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。     

Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of paratuberculosis in lactating dairy cows

OBJECTIVE: To determine whether results obtained for milk and serum samples with ELISAs intended for diagnosis of paratuberculosis in dairy cows were comparable to results obtained by means of mycobacterial culture of fecal samples. DESIGN: Cross-sectional study. ANIMALS: 689 lactating dairy cows in 9 Ontario herds. PROCEDURE: Milk, serum, and fecal samples were obtained from all cows. Fecal samples were submitted for mycobacterial culture. Serum samples were tested with a commercially available ELISA for antibodies against Mycobacterium paratuberculosis, and preserved milk samples were tested with an indirect ELISA for antibodies against M paratuberculosis. RESULTS: Results were positive for 130 of the 689 (18.9%) serum samples, 77 of the 689 (11.1%) milk samples, and 72 of the 689 (10.4%) fecal samples. The level of agreement between results for milk and serum samples was only moderate. Proportions of positive results for serum and fecal samples were significantly different, but proportions of positive results for milk and fecal samples were not significantly different. In addition, results for milk samples had a higher level of agreement with results of mycobacterial culture than did results for serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the indirect ELISA used on milk samples may be a convenient method of detecting paratuberculosis in dairy herds.     

Incidence of mycoplasma mastitis in Czechoslovakia

By the mycoplasmic examination of 143 dairy cows exhibiting clinical signs of mastitis, Mycoplasma californicum and M. bovigenitalium were detected as the causal agents of the disease at two localities. The occurrence of specific antibodies in milk, and/or blood serum, was characteristic of the course of the disease, as documented by the results of the examination of more than 300 samples. In some milk samples taken from cows suffering from mastitis of obscure etiology and obtained from State Veterinary Institutes, antibodies to some of the five species of bovine Mycoplasma involved in the disease were found at 22 localities. The antibodies were detected by means of the indirect haemagglutination test and ELISA method. Both these tests are suitable for such an examination, as indicated by the comparison of the results.     

Evaluation of enzyme-linked immunosorbent assay using milk samples as a potential screening test of bovine tuberculosis of dairy cows in Korea

An enzyme-linked immunosorbent assay (ELISA) using bulk tank milk samples was evaluated as a screening test for bovine tuberculosis (TB), a contagious chronic disease of cattle. An ELISA with MPB70, a major antigen of Mycobacterium bovis was performed using paired sets of milk and sera samples from 33 tuberculin-positive and 43 tuberculin-negative cattle. Anti-MPB70 antibodies were detected in milk samples and there was a significant correlation between seroreactivities of milk and sera samples (R² = 0.83). Using the tuberculin skin test as the reference test, the sensitivities of ELISA using milk and sera samples were 87.8% and 81.8%, respectively, and the specificities were 97.7% and 100%, respectively.In the screening test using bulk tank milk samples from 931 dairy herds in Whasung, Gyeonggi-do, Korea, the positive rate for anti-MPB70 antibody was 4.5% (42/931) and the tuberculin-positive rate was 2.8% (26/931). Individual milk samples (n = 253) were collected from randomly selected 8 problematic and 3 negative herds (positive and negative in the screening test by MPB70 ELISA using bulk tank milk samples, respectively) and tested by MPB70 milk ELISA. In the problematic herds, positive rates were 10.5% (20/190) for anti-MPB70 antibodies in milk ELISA and 2.1% (4/190) in the tuberculin skin test. More than one dairy cows were positive by milk ELISA among the problematic herds, and all tuberculin-positive dairy cows were positive in the milk ELISA. Further, no positive cows were detected in negative herds both by milk ELISA and tuberculin skin test. These results suggest that an ELISA, using bulk tank milk samples, might be a potential efficient screening test for bovine TB of dairy cows.     

Evaluation of an O antigen enzyme-linked immunosorbent assay for screening of milk samples for Salmonella dublin infection in dairy herds.

Levels of antibodies to the O antigens (O:1,9,12) of Salmonella dublin were tested in 1355 serum, 1143 cow milk and 160 bulk milk samples from dairy herds using an enzyme-linked immunosorbent assay (ELISA). In order to define the background reaction, milk samples from all lactating cows and serum samples from 9 animals were collected in each of 20 salmonellosis-free herds located on the island of Bornholm, where cattle salmonellosis has not been reported. Similar samples were collected from all stalled animals in 10 herds with recent (< 6 months) outbreaks of salmonellosis located in Jutland, where salmonella infection is enzootic. Using herd history of salmonellosis, herd location and clinical status of the herds as criteria, the optimal cutoff in the milk ELISA was determined as being at least 5% of the samples having optical density > 0.5, resulting in herd sensitivity of 1.0 and herd specificity of 0.95. While none of the sera in the herds from Bornholm was ELISA positive, 2 herds had a few reactors in the milk ELISA. Using the same cutoff, all but 1 bulk milk sample from 150 herds on Bornholm was ELISA-negative, and all 10 salmonellosis-positive herds from Jutland were ELISA-positive. A significant correlation was found between ELISA reactions in milk and in serum of cows (34% and 32% respectively, rs = 0.69, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of paratuberculosis on culling, milk production, and milk quality in dairy herds

OBJECTIVE: To determine the effect of paratuberculosis on culling, milk production, and milk quality in infected dairy herds. DESIGN: Cross-sectional study. ANIMALS: 689 lactating dairy cows in 9 herds. PROCEDURE: Milk, blood, and fecal samples were obtained from all cows. Fecal samples were evaluated via mycobacterial culture. Serum samples were tested with a commercially available ELISA for antibodies against Mycobacterium avium subsp paratuberculosis, and preserved milk samples were tested with an ELISA for antibodies against M paratuberculosis. Mixed effect and proportional hazards models were used to determine the effect of paratuberculosis on 305-day milk, fat, and protein production; somatic cell count linear score; and the risk of culling. RESULTS: Cows with positive results of bacteriologic culture of feces and milk ELISA produced less milk, fat, and protein, compared with herdmates with negative results. No difference in 305-day milk or fat production was detected in cows with positive results of serum ELISA, compared with seronegative cows. The 3 survival analyses revealed that cows with positive results of each test were at higher risk of being culled than cows with negative results. Paratuberculosis status, as determined by use of all 3 diagnostic tests, was not associated with milk somatic cell count linear score. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that for the 9 herds in this study, paratuberculosis significantly decreased milk production and cow longevity.     

Use of enzyme-linked immunosorbent assay to measure bovine milk and serum antibodies to alpha toxin, beta toxin, and capsular antigens of Staphylococcus aureus

An enzyme-linked immunosorbent assay (ELISA) procedure was used to quantitate milk and serum antibodies (IgG) to Staphylococcus aureus alpha and beta toxins, and S. aureus 2-8 and Smith diffuse strain capsular antigens. Milk samples were collected on two occasions. A comparison was made between levels of milk antibodies specific for the two toxins and capsular antigens for 41 cows that were infected with S. aureus on both sampling dates, and 18 cows not S. aureus-infected on either date. Staphylococcus aureus-infected cows were grouped according to somatic cell counts. All groups of infected cows, regardless of somatic cell counts, had significantly higher milk antibody levels to alpha and beta toxins than did the non-infected cows (P less than .002). Serum samples taken for 13 infected and 4 non-infected cows also indicated that significant elevations in anti-alpha toxin and anti-beta toxin IgG were present in S. aureus-infected cows, compared to non-infected cows. A similar immune response was not seen to capsular antigens, however. No significant differences were present between the two groups of cows for either milk or serum antibodies to Smith diffuse strain capsular antigens. Milk antibodies to 2-8 capsule were significantly elevated only in infected cows with somatic cell counts greater than 10(6)/ml, compared to non-infected cows; no differences were present for serum antibodies to 2-8 capsule between infected and non-infected cows. These results indicate that significant increases in milk (and possibly serum) antibodies to alpha and beta toxins are present in cows with chronic staphylococcal mastitis, apparently resulting from a systemic immune response to these toxins. There does not appear to be a similar immune response to capsular antigens.     

Enzyme-linked immunosorbent assay for detecting antibodies to Brucella abortus in bovine milk and serum

An enzyme-linked immunosorbent assay (ELISA) method was evaluated for the detection of antibodies to Brucella abortus in cows milk. Milk samples from seropositive or -negative cows were sed to determine the distribution of absorbance values to classify milk as ELISA positive or ELISA negative. Brucella abortus was isolated from milk samples from 10 (45%) of the 22 cows whose milk and serum were ELISA positive. The ELISA was evaluated and determined to be an appropriate method for detecting antibodies to B abortus in bovine milk.     

Passive immunity and serological immune response in dairy calves associated with natural Giardia duodenalis infections

In a previous study, Giardia infection patterns were studied in newborn dairy calves over a 4-month period. Chronic Giardia infections were observed in all calves with initial cyst excretion occurring at approximately 1 month of age. In the work presented here, the passive immunity and serological immune response associated with these Giardia infections were examined. Colostrum and milk samples were collected from the dams of these calves, and monthly serum samples were collected from each calf. The colostrum, milk and sera samples were analyzed by ELISA and Western blot for the presence of anti-Giardia IgG antibodies. In addition, the in vitro anti-Giardia activity of milk and colostrum was examined using a miniculture adherence assay. When examined by ELISA, mean anti-Giardia antibody titres were found to be significantly higher in colostrum compared to milk. The monthly mean serum antibody titres in the calves were not found to differ significantly at any time point during the study. Western blot analysis revealed that colostrum from the dams reacted strongly with many different Giardia antigens between 205 and 7.5kDa, while milk reacted with few antigens in the same size range. Sera collected from the calves when 30 and 60 days of age reacted with few Giardia antigens, but as the calves aged, IgG antibodies in their sera began to react with antigens of 21, 50, 65, 73 and 79kDa. The miniculture adherence assay demonstrated that colostrum had significantly more anti-Giardia activity in vitro compared to milk. These results suggest that the calves in this dairy did not mount a significant humoral immune response against Giardia following infection. However, colostrum contained a high level of anti-Giardia antibodies and exhibited anti-Giardia activity in vitro. Therefore, colostrum may have the potential to provide initial protection against Giardia infections in calves, but the lack of a strong, specific humoral immune response by these calves could account for the high prevalence and chronic duration of the infections.     

Evaluation of serum and milk ELISAs for paratuberculosis in Danish dairy cattle

A milk and a serum ELISA for detection of antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) were evaluated against the complement-fixation test (CFT) and culture of faecal samples from 580 cows collected between August 1996 and December 1996. Milk and serum were obtained concurrently from six dairy herds infected with MAP and from two dairy herds without history of infection with MAP.

A cut-off value of 7 OD% was used in the ELISAs. At this cut-off value, all six culture-positive herds were positive in the serum ELISA but one was negative in the milk ELISA. All six culture-positive herds were positive in the CFT. In the two culture-negative herds, the serum and the milk ELISA deemed all serum samples negative at this cut-off value, whereas four serum samples from one of these herds were positive in the CFT. The highest cut-off value enabling the milk ELISA to record all six culture-positive herds as positive was 4 OD%. The highest cut-off value enabling the serum ELISA to record all six culture-positive herds as positive was 17 OD%. Individual-sample relative sensitivities of the ELISAs ranged from 49 to 64% and relative specificities were 80–96% at the cut-off values of 4, 7 and 17 OD%.     

Evaluation of a blocking ELISA for the detection of bovine viral diarrhoea virus (BVDV) antibodies in serum and milk

The performance characteristics of a blocking ELISA test applied to serum and individual milk for the detection of antibodies to bovine viral diarrhoea virus (BVDV) were assessed using 1189 matched milk/serum samples collected from cows of 42 dairy herds located in Brittany (west of France). This test was based on a monoclonal antibody directed against non-structural protein NS2-3 of pestiviruses. All tests were performed blind. For each type of sample, negative/positive cut-off values were determined using receiver operating characteristic (ROC) analysis. Sensitivity and specificity were estimated using the virus neutralisation test as a reference. For sera, the ROC analysis provided a negative/positive inhibition percentage cut-off value of 50% giving a sensitivity and a specificity of 96.9 and 97.8%. For individual milk samples, the cut-off was fixed at 30%, leading to a sensitivity and a specificity of 96.9 and 97.3%. Using this test, a good overall agreement was found between results obtained on matched milk/serum samples (Kappavalue=0.95). The present results indicate that this blocking ELISA test is reliable enough for use in a mass screening and control scheme on BVDV.     

Presence of Neospora caninum specific antibodies in three dairy farms in Georgia and two in Texas

Neospora caninum is known to cause abortion in cattle. This study demonstrated the presence of specific IgG to Neospora in milk and serum samples obtained from three dairy farms in Georgia and two in Texas. Samples from four hundred fourteen dairy cows were examined using a western blot assay of which 362 were milk and 87 were serum. Samples with antibodies to Neospora were identified in 32.1% (105/327) of the examined animals in Georgia, whereas in Texas it was identified in 10.3% (9/87). Positive Georgia samples were found in 24.4% from farm A (28/115), 21.6% from farm B (30/139), and 64.4% from farm C (47/73). In Texas, 13.5% (7/52) of animals in farm D and 5.71% (2/35) from farm E also had specific antibodies to Neospora. The number of animals from Georgia dairy farms with antibodies to Neospora was significantly higher than the Texas dairy farms. This may be related to the age of the animals examined in this study (more than 2 years old). Antibodies present in sera had excellent agreement with the antibodies present in milk. Collection of milk samples for serological testing is easier and less invasive than obtaining bovine sera, therefore offering an alternative for animal testing.     

Comparison of the dot-immunobinding assay with the serum agglutination test, the rose bengal plate test and the milk ring test for the detection of Brucella antibodies in bovine sera and milk.

In this study, Brucella antibodies in bovine sera and milk were detected using the dot-immunobinding assay (DIA), the serum agglutination test (SAT), the Rose Bengal plate test (RBPT) and the milk ring test (MRT). For this purpose, a total of 116 paired blood and milk samples collected at the same time from 56 aborted and from 60 healthy dairy cows was examined. In DIA, a nitrocellulose membrane (NCM) was used as the solid phase. Antigen adsorbed on the NCM was extracted from Brucella abortus S99 by heat treatment. The results obtained by DIA were compared with those of SAT, RBPT and MRT. Of the 116 paired blood and milk samples, 24 were positive and 72 were negative by all tests used. Serum samples of six aborted cows were positive by DIA, SAT and RBPT but the milk samples were negative by DIA and MRT. Serum and milk samples of four aborted cows gave positive reaction only by DIA tests. The remaining six aborted cows were negative only by MRT and two of them were negative by both RBPT and MRT. Four sera of healthy cows were found to be positive only by SAT.     

Prevalence of Borna disease virus antibodies in healthy Japanese black cattle in Kyushu

Epidemiological studies have demonstrated that asymptomatic infection of Borna disease virus (BDV) is found in various species of animals in Japan. Recent reports have also revealed that neurological diseases caused by this virus could exist in horses, cattle, a dog, and cats in this country. In this study, we investigated seroprevalence of BDV antibodies in Japanese black cows reared in Kyushu, the southernmost main island of Japan, using ELISA and Western-immunoblotting. Of 101 serum samples, 11 (10.9%) and 21(20.7%) sera were identified as having antibodies to the BDV N and P antigens, respectively. Among the positive sera, three cows (2.9%) were seropositive for both of the antigens. Furthermore, interestingly, only female cows showed antibodies to P, whereas N antibodies were detected in male and female cows with a comparative ratio. Together with previous studies, our results indicate that BDV might be widely spread in cattle raised in Japan. Furthermore, this is the first report to show that beef cattle, Japanese black cattle, have antibodies against a possible zoonotic pathogen, BDV.     

Detection of Dermatophilus congolensis antibody in the milk of streptothricosis infected cows

Passive haemagglutination and agar gel diffusion tests were used to detect specific antibody to Dermatophilus congolensis antigens in serum and milk of eight streptothricosis infected Friesian milking cows. All the sera and milk samples showed the presence of antibody but titres were higher in sera. Precipitating antibodies were detected only in three sera. A possible implication of this finding is discussed with respect to passive immunity in the young calves from infected dams.     

Control measures in officially acknowledged brucellosis-free and leukosis unsuspected dairy herds on the basis of bulk milk samples in combination with ELISA tests

1. EC- and National Regulations. Since 1988 the EC-regulations accept in addition to the on Agar Gel Immunodiffusion test (AGIDT) based blood serum testing of cattle herds that are filed as "free from Enzootic Bovine Leucosis" the use of ELISA for this purpose. The regular testings in dairy cattle herds can be done alternatively with single or pooled milk samples, in other herds with pooled blood sera using ELISA. General condition is only a minimal sensitivity of the test to detect the European EBL Antibody Standard ("E4") in a dilution of 1:10 in negative serum or 1:250 in negative milk. Adequate national regulations are in preparation. The present limitation of pool sizes, blood maximum 50 animals without preparation steps 20, and milk after concentration treatment 50 cows is neutralized by proceedings in development of higher sensitive ELISA tests. This limitation should be canceled. Herd bulk milk samples without size limitations are accepted to be tested with "Milk Ring Test" by EC for the regular testings in filed "Brucellosis Free Dairy Cattle Herds". The alternative use of more sensitive (and more specific) ELISA tests for this purpose including the technical conditions is in a final discussion. 2. Scientific-Technical Base for Using the Chances of the Proceeding in the EC-Regulations. The realisation of the EC accepted or final discussed ELISA based bulk milk testing to control filed "EBL- and/or Brucellosis Free Herds" depends on some basic conditions like sensitivity, specificity, and variability of the ELISA systems. Field trials of more than 20,000 bulk milk samples in case of Brucellosis and more than 2,000 in case of EBL show the feasibilities and the limits of the ELISA systems in defining the status of the herds. The Brucellosis respectively the EBL situations of the dairy cattle herds tested in this trail were well known by history and by investigation of single animal blood samples using conventional tests. Special test run variations of pretested assays demonstrated the possibilities to define the EBL status of dairy cattle herds up to 50 lactating cows without preparation of the bulk milk sample and up 100 after concentration of the antibodies by the rennet-ammonium sulfate method. The concentration limit for detection of Brucellosis antibodies is 100 lactating cows. The bulk milk of smaller herds can be tested without concentration. On principle the evaluation of the test values bases on defined relations to a "weak positive" reference.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Mycobacterium avium subsp. paratuberculosis ELISA response by parity and stage of lactation

Two cross-sectional studies were carried out to determine the enzyme-linked immunosorbent assay (ELISA) response to Mycobacterium avium subsp. paratuberculosis (Map) by cow characteristics and stage of lactation. One of the studies (referred to as "milk-group") used milk samples from all lactating cows (n=7994) in 108 Danish dairy herds. The other study (referred to as "serum-group") used serum samples collected from all cows (n=5323) in a subset of 72 herds from the 108 herds. These samples were analysed using a similar ELISA for detection of antibodies.The results from the ELISAs were interpreted with two cut-off values as the optimal cut-off value is not known, and as several levels are recommended to be used in practice. The results showed that the probability of being ELISA-positive was two to three times lower for cows in parity 1 relative to cows in other parities using both milk and serum ELISA. At the beginning of the lactation the probability of being positive was highest in the milk ELISA. In the serum ELISA the odds of being positive was highest at the end of lactation. The findings are important in the interpretation of ELISA results at cow level with a subsequent tentative diagnosis and correction for parity and stage of lactation should be considered when providing a diagnosis of paratuberculosis. Some issues related to the pathogenesis are also discussed.     

Feasibility of serological bulk milk testing as a method for BVD surveillance

A commercial ELISA detecting antibodies against bovine viral diarrhoea Virus (BVDV) was analysed for its applicability for bulk-milk screening. Detection limits were analysed using native and concentrated milk samples (milk treated with rennet and ammonium sulfate precipitated) from 10 cows whose sera showed different reactivity levels in the ELISA and from two cows which gave birth to persistently infected calves during the last year. Further this and a second commercial ELISA were used to screen 591 randomly selected bulk-milk samples. To clarify discrepancies thirty-nine herds were included in a follow-up study. A second bulk-milk sample and serum samples from 10 young cattle of 6 to 28 month of age per herd were analysed for antibodies against BVDV. The results of this second testing and the detection of viremic animals in 4 herds confirmed the results from initial bulk-milk testing with both tests. The analysed test is suitable for bulk-milk testing although its application is limited by vaccination.     

Evidence of Schmallenberg virus circulation in ruminants in Greece

During March 2013, we investigated the presence and the levels of Schmallenberg virus (SBV) circulation in three dairy cow herds and three sheep flocks in Central Macedonia, Greece. In two cow herds, a high number of abortions had been observed during the winter. Six bulk-tank milk samples and 147 individual sera were screened for SBV-specific antibodies by ELISA. Positive reactions were obtained from 5 out of 6 bulk-tank milk samples, 58 out of 90 sera from the 3 cow herds, and 2 sera from 2 of the 3 sheep flocks. Twenty-two ELISA-positive sera were tested by serum neutralization test (SNT). SNT confirmed the presence of neutralizing antibodies against SBV in all samples tested, with titers ranging between 1:32 and ≥1:256. No neutralizing antibodies against Akabane virus (AKAV) or Shamonda virus (SHAV) were detected, indicating that neutralizing antibodies against SBV do not cross react with AKAV or SHAV in SNT. ELISA testing of bulk-tank milk samples proved to be convenient and reliable. None of the tested sera was found positive for SBV by real-time RT-PCR, indicating that the sampling was conducted past the viremia stage. This is the first report of SBV circulation in Greece.     

Use of bulk milk for detection of Neospora caninum infection in dairy herds in Thailand

The relationship between the level of Neospora caninum antibodies in bulk milk and the seroprevalence in lactating cows was investigated. Bulk milk was also used to estimate the prevalence of N. caninum infection in dairy herds in the northeast and north Thailand. Bulk milk and individual serum from all lactating cows in 11 herds as well as 220 bulk milk samples from nine milk collection centres were analysed for presence of N. caninum antibodies using an iscom ELISA. In the 11 herds the bulk milk absorbances ranged between 0.04 and 0.89 and the seroprevalences varied between 0 and 46%. Five herds had milk absorbances below 0.20, among those were the two herds housing only seronegative lactating cows. In the remaining three herds with such low bulk milk absorbances one or two cows (5-14%) were seropositive. Six of the investigated herds had bulk milk absorbances above 0.20. In the two herds with the highest bulk milk absorbances more than 30% of the cows were seropositive. Using an absorbance of 0.20 to discriminate between negative and positive herds, 102 (46%) of 220 bulk milk samples were judged positive. There was no significant difference in mean bulk milk absorbance between the milk collection centres within each region. However, the proportion of herds with bulk milk absorbances > or =0.50 in the north was statistically (P < 0.01) higher than that in the northeast. It was concluded that bulk milk antibody testing can be used to identify N. caninum-infected herds and that N. caninum is a common infection in dairy herds in Thailand.