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The development of an alternative in vitro potency test required experimental studies, which were performed in-house and in collaboration with other laboratories (Official Medicines Control Laboratories, Manufacturers), coordinated by EDQM (European Directorate for the Quality of Medicines & HealthCare). This paper provides background information concerning the development of the quantitative ELISA for inactivated Newcastle disease virus (NDV) antigen, which was added in the European Pharmacopoeia monograph as an in vitro batch potency test.

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The correlation between the antigen content of inactivated Newcastle disease (ND) oil emulsion-vaccines and the serological response after immunisation was studied. The haemagglutinin-neuraminidase (HN) and fusion (F) proteins of Newcastle disease virus (NDV) were quantified in 33 inactivated oil-adjuvanted ND vaccines using isopropylmyristate (IPM)-extraction and antigen ELISAs. These commercial vaccines differed in NDV-vaccine strain, the method applied to inactivate the vaccine virus, the vaccine valence and the composition of the oil emulsions. Large differences, up to 100-fold, in the antigen quantities present in different vaccines were found. The NDV-HN content and the NDV-F content of the vaccines both highly correlated with the haemagglutination-inhibiting (HI) antibody titres after immunisation. These correlation's were found over a 10,000-fold range of applied antigen. The antigen content of the oil emulsion-vaccines also highly correlated with virus neutralising antibody titres. The presence of serum HI antibody titres in individual specific pathogens free (SPF)-chickens after immunisation with inactivated ND vaccines was highly indicative for clinical protection against challenge with the virulent NDV-Herts strain. Our results are the first to demonstrate that the protective serological response after immunisation highly correlates with the antigen content of oil-adjuvanted vaccines.

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The efficacy of inactivated infectious bursal disease vaccines was determined by measuring both the antibody response of vaccinated chickens and clinical protection of progeny chicks from vaccinated dams. Similar virus neutralizing (VN) antibody titres were obtained in 4-week-old chickens and mature hens after vaccination with one vaccine dose. VN titres below 10 log 2 increased considerably between the fourth and seventh week after vaccination in 4-week-old chickens as well as in mature chickens. All 2-week-old progeny chicks with serum VN antibody titres of at least 9 log 2 were clinically protected against the classical virulent 52/70 infectious bursal disease virus (IBDV) strain, as well as against the very virulent IBDV (vvIBDV) strain D6948. However, vaccination often did not prevent subclinical infection in these 2-week-old progeny chicks, which often resulted in severe lymphocyte depletion in the bursa of Fabricius. Even a serum VN titre of 11 log 2 was not always sufficient to prevent severe bursal damage. Although 52/70 IBDV and vvIBDV were equally pathogenic in 2-weekold specific pathogen free chickens, significant higher maternal antibody titres were required to prevent the adverse effects of vvIBDV in comparison with 52/70 IBDV. The relation between the serological response of chickens after application of inactivated IBD vaccines and the protection of progeny chicks of vaccinated dams depended on both the virulence of the IBDV challenge strain and the IBDV strain in the vaccine.

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Routine batch control of licensed inactivated viral vaccines for poultry usually includes a potency assay as a measure of vaccine efficacy. Potency assays often consist of vaccination-challenge experiments in the target species or in laboratory animals. Instead of measuring the protection of vaccinated animals against virulent pathogens, the serological response after vaccination can be quantified for some vaccines. In vitro antigen quantification assays would be attractive alternatives for the current potency assays because the time and costs involved could be greatly reduced and animal use could be avoided. Such in vitro assays will only be acceptable when the correlation between results and efficacy or potency has been demonstrated convincingly. The results of our studies on antigen quantification assays indicate that, in principle, quantification of viral antigens from inactivated oil-adjuvanted vaccines is feasible and reproducible using specially developed antigen capture ELISAs in combination with specific software for statistical analysis of the ELISA data. We have developed methods to quantify the haemagglutination-neuraminidase (HN) and fusion (F) proteins of Newcastle disease virus (NDV), the viral protein 3 (VP3) of the infectious bursal disease virus (IBDV), and the spike-1 (S1) protein of the infectious bronchitis virus (IBV). Vaccination experiments with inactivated ND vaccines indicate that the in vitro quantified HN- or F-proteins of NDV are reliable indicators of the serological response after vaccination.

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Knowledge of the dose-response relation of inactivated vaccines and of the factors that influence this relation is essential for the evaluation of existing vaccine potency assays and the development of new potency assays that are based on the antigen content of the inactivated vaccines. We quantified the relation between vaccine dose, serologic response, and clinical protection after vaccination for three different inactivated Newcastle disease (ND) vaccines. Qualitatively, similar dose-response curves were obtained for the three vaccines when either the serologic response or the clinical protection of specific-pathogen-free (SPF) chickens was plotted against the different vaccine doses applied. However, the vaccines differed quantitatively: doses of vaccines that induced similar antibody titers or clinical protection differed 2-8-fold. In contrast with the narrow range of antibody titers induced by a full vaccine dose, a very broad range of titers was obtained after dilution of the vaccines. At least 95% of the SPF chickens with detectable antibody in the serum were protected against a challenge with virulent Herts ND virus. The relation between the dosage of two different ND vaccines and the serum antibody titers remained markedly constant between 3 and 18 wk after vaccination. Vaccination of broilers instead of layers with a dilution series of inactivated ND vaccine resulted in significantly lower antibody levels and less clinical protection against virulent challenge. In conclusion, despite quantitative differences, we found comparable dose-response relations for the three inactivated ND vaccines studied.

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We evaluated the influence of the use of the Newcastle disease virus (NDV)-strains Ulster and La Sota in the haemagglutination inhibition (HI) assay for the measurement of antibody titres after NDV vaccination. The use of the homologous La Sota antigen in the HI assay after Clone30 and La Sota vaccination of SPF-chickens, resulted in significantly higher titres than the use of the heterologous Ulster virus. The mean difference was 1.4 on a log 2 scale (2.6-fold). A significant difference was also found in virus neutralization (VN) assays. The virus strain in the HI or VN test did not influence the resulting titres after Ulster vaccination. When HI antibody titres after vaccination were related to VN titres measured with virulent Herts NDV or to survival after virulent challenge, it was found that the use of La Sota antigen in the HI assay after vaccination with Clone30 or La Sota resulted in an overestimation of protective serum antibody titres. Also in commercially derived White Leghorns vaccinated with Clone30, significantly higher HI titres were obtained when La Sota antigen was used in the HI test. Our data have direct implications for potency testing of inactivated vaccines as the European Pharmacopeia does not prescribe the antigen to be used in the HI test.

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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.

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Five live virus vaccines against avian infectious laryngotracheitis were studied with regard to safety, immunogenicity and route of administration. Significant differences in virulence between the vaccine strains were found. Reduced virulence was accompanied by a reduction of immunogenicity and capacity to spread. After eyedrop application, a low virulent vaccine induced 90-100% flock immunity for the first 10 weeks after vaccination (PV), followed by a slow decline to 50% at 31 weeks PV, whereas flock immunity induced with the more virulent types remained at about 90% till the end of the experiments (24 and 48 weeks PV). Aerosol vaccination induced 70-100% flock immunity but vaccine reactions were severe. Application of vaccine in a coarse spray did not result in adverse vaccine reactions but induced a maximal protection rate of only 50%. Microneutralisation titres provided a useful indicator of immunity from the onset of immunity until immunity started to decline. A vaccine virus carrier state was demonstrated by means of sentinel birds.

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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.

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Four trials employing 50% protective dose (PD50) assays were performed to compare the efficacy of Marek's disease vaccine after intramuscular (i.m.) and subcutaneous (s.c.) inoculation of specific-pathogen-free (SPF) chickens. Female chickens were employed in trials 1 and 2. In trials 3 and 4 the results were separately recorded for both sexes. Cell-associated Marek's disease virus serotype 1 vaccines derived from strain CVI-988 were used. Intramuscular administration yielded PD50 estimates which were below the PD50 obtained by the s.c. route in all five pairs of comparable assays. A 4-fold difference of PD50 estimate, in favour of i.m. inoculation, was observed between the two series of female chickens of the trial which most properly fulfilled the requirements for a PD50 assay. By comparing 25 individual pairs of vaccine dose groups, the best protection was observed in 24 groups of chickens which received the vaccine by i.m. route. These data were statistically analysed by comparing the average degrees of protection provided by i.m. or s.c. inoculation. In trials 1 and 2 the i.m. vaccination provided significantly better protection than the s.c. route. Although not significantly different, the differences in trials 3 and 4 were in the same direction. It is recommended that the potency of the minimally acceptable field dose should be increased if s.c. administration becomes the normal practice.

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The sporicidal and fungicidal activity of disinfectants was studied in a suspension test. Glutaraldehyde 4%, sodium-dichloroisocyanurate-dihydrate (2400 ppm active chlorine) and peracetic acid 0.25% demonstrated after 30 min of exposure at 20 degrees C in the presence of 4% horse serum a clear activity against spores of Bacillus cereus. Under the same conditions formaldehyde 4% and glutaraldehyde 2% were also found to be sporicidal, but only after a longer time of exposure. Spores of Bacillus anthracis and B. cereus appeared to be comparably resistant against the investigated disinfectants, whereas conidiospores of Aspergillus fumigatus and Aspergillus niger were less resistant. Of the micro-organisms tested Candida albicans proved to be slightest resistant, while spores of Bacillus subtilis were found the most resistant.

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The activity of disinfectants with regard to spores of Bacillus anthracis was determined in a suspension test. Creoline (10%) and also several other disinfectants for veterinary use showed no activity against spores of B. anthracis. Natriumdichloorisocyanuraat-dihydrate (2400 ppm active chlorine) and peracetic acid 0,25% demonstrated after 30 minutes of exposures at 20 degrees C in the presence of 4% horse serum a significant sporicidal effect. Under the same conditions formaldehyde 4% and glutaraldehyde 2% were also found to be sporicidal but only after 2 hours of exposure.

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Four live virus vaccines against Infectious Bursal Disease (IBD) were studied with regard to their safety, immune response and applicability. None of the vaccines caused clinical symptoms or had an adverse impact on bodyweight. Differences between these vaccines were observed in their effect on the Bursa/Bodyweight Ratio and the severity of the microscopical lesions of the bursa Fabricii. The immunosuppressive effect of IBD vaccination at one day of age on the response to Newcastle disease vaccine applied was rather low. Three of the four vaccines induced antibodies associated with protection against challenge. Vaccination of SPF rearing chickens by drinking water at an age of 15 weeks produced an antibody response (Agar Gel Precipitin Test) whereas at 15 weeks produced an antibody response (Agar Gel Precipitin Test) whereas at an age of 23, 32 and 60 weeks it did not. Chickens of all age groups responded serologically to an intramuscular vaccination. A correlation was found between the immunological response and the effect of the vaccines on the bursa Fabricii.

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Summary Four live virus vaccines against Infectious Bursal Disease (IBD) were studied with regard to their safety, immune response and applicability. None of the vaccines caused clinical symptoms or had an adverse impact on bodyweight. Differences between these vaccins were observed in their effect on the Bursa/ Bodyweight Ratio and the severity of the microscopical lesions of the bursa Fabricii. The immunosuppressive effect of IBD vaccination at one day of age on the response to Newcastle disease vaccine applied was rather low. Three of the four vaccines induced antibodies associated with protection against challenge. Vaccination of SPF rearing chickens by drinking water at an age of 15 weeks produced an antibody response (Agar Gel Precipitin Test) whereas at an age of 23, 32 and 60 weeks it did not. Chickens of all age groups responded serologically to an intramusculair vaccination. A correlation was found between the immunological response and the effect of the vaccines on the bursa Fabricii.

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The registration, licensing and investigation of veterinary vaccines are discussed with reference to the producer, the user and the authorities. The significance of basic research by the producer, of testing by the authorities, of quality control and of evaluation (laboratory and field studies) in the control of animal diseases are examined more closely. The fact is stressed, that legislation concerning veterinary medicines in general and vaccines in particular should be well defined. All this is regarded as an integral part of recent developments in animal farming in the Netherlands.

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