【摘要】：奶牛乳房炎是危害奶牛養(yǎng)殖業(yè)健康發(fā)展的常見疾病之一。該病增加了奶牛淘汰率與保健治療費(fèi)用,給奶業(yè)造成嚴(yán)重的經(jīng)濟(jì)損失。據(jù)有關(guān)部門統(tǒng)計(jì),在中國僅因奶牛乳房炎淘汰的奶牛占總淘汰牛的9%-11%,每年造成的經(jīng)濟(jì)損失超過135億元。引起奶牛乳房炎的病原微生物極為復(fù)雜,而鏈球菌和葡萄球菌是奶牛乳房炎的主要致病菌,鏈球菌中又以無乳鏈球菌占主要比例。無乳鏈球菌按蘭氏劃分法屬于B群鏈球菌,該菌不但可引起奶牛、山羊、綿羊等動(dòng)物的乳房炎,且可導(dǎo)致人類新生兒敗血癥、腦膜炎和肺炎等,嚴(yán)重危害動(dòng)物及人類的健康。因此快速準(zhǔn)確地進(jìn)行診斷以及研究出有效的防治制劑和方法對(duì)控制奶牛乳房炎具有重大而深遠(yuǎn)的意義。1.本實(shí)驗(yàn)從銀川市及周邊地區(qū)牛場(chǎng)采集大量乳房炎乳樣,通過細(xì)菌培養(yǎng)、形態(tài)學(xué)觀察和生化試驗(yàn)等一系列檢測(cè)方法,分離出266株鏈球菌。之后結(jié)合分子生物學(xué)鑒定法,共鑒定出無乳鏈球菌22株,停乳鏈球菌19株,乳房鏈球菌15株。試驗(yàn)結(jié)果表明寧夏地區(qū)奶牛乳房炎鏈球菌感染中,無乳鏈球菌的分離率最高。因此,無乳鏈球菌是寧夏地區(qū)奶牛乳房炎鏈球菌感染的主要流行菌株。2.采用CLSI推薦的紙片擴(kuò)散法,對(duì)22株臨床型奶牛乳房炎無乳鏈球菌分離株進(jìn)行了19種常用藥物的敏感試驗(yàn)。結(jié)果表明,分離菌株對(duì)19種抗菌藥物具有不同程度的耐藥性,其中對(duì)磺胺異惡唑的耐藥率達(dá)到95.5%；對(duì)復(fù)方新諾明的耐藥率達(dá)到77.3%；對(duì)萘啶酸和氨芐青霉素的耐藥率達(dá)到68.2%；對(duì)四環(huán)素和林可霉素的耐藥率達(dá)到59.1‰對(duì)頭孢他啶的耐藥率達(dá)到54.5%；對(duì)克林霉素、羧芐西林、諾氟沙星的耐藥率均達(dá)到50%；對(duì)阿莫西林的耐藥率也達(dá)到45%以上。另外22株無乳鏈球菌共出現(xiàn)18種耐藥譜,最高的達(dá)到13耐。其中耐藥類型最少的是2耐,僅1株(4.5%),最多的是9耐,達(dá)到8株(36.4%)。3.根據(jù)GenBank公開的相關(guān)序列,設(shè)計(jì)磺胺類sull、sul2、sul3和四環(huán)素類tetE的PCR反應(yīng)特異性引物,建立了相應(yīng)的PCR檢測(cè)方法。結(jié)果表明,該方法靈敏可靠,可以用作臨床檢測(cè)方法的使用。對(duì)22株奶牛乳房炎無乳鏈球菌臨床分離株進(jìn)行檢測(cè),4種耐藥基因的檢出率分別為：sull(18.2%)、sul2 (22.7%)、sul3 (27.3%)、tetE (18.2%)。本研究對(duì)寧夏地區(qū)奶牛乳房炎無乳鏈球菌分離株耐磺胺類藥物性和耐四環(huán)素類藥物性進(jìn)行檢測(cè),為預(yù)防奶牛乳房炎無乳鏈球菌耐藥性提供科學(xué)的理論基礎(chǔ)。4.對(duì)22株無乳鏈球菌,分別擴(kuò)增其Sip、cpsE、CAMP三種主要毒力基因并進(jìn)行測(cè)序,之后對(duì)其序列進(jìn)行對(duì)比分析。結(jié)果表明,三種基因與GenBank公開的序列相似性非常高,其中,Sip基因的相似性均高于94%；cpsE基因的相似性為99%；而CAMP基因的相似性均為98%以上。
[Abstract]:Cow mastitis is one of the common diseases that endanger the healthy development of dairy cattle breeding industry. The disease increases the rate of dairy cow elimination and the cost of health care treatment, and causes serious economic losses to the dairy industry. According to the statistics of relevant departments, only the cows eliminated because of mastitis accounted for 9- 11% of the total cattle eliminated in China, resulting in economic losses of more than 13.5 billion yuan each year. The pathogenic microorganisms causing cow mastitis are very complicated, and Streptococcus and Staphylococcus are the main pathogens of dairy mastitis. Streptococcus actinomycetes belongs to group B streptococcus according to the method of Lance's division. This bacterium can not only cause mastitis of cows, goats, sheep and other animals, but also lead to neonatal septicemia, meningitis and pneumonia, and seriously harm the health of animals and human beings. Therefore, rapid and accurate diagnosis and the development of effective preparation and methods for the control of cow mastitis has great and far-reaching significance. In this experiment, a large number of mastitis milk samples were collected from cattle farms in Yinchuan city and surrounding areas. 266 strains of streptococcus were isolated by a series of methods such as bacterial culture, morphological observation and biochemical test. Combined with molecular biological identification, a total of 22 strains of streptococcus lactobacillus, 19 strains of Streptococcus brevis and 15 strains of Streptococcus brevis were identified. The results showed that the isolation rate of streptococcus mastitis was the highest in Ningxia. Therefore, Streptococcus acuminata is the main epidemic strain of Streptococcus mastitis in Ningxia. Using the disk diffusion method recommended by CLSI, the sensitivity tests of 19 commonly used drugs were carried out on 22 clinical isolates of Streptococcus acuminatum from dairy cow mastitis. The results showed that the isolates were resistant to 19 antimicrobial agents to varying degrees, among which the resistance rate to sulfamethoxazole was 95.5.The resistant rate to compound sulfamethoxazole was 77.3%, the resistance rate to nalididinic acid and ampicillin was 68.2%, the drug resistance rate to sulfamethoxazole reached 95.5%, and the resistance rate to compound sulfamethoxazole reached 77.3%, and the resistance rate to ampicillin was 68.2%. The resistance rate to tetracycline and lincomycin was 59.1 鈥，

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