite-bradyzoite interconversion in T. gondii infections. Whereas several cell surface- and dense granule-associated proteins have been identified and characterized in N. caninum tachyzoites, not much is known about antigenic components expressed in N. caninum bradyzoites. In this study, the differential expression of the 2 N. caninum surface proteins Nc-p43 and Nc-p36 and the dense granule protein Nc-p33 (NCDG1) within tachyzoites and bradyzoites of N. caninum has been investigated.

Animals↗