G.F. de Boer

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.

Prevalence of antibodies to equine viruses in the Netherlands

Summary The prevalence of antibodies to various viruses was investigated in a series of serum samples collected from horses in the Netherlands between 1963 and 1966 and from 1972 onwards. Neutralizing antibodies to equine rhinopneumonitis virus, equine arteritis virus and to equine rhinovirus types 1 and 2 were detected in respectively 76%, 14%, 66% and 59% of the equine serum samples tested. The observed incidence of serum samples positive to equine adenovirus in the complement fixation test was 39%. Precipitating antibodies to equine infectious anaemia virus were detected only in serum samples from two horses imported from abroad. Haemagglutination inhibiting antibodies to Myxovirus influenzae A / equi-1, M. Influenzae A / equi-2, and Reovirus types 1, 2, and 3 were present in respectively 82%, 50%, 10%, 33% and 3.6% of the serum samples tested. The most frequently observed incidence of antibodies to the various equine respiratory viruses occurred in the groups of horses having repeatedly contact with other horses.

Detection of chicken anaemia virus by DNA hybridization and polymerase chain reaction.

A clone containing the complete genome of chicken anaemia virus (CAV) was used in hybridizations with DNA from various field isolates of CAV. CAV DNA from all field isolates was detected in a polymerase chain reaction with oligonucleotides derived from the sequence of the cloned CAV DNA as primers. By way of Southern blot analysis with (32)P-labelled DNA probes derived from cloned CAV DNA, all field isolates were shown to contain DNA molecules of about 2.3 kb, i.e. the size of cloned CAV DNA. In a dot-blot assay it was demonstrated that non-radioactively-labelled cloned CAV DNA hybridized specifically to DNA from field isolates. The cloned CAV DNA is highly similar to the DNA of field isolates, as borne out by restriction-enzyme mapping. We conclude that our cloned CAV genome is representative for CAV in the field. The described PCR and hybridization techniques, may, therefore, be used for research and diagnosis of CAV infections.

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 Mareks 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.

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.

The use of ELISA for detection of exogenous and endogenous avian leukosis viral antigens in basic breeding flocks.

An enzyme-linked immunosorbent assay (ELISA) for avian leukosis/ sarcoma virus (ALV) group-specific (gs) antigens was used to study the identification of hens which congenitally excrete exogenous ALV. The sensitivity of this assay was compared with that of the phenotypic mixing test (PMT) and the direct complement fixation test (CFT) by testing limiting dilutions of purified avian myeloblastosis virus (AMV), embryo homogenates and albumens. About 0.4 ng/ml of purified AMV protein could be detected by ELISA and 23.8 ng/ml of AMV protein was demonstrable by the CFT. The lowest levels of gs-antigen detection corresponded with about 100 median tissue culture infectious doses (TCID50) of infectious ALV. Albumens and embryos of three White Leghorn flocks and one White Plymouth Rock flock were tested for the presence of avian leukosis virus (ALV) gs-antigens by the complement fixation test (CFT) and the enzyme-linked immunosorbent assay (ELISA) and exogenous ALV employing the phenotypic mixing test (PMT). The highest number of ALV-gs antigen positive samples of egg albumens was obtained by the ELISA. In embryo homogenates prepared from eggs of the four flocks under study 100% scores for gs-antigens were obtained by ELISA. A differentiation between gs-antigens of endogenous and exogenous ALV was made by testing of supernatant fluids after one chick embryo fibroblast passage of the C/E phenotype. Endogenous viral (ev) genes leading to the expression of endogenous ALV gs-antigens were apparently present in practically all chickens of the four flocks under study. Complete endogenous ALV (subgroup E) was detected in embryos (41%) from the White Plymouth Rock grandparent flock, but was not found in embryos of two White Leghorn basic breeder flocks. The results indicate that both flocks of White Leghorn chickens are endowed with ev 3 genes. Circumstantial evidence was obtained for the prevalence of endogenous ALV gs-antigens in albumen samples of eggs from flocks with gs+chf+ and V-E+ phenotype respectively.

Application of monoclonal antibodies in the avian leukosis virus GS-antigen ELISA.

Five hybridoma cell lines which secrete antibodies to avian leukosis/ sarcoma (ALV) group-specific (gs) antigens (gag gene products) have been established. The hybrid cells resulted from fusion of P3/X63-Ag8.653 myeloma cells with splenocytes of BALB/c mice which had been immunised with purified avian myeloblastosis virus (AMV). Screening of supernatant fluids was performed by an indirect double antibody sandwich enzyme-linked immunosorbent assay (IDAS-ELISA) and the immunoelectroblotting technique. Three hybrid clones secrete monoclonal antibodies (MCA) to 27,000 dalton polypeptides (p27) and two hybridomas produce monoclonal antibody directed to 19,000 dalton phosphoprotein (pp19). All five monoclonal antibodies belong to mouse immunoglobulin isotype IgG(1). MCAs directed to different ALV-p27 epitopes can replace polyclonal rabbit or hamster anti-gs sera in the DAS-ELISA in use for the detection of congenitally ALV-shedding hens. In albumen samples a 16- to 32-fold increase of sensitivity of ALV gs-antigen detection was obtained as compared to the DAS-ELISA employing polyclonal sera. Gs-antigens of a purified AMV preparation were detectable up to minimal concentrations of 13 pg/ml.

Comparison of complement fixation and phenotypic mixing tests for the detection of lymphoid leukosis virus in egg albumen and embryos of individual eggs

Individual eggs, collected from 14 hens over a period of 10 weeks, were tested for lymphoid leukosis (LL) virus by four methods. The chickens were selected from a conventional flock on the basis of virus recovery from embryos 3 months earlier. Albumen samples and extracts of embryos were examined by complement fixation (CFT) and phenotypic mixing tests (PMT). Most eggs positive for LL virus (LLV) and/or group specific (gs)-antigen were detected by testing of embryo extracts by PMT and CFT. Examination of albumens yielded less LLV and gs-antigen positive eggs. However, because a few birds produced eggs with predominantly gs-antigen in the albumen and less frequently with virus in the embryos from the same eggs, the combination of CFT on both albumen and embryo extract proved to be the most sensitive detection system. CFT on both albumen and embryo are easier to perform than PM tests and therefore may be useful in LL eradication programmes. The majority of the hens intermittently transmitted virus and/or gs-antigen to embryos. The results of this study indicate that congenital transmission patterns of LLV infections may be different in individual birds.

On epizootiology and control of lymphoid leukosis in chickens

Abstract Sera and organ extracts from ten different commercial stocks of layer chickens were examined for the presence of lymphoid leukosis (LL) viruses. Virus was recovered from 40.8% of the cockerels between three and six weeks of age. Their female hatch mates were examined at the age of 20 months. A mean of 11.3% of these laying hens was positive in the NP activation test. Lymphoid leukosis was successfully controlled in three inbred strains of White Leghorn chickens and in a commercial White Plymouth Rock line. All flocks were kept in a filtered air positive pressure (FAPP) house during the first two months of life and thereafter transferred to a conventional environment. The control method is based on three elements: • —from an infected flock, hens are selected in whose eggs no avian lymphoid leukosis viruses can be detected by examination of pooled extracts of groups of embryos; • —only eggs from hens that are shown not to shed congenitally virus in their eggs are used for the production of progeny. The offspring are reared in isolation until two months of age at which time the age-related resistance against tumour formation appears to be sufficiently developed; • —the chickens are subsequently intramuscularly inoculated with lymphoid leukosis viruses of subgroups A and B and transferred to a conventional chicken house. The inoculated birds become persistently viremic and resist horizontal virus exposure and intramuscular challenge infections. Horizontal virus transmission was observed to take place when virus-free non-vaccinated chickens were reared in isolation for two months and then exposed under field conditions. Efficiency of virus recovery was considerably improved when washed buffy coat cells were cocultivated with chick embryo fibroblasts or explant cultures were prepared from various tissues before testing with the NP activation test.

Age related resistance to avian leukosis virus. III. Infectious virus, neutralising antibody and tumours in chickens inoculated at various ages

Viraemia and neutralising antibodies were determined in chickens of six age-groups following inoculation with leukosis virus of subgroups A and B at the age of 1 day, and 2, 4, 6, 8 and 10 weeks respectively. The birds were kept in a filtered air positive pressure (FAPP) house. A seventh age-group, accommodated in a separate FAPP-house, was used as an untreated control. Serum samples, received at biweekly intervals between 1-17 weeks post-inoculation, from birds of the groups inoculated at 4, 6, 8 and 10 weeks of age, showed at 1 week post-inoculation a transient viraemia followed by neutralising antibodies at the later sampling times. Neutralising antibody to subgroup A virus was detected in nearly all birds tested; this was not so for antibody to subgroup B. In all four groups the average titre of the former antibody was higher than that of the latter. Midway through the laying period birds of each group inoculated with leukosis virus, and some of the uninoculated controls, were challenged by infection with either subgroup A or B virus. At termination of the experiment survivors from each group were tested for the presence of leukosis virus. The virus recovery was performed with plasma samples, white blood cell preparations and explant cultures of various organs. The plasma samples were all negative; the great majority of blood cell specimens received from birds inoculated early with leukosis virus were positive, whereas the majority of the preparations from the birds inoculated later remained negative. The organ explants from the two youngest age groups were mostly leukosis virus-positive, from the birds inoculated at 4 weeks of age the spleen and kidney explants contained leukosis virus whereas in the groups inoculated at 6, 8 and 10 weeks of age only the spleen explants of birds challenged with subgroup A virus In a subsidiary experiment, started 4 months after the challenge infection, four birds from each group (two challenged with leukosis virus of subgroup A and two with subgroup B) were accommodated in isolators. The birds were challenged again, this time with Rous sarcoma virus (RSV) of the homologous subgroup used for the previous challenge. The tests for virus just prior to the challenge showed leukosis virus only in the white blood cell preparations from the birds in the three youngest age groups; the birds from the older groups were virus-negative. The serological tests after challenge showed neutralising antibodies to both subgroups in birds of nearly all groups. Tumour formation at the site of injection was mainly observed in the chickens challenged with RSV of subgroup B. The virological and serological results as well as the tumour response show that the immune system of birds between 0-4 weeks of age is insufficiently developed to cope with a controlled exposure with leukosis virus, whereas in birds of 4-10 weeks of age an adequate immunological response has developed. The significance of the presence of leukosis virus in sera, plasma, white blood cell preparations and organ explant cultures is mentioned. In programmes for the control of lymphoid leukosis in reproductive stock the use of information on virus and neutralising antibodies is recommended.