禽流感是由甲型流感病毒引起的一种急性、烈性、高致死性的动物传染病.临床上根据其致病性的不同,分为高致病性、中致病性和低/非致病性3种类型.高致病性禽流感因发病急、致死率高,而且已经跨越种属障碍感染人类,被世界动物卫生组织(OIE)列为法定报告动物传染病,也是目前我国唯一一个在家禽和水禽上实施强制免疫的疾病.

随着蛋鸡养殖规模化程度的不断提高,封闭式鸡舍应用越来越多,随之而来的是通风、温湿度调节和光照控制等问题.在解决通风问题时,大型蛋鸡养殖企业采用各型智能环境控制系统来应对烦琐的计算和设备调节,而中小规模场使用简单的时控和变频设备凭经验操作,随意性较强.对于生产管理者来说,无论多大规模的鸡舍,采用什么样的设备,都应懂得基础的通风量计算方法,以便调整设备和设定验证程序.

为了比较3种ALVp27抗原ELISA试剂盒对外源性ALV检测效果的差异,本研究将1株滴度为4.64×104TCID50/mL的ALV-A毒株GD-13分别以100、10-1、10-2、10-3进行倍比稀释(每个稀释度病毒接种含量分别为2.32 ×103 TCID50、2.32×102TCID50、2.32×101TCID50、2.32TCID50)接种于24孔板的含10％ FBS DF-1细胞悬液中,待细胞长满后换为1％ FBS维持液,从更换维持液后第3天开始至第9天每天取细胞上清分别同时用3种不同的ELISA试剂盒(A、B、C)进行检测.结果显示,当病毒接种含量为2.32×103TCID50时,A、B试剂盒最早可在换液后第3天检出病毒p27抗原阳性,C试剂盒最早在第4天才可以检出病毒阳性,检测阳性率均为100％(5/5).当病毒接种含量为2.32×102TCID50时,使用A试剂盒在接种后第6天能够检出,检测阳性率为80％(4/5);使用B试剂盒最早在接种后第5天可检测出,检测阳性率为20％(1/5);使用C试剂盒直到第9天才能检测出病毒,而且检测阳性率为20％(1/5).当病毒接种含量为2.32×101TCID50和2.32TCID50时,使用A、B和C试剂盒均检测不出病毒.本 研究结果表明,对于ALV-A的检测,无论是在检出时间还是检出率上B试剂盒都优于A、C试剂盒,提示不同商品化试剂盒的灵敏度存在一定的差异.本研究可为禽白血病病原学和净化检测提供参考.

对来自江西3个地方鸡品种(崇仁麻鸡、余干乌骨鸡、东乡绿壳蛋鸡)进行禽白血病病毒(ALV)病原学调查.将所采集的血浆接种DF-1细胞,经ALV p27抗原ELISA检测,结果显示这3个江西地方鸡品种均有外源性ALV感染,经鉴定得到4株J亚群禽白血病病毒(ALV-J).基于gp85序列分析表明这4个分离株与ALV-J英国原型株HPR-103 gp85基因核苷酸序列相似性最高(平均为94.6％),而与A、B、C、E亚群ALV gp85基因的核苷酸相似性仅在50.6％～54.5％之间.这是江西地方鸡品种分离和鉴定ALV-J的初次报道,对于我国江西省地方鸡品种的禽白血病净化具有参考价值.

为了解麻黄肉种鸡蛋清中内源性禽白血病病毒(内源性ALV)的分布情况,从广东某麻黄肉种鸡场采集1 038份蛋清,经ALV p27抗原ELISA检测出30份阳性蛋清;将阳性蛋清同时接种DF1和CEF细胞,培养7d后,p27抗原ELI-SA检测到13份样品为DF1-CEF+;以这13份CEF+细胞培养物的基因组DNA为模板,经PCR扩增克隆测序最终得到了1株内源性ALV前病毒序列.将经ELISA、PCR和IFA等方法鉴定的该内源性ALV命名为HN1301株.基于遗传分析表明,HN1301株的前病毒基因序列与已知的E亚群毒株ev1、SD0501的相似性均为99.4％.表明该麻黄肉种鸡品系中存在有复制能力的内源性ALV,因而易干扰基于p27抗原的ELISA检测;本研究对于麻黄肉种鸡禽白血病的防控与净化有参考价值.

为了探讨不同接种途径和日龄对SPF鸡感染J亚群禽白血病病毒(ALV-J)后感染模式的影响,本研究分别经11日龄鸡胚尿囊腔、1日龄口服、点眼滴鼻、腿部肌肉注射、颈部皮下注射、腹腔注射和7日龄腹腔注射途径接种了ALV-J CHN06株,于不同时间点(第1、2、4、6周),分别对各试验组的病毒血症、抗体及排毒进行了动态检测.结果显示,11日龄鸡胚尿囊腔组100％(15/15)感染模式为V+S+.1日龄口服组和点眼滴鼻组感染模式以V-S-为主.1日龄颈部皮下注射组、腿部肌肉注射组和腹腔注射组在第4、6周的感染模式均以V+S+为主.7日龄腹腔注射组在试验期内仅呈现一过性病毒血症,而且7日龄感染组在第6周的抗体阳性率比1日龄感染组高,差异显著(P＜0.05).本研究结果表明,胚胎感染易导致持续性病毒血症,注射途径比非注射途径更易诱导病毒血症和排毒.随着感染日龄的增加,病毒血症持续时间缩短,排毒时间推后,抗体生成水平升高;防止ALV-J早期感染有利于ALV-J的防控和净化.

【目的】评估黄羽种鸡禽白血病的净化工作,比较源于同一鸡群不同样品对禽白血病病毒（ALV）检测结果的影响.【方法】采用ALV抗原ELISA方法对广东一黄羽种鸡场采集的2 691枚种蛋进行检测,根据检测结果采集其中70份不同S/P区间所对应的母鸡抗凝血,分离血浆后分别同时接种于CEF和DF-1细胞,用病毒分离方法进行检测,并用PCR方法进行亚群鉴定.【结果和结论】从送检种蛋蛋清共检出ALV p27阳性样品243份,阳性率为9.03%（243/2 691）;蛋清不同ELISA S/P区间所对应的病毒分离情况分别为:蛋清ELISA S/P≥2.0的鸡只其血样CEF和DF-1细胞病毒分离率均为100%;1.5≤S/P＜2.0的鸡只病毒分离率均为88.9%;1.0≤S/P＜1.5的鸡只病毒分离率均为85.7%;0.2≤S/P＜1.0的鸡只病毒分离率分别为60%~75%（CEF）和40%~50%（DF-1）;0.1≤S/P＜0.2的鸡只病毒分离率为62.5%（CEF）和50%（DF-1）;S/P＜0.1的鸡只分别为27.2%（CEF）和9.1%（DF-1）的病毒分离率.PCR结果显示,所有7份CEF+DF-1-的CEF培养物中有6份为内源性ALV-E.研究表明,在蛋清ELISA检测时,S/P越高的阳性蛋清样品其对应母鸡越可能是外源性ALV病毒血症阳性;有一定比例S/P较低的阳性蛋清样品是由对应母鸡体内的内源性ALV排毒所致;而净化初期少数蛋清S/P为阴性的蛋清,其对应母鸡仍可能检出外源性ALV,说明在黄羽种鸡外源性ALV净化方案中单独使用1次蛋清ELISA检测可能会给禽白血病的净化检测造成一定的"误诊"和"漏检".

【Objective】To investigate the prevalence of breeders infected by avian leukosis virus subgroup A( ALV-A) in Guangdong Province of China. 【Method】A total of 1 561 plasma samples of breeders collected from four different farms( A,B,C,D) were inoculated into DF-1 cell cultures for 7 days; the positive samples were detected by antigen p27 ELISA test and identified via PCR and IFA. 【Result and conclusion】Results showed that 71 positive samples were detected from four farms and the positive rate of exogenous ALV isolation was 4. 6%. Two ALV-A viruses were isolated from antigen p27 ELISA positive samples of the breeder chickens farm C,which were designed as GD13-1 and GD13-2,and the GD13-2was determined for mixed infection with ALV-J. The full length proviral genomes of two isolates were7 721,7 715 bp after PCR amplification and sequencing. In comparison to the LTR and gp85 sequenceof the other ALV-A strains at home and abroad,the isolates had the highest similarity with that of subgroup A strain SDAU09E2. The nucleotide similarity of LTR and gp85 were 96. 9%,97. 2% and98. 4%,98. 7% respectively. In comparison to the LTR and gp85 sequence of ALV-A GD08 isolated from Guangdong five years ago,the similarity of LTR and gp85 were 88. 9%,89. 5% and 98. 5%,98. 8% respectively. The survey results show that ALV-A still exist in some breeder farms,but it is not prevalent any more.

Suspected cases of hemorrhagic enteritis associated with hemorrhagic enteritis virus (HEV) are becoming more frequent among yellow chickens in the Guangdong Province of China. In this study, we have developed a one-step, ecumenical, real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay for the rapid diagnosis of HEV. The RealAmp assay was performed at 63°C and reduced the assay time to 15min, using a simple and portable device, the ESE-Quant Tube Scanner. The detection limit of DNA was 1fg/μl, and the detection was specific only to HEV. We also used nested PCR to evaluate the application of the RealAmp assay. The coincidence rate of the two methods was 100%. Our data indicated that the RealAmp assay provides a sensitive, specific, and user-friendly diagnostic tool for the identification and quantification of HEV for field diagnosis and in laboratory research.

To assess the effect of albumen samples in isolation of avian leukosis viruses(ALV),a total of 1673 albumen samples collected from a local breeder flock of Guangdong province were tested by ALV p27 antigen ELISA,the positive albumen samples were divided into five different S/P value intervals:A≥2.0,1.5≤B2.0,1.0≤C1.5,0.5≤D1.0,0.2≤E0.5,and inoculated into DF-1 cells and chicken embryo fibroblasts(CEF)cells respectively for virus isolation with a control S/P value interval group(F0.2). Twenty DF-1 p27 antigen-positive samples were further identified by PCR.Total of 107 positive samples were detected in this local breeder flock and the ALV p27 positive rate was 6.4%(107/1673). The virus isolation rate in different S/P value intervals were found as follows:both 100%(27/27)in A interval;80%(16/20,CEF)and 75%(15/20,DF-1 cells)in B interval;55.6%(10/18,CEF)and 33.3%(6/18,DF-1cells)in C interval;36.4%(8/22,CEF)and 9.1%(2/22,DF-1 cells)in D interval;5%(1/20,CEF)and 0%(0/20,DF-1cells)in E interval;8.3%(1/12,CEF)and 0%(0/12,DF-1 cells)in control group F interval. PCR results showed that20 samples were all ALV-J positive,and 6 of which were co-infected with ALV-A. The results showed that exogenous and endogenous ALV could be isolated from albumen samples;the higher the ALV p27 antigen ELISA S/P value,the more probable the exogenous ALV could be isolated;the relatively lower S/P value was likely due to endogenous ALV. Exogenous ALV-J still remained in this local breeder flock,reproductive endogenous ALV was also revealed.

ABSTRACT The integration of retroviruses into the host genome following nonrandom genome-wide patterns may lead to the deregulation of gene expression and oncogene activation near the integration sites. Slow-transforming retroviruses have been widely used to perform genetic screens for the identification of genes involved in cancer. To investigate the involvement of avian leukosis virus subgroup J (ALV-J) integration in myeloid leukosis (ML) in chickens, we utilized an ALV-J insertional identification platform based on hybrid capture target enrichment and next-generation sequencing (NGS). Using high-definition mapping of the viral integration sites in the chicken genome, 241 unique insertion sites were obtained from six different ALV-J-induced ML samples. On the basis of previous statistical definitions, MYC, TERT, and ZIC1 genes were identified as common insertion sites (CIS) of provirus integration in tumor cells; these three genes have previously been shown to be involved in the malignant transformation of different human cell types. Compared to control samples, the expression levels of all three CIS genes were significantly upregulated in chicken ML samples. Furthermore, they were frequently, but not in all field ML cases, deregulated at the mRNA level as a result of ALV-J infection. Our findings contribute to the understanding of the relationship between multipathotypes associated with ALV-J infection and the molecular background of tumorigenesis. IMPORTANCE ALV-Js have been successfully eradicated from chicken breeding flocks in the poultry industries of developed countries, and the control and eradication of ALV-J in China are now progressing steadily. To further study the pathogenesis of ALV-J infections, it will be necessary to elucidate the in vivo viral integration and tumorigenesis mechanism. In this study, 241 unique insertion sites were obtained from six different ALV-J-induced ML samples. In addition, MYC, TERT, and ZIC1 genes were identified as the CIS of ALV-J in tumor cells, which might be a putative “driver” for the activation of the oncogene. In addition, the CIS genes showed deregulated expression compared to nontumor samples. These results have potentially important implications for the mechanism of viral carcinogenesis.