本文關(guān)鍵詞： 豬傳染性胸膜肺炎放線桿菌 血清型分型 基因分型 豬呼吸道疾病綜合征 復合PCR　出處：《華中農(nóng)業(yè)大學》2015年碩士論文　論文類型：學位論文

【摘要】：豬傳染性胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae,APP)是豬傳染性胸膜肺炎的主要病原菌。豬感染APP后會導致急性纖維素性肺炎、肺出血性壞死乃至死亡,或者演變成慢性感染,雖無明顯的臨床癥狀但生長緩慢乃至成為僵豬。發(fā)病后幸存下來攜帶病原菌的豬,可能會成為再次爆發(fā)此病的傳染源。近年來,豬傳染胸膜肺炎放線桿菌繼發(fā)感染以及與其它病原菌混合感染的情況越來越嚴重,造成臨床上癥狀愈加復雜而難以區(qū)分。迄今為止,根據(jù)莢膜多糖,可將APP分為15種血清型。根據(jù)培養(yǎng)過程中是否需要加NAD,可將APP分為生物I型和生物II型,我國流行的主要是生物I型。由于APP血清型眾多,現(xiàn)有的商品化疫苗沒有涵蓋所有血清型,加上制備型特異性血清過程復雜且耗時長,導致對該病的診斷和預(yù)防帶來了很大的困難。因此,APP的鑒定及分型,以及進一步根據(jù)病原采取對應(yīng)的疫苗防疫和藥物治療措施,對該病的綜合防控具有重要的意義。本研究利用APP生物I型標準菌株制備了13種標準陽性血清,經(jīng)瓊脂擴散試驗(GDT)測定后,標準陽性血清5型的效價是1:8;標準陽性血清2型、4型、9型和15型的效價是1:16;標準陽性血清1型、3型、6型、7型、8型、10型、11型和12型的效價是1:32。近年來,復合PCR方法因快速、簡便的特點而發(fā)展迅速得到廣泛的應(yīng)用。根據(jù)APP的四種外毒素apx I、apx II、apx III和apx IV設(shè)計了5對特異性引物,建立了復合PCR分子分型方法研究,通過一步復合PCR方法就可以區(qū)分出APP血清型1~13中的7種(1型、3型、4型、5型、8型、10型和12型)。豬呼吸道疾病是導致保育豬和生長豬以及育肥豬死亡的主要原因。豬嗜血桿菌、豬鏈球菌、豬多殺巴氏桿菌和豬傳染性胸膜肺炎放線桿菌是引起呼吸道綜合征(PRDC)的主要病原菌。本研究針對副豬嗜血桿菌的16S r RNA基因、豬鏈球菌的gdh基因、豬多殺巴氏桿菌的kmt基因和豬傳染性胸膜肺炎放線桿菌的apx IV基因設(shè)計出四對特異性引物,分別擴增出了長度為821bp、689 bp、457 bp、319 bp的特異性片段。在建立了APP單一PCR方法基礎(chǔ)上,進一步對影響PCR的幾個主要因素(r Taq酶、Mg2+濃度、d NTP濃度和引物濃度)進行優(yōu)化,建立了復合PCR檢測方法。特異性試驗結(jié)果表明,副豬嗜血桿菌、豬鏈球菌、豬多殺巴氏桿菌和豬傳染性胸膜肺炎放線桿菌復合PCR只能擴增出四種特異性的目的條帶,其它結(jié)果為陰性;敏感性試驗中,最低檢測量分別是副豬嗜血桿菌為112.5pg/μL,豬鏈球菌為11.7pg/μL,豬多殺巴氏桿菌為8.7ng/μL,豬傳染性胸膜肺炎放線桿菌為267pg/μL;重復性試驗中,通過9次重復試驗的結(jié)果表明此方法重復性好。利用該方法對湖北省部分地區(qū)采集的579份病料進行了初步檢測,結(jié)果有59株HPS(10.2%),74株SS(12.8%),35株P(guān)m(6%),24株APP(4.1%),與單一PCR方法檢測結(jié)果相比,平均陽性符合率為95%。表明該方法實際可行,也為PRDC病原菌的檢測和疫病防控提供了技術(shù)支撐。
[Abstract]:Actinobacillus pleuropneumoniae is the main pathogen of swine infectious pleural pneumonia. Infection with APP can lead to acute cellulosic pneumonia, pulmonary hemorrhage necrosis or death, or to chronic infection. Although there are no obvious clinical symptoms, but the growth is slow to become stiff pigs. The pigs survived to carry the pathogen after the disease, may become a source of infection for another outbreak of the disease in recent years, Actinobacillus pleuropneumoniae secondary infection and co-infection with other pathogens are becoming more and more serious, resulting in more complex and difficult clinical symptoms. APP can be divided into 15 serotypes. APP can be divided into biological type I and biological type II according to whether it is necessary to add NAD in the course of culture. The existing commercial vaccines do not cover all serotypes, and the process of preparing specific serotypes is complicated and time-consuming, which makes it difficult to diagnose and prevent the disease. Therefore, the identification and typing of app. It is of great significance for the comprehensive prevention and control of the disease to take corresponding vaccine prevention and drug treatment measures according to the pathogen. In this study, 13 standard positive sera were prepared by using APP biotype I standard strain. The titer of standard positive serotype 5 was 1: 8, the titer of standard positive serotype 2, serotype 2, serotype 4, and type 15 was 1: 16, and the titer of standard positive serum type 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, type 1, type 1, type 3, type 1, type 7, type 1, type 10, type 10, type 11, and type 12, type 12, were 1: 32 in recent years. The compound PCR method has been widely used because of its rapid and convenient characteristics. Five pairs of specific primers were designed according to apx Iapx IIAPX III and apx IV of four foreign toxins of APP, and the molecular typing method of compound PCR was established. By using one-step composite PCR method, we can distinguish seven types of APP serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, serotype 1, type 1, type 3, type 4, type 5, type 8, type 8, type 10, and type 12. Respiratory diseases in pigs are the main causes of mortality in the care of pigs, growing pigs and fattening pigs, Haemophilus suis, and Haemophilus suis. Streptococcus suis, Pasteurella multocida and Actinobacillus pleuropneumoniae are the main pathogens causing respiratory syndrome. In this study, the 16s r RNA gene of Haemophilus parahaemophilus and the gdh gene of Streptococcus suis were studied. Four pairs of specific primers were designed from the kmt gene of Pasteurella multocida and the apx IV gene of Actinobacillus pleuropneumoniae, respectively, and the specific fragment was amplified from 821 BP, 689 BP, 457 BP, 319bp, respectively. Based on the APP single PCR method, a single PCR method was developed. Furthermore, several main factors affecting PCR, such as NTP concentration and primer concentration, were optimized, and a complex PCR detection method was established. The specific test results showed that Haemophilus parais and Streptococcus suis were detected. Pasteurella multocida and Actinobacillus pleuropneumoniae combined with PCR could only amplify four specific target bands, but the other results were negative. The lowest detections were 112.5 PG / 渭 L for Haemophilus parais, 11.7 PG / 渭 L for Streptococcus suis, 8.7 ng / 渭 L for Pasteurella multocida and 267pg / 渭 L for Actinobacillus pleuropneumoniae. The results of 9 repeated experiments showed that the method was reproducible. The method was used to detect 579 samples collected from some parts of Hubei Province. The results showed that there were 59 strains of HPSN 10.2 and 74 strains of PMS 12.80.35 strains of APP 4.1B, compared with the results of single PCR method. The average positive coincidence rate was 95. The results showed that the method was feasible and provided technical support for the detection of pathogenic bacteria of PRDC and the prevention and control of epidemic diseases.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.61

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本文編號：1545124