【作者】 林琳；

【导师】 吴异健； 李文迹；

【作者基本信息】 福建农林大学 ， 兽医硕士（专业学位）， 2018， 硕士

【摘要】 鸡毒支原体(Mycoplasma gallisepticum,MG)可以引起鸡的慢性呼吸道疾病(Chronic respiratory disease,CRD),临床主要表现为咳嗽、流浆液性鼻液、呼吸困难、湿性啰音,剖检可见到鼻道、气管卡他性渗出物和严重的气囊炎。该病病程长,发展缓慢,若无其他病原协同感染时,其死亡率较低,与新城疫病毒、传染性支气管炎病毒和大肠杆菌等病原交叉感染时可引起较高的发病率和死亡率,是目前困扰我国规模化鸡只养殖的一大难题。闽北地区是福建省最大的白羽肉鸡养殖地区,但该地区尚未见MG感染病原学调查的相关报道,给该地区有效防制MG感染造成极大的盲目性。因此,本研究对闽北肉鸡养殖区开展MG感染调查,同时进行流行菌株的分离鉴定,通过分离菌株的药物敏感性试验和pvpA基因序列的测序分析比较,为其防制工作提供参考依据。方法:本实验首先采用Real-time PCR对闽北地区白羽肉鸡MG感染情况开展调查;其次,选择适宜的支原体培养基从临床疑似CDR病鸡喉气管黏液棉拭子中分离培养及鉴定MG,并对临床分离株进行序列测定与比较分析;最后,选择临床常用的7种抗菌药物进行最低抑菌浓度试验(MIC)筛选临床MG分离株敏感的药物。结果如下:(1)比较Real-time PCR检测喉气管拭子及肺脏组织样品的MG阳性率,结果显示喉气管拭子和肺脏组织样品的阳性检出率分别为28.12%和3.12%,表明喉气管拭子样品更适宜MG Real-time PCR检测。(2)自2017年3月至2017年11月期间,从闽北地区对不同免疫背景及不同用途的90个白羽肉鸡养殖场采集1800份喉气管拭子样品,采用Real-time PCR进行MG检测,结果显示:进行过MG免疫的祖代场和父母代场种鸡场均未受到MG感染,未进行MG疫苗免疫的商品代白羽肉鸡场的MG感染阳性场的比率为33.33%,样本阳性率为13.17%,其中阳性场的阳性样本率最高为100%,最低为5%,平均值为39%。(3)从临床样本中成功分离培养了 2株支原体,经鉴定为MG,命名为MX分离株和HY分离株。(4)通过pvpA基因部分核苷酸序列比对结果显示,MX和HY分离株的该段序列完全一致,与F株的核苷酸序列同源性为96.7%,有10处碱基不一致并且出现一处缺失3个碱基,推测的氨基酸序列同源性为92.2%,有10个氨基酸差异,其中缺失一个苏氨酸;与R株的核苷酸序列同源性为98.9%,推测的氨基酸序列同源性为98.0%,缺失DR1、DR2之间77个氨基酸序列;与CG5株的核苷酸序列同源性为90.9%,推测的氨基酸序列同源性为86.3%。遗传进化分析表明MX分离株和HY分离株与R株亲缘关系最近。(5)动物回归试验结果显示,接种MX分离株液体培养液和HY分离株液体培养液的两组鸡只均出现明显的呼吸道症状,气囊均不同程度地表现出混浊、增厚和产生泡沫的病理变化;接种F疫苗株的组和空白对照组的鸡只生长发育良好,均未表现出明显的临床症状。(6)根据体外药物敏感性试验结果显示,泰妙菌素均对MX分离株和HY分离株的抑菌效果最好,其次为泰乐菌素和氟苯尼考。结论:闽北地区商品代白羽肉鸡MG感染的情况较为严重;从此地区分离的MX和HY分离株均具有一定的致病性;泰妙菌素、泰乐菌素和氟苯尼考为此地区防治CRD的首选药物。更多还原

【Abstract】 Mycoplasma gallisepticum(MG)can cause Chronic respiratory disease(CRD)of chicken.Clinical symptoms are mainly coughing,serous rhinorrhea,breathing with difficulty and moist rale.Catarrhal inflammation exudate stuck in nasal meatus and trachea and serious air sacculitis can be seen in the dissection.This disease has a long corse of disease and develop slowly,the death rate is relatively low if there is no other pathogen co-infecting.The cross-infection with Newcastle Disease Virus(NDV)or Infectious Bronchitis Virus(IBV)or Escherichia coli(E.coli)can lead to a higher incidence and death rate,which is a tough problem of large-scale chicken breeding in China.The north of Fujian is the largest breeding district of White Meat-type Chicken in Fujian province.However,there is no relative report of MG infection situation investigation in this area,which makes the effectively prevention of MG infection aimless.Therefore,this research contain the investigation of MG infection situation in the north of Fujian from chicken farming area,at the same time,the isolation and identification of epidemic strains were carried out,the basis of drug sensitivity test of isolated strains and sequence analysis and comparison of pvpA gene sequences,It provides a reference for their prevention and control.Methods:First,Real-time PCR method was used in the investigation of the MG infection situation of White Meat-type Chicken in the north of Fujian province.Secondly,proper Mycoplasma culture medium was chosen from chicken laryngeal tracheal mucus swabs with clinically suspected CDR disease to isolation cultivate and identification for MG,and the clinical isolates were sequenced and compared.At last,choosing 7 clinically-used antibacterials were selected for the Minimal Inhibitory Concentration Test(MIC)to screen sensitive drugs for clinical MG isolates.Result:(1)The positive rate of MG in laryngealtracheal swabs and lung tissue samples was compared by Real-time PCR.The results showed that the positive rates of laryngealtracheal swabs and lung tissue samples were 28.12%and 3.12%,respectively,indicating that laryngealtracheal swabs sampling is more suitable for MG Real-time PCR detection.(2)From March to November 2017,collect 1800 laryngotracheal swab samples from the 90 White Meat-type Chicken farms with different immune backgrounds in north of Fujian Province,The Real-time PCR was used for MG detection,and the results showed that the inoculated with MG vaccine breeder chicken farms were not infected by MG,the MG infection positive rate of commercial broiler farms without MG vaccine immunization is 33.33%,the positive sample rate is 13.17%.The maximum positive sample rate of is 100%,the minimum rate is 5%,and the average rate is 39%.(3)Two strain of Mycoplasma were isolated from laryngotracheal swabs,which were identified as MG,and named as MX isolate and HY isolate.(4)The comparing result of pvpA gene nucleotide sequence showed that the MX isolate and HY isolate are the same.The nucleotide sequence homology with the F strain was 96.7%,there were 10 bases inconsistencies and a loss of 3 bases.The conjectural amino acid sequence homology was 92.2%,and there were 10 differences in which one threonine was deleted.The nucleotide sequence of the R strain was 98.9%,the conjectural amino acid sequence homology was 98%,and the 77 amino acid sequences were missing between DR1 and DR2.The nucleotide sequence of the CG5 strain was 90.9%,the conjectured amino acid sequence homology was 86.3%.Phylogenetic analysis showed that MX isolates and HY isolates were closely related to R strains.(5)The results of animal regression experiments showed that two group of chickens that was inoculated with MX isolate and HY isolate both showed obvious respiratory symptom and the air float both showed pathological changes,like opacity and thickened and foamed,in different level.The chicken in the group that was inoculated with F vaccine strain and the blank control group both were well-developed and no showed obvious clinical symptom.(6)In vitro drug sensitivity test of result showed that the Tiamulin have the best antibacterial effect on MX isolate and HY isolate,followed by Tylosin and Florfenicol.Conclusion:The MG infection situation of commercial broiler of is comparatively severe in the north of Fujian Province.The MX isolate and HY isolate that separated from this district both have pathogenicity.Tiamulin,Tylosin and Florfenicol are the preferred drug to prevention and treatment of CRD.更多还原