Dose-dependent inhibition of virus rescue from lymphocytes latently infected with turkey herpesvirus or Marek's disease virus

The number of plaque-forming units (PFU) of turkey herpesvirus (HVT) isolated per 10(6) latently infected splenic lymphocytes was determined by co-cultivation on permissive monolayer cultures in 35-mm-diameter Petri dishes. Doses of 1 x 10(6) spleen cells or less per culture gave uniform dose-related titers, whereas doses of 8 x 10(6) cells often yielded less than 1-2% of the expected number of PFU. Intermediate doses gave proportionally reduced virus yields. This dose-dependent inhibition was observed with spleen cells from birds within a week after infection and became more marked with time. A similar phenomenon occurred with a non-oncogenic Marek's disease virus (MDV) isolate (SB-1) but not with oncogenic MDV isolates (CU-2, JM-10, GA-5), except in genetically resistant birds. High numbers of uninfected spleen cells mixed with low numbers of HVT-infected cells during assay reduced titers only slightly. Immunosuppression by combined neonatal thymectomy and cyclophosphamide treatment before HVT infection prevented the inhibition, but embryonal bursectomy had no effect.     

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.     

A simple in vitro differentiation between turkey herpesvirus and Marek's disease virus

The growth and plaque formation by turkey herpesvirus (HVT) amd Marek's disease herpesvirus (MDHV) were examined in QT35 cells, a continuous fibroblast cell line derived from chemically induced tumors of Japanese quail. HVT grew and formed plaques consistently in QT35 cells when inoculated with cell-culture-propagated virus or peripheral mononuclear leukocytes (PML) from chickens that had been inoculated with HVT. Both oncogenic and nononcogenic strains of MDHV, however, failed to grow and induced neither plaques nor cytopathic effects in QT35 cells, whether inoculated with cell-culture-grown virus or heavily infected PML. When PML from chickens infected with both HVT and MDHV were assayed, only HVT plaques had developed, despite the presence in the inocula of high levels of MDHV with less HVT. The QT35 cell line provides a simple in vitro system for differentiating between HVT and MDHV and for selective isolation and identification of HVT from chickens infected with both HVT and MDHV.     

Effect of acyclovir on the replication of turkey herpesvirus and Marek's disease virus

The toxicity of acyclovir for chick embryo fibroblasts and its effect on the replication of turkey herpesvirus (strain FC 126) and Marek's disease virus (strain HPRS 16) multiplied on fibroblast culture was studied. The influence of using acyclovir on the development of the tumour process in birds infected with a virulent Marek's disease virus was also determined. Acyclovir used in doses below 12.5 micrograms ml-1 proved to be nontoxic for chick embryo fibroblast culture. It inhibited in vitro replication of turkey herpesvirus and Marek's disease virus. It was also shown to diminish the development of tumours in birds infected with Marek's disease virus.     

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.     

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.     

Detection of chicken anemia virus DNA in the thymus of naturally infected chicks in turkey.

In this study, 94 clinically ill 11-28-day-old chicks belonging to eight broiler units from the Marmara region were investigated clinically for changes in hematocrit values and for the presence of chicken anemia virus (CAV) DNA. CAV DNA was detected by polymerase chain reaction in the thymus of 8.5% of the chicks. These chicks showed clinical signs of diarrhea, anorexia, depression, and growth retardation. The hematocrit values of these chicks were between 24% and 38%. At necropsy, hemorrhages were observed in the leg and pectoral muscles. Atrophy was noted in the thymus and in the bursa of Fabricius of positive chicks, and hemorrhages in the proventriculus of one positive chick were observed. This report describes the first detection of CAV DNA in chicks in Turkey.     

Replication of turkey herpesvirus and Marek's disease virus in chick embryo skin organ culture.

Turkey herpesvirus (HVT) and an attenuated Marek's disease virus (MDV) replicated in organ cultures of chick embryo skin as assessed by immunofluorescence and/or electron microscopy. HVT-specific immunofluorescent antigen was detected in the feather follicle epithelium (FFE) and in the surface layer of the skin epidermis. Electron microscopy of infected explants revealed herpes-type cytopathology. Immature particles of both viruses appeared first in the nucleus. Oval or horseshoe-shaped non-enveloped particles of HVT and enveloped virions of MDV were seen in the cytoplasm of some transitional cells. The difference in the ability of HVT and MDV to form an envelope was believed to account for the difference in their transmissibility in chickens. The results indicated that HVT replicated in the FFE and in the epidermis of the skin. However, attempts to localise the site(s) of MDV replication by electron microscopy were unsuccessful.     

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.     

Persistence of inclusions and antigens of chicken anemia virus in Marek's disease lymphoma

The pathogenesis of the co-infection of CAV to MDV is complicated. In order to investigate the impact of CAV on the transformation phase of MD, MDV and, subsequently, CAV, were inoculated at 1day and 4weeks of age, respectively. Chickens were divided into six groups; vvMDV, vvMDV-CAV, vMDV, vMDV-CAV, CAV and a control group. The CAV inclusions and antigens were continuously detected in MD lymphomas in the vMDV-CAV and vvMDV-CAV groups in large bizarre-shape (presumably CD4(+) T cells) and small MD lymphoid cells (presumably CD8(+) T cells). The MD lymphomas were composed primarily of CD4(+) T cells, but CD8(+) T cells were infiltrated singly or in clusters. CAV enhanced the MDV-induced brain lesions in the vMDV-CAV group. The lymphoproliferative lesion (LP) in the vvMDV-CAV and vMDV-CAV groups was non-significantly higher than those in vvMDV and vMDV groups, respectively. CAV significantly increased the LP lesion in sciatic nerves. In conclusion, MD lymphomas enabled CAV replication and dissemination. The depletion of CTLs by CAV did not significantly affect progression of MD lymphoma, although they are essential for possible transition of lymphomatous to inflammatory lesion.