Effect of Different Test Materials on the Detected Results of Avian Leukosis Virus-p27 Antigen by ELISA

In order to study the effect of different test materials on the detected results of avian leukosis virus (ALV)-p27 antigen by ELISA, the cloacal swabs ALV-p27 antigen were detected by ELISA in 180-day local breeds hen, and the parts of the positive and negative chicken was selected to group test, the serum, egg whites and cell supernatant ALV-p27 antigen were detected by ELISA, the specific genes of ALV were detected by RT-PCR in blood.The results showed that serum, egg white and cell supernatant as ELISA test materials, the positive rate lower of than cloacal swabs, and serum ALV-p27 positive samples include all egg whites and cell supernatant positive samples in positive group.It was a significant correlation between ELISAs with serum and cell supernatant (linear equation:Y=1.8439X-0.1469, the correl was 0.937).In positive group, ALV-p27 gene positive rate lower than cloacal swabs ELISA, but higher than the serum, egg and cell culture medium, and ALV-p27 gene positive samples include serum positive samples by ELISA.ALV-J gp85 gene positive rate of 29.17%, and all positive samples were included in the serum ALV-p27 positive samples.The results suggested that the cloacal swabs as test material may occur false positive results, and egg whites and cell supernatant may occur undetected by ELISA ALV-p27 antigen assay in adult chicken, serum as test material in ELISA could more accurately reflect the state of adult chickens infected with ALV.     

不同检测材料对ELISA检测禽白血病病毒-p27抗原结果的影响

为探讨不同检测材料对ELISA检测禽白血病病毒(ALV)-p27抗原结果的影响,试验首先对180日龄地方品种母鸡的泄殖腔棉拭子ALV-p27抗原进行ELISA检测,选取部分阳性鸡和阴性鸡进行分组试验,采用ELISA对试验鸡血清、蛋清和病毒分离的细胞培养液进行ALV-p27抗原检测,采用RT-PCR方法检测血液ALV特异性基因。结果显示,阳性组中以血清、蛋清和细胞培养液作为ELISA ALV-p27抗原检测材料,其检测阳性率均低于泄殖腔棉拭子,血清检测的阳性个体包含全部蛋清和细胞培养液ELISA检测的阳性个体。ELISA检测数据的相关性分析显示,只有血清和细胞培养液检测数据间存在显著性相关,线性关系方程为Y(细胞上清)=1.8439X(血清)-0.1469,R²=0.937;阳性组中ALV-p27基因检测阳性率低于泄殖腔棉拭子,但高于血清、蛋清和细胞培养液,其包含所有血清ELISA检测的阳性样品;外源性ALV-J gp85基因阳性率仅为29.17%,且阳性样品均属于血清ELISA阳性样品。综上所述,成年鸡以泄殖腔棉拭子作为ELISA ALV-p27抗原检测材料存在假阳性结果,蛋清和细胞培养液作为检测材料存在漏检的可能,血清作为ELISA检测材能够更准确地反映成年鸡群ALV感染状态。     

Replication-competent endogenous avian leukosis virus in commercial lines of meat chickens

Group-specific (gs)-antigen-positive egg albumen in seven commercial lines of meat chickens was found to result from the presence of endogenous avian leukosis virus (ALV); these lines had resisted selection attempts to reduce the shedding rate. In two meat lines, exogenous as well as endogenous ALV contributed to the gs-antigen shedding. All hens that produced gs-antigen-positive albumen transmitted endogenous ALV to a high proportion of their embryos (20 to 100%). Hens shedding gs-antigen to albumen were negative for endogenous ALV in vaginal swabs and had no detectable antibody to subgroup E virus. Chickens hatched from these dams were negative for endogenous ALV in meconia but were viremic at 2 weeks of age. Replication-competent endogenous ALV was almost uniformly expressed in embryos of hens from nine meat lines that were negative for gs-antigen in albumen. Shedding of gs-antigen to albumen was not related to the level of endogenous ALV expression. Embryos from five meat lines tested were resistant to infection with ALV of subgroup E. The level of endogenous gs-antigen in albumen was consistently lower than the level of exogenous gs-antigen.     

Congenital transmission of avian leukosis virus in the absence of detectable shedding of group specific antigen

Congenital transmission of avian leukosis virus (ALV) in the absence of detectable amounts of group specific (gs) antigen in egg albumen was found to occur in one commercial and one specific pathogen-free (SPF) flock. The prevalence of congenitally transmitting hens which did not excrete gs antigen was particularly high in a commercial flock where 26/27 hens transmitted ALV. Some of the ALV-transmitting hens in the commercial flock had virus in vaginal swabs thus enabling infection to be detected. The reasons for such a high proportion of congenitally transmitting hens which did not shed detectable amounts of gs antigen in the commercial flock was not determined. In the SPF flock, 2/15 hens congenitally transmitted ALV although virus could not be detected in vaginal swabs, whole blood or egg albumen and antibodies to subgroups A or B were not present. This form of ALV-infection persisted in two successive generations. These results indicate the necessity of testing for infectious ALV in embryos, in order to ascertain that a flock is genuinely free of ALV.     

龙岩市地方优良种禽原种场禽白血病p27抗原检测与分析

用酶联免疫吸附试验法对长汀河田鸡原种场、武平象洞鸡原种场、龙岩山麻鸭原种场共686份蛋清样本及801份泄殖腔棉拭子进行禽白血病p27抗原检测。结果为：龙岩山麻鸭的蛋清样本和泄殖腔棉拭子均未检出ALV核酸;鸡蛋清样本阳性率为12.8%（80/626）,明显高于泄殖腔棉拭子阳性率8.9%(66/741);鸡原种场180日龄蛋清样本和泄殖腔棉拭子阳性率均最高,平均阳性率分别为15.0%和11.3%。表明：龙岩市境内地方优良鸡原种场中有禽白血病感染现象。     

基于RNA-Seq筛选ALV-J感染汶上芦花鸡肝差异表达致病相关基因

为挖掘感染ALV-J汶上芦花鸡的肝差异表达致病相关基因。本研究以汶上芦花鸡为试验素材,分别在42和300日龄进行ALV血液病毒分离检测筛选ALV阴性和阳性个体,对ALV阳性个体有病理变化的肝组织和阴性个体肝组织进行PCR检测,分别选取3只ALV阳性（G1组）和阴性（G2组）个体的肝组织,并对G1组PCR产物测序、聚类分析确定ALV亚型,利用RNA-Seq测序技术筛选差异表达基因,并进行功能分析,利用荧光定量qRT-PCR对部分差异表达基因进行验证。结果表明,G1组样品经PCR检测、PCR扩增产物测序和聚类分析确定ALV为J亚型,转录组分析发现,共有42个差异基因在GO和KEGG中富集,其中,上调基因18个,下调基因24个。随机选取的5个差异表达基因qRT-PCR验证结果与RNA-Seq测序结果相一致。GO分析显示,差异表达基因主要涉及细胞过程、代谢过程、对刺激的反应、免疫系统等;KEGG分析显示,差异表达基因主要富集在细胞过程、信号传导、疾病、新陈代谢等信号通路。本研究通过转录组分析发现了影响ALV-J致病性的多个基因和关键信号通路,为深入了解ALV-J致病的分子机制提供了思路。     

Detection of avian leukosis virus antigens by the ELISA and its use for detecting infectious virus after cultivation of samples and partial characterization of specific pathogen-free chicken lines maintained in this laboratory.

An enzyme-linked immunosorbent assay (ELISA) for detecting avian leukosis virus (ALV) antigens was developed with rabbit anti-ALV serum. The ELISA detected purified ALV of subgroups A and B at a concentration of 0.4 ng/well and about 10(3) infectious units/well estimated by a resistance-inducing factor (RIF) test, and antigens in culture fluids from chicken embryo fibroblasts infected with subgroups A, B or E of ALV. These results showed that common antigens among the subgroups were detected by the ELISA. When virus titration was performed, virus infectivity could be determined by the ELISA within 7 days after cultivation. The titer was similar to that obtained by the RIF test on 19 days after 3 subcultures. These results indicate that the ALV-isolation test by the ELISA was superior to the RIF test in rapidity and applicability to large-scale field trials. Four specific pathogen-free (SPF) chicken lines maintained in this laboratory were examined for endogenous ALV antigens by the ELISA. Sera from laying hens had considerably high absorbance (A) values, whereas albumen samples showed low A values except for some samples (7/40 hens). Although most of sera from 1-day-old SPF chicks showed lower A values than those from laying hens, some sera showed A values as high as those from viremic chicks in 2 lines. Endogenous ALV was isolated from sera from laying hens (6/40) and their albumens (4/7) with high A values. Two SPF chicken lines were found to produce endogenous virus at a high frequency.     

禽白血病p27抗原检测阳性与病毒分离的相关性比较

本试验以80只300日龄的A品系蛋鸡为试验对象,分5个日龄段按翅号采集蛋清、泄殖腔棉拭子,无菌抗凝血和血清.用ALV p27抗原检测试剂盒检测蛋清和泄殖腔棉拭子.将无菌抗凝血分离血浆接种DF-1细胞,培养一周后用同样方法检测上清收集液,分析该群鸡只在不同日龄段泄殖腔棉拭子阳性、蛋清样本阳性和病毒分离阳性之间的相关性.用ALV-Ab抗体试剂盒检测各日龄段血清的抗体水平.此外,选取某一日龄段蛋清和泄殖腔拭子样本用4个不同厂家的ALV p27抗原检测试剂盒进行检测比较.结果表明,5个日龄段泄殖腔棉拭子平均阳性率为61％,蛋清样本平均阳性率为72.6％,病毒分离平均阳性率为48.8％.5个日龄段ALV的抗体阳性率一直为零;4个厂家的ELISA试剂盒对同一批样本的检测结果表明,IDEXX试剂盒的敏感度最高.本试验为外源性鸡白血病病毒检测及鸡白血病净化其方法的应用、试剂盒的选择、减少判定的误差、提高净化效果提供了一定的科学依据.     

种鸡场鸡白血病净化的研究(Ⅲ)-ALV检测不同样品种鸡场净化效果的比较

应用双抗体夹心酶联免疫吸附试验 (DAS- EL ISA)在种鸡白血病的净化中 ,针对同一种鸡群 ,同时采用蛋清和肛拭作为试验材料 ,实验反映的结果不同 ;将产第一枚蛋为阳性的鸡隔离饲养 ,检测其以后蛋中 AL V的状况 ,结果发现 :在随后的 2 5天左右的每一枚蛋清中都能检测到 AL V:在不同种鸡场分别采用蛋清、蛋清和肛拭同时作试验样品进行净化 ,跟踪结果发现 :在不同种鸡场中 ,AL V阳性感染率都有不同幅度的下降。另一方面 ,检测雏鸡的胎粪 ,淘汰阳性者 ,到其产蛋期抽检蛋清 ,该群鸡 AL V阳性率有所下降     

High virus titer in feather pulp of chickens infected with subgroup J avian leukosis virus

Subgroup J avian leukosis viruses (ALVs), which are a recombinant virus between exogenous and endogenous ALVs, can spread by either vertical or horizontal transmission. Exogenous and endogenous ALVs can be detected in feather pulp. In this study, virus titers in feather pulp of chickens infected with subgroup J ALV were compared with those of plasma and cloacal swab. All of the broiler chickens inoculated with subgroup J ALV at 1 day old were positive for virus from feather pulp during the experimental period of between 2 wk and 8 wk of age. Virus titers in feather pulp of some broiler chickens infected with subgroup J ALV were very high, ranging from 10(7) to 10(8) infective units per 0.2 ml. Virus titers in feather pulp were usually the highest among the samples of plasma, cloacal swab, and feather pulp tested. In another experiment in which layer chickens were inoculated with subgroup J ALV at 1 day old, virus was detected in feather pulp from 2 wk until 18 wk of age, and virus persisted longer in feather pulp than in plasma. Almost all of the layer chickens tested were positive for virus by polymerase chain reaction (PCR) with DNA extracted from feather pulp samples at 2, 4, and 10 wk of age, and the PCR from feather pulp was more sensitive than virus isolation from plasma, cloacal swab, and feather pulp. All above results indicate that samples of feather pulp can be useful for virus isolation and PCR to confirm subgroup J ALV infection.     

Avian leukosis virus (ALV) infection, shedding, and tumors in maternal ALV antibody-positive and -negative chickens exposed to virus at hatching

Chickens highly susceptible to avian leukosis virus (ALV) infection and tumors, with and without ALV subgroup A maternal antibody (MAB), were infected with a field strain of ALV subgroup A at hatching. Viremia, antibody development, cloacal and albumen shedding, and tumors in chickens with MAB (MAB+) were compared with those in chickens lacking MAB (MAB-). At 18 weeks of age, the incidence of viremia was significantly lower in MAB+ chickens than in MAB- chickens; further, MAB significantly reduced the proportion of tolerantly infected (viremic antibody-negative) chickens. Cloacal shedding of ALV at 22 weeks of age and shedding of ALV group-specific (gs) antigen in albumen of eggs from all laying hens at 30-32 weeks of age were significantly lower in MAB+ hens than in MAB- hens. The incidence of ALV-induced tumors was lower in MAB+ chickens than in MAB- chickens, significantly so in one of three trials conducted. These results suggest that MAB may influence the development of viremia, antibody, and shedding of ALV following massive exposure to virus at hatching.     

Effects of chemically or virus-induced immunodepression on response of chickens to avian leukosis virus

The effects of chemically or virus-induced immunodepression on the infection profile (development of viremia and antibody) and shedding of avian leukosis virus (ALV) were studied in progeny chickens of experimental or commercial breeder flocks. Chickens were infected with ALV subgroup A by contact at hatching and by oral inoculation at 4-5 weeks of age. In the first experiment, chickens were inoculated with a virulent strain of infectious bursal disease virus (IBDV) at 1 day or 6 weeks of age. In the second experiment, chickens were neonatally treated with cyclophosphamide (CY), or were inoculated with strain T of reticuloendotheliosis virus (REV) at hatching, or were inoculated with strain JM of Marek's disease virus (MDV) at 2 weeks of age. The infection profile and cloacal shedding of ALV in chickens exposed to ALV and inoculated with immunodepressive viruses or CY were compared with those in hatchmates exposed only to ALV. In two of four chicken lines tested in the first experiment, shedding of ALV, as determined by virological assays of cloacal swabs at 22 weeks of age, was significantly higher in chickens infected with IBDV at 1 day of age than in uninfected hatchmates. The rate of shedding of ALV in one of these two lines was also significantly higher in chickens infected with IBDV at 6 weeks of age than in uninfected chickens. Further, the frequency of ALV-antibody detection at 22 weeks of age was significantly lower in chickens of these two lines infected with IBDV at 1 day of age than in uninfected chickens. In the second experiment, neonatal treatment with CY significantly increased the frequency of viremic chickens of both experimental and commercial flocks. The frequency of ALV-viremic chickens at 22 weeks of age was considerably higher in the REV- and MDV-inoculated groups (54% and 44%, respectively) than in control hatchmates (29%), but only in chickens of the commercial line. These findings suggest that chemically or virus-induced immunodepression may lead to an increase in rates of viremia and shedding of ALV in chickens infected with virus after hatching, especially in certain genetic lines.     

Influence of maternal antibody on avian leukosis virus infection in White Leghorn chickens harboring endogenous virus-21 (EV21).

Slow-feathering (SF) white leghorn dams harboring the endogenous viral gene ev21, which encodes for complete endogenous virus-21 (EV21), and rapid-feathering (RF) dams lacking EV21 were immunized with a live field strain of avian leukosis virus (ALV) subgroup A. One group of SF dams and one group of RF dams were not immunized and were maintained to produce chicks lacking maternal ALV antibody. When the SF dams were crossed with line 15B1 males, the resulting male progeny were SF, EV21-positive, and the females were RF, lacking EV21 or congenitally infected with EV21. EV21-positive and -negative progeny of immunized and unimmunized SF and RF dams were exposed to ALV at hatching. Viremia, antibody development, cloacal shedding, and tumors in chickens lacking EV21 were compared with those in chickens with EV21. Congenital transmission of EV21 from SF dams to RF female chicks was significantly higher in immunized dams than in unimmunized dams. Maternal ALV antibody delayed infection with ALV and reduced viremia and cloacal shedding of virus in progeny. The effect of maternal antibody on ALV infection was much more pronounced in progeny lacking EV21 than in progeny harboring EV21. The data suggest that the development of ALV infection and tumors may be influenced by status of infection with EV21 and by the immune status of dams.     

2009—2010年中国部分地区进口鸡群禽白血病流行病学调查

为了解禽白血病(avian leukemia, AL)在中国进口品种鸡群中的感染状况,2008年12月至2010年6月,在4个省市各选择1个进口品种鸡场进行禽白血病流行病学调查。此次调查共涉及4个场、5个品种、不同代次、不同生长阶段的115个鸡群共计7000余份样品,分别采集泄殖腔拭子进行p27抗原的检测和血清中的J抗体、A/B抗体的检测。结果表明,中国进口品种鸡群中存在不同程度的ALV-J亚群、ALV-A/B亚群感染,J抗体阳性率普遍高于A/B抗体阳性率,且肉鸡品种的J抗体阳性率高于蛋鸡品种;此外,本次调查结果还显示,产蛋初期的鸡群p27抗原阳性率和J抗体阳性率较高。     

地方品种HR土鸡不同亚型禽白血病病毒的共感染

为研究HR土鸡中存在的不同亚型禽白血病病毒(ALV)共感染的情况,本实验分别采集46只HR土公鸡的泄殖腔棉拭子和455枚鸡蛋卵白样品,采用ELISA试剂盒检测p27抗原;并采用相应的ELISA试剂盒分别检测卵黄中J亚型ALV(ALV-J)和AB亚型ALV(ALV-AB)抗体。结果表明:HR土公鸡泄殖腔棉拭子样品中p27检出阳性率为87%(40/46),卵白检出率为74.7%(340/455);而卵黄中ALV-J和ALV-AB抗体阳性率分别为0(0/30)和80%(24/30)。无菌采集初步筛选p27抗原检测为阳性的5只HR土公鸡的抗凝血接种CEF,采用抗ALV-J和ALV-A的单克隆抗体进行IFA检测,结果显示5份样品中ALV-A和ALV-J的阳性率均为100%(5/5)。同时选取HR土鸡分离株HR332进行PCR扩增鉴定,结果表明分离株HR332存在ALV-J(HR332J)和ALV-A(HR332A)。其中,HR332J与11株ALV-J国内外参考株的同源性为92.4%～97.9%;HR332A与ALV-A参考株RSA-A、MQNCSU的同源性分别为90.1%和89.7%,与国内分离株SDAU09E2的同源性为99.0%。本研究显示,地方品种HR土鸡存在不同亚型ALV共感染,同时ALV-A和ALV-J共感染同一个鸡的现象已经存在。     

Development of an endogenous virus-free line of chickens susceptible to all subgroups of avian leukosis virus

Primary chicken embryo fibroblasts (CEF) from special specific pathogen-free chicken lines are used for detection of contamination of adult or embryonic tissues, meconium, or tissue culture fluids with avian leukosis viruses (ALV). The suitability and efficiency of such tests depend on the susceptibility of CEF to the various subgroups of exogenous as well as endogenous ALV. The ideal CEF for such tests should be not only susceptible to all retroviruses, but also free of endogenous viruses so that such tests are immune to any interference that may occur between the endogenous and the tested (exogenous) viruses. CEF and/or chickens free of endogenous viruses are also desirable for gene transfer studies using retroviral vectors, such as RNA interference (RNAi) experiments and transgenic work. The absence of ev genes in CEF or chickens can empower clean detection of successful RNAi construct delivery or gene transfer. CEF free of ev genes are also essential reagents routinely used in growing and detecting unknown retroviruses in varied viral assays. This report documents the development of a new line of chickens, 0.TVB*S1, that is free of endogenous viruses and susceptible to all subgroups of ALV identified in chickens.     

Isolation and characterization of an adventitious avian leukosis virus isolated from commercial Marek's disease vaccines

Commercial Marek's disease (MD) vaccines produced by two manufacturers were tested for possible contamination with avian leukosis virus (ALV). Samples of MD vaccines manufactured by two companies (A and B) were received from a breeder company; samples were also received directly from vaccine company B. Using virus isolation tests, samples initially tested positive for subgroup E (endogenous) ALV. However, upon repassage, the vaccines also tested positive for exogenous ALV. The isolated exogenous ALV proved to be a subgroup A virus, as determined by flow cytometry using polyclonal chicken antibodies specific for various subgroups of ALV, and by DNA sequencing of the envelope glygoprotein (gp85). The exogenous ALV isolated from MD vaccines was inoculated in chickens from ADOL lines 15I(5) x 7(1) and 0 to determine its pathogenicity and compare it with that of Rous-associated-virus-1 (RAV-1), the prototype strain of ALV-A. Each chicken from each line was inoculated with approximately 10,000 infectious units of RAV-1 or the ALV-A isolated from vaccines termed B-39 virus at 7th day of embryonation. At hatch, and at 4, 8, and 16 wk of age, chickens were tested for viremia and cloacal shedding; chickens were also observed for ALV-induced tumors within 16 wk of age. Viremia and cloacal shedding results suggest that chickens from both lines were susceptible to infection with either virus. Within 16 wk of age, the proportion of ALV tumors induced by strain B-39 in line 0 and line 15I5 x 7(1) chickens was 0% and 12%, respectively, compared with 62% and 67% in chickens inoculated with RAV-1. The data indicate that commercial MD vaccines produced by two manufacturers were contaminated with endogenous subgroup E and an exogenous subgroup A ALV. Further, data from biological characterization suggest that the ALV-A isolated from commercial MD vaccines is of low oncogenicity, compared with that of RAV-1. GenBank accession numbers: The gp85 gene sequences of ALV isolated from commercial Marek's disease vaccines have been deposited in GenBank and assigned the following accession numbers: A46 subgroup A, DQ412726 ; B53 subgroup A, DQ412727; A46 subgroup E, DQ412728; B53 subgroup E, DQ412729.     

Expression of Enhanced Green Fluorescent Protein (EGFP) Gene Carried by a Lentiviral Vector RCASBP in DF-1 Cell

To construct a lentiviral vector RCASBP carrying the enhanced green fluorescent protein (EGFP) gene which could be expressed stably in DF-1 cell,EGFP gene was amplified by PCR and then inserted into the lentiviral vector RCASBP after digested with the restriction endonuclease ClaⅠto construct recombinant lentiviral vector RCASBP-EGFP.The recombinant vector was transfected into DF-1 cells by Lipofectamine^TM 2000.Avian leukosis virus (ALV) p27 antigen ELISA test was performed after four passages of the transfected cells and the positive results of ELISA suggested the success rescue of the virus.The expression of EGFP was observed in more than 80 percentages of DF-1 cells under fluorescence microscope.The proviral genome PCR showed EGFP gene carried by the recombinant lentiviral vector RCASBP-EGFP had been integrated into the genome of DF-1 cells.The RCASBP lentiviral-mediated expression system provided a basis for study of the structure and function of ALV genes.