Mannitol agar for microbiologic diagnosis of bovine mastitis

A medium containing mannitol (mannitol agar) was developed and evaluated as a tool for the microbiologic diagnosis of bovine mastitis. Mannitol agar supported growth of all important bacterial mastitis pathogens (staphylococci, streptococci, coliforms, and pseudomonads) except Corynebacterium pyogenes. Color change around colonies in the agar permitted the differentiation of pathogenic from nonpathogenic staphylococci. Most Staphylococcus aureus strains and some Staphylococcus epidermidis strains produced yellow zones. These yellow zone-producing strains (mannitol fermenters) of staphylococci were obtained from quarters with significantly elevated (P less than 0.05) somatic cell counts (SCC) in the milk, as compared with uninfected quarters and, therefore, would be considered pathogens. Mannitol-negative strains of S epidermidis (those with red zones) were obtained from quarters with SCC similar to those of uninfected quarters. The streptococci could be divided into 2 groups on the basis of color change around the colonies: Streptococcus agalactiae, Str dysgalactiae, and group G streptococci produced red zones; Str uberis, Str bovis, and enterococci produced yellow zones. Pathogenic streptococci (Str agalactiae, Str dysgalactiae, Str uberis, and group G streptococci) were obtained from quarters with SCC significantly higher (P less than 0.01) than those of uninfected quarters. Streptococcus bovis and enterococci were obtained from quarters with SCC similar to those of uninfected quarters and were considered nonpathogenic. Pathogenic streptococci were found in much higher concentration than nonpathogenic streptococci and could be differentiated on that basis.     

Frequency of isolation of environmental mastitis-causing pathogens and incidence of new intramammary infection during the nonlactating period

Quarter samples (n = 6,328) of mammary secretions were collected from 160 cows during physiologic transitions of the udder to determine the frequency of isolation of mastitis-causing pathogens and the incidence of new intramammary infections (IMI) during the nonlactating period. None of the cows in the herd was infected with Streptococcus agalactiae, and the prevalence of Staphylococcus aureus was low. Cows were not treated with antibiotics at cessation of milking. A threefold increase in the percentage of quarters infected with major mastitis-causing pathogens developed from late lactation to early involution. Coliforms and streptococci other than Str agalactiae accounted for 94% of major pathogen infections. The number of quarters infected with coagulase-negative staphylococci increased slightly from late lactation to early involution, whereas the number of quarters infected with Corynebacterium bovis decreased markedly. Major pathogens caused 101 of 153 IMI at parturition and greater than 90% were caused by streptococci and coliforms. At parturition, 51 of 52 minor pathogen IMI were caused by coagulase-negative staphylococci. During early lactation, there was a marked decrease in quarters infected with major pathogens; however, the number of quarters with major pathogen IMI during early lactation was 2.3 times higher than the number of quarters infected before cessation of milking. The number of quarters with minor pathogen IMI during early lactation was the same as at parturition, but a marked decrease in quarters infected with coagulase-negative staphylococci and a marked increase in C bovis IMI developed from parturition to early lactation.     

Duration of bovine intramammary infections in commercial dairy herds

Data on the infection status of cows on seven commercial dairy farms were collected over 492 full lactations. Foremilk samples were taken at an average interval of five weeks. A total of 249 streptococcal and 433 staphylococcal infections were diagnosed. Spontaneous elimination occurred in 49 per cent of all streptococcal infections and in 54 per cent of Staphylococcus aureus infections. The average duration of spontaneously eliminated infections was 10.8 weeks for Streptococcus agalactiae, 9.9 weeks for Strep dysgalactiae, 10.4 weeks for Strep uberis and 12.8 weeks for Staph aureus. The average duration of infections persisting until drying off was 19.3 weeks for Strep agalactiae, 18.7 weeks for Strep dysgalactiae, 18.5 weeks for Strep uberis and 25.2 weeks for Staph aureus. The method and rate of elimination of infection as found in this analysis are of value for estimating new infection rates and selecting quarters for dry cow therapy.     

The susceptibility of bovine udder quarters colonized with Corynebacterium bovis to experimental infection with Staphylococcus aureus or Streptococcus agalactiae.

Twenty bovine udder quarters colonized with Corynebacterium bovis SR6 and 20 uncolonized quarters were challenged by inoculation of Staphylococcus aureus Newbould 305 (ATCC 29740) into the teat cistern. The percentage of infection in quarters colonized with C. bovis (50%) was significantly lower than that in controls (100%). By similar challenge no significant difference was observed between the percentage of infection with Streptococcus agalactiae ATCC 27956 in 33 quarters colonized with C. bovis (70%) compared to 33 controls (87.9%). A total of 37 quarters colonized with C. bovis and 37 control quarters were challenged with Staph. aureus Newbould 305 (ATCC 29740) and Maxi (ATCC 27543) and Strep. agalactiae (ATCC 27956) by exposure of the teat orifice. The percentage of teat ducts colonized with C. bovis which became infected with either pathogen was not different from that for controls.     

Development of a novel biochip for rapid multiplex detection of seven mastitis-causing pathogens in bovine milk samples

To efficiently prevent and treat bovine mastitis and minimize its effect on the dairy industry, a sensitive, rapid, and specific test is required for identifying the mastitis-causing pathogens. In this study, a biochip capable of detecting 7 common species of mastitis-causing pathogens, including Corynebacterium bovis, Mycoplasma bovis, Staphylococcus aureus, and the Streptococcus spp. S. agalactiae, S. bovis, S. dysgalactiae, and S. uberis, within 6 hr was developed. The technique is based on DNA amplification of genes specific to the target pathogens and consists of 4 basic steps: DNA extraction of bacteria, polymerase chain reaction, DNA hybridization, and colorimetric reaction. To examine the accuracy and specificity of this biochip, a preliminary test with 82 random quarter milk samples were analyzed and compared with results from conventional microbiological methods conducted simultaneously. Results from all but 1 sample analyzed by the biochip were in agreement with those analyzed by bacteriology. The biochip could be a feasible tool for rapidly diagnosing mastitis-causing pathogens in milk and providing information for a more effective treatment to cure mastitis.     

Rapid identification of bovine mastitis pathogens by high-resolution melt analysis of 16S rDNA sequences

Accurate identification of mastitis pathogens is often compromised when using conventional culture-based methods. Here, we report a novel, rapid assay tested for speciation of bacterial mastitis pathogens using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time PCR amplification of 16S rRNA gene fragment, spanning the variable region V5 and V6 was performed with a resulting amplicon of 290bp. First, a library was generated of melt curves of 9 common pathogens that are implicated in bovine mastitis. Six of the isolates, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus uberis, Staphylococcus aureus and Mycoplasma bovis, were type strains while the other 3, Arcanobacterium pyogenes, Corynebacterium bovis and Streptococcus dysgalactiae, were bovine mastitis field isolates. Four of the type strains, E. coli, S. agalactiae, K. pneumoniae and S. aureus, were found to be of human origin, while the other 3 type strains were isolated from bovine infections. Secondly, the melt curves and corresponding amplicon sequences of A. pyogenes, E. coli, S. agalactiae, S. dysgalactiae, K. pneumoniae, S. uberis and S. aureus were compared with 10 bovine mastitis field isolates of each pathogen. Based on the distinct differences in melt curves and sequences between human and bovine isolates of E. coli and K. pneumoniae, it was deemed necessary to select a set of bovine strains for these pathogens to be used as reference strains in the HRMA. Next, the HRMA was validated by three interpreters analyzing the differential clustering pattern of melt curves of 60 bacterial cultures obtained from mastitis milk samples. The three test interpreters were blinded to the culture and sequencing results of the isolates. Overall accuracy of the validation assay was 95% as there was difficulty in identifying the streptococci due to heterogeneity observed in the PCR amplicons of S. uberis. The present study revealed that broad-range real-time PCR with HRMA can be used as a powerful, fast and low-cost tool for the differentiation of clinically important bacterial mastitis pathogens.     

Microbial pathogens from goat mastitis and phage-typing of Staphylococcus aureus isolates

Examination of milk from goats yielded 41 strains from 40 clinically affected halves; 15 were Staphylococcus aureus, 6 Staph. epidermis, 1 Streptococcus agalactiae, 2 Strept. dysgalactiae, 5 Strept. uberis, 2 Corynebacterium pyogenes, 3 Escherichia coli, 3 Pasteurella spp. and 4 Mycoplasma spp. One half had dual infection of Staph. aureus and Strept. dysgalactiae. Twenty two of the 297 milk samples from apparently normal halves also harboured pathogens comprising of 9 Staph. aureus, 1 Strept. agalactiae, 2 E. coli, 2 Pasteurella spp., 2 Candida albicans and 6 Mycoplasma spp. Most of the bacterial isolates were sensitive to many broad spectrum antibiotics. Twenty of the 24 Staph. aureus isolates were phase typable by a set of 23 human Staphylococcal International Phages suggesting the utility of these phages for the typing of goat strains. The isolates were grouped into 15 phage-types, many of which have been reported from human infections in Iraq. This indicates the possibility of association of human strains of Staph. aureus in caprine mastitis. No definite correlation could be noted between antibiogram and phage types of Staph. aureus strains.     

2018年天津市原料奶细菌总数、体细胞数及奶牛乳房炎病原菌、耐药基因调研报告

本研究旨在调查天津市原料奶细菌总数、体细胞数及乳房炎病原菌、耐药基因,了解全市原料奶的质量状况及引起奶牛乳房炎发生的主要原因。采集天津市5家乳品加工企业奶罐车的原料奶样品,用于检测体细胞数和菌落总数;采集天津市奶牛养殖场储奶罐奶样品,用于检测体细胞数;采集奶牛场临床型乳房炎发病乳区的牛奶样品,用于检测乳房炎病原菌及耐药基因。结果显示:2018年天津市乳品加工企业原料奶体细胞数平均值为44.65万/mL,标准差42.41万/mL,变异系数94.97%,最大值为225.50万/mL,最小值为1.20万/mL,SCC≤50万/mL的样品占74.37%,50万/mL200万/mL的样品占3.13%;细菌总数平均值11.54万CFU/mL,标准差26.28万CFU/mL,变异系数227.66%,最大值190.00万CFU/mL,最小值0.095万CFU/mL,细菌总数≤10万CFU/mL的样品占74.37%,10万CFU<细菌总数≤50万CFU/mL的样品占21.25%,50万CFU<细菌总数≤100万CFU/mL的样品占1.88%,100万CFU/mL<细菌总数≤200万CFU/mL的样品占2.50%。2018年天津市奶牛养殖场原料奶体细胞数平均值为38.81万/mL,标准差36.49万/mL,变异系数94.03%,最大值为210.00万/mL,最小值为0.80万/mL,SCC≤50万/mL的样品占79.17%,50万/mL200万/mL的样品占0.83%。乳房炎病原菌检测结果显示:36个样品检出病原菌,总检出率为94.74%;共检出8种病原菌,检出率最高的是乳房链球菌,检出率为73.68%,其他病原体检出率依次为:牛支原体34.21%,牛棒状杆菌13.16%,无乳链球菌10.53%,大肠杆菌5.26%,白色念球菌5.26%,停乳链球菌2.63%,铜绿假单胞菌2.63%。14个样品检出耐药基因,总检出率为36.84%;2种耐药基因的检出率分别为β-内酰胺耐药基因CTX-M934.21%,耐甲氧西林葡萄球菌耐药基因MecA 2.63%。研究表明,2018年天津市原料奶SCC及细菌总数大部分接近欧盟标准,但仍有待进一步提高;引起天津地区临床型乳房炎的3种主要致病菌为乳房链球菌、牛支原体和牛棒状杆菌。     

"乳康2号"对奶牛细菌性乳房炎的疗效研究

用＂乳康2号＂治疗临床型乳房炎奶牛37头。结果表明,治愈28头,有效6头,无效3头,总有效率为91.89%,平均治疗时间4.7d。对采集的37头奶牛治疗前的42个乳区奶样,进行细菌学检查,有33个乳区检出与乳房炎有关的病原菌,占78.57%。检出病原菌5种,主要为无乳链球菌、停乳链球菌、金黄色葡萄球菌、乳房链球菌和大肠埃希菌。停药后采集33个乳区奶样,结果有14个乳区细菌转阴,细菌转阴率为42.42%。在转阴的病原菌中,大肠埃希菌转阴率最高（100%）,其次是混合感染（60.00%）和乳房链球菌（50.00%）。     

多重PCR检测奶牛乳腺炎金黄色葡萄球菌、无乳链球菌、停乳链球菌和酵母菌方法的建立与应用

参照GenBank发表的序列，在金黄色葡萄球菌、无乳链球菌和停乳链球菌16SrRNA与23SrRNA之间的区域设计了3对引物，参照念珠菌和隐球菌的18SrRNA的序列设计1对引物，建立了检测金黄色葡萄球菌、无乳链球菌、停乳链球菌和酵母真菌4种乳腺炎主要致病菌的多重PCR方法。参照Skladny的方法制备模拟了细菌感染l临床标本。结果表明：本试验建立的多重PCR方法具有较好的特异性，多重PCR方法检测乳样中的金黄色葡萄球菌的细菌最小浓度为10^4CFU／mL，检测无乳链球菌、停乳链球菌和酵母真菌的细菌最小浓度分别为10^4CFU／mL、10^3CFU／mL和10^3CFU／mL。通过对采自临床型乳腺炎（46个）和隐性乳腺炎（167个）动物共计213个乳样分别用传统细菌学培养法和多重PCR方法进行检测，多重PCR对金黄色葡萄球菌和酵母真菌的检测具有更高的检出率（P〈0．01），但该方法对无乳链球菌和停乳链球菌的检出率与培养法差异不显著（P〉0．05）。     

奶牛急性乳房炎病原菌的分离与鉴定

[目的]为了掌握奶牛急性乳房炎致病菌的种类.[方法]通过采集病牛乳汁样品3份,分别对其病原茵进行分离与鉴定.检测[结果]表明:检出4种共13株细菌,其中致病性金黄色葡萄球菌6株;停乳链球菌5株;大肠埃希茵1株,乳房链球茵1株.[结论]该场奶牛的急性乳房炎是由致病性金黄色葡萄球菌、停乳链球菌、乳房链球菌和大肠杆菌共同引起...     

Udder shape and teat-end lesions as potential risk factors for high somatic cell counts and intra-mammary infections in dairy cows

The association of common bacterial pathogens in milk samples during calving with udder shape or the presence of 'teat-end' lesions was investigated in 240 dairy cows from two herds. Sixty-three of 120 cows (53%) in one herd (herd A) and 54/120 animals (45%) in a second herd (herd B) had normal-shaped udders. The remaining animals had udder shapes defined as follows: large pendulous (18% herd A, 26% herd B); large between hindquarter (10% herd A, 17% herd B); overall small (8% herd A, 5% herd B); or small but pendulous (11% herd A, 7% herd B). At calving teat-end lesions were present in 63% and 76% of the quarters of herd A and B animals, respectively. There was no herd effect on udder shape or teat-end lesions. Analysis of variance revealed that udder shape and teat-end lesions did not have a significant association with quarter somatic cell count. However there was some association between mammary infection and udder shape and teat-end lesions. Compared to other udder shapes, cows with large between hindquarter shape had significantly less Staphylococcus aureus and Streptococcus uberis infection (P<0.001). There was a similar albeit less significant negative association with Escherichia coli infection (P<0.01). Infection with Streptococcus agalactiae and Streptococcus dysgalactiae was more frequent in cows with large pendulous and overall small udder conformations. The results also suggest an association between intra-mammary infection at calving and the presence of hyperkeratotic teat-end lesions, given that S. aureus, coagulase-negative staphylococci, S. uberis, S. agalactiae and E. coli were cultured from significantly more quarters with such lesions than from quarters without lesions or with other types of lesion (P<0.001).     

Incidence and types of clinical mastitis in dairy herds with high and low somatic cell counts

Eighteen dairy herds were studied, 12 with a 12-month Dairy Herd Improvement Association herd mean somatic cell count (SCC) less than or equal to 150,000 cells/ml (low SCC) and 6 with a 12-month mean SCC greater than 700,000 cells/ml (high SCC). At the outset of the study, quarter samples for bacteriologic culture were collected (in duplicate) from all quarters of all lactating cows (whole herd culture). Subsequently, quarter milk samples for culture from all cows with clinical mastitis were collected for a period of 6 months. In the herds with low SCC, results of whole herd culture revealed low prevalence of intramammary infection attributable to all major pathogens (less than 4% of all quarters). Prevalence of infection with Streptococcus agalactiae (22.2% of all quarters) and Staphylococcus aureus (6.6% of all quarters) was significantly (P less than 0.05) higher in the herds with high SCC. Mean incidence of clinical mastitis in the herds with low SCC was 4.23 infections/100 cows/month (range, 0.42 to 10.25 infections). In the herds with high SCC, mean incidence was 2.91 infections/100 cows/month (range, 1.33 to 3.92 infections). In the herds with low SCC, infection type, as mean percentage of total clinically infected quarters sampled for culture/herd, was 0.0%, 2.2%, 12.3%, 43.5%, and 28.6% for Str agalactiae, S aureus, streptococci other than Str agalactiae, coliforms, and organisms not isolated, respectively. Respective percentages for the herds with high SCC were 41.5%, 18.3%, 12.6%, 8.0%, and 8.8%. During the study period (from April through January), incidence of clinical mastitis and clinical mastitis caused by coliform bacteria were highest in July and August for herds with low SCC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adherence as a Prerequisite for Infection of the Bovine Mammary Gland by Bacteria

Using a simple in vitro test it was demonstrated that staphylococci, Streptococcus agalactiae, and micrococci, the species of bacteria which are commonly isolated from udder infections, adhered to mammary gland epithelial cells readily and in large numbers. Some strains of organisms which are associated with sporadic outbreaks or occur less commonly, like Str. dysgalactiae and Str. uberis, adhered moderately. Escherichia coli, Klebsiella spp., Corynebacterium pyogenes, C. bovis, Str. bovis, and Str. faecalis, species which are isolated occasionally, adhered poorly. From these studies, it appears that selective adherence of bacteria to the epithelial cells is a factor contributing to the ability of organisms to infect the mammary gland and may, therefore, be considered an important stage in the pathogenesis of bovine mastitis.     

Occurrence of clinical and sub-clinical mastitis in dairy herds in the West Littoral Region in Uruguay

Twenty-nine dairy farms were selected to determine the incidence of clinical mastitis, prevalence of sub-clinical mastitis and bacterial aetiology in the West Littoral Region of Uruguay. In samples taken by the owner and frozen at -20 degrees C during a week the incidence rate of clinical mastitis was determined as 1.2 cases per 100 cow-months at risk. Staphylococcus aureus was the most common isolated pathogen in 37.5% of 40 milk samples from clinical cases obtained in 1 month. No bacteria grew in the 32.5% of the total samples. A sub-sample including 1077 dairy cows from randomly selected farms was used to determine the prevalence of sub-clinical mastitis. These samples were taken on one visit to each farm. The prevalence was 52.4% on a cow basis and 26.7% on an udder quarter basis. In 55.1% of the quarters of the selected animals with more than 300,000 cells/ml there was no growth. The isolated pathogens from sub-clinical cases and their relative frequencies were: Staphylococcus aureus 62.8%, Streptococcus agalactiae 11.3%, Enterococcus sp. 8%, coagulase-negative staphylococci 7.4%, Streptococus uberis 6.4%, Streptococcus dysgalactiae 1.8%, Escherichia coli 1.5% and Staphylococcus hyicus coagulase-positive 0.6%.     

Investigating clustering in interval-censored udder quarter infection times in dairy cows using a gamma frailty model

Udder infections in dairy cows are observed at udder quarter level. Therefore, the best strategy to study infection dynamics of particular bacteria causing mastitis is to follow up and model individual udder quarter infection times. Udder quarter infection times, however, are not independent as they are clustered within a cow and herds. Another challenge in modelling infection times is that the exact infection time is unknown; it is only known that the infection has taken place in the interval between the last negative and the first positive sample. We applied a technique based on the gamma frailty model which handles the clustering and interval censoring simultaneously. Parameter estimates can be obtained analytically and their variance is obtained by the inverse of the hessian matrix. The proposed technique was applied to udder quarter infection times for Corynebacterium bovis, Staphylococcus aureus and Streptococcus uberis. Multiparous cows were more likely to get infected earlier in lactation with C. bovis or S. uberis than primiparous cows. The times to infection of all three bacteria were highly clustered at cow level and the results of a stratified model on a subset of herds suggested a high clustering on herd level for C. bovis and S. uberis.     

通用基因芯片对奶牛乳房炎主要致病茵的检测

应用生物信息学手段和查阅文献资料设计了金黄色葡萄球菌、大肠埃希菌、无乳链球菌、绿脓杆菌、停乳链球菌、乳房链球菌6种奶牛乳房炎主要致病菌的通用引物和金黄色葡萄球菌、大肠埃希菌、绿脓杆菌的寡核苷酸探针及无乳链球菌、停乳链球菌、乳房链球菌的特异引物,并用这3种特异引物扩增片段的纯化产物作为这3种链球菌的检测探针。在引物对样品中细菌的相应基因片段扩增的同时进行靶基因的生物素标记,扩增的产物与硝酸纤维素膜上的探针进行杂交,酶联、显色后根据芯片扫描仪的判读结果来确定奶牛乳房炎致病菌感染的种类。结果表明,建立的以16S rDNA为对象的基因芯片技术可以快速的检测出以上6种细菌,整个检测过程需要6h~7h,灵敏度高,特异性好,能快速的对奶牛乳房炎的主要致病菌做出诊断。     

The major bovine mastitis pathogens have different cell tropisms in cultures of bovine mammary gland cells

We previously showed that Staphylococcus aureus cells adhered mainly to an elongated cell type, present in cultures of bovine mammary gland cells. Moreover, we showed that this adhesion was mediated by binding to fibronectin. The same in vitro model was used here, to study adhesion of other important mastitis pathogens. Like the S. aureus strains, the Streptococcus dysgalactiae strains adhered mainly to elongated cells, which seemed to be mediated by fibronectin binding. In contrast, Streptococcus uberis strains adhered mainly to cubic cells. Since the cubic cells did not express fibronectin and S. uberis cells bound fibronectin less efficiently, the adhesion of S. uberis cells was independent of fibronectin binding. Streptococcus agalactiae strains adhered poorly to both cell types. The specificity and efficiency of adhesion of Escherichia coli strains was strongly strain dependent. None of the S. agalactiae and E. coli strains tested was able to bind fibronectin efficiently. The results suggest that the different mastitis pathogens have different target cell specificities and use different mechanisms to adhere to cells of the bovine mammary gland.     

Experimental infection of lactating bovine mammary glands with Streptococcus uberis in quarters colonized by Corynebacterium bovis

Twenty-seven quarters of 18 lactating dairy cows were inoculated intramammarily with 3.6 X 10(4) colony-forming units (CFU) of a strain of Streptococcus uberis isolated from a cow with clinical mastitis. Before quarters were inoculated, 22 were considered as naturally colonized with Corynebacterium bovis, and 5 were considered bacteriologically negative. Streptococcus uberis was isolated from all quarters within 2 days after inoculation, and all quarters developed clinical mastitis by 3 days after inoculation. Mastitis was acute, and most cows had increased rectal temperatures. The number of somatic cells increased significantly (P less than 0.05), and milk production decreased significantly. In many cows, rectal temperatures remained increased, and Str uberis was isolated from infected glands after intramammary and systemic antimicrobial treatments were given. A decreased number (110 CFU) of the same strain of Str uberis caused equally severe mastitis in 3 quarters colonized with C bovis and in 1 bacteriologically negative quarter in 2 cows. Streptococcus uberis was isolated from all inoculated quarters, and all quarters developed clinical mastitis by 2 days after inoculation. Two quarters colonized with C bovis and 2 bacteriologically negative quarters were inoculated once with 25 CFU and once with 240 CFU of a different strain of Str uberis (ATCC 27958). Streptococcus uberis was never isolated from inoculated quarters, and changes in milk yield or number of somatic cells were not observed.     

连翘酯苷在体外与体内的抑菌效果研究

本文提取制备了连翘酯苷,并对其进行了体内和体外的抑菌试验。试验结果表明,连翘酯苷在体外和体内对引发奶牛乳房炎的主要致病菌金黄色葡萄球菌、停乳链球菌、无乳链球菌均有较好的抑制作用。