In vitro isolation and characterization of bovine Neospora caninum in Korea

The brains of nine aborted bovine fetuses and two newborn calves born from dams suspected to be infected with Neospora caninum were homogenized and inoculated into Vero cells. All fetuses and calves were from cows determined as seropositive to N. caninum by an IFA test. Sera and thoracic fluids of all fetuses and calves also revealed high antibody titer to N. caninum by IFAT ranging from 1:800 to 1:3200. N. caninum was isolated from the brains of one aborted fetus and one newborn calf when the brain homogenates were grown continuously in Vero cell culture. N. caninum tachyzoites, giemsa-positive, were first observed on Days 45 and 56 postinoculation in the newborn calf and the aborted fetus, respectively. The isolates (KBA-1 and KBA-2) were morphologically and ultrastructurally similar to previously published Neospora isolates. The isolated parasites were confirmed as N. caninum by means of the antigenic reactivities, immunostaining, PCR and southern blotting, and electron microscopy.     

Risk of abortion associated with Neospora caninum during different lactations and evidence of congenital transmission in dairy cows

OBJECTIVE: To examine the relationship between exposure to Neospora caninum and abortion in dairy cows during their first, second, third, and fourth or later lactations and to establish the main mode of transmission in female calves from birth until their first pregnancy was terminated by abortion or parturition. DESIGN: Prospective observational study. ANIMALS: 460 Holstein cows and 79 female calves. PROCEDURE: Cows were classified as seropositive or seronegative to N caninum within 7 days after calving; incidence of abortion was compared between groups during different lactations. Blood samples were collected from female calves before ingestion of colostrum and every 6 months until their first pregnancy was terminated by abortion or parturition; number of seropositive calves was compared between seropositive and seronegative dams. RESULTS: During the first pregnancy of their second lactation, risk of abortion for seropositive cows was 2.8 times that of seronegative cows. Among 10 calves born to seropositive cows, 4 were classified as seropositive at birth and thereafter. Among 69 calves born to seronegative cows, all were classified as seronegative at birth; 67 calves remained seronegative thereafter. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure to N caninum alone was not significantly associated with abortion in cows during the first, third, and fourth or later lactations. Seropositive cows that have aborted previously may have subsequent abortions attributable to N caninum. Congenital infection was the main mode of N caninum transmission in a cohort of female calves.     

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.     

Experimental studies on the transmission of Neospora caninum between cattle

Three studies were conducted to investigate the transmission of Neospora caninum between cattle by the oral route. In the first study, six calves were dosed with 10(7)N caninum tachyzoites (NC LivB1) in colostrum and/or milk replacer on four occasions. In the second study, two calves and two cows were fed placental tissues from N caninum -infected cows, and, in the third study, seven uninfected calves were fostered onto N caninum -infected dams. In the first study, all six calves developed antibody responses and five calves developed antigen-specific lymphoproliferation responses, including two calves initially challenged at 1 week of age. No evidence of N caninum infection was found in the brain or heart of these calves by histology or polymerase chain reaction (PCR). In the second and third studies, there was no evidence of N caninum infection in any of the calves and cows. The results confirm that calves up to 1 week of age can be experimentally infected via the oral route, but suggest that this is not an important natural route of transmission for N caninum between cattle.     

Economic considerations for diagnostic and control options for Neospora caninum-induced abortions in endemically infected herds of beef cattle

OBJECTIVE: To compare economic outcome for herds not exposed to Neospora caninum with that for herds with various seroprevalences of N caninum infection and evaluate 3 control strategies. DESIGN: Economic simulation model. SAMPLE POPULATION: Beef herds with various seroprevalences of N caninum infection. PROCEDURE: A 5-year simulation model was used. Control strategies that were evaluated included culling females that fail to calve, selling seropositive females and purchasing seronegative replacements, and excluding the daughters of seropositive dams as potential replacements. RESULTS: For a 5-year period with low prices for feeder calves, endemic N caninum infection decreased mean return to fixed assets by 22.2% when true seroprevalence was 10% and by 29.9% when true seroprevalence was 70%. Percentage decrease in return to fixed assets was less dramatic when a 5-year period with high prices for feeder calves was evaluated. Analysis indicated that 2 control strategies (culling females that fail to give birth to a calf and selling seropositive female cattle and purchasing seronegative replacement female cattle) were not likely to be economically beneficial. The third control strategy (testing the entire herd for N caninum infection and excluding the female offspring of seropositive dams as replacements) appeared to be a reasonable control strategy. CONCLUSIONS AND CLINICAL RELEVANCE: For the assumptions in the model, endemic N caninum infection decreases return to fixed assets for cow-calf herds. Of the potential control strategies evaluated, testing the entire herd for N caninum infection and excluding the daughters of seropositive dams as potential replacements provided the best economic return.)     

Application of the Neospora caninum IgG avidity ELISA in assessment of chronic reproductive losses after an outbreak of neosporosis in a herd of beef cattle.

Point-source infections are most likely the cause for Neospora caninum-induced abortion outbreaks in cattle, whereas an increased annual abortion rate may be a consequence of vertical transmission. The aims of the present study were to examine the reproductive effects of neosporosis in a beef herd for 3 years, after a point-source outbreak and to use IgG avidity serology to examine the chronicity of infections and patterns of transmission. During the study, 76-78% of animals were seropositive for N. caninum. The pregnancy rate varied from 88% to 94%, without any reduction in the pregnancy rate of seropositive cows compared with seronegative cows. The annual abortion rate was 2.5-5.5%, and all but 1 abortion occurred in seropositive dams. The efficiency of vertical transmission was estimated to be 85%. Several calves, born to seronegative dams, were seropositive at 6-13 months of age, indicating a 22% mean annual rate of horizontal transmission. The mean avidity in seropositive cows increased from 30 during the initial outbreak to 74 after 3 years. The mode of IgG avidity was 21-40 during the initial abortion outbreak, 41-60 after 1 year, and 61-80 after 2 and 3 years. The results reveal high annual rates of both vertical and horizontal transmission of N. caninum in a herd of beef cows and provide further validation on the ability of the N. caninum IgG avidity ELISA to accurately assess the chronicity of infection.     

Studies on Neospora caninum DNA detection in the oocytes and embryos collected from infected cows

Neosporosis is a major cause of abortion in cattle over the world. One of the methods of preventing vertical transmission within the herd is to avoid breeding replacement heifers from infected dams. Another procedure suggested and recommended by the International Embryo Transfer Society (IETS) is embryo transfer (ET) from infected dams into uninfected recipients. Oocytes and embryos taken from seropositive cows were examined for the presence of Neospora caninum DNA. A modified PCR protocol using Np21 and Np6 primers was applied to detect parasite DNA in the samples. The expected 328 bp product was not obtained in oocytes and/or embryos collected from seropositive dams. The results confirmed that transfer of the embryos from seropositive donors into seronegative recipients is an appropriate method to eliminate vertical transmission of neosporosis in a herd. The present study demonstrated that oocytes and embryos are not exposed to N. caninum in the uterine cavity of seropositive dams.     

Postnatal neosporosis in dairy cattle in northeast Thailand

A total of 83 dairy cows in Loei Province (Muang) and Nong Bua Lamphu (NBL) Province, northeast Thailand were sampled three times within 6 months in 1998 and their sera were examined for antibodies to Neospora caninum at a dilution of 1:100 in the indirect fluorescent antibody test (IFAT). In Muang the seroprevalence of N. caninum was 37.5% (June), 60% (August), and 62.5% (November). In NBL the prevalence was 50% (August) and 70% (November). In both areas abortions were observed between 1 and 3 months after the introduction of these cattle from another area. Nine of 14 and seven of 17 calves were descendants of seropositive dams, of which only two calves from Muang and two calves from NBL were positive for N. caninum antibodies. These findings suggest postnatal N. caninum transmission.     

Survey of Neospora caninum antibodies in dairy and beef cattle from five regions of the United States

Serum samples from 4,907 cows from 93 dairy and five beef herds were analyzed for antibodies to Neospora caninum by commercially available ELISA. The herds were recruited from 20 states and the territory of Puerto Rico. A questionnaire assessed the herd's abortion history and exposure risks to N. caninum. Results showed that 788 (16%) cows tested positive for antibodies to N. caninum. At least one positive animal was detected in 90% of the herds tested. The prevalence of cows seropositive to N. caninum varied from 2% to 65% among herds. Abortion rates ranging from 2% to 19% were reported. The epidemiologic data collected from the questionnaire suggested that the presence of farm dogs does not increase the risk of exposure of cows to N. caninum.     

Neospora caninum infection associated with stillbirths in captive antelopes (Tragelaphus imberbis)

Neosporosis was diagnosed in full-term stillborn twin calves of captive antelopes (Tragelaphus imberbis) in a German zoo. In both calves a multifocal non-suppurative encephalitis was present and infection with Neospora (N.) caninum was confirmed by foetal serology and PCR. N. caninum-DNA was detected in brain, heart, lung, liver and spleen of a third full-term stillborn calf. Histopathological lesions were absent in this calf and antibodies against N. caninum were not detected in pleural fluid. Immunohistochemistry failed to demonstrate N. caninum antigen in all three calves.     

Neospora caninum: oocyst challenge of pregnant cows

Three pregnant cows were each orally challenged at 10 weeks of gestation with 600 sporulated oocysts of Neospora caninum. The number of oocysts was limited by those available. In concurrent bioassays, one oocyst per os infected each of two gerbils. Challenged cattle developed Neospora-specific antibody, cell proliferation and gamma-interferon responses. N. caninum specific PCR demonstrated persisting infection in the brains of cows 4 months after calving. Abortion was not induced and there was no evidence of transplacental infection in the healthy calves born at full-term. This experiment suggests that the dose threshold for induction of abortion exceeds 600 oocysts.     

Diagnostic significance of Neospora caninum DNA detected by PCR in cattle serum

A nested PCR that successfully detected Neospora caninum DNA in serum of cattle was used for investigation of selected abortion cases and in a study of healthy pregnant cows at an abattoir. N. caninum DNA was not detected in serum from antibody positive dams that aborted due to N. caninum, but was present in serum of some antibody negative dams that aborted due to other causes. N. caninum DNA was also found in the serum of about half of the animals that aborted of undetermined cause, but was not detected in cow sera from two beef cattle herds in Western Australia with no recent history of abortion. In the abattoir study of 79 dams and their foetuses N. caninum DNA was found in serum of 3 dams and in material from 11 foetuses. The majority of the cows and all foetuses were antibody negative. Our findings suggest that there is no obvious relationship between the presence or absence of N. caninum DNA in serum and the presence of antibodies to N. caninum in dams, the presence of N. caninum DNA in foetuses or abortion due to N. caninum. This is the first report of the detection of N. caninum DNA in serum of cattle rather than the white blood cell fraction. It indicates the presence of free tachyzoites and/or parasite DNA in circulation. The results suggest that persistent infection in the absence of antibodies is a possible outcome of N. caninum infection. Infection of foetuses in the absence of antibodies supports the possibility of persistent infection due to immunotolerance to an early in utero infection. It is therefore important to test for N. caninum DNA as well as antibodies for the detection of exposed and/or infected animals. However, the presence or absence of N. caninum antibodies or DNA did not support nor exclude N. caninum as the cause of abortion. Additional criteria are required for a positive diagnosis of abortion caused by N. caninum.     

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.     

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   

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.     

Evaluation of the embryo transfer procedure proposed by the International Embryo Transfer Society as a method of controlling vertical transmission of Neospora caninum in cattle

OBJECTIVE: To evaluate efficacy of embryo transfer into seronegative recipients, using the procedure proposed by the International Embryo Transfer Society (IETS), for preventing vertical transmission of Neospora caninum in cattle. DESIGN: Prospective clinical trial. ANIMALS: 87 recipient cows and heifers and their embryo transfer calves from 22 donors originating from 9 dairy herds. PROCEDURE: Neospora caninum serologic status of donors and recipients was determined before collection and transfer of embryos. Viable embryos were washed and treated with trypsin. Recipients in experimental groups A (n = 50) and B (29) were seronegative and received embryos from seropositive and seronegative donors, respectively. Recipients in group C (n = 8) were seropositive and received embryos from seronegative or seropositive donors. Antibody titers against N caninum were determined monthly during pregnancy in recipients and in calf blood samples collected at birth. Tissues collected from stillborn calves and aborted fetuses were analyzed histologically and by immunohistochemical (IHC) methods. RESULTS: 76 calves and 11 fetuses and stillborn calves were examined. All calves from groups A and B were seronegative (n = 70) or lacked evidence of infection by use of tissue analysis (9). In group C, 5 of 6 calves were seropositive at birth, and IHC results were positive for 1 of 2 calves. Vertical transmission rate was significantly lower in groups A and B (0%) than in group C (75%). CONCLUSION AND CLINICAL RELEVANCE: Embryo transfer into seronegative recipients, using the procedure proposed by IETS, is an effective way to prevent vertical transmission of N caninum. Results provide support for pretransfer testing of all embryo transfer recipients.     

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.     

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.     

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.     

Herd-level risk factors for Neospora caninum seroprevalence in dairy farms in southern Brazil

A cross-sectional study was used to test the relationship between herd seroprevalence to Neospora caninum and various potential herd-level risk factors in 60 dairy farms located in two distinct regions in southern Brazil. Thirty farms were randomly selected from within each region. A questionnaire was designed to summarize each farm's production system as it might relate to N. caninum transmission. The questionnaire contained 105 closed questions relating to general characteristics of the farms, farm facilities, management, source of food and water, herd health, environment and biosecurity, which included questions relevant to N. caninum transmission, including presence and number of dogs and other animals, purchase of animals and contact with man. Serum samples were collected from 40% of animals in each farm and N. caninum antibodies were detected by immunofluorescent antibody test (IFAT). The association between potential risk factors and the probability of an animal being seropositive was modeled using a generalized estimation equations (GEE) logistic regression model. The model accounted for multilevel correlation of data from multiple animals within herds. The mean (+/-S.D.) number of animals in the 60 herds was 64.5 (+/-45.6), ranging from 20 to 280 females. Blood samples were collected from 1549 animals. The size of the farms varied from 4 to 100 ha (mean 30.1+/-25.9 ha). At least one dog was found in 57 of the 60 dairy farms (95%). The mean number of dogs was 3.1 (+/-1.9), ranging from 0 to 10. All females were raised on pasture. For all cattle sampled, N. caninum seroprevalence was 17.8%. Overall, 93.3% of herds (56/60) had at least one seropositive animal identified. Four variables were significantly associated with N. caninum sero-response in the 57 dairy farms, which were included in the final multivariable model: the number of dogs on the farm, farm area (hectares), feeding pooled sources of colostrum and region. The odds of a cow being seropositive increased 1.13 times for each additional dog present on the farm (P=0.021). Cattle from farms that fed calves colostrum pooled from multiple cows had 1.79 times greater odds for being seropositive for N. caninum (P<0.003). The probability of being seropositive was inverse to the area of the farms, such that cattle had 0.92 times the odds to be seropositive (P=0.014) for each additional 10 ha of farmland. Finally, cattle from farms in region one had 0.71 times the odds to be seropositive than cattle from region two (P=0.035). Results of this study suggest that several risk factors may explain why dairy cattle in Brazil may become exposed to N. caninum. However, further investigation of these factors is necessary because the purpose of this study was to refine and generate hypotheses on N. caninum transmission.