A method for the control of lymphoid leukosis in chickens.

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.

Detection of proviral DNA and viral RNA in various tissues early after avian leukosis virus infection.

Using molecular biological techniques, a study was made of the tissue tropism of avian leukosis virus (ALV) early after infection. Two strains of chickens, one with and the other without endogenous viral genes, were infected with ALV of subgroup A immediately after hatching; specimens of nine tissues and blood samples were analyzed at various times thereafter. A polymerase chain reaction (PCR), specific to ALV subgroup A, was used to detect proviral DNA and viral RNA. In situ hybridization was used to confirm the presence of proviral DNA in tissue samples and to calibrate the PCR. The pattern of detection of proviral DNA and of ALV-RNA in the various tissues was similar for both chicken strains. At 2 weeks of age, ALV-RNA was demonstrated in all tissues tested: bursa of Fabricius, thymus, bone marrow, proventriculus, liver, spleen, kidney, muscle, gonads, and blood samples, and at 4 weeks of age all tissues contained proviral DNA. No tropism for a specific tissue was observed early after an ALV infection.

Transcription of the chicken anemia virus (CAV) genome and synthesis of its 52-kDa protein.

This paper describes the expression of the chicken anemia virus (CAV) genome, a recently characterized single-stranded circular-DNA virus of a new type [Noteborn et al., J. Virol. 65 (1991) 3131-3139]. The major transcript from the CAV genome is an unspliced mRNA of about 2100 nucleotides (nt). Its transcription start point and poly(A)-addition site are located at nt 354 and 2317 of the CAV sequence, respectively. In vitro translation experiments provide evidence that the major CAV open reading frame encodes a 52-kDa protein by using the fifth AUG as a start codon of the unspliced CAV mRNA.

The monoclonal antibody CVI-ChNL-68.1 recognizes cells of the monocyte-macrophage lineage in chickens.

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.

Maedi-visna control in sheep II. Half-yearly serological testing with culling of positive ewes and progeny.

In 1979 a field trial was started to study the feasibility of maedi-visna control in sheep by half-yearly serological testing (by ELISA) with culling of sero-positive ewes and their progeny. In 13 commercial flocks, with a mean initial incidence of serological reactors of 17%, the sero-positive ewes and all their progeny, those of preceding years included, were culled after each half-yearly test. The percentage of sero-positive sheep decreased gradually and at the end of the second year, at the 5th test, all flocks were sero-negative. Also the 6th and 7th test did not yield sero-positive sheep. At the 8th test, however, 3 sero-positive ewes were detected in one of the flocks. A definite conclusion as to the source of infection could not be drawn. The following flock test was negative. In 2 other commercial flocks, which had a mean initial incidence of sero-positive sheep of 53%, those sero-positive and only their suckling lambs were culled. Here too, a gradual decrease in the incidence of sero-positive sheep was observed at the 2nd and 3rd test, but at the 4th test a sharp increase occurred. The programme was continued and a decrease followed until 0% was reached at the 7th test (end of third year). Age analysis of the sero-positive sheep which caused this peak revealed that the majority had been born before the start of the trial. This suggests that a 'second wave' of sero-positive sheep may be prevented and a quicker result obtained if progeny of preceding years are culled as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Maedi-visna control in sheep. I. Artificial rearing of colostrum-deprived lambs.

A field trial to study the practicability and efficacy of maedi-visna control in sheep by artificial rearing of lambs was carried out during the lambing season of 1979. Lambs were immediately separated from the dams at birth, deprived of ovine colostrum, and reared isolated from the parent flock. Bovine colostrum was given instead of maternal colostrum. Eleven farms participated in the experiment. All flocks were severely infected with maedi-visna virus: 63-100% of the ewes were seropositive as demonstrated by ELISA. Artificially reared lambs were serologically tested and positives culled at the age of 6, 12, 18, 24, 30 and 36 months. Only very few positives were found: 1/389, 1/376, 0/337, 1/223, 1/192 and 0/144, respectively. The first two sero-positive lambs occurred in one flock, and it could be ascertained that both had mistakenly been given ovine colostrum probably containing maedi-visna virus. No explanation, other than sub-optimal hygiene and isolation, could be found for the two sero-positive sheep that turned up in another flock at 24 and 30 months of age although, transplacental infection cannot be entirely excluded. It is concluded that artificial rearing of ovine colostrum-deprived lambs is an effective and practicable method for the control of maedi-visna in sheep. The method appears particularly useful when valuable genetic material has to be salvaged.

Prevalence of influenza viruses A-H1N1 and A-H3N2 in swine in the Netherlands.

In the period December 1979-May 1980 a respiratory disease spread rapidly through pig herds in The Netherlands. Surveillance of 12 pig farms resulted in isolation of 22 influenza A-Swine-H1N1 (Hsw1N1) strains from 9 pig herds. The morbidity rate was high but the mortality rate was nil. Retardation in growth was observed. Sera collected from affected pig herds showed a fourfold increase in haemagglutination inhibition (HI) titre against A-Swine-H1N1 virus. Sera collected on five farms showed a geometric mean HI titre against the A-H3N2 virus above 100. A significant HI titre increase against this virus was found in sera collected on three farms. These findings indicated a recent infection by this virus. A-H3N2 virus was not isolated. The Dutch Swine-1980 isolates showed in the cross-HI test a distant antigenic relationship with the classical A/Swine/Iowa/30 (H1N1) virus and one-sided close antigenic relationship with A/New Jersey/76 (H1N1) virus. HI antibody to A/Swine/Nederland/80 (H1N1) virus was found in 4, 0, and 44%, to A/New Jersey/76 (H1N1) virus in 0.5, 0.4, and 42%, and to A/Swine/Iowa/30 (H1N1) virus in 0.5, 1, and 30% of pig sera collected in 1976, 1977, and 1980, respectively. HI antibody to A/Hong Kong/68 (H3N2) virus was detected in 36, 56, and 68%, and to A/Victoria/75 (H3N2) virus in 38, 73, and 68% of these sera, respectively. The results of this study indicate that pigs in The Netherlands, like those in North America, Southeast Asia, Japan, and Western Europe harbour A-Swine-H1N1 and A-H3N2 influenza viruses and are thus potential reservoirs for future human pandemics.

Monoclonal antibodies as probes for defining cellular subsets in the bone marrow, thymus, bursa of fabricius, and spleen of the chicken.

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.

Use of milk samples and monoclonal antibodies directed against BLV-p24 to identify cattle infected with bovine leukemia virus (BLV).

An indirect double-antibody sandwich (IDAS) enzyme-linked immunosorbent assay (ELISA) using milk samples was developed to identify cows infected with bovine leukemia virus (BLV). Two monoclonal antibodies (McAbs) were used. One, which was directed against BLV core protein p24, was used to coat ELISA plates; the other was used to prepare a horseradish peroxidase (HRP) conjugate directed against bovine immunoglobulin. The IDAS-ELISA detected antibodies directed against BLV-p24 in 97% of the milk samples collected from known seropositive cows identified by the agar gel precipitation test (AGTP). Even when milk samples were diluted 1:50, 93% of the seropositive cows were identified. Only 0.43% of the 4000 milk samples collected in The Netherlands reacted nonspecifically. Nonspecific binding disappeared, however, when these samples were diluted 50 times in BLV-negative milk. In a comparative evaluation of BLV test-kits in various European laboratories, our IDAS-ELISA using McAb directed against p24 was one of the most sensitive.

Distribution and function of non-lymphoid cells positive for monoclonal antibody CVI-ChNL-68.2 in healthy chickens and those infected with Marek's disease virus.

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.

Construction of a bovine-murine heteromyeloma cell line; production of bovine monoclonal antibodies against rotavirus and pregnant mare serum gonadotrophin.

Bovine-murine heteromyeloma cell lines were prepared by fusing lymphoid cells from a bovine leukemia virus (BLV)-infected cow with mouse myeloma cells. Selection of hybrid cell colonies was based on the ratio of bovine and murine chromosomes, the presence of cell-surface immunoglobulins and growth characteristics. First-generation fusion partners were compared for fusion efficiency and the number of antigen-specific antibody-producing clones generated. Hybrid cell colonies that initially secreted antibodies were selected from first-generation heteromyelomas to function as second-generation fusion partners. Although fusion efficiencies for both generations did not differ, the second-generation heteromyelomas yielded a higher number of specific antibody-producing clones. Fusion of hteromyelomas with either lymph node cells or splenocytes indicated that fusion with lymph node cells results in a higher number of specific antibody-producing clones, whereas fusion efficiency was found to be higher with splenocytes. The optimal time intervals between the final booster injection and fusion were found to be 4 days for splenocytes and 7 days for lymph node cells. Finally, the characterization of bovine monoclonal antibodies against bovine rotavirus and pregnant mare serum gonadotrophin and their neutralizing capacities in vitro are described.

Pathogenicity studies with plaque-purified preparations of Marek's disease virus strain CVI-988.

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.

Protective efficacy of Marek's disease virus (MDV) CVI-988 CEF65 clone C against challenge infection with three very virulent MDV strains.

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)

In situ detection by monoclonal antibody D-35.1 of cells infected with Marek's disease virus that interact with splenic ellipsoid-associated reticulum cells.

Immuno- and enzyme-histochemical staining procedures were used to investigate in vivo the interaction of Marek's disease virus (MDV) with splenic non-lymphoid cells. The newly developed monoclonal antibody D-35.1, which recognizes all three MDV serotypes, was used to study the localization of MDV at various times after intramuscular inoculation of 1-day-old chicks with MDV strain K. The D-35.1-positive cells were detected in the bursa of Fabricius, spleen, thymus, proventriculus, and cecal tonsils, and the number of chickens showing the cells increased between days 4 and 10. From day 21, the skin of the chickens contained D-35.1-positive feather follicles. The D-35.1 monoclonal antibody did not stain any cells in peripheral blood, nerves, kidney, and gonads at any time. In addition, D-35.1-positive cells were not detected in lymphoproliferative lesions in visceral organs and peripheral nerves. Double staining procedures on serial sections using monoclonal antibody CVI-ChNL-68.2, specific for splenic ellipsoid-associated reticulum cells, revealed that the majority of D-35.1-positive cells were situated in the peri-capillary sheath of reticulum cells at day 10. The sheath of cells detected by monoclonal antibody CVI-ChNL-68.2 was disrupted, and they were clustered around D-35.1-positive cells. These results support the hypothesis that ellipsoid-associated reticulum cells are involved in the early pathogenesis of Marek's disease.

Zwoegerziekte virus, the causative agent for progressive interstitial pneumonia (maedi) and meningo-leucoencephalitis (visna) in sheep.

The final results of experimental infections with virus recovered from the lungs of sheep suffering from progressive interstitial pneumonia (=zwoegerziekte=maedi) are reported. The virus could be reisolated from blood samples of all experimentally infected sheep. Every animal produced antibodies against the virus. The neutralising, complement-fixing and precipitating antibodies remained present in the blood for six years. Fourteen out of 21 intrapulmonarily infected sheep developed clinical and/or histopathological lung lesions and in three a meningo-leucoencephalitis was detected in addition. One of these three developed the clinical and pathological signs of 'visna' 14 months after inoculation. Signs of visna were seen in eight of 10 sheep that had been inoculated intracerebrally. Furthermore, nine of these sheep suffered from progressive interstitial pneumonia. Hence the name maedi-visna virus is proposed for the agent which causes both disease entities. Three sheep that yielded virus after infection and in which antibodies were detected, did not develop histopathological lesions.

Studies in epidemiology of maedi/visna in sheep.

Lambs born to ewes from flocks with a high incidence of maedi/visna were separated from their dams at birth (group 1), or after 10 h (group 2), six weeks (group 3) or one year (group 4) and were observed for periods of up to eight years. Group 1 lambs remained free of infection while 28 per cent, 76 per cent and 81 per cent respectively of lambs in the other groups developed clinical, serological or histopathological evidence of infection during the observation period. It is therefore concluded that vertical transmission, if it occurs at all, is of little significance in the epidemiology of the disease. The number of serologically, virologically and histopathologically maedi/visna positive sheep, the time of onset of disease and the severity of lesions were related to the duration of exposure to the parent flock. In a separate trial no evidence was obtained for the transmission of maedi/visna by Muellerius capillaris larvae.

Chicken anaemia virus influences the pathogenesis of Marek's disease in experimental infections, depending on the dose of Marek's disease virus.

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.

Biological characteristics of Marek's disease vaccine CVI-988 clone C1.

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.

Interference of maternal antibodies with the immune response of foals after vaccination against equine influenza.

The purpose of the study was twofold. First, using two groups of 22 foals each, we investigated the extent to which maternal antibodies interfere with the humoral response against equine influenza. The foals were born to mares that had been vaccinated twice yearly against influenza since 1982. Foals of group I were vaccinated three times at early ages (12, 16, and 32 weeks of age), and foals of group II were likewise vaccinated but a later ages (24, 28, and 44 weeks of age). After the first and second vaccinations, neither group showed an increase in antibodies that inhibit haemagglutination. Group II foals, however, had a significantly stronger antibody response against nucleoprotein after the second vaccination than the foals of group I. After the third vaccination, group II foals had a significantly stronger and longer lasting antibody response against haemagglutinin than the foals of group I. However, the antibody response to nucleoprotein was comparable in both groups. Second, the foals of group II were studied to determine the persistence of maternal antibodies directed against a common nucleoprotein and the haemagglutinin of two strains of equine influenza A virus. Biological half-lives of 39, 32, and 33 days were calculated for maternal antibodies directed against haemagglutinin of strains H7N7 Prague and H3N8 Miami, and against the nucleoprotein respectively. Maternal antibody titres at the time of vaccination were closely related to the degree of interference with the immune response. Because even small amounts of maternal antibodies interfered with the efficacy of vaccination, we conclude that foals born to mares vaccinated more than once yearly against influenza virus should not be vaccinated before 24 weeks of age.

Inactivation of chicken anaemia virus in chickens by heating and fermentation.

The transmission of pathogenic microorganisms such as viruses by the use of animal products in animal feed constitutes a potential risk to the health of livestock. To reduce the risk, it is necessary to understand the survival of viruses during the processing of animal products to feed-stuffs. Since chicken anaemia virus (CAV) is very resistant to inactivation, we used it as a model for the inactivation of pathogenic viruses during treatment of animal products. It is concluded that fermentation of CAV viraemic tissue did not affect the inactivation of CAV, however, heating at a core temperature of 95 degrees C for 30 min or 100 degrees C for 10 min is sufficient to inactivate CAV. Compared with the conditions for inactivation reported in the literature for other pathogenic viruses, our treatment is more stringent. CAV viraemic chickens are thus suitable as a model to test the heat inactivation of pathogenic viruses.