奶牛新孢子虫病血清流行病学调查

本研究用酶联免疫吸附试验（ELISA）对采集自北京地区的94份奶牛血清（随机采集）和河北地区的55份奶牛血清（有流产史奶牛），进行Neospora caninum血清抗体检测。结果发现，北京地区随机采集的奶牛血清N．caninum抗体阳性率为18．1％（17／94），河北地区有流产史的奶牛N．caninum血清抗体阳性率为23．6％（13／55）。采用牛奶记录体系（DHI）对北京地区17头N．caninum血清抗体阳性牛进行了日产奶量、乳中蛋白率和乳脂率的测定，并与同群牛中134头阴性牛比较。结果表明，N．caninum血清抗体阳性牛日产奶量比阴性牛降低9．7％，乳中蛋白率和乳脂率分别降低20％和15．4％。初步证明N．caninum血清抗体阳性奶牛产奶量降低及奶品质的下降。对不同N．caninum抗体滴度阳性牛的泌乳期主要生产性能比较发现，其生产性能的变化与抗体滴度无明显相关性。     

Induced transplacental transmission of Neospora caninum in cattle.

Three Jersey cows were inoculated SC and IM with 26 million Neospora caninum tachyzoites at 129 (cow 1), 126 (cow 2), and 81 (cow 3) days after mating. Cows remained clinically normal for at least 1 month after inoculation of N caninum. Cow 1 was euthanatized 32 days after inoculation because of gangrenous mastitis. Cow 1 had a live fetus with no gross lesions; however, microscopic lesions were seen in the fetus and consisted of severe nonsuppurative necrotizing encephalitis of the cerebral white matter. Neospora caninum was identified in lesions by staining with anti-N caninum serum in an immunohistochemical test, by bioassays in mice, and by inoculation of bovine monocyte cultures with fetal tissue homogenate. Neither N caninum nor lesions were associated with infection with the protozoon identified in tissues of cow 1. Cows 2 and 3 aborted small autolysed fetuses 101 and 74 days, respectively, after inoculation with N caninum; the fetuses and attached placenta were unsuitable for laboratory investigations. Cows 2 and 3 remained clinically normal 4 months after abortion. Results of this study indicated that N caninum can be transmitted transplacentally in cattle.     

Neospora caninum serostatus and culling of Holstein cattle

OBJECTIVE: To determine whether time until culling or risk of culling was associated with Neospora caninum serostatus among Holstein cattle in dairy herds in Ontario. DESIGN: Retrospective cohort study. ANIMALS: 3,412 cows in 56 herds. PROCEDURE: Blood samples were collected, and serum was tested for antibodies against N. caninum. Information on cows that were culled was collected during the 1- to 2-year period that producers were unaware of serostatus of individual cows in their herds. RESULTS: Herd prevalence of N. caninum-seropositive cows ranged from 0 to 68.3% (median, 7.0%). During the time of the study, 184 of 359 (51.3%) N. caninum-seropositive cows were culled, compared with 1,388 of 3,053 (45.5%) seronegative cows. Mean time from blood sample collection to culling for seronegative cows (289 days; 95% confidence interval, 280 to 299 days) was not significantly different from mean time for seropositive cows (296 days; 95% confidence interval, 269 to 323 days). Survival analysis indicated that N. caninum serostatus was not associated with time to culling or risk of culling. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that N. caninum serostatus of Holstein cows in Ontario was not significantly associated with either time to culling or risk of culling. Thus, N. caninum serostatus alone should not be used to determine whether cows should be culled.     

Neospora caninum seroprevalence in dairy cattle in central Thailand

The seroprevalence, in dairy cattle, of antibodies to Neospora caninum, the relationship between seropositivity and age (heifer versus cow), the relationship of herd infection with herd size and the relationship of herd infection with the presence of dogs on the farm were studied. The study involved 549 cows and 82 dogs in 59 dairy herds in Nakhon Pathom, Thailand. A competitive enzyme-linked immunosorbent assay (cELISA) with NC-specific monoclonal antibody was used to detect the NC antibodies in the sera. Individual and herd seroprevalence of NC were 5.5% (30/549) and 34% (20/59), respectively. No significant relationships between NC seropositivity with the age of the cows (heifer versus cow; P > 0.05) and between herd infection and the presence of dogs on the farm (P > 0.05) were found. Herd size significantly affected herd infection (P < 0.05) with higher infection in large than small herds (> or = 21 versus < or = 20 cows). Of 12 cows with a history of abortion, one was seropositive to NC. The seroprevalence of NC antibodies in dogs was 1.2% (1/82). This is the first NC seroprevalence study in dogs in Thailand. It was concluded that Neospora infection was more common at the herd level rather than the individual level in Thailand and the presence of dogs on the farm was not related to the level of herd infection. Caution should be taken in the interpretation of serological tests from the farm dogs.     

Monensin use against Neospora caninum challenge in dairy cattle

Using a randomized controlled trial design, a randomly allocated intervention group of 15 cows received a slow-release bolus that delivered 100 days of monensin. The negative control group of 15 cows received a placebo bolus that was identical to the monensin bolus, except without the monensin. Two weeks after bolus administration, all cows were challenged with a 2 ml subcutaneous injection of a live tachyzoite suspension. Whole blood and serum samples were collected from each cow every week for the first month post-challenge, and then every 2 weeks for the next 2 months. The extracted DNA from whole blood was tested for the Nc-5 gene fragment of Neospora caninum using a quantitative real time polymerase chain reaction. Serum was tested for antibodies to N. caninum using the IDEXX ELISA. Cows treated with monensin boluses had a significantly lower humoral immune response than cows treated with placebo boluses at one time point post-challenge (week 4 post-challenge). However, when adjusting for repeated measures within cows, the P value for this humoral difference was 0.098. No DNA for N. caninum was detected in either group, likely due to study design features.     

Udder health in dairy cattle infected with Neospora caninum

Blood samples were collected from 3449 cows on 57 representative Ontario dairy herds during the summer of 1998 and analysed for antibody to Neospora caninum using an ELISA. Forty-eight herds (2742 cattle) contained at least one N. caninum-seropositive animal. Two composite milk samples were collected from all cattle: the first on the day of blood collection and the second 68 to 365 days later. All milk samples were submitted for bacteriological culture. Ontario Dairy Herd Improvement Corporation (DHI) data were available for 3162 cattle in the 57 herds at the time of bleeding. Furthermore, complete DHI data were available for 1658 cattle that were culled between 12 and 24 months following blood collection. Using a standardised ELISA sample-to-positive (S/P) cut-off of ≥0.45, the corrected seroprevalence was 8.2% overall and 10.1% within seropositive herds. At blood collection the odds of N. caninum-seropositive cows having a high linear score (≥4.0; equivalent to a somatic cell count ≥200,000 cells/ml) was 27% less than for seronegative animals. Similarly, at the time of culling, the odds of having a high linear score was 22% less in N. caninum-seropositive cattle. Overall, linear score was lower in N. caninum-seropositive cattle at culling. After controlling for herd, parity, days in milk, and the interval between collection of milk samples, the odds of N. caninum-seropositive cattle testing positive for an environmental pathogen (i.e. environmental Streptococcus species and coliforms) on the second milk sample was 56% less than for seronegative animals. The odds were 83% less at a higher ELISA S/P cut-off of ≥0.70. Finally, the odds of N. caninum-seropositive cattle developing a new infection with a major pathogen (environmental or contagious) were 60% less than seronegative cows using the higher ELISA S/P cut-off.     

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.     

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.     

Seroprevalence of Neospora caninum in beef cattle in northern Alberta

Blood samples were collected from 1806 pregnancy-tested cows from 174 herds at a northern Alberta auction mart in the fall of 1998. One hundred sixty-two (9.0%) of these samples were positive for antibodies to N. caninum. Thirty-five of 260 samples (13.5%) collected from the same region in the 1980s were also serologically positive for N. caninum.     

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.     

Seroprevalence of Neospora caninum in aborting dairy cattle in the Czech Republic

A serological survey for antibodies against Neospora caninum in aborting cattle was carried out in the Czech Republic. Serum samples from 463 aborting dairy cows originated from 137 farms from different parts of the Czech Republic were tested for presence of N. caninum antibodies by use of an enzyme-linked immunosorbent assay (ELISA) and an indirect fluorescent antibody test (IFAT). Antibodies (> or = 1:640) to N. caninum were found in 18 (3.9%) of 463 aborting cows. Farm prevalence in aborting cows was 12.4% (17/137). The antibody titres of cows were 1:200 (9 cows), 1:640 (7 cows), 1:1280 (3 cows), 1:2560 (3 cows), 1:5120 (3 cows), 1:10,240 (2 cows) and 1:20,480 (0 cow). A case-control study was conducted to estimate the association of N. caninum infection and abortion. For this 407 serum samples were collected from cows on five dairy farms with repeated occurrence of endemic and sporadic abortion of unidentified etiology. These samples were obtained from aborting cattle (n=44) and normally calving cattle (control group; n=363) and tested for N. caninum antibodies by an immunofluorescent antibody test (IFAT). Overall, 3.19% (13/407) of cows sampled had positive N. caninum fluorescence with a cut-off titre of 1:200. The prevalence of N. caninum was significantly higher (P<0.05) in the aborting group (13.64%; 95% confidence interval (CI): 5.2, 27.4) than in the control group (1.93%; 95% CI: 0.8, 3.9). A strong association between seropositivity and abortion was found, with seropositive cows being eight times more likely to abort than seronegative cows (odds ratio=8; 95% CI: 2.6, 25.1). This first report on the serological prevalence of N. caninum in cows in the Czech Republic verified a strong association between N. caninum infection and abortions in five dairy farms. Thus, the neosporosis should be considered in differential diagnosis of bovine abortion.     

Characterisation of the first Australian isolate of Neospora caninum from cattle

OBJECTIVE: To isolate Neospora caninum from a congenitally infected calf. PROCEDURE: A calf was obtained from a N. caninum infected dam maintained in a dairy herd of Holstein-Friesian cattle located on the south coast of NSW near Nowra. The calf was euthanased and samples collected for serology and pathology. Samples of brain and spinal cord of the calf were homogenised and injected into immunocompromised mice in an attempt to recover protozoa by in vivo culture. Sequential passage of brain homogenate through IFNgammaPKO mice was performed and tissue culture flasks were inoculated with brain homogenate. Parasites were identified by electron microscopy and DNA sequencing. The antigen profile of the isolate was analysed using Western blotting. Pathogenicity was examined in BALB/c mice and transmission of the parasite during pregnancy was examined in Qs mice. RESULTS: The calf was seropositive for N. caninum and histopathological examination of sections of cerebrum identified lesions consistent with a very mild infection with N. caninum. The parasites isolated using tissue culture were identified as N. caninum, based on the sequence of the ribosomal DNA and electron microscopy. The antigen profile of the new isolate was similar to that of the NC-Liverpool isolate, but quite different from that of Toxoplasma gondii. In BALB/c mice inoculated with the new isolate, severe clinical signs developed in only three of ten infected mice, compared with six of ten mice infected with NC-Liverpool. Mild to moderate nonsuppurative encephalitis was observed in BALB/c mice infected with the new isolate, compared with mice infected with NC-Liverpool, that developed severe nonsuppurative encephalitis. Transplacental transmission of the isolate arising from an acute infection during pregnancy occurred in about 87% of pups. CONCLUSION: This is the first isolation of bovine Neospora caninum in Australia. This isolate, called NC-Nowra, appears to be a less virulent form and may prove to be a suitable candidate for vaccine development.     

Serological evidence of Neospora caninum in dual-purpose cattle herds in Venezuela

Bovine neosporosis is a parasitic disease produced by Neospora caninum that induces abortion in cows, and consequently has a negative impact on the herd's reproductive efficiency. The main objective of this research was to determine the serological evidence of N. caninum in cattle herds from Venezuela using an indirect antibody capture ELISA test. Four hundred and fifty-nine (459) serum samples from crossbred adult cows were collected to be tested for Neospora antibodies. The sampled cows came from 15 large farms located in eight important cattle states that have predominant dual-purpose production systems (cattle from these farms are used for both milk and meat production). Fifty-two cows (11.3%) were seropositive to N. Caninum. Thirteen (86.7%) of 15 studied herds had cows seropositive to N. caninum. The average within-herd seroprevalence was 11.5% (range 3.8-36.7%). Cows that aborted in some of these farms had 2.71 (P: 0.009) greater odds to be seropositive when compared to cows that did not abort. Each one of the eight states represented in our study had seropositive animals. These results are the first evidence of exposure to N. caninum in Venezuelan cattle herds, indicating the possible circulation of this pathogen in the country. Further epidemiological studies should be granted to determine the spread of the disease in the Venezuelan cattle industry and its associated risk factors.     

Recent advances in Neospora and neosporosis.

Neospora caninum has emerged as a major cause of abortion in cattle in many countries. This paper reviews recent advances in the life cycle and biology of Neospora with the emphasis on bovine neosporosis. The role of the recently discovered oocyst stage of N. caninum in the epidemiology of neosporosis is discussed. Progress made in serologic diagnosis of N. caninum infection is discussed. There is no vaccine for preventing Neospora-induced abortions in cattle or to prevent oocyst shedding in dogs.     

Prevalence of antibodies to Neospora caninum in Korean native beef cattle

A total of 438 sera from Korean native beef cattle in 9 provinces were tested for Neospora caninum antibodies using an immunofluorescent antibody test (IFAT). Eighteen (4.1%) cattle were positive by IFAT. The titers ranged from 1:200 (10 animals), 1:400 (5 animals), 1:800 (2 animals) to 1:1,600 (1 animal). Although the seroprevalence was slightly higher in Chungnam (8.9%), this was not significantly different from those noted in Kyunggi, Kangwon, Kyungbuk, Kyungnam, and Cheju provinces. Sera obtained from beef cattle in the provinces of Chungbuk, Jeonbuk and Jeonnam were all negative. Neospora positive sera were also tested for anti-Toxoplasma gondii antibodies using a commercial latex agglutination test (LAT). Antibody to T. gondii was detected in only 1 (5.6%) of 18 N. caninum positive sera. These results indicate that N. caninum and T. gondii infection are present at a low level in the Korean native beef cattle.     

Isolation of Neospora caninum from dairy zero grazing cattle in Israel

First Israeli Neospora caninum isolates were obtained from brain tissues of aborted fetuses (NcIs491 and NcIs580) from dairy farms endemic for neosporosis and maintaining cattle on zero grazing. Tissues from different parts of the fetus brains were used to infect Vero cells. Tachyzoites of N. caninum were first observed in cultures from days 30 and 32 after infection. To confirm the identity of the isolated parasites, DNA extracts from brains and cultures were tested by PCR with specific primers based on the Nc5 gene. Specific fragments were amplified by PCR from infected cultures of both fetuses on day 25. Susceptible seronegative gerbils (Meriones tristrami) were inoculated intraperitoneally with 10(3) to 10(5) tenfold dilutions of subculture tachyzoites. The inoculated gerbils developed specific antibodies to N. caninum, with end-point serum dilution of 1:4096 in the IFA assay, whereas no neurological signs or deaths were seen during 4 months of observation.     

IgG avidity pattern in cattle after ingestion of Neospora caninum oocysts

The avidity (functional affinity) of specific antibodies are being used to estimate duration of bovine Neospora caninum infection. Here, we report for the first time the avidity pattern in cattle orally inoculated with N. caninum oocysts. In all, 16 pregnant cows and 7 calves were administered N. caninum oocysts. In the cows, the avidity increased during the early course of infection. In all but one, the avidity was < or = 35 during the first 6 weeks after infection and no cow had an avidity value >50 until week 9. The calves were sampled either week 6 (n = 3) or week 9 (n = 9) after infection, and by then had avidities between 2 and 17. The results are in agreement with results from previous investigations of naturally infected cattle, and calves that were experimentally infected with tachyzoites. They further validate the ability of the N. caninum iscom avidity ELISA to accurately assess the duration of infection.     

Neospora caninum and Leptospira serovar serostatus in dairy cattle in Ontario

No significant association existed between Neospora caninum titer and serostatus to Leptospira serovar hardjo, icterohaemorrhagiae, or pomona in cattle on 78 dairy herds in Ontario. Leptospira titer increased with parity. Amongst herds not vaccinated against Leptospira, the proportions of herds with > or = 1 animal seropositive to serovar hardjo, icterohaemorrhagiae, or pomona were 45%, 42%, and 58%, respectively.     

Seroprevalence of Neospora caninum in dairy cattle in Bahia, Brazil.

Sera collected from 447 dairy cattle on 14 dairy farms were tested for Neospora caninum antibodies by use of an immunofluorescent antibody technique. Positive reactions with titres > or =1:200 were found in 63 (14.09%) of animals. Neospora positive sera were also tested for Toxoplasma gondii antibodies by using a commercial latex agglutination test. Antibodies to T. gondii were detected in 3 (4.76%) of 63 N. caninum positive sera. These results indicate that N. caninum infection is widespread among dairy cattle in Bahia state.     

Isolation and molecular characterisation of Neospora caninum in cattle in New Zealand

AIM: To isolate Neospora caninum from the brains of naturally infected cattle and use molecular techniques to characterise the isolates. METHODS: Neospora caninum tachyzoites were isolated in Vero cell culture from the brains of a cow and two calves. The isolates were characterised using polymerase chain reaction (PCR) methods, DNA sequencing, an immunofluorescent antibody test (IFAT), transmission electron microscopy (TEM), and immunohistochemistry (IHC). The brains of the three cattle were subjected to histopathological examination. A pathogenicity study was conducted in 120 BALB/c mice. RESULTS: Neospora caninum tachyzoites were isolated from all three cases and first observed in vitro between 14 and 17 days post-inoculation. Parasites were sub-cultured and maintained in Vero cell culture for more than 6 months. PCR products were generated for all three isolates, using two different primers. Sequencing of the PCR products and a subsequent BLAST search identified the isolates as N. caninum. In addition, the isolates tested positive using IFAT and IHC, and ultrastructure revealed by TEM was characteristic of N. caninum. Histopathological examination revealed lesions characteristic of N. caninum in 1/3 brains. In the pathogenicity study using BALB/c mice, the mortality rate was 3-7%. CONCLUSION: This was the first successful isolation of N. caninum in New Zealand confirmed using molecular characterisation tests.