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Eight groups of 1-day-old chickens were inoculated with 0, 250, 5000, or 100,000 white blood cells of chickens infected with Marek's disease virus strain K (MDV-WBC). Four of these groups were additionally infected with 10(5) TCID50 chicken anaemia virus (CAV). At day 14 after inoculation, chickens infected with CAV had reduced haematocrit levels, reduced body weights, and depletion of the thymic cortex and bone marrow. Semi-quantitative immunohistochemical examination of nerves and visceral organs was performed at day 28 by immunoperoxidase staining in which a monoclonal antibody specific for leucocytes was used. CAV significantly enhanced the number of lymphoproliferative lesions induced by 5000 MDV-WBC. In contrast, CAV significantly reduced the number of lymphoproliferative lesions induced by 100,000 MDV-WBC. Comparable results were found at day 61 after macroscopic examination of nerves and visceral organs. These findings show that the pathogenesis of MD in experimental infections appears to be enhanced or inhibited by CAV, depending on the dose of MDV.

To investigate the age-dependent mechanism of susceptibility for chicken anemia virus (CAV) infection, we inoculated embryos and chickens of ages between day 9 of embryonic development and day 28 after hatching with CAV. Chicken embryos inoculated at days 9 and 11 of development showed no CAV-infected cells in the thymus, nor in other lymphoid organs. Many CAV-infected cells were detected in the thymic cortex of all chicken embryos inoculated at days 13 and 16 of development and of all chickens inoculated 1, 3, and 7 days after hatching. All embryos and chickens that contained CAV-infected cells in the thymus also contained CAV-infected cells in the bone marrow, but not in the bursa of Fabricius or the spleen. In chickens inoculated at days 14 and 21, only few CAV-infected cells were detected in the thymus, whereas these cells were not detected in thymi of 28-day-old inoculated chickens. Depletion of the thymic cortex was only detected in chickens inoculated from day 16 of embryonic development till day 21 after hatching. Only hematocrit values of the chickens inoculated 1 and 3 days after hatching were below normal. The rationale for the simultaneous susceptibility of cells of the T-cell lineage and cells of the erythrocyte lineage is discussed. As far as the thymus is concerned, the absence of clinical and microscopical signs of CAV infection in older chickens and the inability of CAV to infect embryos at days 9 and 11 of embryonic development may be caused by a lack of susceptible thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular double-stranded replication intermediates were identified in low-molecular-weight DNA of cells of the avian leukemia virus-induced lymphoblastoid cell line 1104-X-5 infected with chicken anemia virus (CAV). To characterize the genome of CAV, we cloned linearized CAV DNA into the vector pIC20H. Transfection of the circularized cloned insert into chicken cell lines caused a cytopathogenic effect, which was arrested when a chicken serum with neutralizing antibodies directed against CAV was added. Chickens inoculated at 1 day of age with CAV collected from cell lines transfected with cloned CAV DNA developed clinical signs of CAV. The 2,319-bp cloned CAV DNA contained all the genetic information needed for the complete replication cycle of CAV. The CAV DNA sequence has three partially overlapping major reading frames coding for putative peptides of 51.6, 24.0, and 13.6 kDa. The CAV genome probably contains only one promoter region and only one poly(A) addition signal. Southern blot analysis using oligomers derived from the CAV DNA sequence showed that infected cells contained double- and single-stranded CAV DNAs, whereas purified virus contained only the minus strand. It is the first time that the genome of one of the three known single-stranded circular DNA viruses has been completely analyzed.

Transgenic mice are produced by retroviral insertion, micro-injection in the early embryo, and recently by transfection of embryonic stem cells. Transgenic chickens were only made by retroviral vectors based on avian leukemia virus (ALV) and reticuloendotheliosis (REV) genomes. A replication-defective retroviral vector is preferentially used because these do not induce infectious virus. Since chickens are lacking endogenous REV sequences, a replication defective REV vector is most useful for practical application. Transgenic disease resistance is most likely obtained by blocking of viral receptors. By this approach recently transgenics with resistance against ALV infection were made at the Regional Poultry Disease Laboratory in East Lansing. Inhibition of virus replication by antisense DNA, which is complementary to viral mRNA, is promising for the future. Considerable research efforts still have to be made, however. Production of biomedical proteins will most likely be the first practical use of transgenic chickens. For the time being, vaccines will be used for the control of infectious diseases. The current live-virus vaccines will be replaced by inactivated (sub-unit) vaccines and thereafter by recombinant DNA vaccines based on viral vectors.

The pathogenicity of Marek's disease (MD) strain CVI-988 vaccine, eight plaque-purified preparations originating from this strain, and the vaccine HVT FC126 (based on herpesvirus of turkeys) was determined by intramuscular administration of high virus doses to day-old specific-pathogen-free Rhode Island Red (RIR) chickens, which are extremely MD-susceptible. Paralysis and neuritis were observed in 88% of RIR chickens inoculated with MDV CVI-988 at the cell-passage level of the commercial vaccine. HVT FC126 caused paralysis in two of 39 RIR chickens tested, of which one had an endoneural lymphoma, and another three had endoneural inflammation. Five plaque-purified MDV CVI-988 virus preparations at various cell-culture-passage levels caused no lesions. Of another three clones, two caused inflammatory B-type lesions in the nerves of 1/10 chickens, and the third clone caused inflammatory nonneoplastic MD lesions in the liver of 1/11 chickens.

Two field trials were performed to study possible effects of the lymphoid leukosis (LL) control programme as carried out in the Netherlands, on production characteristics. Progeny groups of selected ALV-negative, congenitally non-shedding, hens and progeny of conventional flocks were divided into flocks either to be reared in conventional conditions or in isolation for a period of eight weeks followed by controlled exposure (vaccination) with ALV. Detrimental effects on production characteristics attributable to the LL control programme were not observed in the two trials. The LL control procedure was successful in the prevention of lymphomas and elimination of congenital ALV shedding. Differences in egg production between LL control groups and conventional flocks were not observed. This was presumably due to the absence or too small a number of ALV-infected hens in the conventional flocks. Relatively low immunoglobulin class IgA levels were observed in serum samples of chickens which were reared in isolation for a period of eight weeks. Quantitative differences in immunoglobulin classes IgG and IgM were not perceptible between samples from chickens which were reared in isolation and those reared in conventional conditions.

Comparative 50% protective dose (PD50) assays were performed using a plaque-purified preparation of Marek's disease virus (MDV) strain CVI-988 at the 65th chicken embryo fibroblast (CEF) passage level (MDV CVI-988 CEF65 clone C) and three commercial MD vaccines: herpesvirus of turkeys (HVT) FC126, MDV CVI-988 CEF35, and a bivalent vaccine composed of HVT FC126 and MDV SB-1. In addition, comparative PD50 assays were performed in groups of chickens with maternal antibody to each of the three vaccines. Three representatives of the newly emerged biovariant very virulent (vv) MDV strains-RB/1B, Tun, and Md5-were employed as challenge virus. The experiments made feasible the differentiation between virulent MDV and vvMDV strains, within serotype 1. Vaccination with CVI-988 clone C vaccine resulted in PD50 estimates of about 5 plaque-forming units (PFUs) against challenge infection with each of the three vvMDV strains. The PD50 estimate of CVI-988 clone C vaccine was 12-fold below the PD50 of HVT FC126. The protective synergism of bivalent vaccine, composed of HVT and SB-1, was confirmed by groups given the lowest vaccine doses. The bivalent vaccine, however, resulted in incomplete protection in groups given the highest vaccine doses. Homologous maternal antibodies to serotype 1 caused a fivefold increase in the PD50 estimate of CVI-988 clone C. Heterologous maternal antibodies against HVT did not interfere with efficacy of CVI-988 clone C vaccination. However, the combination of maternal antibodies against both HVT and SB-1 (serotypes 2 and 3) showed a strong adverse effect on CVI-988 clone C vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphoid leukosis (LL) was eliminated from 3 inbred lines of White Leghorn chickens that were temporarily kept in isolation. The method of control was based on three elements: 1) From an infected flock we selected hens that produced LL virus-free eggs. Pooled extracts from groups of embryos were tested for vertical virus transmission by the nonproducer cell activation test. 2) Only eggs from dams that did not congenitally shed virus to their embryos were used to produce progeny. The offspring were reared in isolation for 2 months (at which time the age-related resistance against tumor formation had developed sufficiently). 3) The birds were subsequently inoculated im with 10(5) mean tissue culture infective doses of LL viruses of subgroups A and B and transferred to a conventional chicken house. The controlled exposure provided immunity against LL virus infection in a highly infect environment. This procedure resulted in birds a) with no boservable LL, and b) producing LL virus-free eggs. To obtain flocks that had no LL and produced virus-free progeny, the procedure had to be repeated for at least two generations due to the intermittent virus excretion that often occurred.

The characteristics of monoclonal antibody CVI-ChNL-68.1, which specifically reacts with a group of chicken non-lymphoid cells, are described. Both tissue distribution shown on cryostat sections using immuno-enzyme histochemistry, and quantitative data obtained on cell suspensions are presented. Functional characteristics of CVI-ChNL-68.1-positive cells, such as antigen uptake and glass adherence, are determined. Results show that CVI-ChNL-68.1 reacts with monocytes, macrophages, and interdigitating cells. Possible relationships between the various non-lymphoid cells are discussed.

Immuno-enzyme histochemistry was used to study the staining pattern and tissue distribution of monoclonal antibody CVI-ChNL-68.2 that specifically reacts with a subset of non-lymphoid cells in healthy chickens and those infected with Marek's disease virus (MDV). Functional characteristics of CVI-ChNL-68.2-positive cells, e.g. antigen uptake, are determined. In the liver CVI-ChNL-68.2 recognizes reticulum cells, whereas in the bursa of Fabricius it detects single cells in the interfollicular connective tissue. In the spleen CVI-ChNL-68.2 reacts selectively with the reticulum cells of the ellipsoid. In some MDV-infected chickens the splenic reticulum cells show a different staining and distribution pattern. Furthermore, the proliferative lesions associated with Marek's disease contain many CVI-ChNL-68.2-positive cells. The possible role of CVI-ChNL-68.2-positive cells in disseminating Marek's disease virus is discussed.

Biological characteristics of Marek's disease virus (MDV) CVI-988 clone C, of importance for vaccine application, are described. CVI-988 clone C was shown to be nonpathogenic for highly MD-susceptible chickens and slightly more effective than prototype CVI-988 vaccine. During plaque purification and serial cell-culture passages, reductions were observed in the release of 'A' antigen from infected cell cultures, in spreading properties and in virus replication in vivo. Pre-licensing batches of CVI-988 clone C vaccine afforded excellent protection against challenge infection with virulent MDV and highly virulent MDV strains. Groups of chickens with bivalent (HVT/SB-1) vaccine-induced maternal antibodies were equally protected by a double dose of CVI-988 clone C vaccine. Field trials performed in the Netherlands and in the United States confirmed the safety and protective efficacy of monovalent CVI-988 clone C vaccine.

The postnatal development of chicken mucosa-associated lymphoid tissues of the eyes, lungs, and intestines were investigated with monoclonal antibodies specific for either all leucocytes, B lymphocytes, mononuclear phagocytes, IgM, IgG, or IgA. Attention has been paid to the relation of lymphoid infiltrates with their surrounding mucosae, the segregation into B-cell and T-cell areas, development of germinal centers, and secretory immunoglobulins. Abundant secretory IgM and IgA was detected in the epithelium of the Harderian glands in the orbits, even though they lacked large leucocyte infiltrates with germinal centers. Lymphoid tissues in the mucosae of lungs and intestines developed separate B-cell and T-cell areas. The proventriculus, Meckel's diverticulum, and Peyer's patches generally contained germinal centers from 12 weeks of age on. Because chickens as young as 2 weeks old had germinal centers in bronchus-associated lymphoid tissue and cecal tonsils, these areas were probably highly stimulated by antigens. Isotype-specific monoclonal antibodies were used to detect IgM-, IgG-, and IgA-bearing follicular cells in the same germinal center.

Using immunohistochemistry, the distribution and characteristics of cells detected by the newly developed monoclonals HIS-CI (B lymphocytes), HIS-C7 (leucocytes), HIS-C12 (IgM), CVI-ChIgM-59.7 (IgM), CVI-ChIgG-47.3 (IgG), and CVI-ChIgA-46.5 (IgA) are described in bone marrow, thymus, bursa of Fabricius, and spleen of chickens of different ages. Furthermore, quantification of cells positive with the described monoclonal antibodies was performed on cytocentrifuge preparations. The specificities of the monoclonal antibodies are discussed.