Comparative studies on pathogenical, virological and serological properties of Marek's disease virus isolated from Japanese quail and chicken

The QM-1 strain of Marek's disease virus (MDV) isolated from Japanese quail (JQ) induced higher mortality (96%) with MD lesions in quail than the JM strain of MDV isolated from chickens (C) (21%). In contrast both viruses induced MD in 90% of chickens. Pathological changes produced in quail inoculated with JQ-MDV or C-MDV were similar. However, the incidence of tumour lesions in the duodenum of quail inoculated with JQ-MDV was significantly higher. Neither virus induced tumour lesions in the duodenum of chickens. Appearance of viraemia in quail inoculated with MDV was delayed and its level was low as compared with chicken cases. MDV antigens in feather tips appeared in quail inoculated with JQ-MDV but not with C-MDV. No MDV was recovered from the MDV-inoculated quail by direct kidney cell cultures, although MDV was recovered by subpassage of the cultures into chicken embryo fibroblasts. No antibodies were detected in the affected quail by agar gel precipitation test.     

Isolation of Marek's disease virus: revisited.

Splenocytes from chickens infected with low-passage stocks of Marek's disease virus (MDV) RB-1B, a very virulent (vv) strain and vv+ RK-1 were used to compare the efficacy of chick kidney cells (CKC), chicken embryo fibroblasts (CEF) and chicken embryo kidney cells (CEKC) for virus isolation. CKC were superior to CEF and CEKC. MDV foci were present at 4 days post infection in CKC but not until 6 days post infection in CEF or CEKC. Virus yield was higher in CKC than in CEF or CEKC at 6 days post infection. Passage of RB-1B in CKC yielded a significantly higher virus increase than with CEF or CEKC. The same was true for RK-1 comparing CKC with CEKC. Interestingly, RK-1-infected CEF were negative or had very low number of foci in passage 1, but virus yield increased 500-fold to 600-fold on passage in CKC, CEF, and CEKC. Recommendations on procedures for successful virus isolation are provided.     

Isolation of Marek's disease virus DNA from infected cells by electrophoresis on polyacrylamide gels

Summary Marek's disease virus DNA isolated from the nuclear fraction of infected chicken embryo fibroblasts and sucrose-purified particles was electrophoresed on 3 per cent polyacrylamide gels and was compared in its electrophoretical behaviour with isolated pseudorabies and herpes simplex DNA, strain HF. The DNA molecules eluted from the gel were identified by their sedimentation coefficient (53–55S) and buoyant density (1.707 g/ml) to be of viral origin. MDV DNA molecules were electrophoretically also detected and identified in DNA preparations of the lymphoblastoid Marek's disease tumour cell line MSB-1 which therefore has to be considered as a producer line. The electrophoresis of DNA preparations from Marek's disease virus-infected cells on polyacrylamide gels provides a semipreparative method for the isolation of MDV DNA.With 5 Figures     

A study of vaccination against Marek's disease with an attenuated Marek's disease virus

Approximately 2,000 day-old white leghorn chickens were distributed into 10 pens and half the pens vaccinated with the attenuated strain of HPRS-16. At 72 weeks of age, mortality from non-specific death and Marek's disease was 17.6% and 15.6% respectively, in vaccinated chickens compared with 14.2% and 51.7% in unvaccinated chickens. Body weights of vaccinated chickens were 5.6% higher at 8 weeks of age than unvaccinated chickens. Vaccinated chickens consumed 2.3% more feed and the hen housed egg production and hen day egg production were 58.7% and 7.0% greater than unvaccinated chickens. Vaccination resulted in a 3.8 fold increase in net margin over food and bird costs per bird housed at 16 weeks. Active antibody production to 'A' antigen occurred later and in a smaller proportion of vaccinated chickens than unvaccinated chickens. Viraemia due to vaccination was detected at 2 weeks of age and viraemia due to field virus was detected at 5 weeks of age. The incidence and titres of viraemia due to field virus were higher in unvaccinated chickens compared with vaccinated chickens. No evidence of spread of vaccine virus to unvaccinated chickens could be found. Acute, classical and apathogenic strains of field virus were isolated from vaccinated chickens and strains of field virus were found to persist throughout the life of the vaccinated chickens. Mild classical and/or apathogenic strains were first apparent at 18 weeks of age and increased in proportion thereafter, forming the majority of isolates from 52 weeks of age. Data on individual birds suggested a direct relationship between virus titre and lesion (or Marek's disease) frequency.     

Structural proteins of Marek's disease virus

Marek's disease virus (MDV) was propagated in roller-bottle cultures of duck embryo fibroblasts and partially purified by sucrose gradient centrifugation. Analysis of viral protein by polyacrylamide gel electrophoresis revealed that at least eight proteins (designated VPI-VPVIII) were present in MDV. The VPI is the major viral protein. At least two viral proteins, VPII and VPIV, with glucosamine label could be detected. These two peaks may represent viral glycoprotein and may be associated with the viral envelope. No host cell proteins coelectrophoresed with any viral proteins. Similar electropherograms were obtained by coelectrophoresis of MDV and herpes simplex virus proteins and of MDV and pseudorabies virus proteins.     

Pathogenesis of infection with attenuated Marek's disease virus strains

Recently, attenuated Marek's disease virus (MDV) became of renewed interest as a component in bi- or polyvalent vaccines. The effect of attenuation on the pathogenesis of infection was investigated. Cloned preparations of the JM-16, BC-1A and RB-1B strains of MDV were attenuated by serial passage in chick kidney cells or chicken embryo fibroblasts. Subclones were obtained from the JM-16 strain at passage (p) 26 (JM-16d) and 50 (JM-16a, b and c). The passage level at which each virus became attenuated was dependent on the virus strain. The highly oncogenic RB-1B strain was still oncogenic after 37 passages, while JM-was already attenuated at p. 27. In ovo infection of high passage JM-16 and RB-1B (p 54 and 55) demonstrated the presence of residual pathogenicity. Attenuated virus failed to induce the early cytolytic infection which is characteristic for the pathogenesis of infection with oncogenic MDV. Low levels of lymphocyte-associated viraemia could be detected after infection with all attenuated viruses except with the subclone JM-16a. This virus was, however, able to induce moderate protection against challenge and antibodies were detectable, suggesting that cells other than lymphocytes became infected. The pathogenesis after in ovo infection with attenuated virus was similar to that after infection of chicks. The in vivo data suggested that attenuation reduced the efficiency of infection of, or virus replication in, lymphocytes. A markedly reduced ability to establish in vitro infection of lymphocytes by exposure to heavily infected lymphocytes was observed, and this supports this hypothesis. The altered characteristic of attenuated virus to infect lymphocytes in vivo or in vitro was not caused by the selection of temperature sensitive or thymidine kinase negative mutants.     

Rabies vaccine prepared from the virus grown in Japanese quail embryo cell cultures

Fixed rabies virus strain MNIIVP-74 was grown in Japanese quail embryo cell cultures, concentrated by ultrafiltration and inactivated with beta-propiolactone. The resulting vaccine was markedly antigenic and immunogenic for laboratory animals. Human volunteers injected with 2.0 ml vaccine on days 0, 3, 7, 14, 30 and 90 exhibited more intensive and longer antibody production than those injected daily for 14 days.     

Disinfection of Marek's disease virus in poultry dust

Dust samples from farms contaminated with Marek's disease virus (MDV) were exposed to various disinfectants under experimental and field conditions. After the disinfection treatment, examination was made for the presence and oncogenicity of surviving MDV by the inoculation of tissue cultures and susceptible chickens. A combination of formaldehyde vapour and a preparation based on iodine bound to organic carriers appeared to be the most effective for disinfection.     

Isolation of chicken anaemia agent and Marek's disease virus from chickens vaccinated with turkey herpesvirus and lesions induced in chicks by inoculating both agents

A disease that was characterised by high mortality, necrotic lesions, severe thymic and bursal atrophy, as well as Marek's disease (MD) lesions, occurred in two young layer chicken flocks vaccinated with turkey herpesvirus (HVT) at hatching. An agent similar to the chicken anaemia agent, designated CAA82-2, and a virulent MD virus strain, designated Md82-2, were isolated from the kidneys of affected chickens. Dual inoculation of young chicks with both agents resulted in early mortality associated with severe necrosis and depletion of lymphocytes in the lymphoid organs and aplasia of the bone marrow. The similarity of this disease induced in chicks to an early mortality syndrome caused by variant MD virus strains was discussed.     

Protection studies against Marek's disease using baculovirus-expressed glycoproteins B and C of Marek's disease virus type 1

Glycoproteins B (MDV1-gB) and C (MDV1-gC) of Marek's disease virus type 1 (MDV 1) expressed by baculovirus recombinants [rAcNPV(s)] were investigated for their ability to confer protective immunity in chickens against virulent MDV1 challenge. Immunization of chickens with rAcNPV-expressed MDV1-gB induced antibody against MDV1 and resulted in strong protection against the lethal challenge with MDV1. In contrast, immunization of chickens with rAcNPV-expressed MDV1-gC induced antibody against MDV1, but failed to protect against challenge with the virulent MDV1. Co-immunization with both rAcNPV-expressed MDV1-gB and MDV1-gC seemed to show no synergistic effect.     

Detection of Marek's disease virus antigens and DNA in feathers from infected chickens

Two novel tests, enzyme-linked immunosorbent assay (ELISA) and dot-blot hybridization, were developed to detect and quantify the antigens and DNA of Marek's disease virus (MDV) in feather tips from infected chickens. In both methods, buffered extracts of the feathers served as the same test material. The ELISA technique was compared to the conventional agar-gel precipitation (AGP) test, using the same convalescent serum from a MDV-infected bird. Of 86 feather samples tested, 34 were negative by both methods, while 6 out of 52 were ELISA positive but AGP negative. Viral antigen detection by the AGP and ELISA methods was compared with the detection of MDV DNA by the dot-blot DNA hybridization technique. At an ELISA reading (OD 405) of 0.3 and above, only 5 out of 48 DNA extracts failed to hybridize with the MDV-DNA probe. The use of the radioactively labelled MDV-DNA probe for hybridization with DNA extracts from feather tips of MDV-infected chickens was both sensitive and specific, and there was good correlation among the different tests.     

Cell culture amplification of a defective Marek's disease virus

A highly amplified 4-kb EcoRI fragment was present in DNA isolated from high cell culture passaged stocks (>93 passages) of 281MI/1, a serotype 2 Marek's disease virus (MDV). The isolated 4-kb fragment is amplified in the presence of MDV, replicating as a high molecular weight, head-to-tail concatemer. When the 4-kb fragment was cloned into pUC18 and cotransfected with MDV DNA into chicken embryo fibroblast cells, the plasmid clone also replicated as a high molecular weight concatemer.     

Sequential skin lesions in chickens experimentally infected with Marek's disease virus

Skin biopsies taken at weekly intervals from the same specific-pathogen free (SPF) chickens inoculated with Md/5 Marek's disease virus revealed two distinctive patterns of perifollicular cutaneous lesions, tumour-associated and non-tumour-associated. The tumour-associated pattern was subdivided into two types. The progressive type was manifested by a continuous increase of lymphoid cell aggregates (LCA) in the skin, resulting in the development of gross skin tumours with or without visceral tumours, and the regressive type showed initially increased and finally regressed LCA in the skin, associated with the development of visceral tumours. The non-tumour-associated pattern was characterized by initial transient small LCA in the skin without evidence of tumour formation. Birds with the tumour-associated pattern, regardless of type, had persistent nuclear inclusions (NI) and positive reactions against MDV1-specific phosphorylated polypeptides in the feather follicle epithelium (FFE) and initial R(1)-type (consisting mainly of small lymphocytes with a few lymphoblasts) to advanced T-type (consisting predominantly of lymphoblasts) feather pulp lesions (FPL). On the other hand, birds with the non-tumour-associated pattern formed transient NI and positive reactions against MDV1-specific phosphorylated polypeptides in the FFE and Ri-type to R(2)-type (consisting mainly of plasma cells with oedema) FPL. Antigen-positive lymphoid cells against MDV1-specific phosphorylated polypeptides were detected in both inflammatory and tumourous lesions, especially in the necrotic tumour lesions in the skin of birds showing the progressive type.     

Effect of reticuloendotheliosis virus and infectious bursal disease virus on Marek's disease herpesvirus latency

Birds infected with reticuloendotheliosis virus (REV) were exposed to Marek's disease virus (MDV) to determine if the establishment of MDV latency was affected by REV-induced immunosuppression, while other chickens, already latently infected with MDV, were challenged with REV or infectious bursal disease virus (IBDV) to determine if the consequent immunosuppression caused a return to cytolytic infection. Immunosuppression was assessed by in vitro mitogen stimulation assays with spleen cells. Latently MDV-infected cells were free of viral internal antigen(s) (VIA) but could be identified by their ability to produce VIA after in vitro cultivation. The results were unexpected: chickens infected with either of these viruses had very low, and often undetectable, levels of MDV infection when compared with appropriate controls. REV infection interfered with early cytolytic MDV infection, and IBDV and REV both failed to activate latent MDV infection in the face of inferred (IBDV) or demonstrated (REV) immunosuppression by these viruses. Apparently, both viruses reduced the number of MDV infected cells since neither cytolytic nor latent infection could be demonstrated. This was based on an absence of cells with VIA either before or after cultivation and, in the case of REV infection, on failure to detect MDV-DNA using a dot-blot hybridisation technique.     

Immunosenescence and age-related susceptibility to influenza virus in Japanese quail

We evaluated juvenile, pubescent, reproductive adult, and aged Japanese quail (Coturnix coturnix japonica) to determine if there were age-related differences in immune function with the hypothesis that aged birds would have weaker immune responses. Immune responses were measured using phytohemagglutinin (PHA) skin test, antibody response to foreign red blood cells and exposure to an H9N2 influenza virus. Adult birds consistently had stronger immune responses than young and aged birds. Aged quail had skin responses 38% lower than adults. Pubescent birds' mean anti-red blood cell response was four-fold lower than adult birds. Adults had greater increase in total anti-viral antibody between primary and secondary infections than all other groups. Our data demonstrate an age-related difference in immune function in Japanese quail that has similarities to age-related immunity in humans; younger and older animals had weaker immune responses compared to young adults.