湖北地区鸡毒霉形体的分离鉴定及分子流行病学研究
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摘要
鸡毒霉形体(Mycoplasma gallisepticum)在鸡群中可引起严重的慢性呼吸道病(Chronic respiratory disease, CRD)。该病在国内鸡群中非常普遍,可导致蛋鸡产蛋率下降,肉鸡出栏期延长。一旦和新城疫病毒、传染性支气管炎病毒、大肠杆菌等病原发生协同感染,则可引起较高的发病率和死亡率。目前,我国针对鸡毒霉形体的鉴定主要是采用病原分离培养及血清学鉴定等方法,但由于这些传统方法既费时又费力,因此无法满足临床快速诊断的要求。而且,针对湖北地区家禽养殖场(户)的鸡毒霉形体流行现状、耐药性和病原性的相关研究尚无系统报道。鉴于此,本课题拟选用16S-23S rRNA基因间隔序列Intergenic spacer regions, ISR)对湖北地区鸡毒霉形体流行毒株进行研究,该段序列兼有保守性和突变性的特点,既可满足对鸡毒霉形体临床感染的快速诊断,也可用于对其分子流行病学和病原学进行系统分析。
本研究应用传统实验室分离培养和鉴定技术,结合限制性酶切长度多态性分析技术,对湖北地区流行的M. gallisepticum野毒株进行了分离鉴定和耐药性分析,建立了针对M. gallisepticum 16S-23S rRNA ISR PCR快速检测技术,并进行了分子流行病学调查和病原性研究。具体研究成果如下。
1、鸡毒霉形体的分离鉴定及分离菌株的耐药性分析
在湖北地区47个养殖场(户)共采集病样172份,经分离培养获得77株疑似霉形体分离物,进一步采用生化试验和代谢抑制试验进行鉴定,其中57株代谢抑制价在1：80以上,并采用16S rRNA PCR技术进一步检测,均扩增出186bp大小的特异性条带,鉴定为鸡毒霉形体,阳性检出率为33.1%,；另20株为非鸡毒霉形体,有待进一步鉴定。从各养殖场分离的鸡毒霉形体分离株中选取部分菌株进行药物敏感性试验,结果表明大部分临床分离株对泰妙菌素、吉它霉素和泰乐菌素高度敏感,而且分离自不同地区的分离株对抗菌药物的敏感性存在一定差异。
2、鸡毒霉形体16S-23S rRNA ISR PCR诊断方法的建立及分子流行病学研究
以鸡毒霉形体16S-23S rRNA基因间隔区为目的基因,建立PCR检测方法,对所有172份样品进行扩增分析,结果从70份病料中扩增出了850bp大小的特异性条带,其中包括57株经传统技术鉴定为阳性的病样,其阳性检出率为40.7%。同时采用该PCR方法及对上述分离获得的20株非鸡毒霉形体分离物进行分析,初步鉴定为家禽霉形体。因此,针对16S-23S rRNA基因间隔区建立的鸡毒霉形体PCR检测方法比传统技术具更高的敏感性。随后,将RFLP技术和PCR技术结合起来,采用限制性内切酶RsaⅠ对PCR扩增片段进行限制性片段长度多态性分析,根据酶切图谱可将所有分离菌株分为两个谱系(R1和R2)。同时,结合流行病学调查, R2基因型比R1基因型的菌株更为流行,且未见同一个场内同时发生两种基因型菌株感染的情况。进一步对分离株16S-23S rRNA基因间隔区进行序列比对和进化分析,来自各不同养殖场的分离菌株可归为三个亚群,有的菌株与参考株S6株为同一亚群,有的与F株为同一亚群,而参考株R株则独自成一个亚群。
3、湖北地区鸡毒霉形体分离株致病性研究
根据上述进化分析结果,选择在进化树上分别与S6和F株亲缘关系较近的XA41株和ML21株作为研究对象,以S6和F株作为参考菌株,分别对4周龄雏鸡进行攻毒实验,8天后以新城疫弱毒苗(Ⅳ系)进行激发感染。结果接种S6和XA41株的实验动物全部发病,有明显的呼吸道症状；通过剖检肉眼观察、组织学病理切片和电镜观察,发病动物主要以上呼吸道病理变化为主；经血清学检测,抗体阳性反应；S6和XA41株引起的气囊损伤程度分别为3和2.8；S6和XA41株对鸡胚致死率均为80%。而攻F株和ML21株的实验动物未出现明显临床症状；通过剖检肉眼观察、病理切片和电镜观察,各部位均无明显病变；气囊损伤程度均为0；抗体检测为阴性；F株和ML21株对鸡胚的致死率均为40%。表明XA41株和S6致病性相似,为强毒株；ML21株和F株相似,为弱毒株。因此,通过致病性研究结果初步确定16S-23S rRNA ISR可作为区分不同基因型鸡毒霉形体菌株致病性的分子工具。
Mycoplasma gallisepticum (M gallisepticum) can cause severe chronic respiratory disease in farm flocks. M. gallisepticum infection is common in our country. It could lead to egg production decreased of layer and slaughter delayed of broiler. Once chickens suffered co-infection with Newcastle virus, Infectious brochitis or Escherichia, which could lead higher motality and incidence. Isolates, culture and serological identification for the detection of M. gallisepticum are mainly used currently in China. But these asssys are laborious and time consuming, it can not satisfy rapid application. However, little is known about epidemiology, drug resistance and pathogenic of Mycoplasma in chicken farms of Hubei Province. To aim directly at aboved-metioned, 16S-23S rRNA intergenic spacer region (ISR) was elected as a tool to investigate M gallisepticum in Hubei Province.The 16S-23S rRNA ISR is a nucleotide sequence with high conservatism and discontinuity. It could satisfy rapid detection. Moreover, it may be a useful molecular epidemiology tool and provide thereunder etiology assay of M. gallisepticum.
In this study, facing the shortage of the present methods used in detecting, vacuity of epidemiology and etiology assay of CRD in poultry. The routine lab technology and Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used. And a PCR detection techonlogy based on the 16S-23S rRNA ISR was established. Moreover, the research of molecular epidemiology and etiology was advanced.
1、Isolates, identification and resistance of M. gallisepticum with routine technology,
A total of 172 field samples were collected from 47 farms in this region and 77 isolates were obtained by cultivation. These isolates were examined by routine microbiological, biochemical test and metabolism inhibition assays. We found that 57 isolates which metabolism inhibition titer was higher than 1:80. Then 16S rRNA PCR was used. The length of 16S rRNA PCR fragments was 186bp. So they were identified as M. gallisepticum. The positive rate of routine technology is 33.1%. In addition, we found that 20 isolates were not M. gallisepticum.The resistance test of some strains of M. gallisepticum showed that, sensitivity of clinical strains to antibiotics have area characteristic and majority strains were sensitive to drugs, such as Tiamulin, Tylosinum, Kitasamycin. (The minimum inhibition concentraion)
2、16S-23S rRNA ISR PCR and molecular epidemiology of M. gallisepticum
A PCR detection technology based on the 16S-23S rRNA intergenic spacer regions (ISR) gene fragments was established. A total of 172 samples were detected. We found that 70 isolates including 57 isolates were detected through routine technology which special fragment length was 850bp The positive rate is 40.7%. In addition, we found 20 isolates were M. gallinarum. Therefore, the PCR detection technology based on ISR have higher specificity than routine technology. Then PCR-RFLP technology was used at the same time. All isolates were divided into two groups through RsaⅠ(R1 and R2). Epidemiological investigation suggested that the R1 and R2 groups of M. gallisepticum appeared not to affect chickens at the same farm simultaneously. The R2 genotype of M. gallisepticum was more prevalent, evidentially by more farms and more samples were infected with this genotype of M. gallisepticum. The R2 group of M. gallisepticum appeared to be prevalent at one location and then changed to the R1 type of M. gallisepticum in the same city. This was the first report on genetic variants of M. gallisepticum in China Further analysis including sequenced, alignment and evolution analyzing of 16S-23S rRNA ISR revealed that there were three subgroups of M. gallisepticum with different genotypes. Some isolates were closely related to the reference strain of S6, some others to the F, and no single isolates to the reference strain of R.
3、The assay of etiology about M. gallisepticum
4-week-old chick were infected artificially with four strains of M. gallisepticum in which XA41 and ML21 strains were isolated in Hubei Province, which closely related to the reference strain of S6 and F in phylogenetic analysis respective, and S6 and F strains were reference strains. Provocative infection was made by New castle disease attenuated vaccine(Ⅳ) eighe days later. Chicks infected with XA41 and S6 were sick, typical respiratory tract symptoms occurred. Pathoanatomical results, Scanning electron microscopy (SEM) and pathological section showed that respiratory were damage in serious degree. Serological reaction was positive. Mean Lesion Score of air sac were 3.0(S6),2.8(XA41)XA41. The virulense of XA41 and S6 for SPF embryoes were 80%. The chicken infected F and ML21 strain did not show any clinical symptom. Pathoanatomical results, SEM and pathological section showed that respitatory tract have not any damage. Mean Lesion Score of air sac were zero(F and ML21). Serological reaction was positive. The pathogenicity of F and ML21 strains for SPF embryoes were 40%. The study on etiology of XA41 and ML21 showed that the fomer was virulent strain that same with S6, and the latter was low virulent strain that same with F. Therefore, base on the study on etiology,16S-23S rRNA ISR could be infered as a useful molecular tool which can discrimination pathogenicity of different genotype of M. gallisepticum.
本研究应用传统实验室分离培养和鉴定技术,结合限制性酶切长度多态性分析技术,对湖北地区流行的M. gallisepticum野毒株进行了分离鉴定和耐药性分析,建立了针对M. gallisepticum 16S-23S rRNA ISR PCR快速检测技术,并进行了分子流行病学调查和病原性研究。具体研究成果如下。
1、鸡毒霉形体的分离鉴定及分离菌株的耐药性分析
在湖北地区47个养殖场(户)共采集病样172份,经分离培养获得77株疑似霉形体分离物,进一步采用生化试验和代谢抑制试验进行鉴定,其中57株代谢抑制价在1：80以上,并采用16S rRNA PCR技术进一步检测,均扩增出186bp大小的特异性条带,鉴定为鸡毒霉形体,阳性检出率为33.1%,；另20株为非鸡毒霉形体,有待进一步鉴定。从各养殖场分离的鸡毒霉形体分离株中选取部分菌株进行药物敏感性试验,结果表明大部分临床分离株对泰妙菌素、吉它霉素和泰乐菌素高度敏感,而且分离自不同地区的分离株对抗菌药物的敏感性存在一定差异。
2、鸡毒霉形体16S-23S rRNA ISR PCR诊断方法的建立及分子流行病学研究
以鸡毒霉形体16S-23S rRNA基因间隔区为目的基因,建立PCR检测方法,对所有172份样品进行扩增分析,结果从70份病料中扩增出了850bp大小的特异性条带,其中包括57株经传统技术鉴定为阳性的病样,其阳性检出率为40.7%。同时采用该PCR方法及对上述分离获得的20株非鸡毒霉形体分离物进行分析,初步鉴定为家禽霉形体。因此,针对16S-23S rRNA基因间隔区建立的鸡毒霉形体PCR检测方法比传统技术具更高的敏感性。随后,将RFLP技术和PCR技术结合起来,采用限制性内切酶RsaⅠ对PCR扩增片段进行限制性片段长度多态性分析,根据酶切图谱可将所有分离菌株分为两个谱系(R1和R2)。同时,结合流行病学调查, R2基因型比R1基因型的菌株更为流行,且未见同一个场内同时发生两种基因型菌株感染的情况。进一步对分离株16S-23S rRNA基因间隔区进行序列比对和进化分析,来自各不同养殖场的分离菌株可归为三个亚群,有的菌株与参考株S6株为同一亚群,有的与F株为同一亚群,而参考株R株则独自成一个亚群。
3、湖北地区鸡毒霉形体分离株致病性研究
根据上述进化分析结果,选择在进化树上分别与S6和F株亲缘关系较近的XA41株和ML21株作为研究对象,以S6和F株作为参考菌株,分别对4周龄雏鸡进行攻毒实验,8天后以新城疫弱毒苗(Ⅳ系)进行激发感染。结果接种S6和XA41株的实验动物全部发病,有明显的呼吸道症状；通过剖检肉眼观察、组织学病理切片和电镜观察,发病动物主要以上呼吸道病理变化为主；经血清学检测,抗体阳性反应；S6和XA41株引起的气囊损伤程度分别为3和2.8；S6和XA41株对鸡胚致死率均为80%。而攻F株和ML21株的实验动物未出现明显临床症状；通过剖检肉眼观察、病理切片和电镜观察,各部位均无明显病变；气囊损伤程度均为0；抗体检测为阴性；F株和ML21株对鸡胚的致死率均为40%。表明XA41株和S6致病性相似,为强毒株；ML21株和F株相似,为弱毒株。因此,通过致病性研究结果初步确定16S-23S rRNA ISR可作为区分不同基因型鸡毒霉形体菌株致病性的分子工具。
Mycoplasma gallisepticum (M gallisepticum) can cause severe chronic respiratory disease in farm flocks. M. gallisepticum infection is common in our country. It could lead to egg production decreased of layer and slaughter delayed of broiler. Once chickens suffered co-infection with Newcastle virus, Infectious brochitis or Escherichia, which could lead higher motality and incidence. Isolates, culture and serological identification for the detection of M. gallisepticum are mainly used currently in China. But these asssys are laborious and time consuming, it can not satisfy rapid application. However, little is known about epidemiology, drug resistance and pathogenic of Mycoplasma in chicken farms of Hubei Province. To aim directly at aboved-metioned, 16S-23S rRNA intergenic spacer region (ISR) was elected as a tool to investigate M gallisepticum in Hubei Province.The 16S-23S rRNA ISR is a nucleotide sequence with high conservatism and discontinuity. It could satisfy rapid detection. Moreover, it may be a useful molecular epidemiology tool and provide thereunder etiology assay of M. gallisepticum.
In this study, facing the shortage of the present methods used in detecting, vacuity of epidemiology and etiology assay of CRD in poultry. The routine lab technology and Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used. And a PCR detection techonlogy based on the 16S-23S rRNA ISR was established. Moreover, the research of molecular epidemiology and etiology was advanced.
1、Isolates, identification and resistance of M. gallisepticum with routine technology,
A total of 172 field samples were collected from 47 farms in this region and 77 isolates were obtained by cultivation. These isolates were examined by routine microbiological, biochemical test and metabolism inhibition assays. We found that 57 isolates which metabolism inhibition titer was higher than 1:80. Then 16S rRNA PCR was used. The length of 16S rRNA PCR fragments was 186bp. So they were identified as M. gallisepticum. The positive rate of routine technology is 33.1%. In addition, we found that 20 isolates were not M. gallisepticum.The resistance test of some strains of M. gallisepticum showed that, sensitivity of clinical strains to antibiotics have area characteristic and majority strains were sensitive to drugs, such as Tiamulin, Tylosinum, Kitasamycin. (The minimum inhibition concentraion)
2、16S-23S rRNA ISR PCR and molecular epidemiology of M. gallisepticum
A PCR detection technology based on the 16S-23S rRNA intergenic spacer regions (ISR) gene fragments was established. A total of 172 samples were detected. We found that 70 isolates including 57 isolates were detected through routine technology which special fragment length was 850bp The positive rate is 40.7%. In addition, we found 20 isolates were M. gallinarum. Therefore, the PCR detection technology based on ISR have higher specificity than routine technology. Then PCR-RFLP technology was used at the same time. All isolates were divided into two groups through RsaⅠ(R1 and R2). Epidemiological investigation suggested that the R1 and R2 groups of M. gallisepticum appeared not to affect chickens at the same farm simultaneously. The R2 genotype of M. gallisepticum was more prevalent, evidentially by more farms and more samples were infected with this genotype of M. gallisepticum. The R2 group of M. gallisepticum appeared to be prevalent at one location and then changed to the R1 type of M. gallisepticum in the same city. This was the first report on genetic variants of M. gallisepticum in China Further analysis including sequenced, alignment and evolution analyzing of 16S-23S rRNA ISR revealed that there were three subgroups of M. gallisepticum with different genotypes. Some isolates were closely related to the reference strain of S6, some others to the F, and no single isolates to the reference strain of R.
3、The assay of etiology about M. gallisepticum
4-week-old chick were infected artificially with four strains of M. gallisepticum in which XA41 and ML21 strains were isolated in Hubei Province, which closely related to the reference strain of S6 and F in phylogenetic analysis respective, and S6 and F strains were reference strains. Provocative infection was made by New castle disease attenuated vaccine(Ⅳ) eighe days later. Chicks infected with XA41 and S6 were sick, typical respiratory tract symptoms occurred. Pathoanatomical results, Scanning electron microscopy (SEM) and pathological section showed that respiratory were damage in serious degree. Serological reaction was positive. Mean Lesion Score of air sac were 3.0(S6),2.8(XA41)XA41. The virulense of XA41 and S6 for SPF embryoes were 80%. The chicken infected F and ML21 strain did not show any clinical symptom. Pathoanatomical results, SEM and pathological section showed that respitatory tract have not any damage. Mean Lesion Score of air sac were zero(F and ML21). Serological reaction was positive. The pathogenicity of F and ML21 strains for SPF embryoes were 40%. The study on etiology of XA41 and ML21 showed that the fomer was virulent strain that same with S6, and the latter was low virulent strain that same with F. Therefore, base on the study on etiology,16S-23S rRNA ISR could be infered as a useful molecular tool which can discrimination pathogenicity of different genotype of M. gallisepticum.
引文
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