Immunoglobulins and anti-Marek's disease virus antibody synthesis in chickens after passive immunization with immunoglobulin Y anti-Marek's disease virus antibody.

The effect of passive immunization with immunoglobulin Y (IgY) antibody against Marek's disease virus (MDV) was examined in MDV-susceptible chickens. The production of IgY, immunoglobulin M, and probably also immunoglobulin A was depressed in passively immunized chickens when compared with that in MDV-exposed chickens which had not been given IgY anti-MDV antibody. In passively immunized chickens, the synthesis of immunoglobulin M and IgY anti-MDV antibodies in response to MDV infection also was delayed as determined by agar gel precipitin and indirect fluorescence antibody tests.     

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.     

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.     

Effects of IgY antibody on the development of Marek's disease.

The effects of passive immunization with immunoglobulin Y (IgY) on the pathogenesis of Marek's disease (MD) were examined in an experimental line of White Leghorn chickens highly susceptible to MD. Purified IgY with anti-MDV antibody activity, when injected into chicks, delayed the development of MDV viremia and lesions until 9 days postinoculation (PI) with Marek's disease virus (MDV). The blastogenic response of spleen cells to concanavallin-A was depressed at 6 days PI in the birds without passive immunization, whereas it was not totally depressed until 17 days in birds passively immunized with IgY anti-MDV antibody.     

Immunopathology of Marek's disease in quails: presence of antinuclear antibody and immune complex.

Antinuclear antibody (ANA), a marker for autoimmune reactions, was detected in the sera of quails with Marek's disease (MD). The autoantibody was detected 3 weeks after infection in quails infected with chicken Marek's disease virus and 4 weeks after infection in quails infected with quail Marek's disease virus. The ANA titers were low and ranged from 10 to 40. A speckled type of nuclear fluorescence was the characteristic staining feature. In addition to the presence of ANA, immune complexes (IC) were also detected in the kidney glomeruli of quail infected with Marek's disease virus. Initially about 25-30% of the glomeruli in the kidneys of infected quails had IC deposits. In subsequent periods, the amount of IC deposit and the number of glomeruli showing IC also increased considerably. The findings of the present study suggested autoimmunity may play a pathogenic role in MD.     

Testing the immunogenicity of the dermal antigen in Marek's disease virus]

An experiment was performed to study the immunogenicity of the dermal antigen of Marek's disease virus, extracted from the skin of 30-day-old chickens, infected with Marek's disease virus on the first day of life. Three kinds of samples were tested: (1) dermal antigen centrifuged at 10 000 g per 0.5 h, (2) dermal antigen centrifugated at 10 000 g per 0.5 h and 100 000 g per 1 h, (3) dermal antigen treated like sample (2) and partly purified by DEAE-cellulose chromatography. Samples (1) and (2) were inoculated to two-day-old chickens and the vaccination was repeated, using complete Freund's adjuvant, 21 days later. Sample (3) was inoculated to two-day-old chickens with DEAE-dextran. All the three groups were challenged together with the controls (non-vaccinated chickens) on the seventh day after the first vaccination. A reduction of mortality was observed in the chickens vaccinated with and re-vaccinated with sample (1) (23.07%) and in the chickens vaccinated with sample (3) (30.76%). The chickens of the latter group were the last to start dying from Marek's disease--only after the 10th week of life. In the chickens which had been vaccinated and revaccinated with sample (2) the mortality was not reduced. The study is continued, with particular emphasis on the relationship of DEAE-dextran to protection against Marek's disease.     

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.     

Monoclonal-antibody-mediated enzyme-linked immunosorbent assay for detection of reticuloendotheliosis viruses

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of reticuloendotheliosis viruses (REVs). The assay uses a mixture of monoclonal antibodies (MCAs) prepared against a 62-kilodalton REV envelope glycoprotein (gp62) to capture antigen, rabbit anti-REV serum as detection antibody, and peroxidase-conjugated anti-rabbit IgG as indicator antibody. The MCAs were reactive with REV strain T, chick syncytial virus, and duck infectious anemia virus but unreactive against Marek's disease and avian leukosis viruses. The ELISA was compared with complement fixation test and REV immunofluorescent assay of infected fibroblasts, plasmas, and egg albumen from infected chickens. The lower limit of gp62 detection was about 120 ng of REV protein. Limit dilution of infectious REV was detected after 7-8 days of cultivation of infected fibroblasts.     

Porcine parvovirus: propagation in microcarrier cell culture and immunogenic evaluation in pregnant gilts

Porcine parvovirus was propagated in PK-15 cells cultured in roller bottles or on microcarrier beads. After inactivation, the virus was used as antigen in the preparation of vaccines. The immunogenic potency and safety of the vaccines were evaluated in specific pathogen free pregnant gilts and guinea pigs. Experimental challenge tests determined the efficacy of the vaccine in preventing porcine parvovirus transplacental infection. Neither viral antigens nor specific antibodies were detected in fetuses from vaccinated gilts. In contrast, fetal death and, or, mummification occurred when unvaccinated gilts were infected. Both virus and, or, antibodies were also detected in fetuses from these unvaccinated gilts. Serum conversion after vaccination was assayed by microserum neutralisation using guinea pig erythrocytes as cell indicators and by haemagglutination inhibition tests. Viral antigens in fetal tissues were detected using ELISA, the immunobeads technique, the haemagglutination test and by virus isolation.     

Comparison of hemagglutination-inhibition, agar gel precipitin, and enzyme-linked immunosorbent assay for measuring antibodies against influenza viruses in chickens

Individual variations in serological response to avian influenza virus infection were demonstrated after experimental infection of specific-pathogen-free chickens with H6N2 influenza virus. Homologous antibodies were detected from the 6th to the 157th day after infection using hemagglutination-inhibition or enzyme-linked immunosorbent assay and from the 11th to the 157th day by agar gel precipitation test.     

Influence of vaccination with avirulent herpesvirus on subsequent infection of chickens with virulent Marek's disease herpesvirus.

Vaccination of chickens with turkey herpesvirus (HVT) or attenuated Marek's disease herpesvirus (aMDHV) blocked infection with virulent MDHV (VMDHV) for approximately 5 weeks after contact exposure. However, there was no apparent blockage of infection when challenge virus was administered intraabdominally (IA). Evidence for infection with VMDHV was based on viral isolation by in vivo assay or by detecting precipitins to "A" antigen associated with virulent virus. The HVT stimulated production of neutralizing antibody against VMDHV in a high percentage of chickens, whereas the aMDHV was a comparatively poor inducer of such antibody. Despite this difference, both of the vaccinal viruses conferred protection against development of Marek's disease.     

Marek's disease virus infection in the brain: virus replication, cellular infiltration, and major histocompatibility complex antigen expression

Marek's disease virus (MDV) infection in the brain was studied chronologically after inoculating 3-week-old chickens of two genetic lines with two strains of serotype I MDV representing two pathotypes (v and vv+). Viral replication in the brain was strongly associated with the development of lesions. Three viral antigens (pp38, gB, and meq) were detected in the brain of infected chickens. Marked differences between v and vv+ pathotypes of MDV were identified for level of virus replication, time course of brain lesions, and expression of major histocompatibility complex (MHC) antigens. Two pathologic phenomena (inflammatory and proliferative) were detected in the brain of chickens inoculated with vv+MDV, but only inflammatory lesions were observed in those inoculated with vMDV. Inflammatory lesions, mainly composed of macrophages, CD4+ T cells, and CD8+ T cells, started at 6-10 days postinoculation (dpi) and were transient. Proliferative lesions, characterized by severe infiltrates of CD4+CD8- T cells (blasts), started at 19-26 dpi and persisted. Expression of MHC antigens in endothelial cells and infiltrating cells within the brain was influenced by MDV infection. Upregulation of MHC class II antigen occurred in all treatment groups, although it was more severe in those inoculated with vv+MDV. MHC class I antigen was downregulated only in those groups inoculated with vv+MDV. These results enhance our understanding of the nature and pattern of MDV infection in the brain and help to explain the neurovirulence associated with highly virulent MDV.     

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.     

Nephropathogenesis of chickens experimentally infected with various strains of infectious bronchitis virus

Four-day-old specific-pathogen-free chickens were inoculated by eyedrop with four different strains (Gray, JMK, CV56b, and Wolgemuth) of infectious bronchitis virus (IBV). Birds were monitored clinically and euthanatized at 1, 4, 7, and 14 days postinfection and tissues were collected for virus isolation, histopathologic examination, in situ hybridization (ISH), and immunohistochemistry (IHC). Clinical disease was severe in chickens infected with Wolgemuth, but no overt disease was observed with the other strains. Virus was isolated from the kidneys of chickens infected with the Gray-, CV56b-, and Wolgemuth-strains of IBV. Histologically, interstitial nephritis was evident in chickens infected with these same 3 strains. However, viral nucleic acid and antigen were detected only with Wolgemuth-infected kidneys by ISH and IHC. These results indicate that the pathological changes in kidneys from chickens infected with Gray and CV56b may not have resulted from the cytolytic action of the virus.     

An enzyme-linked immunosorbent assay for detection of antibodies to exogenous avian leukosis virus

A microplate enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to avian leukosis virus (ALV) of subgroups A and B in infected chickens was developed with the use of Rous-associated virus (RAV)-1 (subgroup A) and RAV-2 (subgroup B) antigens purified by sucrose-gradient centrifugation. The antigen was used for ELISA after treatment with Triton X-100. In the ELISA, the subgroup viral antigen reacted strongly with homologous antiserum but also reacted with heterologous antiserum. Tests with serum absorbed with purified homologous and heterologous virus and tests for antigen-blocking by group-specific antibodies to ALV revealed that the reaction was caused mainly by subgroup-specific antibodies. The ELISA was 8 to 32 times more sensitive than the virus-neutralization (VN) test and detected antibodies to ALV earlier than the VN test in chickens infected experimentally with RAV-1 and RAV-2. In field application of the ELISA, 44.2% of 484 chicken sera were positive for RAV-1 and/or RAV-2 antigen, and 80.4% of flocks were positive. These findings indicate that ELISA is superior to the VN test in sensitivity, simplicity, rapidity, and applicability for large-scale field surveys for ALV infection.     

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%).     

马立克氏病胚胎免疫雏鸡体重和免疫器官增重变化

１８日龄鸡胚免疫接种火鸡疱疹病毒（ＨＶＴ）疫苗后,对出壳后雏鸡及其用马立克氏病（ＭＤ）病毒攻毒鸡体重与免疫器官增长效果的对比研究表明：相同饲养条件下胚胎免疫雏鸡的增长速度高于非免疫对照鸡,而且胚胎免疫组雏鸡免疫器官的生长发育普遍较相应的对照组雏鸡快。     

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。     

Immunoglobulin class distribution of systemic and mucosal antibody responses to Newcastle disease in chickens

In serum, tracheal wash fluid, and bile from chickens that were inoculated with live or inactivated Newcastle disease virus (NDV), the kinetics and immunoglobulin (Ig) class distribution of an antibody response were demonstrated. The Ig classes (IgM, IgG, and IgA) were captured using monoclonal antibodies (MAbs) in enzyme-linked immunosorbent assays (Ig-capture ELISA). The antibody specificity of the captured Ig was confirmed by binding of NDV. After inoculation with live virus, antibodies of the IgG and IgM classes were mainly found in serum. IgM was produced early from day 4 postexposure (PE) onward, IgG was detected later from day 7 PE onward, and in the tracheal wash fluid and bile, all three Ig classes were demonstrated. After inoculation of inactivated virus, a delayed response of all three classes was observed in serum, and only IgM and IgG were recognized in the tracheal fluid and bile. The type of vaccine and the mute of antigen entrance may have determined the immunoglobulin class produced. The Ig-capture ELISA assay developed in this study can be useful for evaluating various strategies to improve the efficacy of Newcastle disease vaccines and to study the evoked immune mechanisms.     

The effect of cyadox and virginiamycin on Marek's disease in chickens

Chickens infected with highly virulent Marek's disease virus were administered in the course of 56-day fattening cyadox at a dose of 20 mg per kg feed mixture (MC group). and virginiamycin at a dose of 10 mg per kg feed mixture (MV group). The weight gains on the 56th day and dressing percentage were highest, in comparison with the control group (711.11 g and 60.62%), in the virginiamycin-treated group. An investigation into the basic chemical composition of meat revealed lower contents of dry matter and proteins in the virginiamycin-treated group (26.9% and 22.76%) if compared with the control group of chickens which were also infected with Marek's disease virus. A biometrical examination of the organs showed a statistically significant decrease in heart weight in the cyadox-treated group, increase in liver and heart weight and intestine shortening in the virginiamycin-treated group, in comparison with the control group. Pathologico-anatomical changes typical of Marek's disease were recorded in a randomly selected number of chickens in 33.3% in the control group, in 28.5% in the cyadox-treated group and in 50.0% in the virginiamycin-treated group.