河北省鸭肝炎病毒BD-Ⅰ株的分离鉴定及弱毒株培育
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摘要
鸭病毒性肝炎是由鸭肝炎病毒(DHV)引起雏鸭的一种传染病,具有发病急、传播迅速、病程短、病死率高等特点。本病在全国各地都有发生,是严重危害养鸭业的主要疾病之一,并常和其他病毒、细菌混合感染,严重影响了养鸭业,造成了巨大的经济损失。临床上主要表现为食欲减退、神经症状、突然死亡和肝脏肿大出血。病原有3种,其中Ⅰ型鸭肝炎病毒在世界范围内流行,Ⅱ型和Ⅲ型鸭肝炎病毒分别局限于英国和美国。不同血清型毒株具有不同的理化和生物学特性,且无免疫交叉反应。近年来国内外报道了Ⅰ型鸭肝炎病毒的变异株,且已完成了病毒基因组的测序工作,并随之出现RT-PCR诊断技术,对雏鸭病毒性肝炎的诊断和防控具有重要意义。
本研究通过鸡胚尿囊腔接种,对河北省保定地区采集的死于疑似鸭病毒性肝炎的病鸭肝组织进行病原分离,获得1个病毒分离株,命名为BD-Ⅰ株。该分离株经病理学检查、理化特性实验、中和试验、动物回归试验等鉴定为血清Ⅰ型鸭肝炎病毒(DHV)。证实引起该鸭场雏鸭发病和死亡的主要原因为DVH所致。
根据GenBank中已发表的Ⅰ型鸭病毒性肝炎病毒(DHV-Ⅰ)的基因序列,应用Primer Premier 5.0软件设计了1对引物,建立了检测DHV-Ⅰ的RT-PCR方法,该法能从DHV-Ⅰ中扩增到506bp的条带。经敏感性、特异性检测,证实为DHV-Ⅰ的特异性条带,而新城疫病毒(NDV)、传染性法氏囊病病毒(IBDV)、鸭瘟病毒(DPV)、番鸭细小病毒(MPV)、鸭病毒性肿头出血症病毒(DSHDV)、鹅细小病毒(GPV)均为阴性。最低检测浓度到128倍稀释的鸭肝毒(相当于500ELD_(50))。RT-PCR对河北保定地区的发病鸭群的13份鸭病毒性肝炎病料进行检测,其检出率为76.92%(10/13)。因此,所建立的RT-PCR检测技术具有特异、快速和敏感的特点,可用于鸭病毒性肝炎病毒的快速诊断。
作者对实验室分离的鸭肝炎病毒经过鸡胚传代致弱后,收集鸡胚尿囊液毒不同含毒量对1日龄雏鸭进行免疫,并于14日龄时用新型鸭肝炎强毒进行攻毒保护试验,结果BD-Ⅰ-40株在500ELD_(50)时,保护率达100%。采集免疫的雏鸭血清,中和试验测定血清的效价,雏鸭在免疫后可检测到中和抗体,第14天时抗体水平达到高峰,然后呈逐渐下降趋势。结果表明,BD-Ⅰ株经过鸡胚传代致弱后在毒力下降的同时仍保留免疫原性,可以作为疫苗的候选株。
Duck viral hepatitis is an acute, highly contagious , viral disease of young ducklings by Duck Hepatitis Virus . It is characterized by high mortality and short incubation period. It is one of the main serious diseases in duck, it exist in many places of our country.Many viruses and bacteria co-infect with the DHV-I, which causes great loss . Clinical signs include anorexia,sudden death,opisthotonos and enlarged liver with haemorrhagic lesions. Three distinct types of duck hepatitis virus have been isolated from diseased ducklings. DHV- I is present in all duck-raising areas of the world , DHV- II and DHV-III are rest ricted to the United Kingdoms and United States of America respectively. Three kinds of serotype strains have no immunological cross-reactions except for different physiochemi-al and molecular characteristics. Recently distinct serologic variants of DHV-I have been described and complete genome sequence of DHV-I has been finished. Subsequence appearance of diagnosis method by RT-PCR is important to prevention and treatment of duck viral hepatitis.
A strain of virus ,which was suspected to be duck hepatitis virus (DHV), was isolated from duckling liver samples through chicken embryo by allantoic sac inoculation . The sample was collected from ducklings diagnosed clinically dying from duck virus hepatitis on the duck farm in Baoding County of Hebei Province.The strain was named DHV BD-I strain. It was confirmed by pathological examination , physical-chemical characterization tests, neutralization test and animal relapsing test that the virus strain was DHV serotype I and had resulted in plenty of ducks to be pathogenetic and dead on the farm.
A RT- PCR method for detecting the duck hepatitis virus I type was established with a pair of primers designed and synthesized based on the gene of GenBank.The primer was designed with help of Primer Premier 5.0 design software .A specific 506 bp fragment was amplified from RNA templates of DHV- I virulence AV2111 strain.And the sensitivity,specificity tests were carried out. The RT- PCR method could detect 128 folds dilution of the liver samples of duck hepatitis virus I .But it couldn' t detect Newcastle disease virus (NDV) ,infectious bursal disease virus(IBDV),duck plague virus(DPV) , Muscovy parvovirus(MPV), duck swollenhead hemorrhagic disease virus(DSHDV),and Goose paramyxoviridae(GPV). The rates were 76.92%(10/13) when liver samples of clinical cases from Hebei province of China stored at -80°C were detected by the RT-PCR.The results showed that the sensitivity and specificity was very high. So the method can be applied for the fastly diagnosis of Duck hepatitis virus I type.
The strain of DHV was attenuated by passage in chicken embryos and designated BD-I strain. Diluted allantotic fluid virus was used to immunize 1-day-old ducklings and the immunized ducklings were challenged with virulent serotype I 14 days after immunization .The result indicated that the protection rate of 500ELD_(50) of BD-I -40 could reach up to 100 percent. The sera of the immunized ducklings were collected and the antibody liters were measured by virus neutralization test. The antibody level reached peak in 14 days post vaccination and then decreased.The results showed that BD-I strain could provide protection against the infection with serotype I DHV and maybe used as the candidate vaccine strain.
本研究通过鸡胚尿囊腔接种,对河北省保定地区采集的死于疑似鸭病毒性肝炎的病鸭肝组织进行病原分离,获得1个病毒分离株,命名为BD-Ⅰ株。该分离株经病理学检查、理化特性实验、中和试验、动物回归试验等鉴定为血清Ⅰ型鸭肝炎病毒(DHV)。证实引起该鸭场雏鸭发病和死亡的主要原因为DVH所致。
根据GenBank中已发表的Ⅰ型鸭病毒性肝炎病毒(DHV-Ⅰ)的基因序列,应用Primer Premier 5.0软件设计了1对引物,建立了检测DHV-Ⅰ的RT-PCR方法,该法能从DHV-Ⅰ中扩增到506bp的条带。经敏感性、特异性检测,证实为DHV-Ⅰ的特异性条带,而新城疫病毒(NDV)、传染性法氏囊病病毒(IBDV)、鸭瘟病毒(DPV)、番鸭细小病毒(MPV)、鸭病毒性肿头出血症病毒(DSHDV)、鹅细小病毒(GPV)均为阴性。最低检测浓度到128倍稀释的鸭肝毒(相当于500ELD_(50))。RT-PCR对河北保定地区的发病鸭群的13份鸭病毒性肝炎病料进行检测,其检出率为76.92%(10/13)。因此,所建立的RT-PCR检测技术具有特异、快速和敏感的特点,可用于鸭病毒性肝炎病毒的快速诊断。
作者对实验室分离的鸭肝炎病毒经过鸡胚传代致弱后,收集鸡胚尿囊液毒不同含毒量对1日龄雏鸭进行免疫,并于14日龄时用新型鸭肝炎强毒进行攻毒保护试验,结果BD-Ⅰ-40株在500ELD_(50)时,保护率达100%。采集免疫的雏鸭血清,中和试验测定血清的效价,雏鸭在免疫后可检测到中和抗体,第14天时抗体水平达到高峰,然后呈逐渐下降趋势。结果表明,BD-Ⅰ株经过鸡胚传代致弱后在毒力下降的同时仍保留免疫原性,可以作为疫苗的候选株。
Duck viral hepatitis is an acute, highly contagious , viral disease of young ducklings by Duck Hepatitis Virus . It is characterized by high mortality and short incubation period. It is one of the main serious diseases in duck, it exist in many places of our country.Many viruses and bacteria co-infect with the DHV-I, which causes great loss . Clinical signs include anorexia,sudden death,opisthotonos and enlarged liver with haemorrhagic lesions. Three distinct types of duck hepatitis virus have been isolated from diseased ducklings. DHV- I is present in all duck-raising areas of the world , DHV- II and DHV-III are rest ricted to the United Kingdoms and United States of America respectively. Three kinds of serotype strains have no immunological cross-reactions except for different physiochemi-al and molecular characteristics. Recently distinct serologic variants of DHV-I have been described and complete genome sequence of DHV-I has been finished. Subsequence appearance of diagnosis method by RT-PCR is important to prevention and treatment of duck viral hepatitis.
A strain of virus ,which was suspected to be duck hepatitis virus (DHV), was isolated from duckling liver samples through chicken embryo by allantoic sac inoculation . The sample was collected from ducklings diagnosed clinically dying from duck virus hepatitis on the duck farm in Baoding County of Hebei Province.The strain was named DHV BD-I strain. It was confirmed by pathological examination , physical-chemical characterization tests, neutralization test and animal relapsing test that the virus strain was DHV serotype I and had resulted in plenty of ducks to be pathogenetic and dead on the farm.
A RT- PCR method for detecting the duck hepatitis virus I type was established with a pair of primers designed and synthesized based on the gene of GenBank.The primer was designed with help of Primer Premier 5.0 design software .A specific 506 bp fragment was amplified from RNA templates of DHV- I virulence AV2111 strain.And the sensitivity,specificity tests were carried out. The RT- PCR method could detect 128 folds dilution of the liver samples of duck hepatitis virus I .But it couldn' t detect Newcastle disease virus (NDV) ,infectious bursal disease virus(IBDV),duck plague virus(DPV) , Muscovy parvovirus(MPV), duck swollenhead hemorrhagic disease virus(DSHDV),and Goose paramyxoviridae(GPV). The rates were 76.92%(10/13) when liver samples of clinical cases from Hebei province of China stored at -80°C were detected by the RT-PCR.The results showed that the sensitivity and specificity was very high. So the method can be applied for the fastly diagnosis of Duck hepatitis virus I type.
The strain of DHV was attenuated by passage in chicken embryos and designated BD-I strain. Diluted allantotic fluid virus was used to immunize 1-day-old ducklings and the immunized ducklings were challenged with virulent serotype I 14 days after immunization .The result indicated that the protection rate of 500ELD_(50) of BD-I -40 could reach up to 100 percent. The sera of the immunized ducklings were collected and the antibody liters were measured by virus neutralization test. The antibody level reached peak in 14 days post vaccination and then decreased.The results showed that BD-I strain could provide protection against the infection with serotype I DHV and maybe used as the candidate vaccine strain.
引文
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