Application of the California mastitis test in intramammary Streptococcus agalactiae and Staphylococcus aureus infections of camels (Camelus dromedarius) in Kenya

A study was conducted on 207 lactating camels in six herds in Kenya to evaluate the California mastitis test (CMT) for the detection of intramammary infections (IMIs) caused by Streptococcus agalactiae and Staphylococcus aureus and to investigate the prevalence of both the pathogens in the camel udder. IMI with S. agalactiae was found in 12% of all camels sampled. IMI with S. aureus was present in 11% of all camels sampled. The herd-level prevalence of IMI varied between 0 and 50% for S. agalactiae and between 0 and 13% for S. aureus. Longitudinal observations over 10–12 months confirmed persistent infections for both pathogens. Observations in one herd suggested that camel pox was a contributing factor in spreading and exacerbating S. agalactiae udder infections.The CMT had quarter-level sensitivities of 77 and 68% for S. agalactiae and S. aureus in camels, respectively. The CMT specificities were 91% for both the pathogens.     

奶牛乳腺炎无乳链球菌毒力相关因子

链球菌、金黄色葡萄球菌和大肠杆菌是引起奶牛乳腺炎的3大病原菌，在链球菌属中无乳链球菌是引起奶牛乳腺炎的重要病原菌之一，由无乳链球菌导致的乳腺炎约占隐性乳腺炎发病率的56.25%。无乳链球菌入侵奶牛乳腺的过程主要包括感染、黏附上皮细胞、侵入上皮细胞、损伤机体和免疫逃避等过程。无乳链球菌的毒力因子具有附着和侵袭机体细胞的作用，使菌体在奶牛乳腺表面形成生物被膜，进而干扰机体的正常免疫功能并引起疾病。本文主要阐述了无乳链球菌在入侵乳腺组织过程中发挥主要作用的毒力因子的种类、作用机制以及调控过程，旨在通过抑制其相关毒力因子的活性，从而阻断无乳链球菌在乳腺中感染和传播，进而为预防和治疗链球菌型乳腺炎提供新的思路。     

Advances on Virulence Factors of Streptococcus agalactiae of Dairy Cows Mastitis

Streptococcus, Staphylococcus aureus, and Escherichia coli are the three major pathogens causing mastitis in dairy cows, and Streptococcus agalactiae (S.agalactiae) in Streptoco-ccus is one of the important pathogens causing mastitis in dairy cows, which accounts for around 56.25% of the incidence of sub-clinic mastitis. The process of S.agalactiae invasion mammary glands of dairy cows mainly includes:infection, adhesion, invasion, and then damage the mammary tissue and entail the inflammation. The virulence factor of S. agalactiae has the function of attaching and invading the mammary gland epithelial cells so that the bacteria could form the biofilm on the surface. After infection, the irritation of the mammary tissues by infectious processes serves to the immune response. In this paper, we summarized the types, mechanism, and regulation process of S. agalactiae virulence factors which plays a major role in the invasion of mastitis, aimed to show the activity of S. agalactiae by inhibiting the activity of its related virulence factors in preventing dairy cow mastitis and provide new ideas for the treatment.     

Epidemiological studies of clinical and subclinical ovine mastitis in Awassi sheep in northern Jordan

Forty-six Awassi sheep flocks selected by stratified random sampling were subjected to a cross-sectional study to determine the prevalence of intramammary infections, to assess the influence of flock size and parity on the prevalence of somatic cell count (SCC) and to identify major udder pathogens. Of the 3472 udder halves examined, 29.8% had over 10⁶ SCC/ml and 0.03% had dry teats due to chronic mastitis. Flocks with 30–49 milking ewes (small flock size) were much younger (P < 0.001) than flocks with 50–99 ewes (medium) and flocks with ≥ 100 ewes (large). Pairwise analysis of the InSCC of both halves of the udders revealed significant mean differences for small and large flock size (P < 0.05), and for medium and large flock size (P < 0.001). Mean InSCC was lower (P < 0.05) in samples obtained from the left half compared with samples of the right half of the udder. Multiparous ewes had higher (P < 0.001) mean InSCC than primiparous ewes. Also, ewes with twin lambs had higher (P < 0.001) mean InSCC in the right half of the udder compared with single-lamb ewes. Samples collected in January (winter) had lower (P < 0.05) mean InSCC compared with samples collected in June. The most common organisms isolated from subclinical mastitis cases were coagulase-negative Staphylococci (17.8%), E. coli (13.6%), Streptococcus agalactiae (6.8%) and Staphylococcus aureus (6.8%). Of the 46 flocks, 20 were monitored monthly for 9 consecutive months to determine the incidence of clinical mastitis diagnosed by shepherds or/and sheep farmers with major pathogens. The incidence of clinical mastitis (expressed as the number of clinical cases per 100 ewe-months) were 2.1 ± 1.9 (SD), 1.9 ± 1.1, and 1.2 + 2.1 for small, medium and large flocks size strata, respectively. The overall population estimate was 1.7 ± 0.02 cases per 100 ewe-months. The most-common clinical isolates were S. aureus (22% of all clinical isolates) and E. coli (14.2%).     

A model to determine sampling strategies and milk inoculum volume for detection of intramammary Staphylococcus aureus infections in dairy cattle by bacteriological culture

A model was developed to evaluate the effects that methods of obtaining milk samples and culture inoculum volumes had on the sensitivity of microbiological culture to detect Staphylococcus aureus intramammary infections (IMI). An assumption was made that milk from mammary quarters infected with S. aureus only contains bacteria intermittently. A modified sine wave function was used to model this intermittent shedding pattern. Specifications for the components of the shedding cycle used in this function were based on quantitative culture results from 54 experimentally infected S. aureus quarters, sampled daily for a period of 30–49 days. The components of the shedding cycle were length in days, peak number of CFU shed per milliliter of milk, and length of time in the cycle when no shedding occurred. These components were used to estimate the model's predicted distribution of S. aureus CFU ml⁻¹ milk when individual quarter milk samples were cultured for S. aureus. The sensitivity of culture for several sampling methods was then calculated. The model predicted that culture of a single quarter milk sample had a sensitivity ranging from 60 to 87% for detection of S. aureus IMI depending on inoculum volume. Quarter milk samples taken on day 1 and repeated either on day 3 or day 4, and cultured separately using 0.1 ml of milk for culture inoculum, were predicted to have sensitivities of 90–95% and 94–99%, respectively. Other milk-sampling strategies examined included culture of a composite milk sample (equal-volume mixture of milk from four separate mammary quarters ) and pooled milk samples in which samples from different milkings (either quarter or composite samples) were mixed together and then cultured. The range of predicted sensitivities of these other sampling strategies was 30–97%. Factors having the greatest impact on the sensitivity of culture, in order of importance were: the type of milk sample, the volume of milk cultured, and the time interval between repeated milk sample collection strategies.     

The Study of Isolation,Identification of S.cerevisiae from Dairy Feed and its Antibacterical Activity in the Fermentation Brothe in vitro

The aim of the study was to isolate and identify S.cerevisiae in the dairy feed and analyze antibacterial activity of S.cerevisiae fermentation brothe in vitro.5 forage samples and 5 feed samples were collected to isolate yeast by YPD solid medium.Biochemical identification and 26S rDNA sequence were used to identify the isolated strains.The antibacterial activity of S.cerevisiae strains was tested by cylinder plate method.A total of 12 yeast belonged to 4 species were isolated and identified, including 3 S.cerevisiae (Y1, Y7 and Y11), 4 Debaryomyces hansenii, 3 Cryptococcus flavescens and 2 Torulaspora debrueckii.The antibacterial activities of the 3 S.cerevisiae fermentation brothe to Staphylococcus aureus, E.coli and Streptococcus agalactiae were different.Y7 and Y11 fermentation brothe showed antibacterial activities to all the tested pathogens, while Y1 inhibit E.coli alone.This study laid the foundations for further research of probiotics containing S.cerevisiae with excellent performance.     

奶牛饲料中酿酒酵母菌的分离鉴定及其发酵液的体外抑菌活性研究

本试验旨在分离鉴定甘肃地区奶牛场饲料中的酿酒酵母菌,进而研究酿酒酵母菌发酵液的体外抑菌活性。通过YPD固体培养基对采集的5份饲草和5份饲料样品中的酵母菌进行分离,分别进行生化鉴定和26SrDNA测序鉴定,进而检测酿酒酵母发酵液的体外抑菌活性。结果表明,从样品中分离出4种共12株酵母菌,分别为3株酿酒酵母菌(Y1、Y7和Y11)、4株汉逊德巴利酵母菌、3株浅黄隐球酵母菌和2株戴尔有孢圆酵母菌;3株酿酒酵母菌的发酵液对奶牛乳房炎金黄色葡萄球菌、大肠杆菌和无乳链球菌的抑菌活性存在差异,其中酵母菌Y7和Y11的发酵液对3种病原菌均具有抑菌活性,而Y1的发酵液只对大肠杆菌具有抑制作用。本试验为进一步筛选性能优异的酿酒酵母菌用于微生态制剂研发奠定了基础。     

Interpretation of California mastitis test scores using Staphylococcus aureus culture results for screening of subclinical mastitis in low yielding smallholder dairy cows in the Dar es Salaam region of Tanzania

Screening of subclinical mastitis under field conditions is done using the California mastitis test (CMT). CMT score of ≥1 corresponding to ≥500,000 somatic cells ml⁻¹ is commonly used as threshold of subclinical mastitis in temperate countries. However, given the innately high physiological level of somatic cells in low yielding dairy cows, this threshold may not apply to low yielding dairy cows. The current study was undertaken to investigate the clinical utility of CMT for screening of Staphylococcus aureus subclinical mastitis in low yielding smallholder dairy cows in Tanzania. A total of 1151 of quarter-milk samples were CMT tested, of these 914-originated from cows with a lactation period of 14–305 days. All samples were screened for subclinical mastitis by the CMT as well as microbiological culture of single, duplicate (two consecutive) and triplicate (three consecutive) samples as a gold standard. For the duplicate and triplicate quarter-samples, cows were considered positive for S. aureus subclinical mastitis if results of microbiologic culture for S. aureus were positive for two of two, and for at least two of the first three consecutive quarter-milk samples collected from that cow, respectively. Using a CMT score of ≥1 would classify 78.6% of the 940 quarter-samples as positive. Eighty-two percent of the samples in which S. aureus was isolated had CMT scores ≥2; this would classify 51.6% of the 940 quarter-samples as positive. For the single sample, this cut-off had sensitivity, specificity and likelihood ratio for S. aureus of 0.87, 0.83 and 4.24, respectively. For the duplicate quarter-milk samples this cut-off had sensitivity, specificity, and likelihood ratio of 0.94, 0.86, and 5.19. While, for the triplicate quarter-milk samples this cut-off had sensitivity, specificity and likelihood ratio of 0.97, 0.92 and 7.47, respectively. Based on these results and practical considerations, it is concluded that CMT score of ≥2 corresponding to ≥800,000 somatic cells Ml⁻¹ is the best cut-off to correctly identify S. aureus intramammary infections in low yielding dairy cows in Tanzania.     

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.     

Risk factors for intramammary infections and relationship with somatic-cell counts in Italian dairy goats

Routine examination of milk was performed on five herds of lactating goats in northern Italy as part of a milk quality-monitoring program in the year 2000. As part of the study, aseptic samples of foremilk were collected monthly from both half udders during the entire lactation for 305 goats, resulting in a total of 4571 samples. The samples were tested with cytological and bacteriological analyses to evaluate the relationship between mammary infections and somatic-cell count (SCC; Fossomatic (TM) method). Prevalence of intramammary infection (IMI) was 40.2% (n = 1837) of all udder-half samples examined. The most-prevalent mastitis agents were coagulase-negative Staphylococci (CNS), 80% (n = 1474 udder-half samples); within this group, Staphylococcus epidermidis was the most-prevalent species (38%). Other prevalence were Staphylococcus aureus 6% (n = 112 udder-half samples) and environmental pathogens 14% of infected udder-half samples (n = 251) with a diverse mixture of species, none of which had a frequency of >4%. Enterococcus faecalis was the most-frequently isolated among this group. Neither Salmonella spp. nor Listeria monocytogenes were detected. The risk (sample level) of infection differed across herds, parities, and stage of lactation according to results from logistic multiple regression. Infection was more common among goats in third and fourth parities and during the later stages of lactation. Of the 2734 samples from uninfected udder halves, the mean log₂ SCC was 3.9 cell/ml; of the 1837 bacteriological positive samples, the mean log₂ SCC was 5.6 cell/ml. According to results from a linear mixed model, concentrations of somatic cells tended to increase with increasing age and days in milk and with the presence of bacteria. Infection with S. aureus was associated with the highest SCS.     

牦牛隐性乳房炎主要病原菌及耐药和毒力基因分布情况

为探明牦牛隐性乳房炎（SCM）主要病原菌及其耐药和毒力基因的分布情况，本研究自甘肃省甘南州夏季牧场收集无乳房炎临床症状牦牛乳样，通过兰州乳房炎试验（LMT）筛选SCM乳样，从中分离病原菌并纯化培养，利用16S rDNA鉴定主要病原菌，通过纸片扩散法判定其药物敏感性，并采用PCR方法对相关耐药及毒力基因进行检测。结果显示，共筛选出牦牛SCM乳样324份，检出率14.43%；主要病原菌为葡萄球菌属、埃希氏菌属和肠球菌属，其中葡萄球菌分离株对青霉素和四环素耐药率最高，分别为59.57%和47.52%；大肠埃希氏菌分离株对四环素和氨苄西林耐药率最高，分别为43.40%和20.75%；粪肠球菌分离株对四环素和红霉素耐药率最高，分别为25.00%和16.67%；59株耐青霉素金黄色葡萄球菌中共检出MRSA 12株，其中7株携带mecA基因，5株含mecC基因；四环素外排泵基因tetK、tetA携带率最高（85.45%、56.36%），核糖体保护基因tetM携带率最低（34.55%）；毒力基因中，clfA、clfB、fib、coa基因检出率较高（87.64%、84.27%、83.15%、82.02%）。研究表明，牦牛SCM的主要病原菌为金黄色葡萄球菌和大肠埃希氏菌，均对青霉素类和四环素类抗生素耐药性较高，其中金黄色葡萄球菌的主要毒力因子为黏附因子和凝固酶。     

Prevalence, risk factors, and major bacterial causes of camel mastitis in Borana Zone, Oromia Regional State, Ethiopia

A cross-sectional study was carried out from November 2010 up to April 2011 to estimate mastitis prevalence and associated risk factors and to assess its bacterial causes in traditionally managed camels in Borana Zone, Southern Ethiopia. Thus, 348 lactating camels were examined clinically, and subclinical cases were checked with California mastitis test (CMT). The overall prevalence of mastitis was 44.8 % (156/348), comprising clinical (19, 5.4 %) and subclinical (137, 39.4 %) cases. The quarter level prevalence of mastitis was 24.0 % (334/1,392). Of the total 1,392 examined teats, 30 were blind, and hence, from the 1,362 non-blind CMT-examined teats, 22.3 % (304/1,362) were CMT positive. Of the 304 CMT-positive samples, 264 were culture positive (197 Gram-positive, 41 Gram-negative, and 26 mixed isolates), and 40 were culture negative. The prevalence of Staphylococcus aureus was found to be the highest at both the animal (12.8 %, 39/304) and quarter level (2.9 %, 39/1,362). Regression analysis revealed higher likelihood of mastitis occurrence among camels from Dharito (OR?=?3.4, 95 % confidence interval (CI)?=?1.8, 6.4), Gagna (OR?=?3.4, 95 % CI?=?1.8, 6.5), and Haro Bake (OR?=?2.6, 95 % CI?=?1.3, 5.1) than camels from Surupha. Likewise, there was higher chance of mastitis occurrence among camels at the early lactation stage (OR?=?2.3, 95 % CI?=?1.1, 4.6) and camels with udder/teat lesions (OR?=?13.7, 95 % CI?=?1.7, 109.4) than among camels at late lactation stage and camels with healthy udder/teats, respectively. In conclusion, this study reveals the current status of camel mastitis in Southern Ethiopia.     

广西罗非鱼无乳链球菌的分离鉴定、毒力基因及耐药性研究

本研究旨在明确中国罗非鱼主养区广西各地罗非鱼无乳链球菌（Streptococcus agalactiae）分离株的血清型分布、毒力基因携带情况和耐药情况,为罗非鱼无乳链球菌病的防控奠定基础。2018-2019年从广西柳州、钦州、南宁、北海等地患无乳链球菌病罗非鱼体内分离了47株无乳链球菌,并对各分离株的血清型、毒力基因分布和耐药情况进行检测和分析。血清型检测结果表明,47株无乳链球菌血清型高度一致,均为Ⅰa血清型。对4种毒力基因检测结果表明,47株无乳链球菌临床分离株cylE、sodA、gapC毒力基因的检出率均为100%,而scpB基因仅在人源参考菌株2603V/R中检出,在所有鱼源分离株中未检出。对11类（31种）常见抗生素的药敏试验结果表明,临床分离株对磺胺异噁唑、新霉素、庆大霉素、卡那霉素的耐药率达到90%以上,对氧氟沙星、左氧氟沙星、氨苄青霉素、阿莫西林、头孢克洛、头孢曲松、头孢哌酮、头孢拉定、土霉素、强力霉素的敏感性均为100%。多重耐药检测结果表明,5重以上耐药菌株占93.62%,其中9重以上耐药菌株为19.15%,且均分离自柳州地区。结果表明,广西地区罗非鱼源无乳链球菌血清型单一,均为Ⅰa血清型,且均携带多种毒力基因,多重耐药现象严重。     

金黄色葡萄球菌对BV2细胞IFN-α生成的影响

旨在探究金黄色葡萄球菌（Staphylococcus aureus，简称金葡菌）对小胶质细胞α干扰素（interferon α，IFN-α）生成的影响，本研究先用金葡菌感染BV2细胞，检测IFN-α的mRNA表达、分泌到细胞培养液上清的量以及TBK1/IRF3通路的激活情况，再分别用TBK1抑制剂BX-795和amlexanox、NF-κB抑制剂IMD-0354处理细胞，检测TBK1/IRF3的激活以及IFN-α水平。结果表明，BV2感染金葡菌后，IFN-α转录水平在3~12 h升高，6~12 h释放量增加，呈剂量依赖性，TBK1和IRF3的转录水平和蛋白水平未发生变化，但在1~12 h其磷酸化水平升高，表明金葡菌激活BV2细胞TBK1/IRF3通路，BX-795、amlexanox和IMD-0354处理细胞后，IRF3磷酸化水平降低，IFN-α生成减少，表明TBK1和NF-κB参与金葡菌促进BV2细胞生成IFN-α。综上所述，金葡菌感染BV2后促进IFN-α生成，且该过程依赖于TBK1和NF-κB，本结果为临床防治中枢神经系统感染金葡菌提供了可能靶点和理论基础。     

Udder health in beef cows and its association with calf growth

Background

Studies outside the Nordic countries have indicated that subclinical mastitis (measured by milk somatic cell count or the California Mastitis Test), intramammary infections (IMI), or blind quarters in beef cows may have negative effects on beef calf growth. Knowledge on prevalence of such udder health problems in Swedish beef cows is scarce. Therefore, the main aim of this study was to investigate subclinical mastitis, IMI and udder conformation in a number of beef cow herds. Production of β-lactamase in staphylococci was also investigated. Associations between certain cow factors and subclinical mastitis and IMI, and associations between cow and calf factors and 200 day calf weaning weight were also studied. The herds were visited once within a month after calving and once at weaning. Udder examination and quarter milk sampling, for somatic cell count and bacteriology, were performed in 8 to 12 cows per herd and occasion.

Results

Approximately 50%, 40% and 10% of the cows had subclinical mastitis, IMI, and at least one blind quarter, respectively, but the prevalence varied markedly between herds. Intramammary infections (mainly due to staphylococci) were identified in 13-16% of the milk samples. Less than 5% of the staphylococcal isolates produced β-lactamase. Approximately 11% of the cows sampled twice had the same IMI (mostly Staphylococcus aureus) at both samplings. Cow factors of importance for subclinical mastitis and/or IMI were teat and udder shape, breed, parity, presence of blind quarters, and cow hygiene. No significant associations were found between udder health parameters studied and calf weaning weights.

Conclusions

Subclinical mastitis and IMI, but not blind quarters, were common in beef cows, but the prevalence varied markedly between herds. Most IMI were caused by staphylococci and more than 95% of those were sensitive to penicillin. Cows with large funnel-shaped teats or pendulous udder after calving, and cows with blind quarters were at risk of having subclinical mastitis and/or IMI. Poor hygiene was also a risk factor for udder health problems. No significant associations were found between udder health and calf weaning weight. More studies on risk factors are warranted to improve advisory services on awareness and prevention of mastitis in beef cows.     

Investigation of Biofilm Formation,Virulence Genes and agr Typing of Staphylococcus aureus from Bovine Mastitis Cases

The objective of the present study was to investigate the biofilm formation, antimicrobial resistance, virulence genes, and agr genotypes of Staphylococcus aureus (S. aureus) isolated from bovine mastitis cases, and to analyze the correlations between agr genotypes and virulence genes. Biofilm formation, antimicrobial resistance, and virulence genes for 336 strains of S. aureus were detected by microtiter plate method, disk diffusion method, and PCR respectively, and the agr typing of tested strains was determined by multiplex PCR. The results showed that all 336 strains of S. aureus from bovine mastitis were biofilm producers, among which 52.1% and 47.9% of isolates tested were moderate (++) and strong (+++) biofilm producers, respectively. Antimicrobial susceptibility testing demonstrated that S. aureus strains were highly resistant to penicillin, with a resistance rate of 91.7%, followed by erythromycin (89.6%), kanamycin (72.9%), clindamycin (66.7%) and gentamicin (60.4%). However, all isolates were sensitive to nitrofurantoin and linezolid. PCR results showed that the prevalence of fnbA gene was the highest (99.7%), followed by icaD (98.2%), icaA (89.6%), clfA (86.0%), cna (56.0%), and bap (14.6%) genes. Moreover, the sea, seb, sec and tst genes were found in 26.5%, 8.3%, 6.8% and 8.3% of the isolates, respectively. The agr typing results showed that S. aureus strains belonging to agr Ⅰ was predominant in our study, accounting for 77.1% of the isolates, and the frequencies of agr Ⅱ, agr Ⅲ and agr Ⅳ genotypes were 14.0%, 4.8% and 2.1%, respectively. Statistical analysis indicated that the strains of S. aureus belonging to agr Ⅰ genotype have the potential to carry more virulence genes, while no toxin genes could be found in any of the strains belonging to agr Ⅳ. The results revealed high antimicrobial resistance to common antimicrobial agents in S. aureus isolated from bovine mastitis milk samples. Moreover, agr Ⅰ was the predominant genotype with diverse toxin genes in S. aureus from bovine mastitis, and the potential hazard should be of concern.     

牛源金黄色葡萄球菌生物被膜与毒素基因检测及agr分型

旨在对牛源金黄色葡萄球菌（Staphylococcus aureus，S.aureus）的生物被膜、耐药性、毒素基因和agr基因型进行研究，并分析agr基因型与毒素基因之间的相关性。分别用微量滴定板法、药敏纸片法和PCR对S.aureus的生物被膜、耐药性和毒素基因进行检测，用多重PCR对S.aureus进行agr分型。结果表明，336株牛源S.aureus均能形成生物被膜，其中，形成中等（++）和强（+++）生物被膜的S.aureus分别占52.1%和47.9%。药敏试验结果显示，S.aureus对青霉素耐药最为严重，耐药率达91.7%，其次是红霉素、卡那霉素、克林霉素和庆大霉素，耐药率分别为89.6%、72.9%、66.7%和60.4%，而所有S.aureus对呋喃妥因和利奈唑胺均表现为敏感。PCR检测结果显示，黏附素基因fnbA检出率最高，达99.7%，其次是icaD、icaA、clfA和cna，检出率分别为98.2%、89.6%、86.0%和56.0%，bap基因检出率最低，为14.6%。肠毒素基因sea的检出率为26.5%，其次是seb（8.3%）和sec（6.8%），毒素基因tst的检出率占8.3%。分型结果显示，agr Ⅰ型S.aureus是主要的流行菌株，占77.1%，agr Ⅱ、agr Ⅲ和agr Ⅳ型S.aureus流行率分别为14.0%、4.8%和2.1%。统计分析结果表明，agr Ⅰ型S.aureus更具有携带多种毒素基因的潜力，而agr Ⅳ型S.aureus无毒素基因携带潜力。综上表明，牛乳腺炎性S.aureus对常见的抗菌药物耐药严重，毒素基因分布多样，agr Ⅰ型是奶牛乳腺炎性S.aureus主要的基因型，且具有携带多种毒素基因的能力，其潜在威胁应引起重视。     

Udder health and risk factors for subclinical mastitis in organic dairy farms in Switzerland

A study was performed in 1997 to estimate the prevalence and to investigate the etiology of subclinical mastitis in Swiss dairy herds managed under guidelines of controlled organic farming. It was planned as a longitudinal study over a period of 1 year and included a stratified random sample of 152 certified organic farms and 1907 cows. Two farm visits (the first from June to October when cows were on pasture, the second from January to March when cows were confined to barns) were performed on each farm. At each visit, farm management and individual-cow data (with emphasis on milking procedures and udder sanitation) were recorded. California mastitis tests (CMTs) were performed on each udder quarter of all cows in lactation. Milk samples with CMT >1+ were submitted for somatic cell counting (SCC), bacteriological examination and to test for antibiotic susceptibility. The SCC and germ-cell counts of monthly bulk-tank milk samples were available through Dairy Inspection and Advisory Services and milk production data of 567 herd-book cows were available from breeding associations. Possible individual and environmental predictors of subclinical mastitis were identified using logistic models adjusted for clustering of the data at herd and cow levels. Data were analyzed separately for cows from 7 to 100 and from 101 to 305 days post partum. Prevalences of subclinical mastitis at the quarter level were 21.2% for lactation period 7–100 days and 34.5% for 101–305 days post partum. The geometric mean SCC in bulk-tank milk samples was 85.6×10³ cells/ml. Samples at 7–100 and 101–305 days post partum were positive for Staphylococcus aureus in 16.0 and 7.4%, for coagulase-negative Staphylococci in 51.5 and 50.6%, for Streptococcus agalactiae in 0.0 and 0.8%, for other Streptococci in 19.4 and 15.6%, for E. coli in 1.0 and 0.4%, and for Corynebacterium bovis in 25.7 and 45.1%, respectively. Risks of subclinical mastitis increased significantly with increasing days post partum and advancing age of cow. Cows that were sampled when staying in alpine dairies had considerably higher risks of subclinical mastitis than cows staying in home barns. Significantly lower risks of subclinical mastitis were observed in farms where CMT was performed regularly as a control measure. Bacteria in milk from cows with mastitis exhibited antibiotic resistance at a comparable frequency as found previously in conventional farms.     

金黄色葡萄球菌处理对奶牛乳腺上皮细胞外泌体表征的影响

【目的】本研究旨在从金黄色葡萄球菌(Staphylococcus aureus,S.aureus,以下简称金葡菌)处理奶牛乳腺上皮细胞(bovine mammary epithelial cell,BMEC)上清中分离外泌体,探究金葡菌与BMEC数量比值(MOI)和处理后培养时间对外泌体总浓度的影响,并对金葡菌诱导BMEC释放的外泌体进行分离和鉴定。【方法】采用金葡菌临床分离菌株以MOI＝1和MOI＝10分别处理BMEC 3 h,对已处理的BMEC继续无外泌体培养9、12、24 h,同时设空白对照组,通过扫描电镜观察金葡菌处理对BMEC造成的超微结构损伤。采用超速离心法分离金葡菌诱导的BMEC上清液中的外泌体,分别用透射电镜、纳米颗粒追踪分析技术及Western blotting法对外泌体形态、颗粒大小和特异性标志蛋白进行分析鉴定。通过检测外泌体总蛋白浓度评估不同MOI和处理后培养时间对外泌体总量的影响,明确金葡菌诱导BMEC释放外泌体的最佳条件。【结果】通过扫描电镜观察发现,金葡菌处理BMEC后,细胞微绒毛脱落,细胞骨架破坏。透射电镜观察分离的外泌体为圆形的双层膜囊泡,直径在30~150 nm,形态均一。通过检测外泌体总蛋白浓度显示,在MOI＝10时,培养9、12和24 h金葡菌诱导BMEC释放外泌体蛋白浓度均高于MOI＝1,而培养12 h时释放外泌体蛋白浓度最高。对金葡菌诱导BMEC释放的外泌体进一步鉴定,其平均直径约为116 nm;Western blotting检测结果显示外泌体标记物CD9、CD81和TSG101表达阳性。【结论】本研究从形态学和分子生物学特征等方面证实获得的分离物为外泌体;金葡菌可以诱导BMEC释放外泌体,当MOI＝10时,金葡菌处理细胞3 h后继续无外泌体培养12 h,收获的细胞上清中外泌体蛋白含量最高。     

畜禽粪污中金黄色葡萄球菌和大肠埃希氏菌的LAMP检测及其耐药性分析

为深入了解畜禽粪污中金黄色葡萄球菌和大肠埃希氏菌的流行及耐药状况,采用建立的畜禽粪便中金黄色葡萄球菌和大肠埃希氏菌的环介导等温扩增技术（LAMP）检测方法对山东省内牛粪污、猪粪、鸡粪、鸭粪中的金黄色葡萄球菌和大肠埃希氏菌进行检测,进而对检测阳性样品进行分离,并对药敏试验中多重耐药性菌株进行耐药基因预测。结果表明,LAMP方法检测畜禽粪污中金黄色葡萄球菌和大肠埃希氏菌与国家标准方法检测结果符合率为100%,重复性好,特异性高。从80份畜禽粪污样品中分离到的3株金黄色葡萄球菌均对青霉素耐药;34株大肠埃希氏菌对氟苯尼考、利福平的耐药比例高于50%,高于25%以上的还有氨苄西林、四环素、多西环素、磺胺异噁唑、头孢噻吩和头孢噻呋;耐药基因预测结果显示,鸡粪、鸭粪、牛粪和猪粪中的4株大肠埃希氏菌分别携带10、8、20和15种耐药基因;鸡粪中的1株金黄色葡萄球菌携带11种耐药基因。说明山东地区畜禽粪污中金黄色葡萄球菌和大肠埃希氏菌耐药状况严峻,菌株普遍多重耐药,且携带多种耐药基因。