安徽地区猪圆环病毒2型的流行病学调查及地方株的特性研究
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摘要
猪圆环病毒病是由猪圆环病毒2型(Porcine circovirus type 2,PCV2)引起的猪的一系列疾病的总称,主要包括断奶仔猪多系统衰竭综合征(Postweaning multisystemic wasting syndrome,PMWS)和猪皮炎与肾病综合征(Porcine dermatis and nephropathy syndrome,PDNS)等。由于PCV2主要侵害机体的免疫系统,导致免疫抑制,造成继发感染,对养猪业造成的直接、间接损失十分巨大,从而备受世界兽医界的关注。为了解PCV2在安徽省的流行情况,以及PCV2安徽地方株的生物学特性,本研究在2008～2009年间对从安徽省内各地采集来的猪血清样本进行ELISA检测,分离鉴定安徽地区PCV2流行毒株,并进行ORF2基因测序以及动物致病性试验。从而为掌握安徽省猪群中PCV2的感染状况、地方流行株的相关特性、疫苗株的筛选提供科学依据,为有效防控猪圆环病毒病奠定理论基础。
1.应用酶联免疫吸附试验(ELISA)对采自安徽省14个市(县)的468份血清样品进行PCV2抗体检测,并运用PCR技术同步进行PCV2核酸检测。安徽省PCV2抗体检测的平均阳性率为74.36%,且阳性率随猪的日龄增长而升高,皖南地区的阳性检出率明显高于淮北和江淮两个地区,PCR与ELISA检测结果的符合率为99.37%。安徽省猪群中普遍存在PCV2的感染,病毒血症与抗体共存现象显著。
2.运用PCR技术对采自肥东、定远、白湖、宣城、肥西等县市的不同猪场疑似PMWS发病仔猪的组织病料进行PCV2检测,筛选阳性样品经处理后接种PK-15细胞进行PCV2的分离增殖,根据PCR、间接免疫荧光试验、电镜技术进行PCV2分离株的鉴定。PCR检测均扩增出的PCV2特异性目的条带,电镜下可见直径在17nm左右的病毒粒子,并可在胞浆内形成大包涵体,间接免疫荧光试验在细胞核、细胞质中可见特异性荧光,证实获得5株PCV2,分别命名为FD0801、DY0801、BH0801、FX0901和XCh0901。
3.比较5株PCV2分离株ORF2基因第1088-1718位核苷酸序列(630bp)与GenBank中国内外27株参考株的同源性为87.3%~99.6%;5株分离毒株彼此之间的同源性介于92.1~99.6%,传代30次ORF2基因变异较小;进一步建立的系统进化树显示PCV2的ORF2基因核苷酸序列比较稳定,其进化不存在明显的地域相关性,5株分离毒株可能属于PCV2b基因亚型。分离毒株进行小白鼠致病性试验,PCV2抗体检测表明0.8mL病毒液、腹腔注射结合滴鼻灌胃的攻毒方式可以提高PCV2的感染机率,根据脏器病变程度筛选出致病力较强的BH0801株进行本动物致病性试验,攻毒猪血清PCV2抗体呈阳性,且肺脏有暗红色斑块,肾脏表面有出血点,以及淋巴小结增多,出现大量多核细胞等典型PCV2感染的宏观和微观病变,该毒株有望成为疫苗株。
Pig circovirus disease is the general term for a series of pig diseases caused by porcine circovirus type 2 (PCV2)which mainly include postweaning multisystemic wasting syndrome (PMWS) and porcine dermatis and nephropathy syndrome (PDNS) and so on. PCV2 mainly against immune system leading to immune suppression and secondary infection of the pig. PCV2 provoked particular interest among world veterinary community, because the virus caused enormous losses direct and indirect on pig husbandry. In order to understand the prevalence and biological characteristics of PCV2, swine serum samples were collected from different areas in Anhui Province from 2008 to 2009. The pig serum samples were detected by ELISA to isolate and identify PCV2 epidemic strains. The ORF2s were sequenced and analyzed. Animal pathogenicity test was also conducted. This study is a scientific basis for understanding PCV2 infection and relevant characteristics of the local epidemic strains in Anhui. This study also provided scientific basis on choosing vaccine strain and theoretical basis for controlling of porcine circovirus.
1. 468 serum samples from 14 areas in Anhui were detected for antibodies and nucleic acid against PCV2 by ELISA and PCR respectively. The average positive ratio of antibodies against PCV2 in 468 serum samples was 74.36%. The positive ratio of antibodies increased as pig growing up. The positive ratio of antibodies in southern Anhui was significantly higher than areas in north of the Huai River and areas between Yangtze River and Huai River. The compliance rate of PCR and ELISA was 99.37%. The infection of PCV2 existed widespreadly in pigs in Anhui. There was a co-existence phenomenon between viremia and antibody against PCV2.
2. The disease tissues collected from suspected PMWS piglets in Feidong, Dingyuan, Baihu, Xuancheng and Feixi were conducted PCV2 detection using PCR amplification on specific DNA fragment of the virus. Positive samples were inoculated on PK-15 cells to isolate viruses. PCR, electron microscopy and indirect immunofluorescence test (IFA) were used to identify virus. Five PCV2 strains were confirmed by followed tests. Specific 630bp fragment was detected by PCR. 17nm virus particlesin diameter was seen by electron microscopy. The indirect immunofluorescence test showed specific fluorescence in the nucleus and cytoplasm. Then, the Five PCV2 isolates were named as FD0801, DY0801, BH0801, FX0901 and XCh0901.
3. Nucleotide sequence form 1088 to 1718 (630bp) of ORF2 gene of five strains were compared with 27 reference strains registered in GenBank, the homology were 87.3% to 99.8%. The homology between five virus strains were 92.1% to 99.6%, The mutation of ORF2 gene was small after 30 passages. The phylogenetic tree showed the ORF2 gene nucleotide sequence of PCV2 is relatively stable. Its evolution is not showing obvious geographical correlation. The five virus strains may belong to PCV2b subtypes. The five isolated strains were conducted mouse pathogenicity test. The result of PCV2 antibody testing showed that the 0.8mL virus solution intraperitoneal injection combined with nose and mouth attacked can raise the infection probability of PCV2. The BH0801 strain was choosed according to organ lesions to conduct animal pathogenicity test. The serum of challenged pig showed antibody positive of PCV2, The organ lesions of poison attack pig were dark red patches in the lung, bleeding kidney surface, increased lymph nodules, and the appeared large number of multinucleated cells which were typical macro-and micro lesions of PCV2 infection. So, The BH0801 strain is expected to become the vaccine strain.
1.应用酶联免疫吸附试验(ELISA)对采自安徽省14个市(县)的468份血清样品进行PCV2抗体检测,并运用PCR技术同步进行PCV2核酸检测。安徽省PCV2抗体检测的平均阳性率为74.36%,且阳性率随猪的日龄增长而升高,皖南地区的阳性检出率明显高于淮北和江淮两个地区,PCR与ELISA检测结果的符合率为99.37%。安徽省猪群中普遍存在PCV2的感染,病毒血症与抗体共存现象显著。
2.运用PCR技术对采自肥东、定远、白湖、宣城、肥西等县市的不同猪场疑似PMWS发病仔猪的组织病料进行PCV2检测,筛选阳性样品经处理后接种PK-15细胞进行PCV2的分离增殖,根据PCR、间接免疫荧光试验、电镜技术进行PCV2分离株的鉴定。PCR检测均扩增出的PCV2特异性目的条带,电镜下可见直径在17nm左右的病毒粒子,并可在胞浆内形成大包涵体,间接免疫荧光试验在细胞核、细胞质中可见特异性荧光,证实获得5株PCV2,分别命名为FD0801、DY0801、BH0801、FX0901和XCh0901。
3.比较5株PCV2分离株ORF2基因第1088-1718位核苷酸序列(630bp)与GenBank中国内外27株参考株的同源性为87.3%~99.6%;5株分离毒株彼此之间的同源性介于92.1~99.6%,传代30次ORF2基因变异较小;进一步建立的系统进化树显示PCV2的ORF2基因核苷酸序列比较稳定,其进化不存在明显的地域相关性,5株分离毒株可能属于PCV2b基因亚型。分离毒株进行小白鼠致病性试验,PCV2抗体检测表明0.8mL病毒液、腹腔注射结合滴鼻灌胃的攻毒方式可以提高PCV2的感染机率,根据脏器病变程度筛选出致病力较强的BH0801株进行本动物致病性试验,攻毒猪血清PCV2抗体呈阳性,且肺脏有暗红色斑块,肾脏表面有出血点,以及淋巴小结增多,出现大量多核细胞等典型PCV2感染的宏观和微观病变,该毒株有望成为疫苗株。
Pig circovirus disease is the general term for a series of pig diseases caused by porcine circovirus type 2 (PCV2)which mainly include postweaning multisystemic wasting syndrome (PMWS) and porcine dermatis and nephropathy syndrome (PDNS) and so on. PCV2 mainly against immune system leading to immune suppression and secondary infection of the pig. PCV2 provoked particular interest among world veterinary community, because the virus caused enormous losses direct and indirect on pig husbandry. In order to understand the prevalence and biological characteristics of PCV2, swine serum samples were collected from different areas in Anhui Province from 2008 to 2009. The pig serum samples were detected by ELISA to isolate and identify PCV2 epidemic strains. The ORF2s were sequenced and analyzed. Animal pathogenicity test was also conducted. This study is a scientific basis for understanding PCV2 infection and relevant characteristics of the local epidemic strains in Anhui. This study also provided scientific basis on choosing vaccine strain and theoretical basis for controlling of porcine circovirus.
1. 468 serum samples from 14 areas in Anhui were detected for antibodies and nucleic acid against PCV2 by ELISA and PCR respectively. The average positive ratio of antibodies against PCV2 in 468 serum samples was 74.36%. The positive ratio of antibodies increased as pig growing up. The positive ratio of antibodies in southern Anhui was significantly higher than areas in north of the Huai River and areas between Yangtze River and Huai River. The compliance rate of PCR and ELISA was 99.37%. The infection of PCV2 existed widespreadly in pigs in Anhui. There was a co-existence phenomenon between viremia and antibody against PCV2.
2. The disease tissues collected from suspected PMWS piglets in Feidong, Dingyuan, Baihu, Xuancheng and Feixi were conducted PCV2 detection using PCR amplification on specific DNA fragment of the virus. Positive samples were inoculated on PK-15 cells to isolate viruses. PCR, electron microscopy and indirect immunofluorescence test (IFA) were used to identify virus. Five PCV2 strains were confirmed by followed tests. Specific 630bp fragment was detected by PCR. 17nm virus particlesin diameter was seen by electron microscopy. The indirect immunofluorescence test showed specific fluorescence in the nucleus and cytoplasm. Then, the Five PCV2 isolates were named as FD0801, DY0801, BH0801, FX0901 and XCh0901.
3. Nucleotide sequence form 1088 to 1718 (630bp) of ORF2 gene of five strains were compared with 27 reference strains registered in GenBank, the homology were 87.3% to 99.8%. The homology between five virus strains were 92.1% to 99.6%, The mutation of ORF2 gene was small after 30 passages. The phylogenetic tree showed the ORF2 gene nucleotide sequence of PCV2 is relatively stable. Its evolution is not showing obvious geographical correlation. The five virus strains may belong to PCV2b subtypes. The five isolated strains were conducted mouse pathogenicity test. The result of PCV2 antibody testing showed that the 0.8mL virus solution intraperitoneal injection combined with nose and mouth attacked can raise the infection probability of PCV2. The BH0801 strain was choosed according to organ lesions to conduct animal pathogenicity test. The serum of challenged pig showed antibody positive of PCV2, The organ lesions of poison attack pig were dark red patches in the lung, bleeding kidney surface, increased lymph nodules, and the appeared large number of multinucleated cells which were typical macro-and micro lesions of PCV2 infection. So, The BH0801 strain is expected to become the vaccine strain.
引文
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