Natural killer cell activity of chicken intraepithelial leukocytes against rotavirus-infected target cells

Intraepithelial leukocytes (IEL) and splenocytes collected from uninfected and rotavirus-infected chickens were evaluated for cytotoxic activity against a natural killer (NK) cell-susceptible lymphoblastoid cell line (LSCC-RP9) and against rotavirus-infected chick kidney cells in 4-h chromium-release assays. Both splenocytes and IELs from uninfected and rotavirus-infected chickens were cytotoxic for LSCC-RP9, and the levels of this NK cell activity were not altered by infection of the host with rotavirus. IELs but not splenocytes from uninfected and rotavirus-infected chickens were cytotoxic for rotavirus-infected but not for uninfected chick kidney cell targets. Because this cytotoxic activity was not induced nor altered by rotavirus infection of the host, and was not major histocompatibility complex-restricted, it was considered to be due to NK cell activity. The cytotoxicity of IELs against rotavirus-infected target cells was dose-dependent; however, there was some suppression of cytotoxic activity at high effector to target cell ratios. There were no differences in the cytotoxic activities of IELs collected from the duodenum versus the jejunum. The in vitro cytotoxic activity of IELs against rotavirus-infected target cells suggested that NK cell activity may be an important immune response to rotavirus infections in vivo. The absence of cytotoxic activity by splenocytes against rotavirus-infected target cells indicated that there may be different subpopulations of NK cells in the spleen and intestinal epithelium of chickens.     

Antibody directed against Marek's disease-associated tumor surface antigen can be eluted from Marek's disease tumor cells.

Antibody directed against Marek's disease-associated tumor surface antigen (MATSA) was eluted from tumor cells of lymphomas and peripheral blood lymphocytes that were isolated from Marek's disease virus-infected chickens. Feather follicular Marek's disease virus (MDV) antigen could not be demonstrated with this antibody by indirect immunofluorescent (IF) staining. Monoclonal antibody directed against MATSA could completely block the activity of eluted antibody and vice versa. By indirect IF staining using eluted antibody and fluorescein isothiocyanate (FITC) labelled antichicken globulin conjugate. MATSA-bearing cells were detected in MDV infected and herpes virus of turkey (HVT) vaccinated birds. Blocking of immunoglobulin molecules present on B-cells by anti-chicken globulin is critical in this test.     

Presence of adherent cytotoxic cells and non-adherent natural killer cells in progressive and regressive Marek's disease tumors

Marek's disease virus-induced progressive tumors and Marek's disease transplantable local regressive tumors were dispersed by treatment with collagenase and cells were examined in vitro for cytotoxic effector functions against target cells of tumor lines. Both types of tumors had adherent and non-adherent cytotoxic cells. The cytotoxicity of adherent cells was detected in an 18-hour but not in a 4-hour 51Cr-release assay. The adherent effector cells from progressive tumors were inactivated by pretreatment with carbonyl iron and carrageenan whereas the adherent effector cells from the regressive tumors were refractory to these treatments. In the progressive tumors, the 18-hour cytotoxic activity of cells of tumors and of spleens of tumor-bearing chickens was compared; the activity was higher in the tumor than in spleen. The nonadherent cell cytotoxicity detectable in a 4-hour 51Cr-release assay was associated with anti thymocyte serum-resistant natural killer cells. The incidence and levels of natural killer cell reactivity were greater in the regressive tumors than in the progressive tumors. In the regressive tumors, the natural cytotoxicity levels were higher in the tumor than in the spleen of tumor-bearing chickens. The differences in characteristics of adherent cytotoxic cells in virus-induced progressive tumors and transplantable regressive tumors and elevated levels of NK cells in regressive but not in progressive tumors may indicate a role for intratumoral immunity in tumor regression.     

Activation of natural killer cells in newborn piglets by interferon induction

Natural killer (NK) cell activity in the peripheral blood lymphocytes (PBL) of newborn piglets, normally negligible, was stimulated by in vitro treatment with porcine type I interferon (IFN), and the NK activity of PBL from weaned piglets was augmented by the same treatment. Binding of the PBL to the PK-15 targets used in the single cell cytotoxicity assay for NK activity was not affected by age or by IFN treatment. When newborn piglets were treated with a single intravenous dose at 2 days of age of 0.5 mg/kg of polyinosinic:polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose (poly ICLC), a synthetic IFN inducer, their IFN levels peaked at 6 h post-induction, and NK activity in their PBL peaked at 24 h post-induction at the level normally found in weaned piglets. The NK activity then declined until 7 days post-induction, when it increased again in a similar manner to that in untreated control piglets. Target-binding of the PBL was not affected by poly ICLC treatment of the piglets. Newborn piglets treated with poly ICLC and subsequently exposed to infection with transmissible gastroenteritis (TGE) virus showed a delay in onset of clinical signs of TGE compared with untreated control piglets. It was concluded that NK cells in newborn piglets can be activated by treatment of the piglets with poly ICLC, and that the presence of active NK cells is associated with some increase in resistance to challenge with TGE virus.     

Immunization against Marek's disease transplantable cell lines

Continuous passage of MDCC-RP1, a highly tumorigenic Marek's disease (MD) lymphoblastoid cell line, in cell culture resulted in a gradual loss in ability of the cell line to cause progressive tumors in susceptible day-old chicks. Inoculation of day-old chicks with high-cell-culture-passaged (187th to 417th) nontumorigenic MDCC-RP1 cells gave excellent protection against challenge at 8 days with low-passaged tumorigenic MDCC-RP1 cells but failed to protect against primary tumors caused by inoculation with MD virus. Vaccination with the herpesvirus of turkeys, on the other hand, protected the chickens well against primary tumors caused by MD virus and against transplantable tumors caused by tumorigenic MDCC-RP1 cells, but it did not protect as well against another MD lymphoblastoid cell line, MDCC-RP4. It is unlikely, therefore, that vaccines prepared from passaged MDCC-RP1 cell lines will have value for protecting chickens against MD in the field.     

Major histocompatibility complex class I is downregulated in Marek's disease virus infected chicken embryo fibroblasts and corrected by chicken interferon

The major histocompatibility complex (MHC) is a part of the immune system which presents epitopes of intracellular antigens on the cell surface. MHC molecules have receptor-ligand binding affinities with T lymphocytes, permitting the latter to detect foreign intracellular infectious agents. Some pathogens, such as herpesviruses, have developed strategies of evading the host response by MHC. This pressure on the immune system brought, in turn, improvements in the antigen-presenting pathway, for example through the effect of interferon (IFN), which can upregulate MHC expression. The main objective of this work was on the one hand, to determine the abilities of three strains of Marek's disease virus (MDV), a chicken herpesvirus, in interfering with the expression of MHC class I molecules in chicken embryo fibroblasts. On the other hand, we analyzed the ability of IFN to reinstate this important immune capability to the infected cells. Our results show that only an oncogenic serotype 1 strain of MDV (RB1B) was able to markedly decrease MHC class I expression, and that addition of IFN reversed this MDV effect.     

Development and functional characterization of monoclonal antibodies recognizing chicken lymphocytes with natural killer cell activity.

We have developed two monoclonal antibodies which detect cell surface antigens present on chicken lymphocytes mediating natural killer (NK) cell activity against the avian tumor cell target. The monoclonal antibodies, K-14 and K-108, stained 17 and 6% of splenic lymphocytes, and 11 and 14% of peripheral blood lymphocytes (PBL), respectively, and fewer than 5% of thymic and bursal lymphocytes. Neither of these monoclonal antibodies stained adherent macrophages or the MC29-virus transformed monocytic cell line. Both monoclonal antibodies significantly inhibited NK cell activity in a standard 4 h 51Cr-release cytotoxicity assay using the LSCC-RP9 tumor cell line as target cells at an effector to target ratio of 50:1. Pretreatment of splenocytes with either monoclonal antibody in the presence of rabbit complement (C) resulted in a significant reduction in NK cell activity. However, the monoclonal antibody K-1 which detects normal chicken macrophages did not interfere with NK cell activity. The monoclonal antibody K-108 significantly blocked Fc receptor-mediated rosette formation of sheep red blood cells coated with IgG antibodies (EA) by 56% while the monoclonal antibody K-14 did not show a significant blocking. These results indicate that the monoclonal antibodies K-108 and K-14 identify different epitopes present on the surface of chicken splenic lymphocytes which mediate spontaneous NK cytotoxicity.     

Comparative susceptibility of Marek's disease cell lines to chicken infectious anemia virus

Chicken infectious anemia virus (CIAV) is known to infect and replicate in various Marek's disease chicken cell lines (MDCCs) derived from Marek's disease (MD) tumors. One line, MDCC-MSB1, has been the substrate used in most studies. We compared a total of 26 MDCCs, including two sublines of MDCC-MSB1, MSB1 (L) and MSB1 (S), four other MD tumor-derived lines, and 20 lines derived from MD virus-induced local lesions, for susceptibility to the Cux-1 and CIA-1 strains of CIAV. The cell lines represented six phenotypic groups of T cells based on the expression of CD4, CD8, and TCR-2 and -3 surface markers. Susceptibility was measured by the number of cells positive for viral antigen in immunofluorescence (IF) tests at 3-10 days postinfection. No clear-cut differences were found in susceptibility related to phenotype, although CD4-/8+ lines and CD4-/8- lines might be more susceptible than CD4+/8- lines. However, several individual lines were more susceptible to Cux-1 than the two MSB1 sublines tested. Contrary to an earlier report, cells of MDCC-CU147, a CD8+, TCR3+, local-lesion derived line, were found to be susceptible to CIA-1. In fact, CU147 was distinguished by very high susceptibility to both CIAV strains. In direct comparisons with MSB1, CU147 detected approximately 10-fold lower doses of virus. Also, virus spread was faster (P < 0.05) in CU147 than in MSB1 and other lines. Results from polymerase chain reaction (PCR) tests to detect infection in titrations were in general agreement with IF test results although PCR detected infection in a few terminal dilution cultures that were negative by IF.     

银杏叶多糖对雏鸡马立克HVT疫苗免疫效果影响

银杏叶提取物具有广泛的药理学作用，银杏提取物的生物活性，如免疫调节、抗肿瘤、改善心血管功能等已有报道。高媛等以S180种鼠建立模型，研究银杏叶多糖对实体瘤、腹水瘤的作用，结果证实（GBLP）可明显抑制实体瘤、腹水瘤的生长，延长荷瘤水鼠的存活时间。陈群等也对银杏叶提取物的抗肿瘤作用进行研究，结果与高嫒等的报道基本一致。     

马立克氏病的研究进展

鸡马立克氏病(Marek'sDisease,MD)是由马立克病毒引起鸡的一种淋巴组织增生性疾病.以病鸡的外周神经、性腺、虹膜、各种内脏器官、肌肉和皮肤发生单核细胞浸润、形成淋巴肿瘤为特征.本病传播速度快,传播面积广,潜伏期长(1～6个月不等).患急性内脏型鸡马立克氏病的鸡群淘汰及死亡率高达8%一30%.严重发病的鸡群可造成全群覆灭,OIE将其列为B类疫病.     

用鸡胚检测鸡马立克氏病疫苗的免疫效果

将火鸡疱疹病毒（ＨＶＴ）疫苗接种于鸡胚卵黄囊、然后检查绒毛尿囊膜（ＣＡＭ）上病毒痘斑（以下简称“痘斑”）生长情况,以此作为ＨＶＴ疫苗免疫前效力检测的手段,得到满意效果。１材料与方法１．１ＭＤ强毒（ｖＭＤＶ）分离自本公司某鸡场严重感染ＭＤ鸡的血液,经原北京农业大学鉴定为一株ｖＭＤＶ。历年采购的国产和进口ＨＶＴ疫苗３０批次,分甲、乙、丙３组进行观察。１．２经血清学检查（ＭＤ-ＡＧＰ）并证明无ＭＤ抗体的种鸡所产种蛋,按常规方法,孵育到４～５ｄ的鸡胚用于接种;卵黄囊接种已稀释好的ＨＶＴ疫苗,每份ＨＶＴ疫苗分１,２…     

Proliferation of chicken lymphoblastoid cells after in vitro infection with Marek's disease virus.

Activated, thymus-derived (T) lymphoblasts were exposed to Marek's disease virus and cultivated in attempts to induce in vitro transformation. After 9 to 15 days, colonies or small clusters of proliferating lymphoblasts were observed in cultures from three of a total of 122 attempts. These developed into proliferating cell cultures that resembled conventional Marek's disease (MD) lymphoblastoid cell lines in terms of growth characteristics and morphology. All proliferative cultures were unusual in that 1) the expression of viral internal antigens consistently or periodically was very high (up to 30% of all cells) and 2) the cells deteriorated and/or proliferation ceased in all cases after culture periods of 45-176 days. The proliferative cultures were all characterized as CD2+ and CD3+, Ia-bearing T cells; one was CD4+/CD8- and TCR2+, the other two were CD4-/CD8- and TCR1+. The latter two are the only cultures of MD-infected cells known to be TCR1+.     

Induction of apoptosis by canine natural killer cells

Besides a secretory pathway of canine natural killer (NK) cells, which results in necrosis of the target cell, a second pathway was demonstrated, which results in apoptosis of the target cell. Comparing the Chromium Release Assay (CRA) and the Rose Bengal Assay (RBA) for quantification of in vitro canine NK cell activity, a constant 10% higher NK cell activity was found in the RBA compared with the CRA. To find out the mechanism responsible for the different results of both tests, morphological studies of in vitro canine NK cell activity against epithelial and mesenchymal allogenic target cell lines were performed. Most target cells were undergoing necrosis as a result of NK cell killing, which was evidenced by transmission electron microscopy. However, besides necrotic target cells, shrunken target cells with dense cytoplasm, fragmented nuclei and disruption into membrane-bound bodies were detected, which are known as signs of apoptosis. Additionally, using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method, 13-23% of target cells presented a positive staining, indicative of apoptosis. These findings give evidence for the ability of canine NK cells to kill their target cells via two different pathways, which results either in apoptosis or necrosis.