安徽省五华鸡J亚群禽白血病的鉴定及其病理学观察

为调查安徽省五华鸡J亚群禽白血病(Avian leukosis virus subgroup J,ALV-J)的感染情况,采用ELISA对五华鸡进行P27抗原和ALV-J抗体检测.挑选5只抗原抗体阳性鸡进行PCR检测,同时将5只抗原抗体阳性鸡和5只抗原抗体阴性鸡进行剖检,制作病理切片.其中1只鸡PCR检测为阳性,能扩增出545 bp条带,PCR检测阳性的鸡其心脏有肿瘤、脾脏肿大等病理学变化;组织切片发现心脏、肝脏、脾、肾、肺等组织内有弥漫性髓细胞样瘤细胞或髓细胞瘤病灶,髓细胞样瘤细胞的细胞质内可见嗜酸性颗粒.结果表明五华鸡已经感染了ALV-J,且部分鸡个体已经发病.     

Acantholytic folliculitis and epidermitis associated with Staphylococcus hyicus in a line of white Leghorn laying chickens

Several mature Leghorn-type hens with the same genetic background experienced skin and feather problems in a breeder flock. There was almost-total feather loss on the head and neck, as well as thickened, scaly skin, and follicular ostia were plugged with keratin debris. Other individuals exhibited prominent subcutaneous nodules multifocally on the head. Histologic examination of the skin revealed a severe hyperplasia of follicular epithelium with hyperkeratosis and cystic dilation. Numerous clefts and vesicles were detected along the epidermis and follicular epithelium, some containing acantholytic keratinocytes. A mild heterophilic inflammation was associated with these lesions, and few gram-positive cocci were present in the keratin plugs. Bacterial culture of the skin yielded a variable amount of Staphylococcus hyicus. Immunochemistry looking for chicken IgY revealed no intercellular staining in the epidermis or follicular epithelium. All these findings supported a diagnosis of Staphylococcus-associated acantholytic epidermitis and folliculitis. This case suggests that S. hyicus could be a significant pathogen in poultry production. The close genetic relationship among affected individuals could indicate a hereditary predisposition in this line of White Leghorn laying chickens.     

鸡实验性淋巴细胞性白血病的病理学研究

给３５只１日龄伊莎褐蛋母鸡雏腹腔接种淋巴细胞性白血病病毒ＲＡＶ－１株，应用常规病理技术，对接毒后第１５天、１、２、３、４、５、６个月７个批次的实验鸡做了病理学研究。结果：接毒后１５ｄ和１个月，部分实验鸡发生了成髓细胞性白血病，主要表现为骨髓成髓细胞大量增生，或形成成髓细胞性肿瘤结节，肝、心、肾、法氏囊等内脏器官出现成髓细胞聚集；接毒后２～６个月，实验鸡发生了淋巴细胞性白血病，主要表现为法氏囊髓质淋巴细胞发生转化，成淋巴细胞克隆增殖形成成淋巴细胞克隆增殖灶，在肝、心、肾、脾、腺胃等器官中形成成淋巴细胞性肿瘤结节。据此，可对鸡淋巴细胞性白血病做出病理组织学诊断。     

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.     

Importance of the medullary macrophage in the replication of lymphoid leukosis virus in the bursa of Fabricius of chickens

Electron microscopy and immunocytochemistry were used to study the development of lymphoid leukosis virus infection in the bursa of Fabricius of experimentally infected chicken embryos and chickens. In embryos infected at 7 days of incubation and killed 10 days later, virus particles and group-specific viral antigen were confined mainly to the connective tissue of the lamina propria of the bursal mucosal folds; a few developing follicles had discrete virions and group-specific antigen between cells. In chickens infected at 1 day of age, infection (as determined by use of electron microscopy and immunocytochemistry) was maximal in 1- to 4-month-old birds, and the greatest concentration of virus and group-specific viral antigen was in the medulla of the follicles. Although lymphoid leukosis virus was released from lymphocytes, epithelial cells, and macrophages, virus replication in the medullary macrophages was more active than that in the other cells. Normal medullary macrophages had cell membrane vesicles (50 to 80 nm in diameter) that covered part of all of the cell membrane surface. In infected chickens, virus particles frequently developed within these vesicles. Comparable vesicles were not found on cortical macrophages. Results of the present study indicated that the medullary macrophage was the principal host cell for replication of lymphoid leukosis virus in the bursa of Fabricius of the chicken.     

Polymerase chain reaction for detection of avian leukosis virus subgroup J in feather pulp

Feather pulp from experimentally infected chickens was used as a source of DNA for polymerase chain reaction (PCR) amplification of avian leukosis virus subgroup J (ALV-J) proviral DNA. A primer set that produces a large amplicon (approximately 2,125) was used to detect ALV-J proviral DNA. This primer set was used in lieu of previously published primers because it allows for sequencing of the entire envelope gene and because it was able to detect diagnostically a number of North American ALV-J isolates that could not be detected with previously published primers and PCR conditions. ALV-J proviral DNA was detected in feather pulp at 7 days of age in more than 90% of birds infected as embryos and 7 days postinoculation in over 50% of chickens infected at 3 days of age. The results obtained with PCR on feather pulp were compared with those of virus isolation. In the embryo-inoculated birds, the percentages of agreement between PCR and virus isolation were 92.5% at 7 days of age and 100% at 28, 42, 49, and 56 days of age. However, the overall sensitivity of virus isolation in embryo-infected birds was higher, particularly at 7 and 56 days of age. In chickens inoculated at 3 days of age, the percentages of agreement of detection between PCR and virus isolation ranged from 75% at 10 days of age to 100% at 42 days of age. Agreement of negative results of ALV-J detection by PCR and virus isolation in chickens infected posthatch ranged between 66.6% and 100% between the ages of 10 and 42 days. Virus isolation requires chicken embryo fibroblasts of specific genetic lines, and the process takes onaverage 7-9 days. Aseptic collection of blood and tissues for virus isolation and molecular detection of ALV-J requires sterile necropsy instruments as well as syringes and needles for each individual chicken, whereas sterile microcentrifuge tubes and gloves are the only equipment necessary for aseptic feather pulp collection for ALV-J detection by PCR. PCR-based detection of ALV-J in feather pulp is especially suitable when ALV-J infection must be diagnosed rapidly and unequivocally without killing the chicken(s) and in situations where crucial reagents or suitable virus propagation substrates are not readily available for isolation and propagation of ALV-J in cell culture.     

散养淮南王土鸡J亚群禽白血病的诊断

对湖北省某农户散养的12周龄淮南王土鸡病鸡进行剖检,发现病鸡肝脏有白色肿瘤;心肌增厚,有疑似肿瘤赘生物;脾脏萎缩,切面呈暗色。用J亚群禽白血病病毒（ALV-J）特异性引物进行PCR检测,扩增出了约545 bp的条带,病鸡组织内含有ALV-J病毒核酸序列,表明该病例为ALV-J感染。     

Marek's disease virus-induced skin leukosis in scaleless chickens: tumor development in the absence of feather follicles

Marek's disease virus (MDV) is an oncogenic cell-associated herpesvirus that causes T-cell lymphoma in chickens. Lymphoproliferative neoplasms in Marek's disease (MD) occur in various organs and tissues, including the viscera, peripheral nerves, skin, gonads, and musculatures. MDV is restrictively produced in the feather follicle epithelial (FFE) cells, and it gains access to the external environment via infected cells or as infectious enveloped cell-free virus particles. The goals of the present study were to 1) determine whether the MDV-induced skin lesions are neoplastic in nature or inflammatory reactions to viral infection, 2) determine whether physical presence of feather follicles (FF) is necessary for skin tumor development, and 3) study the role of skin epithelial cells not associated with feathers or FF in the replication and dissemination of infectious virus particles. Scaleless chickens that produce only a few scattered feathers and no sculate scales along the anterior metatarsi were used as a unique model to study the pathogenesis of dermal lesions. Histologic and immunohistochemical analysis revealed that the cutaneous lesions were tumorous as was manifested by massive accumulation of lymphoblasts and extensive activation of meq oncoprotein, the hallmark of MDV oncogenesis, within the skin lesions. Neoplastic cutaneous lesions in the scaleless chickens indicate that feather follicles are not necessary for skin tumor development. Finally, our preliminary data indicate that inoculation with supernatant fluid from homogenized and sonicated skin samples of MDV-infected scaleless chickens induces MD in susceptible birds, suggesting that skin epithelial cells not associated with FF also harbor infectious viral particles.     

某海兰褐鸡场祖代、父母代及商品代鸡血管瘤型ALV-J的分离与鉴定

从山东省某海兰褐鸡场祖代、父母代种鸡和商品代蛋鸡中获得疑似血管瘤型禽白血病(Avian leukosis,AL)病料.采用病理剖检、IFA、分子生物学检测,确定为J亚群禽白血病.从祖代、父母代病料中各分离到1株J亚群禽白血病病毒(J subgroup of avian leukosis virus,ALV-J),从商品代蛋鸡中分离到4株ALV-J.根据原型毒株HPRS103设计1对gp85基因引物,获得gp85基因序列.获得的gp85基因序列与各亚群参考毒株序列核苷酸同源性比对,结果显示:分离自商品代蛋鸡的Commercial03株、Commercial04株、Commercial06株和父母代分离株Parent02株位于同一分支,同源性在97.2％～97.9％,与HPRS103株同源性94.7％～95.2％;Commercial05株与祖代分离株Grandparent01株在同一分支,与HPRS103株同源性为98.3％,4株分离自商品代的ALV-J同源性为95.0％～99.9％.表明商品代蛋鸡中的ALV-J可能来自父母代或祖代种鸡的垂直传播,也可能来自于其他来源的水平传播.从同一鸡场祖代、父母代及商品代鸡中分离得到ALV-J,这在我国还是首次.对后续研究其基因突变、致瘤机制等奠定了良好的基础.     

Pathology of spontaneous tumour lesions in pullets and adult chickens in commercial farms - Short communication

Twenty pullets and adult chickens, aged 100 to 403 days, from several commercial chicken farms were examined by gross and histopathology. Grossly, all chickens had white-greyish masses in the visceral organs with or without enlargement of the peripheral nerves. Histopathological examination revealed Marek's disease (MD) lymphoma, lymphoid leukosis (LL) and myeloid leukosis (ML) in 14/20, 5/20 and 1/20 of the chickens, respectively. Lesions of the sciatic nerves in chickens diagnosed as having MD lymphoma were various. No neoplastic and/or inflammatory cells were noted in the peripheral nerves of chickens diagnosed as having LL and ML. These results indicated that MD lymphoma could also develop in older chickens; thus, microscopic examination is needed to identify MD in older chickens showing lymphocyte-derived tumours.     

Testing and management of a specific pathogen free chicken flock with special reference to avian leukosis virus infections.

A specific pathogen free (SPF) chicken flock was reared in isolation under laboratory conditions during five years and continuously tested for presence of specified avian pathogens. The potential occurrence of avian leukosis virus (ALV) was most thoroughly examined. The RIF and neutralization tests were unequivocally negative. Radioimmunoassay was used for detecting the presence of the major protein (gs-a) of the group-specific antigen of avian onoorna viruses. This test seemed to he well suited for checking ALV infections in chicken flocks whereas the COFAL (complement fixation avian leukosis) test was considered unreliable for this purpose. Yolk and serum from SPF chickens were negative for anti-gs-a antibodies measured by the radioimmunoassay; immunized or naturally infected birds showed anti-gs-a amounts correlating with the neutralizing titre. Besides, the flock was regularly tested for presence of seven other contagious avian pathogens. There was no evidence of infection.SPF chicken flock; avian leukosis; laboratory diagnosis of avian leukosis virus infections.     

Heterophil function and resistance to staphylococcal challenge in broiler chickens naturally infected with avian leukosis virus subgroup J

Avian leukosis virus subgroup J has a high tropism for myeloid lineage cells and frequently induces neoplastic transformation of myelocytes. The impact of congenital avian leukosis virus subgroup J infection on the function of circulating heterophils and susceptibility to staphylococcal infection was investigated. Six-week-old broiler chickens negative for exogenous avian leukosis viruses or congenitally infected with avian leukosis virus subgroup J were inoculated intravenously with 10(6) colony-forming units of Staphylococcus aureus, and pre- and postinoculation heterophil function was assessed. All chickens developed a leukocytosis with heterophilia after inoculation, but total leukocyte and heterophil counts were significantly higher in leukosis-negative chickens than in virus-infected chickens. Tenosynovitis was more severe in leukosis-negative chickens, and 2/10 (20%) of the virus-infected chickens had no histologic evidence of tenosynovitis. Osteomyelitis in the tibiotarsus or tarsometatarsus developed in 5/10 (50%) of the chickens in each group. S. aureus was recovered from the hock joint of 6/10 (60%) of the chickens in each group. Heterophils from all chickens exhibited similar phagocytic ability pre- and postinoculation. Heterophils from virus-infected chickens exhibited less bactericidal ability preinoculation than did heterophils from leukosis-negative chickens. However, postinoculation bactericidal ability was similar in both groups. Avian leukosis virus subgroup J provirus was present in heterophils isolated from congenitally infected chickens. Heterophils isolated from broiler chickens congenitally infected with avian leukosis virus subgroup J exhibit no significant functional deficits, and infected and uninfected chickens exhibit similar susceptibility to staphylococcal infection.     

用ALV-J gp85单克隆抗体证明蛋鸡存在J亚群禽白血病

采用免疫组化法，对病理学初步诊断为蛋用型鸡J亚群白血病的自然发病鸡的肿瘤、骨髓、肝脏、脾脏、肾脏、肺脏、心脏、胰脏、输卵管、卵巢、腺胃、骨骼肌、大脑、坐骨神经，用特异性抗J亚群禽白血病病毒(ALV—J)囊膜糖蛋白gp85的单克隆抗体进行检测，待检的组织切片中均检出阳性抗原，免疫组化的研究结果与病理学诊断结果相一致。在国内外首次发现并报道蛋用型鸡J亚群禽白血病的自然病例。     

乌蒙凤鸡肉用专门化品系选育初探

本研究旨在对不同类型乌蒙凤鸡的生长性能进行测定分析,为乌蒙凤鸡肉用专门化品系的选育奠定基础。实验测定了乌蒙凤鸡不同羽色(黄羽、麻羽)、肤色(乌骨、普通)及不同羽色与肤色的组合(黄羽普通、黄羽乌骨、麻羽普通、麻羽乌骨)4个类型鸡群4～24周龄的生长性能。结果表明:按羽色或肤色的单一性状进行划分,乌蒙凤鸡黄羽和麻羽、普通和乌骨之间生长性能差异不显著;黄羽公鸡4～16周龄生长性能显著高于麻羽公鸡;不同羽色和肤色的公鸡生长性能均高于母鸡(P<0.01);黄羽普通鸡群体生长性能高于其他类型鸡群。根据分析测定结果,建议将黄羽普通鸡作为乌蒙凤鸡肉用专门化品系选育的候选类型。     

Molecular and biological characterization of a naturally occurring recombinant subgroup B avian leukosis virus with a subgroup J-like long terminal repeat

Infection of broiler chickens with subgroup J avian leukosis virus (ALV) results in the induction of myeloid tumors. However, although egg-type chickens are susceptible to infection with ALV-J, the tumor incidence is very low, and on rare occasions the tumors observed are of the myeloid lineage. We recently described the isolation of an ALV (AF115-4) from commercial egg-type chickens suffering from myeloid leukosis. AF115-4 was initially identified as an ALV-J isolate based on PCR analysis of the long terminal repeat (LTR). However, further characterization of the viral envelope indicated that the virus is recombinant with subgroups B envelope and J LTR. Here we further characterize this recombinant virus at both the molecular and biological levels. We show that the AF115-4 isolate expresses a recombinant envelope glycoprotein encoded by a subgroup B gp85 region and a subgroup E gp37 region. The host range ofAF115-4 was analyzed using cells resistant to infection by subgroups A/B, J, or E; this shows that no ALV-J was present in the isolates obtained from the affected chickens. Additional antigenic characterization of AF115-4 using chicken sera specific for subgroups B or J indicated that no ALV-J was present in the samples examined. Inoculation of AF 115-4 into ALV-susceptible 1515 X 71 chickens resulted in the induction of lymphoid leukosis but not the expected myeloid leukosis affecting the commercial chickens. These results suggest that differences in the genetic makeup of the chickens from which AF115-4 was isolated and the line 1515 X 71 used in the present experiments may be responsible for the observed differences in pathogenicity. In addition, the results suggest that ALV-J continues to evolve by recombination, generating new viruses with different pathological properties.     

J亚群禽白血病病毒的免疫荧光检测效果

应用特异性抗J亚群禽白血病病毒（ALV－J）的单克隆抗体JE9建立了免疫荧光检测ALV－J抗原和抗体的方法。结果表明，在ALV－JADOL－Hcl感染鸡胚成纤维细胞（CFF）24小时后，可以用1FA检测出阳性细胞，感染72小时后，可以检测出最小病毒感染量≥1．25TCID50／孔。对人工感染ALV－J鸡肾脏冰冻切片检测结果证明，免疫荧光法可以检测出组织中的ALV－J抗原，表现强阳性反应。用重组病毒rBac4817－env感染的Sf9细胞作抗原，可以检测出鸡血清中抗ALV－J的特异性抗体。该方法在ALV－J的控制中必将产生重要作用。     

商品代肉鸡J亚群禽白血病的病理及病毒分离鉴定

通过对山东某肉鸡养殖场发病的商品肉鸡组织病理学检查和将病料接种鸡胚成纤维细胞 (CEF) ,我们在国内首次从商品代肉鸡中分离到了 J亚群禽白血病病毒 (AL V- J)。从组织切片中可以看到肝脏、脾脏等器官的髓细胞瘤细胞 ,呈散在或成簇 ,髓细胞瘤细胞的细胞质内显现嗜酸性颗粒。用抗 AL V- J囊膜糖蛋白的单克隆抗体进行的 IFA中 ,病料接种 CEF后进行的 IFA呈现强阳性     

On epizootiology and control of lymphoid leukosis in chickens

Sera and organ extracts from ten different commercial stocks of layer chickens were examined for the presence of lymphoid leukosis (LL) viruses. Virus was recovered from 40.8% of the cockerels between three and six weeks of age. Their female hatch mates were examined at the age of 20 months. A mean of 11.3% of these laying hens was positive in the NP activation test. Lymphoid leukosis was successfully controlled in three inbred strains of White Leghorn chickens and in a commercial White Plymouth Rock line. All flocks were kept in a filtered air positive pressure (FAPP) house during the first two months of life and thereafter transferred to a conventional environment. The control method is based on three elements:

• —from an infected flock, hens are selected in whose eggs no avian lymphoid leukosis viruses can be detected by examination of pooled extracts of groups of embryos;

• —only eggs from hens that are shown not to shed congenitally virus in their eggs are used for the production of progeny. The offspring are reared in isolation until two months of age at which time the age-related resistance against tumour formation appears to be sufficiently developed;

• —the chickens are subsequently intramuscularly inoculated with lymphoid leukosis viruses of subgroups A and B and transferred to a conventional chicken house. The inoculated birds become persistently viremic and resist horizontal virus exposure and intramuscular challenge infections.

Horizontal virus transmission was observed to take place when virus-free non-vaccinated chickens were reared in isolation for two months and then exposed under field conditions.Efficiency of virus recovery was considerably improved when washed buffy coat cells were cocultivated with chick embryo fibroblasts or explant cultures were prepared from various tissues before testing with the NP activation test.     

鸡传染性支气管炎病毒LH2/01/10的分离鉴定

2001年在我国黑龙江省某鸡场疑似鸡传染性支气管炎的发病鸡群中，分离到一株病毒，将该病毒接种10日龄SPF鸡胚，取72h的尿囊液进行电镜观察并用1％胰酶处理，结果发现尿囊液中存在典型的冠状病毒，用胰酶处理过的尿囊液能凝集SPF鸡的红细胞，初步鉴定为鸡的传染性支气管炎病毒。将该病毒尿囊液再次接种10日龄SPF’鸡胚，通过病毒对鸡胚的致病作用、病毒超微形态特征以及病毒凝集鸡红细胞的特性等对该毒株进行研究，结果表明：经胰酶处理后的病毒尿囊液可凝集鸡红细胞。鸡胚的第二代病毒尿囊液(命名为LH2,／01／10)分别接种1日龄和15日龄的SPF鸡，发现对不同日龄的鸡表现不同的致病性，对1日龄接种鸡和同笼饲养的同居对照鸡致病力高，发病率为11／11，致死率分别为4／6和2／5；15日龄接种和同居感染鸡发病率为9／9，致死率分别为1／5和1／4。实验应用反转录一聚合酶链式反应技术对LH2／01／10的膜蛋白基因进行扩增、克隆和序列测定，结果表明该基因具有IBVM基因的共有分子特征．与IBV标准株M41的M基因核苷酸的同源性为90％，氨基酸的同源性为91％。这从分子水平进一步证实引起鸡群死亡的病毒为鸡传染性支气管炎病毒。     

Functional role of type VI collagen during early feather development of the chicken embryo in vitro

The regulatory function of type VI collagen during early feather development in embryonic chickens was investigated at the cellular and organ levels. Immunohistochemical studies of embryonic chicken skin showed that type VI collagen was distributed in spatial‐specific and temporal‐specific manners related to early feather development. To clarify the role of type VI collagen, we studied the feather development in intact, reconstituted and reconstituted gel skin cultures. When ethyl‐3,4‐dihydroxybenzoate (EDHB) was added to the medium of intact skin as an inhibitor of type VI collagen synthesis, the feather buds did not elongate and the number of neural cell adhesion molecule (NCAM)‐positive cells was reduced. However, the magnitudes of both suppressive effects of EDHB were reduced by the addition of liquid type VI collagen. Similar improvement was also observed in the reconstituted skin with liquid type VI collagen and in the reconstituted gel skin with solid type VI collagen at a low concentration. Moreover, type VI collagen promoted feather bud development in the absence of EDHB. However, a high concentration of solid type VI collagen in the reconstituted gel skin arrested the feather bud elongation, and antitype VI collagen antibodies caused feather buds to become longer and smaller in the reconstituted skin. At the cellular level, type VI collagen affected the proliferation, migration and NCAM expression of mesenchymal cells. These results suggest that type VI collagen regulates early feather development by controlling mesenchymal cell behavior.