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.     

影响马立克氏病毒感染的因素及疫苗免疫策略

马立克氏病毒是高度细胞结合性、嗜淋巴组织的α疱疹病毒,其致病过程包括淋巴细胞的溶细胞感染和潜伏感染以及易感鸡体内CD 4 T细胞的致瘤性转化。宿主的遗传抗性、细胞免疫、体液免疫以及相关细胞因子和淋巴细胞等对M DV的感染过程有重要影响。针对M D肿瘤抗原的靶向免疫应答的保护性抗原分子和宿主细胞因子疫苗是今后疫苗研究的方向。     

Antibody response of chickens to serotype 1, 2, or 3 Marek's disease vaccines based on ELISA with infected cells as antigen

An enzyme-linked immunosorbent assay (ELISA) was applied to evaluate the antibody response of commercial White Leghorn chickens to vaccination against Marek's disease (MD) at hatch (day 0) with serotype-1 (Rispens), -2 (SB-1), or -3 (turkey herpesvirus, HVT) vaccine virus and to challenge on day 21 with MD virus. Antigens for the test were whole chicken embryo fibroblast cells infected with Rispens, SB-1, or HVT. The chickens were progeny of stock that had been vaccinated with HVT, and on day 21 the nonvaccinated group had higher levels of maternal antibodies to HVT than to other antigens (P < 0.05). Only SB-1 vaccine had induced antibodies by day 21, and this was detected only against homologous antigens. On day 49, all three vaccines had induced higher levels of antibodies to homologous than to heterologous antigens. Marek's Disease virus (MDV) induced antibodies to all three antigens, but challenging vaccinated chicks did not significantly increase levels of antibodies on day 81 to any of the three antigens. It was concluded that an ELISA using whole cells as antigens would have potential value for monitoring the antibody response induced by MD vaccines and virulent MDV.     

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.     

Immunological aspects of Marek's disease in chickens with maternal antibodies]

The dynamics of the production of immunoprecipitation antibodies to Marek's disease virus was studied in the serum of chickens with maternal antibodies in relation to the occurrence of the immunoprecipitation antigens of Marek's disease virus in feather follicles. One-day-old chickens were infected by the contact method with Marek's disease virus. The first occurrence of immunoprecipitation antigen was detected on the 14th day after infection and this occurrence persisted throughout the experiment, i. e. until the 112th day after infection. The antibodies were first detected the 28th day after infection and their titre kept rising until the 98th day after infection. Immunoprecipitation antibodies and antigens of Marek's disease virus were detected in some tumorously changed kidneys. Immunoelectrophoretic examination revealed in the same kidneys immunoglobulins of the class IgY, IgA and beta-globulin. The slowest-migrating fraction of IgY, together with IgA, beta-globulin and C-reactive protein were detected in the skin extracts from infected poultry. Indirect haemagglutination enabled the detection of the presence of haemagglutination antibodies in rabbit immunoglobulin to the skin antigen of Marek's disease virus, and in avian immunoglobulin to the same virus. Haemagglutination antigen was revealed in the extract from tumorously changed kidneys.     

Cell-mediated cytolysis of lymphoblastoid cells expressing Marek's disease virus-specific phosphorylated polypeptides.

Cell-mediated immune responses against Marek's disease virus (MDV)-antigens were examined using reticuloendotheliosis virus (REV)-transformed lymphoblastoid cell line CU91 and three cell lines derived from CU91. CU210 was established by establishing a latent MDV infection in CU91. Transfection of CU210 with pNL1, a selectable plasmid or with pNL1 and the cloned BamHI A fragment of MDV DNA resulted in the establishment of CU212 and CU211, respectively. CU211 expressed a MDV-specific phosphorylated polypeptide, while CU210 and CU212 were negative for MDV antigens. Only CU211 was lysed by MDV-specific effector cells. All cell lines were lysed by syngeneic REV-specific effector cells, although high levels of expression of the phosphorylated protein reduced the level of REV-specific lysis.     

Protection of chickens from Newcastle and Marek's diseases with a recombinant herpesvirus of turkeys vaccine expressing the Newcastle disease virus fusion protein.

Recombinant strains of herpesvirus of turkeys (HVT) were constructed that contain either the fusion protein gene or the hemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) inserted into a nonessential gene of HVT. Expression of the NDV antigens was regulated from a strong promoter element derived from the Rous sarcoma virus long terminal repeat. Recombinant HVT strains were stable and fully infectious in cell culture and in chickens. Chickens receiving a single intra-abdominal inoculation at 1 day of age with recombinant HVT expressing the NDV fusion protein had an immunological response and were protected (> 90%) against lethal intramuscular challenge at 28 days of age with the neurotropic velogenic NDV strain Texas GB. Recombinant HVT expressing the NDV hemagglutinin-neuraminidase provided partial protection (47%) against the same challenge. Chickens vaccinated with recombinant HVT vaccines had low levels of protection against NDV replication in the trachea when challenged ocularly. Recombinant HVT vaccines and the parent HVT strain provided similar levels of protection to chickens challenged with the very virulent RB1B strain of Marek's disease virus, indicating that insertion of foreign sequences into the HVT genome did not compromise the ability of HVT to protect against Marek's disease.     

Orientation of bovine CTL responses towards PIM, an antibody-inducing surface molecule of Theileria parva, by DNA subunit immunization

East Coast fever, an acute lymphoproliferative disease of cattle, is caused by the apicomplexan parasite Theileria parva. Protective immunity is mediated by CD8(+) cytotoxic T lymphocytes directed against schizont-infected cells. The polymorphic immunodominant molecule, although an antibody-inducing surface molecule of the schizont, has been hypothesized to play a role in protective immunity. In order to evaluate the immunogenicity of PIM for inducing CTL, cattle were immunized with PIM in isolation from other T. parva antigens, forcing the presentation of PIM-derived epitopes on the MHC class I molecules. Although parasite-specific cytotoxicity was induced in both vaccinated animals, their immune response was clearly different. One animal generated MHC-restricted parasite-specific CTL against PIM while the other calf exhibited a strong PIM-specific proliferative response but non-MHC-restricted parasite-specific cytotoxicity. Only calf 1 survived a lethal sporozoite challenge. This DNA immunization technique with an antigen in isolation of CTL-immunodominant antigens might open possibilities for directing CTL responses against predefined antigens, such as strain cross-reacting CTL antigens.     

Anti-idiotype antibodies to Marek''s disease-associated tumour surface antigen in protection against Marek''s disease

Marek's disease-associated tumour surface antigen (MATSA) removed by enzymatic (papain) digestion of Marek's disease tumour cells was fractionated by gel filtration chromatography. The first peak (F1) was used to raise antibody in rabbits. Monoclonal antibody (RPH-6) directed against MATSA and the anti-F1 IgG were used as idiotypic antibodies to raise polyclonal anti-idiotype serum in heterologous hosts; rabbit and goat, respectively. The anti-idiotypes (anti-Id) were purified by affinity chromatography and characterized by competitive binding assay using immunofluorescent (IF) tests.

Day-old white Leghorn chicks were immunized with anti-Id to MATSA (Group 1) or anti-Id to F1 (Group 3) and challenged with virulent Marek's disease virus (MDV) on the tenth day post immunization. In positive control groups, the day-old chicks were inoculated with anti-BALB/c mouse globulin (Group 2) and anti-rabbit globulin (Group 4) and challenged with virulent MDV on the tenth day post inoculation. As compared with positive control groups, the vaccinated groups (1 and 3) had considerably lower level of MATSA positive cells during the post challenge observation period. The protection level against MD in the immunized groups was 66.6% (Group 1) and 86.6% (Group 3).     

Anatomical distribution of T- and B-lymphocytes in Marek's disease--an immunohistochemical study

Neural lesions of Marek's disease, Marek's disease tumours in the ovary, liver, and kidney, as well as spleen and bursa of Fabricii of chickens bearing Marek's disease tumorous infiltrations, were examined by a new immunohistochemical technique basing upon Sternbergers unlabelled antibody enzyme method which allows the exact localization of lymphoid cells based on their surface antigens. Type C neural lesions contained T-lymphocytes almost exclusively. Type B neural lesions had relatively high proportions of T- and B-lymphocytes, and severe type A neural lesions possessed one part of heavily labelled T-lymphocytes and a number of cells stained weakly by rabbit-anti-chicken-T-cell-globulin. Tumorous infiltrations had similar characteristics as type A neural lesions. Spleen and interfollicular spaces of bursa of Fabricius were infiltrated by T-lymphocytes.     

Immunization trials with virus-free antigens against Marek''s disease

Additional immunization trials were performed to study the immunogenicity of the purified skin antigen of Marek's disease virus which was inoculated, together with Freud's complete adjuvant, into one-day-old chicks. As compared to non-vaccinated chickens and also to chickens vaccinated by herpesvirus turkey (which reduced the mortality by 45.54%) the purified skin antigen reduced the mortality by 69.50%.

In the case of immunization with protein extract from the lymphoblastoid cell line of Marek's disease lymphomes mixed with natural dsRNA showed 38.99% reduction of mortality. DEAE-dextran which had exerted an adjuvant effect in our previous report did not by itself reduce mortality caused by Marek's disease.

Groups of chickens vaccinated with the turkey herpesvirus with protein extract mixed with dsRNA, and a group of chickens inoculated with 0.04 g.ml⁻¹ DEAE-dextran, had a higher whole complement activity in pooled serum from 107 days after challenge than chickens free of Marek's disease.     

Cellular and cytokine responses in feathers of chickens vaccinated against Marek's disease

In Marek's disease virus infection, feather follicle epithelium (FFE) constitutes the site of formation of infectious virus particles and virus shedding. The objective of this study was to characterize cellular and cytokine responses as indicators of cell-mediated immune response in FFE and associated feather pulp following immunization against Marek's disease. Analysis of feather tips collected between 4 and 28 days post-immunization (d.p.i.) from chickens vaccinated post-hatch with either CVI988/Rispens or herpesvirus of turkeys revealed that replication of these vaccine viruses started at 7d.p.i., peaked by 21d.p.i., and subsequently, showed a declining trend. This pattern of viral replication, which led to viral genome accumulation in feather tips, was associated with infiltration of T cell subsets particularly CD8+ T cells into the feather pulp area and the expression of cytokine genes such as interferon-gamma, which is an indication of elicitation of cell-mediated immune responses at the site of virus shedding.     

The early pathogenesis in chicken inoculated with non-pathogenic serotype 2 Marek's disease virus.

A newly cloned serotype 2 Marek's disease virus (MDV), strain ML-6, was inoculated via the nasal cavity in specific-pathogen-free chicks to examine early virus replication and the expression of Marek's disease (MD)-related antigens. Following inoculation, viral intracellular antigens (VIAs) were detected in lymphoid organs (bursas and spleens) between 5 and 14 days post inoculation (PI), in feather follicles between 14 and 30 days PI, and in lungs at 3 days PI by the immunohistopathological staining of avidin-biotin-peroxidase complex method. But, very few VIAs were expressed in the thymuses between 5 and 14 days PI. However, MD tumor-associated surface antigens were not detected in any organs. Viruses were isolated from separated spleen cells at 14 and 30 days PI. Fluorescent antibodies of convalescent sera were also detected after 10 days PI. As most of the VIAs were detectable in B-cells in bursas and spleens. B-cells were considered to be the main first target cells for the serotype 2 MDV infection.     

Production and characterization of monoclonal antibodies against chicken lymphocyte surface antigens

A panel of monoclonal antibodies (mAbs) with specificity for chicken lymphocyte surface antigens was established and characterized based on their reactivities against chicken lymphoid cells and tumor cell lines on flow cytometry. Three mAbs (7-3G-2, 7-2E-8, and JB-2) reacted preferentially with thymocytes, however, none of them reacted with Marek's disease derived T lymphoblastoid cell lines. Four mAbs (6-27A-1, 4-5C-5, Lc-4, and Lc-6) reacted with spleen cells and peripheral blood leukocytes as well as thymocytes. All seven mAbs reacted with chicken embryonic thymocytes from day 12 of embryonic life onward. All mAbs showed no reactivity against bursal lymphocytes.     

Indications of immunodepression in chickens infected with various strains of Marek's disease virus

The effect of infection by various strains of Marek's disease virus (MDV) on the immune function of 3-week-old chickens was examined. MDV strains of low (CU-2, RB-7) and high (RB-3, MD-5, and MD-11) pathogenicity were compared with prototype JM-10 strain of moderate pathogenicity. Mortality, whole body weight, relative weights of lymphoid organs, histopathology, humoral antibody responses to thymus-dependent and -independent antigens, and in vitro lymphocyte responses to mitogen stimulation were investigated at 1, 2, and 3 weeks postinfection. MDV strains of high pathogenicity significantly depressed responses at 3 weeks postinfection, seeming to indicate the ability of these viruses to induce severe immunodepression. However, the fact that the moderately pathogenic and even some of the low-pathogenicity strains induced immunodepression suggests that other viral mechanisms are also important in its determination.     

人参皂苷及其衍生物体内抗马立克氏病毒的间接免疫荧光观察与PCR检测

为了探讨人参皂苷及其衍生物体内抗马立克氏病毒的作用机理.用马立克氏病毒人工感染雏鸡模型,采取人参皂苷及其衍生物口服,通过间接免疫荧光试验动态观察人参皂苷及其衍生物能否减少MDV抗原在组织中的分布;通过PCR检测看人参皂苷及其衍生物能否减少MDV的出现.结果显示:人参皂苷组、衍生物组被检组织的阳性细胞数量明显比盐酸吗啉胍阳性对照组被检组织的阳性细胞数量少;MDV病毒核酸的PCR检测显示,药物没有阻止病毒对组织的感染.结果表明,人参皂苷及其衍生物抗马立克氏病毒效果要优于盐酸吗啉胍.     

Bovine adenoviruses—IV. Two mixed antigens for routine serodiagnosis by complement fixation reaction

The frequency of bovine adenovirus infections occurring in cattle has been grossly underestimated up to the present day. Primary isolation of serotypes belonging to bovine subgroup II is cumbersome. Hardly any laboratory has used all 8 officially-recognised bovine serotypes in seroneutralisation-tests, which should be done when this type-specific method is used for serodiagnosis. The complement fixation test, known as group-reactive from human adenovirus serology, has failed to disclose the true incidence of bovine infections, as until recently the importance of a novel additional group-reactive bovine adenovirus antigen had been undisclosed. Here we describe production, composition and performance of two mixed antigens for complement fixation reactions, which take into account recent findings by our team on peculiarities of bovine adenovirus antigens. Mixed antigen 1 contains bovine serotypes 1, 2 and 3 and detects group-specific antibodies of the classical mastadenoviruses. Mixed antigen 2 contains bovine serotypes 4, 5, 6, 7 and 8 and determines group-specific antibodies against the novel paramastadenoviruses. In addition, each antigen is capable of demonstrating complement-fixing type-specific antibodies in sera against the respective types included in each mixed antigen, with a net enhancing effect produced by mixing. Use of the 2 mixed antigens promptly shows whether bovine adenoviruses, presently recognised as well as unclassified, are involved in a given outbreak of respiratory or enteric disease of cattle. If needed, the responsible type may be determined in a second step of serological investigation.     

Immunogenicity of eight Mycobacterium avium subsp. paratuberculosis specific antigens in DNA vaccinated and Map infected mice

Mycobacterium avium subsp. paratuberculosis (Map), the etiological agent of chronic enteritis of the small intestine in domestic and wild ruminants, causes substantial losses to livestock industry. Control of this disease is seriously hampered by the lack of adequate diagnostic tools and vaccines. Here we report on the immunogenicity of eight Map specific antigens, i.e. MAP1693c, Ag3, MAP2677c (identified by post-genomic and immunoproteomic analysis of Map secretome) and Ag5, Ag6, MAP1637c, MAP0388 and MAP3743 (identified by bioinformatic in silico screening of the Map genome). Strong, antigen-specific IFN-γ responses were induced in mice vaccinated with plasmid DNA encoding MAP1693c, MAP1637c, MAP0388 and MAP3743. In contrast, T cell responses in Map infected mice were directed preferentially against Ag5 and to a lesser extent against MAP3743. None of the tested DNA vaccines conferred protection against subsequent challenge with Map.     

Marek's disease virus-transformed chicken T-cell lines respond to lymphokines.

Current assays for chicken interleukin-2 (IL-2) utilize mitogen-activated lymphocytes. However, very high inter-assay variability and sporadic high background proliferation limit their usefulness. In view of the above, several Marek's disease virus (MDV)-transformed T-cell lines (which grow well in a serum-supplemented medium) were tested for a response to chicken IL-2 when grown in serum-free media. Five of six lines examined showed a dose-dependent proliferative response to chicken T-cell conditioned media. One line, MDCC-CU14, was chosen for further studies. In addition to the tumor cells' dose-dependent responses to semi-purified chicken IL-2, they expressed T-cell activation antigens on the cell surface. Furthermore, the level of surface expression was enhanced on cells provided IL-2. Co-incubation of the tumor cells with monoclonal antibody INN-CH-16 (specific for an antigen on the surface of activated T-cells) and IL-2 resulted in a modulation of lymphokine-induced proliferation. Together, these data suggest that signalling mechanisms in MDV T-cell tumors are intact and that these lines can be used as an assay for chicken T-cell lymphokines. Furthermore, they provide an interesting model for the study of avian and mammalian T-cell transformation. Implications for the study of Marek's disease are also discussed.     

Herpes virus of turkey vaccine: viraemias in field flocks and in experimental chickens

In a field survey of viraemias due to vaccination of chickens with herpes virus of turkey, variation was encountered in titres and percentages of birds viraemic. The incidence of viraemias was much lower in sick than in healthy birds in flocks undergoing mortality from Marek's disease. In a concurrent experiment the same strain of chicken and the same commercial vaccine were used as in the field flocks affected with Marek's disease. A high incidence of viraemia and 84.6 per cent protection against Marek's disease were obtained with single vaccination at one day of age. Revaccination at 21 days of age produced no measurable benefits in the same experiment.