不同药物体外抑制新孢子虫的比较

应用青蒿素、桦褐孔菌多糖、黄芩苷及乙胺嘧啶等药物对Vero细胞中培养的犬新孢子虫进行了体外抑制试验。结果显示,青蒿素、桦褐孔菌多糖、黄芩苷、乙胺嘧啶均有抑制虫体的效果,尤其青蒿素在高浓度试验组中抑虫效果最为明显,质量浓度为20 mg/L时的抑制率为95.17%,Vero细胞形态较为完整,虫体组织包囊体积最小、数量均最少;桦褐孔菌多糖(25 mg/L)、黄芩苷(20 mg/L)和乙胺嘧啶(1.25 mg/L)高质量浓度时的抑虫效果相近。结果表明,青蒿素、桦褐孔菌多糖、黄芩苷和乙胺嘧啶均可以作为抗新孢子虫的潜在药物,其中桦褐孔菌多糖为天然药物提取物,在抗新孢子虫药物中更具有研究价值。     

Evidence of point-source exposure to Neospora caninum and protective immunity in a herd of beef cows

OBJECTIVE: To determine whether cows with evidence of previous infection with Neospora caninum were less likely to abort or give birth prematurely during an outbreak of neosporosis, compared with herdmates with evidence of primary infection. DESIGN: Cohort study. ANIMALS: 208 pregnant beef cows. PROCEDURES: Aborted fetuses and calves born prematurely were examined during an outbreak of neosporosis in a herd of beef cows. Sera were collected from all cows during the outbreak and again 71 days later. Cows were classified into groups on the basis of normal and abnormal reproductive outcomes. Sera were examined, using an avidity ELISA procedure for N caninum, and results were compared between groups and among time points. RESULTS: Antibody concentrations decreased significantly and IgG avidity values increased significantly over time. During the outbreak, cows with normal reproductive outcomes were significantly more likely to have high IgG avidity values than cows with abnormal reproductive outcomes. CONCLUSIONS AND CLINICAL RELEVANCE: The herd had numerous abortions and premature births with evidence of recent point-source exposure to N caninum. Therefore, to reduce risk of transmission of N caninum to cattle, attempts should be made to prevent canine feces from contaminating feed, especially feedstuffs used to prepare mixed rations for cattle. Cows with evidence of previous exposure to N caninum were less likely to abort or give birth prematurely during the outbreak than cows with primary infections with N caninum; this finding suggests development of protective immunity in previously infected cows.     

Survey for natural Neospora caninum infection in wild and captive birds

Neospora caninum is a protozoan parasite that presents worldwide distribution and is mainly implicated as responsible for bovine abortion. Although the presence of birds in cattle-raising properties is positively correlated to higher infection rates, very little has been described about the role of these animals in the parasite's life cycle. In that sense, this work aimed to investigate the serological and histological positivity of different avian species sampled in its natural habitat or in captivity. No serological positivity was observed in the 294 tested serum samples. On the other hand, Apicomplexa-like cysts found in muscular tissues of two Psittaciformes were immunostained with N. caninum antisera. These findings indicate that N. caninum may infect a wider range of hosts than described to date, and that further studies should be performed in order to determine the presence of the infection in different avian species.     

The role of CD4(+) or CD8(+) T cells in the protective immune response of BALB/c mice to Neospora caninum infection

The role of CD4(+) or CD8(+) T cells in the immune response of BALB/c mice against Neospora caninum infection was examined by using anti-CD4 and/or anti-CD8 monoclonal antibodies (mAbs). Mice were intraperitoneally inoculated with anti-CD4 and/or anti-CD8 mAbs before and after infection with N. caninum and observed for 30 days after infection. Most of the anti-CD4 mAb-treated mice and all of the anti-CD4 and anti-CD8 mAbs-treated mice died within 30 days post-infection (p.i.). In contrast, 100% of PBS-treated mice and 70% of anti-CD8 mAb-treated mice survived more than 30 days. When compared with phosphate-buffered saline (PBS)-treated mice, the weight of mice treated with mAbs tended to decrease. From these results CD4(+) T cells, but not CD8(+) T cells, have an important role for protection of mice against N. caninum infection. Serum antibody levels to N. caninum in infected-mice treated with anti-CD4 mAb or a mixture of anti-CD4 and anti-CD8 mAbs were lower than those in the infected mice treated with anti-CD8 mAb or PBS. The mice treated with anti-interferon-gamma (IFN-gamma) mAb produced high antibody levels to N. caninum, but all mice died within 18 days p.i. These results indicated that IFN-gamma is an important cytokine for protection against N. caninum infection at the early stage of infection. However, since CD4(+) T cells against N. caninum were essential to the production of specific antibody, these antibodies might have important roles in host protection at the later stage of infection.     

Diagnosis and treatment of Neospora caninum infection in a dog

Neospora caninum, a protozoan organism, caused extensor rigidity of the pelvic limbs in a 12-week-old dog. Diagnosis was based on results of muscle biopsy, neuroelectrodiagnostics, serotesting, and cell culture. Indirect fluorescent antibody (IFA) titer to N caninum was 1:800 at time of admission and 1:3,200 after 4 and 6 weeks. A reciprocal IFA titer of 50 to N caninum was also found in the CSF. Serotesting for T gondii was negative. Treatment with clindamycin followed by sulfadiazine and trimethoprim did not change the pelvic limb extensor rigidity, but other signs of minor neurologic dysfunction improved.     

Neospora caninum serostatus and milk production of Holstein cattle

OBJECTIVE: To determine whether Neospora caninum serostatus was associated with milk production among Holstein cattle in Ontario. DESIGN: Case-control study and cross-sectional observational study. ANIMALS: 3,702 Holstein cows in 83 herds (case-control study) and 3,162 Holstein cows in 57 herds. PROCEDURE: Herds in the case-control study were grouped on the basis of N. caninum abortion status. Herds in the observational study were considered representative of Ontario dairy herds. The N. caninum serostatus of individual cows was determined with a kinetic ELISA. Milk production was modeled to compare seropositive with seronegative animals while controlling for parity, days since parturition, and herd clustering. RESULTS: In the case-control study, 305-day milk production of seropositive cows was significantly less than milk production of seronegative cows in herds with abortions attributable to N. caninum infection and in herds with abortions attributable to pathogens other than N. caninum, but not in herds without abortion problems. In the observational study, 305-day milk production for seropositive cows was not significantly different from milk production of seronegative cows. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the association between N. caninum serostatus and milk production in Ontario Holstein dairy cattle may depend on abortion status of the herd. In herds with abortion problems, regardless of cause, N. caninum-seropositive cattle produced less milk, whereas in herds without abortion problems, N. caninum-seropositive cattle produced the same amount of milk as seronegative cattle.     

A long-term study of Neospora caninum infection in a Swedish dairy herd

A longitudinal study was performed in a Swedish dairy herd where Neospora caninum had been isolated from a stillborn calf. Starting in autumn 1994, blood samples from all female animals in the herd were collected once yearly until 1999. The sera were analysed for presence of IgG1 antibodies to N. caninum by the iscom ELISA, and by an avidity ELISA to establish the timing of infection. In addition, data on reproductive performance were compiled. During the study the percentage of seropositive female animals increased from 63% to 87%. In 1994 a large number of young animals tested seropositive although their dams were seronegative, indicating that a transmission of the parasite other than the vertical had recently occurred. Low avidity values supported this assumption. The annual abortion rate increased from a mean of 2% before the initiation of the study to 9% in 1994-1998. During the same time, as judged by the avidity data, a large proportion of the animals shifted from being recently to being chronically infected. The source of the external infection in the herd could not be identified.     

Different humoral mechanisms against Neospora caninum infection in purebreed and crossbreed beef/dairy cattle pregnancies

The antigen-specific IgG subclass response may be a convenient indicator of the underlying nature of T helper cell regulation. The aim of the present study was to identify possible differences in Neospora caninum-specific total plasma IgG, IgG1 and IgG2 antibody levels in purebreed and crossbreed pregnancies throughout gestation in beef and dairy cattle chronically infected with N. caninum. Comparisons were also made between aborting and non-aborting dams. The population examined comprised 96 pregnant parous cows seropositive for N. caninum. Plasma antibodies were determined on Days 90, 120, 150, 180 and 210 of gestation or until abortion. Of the 96 pregnancies examined, 12 ended in abortion. None of the 14 Holstein-Friesian (HF) cows inseminated with HF semen (HF-HF group) aborted, whereas 6 (11.0%) of the 54 HF cows inseminated with Limousin semen (HF-L group) and 6 (21.4%) of the 28 Rubia Gallega (RG) beef cows inseminated with RG semen (RG-RG group) aborted. In the 84 non-aborting cows, a significant positive effect of gestation day was observed on total IgG, IgG1 and IgG2 antibodies levels (P<0.0001 for the three variables). In RG-RG cows, significantly higher levels of IgG (P=0.003; d.f.=2; F-value=6.41), IgG1 (P<0.001; d.f.=2; F-value=10.55) and IgG2 (P=0.004; d.f.=2; F-value=5.82) antibodies against N. caninum were recorded throughout gestation compared to the other groups, whereas the levels of these antibodies were significantly lower in HF-HF on Days 180 and 210 of gestation. In aborting cows, significantly lower IgG (P=0.001; d.f.=1; F-value=25.21) and IgG2 (P=0.001; d.f.=1; F-value=20.39) antibody levels were observed in the RG-RG cows compared to the HF-L cows, whereas no significant effect on IgG1 antibody levels was detected in the two groups with aborting animals (RG-RG and HF-L). Our findings indicate that humoral mechanisms against N. caninum infection and abortion differ in purebreed pregnancies and crossbreed pregnancies in beef/dairy cattle.     

Epidemiological survey of Neospora caninum infection in dogs from Romania

Native cathepsin-L cysteine proteinase (28 kDa) was purified from the excretory secretory products of Fasciola gigantica and was used for sero-diagnosis of F. gigantica infection in buffaloes by Dot-enzyme-linked immunosorbent assay (Dot-ELISA). The test detected F. gigantica field infection in these animals with a sensitivity of ～ 90%. No specific IgG antibody binding was displayed by sera obtained from 76 buffaloes considered to be Fasciola and other parasite-free by microscopic examination of faeces and necropsy examination of liver, rumen and intestine. Additionally, sera from 156 Fasciola-free buffaloes, yet infected with Gigantocotyle explanatum, Paramphistomum epiclitum, Gastrothylax spp., Strongyloides papillosus and hydatid cyst were all negative, indicating that F. gigantica cathepsin-L cysteine proteinase does not cross-react with these helminth parasites in natural infection of the host. The data indicated that cathepsin-L cysteine proteinase based Dot-ELISA reached ～ 90% sensitivity and 100% specificity with relation to above parasites in the detection of bubaline fasciolosis. The present Dot-ELISA diagnostic assay is relevant to the field diagnosis of F. gigantica infection in buffaloes.     

A longitudinal study of Neospora caninum infection on three dairy farms in Brazil

Neospora caninum is an apicomplexan protozoan parasite that is one of the most important infectious causes of abortion in both dairy and beef cattle in many countries. The objectives of this longitudinal study were to determine the prevalence, rates of vertical and horizontal transmission of N. caninum and hazard for culling of N. caninum-seropositive animals in three Brazilian dairy herds. Blood samples from all animals were collected nine times at each of the three farms over a two-year period. Serum was tested for antibodies against N. caninum using the indirect fluorescent antibody test with a cutoff value of 1:100. The percentage of N. caninum-positive samples at each sampling time ranged at Farm I from 3.32% to 11.71%, at Farm II from 3.90% to 22.06% and at Farm III from 3.90% to 22.06%. The number of positive serological reactions varied in relation to the number of repeated samples taken from individual animals at each farm. In all herds, there was a high degree (P<0.05) of association between the N. caninum serological status of dams and daughters. The seropositive conversion rate was estimated as 0.37%, 3.00% and 6.94% per 100 cow-years at Farms I, II and III, respectively. The seronegative conversion rate was estimated as 31.58% and 11.11% per 100 cow-years at Farms I and III, respectively. In all herds, there was no difference (P>0.05) in the culling rate between the cattle that were seropositive cattle and seronegative for N. caninum infection. The results from this study confirm the importance of vertical transmission in the epidemiology of N. caninum. Although a few positive seroconversions indicated horizontal transmission, it does not appear to be the major route of infection for N. caninum.     

Serological investigation of aborted sheep and pigs for infection by Neospora caninum

Serology for Neospora caninum was undertaken using direct ELISAs on sera from 660 aborted sheep and 454 breeding sows, which had aborted or were considered infertile. All ovine sera were further tested by indirect fluorescent antibody test (IFAT) for N. caninum, and a latex agglutination test (LAT) for Toxoplasma gondii was performed on 423 of the samples, including all those positive by ELISA. ELISA-positive porcine sera were tested by IFAT and an inhibition ELISA for antibodies to N. caninum and by LAT for T. gondii. Only 3 (0.45%) of the ovine sera were seropositive for N. caninum by both ELISA and IFAT whereas although 40 porcine sera were seropositive by ELISA all were negative by IFAT. The results suggest that environmental exposure to N. caninum occurs rarely in sheep and pigs.     

Sero-epidemiological survey of Neospora caninum infection in dogs in north-eastern Italy

Risk factors associated with Neospora caninum seroprevalence in north-eastern Italy in healthy dogs were assessed. Antibodies to N. caninum were found in 10.9% of 707 kennel and owned dogs by a commercial competitive ELISA (VMRD Inc.). All dogs were negative for Leishmania infantum by indirect fluorescent antibody test indicating no cross reactivity or association between the two protozoa in this area. Seroprevalence association with breed and age of dogs and other factors are discussed.     

Comparison of serological methods for the diagnosis of Neospora caninum infection in cattle

The aims of this study were to evaluate the performance and agreement of various commercial and in-house Neospora caninum antibody assays used in dairy cattle in North America, and to investigate reproducibility of two assays performed in different laboratories. From 1998 to 2005, three enzyme linked immunosorbent assays (ELISAs, a competitive ELISA-VMRD Inc., an indirect ELISA-Biovet Inc., and another indirect ELISA-Herdchek IDEXX Corp.), two indirect fluorescent antibody tests (IFATs, VMRD Inc., and in-house USDA) and one N. caninum agglutination test (NAT, in-house USDA) were utilized to test 397 randomly selected dairy cattle serum samples from 34 herds in eastern Canada for antibodies to N. caninum. The manufacturers' recommended cut-off values were used to evaluate test performance and agreement between tests. One IFAT (VMRD Inc.) performed well (sensitivity and specificity: 0.97 and 0.97, respectively) using reference sera (n = 452), therefore, results from this IFAT on the 397 samples could subsequently be used as the reference standard to calculate test characteristics for the other assays. Only 11% of the 397 sera were found to be N. caninum-positive with the IFAT. Prevalence-adjusted bias-adjusted kappa (PABAK) ranged from 0.06 to 0.99. Positive agreement was moderate to very good (P(pos) = 0.25-0.96). Negative agreement was very good for all assays (P(neg) > 0.94) except NAT (P(neg) = 0.66). Sensitivity was > or =0.89 for all assays except the NAT, which had a significantly lower sensitivity (0.66). Specificity was high (>0.94) for all assays except for one indirect ELISA (specificity = 0.52). This indirect ELISA did not perform satisfactorily when used in 1998, but an improved version of the ELISA performed as one of the best assays in 2004. Reproducibility of the competitive ELISA was excellent, but the reproducibility of the indirect ELISA that was improved was low (concordance correlation coefficient = 0.90 and 0.36, respectively). The performance characteristics observed for most assays in this study make them useful for screening antibodies to N. caninum in cattle.     

牛新孢子虫抗体Dot-ELISA检测方法的建立

新孢子虫病(Neosporiasis)是由犬新孢子虫(Neospra caninum)寄生于犬、牛、羊等多种哺乳动物细胞内而引起的一种原虫病[1-2].该病主要引起母畜的流产、死胎或新生胎儿的运动障碍和神经系统等症状,该病对牛的危害尤为严重,可垂直传播,带虫母牛可将虫体直接传给新生犊牛,已证实该病是造成养牛业严重经济损失的一个重要原因[3].     

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.     

A nationwide survey on seroprevalence of Neospora caninum infection in beef cattle in Uruguay

Bovine abortions due to Neospora caninum infection have been reported worldwide and its economic impact on the beef industry has been acknowledged as a problem. Uruguay has the largest export value of beef per acre in South America. However, no data on the prevalence of N. caninum infection have been available in this country. The objective of this study was to estimate the prevalence and distribution of N. caninum infection in beef cattle in Uruguay through a nationwide survey. A two stage sampling design was used with farms being selected in stage one and animals being selected in stage two. A brief questionnaire was administered on each farm. Seroprevalence of N. caninum in 4444 beef cattle from 229 farms in all the counties, except Montevideo, of Uruguay was determined by an ELISA. The data were then analyzed to identify associations between infection and variables such as type of animal (cow or heifer), herd size, use of veterinary advice, productivity of the soil in relation to the national average, use of improved grass, use of mineral salts, use of supplemental feed, and presence of a dog(s) on the farm. The estimated proportion of positive farms for all the beef cattle operations was 69.2% (95% confidence interval [CI], 53.7-84.7). The overall cattle seroprevalence was estimated as 13.9% (95% CI, 11.6-16.3). The prevalence estimation by animal category was 14.3% (95% CI, 11.4-17.2) for beef cows and 12.9% (95% CI, 10.0-15.8) for beef heifers. There was no significant difference in the estimated prevalence between the two animal types. There was no significant difference in the animal level prevalence of N. caninum infection among different herd sizes. None of the herd demographic or management variables was significantly associated with the seropositivity to N. caninum infection. In conclusion, these results show that N. caninum infection is common among beef herds across Uruguay. Since the beef industry is one of the key industries in Uruguay, the economic effect and risk factors of N. caninum infection among beef cattle in this country should be further evaluated in the near future.     

Seroprevalence of Toxoplasma gondii and Neospora caninum infection of cats in Hungary

Blood samples were collected from 330 cats in Hungary in order to evaluate their seroconversion to Toxoplasma gondii and Neospora caninum using the indirect fluorescent antibody test (IFAT). The overall prevalence of toxoplasmosis was 47.6%, the prevalence being 22.4% among urban, 50% among suburban and 61.3% among rural animals. Significantly more cats had high IFAT titres (1:640 to 1:5120) in the countryside. Female cats were more frequently infected with T. gondii than males (53.3% vs. 39.3%), and seropositivity increased with the age of animals. The prevalence (0.6%) and titre (1:40) of antibodies to N. caninum was low. Sixty-two cats were also screened for seroconversion to feline infectious peritonitis (FIP) virus. Higher titres to T. gondii were more frequently detected among FIP-positive cats, but this difference was non-significant due to the small number of cats with concurrent infection.     

A longitudinal study of Neospora caninum infection on a dairy farm in New Zealand

A 600-cow New Zealand dairy herd experienced an abortion storm in 1997 and was monitored (blood sampling at about 3-month intervals) from May 1997 until January 1999. Abortion risk reached 9% in 1997 and was highest in heifers at 19%. The abortion risk decreased in 1998 to 3.2% (still somewhat higher than during the years prior to the outbreak). The serological reaction pattern for Neospora caninum showed an association with abortion risk only around the time of the 1997 outbreak when seropositive cows were 4.2 times more likely to abort than negative ones. Over the whole study period, only 27% of cows that were sampled on all nine visits always tested negative. Offspring from dams which had positive tests for Neospora caninum were 2.4 times more likely to abort than those from dams testing consistently negative. Controlling for age and breed, seropositive cows produced more milk than those that were consistently negative. Infection might have been present endemically within this herd prior to the epidemic, but in 1997 an additional factor appeared to have triggered the outbreak.