吸附ELISA检测奶牛副结核病

应用吸附ELISA对2批澳大利亚及1批新西兰进口奶牛进行副结核病的检测,结果2头阳性反应,其余为阴性反应。特异性试验和重复性试验表明吸附ELISA在进口奶牛副结核病的检测中,结果准确、特异性高、重复性好,且具有快速、灵敏、操作简单的特点,适合应用于大批量样品副结核病的检测。     

Characterization of the long-term immune response to vaccination against Mycobacterium avium subsp. paratuberculosis in Danish dairy cows

Vaccination of cattle against Mycobacterium avium subsp. paratuberculosis (MAP) provides partial protection by delayed shedding of MAP and reduced numbers of clinically affected animals. The duration of vaccine induced immune response is not known. The primary objective of this study was therefore to characterize the long-term effect of whole-cell based vaccination against MAP on the immune response. A secondary objective was to evaluate whether immunodiagnosis of MAP and Mycobacterium bovis infections is affected by MAP vaccination. Two studies were performed: (1) A retrospective longitudinal study including 895 vaccinated and 2526 non-vaccinated dairy cows in 9 Danish dairy herds aiming at characterizing the long-term antibody-response to vaccination; and (2) a cross-sectional study of responses in the IFN-γ assay carried out in 140 vaccinated animals in two herds to evaluate the effect of vaccination on the cell-mediated immune response and to evaluate a possible interference with the diagnosis of M. bovis infections. The results showed that 37% of samples from vaccinated animals and 5% of samples from non-vaccinated animals, respectively, were test positive in the milk antibody ELISA. The prevalence of antibody responses of the vaccinated animals was relatively constant from 2 to 6 years of age, but decreased in older animals. Among the 140 vaccinated animals 88% tested positive with the IFN-γ test to johnin PPD and 50% responded to PPDb with IFN-γ production above a similar cut-off. Although Denmark is free of M. bovis, two of the vaccinated animals responded with higher IFN-γ levels when cultured with PPDb compared to PPDa. In conclusion, immunization with whole-cell MAP vaccines elicits both humoral and cell-mediated immune reactions, which may interfere with surveillance and diagnosis of both MAP and M. bovis infections using currently available tests.     

Risk factors for metritis in Danish dairy cows

A retrospective longitudinal study of metritis was conducted in Denmark on data collected during 1993-1994. Data on herd size, breed, parity, and treatment of disease were obtained from the Danish Cattle Database. Management and production-facility data were collected using a questionnaire, conducted as a telephone interview in 1994. The study included 2144 herds from three regions in Denmark (102,060 cows). Herd-level variables included were: herd size, housing, flooring, grazing, calving measures, and calving supervision. Cow-level variables were: parity, breed, calving season and whether the cow had been treated by a veterinarian for dystocia or the diseases: retained placenta, reproductive disease, ketosis, milk fever, or dry cow mastitis.Marginal multivariable logistic-regression analyses were performed. The cow with highest odds of metritis was a first or greater than or equal to third parity cow, of large breed, that calved during November-April, in a zero-grazing herd. The cow had been treated for dystocia, retained placenta, and at least one other reproductive disease, but not for ketosis.     

Loser cows in Danish dairy herds: risk factors

The objective of our study was to evaluate risk factors for the loser cow state in Danish dairy herds. A loser cow was defined on the basis of a clinical examination of the individual cow. Recordings of clinical signs were converted into a loser cow score and all cows with a loser cow score of 8 or more were defined as loser cows. We used correspondence analysis to give a first indication of the associations between the proportion of loser cows in the herd and potential risk factors. Risk factors were evaluated both at the herd level (39 herds) and at the cow level (6451 cows) using logistic regression. Conventional herds with a high average somatic cell count, hard cubicles and no grazing seem to be associated with a high proportion of loser cows. Odds ratio for the loser cow state was 4.18 in conventional herds compared to organic herds. Odds ratio for the loser cow state was 1.87 for an increase in average weighted mean bulk tank somatic cell count of 100,000 cells per ml. We found a statistically significant interaction between stall surface and the use of grazing. Odds ratio for the loser cow state in herds with soft cubicles and the use of grazing was 0.09 compared to herds with hard cubicles and no grazing. At the cow level, odds ratio for the loser cow state increases significantly with increasing parity. Odds ratios for the loser cow state were 1, 2.59 and 6.74 for parity 1, 2, and 3 or older, respectively. Odds ratio for the loser cow state was significantly higher (3.38) for cows giving birth to twins at the last calving before the scoring. Additionally, cows scored on pasture had a significantly lower odds ratio (0.26) for the loser cow state compared to cows scored in the barn.     

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%.     

Euthanasia of Danish dairy cows evaluated in two questionnaire surveys

Background

Mortality risk in Danish dairy cows has more than doubled since 1990 (from 2% in 1990 to 5% in 2005). Until now, registrations about dead cows in the Danish Cattle Database have not included information about whether the cow died unassisted or was euthanized.

Methods

We interviewed a random sample of 196 Danish dairy farmers that had reported a dead cow to the Danish Cattle Database in 2002 and 196 dairy farmers that had reported a dead cow in 2006. Our objectives were to evaluate the proportion of euthanized cows, changes in the behaviour of farmers regarding euthanasia of cows over the years and possible reasons for these changes.

Results

It seems that the threshold for euthanasia of cows among farmers has changed. Farmers generally reported a lower threshold for euthanasia compared to 5–10 years ago.

Conclusion

The threshold for euthanasia of cows has, according to the dairy farmers, become lower. This might have positive impacts on animal welfare as more seriously ill cows are euthanized in the herds and not put through a period of suffering associated with disease and treatment or transported to a slaughterhouse in poor condition.     

Fecal shedding of Mycobacterium avium subsp. paratuberculosis by dairy cows

Between 1982 and 2000, fecal samples were obtained from 786 cows that were shedding Mycobacterium avium subsp. paratuberculosis (Map). These cows were resident on 93 Pennsylvania dairies (mean herd size, 64 milk cows) that had no or minimal previous testing for Map. Feces were cultured on four tubes of Herrold's egg yolk medium and the distribution of mean Map colony forming units (CFU) was evaluated. Most cows were light (< 10 CFU/tube, 51.4%) or high (> 50 CFU/tube, 30.8%) fecal shedders with fewer cows in the moderate category (10-50 CFU/tube). Of the 786 cows, 192 (24.4%) had colonies in only one of four tubes. In the multivariable negative binomial model, there were significant associations between mean CFU/tube and prevalence, herd size, and season and an interaction between herd size and season. The linear mixed model of continuous tube counts with a random herd effect yielded similar findings with associations with herd size as a continuous variable, season, and an interaction between categorized prevalence and continuous herd size. Variability in CFU/tube was greatest among cows in the same herd, intermediate for replicate tubes from the same cow, and smallest among cows in different herds. Reduction in the number of replicate tubes from four would have reduced the sensitivity of fecal culture for Map by approximately 6% (for three tubes) to 12% (for two tubes).     

Bulk-tank milk ELISA antibodies for estimating the prevalence of paratuberculosis in Danish dairy herds

Paratuberculosis (Johne's disease) has been widespread in Danish dairy herds for a long time but the herd-level prevalence has never been determined precisely. To evaluate the prevalence of paratuberculosis in Danish dairy herds in various regions, an ELISA based on a commercially available antigen was adapted for testing bulk-tank milk for the presence of antibodies to Mycobacterium avium subsp. paratuberculosis. Bulk-tank milk samples were collected from six milk-collecting centres from six different areas of the country. Samples from 900 herds (about 7.5% of all Danish dairy herds) were examined, and 70% were positive at the statistically optimal cut-off (sensitivity 97.1%; specificity 83.3%). The technical performance of the ELISA was not sufficient to provide a tool for surveillance because even slight changes in optical density for the samples would change the classification of some samples. The infection is more widespread than previous investigations have shown.     

Spatial differences in occurrence of paratuberculosis in Danish dairy herds and in control programme participation

Paratuberculosis is a chronic infection of economic importance to the cattle industry and a voluntary control programme is offered to Danish dairy farmers. Our objective was to evaluate spatial differences in both control programme participation and paratuberculosis prevalence in Denmark. The study included 4414 dairy herds: 1249 were participating in the control programme, and 1503 were tested for antibodies to Mycobacterium avium subsp. paratuberculosis (MAP). Spatial differences were evaluated by kernel smoothing, kriging, and cluster analysis. Participation was lowest among herds on the island Zealand (≤23%). The risk of a herd being infected with MAP was found to be high on most of Zealand, but the uncertainty of this result was large due to a limited number of tested herds. In the rest of the country, the south western part of the peninsula Jutland had the highest risk of MAP (≥91%). The risk of MAP was also high (86-91%) in the northern part of both Jutland and Funen. The predicted apparent within-herd prevalence was highest (5-8.5%) in some local areas across Jutland, in the north western part of the island Funen, and in the south and western part of Zealand. Scan statistics located the primary cluster of herds with high apparent within-herd prevalence in the western part of Funen. Furthermore, a number of significant clusters were found in Jutland and a single significant cluster in Zealand. Consistency was found between kriging and scan statistics results with respect to location of areas with high apparent within-herd prevalence of MAP. Potential explanations for differences in participation include herd size and local herd health advisers, whereas for example soil characteristics might influence prevalence. Further studies are needed to evaluate these and other risk factors.     

Evaluation of test-strategies for estimating probability of low prevalence of paratuberculosis in Danish dairy herds

Paratuberculosis is a chronic infection affecting cattle and other ruminants. In the dairy industry, losses due to paratuberculosis can be substantial in infected herds and several countries have implemented national programmes based on herd-classification to manage the disease. The aim of this study was to develop a method to estimate the probability of low within-herd prevalence of paratuberculosis for Danish dairy herds. A stochastic simulation model was developed using the R^® programming environment. Features of this model included: use of age-specific estimates of test-sensitivity and specificity; use of a distribution of observed values (rather than a fixed, low value) for design prevalence; and estimates of the probability of low prevalence (Pr_Low) based on a specific number of test-positive animals, rather than for a result less than or equal to a specified cut-point number of reactors.

Using this model, five herd-testing strategies were evaluated: (1) milk-ELISA on all lactating cows; (2) milk-ELISA on lactating cows ≤4 years old; (3) milk-ELISA on lactating cows >4 years old; (4) faecal culture on all lactating cows; and (5) milk-ELISA plus faecal culture in series on all lactating cows.

The five testing strategies were evaluated using observed milk-ELISA results from 19 Danish dairy herds as well as for simulated results from the same herds assuming that they were uninfected.

Whole-herd milk-ELISA was the preferred strategy, and considered the most cost-effective strategy of the five alternatives. The five strategies were all efficient in detecting infection, i.e. estimating a low Pr_Low in infected herds, however, Pr_Low estimates for milk-ELISA on age-cohorts were too low in simulated uninfected herds and the strategies involving faecal culture were too expensive to be of practical interest. For simulated uninfected herds, whole-herd milk-ELISA resulted in median Pr_Low values >0.9 for most herds, depending on herd size and age-structure. None of the strategies provided enough power to establish a high Pr_Low in smaller herds, or herds with a younger age-structure. Despite this, it appears as if the method is a useful approach for herd-classification for most herds in the Danish dairy industry.     

Herd-level risk factors for the mortality of cows in Danish dairy herds

The factors affecting the mortality of cows in Danish dairy herds were investigated by analysing data from 6839 herds. The mean risk of mortality during the first 100 days of lactation was 2.5 per cent. The risk of mortality increased with increasing herd size (odds ratio [OR] 1.05 for an increase in herd size of 50 cows), with the proportion of purchased cows (OR 1.05 for an increase in the proportion of purchased cows of 0.1), and with increases in the somatic cell count (OR 1.16 for an increase in average weighted mean somatic cell count of 100,000 cells/ml). The risk decreased with increasing average milk yield per cow (OR 0.93 for an increase in mean yield per cow-year of 1000 kg). The risk was lower in free-stall barns with deep litter (OR 0.79) than in barns with cubicles (OR 1) or tie-stalls (OR 1.04). Herds with Danish Holstein (OR 1) or Danish Jersey (OR 0.93) cows as the predominant breed had a higher risk of mortality than herds of the Danish red dairy breed (OR 0.67). The risk of mortality was lower in organic herds (OR 1) than in conventional herds (OR 1.17), and lower in herds that grazed pasture during the summer (OR 0.78).     

Monensin might protect Ontario, Canada dairy cows from paratuberculosis milk-ELISA positivity

Our objective was to define the role of monensin sodium in protecting cows from being milk-ELISA positive for paratuberculosis in Ontario, Canada dairy herds. In total, 4933 dairy cows from 94 herds were enrolled in this cross-sectional study. Forty-four of the enrolled herds were selected purposively by their herd veterinarian and another 50 herds were randomly selected from a local milk production-recording agency. A herd-management survey was completed on each farm during the months of May through August 2003. During this same time-period, composite milk samples were collected from all lactating cows and tested with a milk-ELISA for antibodies to Mycobacterium avium subspecies paratuberculosis. Analyses were stratified according to the paratuberculosis history of the herds. In the 48 herds in which paratuberculosis had not been diagnosed before, the use of calf hutches and monensin in milking cows were both associated with reduced odds of a cow testing positive (OR = 0.19 and 0.21, respectively). In the 46 herds with a prior history of paratuberculosis, feeding monensin to the breeding-age heifers was associated with decreased odds of a cow testing positive (OR = 0.54). Monensin use might be associated with milk-ELISA positivity, but its impact on the transmission of paratuberculosis remains unknown.     

Bisection of bovine morulae and blastocysts from superovulated Danish dairy cows

Sixtyfour compacted morulae and blastocysts were bisected with a microscalpel. The majority of the demi-embryos (n = 122) were reinserted into separate zona pellucidae (ZP) before non-surgical transfer to 113 synchronized recipients, as singles (n = 98) (DE-S) or in pairs (n = 30) (DE-P). Thirty non-manipulated embryos (E) were transferred during the same period and served as controls. Pregnancies were diagnosed by rectal palpation 4-7 weeks after transfer. The pregnancy rates for DE-S, DE-P and E were 32%, 53% and 40%, respectively (P greater than 0.05). A substantial number of abortions were recorded between 50 and 250 days of pregnancy among the recipients with DE-S. The fetal survival rate for DE-S was reduced to 21% and significantly lower (p less than 0.05) than the survival rates of DE-P (43%) and E (40%). The quality of DE and the presence of ZP did not significantly influence the results. No conclusive reasons for the fetal loss could be found but different possibilities are discussed.     

Serological cross-sectional study of paratuberculosis in cattle in Austria.

From 1995 to 1997, the prevalence of serum antibodies against Mycobacterium avium subspecies paratuberculosis (M. av. ssp. ptbc.)--the causal agent of paratuberculosis (Johne's Disease)--was examined in 11,028 Austrian cattle. Samples from the four oldest cattle on 2757 farms were collected according to a specific sampling schedule for this epidemiological study. District, age and breed of animals were included as variables in this study. For antibody screening against M. avium subspecies paratuberculosis, a modified, commercially available ELISA (ALLIED Monitors, Fayette, USA) was employed. A total of 2253 samples that were found to be positive or questionable were subjected to further testing with a more specific ELISA (Institute of Microbiology and Infectious Animal Diseases). Results of this study were used for statistical analysis. The average prevalence of antibodies to M. avium subspecies paratuberculosis was 1.99% in Austria. The highest prevalence was seen in 6-year-old cattle (2.84%) and Holstein Frisian cattle (3.51%). Sero-positive animals were found on 6.96% of farms tested, and the prevalence was highest in Vorarlberg, followed by Salzburg, the Tyrol, Styria and Carinthia. This study is unique in Europe in the use of an adequate random sampling plan for an investigation of this magnitude.     

Loser cows in Danish dairy herds: definition, prevalence and consequences

During the last few years, many Danish dairy farmers have expressed increasing concerns regarding a group of cows, which we have chosen to term ‘loser cows’. Until now, a loser cow has not been described scientifically. We defined a loser cow on the basis of a clinical examination of the cow. A total of 15,151 clinical examinations were made on 6,451 individual cows from 39 randomly selected, large Danish dairy herds with loose-housing systems using a clinical protocol. Scores for the clinical signs lameness, body condition, hock lesions, other cutaneous lesions, vaginal discharge, condition of hair coat and general condition were converted into a loser cow score. Cows with a loser cow score of 8 or more were classified as loser cows. The overall prevalence of loser cows was 2.15%, 4.50% and 2.98% during the first, second and third round of herd visits, respectively.

The associations between the loser cow state and milk production, mortality, morbidity, culling and workload for the farmer were evaluated using data from herd visits and from the Danish Cattle Database and a number of different statistical techniques. It was concluded that the loser cow state has significant negative consequences for both the farmer and the cow. On average, loser cows yielded 0.61 to 2.24 kg energy corrected milk less per day than non-loser cows depending on parity. Hazard ratio for death or euthanasia was 5.69 for loser cows compared to non-loser cows. Incidence rate ratio for disease treatments was 0.69 for non-loser cows compared to loser cows. Loser cows were often culled in an ‘unfavourable’ way and generally caused extra workload for the farmer.

A simplified version of the loser cow score was evaluated and is recommended for future research and use in practice.     

Critical differences of clinical-chemical parameters in blood from Red Danish dairy cows.

The purpose of the present study was to calculate the critical differences between two analytical results for 23 routinely used bovine clinical-chemical parameters. The critical difference can be used to judge whether the difference between two consecutive analytical results from the same animal is due to natural variation or not. From 20 cows, blood samples were collected once daily for five days, and the interindividual variance, the intra-individual variance, and the analytical variance were calculated using nested analysis of variance. The critical difference both in absolute values and in percentages was calculated from these variances. The main conclusions were that the critical difference in percentages was in general between 10 and 40 per cent but varied from 10.6 per cent for sodium to 280.2 per cent for lactate and that the analytical variation was generally in accordance with accepted standards, although the analytical variation for alkaline phosphatase, total serum protein, urea and fructosamine should be improved.     

Risk of removal and effects on milk production associated with paratuberculosis status in dairy cows

OBJECTIVE: To determine effects on production and risk of removal related to Mycobacterium avium subsp paratuberculosis (MAP) infection at the individual animal level in dairy cattle. DESIGN: Longitudinal study. ANIMALS: 7,879 dairy cows from 38 herds in 16 states. PROCEDURE: A subset of dairy cattle operations that participated in the National Animal Health Monitoring System Dairy 2002 study was evaluated via a serum ELISA for antibodies against MAP and categorized according to ELISA score. Dairy Herd Improvement Association records were obtained to collect current and historical lactation data and removal (ie, culling) information. Production variables were evaluated on the basis of serum ELISA category. RESULTS: Cows with strong positive results had mature equivalent (ME) 305-day milk production, ME 305-day maximum milk production, and total lifetime milk production that were significantly lower than cows in other categories. No differences were observed for ME 305-day fat and protein percentages, age, lactation, and lactation mean linear somatic cell count score between cows with strong positive results and those with negative results. After accounting for lactation number and relative herd-level milk production, cows with strong positive results were significantly more likely to have been removed by 1 year after testing. CONCLUSIONS AND CLINICAL RELEVANCE: Without management changes designed to reduce the farm-level prevalence of MAP infection, paratuberculosis will continue to reduce farm income by decreasing milk production and potentially increasing premature removal from the herd.     

Crossbreeding experiment with beef and dual-purpose sire breeds on Danish dairy cows

The experimental design and objectives are outlined for a crossbreeding experiment with European beef breeds as sire breeds and RDM (Danish Red), SDM (Black Pied Danish) and Danish Jersey as dam breeds.Results from a calving survey of 1006 single calvings are presented. Gestation length, birth weight and calving performance were strongly influenced by breed of sire, breed of dam and sex of calf. The variation in gestation length seemed to have only a slight effect on birth weight. The variation in birth weight had a strong effect on calving performance.The ranking of the sire breeds in descending order according to the frequency of unasisted calvings (which varied from 70 to 26 percent) was as follows: Hereford, Limousin, Danish Red and White Cattle, Simmental, Blonde d'Aquitaine, Chianina, Charolais and Romagnola.     

Risk factors associated with Mycobacterium avium subspecies paratuberculosis seropositivity in Canadian dairy cows and herds

Our objective was to determine the risk factors associated with the seroprevalence of Mycobacterium avium subspecies paratuberculosis (MAP) in a large number of randomly selected Canadian dairy herds, controlling for important confounding variables and co-infections with bovine leukemia virus (BLV), bovine viral diarrhea virus (BVDV) and Neospora caninum (NC). Serum samples from 30 randomly selected cows, where available, in 315 herds from seven provinces were tested for antibodies against BLV, MAP and NC using commercially available enzyme-linked immunosorbant assay (ELISA) test kits, while five unvaccinated cattle >6 months old from each herd were tested for antibodies to BVDV. We used a zero-inflated negative-binomial (ZINB) multivariable model to determine simultaneously the risk factors associated with the count of MAP-seropositive cows in a herd, and the odds of herds having no MAP-seropositive cows as compared to having one or more MAP seropositive cows in a herd. The following factors were significantly positively associated with the count of MAP-seropositive cows: "more than one cow in the maternity pen", "group-housing for pre-weaned calves in winter", "open heifers purchased during the last 12 months", "beef cattle direct (nose-to-nose) contact", "BVDV-seropositive herds (> or = 1 animal with > or = 1:64 titer)" and "BVD vaccination not done properly in calves" (i.e. after 6 months old, animals were not boostered 2-4 weeks after their first killed vaccine, or not given modified live vaccine), with count ratios of 1.7, 2.0, 2.3, 1.9, 1.4 and 1.8, respectively. The variable "BVDV vaccination (modified live) done properly in calves" (i.e. received another modified live vaccination after 6 months as well) was associated with 0.4 times fewer MAP-seropositive cows.