Distribution and function of non-lymphoid cells positive for monoclonal antibody CVI-ChNL-68.2 in healthy chickens and those infected with Marek's disease virus

Immuno-enzyme histochemistry was used to study the staining pattern and tissue distribution of monoclonal antibody CVI-ChNL-68.2 that specifically reacts with a subset of non-lymphoid cells in healthy chickens and those infected with Marek's disease virus (MDV). Functional characteristics of CVI-ChNL-68.2-positive cells, e.g. antigen uptake, are determined. In the liver CVI-ChNL-68.2 recognizes reticulum cells, whereas in the bursa of Fabricius it detects single cells in the interfollicular connective tissue. In the spleen CVI-ChNL-68.2 reacts selectively with the reticulum cells of the ellipsoid. In some MDV-infected chickens the splenic reticulum cells show a different staining and distribution pattern. Furthermore, the proliferative lesions associated with Marek's disease contain many CVI-ChNL-68.2-positive cells. The possible role of CVI-ChNL-68.2-positive cells in disseminating Marek's disease virus is discussed.     

Comparative pathogenesis studies with oncogenic and nononcogenic Marek's disease viruses and turkey herpesvirus.

An apparently nononcogenic Marek's disease virus (SB-1) and turkey herpesvirus could be readily isolated from spleen, bursa of Fabricius, thymus, and peripheral blood lymphocytes of chickens beginning 4 to 6 days after inoculation, but unlike infections with two isolates of oncogenic Marek's disease virus (JM-10 and CU-2), virus replication in these cells was rare, and necrosis in the organs was essentially absent. Splenic enlargement was observed regularly during the first 4 to 11 days after inoculation, and Marek's disease tumor-associated surface antigen was observed on splenic and other lymphocytes in the four viral inoculation groups. Cellular cytotoxicity of splenic lymphocytes was demonstrated in vitro with cultured Marek's disease tumor cells (MSB-1 lymphoblastoid cell line) as the target in a chromium-release assay. The four viral infections induced sensitized lymphocytes.     

Cyclophosphamide-induced amelioration of Marek's disease in Marek's disease-susceptible chickens.

Bursa- and thymus-dependent functions were examined in Marek's disease (MD)-susceptible normal chickens and in chickens treated with 5 and 16 mg of cyclophosphamide (CY) at the time of hatching. Chickens not exposed to Marek's disease virus (MDV) and treated with CY temporarily lost mitogenic response to concanavalin A but regained full response after 5 weeks. Bursa-dependent functions, such as presence of germinal centers in spleen and cecal tonsils, morphologic features of bursa, and sheep red blood cell antibody response were completely lost in chickens treated with 16 mg of CY and only partly retained in chickens treated with 5 mg of CY. In chickens exposed to MDV, the degree of thymus-dependent spleen cell mitogenic response was directly related to frequency and severity of MD. Chickens treated with 16 mg of CY had a mild mitogenic depression and low frequency and severity of MD lesions, whereas those treated with 5 mg of CY and those not treated had marked mitogenic depression and high frequency and severity of MD. Suppressions of bursa- and thymus-dependent functions by MDV alone were also evident when comparing MDV-exposed and nonexposed chickens. The results also indicate that presence of small, residual amounts of humoral factor(s) may enhance MDV oncogenesis.     

鸡血液激素水平与抗病力相关性研究

本试验以ＡＡ肉鸡父母代和商品代为试验材料，对其血浆中皮质酮、三碘甲腺原氨酸（Ｔ３）、甲状腺素（Ｔ４）和生长激素（ＧＨ）进行了测定，并对部分鸡进行马立克强毒接种，以探讨血浆激素与抗病力关系。结果表明，７周龄不同基因型鸡间存在明显差异。血液Ｔ淋巴细胞活性在商品代明显高于父母代。马立克强毒接种后血浆高、低皮质酮水平间Ｔ淋巴细胞活性有明显差异。相关估计结果表明，血浆激素水平大多与Ｔ淋巴细胞呈低正相关，而皮质酮与Ｔ淋巴细胞呈负相关（Ｐ＜０．０５）。本研究结果指出，激素受环境因素影响较大，应用于抗病育种仍需进一步研究。     

In situ detection by monoclonal antibody D-35.1 of cells infected with Marek's disease virus that interact with splenic ellipsoid-associated reticulum cells

Immuno- and enzyme-histochemical staining procedures were used to investigate in vivo the interaction of Marek's disease virus (MDV) with splenic non-lymphoid cells. The newly developed monoclonal antibody D-35.1, which recognizes all three MDV serotypes, was used to study the localization of MDV at various times after intramuscular inoculation of 1-day-old chicks with MDV strain K. The D-35.1-positive cells were detected in the bursa of Fabricius, spleen, thymus, proventriculus, and cecal tonsils, and the number of chickens showing the cells increased between days 4 and 10. From day 21, the skin of the chickens contained D-35.1-positive feather follicles. The D-35.1 monoclonal antibody did not stain any cells in peripheral blood, nerves, kidney, and gonads at any time. In addition, D-35.1-positive cells were not detected in lymphoproliferative lesions in visceral organs and peripheral nerves. Double staining procedures on serial sections using monoclonal antibody CVI-ChNL-68.2, specific for splenic ellipsoid-associated reticulum cells, revealed that the majority of D-35.1-positive cells were situated in the peri-capillary sheath of reticulum cells at day 10. The sheath of cells detected by monoclonal antibody CVI-ChNL-68.2 was disrupted, and they were clustered around D-35.1-positive cells. These results support the hypothesis that ellipsoid-associated reticulum cells are involved in the early pathogenesis of Marek's disease.     

A TRANSMISSIBLE CHICKEN TUMOUR ASSOCIATED WITH RETICULOENDOTHELIOSIS VIRUS INFECTION

Histiocytic lymphosarcomas of the intestine, liver, spleen and sciatic nerve were found at necropsy in a 36-week-old laying hen that was culled from a flock of 1800 birds because of emaciation. Type C particles were observed in ultrathin sections of liver and spleen. The serum of the hen contained reticuloendotheliosis virus (REV) antigen, and antibody against REV, but lacked antibodies reactive with Marek's disease virus or subgroups A and B of Rous sarcoma virus. The tumour was transmitted to chickens using a suspension of the initial tumours. These experimental tumours were then transmitted to further chickens, using cultured spleen cells, viable spleen cells that had been stored frozen, and disrupted spleen cells. The tumours, which developed after incubation periods as short as 2 weeks, were histologically similar to those in the original hen. A few chickens also developed feather abnormalities. The chickens with experimentally transmitted tumours developed antibody against REV and REV antigen was demonstrated in cultured cells from these chickens. The chickens failed to develop antibody against Rous sarcoma virus and only 1 of 29 developed antibody against Marek's disease virus.     

Embryo vaccination with infectious bursal disease virus alone or in combination with Marek's disease vaccine

Studies with specific-pathogen-free chickens revealed that chicks hatching from eggs inoculated at the 18th day of embryonation with infectious bursal disease (IBD) vaccine viruses of low virulence (isolates TC-IBDV and BVM-IBDV) developed antibody against IBD virus (IBDV) and resisted challenge with virulent IBDV at 3 weeks of age or older. Embryo vaccination did not adversely affect hatchability of chicks or survival of hatched chicks. Chicks embryonally vaccinated with TC-IBDV had transient histologic lesions in the bursa of Fabricius at hatch. Similar but milder lesions were also noted in chickens that received TC-IBDV at hatch. The level of protection following embryo vaccination with TC-IBDV and BVM-IBDV was similar to that following vaccination with the same vaccines at hatch. Vaccine viruses of moderate virulence (isolates BV-IBDV and 2512-IBDV) were not suitable as vaccines in embryos lacking maternal antibody to IBDV, because the vaccinated chicks developed acute IBD after hatch. Isolate 2512-IBDV was not pathogenic for embryos bearing maternal antibody to IBDV. Maternal antibody against IBDV interfered with efficacy of embryo vaccination with BVM-IBDV but not with 2512-IBDV. Embryo vaccination with a mixture of vaccines against IBD and Marek's disease resulted in protection of hatched chicks against challenge with virulent IBDV and Marek's disease virus.     

Efficacy of a bivalent vaccine against Marek's disease

A bivalent vaccine was prepared by combining inactivated Marek's disease virus and turkey herpesvirus. The efficacy of this vaccine, compared to turkey herpesvirus and inactivated Marek's disease virus separately, was studied in unsexed White Leghorn chicks which were vaccinated at one day old and then challenged at 21 days old with fowl blood infected with virulent Marek's disease virus. The bivalent vaccine appreciably delayed mortality resulting from Marek's disease and elicited the highest protective efficacy as judged on the basis of Marek's disease-specific mortality and percentage occurrence of lesions. The occurrence, extent and severity of gross lymphomas and microscopic lymphoproliferative lesions in various organs of the bivalent vaccinated birds were less than in the other challenged groups. In addition, the level of viraemia remained consistently and significantly lower in the bivalent vaccinated birds.     

雏鸡感染马立克氏病病毒后免疫器官组织抗体生成细胞的变化

以马立克氏病病毒（ＭＤＶ）感染１日龄肉用雏鸡，在感染后５、２５、４５ｄ采取法氏囊、脾脏、盲肠扁桃体和哈德尔腺，用彩色免疫金银染色法检查免疫器官组织中ＩｇＧ、ＩｇＭ和ＩｇＡ抗体生成细胞数量的动态变化。结果：ＭＤＶ感染雏鸡的法氏囊、脾脏和哈德尔腺中以ＩｇＧ抗体生成细胞居多，ＩｇＧ、ＩｇＭ和ＩｇＡ抗体生成细胞均较正常对照鸡显著减少；盲肠扁桃体中以ＩｇＡ抗体生成细胞居多，ＩｇＡ、ＩｇＧ和ＩｇＭ抗体生成细胞数量显著低于正常对照鸡。由此表明，ＭＤＶ感染鸡全身免疫器官和消化道、呼吸道局部粘膜体液免疫机能明显抑制。     

人参皂苷及其衍生物抗马立克病病毒作用的超微结构和病理变化观察

为了探讨人参皂苷及其衍生物抗马立克氏病病毒的作用机制.采用马立克氏病毒感染的雏鸡作为模型,人参皂苷及其衍生物口服给药途径,动态观察不同时间段,试验鸡的器官病理变化和脾脏、法氏囊的组织超微结构改变.结果发现人参皂苷及其衍生物组的器官病变程度明显低于病毒对照组,法氏囊和脾脏的超微结构病变轻于病毒对照组,说明人参皂苷及其衍生物在体内可以通过保护机体免疫器官来发挥抗病毒作用.     

INFECTION STUDIES ON A RETICULOENDOTHELIOSIS VIRUS CONTAMINANT OF A COMMERCIAL MAREK''S DISEASE VACCINE

Reticuloendotheliosis virus (REV) was isolated in cell cultures from commercial Marek's disease (herpesvirus of turkeys) vaccine and re-isolated from the organs of vaccinated chickens. Runting and feathering abnormalities were produced when 1-day-old specific pathogen free chickens were inoculated with REV. Histopathological lesions in infected chickens were hypoplasia of the thymus, bursa and spleen, and inflammation of the proventriculus, kidneys and liver. Serological responses to REV were detected by the indirect immunoflorescence test in chickens directly inoculated with contaminated vaccine, and spread of REV infection to in-contact chickens was demonstrated by histopathological and serological investigations.     

Isolation of very virulent Marek''s disease viruses from vaccinated chickens in Australia

Very virulent Marek's disease viruses (vvMDV), defined as isolates against which the herpesvirus of turkey (HVT) vaccine provide poor protection, have been isolated from poultry flocks in both the United States and Europe. Twenty-one samples from vaccinated Australian flocks, experiencing problems with excessive Marek's disease (MD), were tested for the presence of transmissible MD viruses (MDV). Of the 16 samples which contained a transmissible agent, 14 were pathogenic in chickens, based on the development of MD lesions or depression of the bursa/body weight ratio. Of the pathogenic isolates which have been successfully typed 10 were serotype 1, and one was serotype 2 MDV. Pathogenicity of isolates varied. Several isolates caused tumours in 20-30% of both vaccinated and unvaccinated chickens. Two isolates, MPF6 and MPF23, caused tumours in more than 50% of chickens. When MPF6 and MPF23 were tested in vaccine trials bivalent vaccine gave no better protection against development of MD lesions than a monovalent vaccine. Isolate MPF23 was so pathogenic that lesions were produced in all chickens, regardless of the vaccine protocol used. Therefore vvMDV have been isolated in Australia, and unlike the vaccines tested overseas, bivalent Australian vaccines do not appear to provide greater protection against these vvMDV.     

The occurrence of Marek's disease in genetically different groups of chickens

The occurrence of Marek's disease was studied under laboratory conditions in four genetically different groups of chickens (Brown Leghorn, F1 hybrids of the CB x IA inbred lines, pullets of the paternal branch of the grandparent stock of the Hybro meat type, and final hybrids of the White Hisex layer type) after infection with the Georgia strain of Marek's disease (MD) virus, used in two doses (1600 and 16,000 PFU per one bird). MD was diagnosed on the basis of the occurrence of macroscopic tumours; when these tumours were absent in birds which had died within 105 days from infection, the dead bodies were subjected to microscopic examination (peripheral nerves, gonads, skin, bursa of Fabricius, thymus, spleen). The size of the parenterally administered dose of the virus had no significant effect on mortality, occurrence of tumours and the MD virus in any of the groups of chickens tested. However, there was a time shift in the mortality curve in chickens infected with a lower dose. The significantly highest occurrence of MD was recorded in BrL chickens (100%). A somewhat lower occurrence of MD was recorded in the Hisex White chickens (87.8%) and in the CB x IA hybrids (73.8%). However, the dead CB x IA chickens had a higher occurrence of tumours (96.6%) than the Hisex White chickens (77.1%). The lowest MD occurrence was recorded in the pullets of the Hybro meat type (25.5%). The organ most frequently affected by tumours after infection of the birds with the Georgia strain was the liver (24.1%).     

Mortality of one-week-old chickens during naturally occurring Marek's disease virus infection

Marek's disease (MD) is a disease of chickens that occurs worldwide and has serious economic consequences. MD can present as one of several forms, with the most commonly occurring forms being the lymphoproliferative diseases. Under experimental conditions, an early mortality syndrome has been recognized following infection by some but not all strains of MD virus (MDV). This is the first report of a confirmed case of mortality due to naturally occurring MDV infection in 1-week-old, nonvaccinated, chickens. Necrotizing lesions were observed in the bursa of Fabricius, lung, duodenum, jejunum, and proventriculus, and large intranuclear inclusion bodies were a striking feature in tissues with lesions in all birds. Immunohistochemical staining for the pp38 protein of MDV revealed abundant pp38 antigen in the affected tissues, confirming the presence of MDV within the lesions. PCR yielded an amplicon with 97% homology to the meq gene of MDV. No evidence of co-infection by either of the immunosuppressive agents chicken anemia virus and infectious bursal disease virus was detected.     

Marek's disease in Japanese quails (Coturnix coturnix japonica): a study of natural cases

Marek's disease was observed in quails. Gross lesions were confined mostly to the spleen and liver. Microscopic lesions were commonly seen in spleen, proventriculus, liver, and duodenum. Skin, peripheral nerves, and other visceral organs were also involved. Of 123 quails examined, 39 had serum antibodies against Marek's disease. These antibodies were detected from 11 to 17 weeks of age; the highest incidence was recorded at 15 weeks. Feather follicular antigen detected in 30 of the 95 quails was comparable to that of chicken. The disease was experimentally reproduced in susceptible quails. Marek's-disease-tumor-associated surface antigens (MATSA) were demonstrated in the peripheral leukocytes and spleen cells of affected quails. The possible source of infection and its epidemiological importance are discussed.     

Tumor histogenesis and macrophage content in lymphomas in Marek's disease of chickens

Histological and to some extent additional enzyme-histochemical studies were conducted into 28 cases of Marek's disease with minor, moderate, and intensive lymphoma formation. The investigations were undertaken with a view to differentiating T-lymphocytes and macrophages by differentiated activity on acid unspecific alpha naphthylacetate esterase. Lymphoma specimens were cytologically evaluated under semi-quantitative or quantitative aspects. Evidence was provided to the existence of correlations between intensity of Marek's disease lymphomas and their relative contribution to lymphocytes, lymphoblasts, and macrophages. The amount of lymphoblasts was found to go up along with advancing lymphoma proliferation, while the amounts of lymphocytes and macrophages went down. Moderately pronounced lymphomas contained 16 percent macrophages on average, whereas only about 8 percent were recordable from highly pronounced lymphomas. The high presence of macrophages in less developed lymphomas is considered to reflect unspecific defence reaction against neoplastic lymphatic cells in the phase of low tumour malignancy.     

Histological evaluation of immune organs in chicken embryos inoculated with Marek's disease virus and lymphokines

The aim of the present study was to evaluate the presence of lymphocytes and granulocytes in different stages of embryonic development and on the first posthatching day. The lymphocytes present in the bursa of Fabricius and thymus were evaluated by histological analysis of the yolk sac, bursa of Fabricius, thymus, liver and bone marrow of 100 chicken embryos divided into groups and treated with: (I) Marek's disease vaccine as viral antigen, (II) Marek's disease vaccine plus lymphokines, (III) lymphokines, and (IV) vaccine diluent. Group V was not treated. Samples were taken on days 14, 17 and 20 of incubation and on the first posthatching day. An increase in the number of epithelial matrix as precursors of lymphoid follicles was observed in the bursa of Fabricius of embryos inoculated with lymphokines compared to embryos in all the other groups (p < 0.05). In addition, a higher amount of granulocytes was found in the yolk sac and liver of embryos inoculated with lymphokines than in the embryos of all other groups (p < 0.05). In the bone marrow, no significant difference was observed among the treated groups concerning the amount of granulocytes. The results suggest that administration of antigens or protein molecules at an early stage of embryonic development increases the presence of granulocytes in the liver and granulopoiesis in the yolk sac, and also increases the number of epithelial matrixs in the bursa of Fabricius.     

Infectious bursal disease virus infection in the quail-chicken hybrid

Hybrids produced from crossing Cornell K-strain white leghorn chickens and Line II Japanese quails were studied for susceptibility to infection with infectious bursal disease virus (IBDV). Quail-chicken hybrids were infected successfully following inoculation with IBDV at 14, 21, or 52 days of age. In most cases, precipitating antibodies were detected in serum by 10 days postinoculation (PI). Although no clinical signs or gross lesions were evident in the bursa of Fabricius of hybrids, histologic changes in the bursa were detected upon microscopic examination using hematoxylin and eosin staining. Chickens were successfully infected also; they had gross and microscopic lesions in the bursa and produced precipitating antibodies. In addition, staining of bursal sections with low concentrations of peroxidase-conjugated concanavalin A revealed a rearrangement of a leukocyte cell type (probably macrophages) in infected chickens and hybrids. Japanese quails were refractory to infection; they showed no bursal changes and did not form precipitating antibodies.     

Induction of specific cytotoxic T-cell activity against xenogeneic target cells in carp (Cyprinus carpio)

OBJECTIVE: To investigate the induction of cytotoxic T cells in carp (Cyprinus carpio) after inoculation of fish with 2 xenogeneic line cells and to examine specificity of the cytotoxic activity. ANIMALS: 22 carp. PROCEDURE: Fish were inoculated with mouse myeloma line cells P3.NS-1/1Ag4.1 (NS-1) or chicken Marek's disease tumor-derived lymphoma line cells (MDCC MSB-1). Cytotoxic activity of immune lymphocytes was evaluated by incubating effector cells with homologous and heterologous target cells. Populations of effector cells were identified by blocking T-lymphocytes from effector cells, using anti-carp T-cell monoclonal antibody and complement. RESULTS: Lymphocytes in blood, spleen, and head kidney of carp inoculated with NS-1 cells or MDCC MSB-1 cells had dose-dependent cytotoxic effects against homologous target cells but not against heterologous target cells. Lymphocytes from noninoculated carp did not have cytotoxic effects. Depletion of T-lymphocytes in spleen cells from NS-1-inoculated carp resulted in a decrease of cytotoxic activity against NS-1 cells. Cytotoxic activity of spleen lymphocytes from NS-1-inoculated or noninoculated carp was not evident when cytotoxic tests were performed after addition of anti-NS-1 carp serum. CONCLUSIONS AND CLINICAL RELEVANCE: Inoculation with xenogeneic target cells induces a specific cytotoxic T-cell response in carp. Thus, cell-mediated immunity plays a role in defense against infection of parasitic organisms such as protozoa and helminths.     

雏鸡马立克氏病强毒感染后脂质过氧化物的含量变化

１日龄雏鸡马立克氏病强毒（ｖＭＤＶ）人工感染马立克氏病（ＭＤ）发病率６２％,ＭＤ死亡率３４％;脾脏、胸腺、法氏囊、心脏、肝脏、肾脏、性腺脂质氧化物（ＬＰＯ）含量高于（Ｐ＞０．０５）或显著高于健康对照雏鸡（Ｐ＜０．０５）。