羊肠道病毒与小反刍兽疫病毒混合感染及流行病学调查

本实验室2014年从吉林省某地暴发严重腹泻的病羊群分离出肠道病毒(CEV-JL14)和小反刍兽疫病毒,表明这2种病毒在该次疫病的暴发中可能发挥重要作用。为进一步了解羊群中小反刍兽疫病毒与羊肠道病毒混合感染情况,本研究应用建立的检测小反刍兽疫病毒抗原的夹心ELISA试剂盒和检测羊肠道病毒抗原的双抗体夹心ELISA试剂盒,对采自吉林省和内蒙古地区的共计1 167份羊粪便分别进行检测,发现被检羊群存在较为严重的小反刍兽疫病毒与羊肠道病毒混合感染。羊群混合感染小反刍兽疫病毒和羊肠道病毒在国内外属首次报道,该发现为今后上述病毒引起的疫病的诊断、防治提供流行病学理论依据。     

羊肠道病毒单克隆抗体的制备及鉴定

根据本实验室分离的国内首株羊肠道病毒CEV-JL14的核苷酸基因组序列,设计并合成了羊肠道病毒VP1基因的引物,并应用RT-PCR扩增了VP1基因片段。扩增的VP1基因片段克隆到原核表达载体pGEX-4T-1的EcoRⅠ/BamHⅠ的酶切位点。重组质粒经酶切鉴定和序列测定正确后,转化到BL21,并以IPTG诱导表达了VP1重组蛋白。重组蛋白纯化后以弗氏完全佐剂乳化,按一定的程序免疫BALB/c小鼠,加强免疫3次后,取小鼠脾细胞与骨髓瘤细胞融合,经过3次亚克隆获得了5株表达VP1单克隆抗体的杂交瘤细胞株。以ELISA对杂交瘤分泌的IgG亚类进行了鉴定,结果4株为IgG1亚类,1株为IgG2b。以免疫酶单层细胞技术鉴定了单克隆抗体,结果显示,获得的单克隆抗体可以特异性检出羊肠道病毒抗原。本试验在国内首次获得了抗羊肠道病毒的单克隆抗体,将为羊肠道病毒的致病机理、诊断及流行病学研究奠定基础。     

宁夏地区牛场E种肠道病毒的分离与鉴定

牛肠道病毒感染是近年来国内新报道的一种传染病,其病原体为微RNA病毒科肠道病毒属中的肠道病毒。本研究应用双抗体夹心ELISA方法对宁夏地区的部分牛群肠道病毒感染进行了调查,其感染率为9.85%、27%、28.2%。应用Vero细胞从采自宁夏牛群的粪便样品中分离出2株病毒,采用病毒学技术、过氧化物酶单层细胞试验和分子生物学技术对分离毒鉴定发现,2株病毒均为肠道病毒。应用PCR技术扩增分离毒的部分核苷酸序列,并对其进行比对分析发现,分离的2株病毒均为E种肠道病毒。该结果将为宁夏地区肠道病毒感染的防控提供依据。     

从暴发严重腹泻羊群中检出G种肠道病毒

从吉林省某羊场暴发的临床上以严重腹泻、发热、流涎、口腔黏膜严重溃疡为特征的未明疫病病羊体内分离到大小为20~30nm病毒粒子。病毒生物学特性、理化学特性和分子生物学特性鉴定发现,该病毒粒子为肠道病毒,命名为OEV-JL14毒株。核苷酸序列测定与比对分析发现,OEV-JL14与OEV-TB4的同源性最高,只有68.8%。进化树分析表明OEV-JL14为G种肠道病毒。本研究首次在国内从病羊体内分离鉴定出羊肠道病毒,该结果将为羊新发肠道病毒感染的诊断及防制提供理论依据。     

1株羊肠道病毒的分离与鉴定

本研究利用Vero细胞分离羊肠道病毒(CEV)流行毒株,并进行分离毒株病毒含量(TCID50)测定、生物学特性检测、5'-UTR基因序列分析、致病性检测等试验.从临床病料中分离出1株CEV,命名为GS株;GS株可在Vero细胞上形成圆缩、拉网、折光性增强等典型病变;TCID50为10-6.88/0.1 mL;对酸性不敏...     

从吉林省肉牛场新发疫病分离出E种肠道病毒

从吉林省某牛场发生的临床上以发热、咳嗽、呼吸困难、严重腹泻、高发病率和死亡率为特征的新发疫病病牛体内分离到大小为20³0nm和15030nm和150²50nm的2株病毒。病毒生物学特性、理化学特性和分子生物学特性显示,20250nm的2株病毒。病毒生物学特性、理化学特性和分子生物学特性显示,20³0nm的病毒粒子为牛肠道病毒。该病毒命名为HY12毒株。病毒5′端非编码区的核苷酸序列分析结果表明,HY12毒株与国外分离株SL305的同源性最高,为90%;而与国内分离毒株的同源性只有75%。系统进化树分析发现,HY12毒株属于肠道病毒属的E种肠道病毒。本研究首次在国内从临床表现为呼吸道和消化道症状的病牛体内分离出E种肠道病毒,该结果将为牛肠道病毒感染的诊断及防治提供依据。     

吉林省不同地区牛肠道病毒遗传变异分析

为了解近年来吉林省牛肠道病毒(bovine enteroviruses,BEV)感染流行情况及病毒株分子变异特征,本研究利用双抗体夹心ELISA方法,对采集于吉林省不同地区的326份疑似BEV感染的牛粪便样品进行检测,并对ELISA检测出的阳性样品进行病毒分离培养;同时对分离毒株5′UTR基因序列进行扩增、序列比对分析。结果表明,不同地区的牛群均存在程度不同的肠道病毒感染;BEV分离毒株的核苷酸序列与本实验室2012年分离的国内首株E种牛肠道病毒HY12基因序列的同源性为86.2%~96.9%。其中从公主岭地区分离的毒株GZL-6与HY12毒株差异性最大,其同源性只有86.2%;而与HY12同地区分离出的HY68毒株同源性只有86.3%。国内外现有BEV毒株序列遗传进化树分析结果显示,从吉林省新分离到11株BEV与HY12处在同一个分支。上述结果表明,新近分离的BEV毒株,无论源于HY12毒株的原地区还是其他不同地区,均有不同程度的核苷酸序列差异。本研究为BEV感染的诊断、防治及肠道病毒进化变异研究打下基础。     

山东省某地区牛、羊肠道病毒感染的流行病学调查

牛、羊肠道病毒感染为国内新近报道的动物传染病,其流行病学本底在国内缺乏。为了解牛、羊肠道病毒感染的情况,本研究应用双抗体夹心ELISA方法对采集于山东省某地区的奶牛、肉牛、绵羊和山羊粪便样品进行了肠道病毒的检测。同时对ELISA检测为阳性的部分样品进行了病毒分离,并以免疫过氧化物酶单层试验对分离毒株进行了鉴定。结果发现,奶牛的肠道病毒感染率为25.0%,肉牛群的感染率为7.6%,绵羊的肠道病毒感染率58.6%,山羊的肠道病毒感染率为48.5%,该结果为肠道病毒感染的防制提供流行病学的理论依据。     

双抗体夹心ELISA检测羊肠道病毒抗原方法的建立及病原流行病学调查

应用本实验室分离的国内首株羊肠道病毒(CEV)-JL14VP1重组蛋白制备的兔源多克隆抗体和抗CEV-JL14病毒的单克隆抗体,建立了检测CEV抗原的双抗体夹心ELISA方法,并对吉林省和内蒙古地区羊群感染CEV进行了病原流行病学调查。方阵试验确定了CEV VP1鼠源IgG捕获抗体的最佳包被量为0.2μg/孔,酶标抗体的最佳稀释倍数为1∶1 000。对大量CEV阴性粪便样品进行检测与统计学处理,确定了双抗体夹心ELISA检测CEV的判定标准为样品D490值≥0.216判定为阳性。特异性、敏感性和重复性试验结果表明,所建立的检测CEV抗原方法具有特异、敏感、快速和重复性好等特点。以建立的双抗体夹心ELISA方法,对吉林省和内蒙古不同地区的羊群粪便样品进行了检测,发现不同地区的羊群都存在着严重的CEV感染,感染率高达12%~100%。本研究为今后CEV感染的诊断、检疫及防制提供了有效的手段和流行病学理论依据。     

广西地区水牛E2型牛肠道病毒的分离和鉴定

为了解广西地区水牛感染中肠道病毒(BEV)的情况，本研究在广西南宁某水牛规模养殖场腹泻粪便样品中成功分离出2株BEV,分别命名为GXNN2106和GXNN2102。将2株病毒空斑纯化后测定GXNN2106毒株TCID₅₀为10^7.99/0.1 mL,GXNN2102毒株TCID₅₀为10^7.37/0.1 mL。多步生长曲线结果显示，GXNN2106毒株在MDBK细胞中复制增殖良好。经BEV VP1、5′UTR特异性引物逆转录聚合酶链反应(RT-PCR)鉴定，2株病毒均属于E2型，与其他E2型BEV 5′UTR核苷酸同源性为75.7%～78.6%,VP1核苷酸同源性为87.1%～91.1%。本研究首次在广西地区水牛粪便中分离得到E2型BEV毒株，为广西水牛源牛肠道病毒的流行分布情况和疫苗的研发提供数据支持。     

Isolation of caprine arthritis-encephalitis virus from a goat

Caprine arthritis-encephalitis virus (CAFV) was isolated from the carpal joint of an Anglo-Nubian yearling goat with chronic progressive pneumonia and subclinical carpal arthritis. Anti-CAEV antibody was detected in this goat and in nine of 69 goat sera tested from two herds.     

Isolation of caprine arthritis-encephalitis virus from a goat

Caprine arthritis-encephalitis-virus (CAEV) was isolated from the carpal joint of an Anglo-Nubian yearling goat with chronic progressive pneumonia and subclinical carpal arthritis. Anti-CAEV antibody was detected in this goat and in nine of 69 goat sera tested from two herds.     

A caprine pneumonia outbreak associated with caprine herpesvirus and Pasteurella haemolytica respiratory infections

In a field disease outbreak, 60 female goats died over a 2-3 week period. Necropsies of seven of these does revealed that six had an acute exudative necrotising broncho-pneumonia, and moderate to high numbers of Pasteurella haemolytica were isolated from their lungs. Caprine herpesvirus, identified as Bovid herpesvirus type 6, was isolated from the lungs of two of these does, including one with pneumonia, and from nasal swabs of in-contact goats. Clinical disease was only observed in does, and deaths began 3 weeks after the introduction of a mob of goat hoggets from another farm.     

Association and General Notes

In a field disease outbreak, 60 female goats died over a 2–3 week period. Necropsies of seven of these does revealed that six had an acute exudative necrotising broncho-pneumonia, and moderate to high numbers of Pasteurella haemolytica were isolated from their lungs. Caprine herpesvirus, identified as Bovid herpesvirus type 6, was isolated from the lungs of two of these does, including one with pneumonia, and from nasal swabs of in-contact goats. Clinical disease was only observed in does, and deaths began 3 weeks after the introduction of a mob of goat hoggets from another farm.     

Effect of colostrum deprivation of goat kids on the natural transmission of caprine retrovirus infection

An investigation was conducted to determine if depriving goat kids of colostrum and rearing them away from the herd would prevent transmission of caprine retrovirus infection. Twenty-four newborn goat kids were deprived of colostrum and reared on cow's milk away from their dams from an endemically infected goat herd. Twenty-three colostrum-deprived kids had no evidence of retrovirus infection at birth. One kid had sucked briefly and obtained some colostrum resulting in passive transfer of antibody but it did not develop evidence of infection. Nineteen showed no serological evidence of infection over the 370 days of the study. One colostrum-deprived, segregated goat was subsequently found to be infected and developed arthritis-synovitis. Three had doubtful positive response in one or 2 serological tests during the period but no evidence of infection in leucocyte co-cultures. Cells centrifuged from colostrum of infected goats were co-cultivated with foetal goat synovial membrane cultures. Caprine retrovirus was isolated from cells in the colostrum from the 3 goats examined.     

Molecular characterization of lentiviruses from goats from Poland based on gag gene sequence analysis

Caprine arthritis-encephalitis virus (CAEV) infection in goats is worldwide but with higher prevalence in industrialized countries. While positive serology of CAEV in Polish goats was reported there was no genetic study of this virus. In this study, we described the molecular characterization of lentiviruses isolated from seropositive goats from Poland. We cloned and sequenced a fragment from the gag gene covering part of the coding sequences for the matrix (MA) p17 and for the capsid (CA) p25 proteins. Resulting nucleotide sequences were aligned with those from other ovine/caprine lentivirus isolates. We present data showing that the sequences of most goat lentivirus isolates are closer to the prototypic CAEV-Co isolate, nevertheless from one goat we isolated a virus that is closer to the sheep Maedi Visna virus (MVV) isolate. This might indicate a recent cross-species infection from sheep to goat.     

山羊支原体性肺炎流行病学调查

山羊支原体性肺炎是威胁山羊养殖的重要传染病,为了解其流行情况,对四川省主要山羊养殖地区的山羊支原体性肺炎进行了流行病学调查。从四川省7个地区山羊养殖场采集肺脏和鼻腔棉拭子样本共135份,经过分离鉴定得到42株支原体,其中绵羊肺炎支原体36株,丝状支原体6株;其中6个羊场仅分离到绵羊肺炎支原体,1个羊场同时分离到绵羊肺炎支原体和丝状支原体。本试验结果表明,绵羊肺炎支原体是引起四川省山羊支原体性肺炎的主要病原,个别地方存在绵羊肺炎支原体和丝状支原体混合感染。     

Genetic and antigenic characterization of the matrix protein of two genetically distinct ovine lentiviruses

Small Ruminant Lentiviruses (SRLV) are a group of non-oncogenic retroviruses including Maedi-Visna virus (MVV) and Caprine Arthritis-Encephalitis virus (CAEV), which cause a chronic, multisystemic disease in sheep and goats, respectively. Phylogenetic analyses of SRLV are based in most cases on partial pol sequences. Several reports indicate that the species specificity of these viruses is not as strict as previously thought; MVV-like viruses have been found in goat populations and vice versa. Recently, the sequencing of some Italian ovine isolates has shown the presence of a new cluster more similar to classical caprine isolates (CAEV-like). Few data are available on the variability of structural proteins involved in the antibody response of infected animals. In this study, the gag gene of two genetically distinct ovine isolates, namely the MVV-like It-561 and the CAEV-like It-Pi1, was sequenced and the epitopes of matrix protein (MA) were mapped. Recombinant MAs and their subunits from both ovine aforementioned strains were tested against a panel of sheep and goat sera. Reactive epitopes were found in all three subunits of MA, although the central subunit displayed a more consistent reactivity. Epitope mapping of this subunit demonstrated that the amino acid sequence of at least one immunodominant epitope was quite different in the two strains. This antigenic variability may affect the sensitivity of a single strain-based immunoassay and suggests that both SRLV genotypes should be used in the development of future diagnostic tests, to avoid viral strain selection during the eradication programmes.     

Isolation of transmissible Gastroenteritis virus, pseudorabies virus, and porcine enterovirus from pharyngeal swabs taken from market-weight-swine

Pharyngeal swab samples were collected at a central Iowa abattoir from 6,010 market-weight slaughter hogs from September 1, 1979 to August 31, 1980. The swab samples were examined for cytopathic viruses by inoculation of monolayer cultures of continuous line of swine testicular cells. Of the 6,010 swab samples tested, transmissible gastroenteritis virus was isolated from 91 (1,51%), pseudorabies virus was isolated from 431 (7.17%), and porcine enterovirus was isolated from 21 (0.35%). Although all 3 viruses were identified throughout the year, transmissible gastroenteritis and pseudorabies viruses were found more frequently during the winter and early spring. In contrast, porcine enterovirus was detected more frequently during the spring and summer.     

ATYPICAL PORCINE ENTEROVIRUS ENCEPHALOMYELITIS: POSSIBLE INTERRACTION BETWEEN ENTEROVIRUSES AND ARSENICALS

Porcine enteroviruses were isolated from weaner pigs that had nervous signs and mild non-suppurative meningoencephalomyelitis and ganglioneuritis. The clinical signs and lesions were not typical of enterovirus infection and it is believed that an organic arsenical present in feed enhanced pathogenicity of enteroviruses. Severe non-suppurative polioencephalomyelitis and ganglioneuritis were produced in gnotobiotic pigs by oral inoculation of the viruses.