安徽部分地区鸭疫里氏杆菌的分离鉴定及其生物学特性研究
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摘要
鸭疫里氏杆菌病是由鸭疫里氏杆菌(Riemerella anatipestifer,RA)引起的的一种主要侵害雏鸭、雏火鸡等多种禽类的急性或慢性传染病。该病的发病率和死亡率较高,已成为目前危害世界各国养鸭业最为严重的传染病之一,给养鸭业造成了巨大的经济损失。RA现已发现21个血清型,各血清型之间很少有交叉保护性;对于已感染RA的鸭群,经常用药易产生耐药菌株。因此,建立快速检测RA的方法、了解RA血清型的变化趋势以及开展RA疫苗方面的研究,对于该病的有效防制是十分必要的。
本研究根据RA 16S rRNA基因序列设计一对RA特异性引物,成功地建立了检测RA的PCR方法,特异性强,同时该PCR方法能检出20 pg的RA DNA,灵敏度高,这为RA的检测提供了一种快速、敏感、特异性强的检测方法。应用该PCR方法对病料组织直接进行RA检测,脑组织为首选检测对象,具有良好的实际应用意义。
利用常规方法和建立的PCR方法对安徽省滁州市、六安市、安庆市、合肥市、巢湖市和蚌埠市六个地区108份病/死鸭进行RA检测,六个地区均检测出RA,阳性检出率为24.07%(26/108),其中滁洲市RA分离株占总分离株的57.69%(15/26),显示安徽地区RA的感染较为普遍,滁洲地区感染情况严重。
26株RA分离株中20株为血清1型,占总分离株的76.92%,6株未定型,表明安徽省目前至少存在两种不同的血清型,而2000年以前的分离株均属血清1型,说明安徽省RA流行菌株的血清型存在动态变化,血清型呈现多极化的趋势。
对安徽省RA分离株的16S rRNA基因和cam基因进行扩增和序列测定,并与国内外参考菌株进行同源性比较,结果表明不同血清型、同一血清型的不同菌株之间及不同地区的菌株之间16S rRNA基因序列无明显差异,RA 16S rRNA基因核苷酸序列差异的距离与地理分布、菌株来源无一定的相关性;不同毒力株之间cam基因并无明显差异,根据cam基因无法判断不同菌株的毒力强弱。
通过动物回归试验,测定8株RA分离株的毒力,其中CZ9和AQ2为弱毒菌株,CZ1、AQ1、LA4为中等毒力菌株,CZ2、CZ12、HF1为强毒菌株。将CZ9和AQ2免疫鸭,CZ9对CZ12和HF1攻击的保护率分别为60%和80%,比AQ2的免疫原性好,故将其作为弱毒疫苗株的候选株,进一步测定CZ9的最佳免疫剂量为8.0×10~8CFU/0.1mL,从而为RA弱毒疫苗的研制提供了一定的基础数据。
Riemerella anatipestifer(RA) infection is a acute or chronic contagious disease of domestic young ducks,turkeys,and various other domestic and wild birds.It accounts for significant economic losses due to high morbidity and mortality,known as is a major disease confronting the duck industry through the world.To date,21 serotypes have been reported,little cross reaction was found between different serotypes.In addition,resistant strains appear with the application of antibiotics The iterative antibiotics treatment is responsible for the occurrence of resistant strains in infected ducks.So,it is becoming more and more urgent to diagnose the early RA infection,predict the potential RA serotype change,and seek for effective vaccine in order to control and prevent the RA infectious disease.
In the present study,a pair of primer was designed and synthesized based on 16S rRNA gene sequence from RA strain.PCR was then performed with these primers in the assay of lesion samples from the suspended infection.It demonstrated the PCR amplification is RA specific with 20pg of the minimum DNA amount for detection.The optimal experiment showed that the brain tissue is the preferential tissue in the assay. Notably,the PCR developed in this study is highly specific,much sensitive,and practically useful in monitoring the RA infectious disease.
Out of 108 tissue samples of infected ducks collected in 6 different regions including Chuzhou,Liuan,Anqing,Hefei,Chaohu and Bengbu in Anhui Province,26 of freshly isolated strains were gotten with 24.07%of the positive percentage.Among them,strains isolated from Chuzhou accounts for 57.69%(15/26).Our results showed that the RA infection is commonly distributed in Anhui area,and Chuzhou is one of regions it happens seriously.
20 strains were further identified as serotype 1 which stands for the predominant serotype with 76.92%.The other 6 isolated strains have not been yet identified as certain type.These results demonstrated that there are at least two serotypes being prevalent in Anhui province.Considering the previous reporter that serotype 1 was the unique type in 2000,we speculate that RA infection appears in a trend of diversity.
We next determined the nucleotide sequences of the 16S rRNA and cam genes in the isolates,and compared them with the known data in GenBank.Our results showed that the sequences of the novel strains in our study for the 16S rRNA are completely identical with the previously described sequences.We come to the conclusion that 16S rRNA gene has no obvious variation according to the geographic distribution,serotypes,as well as within the same type but different strains.Likewise,cam gene doesn't show any significant variation among its different serotypes,or within the same serotype but different strains.Specifically, cam gene has no variation among differently virulent strain.In other works,cam gene could tell the discrepancy of virulence between strains.
The virulences of eight isolated strains were determined by animal regression test. Our data show that CZ9 and AQ2 fall into avirulent strains,CZ1,AQ1 and LA4 were mild strains;CZ2,CZ12 and HF1 were virulent strains.Ducks immunized with CZ9 could resist the challenging of CZ12 and HF1 with a survival percentage of 60%and 80%,respectively. CZ9 strain features with stronger immunogenicity than AQ2.Therefore,CZ9 can be chosen as candidate for attenuated vaccine.With it for vaccination,the optimal dose for the induction of the effective protection to recipient duck was determined to be 8.0×108CFU/ 0.1mL.All these preliminary data supplied a solid foundation for further investigation of RA and the relative infectious disease.
本研究根据RA 16S rRNA基因序列设计一对RA特异性引物,成功地建立了检测RA的PCR方法,特异性强,同时该PCR方法能检出20 pg的RA DNA,灵敏度高,这为RA的检测提供了一种快速、敏感、特异性强的检测方法。应用该PCR方法对病料组织直接进行RA检测,脑组织为首选检测对象,具有良好的实际应用意义。
利用常规方法和建立的PCR方法对安徽省滁州市、六安市、安庆市、合肥市、巢湖市和蚌埠市六个地区108份病/死鸭进行RA检测,六个地区均检测出RA,阳性检出率为24.07%(26/108),其中滁洲市RA分离株占总分离株的57.69%(15/26),显示安徽地区RA的感染较为普遍,滁洲地区感染情况严重。
26株RA分离株中20株为血清1型,占总分离株的76.92%,6株未定型,表明安徽省目前至少存在两种不同的血清型,而2000年以前的分离株均属血清1型,说明安徽省RA流行菌株的血清型存在动态变化,血清型呈现多极化的趋势。
对安徽省RA分离株的16S rRNA基因和cam基因进行扩增和序列测定,并与国内外参考菌株进行同源性比较,结果表明不同血清型、同一血清型的不同菌株之间及不同地区的菌株之间16S rRNA基因序列无明显差异,RA 16S rRNA基因核苷酸序列差异的距离与地理分布、菌株来源无一定的相关性;不同毒力株之间cam基因并无明显差异,根据cam基因无法判断不同菌株的毒力强弱。
通过动物回归试验,测定8株RA分离株的毒力,其中CZ9和AQ2为弱毒菌株,CZ1、AQ1、LA4为中等毒力菌株,CZ2、CZ12、HF1为强毒菌株。将CZ9和AQ2免疫鸭,CZ9对CZ12和HF1攻击的保护率分别为60%和80%,比AQ2的免疫原性好,故将其作为弱毒疫苗株的候选株,进一步测定CZ9的最佳免疫剂量为8.0×10~8CFU/0.1mL,从而为RA弱毒疫苗的研制提供了一定的基础数据。
Riemerella anatipestifer(RA) infection is a acute or chronic contagious disease of domestic young ducks,turkeys,and various other domestic and wild birds.It accounts for significant economic losses due to high morbidity and mortality,known as is a major disease confronting the duck industry through the world.To date,21 serotypes have been reported,little cross reaction was found between different serotypes.In addition,resistant strains appear with the application of antibiotics The iterative antibiotics treatment is responsible for the occurrence of resistant strains in infected ducks.So,it is becoming more and more urgent to diagnose the early RA infection,predict the potential RA serotype change,and seek for effective vaccine in order to control and prevent the RA infectious disease.
In the present study,a pair of primer was designed and synthesized based on 16S rRNA gene sequence from RA strain.PCR was then performed with these primers in the assay of lesion samples from the suspended infection.It demonstrated the PCR amplification is RA specific with 20pg of the minimum DNA amount for detection.The optimal experiment showed that the brain tissue is the preferential tissue in the assay. Notably,the PCR developed in this study is highly specific,much sensitive,and practically useful in monitoring the RA infectious disease.
Out of 108 tissue samples of infected ducks collected in 6 different regions including Chuzhou,Liuan,Anqing,Hefei,Chaohu and Bengbu in Anhui Province,26 of freshly isolated strains were gotten with 24.07%of the positive percentage.Among them,strains isolated from Chuzhou accounts for 57.69%(15/26).Our results showed that the RA infection is commonly distributed in Anhui area,and Chuzhou is one of regions it happens seriously.
20 strains were further identified as serotype 1 which stands for the predominant serotype with 76.92%.The other 6 isolated strains have not been yet identified as certain type.These results demonstrated that there are at least two serotypes being prevalent in Anhui province.Considering the previous reporter that serotype 1 was the unique type in 2000,we speculate that RA infection appears in a trend of diversity.
We next determined the nucleotide sequences of the 16S rRNA and cam genes in the isolates,and compared them with the known data in GenBank.Our results showed that the sequences of the novel strains in our study for the 16S rRNA are completely identical with the previously described sequences.We come to the conclusion that 16S rRNA gene has no obvious variation according to the geographic distribution,serotypes,as well as within the same type but different strains.Likewise,cam gene doesn't show any significant variation among its different serotypes,or within the same serotype but different strains.Specifically, cam gene has no variation among differently virulent strain.In other works,cam gene could tell the discrepancy of virulence between strains.
The virulences of eight isolated strains were determined by animal regression test. Our data show that CZ9 and AQ2 fall into avirulent strains,CZ1,AQ1 and LA4 were mild strains;CZ2,CZ12 and HF1 were virulent strains.Ducks immunized with CZ9 could resist the challenging of CZ12 and HF1 with a survival percentage of 60%and 80%,respectively. CZ9 strain features with stronger immunogenicity than AQ2.Therefore,CZ9 can be chosen as candidate for attenuated vaccine.With it for vaccination,the optimal dose for the induction of the effective protection to recipient duck was determined to be 8.0×108CFU/ 0.1mL.All these preliminary data supplied a solid foundation for further investigation of RA and the relative infectious disease.
引文
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