奶牛乳腺炎病原菌的分离鉴定及耐药性分析

2 0 0 2～ 2 0 0 3年采集上海地区 32个规模化奶牛场 433份奶样 ,共分离 2 54株细菌。其中包括金黄色葡萄球菌 48/2 54 (1 8 9% )、表皮葡萄球菌 34/2 54 (1 3 4% )、大肠杆菌 2 8/2 54 (1 1 0 % )、肺炎 /溶血巴氏杆菌 1 1 /2 54 (4 3 % )、无乳链球菌 2 5/2 54 (9 8% )、停乳链球菌 1 6/2 54(6 3 % )、乳房链球菌 1 5/2 54 (5 9% )及酵母菌 8/2 54 (5 9% )。表明葡萄球菌性乳腺炎仍占主导地位。药敏试验结果表明 ,分离菌株对临床常用的多种抗菌药物都产生了耐药性 ,耐药菌株的出现和抗生素的残留给乳腺炎的防治及食品安全带来了隐患     

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.     

重组溶葡萄球菌酶对奶牛乳房炎和子宫内膜炎常见病原菌的体外杀菌试验

从患乳房炎和子宫内膜炎奶牛的奶或子宫黏液中分离菌株,鉴定后选取金黄色葡萄球菌105株、表皮葡萄球菌20株、大肠埃希氏菌20株、链球菌38株(其中停乳链球菌8株,无乳链球菌30株)和牛源多杀巴氏杆菌5株,在体外用重组溶葡萄球菌酶、苯唑西林、氨苄西林、庆大霉素按照微量肉汤稀释法进行了杀菌试验。结果表明,重组溶葡萄球菌酶对奶牛乳房炎和子宫内膜炎分离的金黄色葡萄球菌和表皮葡萄球菌具有良好抑杀效果,对链球菌(包括无乳链球菌和停乳链球菌)和牛源多杀巴氏杆菌具有一定的抑杀效果,对大肠杆菌作用较差;苯唑西林、氨苄西林、庆大霉素都有一定量的耐药菌株产生,而重组溶葡萄球菌酶对苯唑西林、氨苄西林、庆大霉素耐药菌株同样具有良好抑杀效果。     

Prevalence of contagious mastitis pathogens in bulk tank milk in Prince Edward Island

The purpose of this study was to 1) estimate the herd prevalence of contagious mastitis pathogens in bulk milk from Prince Edward Island (PEI) dairy farms, 2) determine the association between bulk milk culture results and mean bulk milk somatic cell count (BMSCC), and 3) investigate the agreement of repeated bulk milk cultures. Three consecutive bulk milk samples were obtained at weekly intervals from all 258 PEI dairy herds and were cultured using routine laboratory methods. Cumulative prevalence of Staphylococcus aureus, Streptococcus agalactiae, and Mycoplasma spp. (M. bovis and M. alkalescens) was 74%, 1.6%, and 1.9%, respectively. Bulk milk somatic cell count of Staph. aureus-positive herds was higher than that of negative herds. Agreement for Staph. aureus isolation between 3 consecutive tests was moderate (kappa = 0.46). Mycoplasma bovis and M. alkalescens in bulk milk are being reported for the 1st time in PEI ever and in Canada since 1972.     

Survey of marbofloxacin susceptibility of bacteria isolated from cattle with respiratory disease and mastitis in Europe

A monitoring programme conducted in Europe since 1994 to survey the marbofloxacin susceptibility of bacterial pathogens isolated from cattle has established the susceptibility of bacterial strains isolated before any antibiotic treatment from bovine mastitis and bovine respiratory disease (BRD) cases between 2002 and 2008. Minimum inhibitory concentration (MIC) was determined by a standardised microdilution technique. For respiratory pathogens, Pasteurella multocida and Mannheimia haemolytica isolates (751 and 514 strains, respectively) were highly susceptible to marbofloxacin (MIC≤0.03 μg/ml for 77.39 per cent of the strains) and only 1.75 per cent of M haemolytica strains were resistant (MIC≥4 μg/ml). Histophilus somni isolates (73 strains) were highly susceptible to marbofloxacin (0.008 to 0.06 μg/ml). Mycoplasma bovis MIC (171 strains) ranged from 0.5 to 4 μg/ml. For mastitis pathogens, the majority of Escherichia coli isolates were highly susceptible to marbofloxacin (95.8 per cent of 617 strains). Staphylococcus aureus and coagulase-negative staphylococci (568 and 280 strains) had a homogenous population with MIC centred on 0.25 μg/ml. Streptococcus uberis and Streptococcus dysgalactiae (660 and 217 strains) were moderately susceptible with MIC centred on 1 μg/ml. Marbofloxacin MIC for these various pathogens appeared stable over the seven years of the monitoring programme and was similar to previously published MIC results.     

Mastitis in lactating camels (Camelus dromedarius) in Afar Region, north-eastern Ethiopia.

Quarter milk samples (n = 543) from 152 traditionally managed lactating camels (Camelus dromedarius) in Afar Region, north-eastern Ethiopia were examined to determine the prevalence of camel mastitis and identify its bacterial causes. Out of 152 camels examined, 19 (12.5%) were diagnosed as clinical mastitis cases based on clinical signs and bacteriological examinations. Of the 257 California Mastitis Test (CMT) positive quarter milk samples 162 (63.0%) yielded pathogenic bacteria. A positive correlation was observed between CMT positive results and presence of major pathogens in camel milk samples. The main mastitis pathogens isolated were Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus agalactiae, S. dysgalactiae, and other species of streptococci, Pasteurella haemolytica and E. coli. Results of the present study suggest that mastitis in Afar camels is prevalent, Gram-positive cocci are the major isolates from camel milk samples and the CMT can be used as a screening test for the detection of mastitis in camels.     

呼和浩特地区奶牛隐性乳房炎主要病原菌的分离鉴定及药敏试验

本试验对113份奶牛隐性乳房炎奶样进行细菌分离鉴定、致病性评估及药敏试验。共分离到3大类5种91株细菌，其中金黄色葡萄球菌72株，溶血葡萄球菌8株，表皮葡萄球菌6株；停乳链球菌1株；大肠杆菌2株，沙雷氏菌1株，克雷伯氏菌1株。经过绵羊血溶血试验及小白鼠动物试验，确定部分菌株为致病菌。8类18种药物的药敏试验结果表明，金黄色葡萄球菌和溶血葡萄球菌均对四环素、氯霉素、庆大霉素、卡那霉素等药物高度敏感；链球菌对洁霉素、红霉素、氯霉素、喹诺酮类以及庆大霉素等均高度敏感，肠杆菌对新霉素、磺胺、四环素、链霉素、丁胺卡那、庆大霉素、喹诺酮类等均高度敏感，而对临床上常用的青、链霉素等药物均产生了不同程度的耐药性。     

A real-time polymerase chain reaction assay for the detection of Mycoplasma agalactiae

AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene. METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay. RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 104 cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%. CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae.     

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）。     

多重PCR快速检测奶牛乳房炎3种主要病原体

奶牛乳房炎是引起奶牛业经济损失的一种重要疫病,目前还没有快速、特异检测奶牛乳房炎主要致病原的方法。本试验根据金黄色葡萄球菌、无乳链球菌、大肠杆菌各自保守的16S或23S rRNA基因序列,合成了3对特异性引物,建立了三重PCR检测方法。特异性试验表明,该方法对所有参与测试的金黄色葡萄球菌、无乳链球菌和大肠杆菌都能扩增出各自的阳性条带,而对所有参与测试的对照菌株则不能扩增出任何条带。敏感性试验表明该方法能检测到4个菌的金黄色葡萄球菌、无乳链球菌和2个菌的大肠杆菌。对送检的乳房炎奶样36份直接进行PCR检测,金黄色葡萄球菌阳性7份,无乳链球菌阳性2份,大肠杆菌阳性6份。     

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.     

Etiological agents of dairy cows' mastitis in western part of Poland

The purpose of the study was to determine the etiological mastitis agents in cows from herds located in the western part of Poland in years 2003-2005. Bacteriological examinations of 18,713 samples taken from California Mastitis Test (CMT) positive quarters were performed according to standard methods. Some Gram-negative bacilli and Gram-positive cocci were examined with the use of API tests (Analytical Profile Index). It was found that 32.7% of samples that were CMT positive were culture negative and 9.3% of samples were contaminated. Streptococcus species (15.7%), coagulase negative staphylococci (CNS) (14.6%), Staph. aureus (8.6%), Gram-negative bacilli (4.0%) and Corynebacterium species (3.8%) were most frequently isolated. Escherichia coli (52.3%) dominated among Gram-negative bacilli followed by Klebsiella pneumoniae (4.1%), Pseudomonas aeruginosa (3.6%), Enterobacter cloacae (3.6%), Serratia marcescens (3.1%), Pasteurella multocida (3.1%), Acinetobacter lwofii (3.1%), and 26 other bacteria species. Stahylococcus xylosus (31.5%), Staph. hyicus (12.2%), Staph. haemolyticus (12.2%), Staph. sciuri (11.6%), Staph. chromogenes (8.8%), Staph. epidermidis (8.3%) and Staph. simulans (6.1%) were the most frequent CNS. Streptococcus uberis (50%), Str. dysgalactiae (19.7%), Str. acidominimus (6.6%) and Enterococcus faecalis (5.3%) were mostly found among CAMP-negative streptococci. An increase in frequency of mastitis caused by non-agalactiae streptococci, Staph. aureus, A. pyogenes and yeast-like fungi and a decrease in that produced by Str. agalactiae in 2005 as compared to years 2003 or 2004 were observed.     

Antimicrobial resistance of bacteria isolated from dairy cow milk samples submitted for bacterial culture: 8,905 samples (1994-2001)

OBJECTIVE: To determine whether antimicrobial resistance patterns of major mastitis pathogens isolated from milk samples from dairy cows have changed over time. DESIGN: Retrospective study. SAMPLE POPULATION: 8905 bacterial isolates obtained from milk samples submitted to the Wisconsin Veterinary Diagnostic Laboratory between January 1994 and June 2001. PROCEDURE: Antimicrobial susceptibility was determined by means of the Kirby-Bauer disk diffusion method. Logistic regression was used to determine whether percentages of isolates resistant to various antimicrobials changed over time. RESULTS: For the gram-positive mastitis pathogens, percentages of isolates resistant to various flactam antimicrobials did not increase over the course of the study. Percentage of Staphylococcus aureus isolates resistant to penicillin decreased from 49 to 30%; percentage of Streptococcus isolates resistant to penicillin decreased from 6 to 1%. Percentage of isolates resistant to erythromycin increased for S aureus, Escherichia coli, Enterobacter spp, Enterococcus spp, and Pasteurella spp. Percentage of isolates resistant to lincomycin increased for S aureus and Staphylococcus spp. Percentage of coagulase-negative Staphylococcus isolates resistant to pirlimycin increased from 6 to 19%. For several pathogens, percentages of isolates resistant to sulfisoxazole and to trimethoprim-sulfamethoxazole decreased. No pathogens had a significant increase in the percentage of isolates resistant to novobiocin-penicillin. CONCLUSIONS AND CLINICAL RELEVANCE: Results did not indicate a trend toward increased antimicrobial resistance among mastitis pathogens isolated from milk samples from dairy cows between 1994 and 2001. Reduced resistance to flactam antimicrobials was identified for several gram-positive mastitis pathogens.     

昆明地区奶牛临床型乳房炎病原菌分离鉴定及药敏试验

对昆明地区多个奶牛场的奶牛临床型乳房炎病原菌进行分离、鉴定,从114份乳样中共分离出细菌13种305株,经培养特性、染色镜检、生化鉴定等,鉴定出金黄色葡萄球菌63株,占20.66%;表皮葡萄球菌26株,占8.52%;无乳链球菌53株,占17.38%;停乳链球菌23株,占7.54%;乳房链球菌19株,占6.23%;乳链球菌14株,占4.59%;粪链球菌15株,占4.92%;化脓链球菌14株,占4.59%;大肠杆菌48株,占15.74%;真菌12株,占3.93%。分别对分离菌株进行17种药物的敏感性试验,除真菌外,所有菌株对庆大霉素、新霉素、环丙沙星、诺氟沙星、林可霉素、乳肿康均高度敏感,对临床常用的青霉素、链霉素、鱼腥草等具有较强的耐药性。     

THE NEW SOUTH WALES MASTITIS CONTROL PROGRAM

Bacteriological examinations were made on quarter samples from cows in 35 herds over a 3 year period to monitor the response in a mastitis control program. Initially, Staphylococcus aureus predominated in 32 of the herds and the mean herd prevalence was 26%. The control measures halved this rate but there was considerable variation in response between herds. The decline occurred rapidly and there was a significant reduction (P less than 0.01) by 3 months. Streptococcus agalactiae predominated in 3 herds and the overall infection rate was 4.9%. Control measures eliminated the infection completely from most herds but reinfection occurred in 2 herds. The greatest decline occurred in the first 6 months and was significant (P less than 0.05). The measures had little effect upon Str. uberis and Str. dysgalactiae which remained fairly consistently at low levels. Initially, strains of Staph. aureus resistant to penicillin were dominant in most herds. In a minority of herds strains resistant to streptomycin predominated and in these herds there was a concurrent resistance to penicillin. These patterns did not change greatly over the control period. Resistance by Str. agalactiae to streptomycin occurred in most herds at the start of the program.     

某奶牛场临床型乳房炎的病原分离及鉴定

从某奶牛场患乳房炎的奶牛乳汁中共分离到 13株细菌 ,经培养特性、菌落形态和生化特性等鉴定表明 ,13个分离菌株中 ,无乳链球菌最为多见 ,占 10株 ;金黄色葡萄球菌 2株及大肠杆菌 1株。将 3种细菌经人工感染小白鼠 ,都具有一定的致病力。药敏试验结果表明 ,氨苄青霉素及丁胺卡那对上述细菌具有较好的抑制作用     

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.     

奶牛临床型乳房炎的细菌分离鉴定与耐药性分析

2011年山西省多个奶牛场发生了较严重的乳房炎,对76份采集的奶样进行细菌分离鉴定并采用药敏纸片法检测主要分离菌的抗生素耐药情况。所分离细菌以革兰氏阳性菌为主,革兰氏阳性球菌和其他革兰氏阳性菌分别占60.67%和23.59%。分离出多种病原菌和机会致病菌,主要的病原菌有链球菌、金黄色葡萄球菌、大肠杆菌等,检出率为2.24%~11.24%,其中无乳链球菌的检出率最高;机会致病菌有粪链球菌、微球菌、克雷伯菌、凝固酶阴性葡萄球菌等,检出率为1.12%~11.24%,其中粪链球菌和微球菌的检出率较大,分别为11.24%和6.74%。药敏试验检测结果显示,在所选的15种药物中,主要分离菌均对丁胺卡那霉素、氟哌酸和恩诺沙星3种药物敏感;大肠杆菌、克雷伯菌、凝固酶阴性葡萄球菌对青霉素类和β-内酰胺/β-内酰胺酶抑制剂类药物产生了极强的耐药性,耐药率均为100%;乳房链球菌对该类药物也产生不同程度的耐药,耐药率为20%~100%;对链霉素产生100%耐药的细菌有大肠杆菌、克雷伯菌、无乳链球菌、乳房链球菌、停乳链球菌等细菌;部分分离菌对卡那霉素、庆大霉素、链霉素、四环素、红霉素、先锋霉素Ⅴ、复方新诺明等药物产生不同程度耐药。被检奶牛场混合感染较为严重,应进一步加强环境卫生管理,临床治疗应合理有效用药。     

The In Vitro Activity of 15 Antimicrobial Agents against Bacterial Isolates from Dogs

The in vitro activity of 15 antimicrobial agents against clinical isolates of Staphylococcus pseudintermedius, Staphylococcus aureus, Escherichia coli, Pasteurella spp. and Streptococcus canis from dogs was investigated. For Staphylococcus spp., the highest frequency of resistance was observed for penicillin, followed by ampicillin, tetracycline and chloramphenicol. The highest frequency of resistance in E. coli isolates was recorded for tetracycline and streptomycin. Pasteurella spp. and S. canis had the highest resistance rate for tetracycline and chloramphenicol. Most isolates showed full susceptibility to low-level resistance to colistin, florfenicol and fluoroquinolones. Further studies using larger number of isolates from both healthy and diseased dogs would provide a broader picture of antimicrobial resistance at a national level and promote prudent use of antimicrobial agents in companion animals.     

奶牛乳房炎类型与病原菌感染之间相关性的研究

通过对 2 858份不同类型乳房炎奶样的细菌学检验 ,结果分得细菌共 2 4种 31 38株 ,其中分离鉴定出与乳房炎有密切关系的病原菌 1 2种 1 96 8株 ,病原菌检出率为 6 2 .72 %,各种类型乳房炎常见病原菌的区系分布总的趋势是一致的 ,绝大部分是以无乳链球菌为主 ,其次是停乳链球菌、金黄色葡萄球菌、大肠杆菌和乳房链球菌等。但不同类型之间、同一类型不同等级之间及隐性乳房炎泌乳期和干奶前之间奶样的细菌分布和细菌种类有所不同。乳房炎的轻重与病原菌检出率呈正相关。奶牛乳房即使在健康的状态下也存在着带菌现象