Improving the sensitivity of the IBR-gE ELISA for testing IBR marker vaccinated cows from bulk milk

The low sensitivity of the IBR-gE ELISA compared to other diagnostic ELISA tests for IBR is a major disadvantage of IBR control programmes based on IBR marker vaccination. Therefore the IBR-gE ELISA is not generally recommended for testing pooled or bulk milk samples.The aim of this study was to determine the performance of a commercially available kit for concentrating and purifying antibodies in milk in order to improve the sensitivity of detecting IBR-gE antibody positive cows from pooled and bulk milk samples. A single IBR-gE positive cow is likely to remain undetected in a pool of 49 negative milk samples without concentration. By contrast, the bulk milk concentration procedure improved sensitivity from 5.4% to 75.7% in a positive herd. Milk samples with a high or moderate positive signal are more likely to be detected after pool milk concentration compared to weak positive samples. Whereas a follow up study involving a monthly testing of bulk milk samples from three marker vaccinated IBR-gE negative herds over a period of seven months yielded negative results each month, bulk milk from a herd containing <5% IBR-gE positive cows always detected positive after concentration. Although the milk concentration procedure had no impact on specificity, it significantly enhanced the sensitivity of the detection of IBR-gE positive milk in pooled and bulk milk samples. After further evaluation this procedure could allow a cost efficient and reliable method of monitoring IBR marker-vaccinated herds for IBR-gE antibodies.     

一步法RT—PCR检测大缸奶在BVDV清除计划中的应用

本文利用一步法RT—PCR对28个牛场共计50份大缸奶样进行了BVDV核酸检测。在与全群抗原检测结果对比后发现．9个已知存有PI牛的泌乳牛群,其大缸奶核酸检测均为阳性,其余41个已知无PI牛的泌乳牛群．除1个因为存在急性感染牛造成阳性结果外,其余40个均为阴性。由此可见,本方法对与泌乳牛群内是否存在PI牛可以做出准确判断,其检测灵敏度和特异性均为100％,阳性预测结果可信度为0．9（9／10）,阴性预测结果可信度为0．98（40／41）。此外．针对50个大缸奶样进行的抗体检测表明,对于已感染牛场,泌乳牛群是否存在PI牛,抗体水平没有明显差异（OD1．12±0．12vsOD1．34±0．23,P〉0．05）。实验室条件下,本试验使用的RT—PCR方法对于阳性乳的最低检出限为50uL/头,因此理论上,最多可从1000份样品中检出阳性乳成分（总体积为50mL）。综上可知,本试验确立的RT—PCR方法灵敏度高、特异性强,可对泌乳牛群是否存在PI牛进行准确预测,相比大缸奶抗体检测更具实际指导意义,联合ELISA—Ag使用时还可大幅度降低泌乳牛群BVDV清除计划的检测成本,因而值得推广使用。     

奶牛乳房炎溶血性病原菌分离培养及药敏试验

本试验旨在确定吉林市某奶牛场奶牛乳房炎的主要致病菌及致病菌对某些抗生素的敏感性。无菌采集奶牛乳房炎病例的奶样,进行细菌分离培养和细菌的药敏试验。试验结果显示,该奶牛场奶牛乳房炎的主要致病菌为2种溶血性的链球菌,分别是α溶血性链球菌和β溶血性链球菌,且致病菌的耐药性较强,对大多数抗生素均有耐药性。试验结果表明,该奶牛场奶牛乳房炎致病菌具有强致病性和耐药性。     

Comparison of an indirect ELISA with the Brucella milk ring test for detection of antibodies to Brucella abortus in bulk milk samples.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Brucella abortus antibodies in bovine bulk milk samples was evaluated. About 31 individual milk samples from B. abortus infected cows were diluted into bulk milk from a brucellosis free herd. Individual milk samples obtained from 96 negative or positive herds to ELISA or Brucella ring test (BRT), were tested by ELISA. All positive cows were bled and serum samples were tested by the complement-fixation test (CFT) which was considered the definitive test. A herd was considered infected if at least, one cow was positive in the CFT. Four samples were negative in the BRT at the dilution 1:10 but positive in the ELISA. For samples positive in both tests, BRT titers ranged from 1:10 to 1:480 while ELISA titers ranged from 1:10 to 1:3200.Using bulk milk samples, the sensitivity of the ELISA (98.1%) was higher than the BRT (72.2%) but the specificity of BRT (90.5%) was not statistically different (P=1.0) from the ELISA (88.1%). The implications of the results for brucellosis control are discussed.     

2017年上海、河北地区奶牛乳房炎克雷伯菌分离鉴定及耐药性分析

为了摸清上海和河北地区奶牛乳房炎的主要致病菌克雷伯菌的分离率及其耐药情况,对采集自上海和河北两地的乳房炎患病奶牛乳样进行克雷伯菌的分离培养和形态学、分子生物学鉴定,并选择临床常用的抗生素对分离得到的克雷伯菌进行药敏试验。经革兰染色、镜检以及分子生物学鉴定发现,采集自上海地区的100份乳房炎患病奶牛乳样中有18份乳样存在克雷伯菌,分离率为18%;采集自河北地区的100份乳房炎患病奶牛乳样中有14份乳样存在克雷伯菌,分离率为14%;且上海和河北地区2017年下半年乳房炎患病奶牛乳样中克雷伯菌分离率均高于上半年。药敏试验结果表明,该试验分离得到的克雷伯菌对所测试的抗生素存在不同程度的耐药性,其中,单一耐药菌株占21.88%,多重耐药菌株占53.13%,全部敏感的菌株占12.50%。由该试验结果可以得出,上海、河北地区奶牛发生的乳房炎是与克雷伯菌感染有关;且分离得到的克雷伯菌对临床常用的抗生素存在不同程度的耐药性。在动物生产和兽医临床上应及时监控克雷伯菌的流行趋势和耐药性变迁,并合理使用抗生素以减少耐药菌株的产生。     

Somatic cell counts, mastitis and milk production in selected Ontario dairy herds.

Somatic cell counts were performed monthly on bulk tank milk samples for all producers in the Ontario counties of Hastings, Lennox/Addington and Prince Edward throughout 1978 and 1979. Other data were obtained via a structured questionnaire and from the records of the Ontario Milk Marketing Board. Many producers have not adopted practices that have been advocated for the integrated control of mastitis. For example, 43.3% of producers surveyed used single service paper towels, 63.3% regularly used teat dip and 56.5% dry cow therapy. The mean of the average monthly somatic cell count for all producers for 1978 was 621.1 x 10(3) cells/mL. This latter value was used to divide the producers into case (higher than average) and control (lower than average) groups. Control herds averaged 95.9 liters more shipped milk per cow per month than case herds. Milk from control herds averaged 0.22 percentage points higher than case herds for each of average fat and lactose, and 0.16 percentage points higher for protein. The linear regression of monthly shipped milk on the respective monthly bulk tank somatic cell count indicated a loss of 13.26 L/cow/month for each 100,000 increase in somatic cell count.     

A case of bovine raw milk contamination with Listeria monocytogenes

ABSTRACT: During routine sampling of bulk raw milk on a dairy farm, the pathogenic bacteria Listeria monocytogenes was found to be a contaminant, at numbers < 100 cfu/ml. A strain with an indistinguishable pulsed-field gel electrophoresis pattern was isolated from the bulk milk two months later. Environmental swabs taken at the dairy environment were negative for the presence of L. monocytogenes, indicating a possible case of excretion of the L. monocytogenes directly into the milk. Milk samples were collected from the individual cows and analysed, resulting in the identification of L. monocytogenes excretion (at 280 cfu/ml) from one of the 4 mammary quarters of one dairy cow out of 180. When the infected cow was isolated from the herd, no L. monocytogenes was detected from the remaining herd. The pulsed-field gel electrophoresis pattern of the strain from the individual cow was indistinguishable from that originally isolated from the bulk milk. The infected cow did not show any clinical signs of disease, nor did the appearance of the milk have any physical abnormalities. Antibiotic treatment of the infected mammary quarter was found to be ineffective. This study shows that there can be risks associated with direct contamination of raw milk with L. monocytogenes.     

A survey to determine relationships between bulk tank milk antibodies against Ostertagia ostertagi and milk production parameters

In temperate climate regions, gastrointestinal nematodes are still widespread in adult dairy cows, but until now there exists no reliable diagnostic tool that can identify herds where the infection interferes with productivity. The objective of this study was to investigate the relationships between levels of antibodies against Ostertagia ostertagi in bulk tank milk and milk production. Bulk tank milk samples of 2553 dairy herds were obtained in spring and 2104 of these herds were sampled a second time in autumn. The antibody levels against O. ostertagi were determined with a milk ELISA and test results were expressed as an optical density ratio (ODR). The effect of bulk tank milk ODR on three different production parameters, kg milk, % and kg fat, % and kg protein was assessed by a multivariable linear regression model on the herds for which production data were available (n = 1063 and 867 in spring and autumn, respectively). The mean and standard deviation for ODRautumn (0.972+/-0.238) were higher than for ODRspring (0.825+/-0.201). Significant negative relationships were found between ODR and milk yield. An increase in ODRspring and ODRautumn from the 25th to the 75th percentile of the available ODR data was associated with a drop in the annual milk yield of 1.1 kg/cow/day, respectively 0.9 kg/cow/day. When a herd's ODR increased between spring and autumn with 0.142, it produced on average 0.4 kg/cow/day less in September than in April, in comparison with herds where the ODR did not increase. A significant negative association was found between ODRautumn and % protein averaged over the period of a year. No significant associations were found between ODR and % fat averaged over the year. When protein and fat production of September were expressed in kg an increase in ODRautumn from the 25th to the 75th percentile was associated with a decrease of 0.037 kg protein/cow/day and 0.042 kg fat/cow/day.     

中药防治奶牛乳房炎的研究进展

乳房炎是奶牛的一种常见病,是导致牛奶产量和质量下降的主要原因之一。目前,奶牛乳房炎的防治多以抗生素为主,但抗生素的滥用不仅导致了乳汁中病原菌耐药性的产生,同时也造成了乳汁中抗生素的残留。中药由于无残留、无毒副作用和较少出现耐药性等特点而备受科研学者的关注。本文主要从奶牛乳房炎的危害、中药防治奶牛乳房炎的研究现状和作用机理三个方面探讨了当前中药防治奶牛乳房炎的研究进展,旨在为临床上奶牛乳房炎的防治提供客观的参考。     

Considerations concerning diagnostic certainties and cut-off values for a bulk milk ELISA for Mycobacterium avium ssp. paratuberculosis

Serological tests for the examination of individual samples from single animals are evaluated based on their ability to detect true positives above a defined threshold value. If results are obtained not from an individual but from a bulk sample this concept usually is adopted such that the threshold is set to allow the detection of a single positive sample within the pool. In conjunction with the development of a diagnostic paratuberculosis ELISA for the examination of bulk milk samples it is discussed which interpolations of this concept are justified when defining the true status of a herd based on the test parameters and the seroprevalence within the herd. Here, bulk milk from up to 50 animals each and the corresponding individual samples of 4241 dairy cows from 28 herds in the state of Brandenburg are investigated, and results are subjected to different evaluation approaches. Based on epidemiological considerations and test parameters a "critical prevalence" is defined which then serves as basis for the deduction of a cut-off value to be used for bulk milk samples. Finally, the practical relevance of this approach is demonstrated by suggesting an initial scheme for paratuberculosis classification of dairy herds with respect to possible control measures.     

Use of clinical information to predict the characteristics of bacteria isolated from clinical cases of bovine mastitis

Farmers recorded the clinical signs of cows with clinical mastitis and submitted milk samples for bacteriological examination, so that the clinical signs could be correlated with the bacteriological findings. Odds ratios for the demeanour of the cow, the appearance of the milk, milk yield, udder texture, and the administration of parenteral antibiotics were calculated for mastitis cases classified in terms of their microbiology as either enterobacteriaceae, major Gram-positive pathogens, minor pathogens, 'no growths' or 'all other pathogens'. Animals infected with enterobacteriaceae had the highest odds of being reported as having a reduced milk yield, swollen or hard udders, watery milk and/or being systemically sick. A logistic regression model was used to predict the Gram-staining characteristics of the bacteria causing clinical mastitis. The clinical findings found to be significant predictors in the model were the demeanour of the cow and its milk yield. The regression model was used as a basis for a predictive test. Using a test data set, the sensitivity of the test was 28 per cent, its specificity was 96 per cent, the positive predictive value was 74 per cent and the negative predictive value was 80 per cent. The overall accuracy of these predictions was 79 per cent.     

Comparison of Milk Fat Globule Membrane Proteins in Milk Samples of Dairy Cow and Goat

In order to compare the difference of milk fat globule membrane（MFGM）proteins between dairy cow and goat milk,30 milk samples were collected in a dairy farm and a goat farm from Anhui area,respectively.Extracted proteins from the MFGM-enriched fractions were identified and quantified by LC/MS approach.The results showed that 284 and 334 proteins of MFGM from dairy cow and goat were identified,the biological processes of MFGM proteins were similar between dairy cow and goat which were mainly related to biological regulation,localization,transport,signal transduction and response to stimulus.Meanwhile,there were some differences in molecular functions,and protein binding and nucleotide binding were the most prevalent molecular functions in dairy cow MFGM proteins,while protein binding and structural molecule activity were the most prevalent molecular functions in goat MFGM proteins.And structural molecule activity was the main molecular functions among the difference proteins.Pathway analysis revealed that tight junction,axon guidance,antigen processing and presentation,complement and coagulation cascades were enrichment in dairy cow MFGM proteins,and regulation of actin cytoskeleton was enrichment in goat MFGM proteins.Those results revealed the protein expression pattern difference between MFGM protein of dairy cow and goat milk,and provide reference data for further exploring the molecular mechanism of synthetic milk fat globule.     

牛奶和山羊奶中乳脂球膜蛋白的比较研究

试验旨在分析牛奶与山羊奶中乳脂肪球膜（milk fat globule membrane,MFGM）蛋白组成及潜在的生物学功能。采集牛奶和山羊奶各30份,离心分离牛奶和山羊奶中脂肪,提取MFGM蛋白,液相色谱串联质谱分析结合数据库搜索鉴定,比较了奶牛和山羊MFGM蛋白的差异,分析了MFGM蛋白参与的生物过程、分子功能及相关代谢通路。结果显示,奶牛和山羊MFGM中分别鉴定了284和334个蛋白,奶牛和山羊中蛋白参与的生物学过程的模式非常相似,主要涉及生物调控、定位、转运、信号转导和应激反应等。分子功能方面二者存在一定的差异,奶牛MFGM蛋白主要涉及蛋白结合和核苷酸结合,而山羊MFGM蛋白主要涉及蛋白结合和结构分子活性。奶牛和山羊中差异表达的MFGM蛋白主要涉及结构分子活性。通路分析发现,奶牛和山羊MFGM蛋白涉及的生物学通路稍有差异,其中紧密接头、轴突导向、抗原加工与递呈、补体和凝血级联反应通路在奶牛MFGM蛋白中得到富集,而调节肌动蛋白细胞骨架通路在山羊奶中得到富集。研究结果展示了奶牛和山羊MFGM蛋白的表达模式及差异,为进一步探索两种奶畜乳腺合成脂肪球的分子机制的异同提供科学依据。     

Use of the Lachema Bio-La-Test for the determination of urea in milk

The content of urea in milk was studied as a parameter of dairy cow nutrition. It is possible to use for the analysis whole milk where the values are lower by 5.96% than in defatted milk (this fact must be borne in mind when the results are interpreted). Protein can be removed from whole milk within 24 hours after sampling provided that it was placed in a refrigerator within four hours from milking to be stored there at 4 degrees C. In such a case the decrease in urea level is not greater than 4%. No significant differences were found between urea concentration in milk collected by stripping and that in bulk milk. It is not recommended to take samples at the end of milking because such milk contains less urea. Urea concentration in the bulk milk samples corresponds to the average urea concentration in the milk samples taken from dairy cows in the stable.     

Mycoplasmal mastitis in a dairy herd

In October 1985, mycoplasmas were isolated from bulk tank milk samples in a large Florida dairy (greater than 1,400 lactating cows). At that time, measures to isolate and control the spread of infection were instituted. In an initial screening test, Mycoplasma bovis was isolated from 21 of 153 milking string samples (milk from all quarters of 10 cows/string). Composite quarter milk samples from all quarters of every individual lactating cow in the herd were obtained for culture in November 1985 and December 1985. In October, 88 of 1,535 (5.7%) cows were identified as Mycoplasma-positive. An additional 31 Mycoplasma-infected cows were identified in December. The dairy elected to maintain the infected cows in a separate Mycoplasma-positive subherd, which would be milked at the end of each milking session. Seven additional Mycoplasma-positive cows were identified at initiation of lactation. All newly identified infected cows were transferred to the Mycoplasma-positive subherd. After segregation of Mycoplasma-positive cows, bulk tank milk samples obtained routinely from the main herd remained culture negative throughout the study. From February 1986 to October 1986, quarter milk samples were obtained monthly from cows in the Mycoplasma-positive subherd. Any cow that developed clinical mastitis or substantial decrease in milk production was, at the discretion of the herdsman, culled. Of the 126 cows in the subherd, 22 (17.5%) were culled for mastitis, 35 (27.8%) were culled for low production, and 9 (7.1%) were culled for other reasons. Of the remaining 60 cows, 16 (12.7% of the 126 cows) were Mycoplasma-positive on the basis of results from one or more samples obtained after February 1986.(ABSTRACT TRUNCATED AT 250 WORDS)     

Automated detection of estrus and mastitis in dairy cows

The development and test of detection models for oestrus and mastitis in dairy cows is described in a PhD thesis that was defended in Wageningen on June 5, 2000. These models were based on sensors for milk yield, milk temperature, electrical conductivity of milk, and cow activity and concentrate intake, and on combined processing of the sensor data. The models alert farmers to cows that need attention, because of possible oestrus or mastitis. A first detection model for cows, milked twice a day, was based on time series models for the sensor variables. A time series model describes the dependence between successive observations. The parameters of the time series models were fitted on-line for each cow after each milking by means of a Kalman filter, a mathematical method to estimate the state of a system on-line. The Kalman filter gives the best estimate of the current state of a system based on all preceding observations. This model was tested for 2 years on two experimental farms, and under field conditions on four farms over several years. A second detection model, for cow milked in an automatic milking system (AMS), was based on a generalization of the first model. Two data sets (one small, one large) were used for testing. The results for oestrus detection were good for both models. The results for mastitis detection were varying (in some cases good, in other cases moderate). Fuzzy logic was used to classify mastitis and oestrus alerts with both detection models, to reduce the number of false positive alerts. Fuzzy logic makes approximate reasoning possible, where statements can be partly true or false. Input for the fuzzy logic model were alerts from the detection models and additional information. The number of false positive alerts decreased considerably, while the number of detected cases remained at the same level. These models make automated detection possible in practice.     

An investigation of risk factors for nocardial mastitis in central Alberta dairy herds

A case-control study was undertaken during the summer of 1989 in central Alberta dairy herds to identify independent predictors of nocardial mastitis. Thirty-seven herds with nocardial mastitis were matched with control herds based on herd size, milk production, and enrolment in Alberta Dairy Herd Improvement Services. Control herds were considered free of nocardial mastitis based on negative cultures of four weekly bulk tank milk samples and one composite milk sample collected during the same period from each lactating cow in the herd. A detailed questionnaire on herd management was completed during farm visits. The use of blanket dry cow therapy was not found to be a risk factor for nocardial mastitis. Dry cow therapy with intramammary products containing neomycin and the use of multidose vials of dry cow medications were the only predisposing factors identified as being significantly associated with nocardial mastitis in central Alberta dairy herds. Use of neomycin as a dry cow therapy increased the odds of nocardial mastitis occurring in these dairy herds by 169 times.     

Detection and identification of Mycoplasma from bovine mastitis infections using a nested polymerase chain reaction.

A polymerase chain reaction (PCR) test was compared with culture for the detection and diagnosis of bovine Mycoplasma intramammary infection. The PCR test was applied to 24-hour Mycoplasma enrichment cultures of milk from cows with suspected mastitis and from bulk tank milk. In comparison to culture, the sensitivity and specificity of the PCR method were 96.2% and 99.1% for individual cow milk and 100% and 99.8% for the bulk tank milk, respectively. However, in discrepant cases where PCR was positive and culture was negative, the PCR test was correct; subsequent PCR tests and culturing of the individual cow's milk yielded positive results. The PCR test simultaneously detected and differentiated among 11 bovine Mycoplasma species.     

Evaluation of an iscom ELISA used for detection of antibodies to Neospora caninum in bulk milk

The intracellular parasite Neospora caninum is increasingly recognized as an important cause of abortion and stillbirth in cattle. Presence of specific antibodies indicates infection, and the immunostimulating complex (iscom) enzyme-linked immunoassay (ELISA) has previously been evaluated for use on individual milk and sera. In the present study, this test is investigated for use on bulk milk. In this study, 124 herds were used to analyse the relationship between within-herd prevalences based on individual sera and bulk milk optical densities. The individual test results were translated into a herd-level result, which enabled comparison of the bulk milk test result to the aggregate of individual serum results. The relative contribution of milk from cows with different milk yield and antibody status to the tank, i.e. its composition, was expected to influence the outcome of the bulk milk test. Therefore, sensitivity and specificity were calculated at different cut-off levels, not only using a standard cross-tabulation technique, but also a logistic regression model. By using the latter method, the sensitivity and specificity could be estimated adjusting for milk yield covariates. Specificity was estimated to be high ( approximately 98%) at the 0.20 cut-off, which can be used as a decision threshold to rule in infection. With more equal emphasis on sensitivity and specificity, a lower cut-off should be used. Although infection cannot be completely ruled out, herds with test results below 0.05 are highly likely to be non-infected. The within-herd prevalence of false negative herds is probably less than 10-15% at this level. From what is known about test performance at the individual level and the prevalence of infection, the estimate of the specificity of the bulk milk test should be quite accurate while the sensitivity is likely to be underestimated. We confirmed that the performance of the bulk milk test depends on the milk tank composition. In particular the milk yield of cows with high antibody levels affects the probability of a positive outcome of the bulk milk test.