ALV-J和REV诱导雏鸡胸腺细胞凋亡

应用原位末端标记法和HE染色法对人工感染J亚群禽白血病病毒(ALV-J)和禽网状内皮增生症病毒(REV)的SPF雏鸡胸腺细胞的凋亡情况进行了检测,同时辅以电镜超薄切片观察。结果表明,ALV-J和REV均可诱导雏鸡胸腺细胞发生凋亡,混合感染诱导的细胞凋亡更加严重;切片中可出现局灶状凋亡,凋亡细胞多于坏死细胞。研究结果表明,细胞凋亡是导致感染鸡胸腺萎缩的主要原因。     

鸡胚和雏鸡接种ALV-J相关急性纤维肉瘤浸出液的致病性比较

为了比较鸡胚接种ALV-J相关急性纤维肉瘤浸出液对胚体及雏鸡的致病性,将含ALV-J相关病毒的肉瘤浸出液分别经5日龄胚卵黄囊、11日龄胚绒毛尿囊膜、1日龄雏鸡腹腔接种,比较不同接种方式对SPF鸡胚及雏鸡的致病性.结果表明,5日龄卵黄囊接种的鸡胚在18～22日龄死胚率为14/30,肿瘤发生率为8/14; 11日龄绒毛尿囊膜接种的鸡胚在18～22日龄引起鸡胚死亡率为17/30,肿瘤发生率为6/17.对雏鸡的致病性比较表明,绒毛尿囊膜接种的13只出壳雏鸡全部死亡,有11只出现肿瘤.结果提示,绒毛尿囊膜接种的致病性不仅高于卵黄囊接种,也高于1日龄雏鸡接种.绒毛尿囊膜接种不仅肿瘤发生率高,且发生得更早、更快,可作为这种急性纤维肉瘤进一步作人工造病的实验模型.     

应用PCR从AA肉种鸡鸡胚和1日龄雏鸡体内检测出CIAV和ALV-J

为了了解鸡传染性贫血病病毒（CIAV）和J亚群禽白血病病毒（ALV-J）对AA肉种鸡鸡胚和1日龄雏鸡的感染情况,试验直接从山东省3个不同AA肉种鸡场的鸡胚和1日龄雏鸡的肝、脾、肾、胸腺、骨髓、法氏囊等组织（器官）提取DNA,进行了PCR扩增及PCR产物的克隆和序列测定。结果显示,被检的3个肉种鸡场的鸡胚和1日龄雏鸡体内均有这2种病毒核酸的检出,其中CIAV的阳性率是20.42%,ALV-J的阳性率是15.83%,二者共感染的阳性率是6.25%;在阳性检出率中弱雏〉死胚〉健康雏〉正常胚。病毒核酸在各感染组织（器官）的含量也有所差异,CIAV以脾的含量最多,ALV-J以肾的含量最多。对肝进行细菌分离鉴定时发现,3个鸡场还存在大肠杆菌、沙门氏菌和霉形体的混合感染。结果提示,在AA肉种鸡鸡胚和雏鸡体内存在CIAV、ALV-J的感染和二者的共感染以及继发性大肠杆菌、沙门氏菌和霉形体的混合感染。     

REV和ALV-J共感染鸡病毒血症及抗体反应的相互影响

为了研究禽网状内皮增生病痛毒（REV）、禽J亚群白血病病毒（ALV—J）共感染时对彼此病毒血症及抗体反应的影响，2日龄SPF鸡分别接种REV、ALV—J或同时2种病毒造成共感染，对各感染组7周内的病毒血症及抗体反应进行动态检测。相对于单一感染，共感染时，2种病毒的病毒血症水平动态均无明显变化。在单独感染ALV—J后5周有30％（5／16）的鸡产生抗体，而有REV共感染时，没有鸡产生针对ALV—J的抗体（o／16），表明REV感染可明显抑制鸡体对ALV—J抗体的反应，而REV与ALV—J共感染对REV抗体反应无明显影响。     

鸡感染J亚群禽白血病病毒的免疫抑制机理

通过人工接种建立了雏鸡感染J亚群禽白血病病毒（ALV-J）后发生免疫抑制的病理模型。对免疫抑制的机理进行了初步研究。结果表明：ALV-J感染早期可诱导胸腺、法氏囊的淋巴细胞凋亡，这种早期的淋巴细胞凋亡是胸腺和法氏囊萎缩的重要原因之一。病理组织学动态观察表明。ALV-J主要引起骨髓的髓系细胞灶状或弥漫性增生，病变导致骨髓机能受损，使机体免疫机能下降。是引起免疫抑制的根本原因。病毒感染组免疫器官均发生了严重的实质萎缩性病变．这种病变的发生除与骨髓的病变及淋巴细胞凋亡有关外。还与中后期淋巴细胞的坏死有关．从而导致严重的免疫抑制。ALV-J感染可引起免疫器官及部分内脏器官中嗜酸性粒细胞样的瘤细胞浸润增生，这可以作为病毒感染早期的病理诊断指标。     

禽白血病病毒J亚群(ALV-J)的攻毒试验

将禽白血病病毒 J亚群 (AL V- J)内蒙株 NM876 1和 NM9996人工接种于 1日龄爱维茵肉种鸡 ,通过眼观、病理组织学检查观察了攻毒鸡的病理学特征 ;通过 PCR和 EL ISA技术检测了病毒感染率、抗体变化规律以及病毒和抗体的相关性。结果显示 ,攻毒鸡从第 3周开始即可检出病毒 ,NM876 1株和 NM9996株病毒感染率分别为 71.4 %和6 4 .3% ;从第 5周开始 ,出现较明显的病理学变化 ,病变特征以骨髓、肝脏、心脏、脾、卵巢等组织内髓细胞增生为主 ,而法氏囊、脑、坐骨神经无变化 ;攻毒鸡抗体消长变化有一定的规律 ,一般在 7～ 8周龄和 18～ 2 0周龄时分别出现 1次高峰 ,而在 4～ 6周龄、10周龄和 2 3周龄分别出现 1次低谷 ,提示鸡场进行 EL ISA检测时要避开这一时期 ;攻毒鸡产生耐受性病毒血症 ,即病毒阳性而抗体阴性 (V A- )的比例较高 (7/14 ,9/14 )。通过以上的研究证明 ,AL V- J内蒙株疾病模型复制成功 ,NM876 1、NM9996可作为原型株进行相关研究     

应用组织芯片免疫组化法检测ALV-J

禽白血病J亚群（ALV-J）是英国的Payne和他的同事们在20世纪90年代初从肉鸡中分离出来的新亚群，主要引起肉鸡的骨髓瘤白血病。自1999年，我国一些肉用型种鸡场陆续发生了禽白血病J亚群。并且近几年来，ALV-J已从最初只引起肉种鸡发病开始向蛋鸡及中国地方种鸡蔓延。组织芯片技术是一种新型特殊的生物芯片技术，它能明显提高工作效率，减少实验误差。本研究将组织芯片技术和免疫组化染色结合起来，用特异性抗ALV-J囊膜蛋白gp85的单克隆抗体来检测发病鸡只的各组织器官的组织切片。在肝脏、脾脏、肾脏、卵巢、腺胃、骨髓、髓细胞瘤组织均检出病毒阳性抗原。结果表明组织芯片技术和免疫组化染色相结合为临床诊断ALV-J提供了一个高通量、敏感的检测方法。     

ALV-J不同接种途径诱导SPF鸡免疫抑制状况的比较分析

将1日龄SPF鸡分别通过口服、点眼、腹腔注射、肌肉注射的方式感染J亚群禽白血病病毒(subgroup J avian leucosis virus,ALV-J),监测各感染组鸡的生长性能、带毒和排毒规律,并对其病毒血症、相关免疫学指标及抗体反应进行动态检测。结果显示:各感染组鸡的生长性能和免疫应答水平存在明显差异。从感染后第2周开始腹腔注射和肌肉注射组可检出泄殖腔排毒和病毒血症,此后持续带毒、排毒;体质量、免疫器官指数以及淋巴细胞转化率、CD4+和CD8+T淋巴细胞数、细胞因子(IL-2、IL-4和IFN-γ)的分泌量显著低于对照组;抗体转阳不明显。而口服、点眼感染组与对照组相比引起的免疫抑制程度并不显著。结果表明:1日龄雏鸡感染ALV-J容易造成免疫抑制,其中腹腔注射和肌肉注射感染造成鸡体带毒、排毒和免疫抑制的能力最强。本试验明确了鸡群ALV-J水平传播的不同途径与鸡的感染力、免疫抑制力之间的关系,为研究ALV-J的感染机制、种群净化以及防控提供了理论依据。     

蛋种鸡ALV-J与REV共感染病变新特点

2009年8月,山西榆次某鸡场蛋种鸡群产蛋量急剧下降,部分鸡产蛋停止.病理组织学观察发现,病鸡肝脏、肾脏、肺脏、心肌等组织中出现小灶状淋巴细胞瘤,肿瘤细胞为成熟的淋巴细胞;聚合酶链式反应(PCR)、酶联免疫吸附试验(ELISA)和间接免疫荧光(IFA)检测显示,被检鸡ALV-J感染率为8/8,而且7/8为病毒血症,与REV共感染率为2/8,未出现REV单感染;从发病情况看,淋巴细胞瘤均出现于共感染病例,特别是ALV-J病毒血症的共感染鸡病变最为严重,但未见ALV-J的特征性髓细胞瘤.囊膜蛋白氨基酸同源性分析显示,ALV-J毒株与英国HPRS-103原型株同源性最高(96.9％以上),共感染的REV毒株与suv-env株同源性最高(93.8％).本病例揭示,先天传播的ALV-J引起的病毒血症状态可能为REV的致瘤创造了环境,使临床罕见的REV淋巴瘤早于髓细胞瘤出现,因此,严格种鸡群的ALV-J净化是防控此病的重中之重.对于ALV-J与REV协同的机制还需进一步研究.     

ALV-J诱发鸡成红细胞白血病的临床病例分析

2010—2011年检测3批病例,分别来自泰安新泰的110日龄麻鸡、济宁泗水的90日龄麻鸡和济南20日龄蛋雏鸡。病鸡均表现消瘦、精神萎靡、嗜睡、鸡冠稍苍白或发绀等,死亡率分别为20%、12%和18%。剖检,3批病鸡均可见肝脏、脾脏及肾脏严重肿大,胸腺、肌肉、腺胃等组织器官出血。新泰病鸡肝脏和肺脏表面有明显的白色肿瘤结节。血液涂片与骨髓涂片观察发现大量红细胞转变为体积较大,胞质丰富蓝染,胞核大呈圆形、核内有很纤细的染色质、核周围有空泡的成红细胞,以晚幼成红细胞为主;组织学检查发现,在所有组织脏器血管及间质内均可观察到大量聚集的与血涂片中形态相一致的成红细胞,并且可观察到核分裂相,但未见淋巴细胞聚集。通过血液学和组织学观察不仅排除马立克氏病和网状内皮组织增生症,而且排除中毒及代谢性疾病的可能。以肝脏组织DNA为模板,在3批病鸡中利用PCR检测均扩增出J亚群禽白血病病毒(ALV-J)gp85基因924bp的特异性片段,而A亚群禽白血病病毒(ALV-A)、B亚群禽白血病病毒(ALV-B)阴性。免疫组织化学检测结果表明,发病鸡肺脏和脾脏等含血量丰富的组织内发现组织细胞及成红细胞胞质呈ALV-J抗原阳性。根据以上检测结果确定此3批患有成红细胞白血病病鸡均由ALV-J感染引起。ALV-J引起单纯鸡成红细胞白血病在国内为首次发现。ALV-J在我国鸡群中的多潜能致瘤机制需要进一步的研究。     

J亚型禽白血病病毒的分离与鉴定

本研究分别从广西的地方肉鸡及河北、辽宁的蛋鸡中分离到了3株禽白血病病毒.剖检疑似发病鸡只.采集病变的肝脏、脾脏组织,经过RT-PCR检测确定为ALV感染.将病变组织接种CEF细胞,连续传代2次,将细胞上清接种DF-1细胞,p27抗原检测为阳性.同时提取病毒基因组,用H5/ADI和H5/H7两对特异性引物进行PCR扩增,结果3株为ALV-J亚型.进一步设计ALV-J亚型gp85特异性引物进行PCR扩增并测序.结果确认分离到的3株病毒为ALV-J亚型.其gp85序列与标准毒株HPRS-103同源性为96%.同时,用p27单抗进行间接免疫荧光试验,测定毒力.综上所述,我们从广西地方肉鸡巾分离到1株J亚型禽白血病病毒,从河北及辽宁的蛋鸡中分离到2株J亚型禽白血病病毒.     

Myotropic avian leukosis virus subgroup J infection in a chicken

The study describes a highly productive myotropic avian leukosis virus infection (ALV) in a 3-month-old female chicken. At necropsy, ascites, hepatic fibrosis and cardiomegaly were seen. Histologically, the most striking lesion was the presence of cytoplasmic basophilic inclusions in myocardial fibers. Immunostaining for ALV group specific antigen p27 revealed a diffuse presence of virus antigen in cardiac myofibers, in smooth muscle fibers of most of the organs, and in rare, pancreatic and ovarian theca cells. Ultrastructurally, myocardial inclusions consisted of clusters of 50-60 nm round particles with interspersed ribosome-like granules. Numerous C-type particles were found in intercellular spaces of ALV p27 positive tissues. PCR analyses revealed the presence of both ALV-E and ALV-J related sequences. In chicken genome, ALV-E is usually present as endogenous provirus therefore, the pathological findings observed in this case are considered to be related with the ALV-J infection. The results of this report further confirm that ALV-J may be responsible for highly productive myotropic infections.     

Tissue tropism and bursal transformation ability of subgroup J avian leukosis virus in White Leghorn chickens

In Experiment 1, a monoclonal antibody against the envelope glycoprotein (gp85) of subgroup J avian leukosis virus (ALV-J) was used to study the distribution of ALV-J in various tissues of White Leghorn chickens inoculated as embryos with the strain ADOL-Hcl of ALV-J. At 2 and 6 wk of age, various tissues from infected and control uninfected chickens were tested for the presence of ALV-J gp85 by immunohistochemistry. In Experiment 2, using the methyl green-pyronine (MGP) stain, sections of bursa of Fabricius (BF) from chickens of line 15I5 x 7(1), inoculated with ALV-J or Rous-associated virus-1 (RAV-1), a subgroup A ALV, at hatch were examined for transformation of bursal follicles at 4 and 10 wk of age. In Experiment 1, specific staining indicative of the presence of ALV-J gp85 was noted at both 2 and 6 wk of age in the adrenal gland, bursa, gonads, heart, kidney, liver, bone marrow, nerve, pancreas, proventriculus, spleen, and thymus. In Experiment 2, by 10 wk of age, transformed bursal follicles were detected in MGP-stained sections of BF in only one of five (20%) chickens inoculated with ALV-J at hatch, compared with five of five (100%) chickens inoculated with RAV-1. The data demonstrate distribution of ALV-J gp85 in various tissues of White Leghorn chickens experimentally inoculated as embryos with the virus. The data also confirm our previous observation that ALV-J is capable of inducing transformation of bursal follicles, albeit the incidence is less frequent than that induced by subgroup A ALV.     

Response of white leghorn chickens to infection with avian leukosis virus subgroup J and infectious bursal disease virus

The effects of viral-induced immunosuppression on the infectious status (viremia and antibody) and shedding of avian leukosis virus (ALV) were studied. Experimental white leghorn chickens were inoculated with ALV subgroup J (ALV-J) and infectious bursal disease virus (IBDV) at day of hatch with the ALV-J ADOL prototype strain Hcl, the Lukert strain of IBDV, or both. Appropriate groups were exposed a second time with the Lukert strain at 2 wk of age. Serum samples were collected at 2 and 4 wk of age for IBDV antibody detection. Samples for ALV-J viremia, antibody detection, and cloacal shedding were collected at 4, 10, 18, and 30 wk of age. The experiment was terminated at 30 wk of age, and birds were necropsied and examined grossly for tumor development. Neoplasias detected included hemangiomas, bile duct carcinoma, and anaplastic sarcoma of the nerve. Control birds and IBDV-infected birds were negative for ALV-J-induced viremia, antibodies, and cloacal shedding throughout experiment. By 10 wk, ALV-J-infected groups began to develop antibodies to ALV-J. However, at 18 wk the incidence of virus isolation increased in both groups, with a simultaneous decrease in antibody levels. At 30 wk, 97% of birds in the ALV-J group were virus positive and 41% were antibody positive. In the ALV-J/IDBV group, 96% of the birds were virus positive at 30 wk, and 27% had antibodies to ALV-J. In this study, infection with a mild classic strain of IBDV did not influence ALV-J infection or antibody production.     

实时荧光定量RT-PCR检测禽白血病病毒J亚群

将禽白血病病毒J亚群(ALV-J)NX0101毒株接种1日龄和7日龄SPF雏鸡并设阴性对照组,采用实时荧光定量RT-PCR方法,定期检测病毒在体内的复制情况。根据GenBank发表的ALV-Jenv基因保守序列(AY897227)设计1对特异性引物扩增目的基因;根据鸡的3-磷酸甘油醛脱氢酶(GAPDH)基因序列(K01458),在保守区内设计1对引物扩增内参照基因,分别克隆入质粒作为标准品制作标准曲线,采用SYBR GreenⅠ染料建立荧光定量PCR法,并对方法的特异性、敏感性和重复性进行评价。结果显示,标准曲线的Ct值与标准品浓度的对数值之间存在线性关系;最低每个反应可检测到60个拷贝的病毒数,比常规PCR灵敏度高1 000倍。检测结果分别采用绝对定量法和相对定量法进行分析,都达到了良好的效果。通过对病毒含量变化的检测发现,在雏鸡4周龄时,2个接毒组ALV-J病毒突然呈对数式增长。据此分析ALV-J病毒在体内经过3~4周潜伏后,突然呈暴发式增长,这种情况可能和临床表现的免疫抑制直接相关。结果表明,本试验建立了一种特异性强、敏感性高、可定量分析ALV-J病毒增殖的方法,为进一步相关研究奠定了基础。     

重庆市禽白血病及禽网状内皮组织增生症的血清流行病学调查

禽白血病和禽网状内皮组织增生症均为禽的肿瘤性免疫抑制疾病,是危害养鸡业的两种非常重要的病毒性传染病。为调查重庆市肉鸡中禽白血病及禽网状内皮组织增生症的流行情况,在北碚区、涪陵区、开县、垫江县、潼南县五个区（县）的8个活禽交易市场采集260份血清样本,采用酶联免疫吸附试验检测了所有血清中禽白血病病毒（ALV）和禽网状内皮组织增生病毒（REV）的抗体。检测结果显示：禽白血病病毒和禽网状内皮组织增生病毒抗体阳性率分别为16.5%（43/260）和5%（13/260）,双抗体阳性率为4.61%（12/260）。与全国其他地区相比,重庆市肉鸡中这两种病原的感染率相对要低,但仍应重视这两种疾病的防控,以控制病原的进一步传播。     

The effects of avian leukosis virus subgroup J on broiler chicken performance and response to vaccination

The effects of avian leukosis virus subgroup J (ALV-J) infection on meat-type chickens reared in a simulated commercial setting were evaluated. Each of three ALV-J isolates was evaluated with both simulated horizontal transmission (SHT) and simulated vertical transmission (SVT). Mortality, morbidity, disease condemnations, and feed conversions were increased and body weights at processing were decreased in ALV-J infected birds as compared to sham inoculated hatch mates. The adverse effects of ALV-J infection were more severe in birds exposed by SVT than in birds exposed by SHT. At 8 weeks of age response to vaccination for infectious bronchitis virus and Newcastle disease virus or prior exposure to a pathogenic reovirus was assessed in the ALV-J and sham inoculated broiler chickens by challenge studies. Although not statistically significant, an overall trend of decreased protection to challenge after vaccination, or prior exposure, was observed in the ALV-J inoculates as compared to sham inoculated hatch mates. Differences in vaccine response were most evident in groups inoculated with ALV-J by the SVT route.     

Haemangiomas, leiomyosarcoma and myeloma caused by subgroup J avian leukosis virus in a commercial layer flock

An outbreak of simultaneously occurring haemangiomas, leiomyosarcoma and myeloma was observed in a commercial layer flock in China. The sick chickens were extremely thin and dehydrated. Scattered haemangiomas were found on the claws, breast and wings. At necropsy, haemangiomas and some other nodular tumours were also found in the internal organs. In addition, diffuse enlargement of the liver and spleen appeared in some birds. Histopathologically, haemangiomas were typically cavernous haemangiomas and haemangioendothelioma. In the diffusely swollen liver and spleen, multifocal or widespread marrow tumour cells filled with ball-like acidophilic particles in cytosol were observed, which are the characteristic pathological changes of avian myelocytomatosis. The nodular tumour cells formed by muscle bundles were of variable size, irregular shape, poorly differentiated and malaligned. Immunohistochemistry for vimentin, cytokeratin, actin (smooth muscle) and actin (sarcomeric) and Masson's staining confirmed the different cell lineage of the nodular tumour, thus leading to the diagnosis of leiomyosarcoma. The seroprevalence of avian leukosis subgroup J (ALV-J) antibodies was 13.46% (7/52), while ALV-A/B and reticuloendotheliosis virus (REV) antibodies were not detectable. The DF-1 cells inoculated by virus extracted from liver samples from 24 infected chickens were cultured and the group-specific antigen (GSA) was identified by ELISA. All samples were positive for ALV, which was further identified as ALV-J by immunofluorescence assay (IFA). PCR analysis revealed that three isolates of ALV-J proviral sequence were close to the HPRS-103 prototype strain and other Chinese field strains isolated in recent years, while one isolate (DP01) had a lower homology with them. This is the first report that ALV-J infection caused the simultaneous occurrence of haemangiomas, leiomyosarcoma and myeloma in a commercial layer flock.     

禽白血病病毒J亚群诱导CD117^+chB6^+前体B细胞克隆无能引起机体免疫耐受

禽白血病病毒J亚群(ALV-J)属于反转录病毒科α-反转录病毒属的第10个亚群,由英国学者Payne于1991年分离于肉鸡,之后迅速向全世界传播,在1997年~1998年,ALV-J诱导的髓细胞瘤病在欧美肉种鸡群全面爆发,造成了 1/3肉种鸡的损失,有些地区甚至到了无鸡可养的地步。ALV-J垂直传播和水平传播能力均高于其它亚群,其中垂直传播是其扩散的主要途径,病毒经由卵进入子代后,可引起机体产生4种状态,即病毒阳性抗体阴性(V+A)、病毒阳性抗体阳性(V+A+)、病毒阴性抗体阳性(V+A+)和病毒阴性抗体阴性(V-A-),其中V+A-是感染鸡群的主要状态.