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Serology-based potency test for inactivated Newcastle disease vaccines
Serology-based potency test for inactivated Newcastle disease vaccines R.D. Goddard, R.A.J. Nicholas and P.R. Luff Twenty-seven inactivated oil-emulsion Newcastle disease vaccines were tested for potency in chickens. Serum samples from groups given 1/50 dose o f vaccine were examined by the haemagglutination inhibition (HI) test and the indirect enzyme-linked immunosorbent assay (ELISA). Good correlations were observed between potency and H I titres and between potency and ELISA absorb. ance. It is recommended that a serology-based potency test replaces the challenge test, reserving challenge only f o r those batches of vaccines where a clear pass is not indicated. Keywords: Newcastle disease; potency test; challenge test
Introduction The current method for determining the potency of inactivated Newcastle disease (ND) vaccines in the UK is that prescribed by the British Pharmacopoeia (Veterinary) 1. In this test three groups of chickens are given graded doses of the vaccine under test and, together with unvaccinated controls, challenged with virulent virus 17-21 days later. The potency of the vaccine is calculated from the ratio of protected and unprotected birds within the groups after observation for 10 days. This method has a number of disadvantages: it is expensive, it requires high-security housing, it is timeconsuming and it causes stress. Whilst a non-animal test would be ideal, eliminating the challenge step from the existing test would be an improvement both in terms of economics and animal welfare. The haemagglutination inhibition (HI) test has been shown to be a reliable indicator of flock protection and of vaccine immunogenicity2-4. Wilson et al. 5 demonstrated enzyme-linked immunosorbent assay (ELISA) results that corresponded directly with survival rates of challenged birds. Charan et al. 6 and Snyder et al. 7 found a significant correlation between ELISA and HI, whereas Miers et al. 8 failed to demonstrate a direct correlation due to the wide range of ELISA results for each HI titre. Marquardt et al. 9 pointed out that, although the ELISA/HI correlation is generally good, it can vary with the vaccination and sampling regime. The work presented here examines the serological response of chickens, using the ELISA and the HI test, under the specified conditions of potency testing, to see whether a sound basis for a serology-based potency test exists.
Materials and methods Vaccines
Twenty-five batches of commercially available inactivated oil-emulsion Newcastle disease (ND) vaccines Central Veterinary Laboratory, New Haw, Weybridge, Surrey, KT15 3NB, UK. (Received 19 February 1988; revised 15 July 1988) 0264-410X/88/060530-03 $03.00 ~) (British) Crown copyright
530 Vaccine, Vol. 6, December 1988
from five different manufacturers were tested. Six were monovalent; the others contained, in addition, one or more non-ND antigens. P o t e n c y test
The method set out in the British Pharmacopoeia 1 was used, with challenge 21 days after vaccination. The suggested 1/25, 1/50 and 1/100 dose pattern was used initially, and some later batches of vaccines were tested at 1/50, 1/100 and 1/200 dose. Vaccines were tested in groups of 3 to 7 and the potency of each vaccine was calculated by probit analysis. Serology
Birds from the 1/50 dose group were bled from a wing vein 20 days after vaccination, the day before challenge. All sera were subjected to the HI test and the sera from 23 vaccine groups were tested by indirect ELISA.
Haemagglutination inhibition test The HI test was performed following the method of Allan and Gougha° but using Nunc 96-well roundbottomed microwell plates and manual dispensing and dilution using Titertek multichannel pipettes. The incubation time was 1 h at 4°C. The test was considered valid if the recommended result of 25-26 was obtained for the international reference preparation of anti-Newcastle disease serum and 22 or less with the unvaccinated controls. Indirect E L I S A
The Hitchner B1 strain of ND virus, used as coating antigen in the ELISA, was grown in the chorio-allantois of 10-day-old SPF embryonated eggs for 72 h. All subsequent steps were carried out at 4°C. After cooling the eggs overnight the infected fluid was removed and clarified by centrifugation at 1600g for 30 min. The supernatant was pelleted by centrifugation at 30 000g for 45 min. The pelleted virus was resuspended in 1/100th of the original volume in phosphate-buffered saline (PBS, pH 7.4) and was layered on top of a discontinuous gradient of 1 ml 20% and 1 ml 60% (w/w) sucrose. After centrifugation at 45 000g for 3 h, virus at the
Potency test for ND vaccines: R.D. Goddard et al. Table 1
Results obtained from groups of 25 chickens given 1/50 dose of inactivated Newcastle disease vaccines PDso per dose
Vaccinated
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
> 100 >100 >100 37 (31-46) 8 58 (47-72) >100 > 100 >100 25 (22-30) 45 (36-56) 33 (27-40) >100 > 100 >200 >200 >200 >200 > 100 >200 162 (133-219) >200 191 (141-377) >200 196 (148-364) >100
4.36 3.92 4.16 2.20 2.64 4.76 4.60 4.32 2.00 2.60 2.40 4.90 4.40 4.80 4.96 5.00 4.52 3.84 4.64 4.56 4.64 4.52 4.36 4.69 5.76
" 9 5 % confidence limits
Mean ELISA absorbance
Log2 mean HI titre
Vaccine No.
Control
Vaccinated
Control
Ratio vaccinated/control
1.40 1.40 1.40 1.57 1.57 1.57 1.57 1,57 1.90 1.90 1.90 1.90 1.90 2.00 2.00 2.00 2.00 2.00 1.35 1.35 1.35 1.35 1.35 1.35 1.35
1.24 1.13 1.16 0.50 0.82 1.05 0.98 0.97 0.33 0.69 0.40 1.10 1.04 1.23 1.37 1.10 1.12 0.91 1.01 1.05 0.89 -
0.16 0.16 0.16 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.22 0.22 0.22 0.22 0.22 0.18 0.18 0.18 -
7.75 7.06 7.25 2.78 4.56 5.83 5.44 5.39 1.83 3.83 2.22 6.11 5.78 5.59 6.23 5.00 5.09 4.14 5.61 5.83 4.94 -
- , Not done
interface was collected, dissaggregated by passing up and down a pipette several times and diluted 1/5 in PBS. Antigen was stored at -70°C until needed. Unless otherwise stated, all volumes dispensed in the ELISA were 50 ~tl. Antigen was diluted 1/400 in carbonate/bicarbonate buffer pH 9.6 and dispensed in all wells of the polyvinyl chloride microtitre plates (Becton Dickinson). The plates were stored overnight at 4°C and then washed five times in PBS containing 0.05% Tween 20. Serum samples were diluted 1/100 in PBS/ Tween and tested in duplicate. The plates were kept at 37°C for 1 h and washed as before. Rabbit anti-chicken IgG conjugated to alkaline phosphatase (Bio Yeda) was added to all wells at a dilution of 1/1000. The plates were incubated and washed as before. The substrate, p-nitrophenyl phosphate (5 mg dissolved in 5 ml diethanolamine buffer) was added to all wells and incubated for 45 min, after which time the reaction was stopped by the addition of 2 M sodium hydroxide. The absorbance was read at 410 nm using a Dynatech MR600 microplate reader. The variations from plate to plate and test to test were controlled by the use of a positive reference preparation titrated in duplicate on each plate at dilutions of 1/100, 1/500, 1/2500 and 1/12 500. The results of test sera on a single microtitre plate were considered valid if the mean slope of the standard tested on that plate was within the 95% tolerance limits set in earlier work (data not shown). The system had previously been tested to ensure that no cross-reactions occurred with sera from birds inoculated with inactivated oil-emulsion vaccines containing infectious bursal disease virus, infectious bronchitis virus or egg-drop syndrome virus, the antigens contained in combination with ND virus in the multivalent vaccines tested.
Results Results obtained from the potency test, the HI test and ELISA are given in Table 1. Of the 25 vaccines
tested for potency, four had less than the required 50 PDs0 (median protective dose) per dose. Seventeen of the vaccines had potencies too high to allow adequate statistical analysis from the dilutions tested. Regression analysis of the remainder showed correlation coefficients of 0.988 (p=0.001) between HI titres and PDs0 and 0.861 (p=0.01) between ELISA absorbance and PDs0. Mean ELISA absorbance values ranged from 0.82 to 1.37 for vaccines that clearly passed the potency test and from 0.33 to 0.69 for those that failed to reach the pass mark. The corresponding HI titres w e r e 2 2.64 to 2 5.76 and 22 to 22.6 . Statistical analysis after about onehalf of the vaccines had been tested indicated that adequate vaccines should yield mean HI titres in groups of 25 birds of not less than 2381 in 95% of tests. Although this was increased in the final analysis by the inclusion of several vaccines of very high potency, 20 of the 21 adequate vaccines (95.2%) gave a mean titre of >2381. For 11 of the vaccines, all three dose groups (1/25, 1/50 and 1/100) were subjected to HI test and mean titres of 2 4'39, 2 3.53 and 22.83 (r=0.998) indicated a clear dose-related response. Similarly, all three groups from five vaccines were subjected to the ELISA and mean absorbance values of 1.026, 0.862 and 0.767 (r=0.988) showed a clear dose-related response.
Discussion The direct correlation between both the HI titre and ELISA absorbance with the PDs0 reported here for products from five different manufacturers provides a sound basis for using serology as a measure of ND vaccine potency. Since the majority of ND vaccines produced greatly exceed the minimum potency, it is recommended to use a threshold titre of 24, making the possibility of an inadequate vaccine passing the test even less likely, whilst having little effect on the adequate ones. On this basis three vaccines (11% of those tested) which performed well when challenged would also fail the HI-based test. This closely matches the V a c c i n e , Vol. 6, December 1988
531
P o t e n c y test for N D v a c c i n e s : R.D. G o d d a r d et al.
observations of Balla et a L e on both commercially farmed and experimental chickens. A similar observation is seen with the E L I S A . From H I / E L I S A regression analysis, a HI titre of 24 was equivalent to an absorbance of 0.96. Using this figure as a cut-off point, the E L I S A , like the HI test, correctly identifies the inadequate vaccines, but also wrongly includes three vaccines which performed well after challenge. If, however, the E L I S A values are expressed as a ratio of the mean absorbance for vaccinated to unvaccinated, a ratio of 4:1 would correctly classify all the vaccines listed: those with a ratio < 4 would fail and those with more would pass. Indeed, in view of the inherent difficulties in standardizing E L I S A between different laboratories, the use of a ratio would be a more practical, more robust and, from this study, a more accurate measure of vaccine potency. From the results obtained here it is reco m m e n d e d that, for the estimation of the potency of inactivated oil-emulsion N D vaccines, serology is used as a screen to eliminate the need to challenge birds vaccinated with obviously efficacious vaccines. Only those vaccines that at 1/50 dose do not evoke the required serological response (HI titre I>24 or an E L I S A ratio t>4) would need to be challenged.
Acknowledgements The authors are grateful to Ms B. Westbury and Mr A. Ream for invaluable technical assistance.
532
V a c c i n e , Vol. 6, D e c e m b e r 1 9 8 8
References 1 British Pharmacopoeia (Veterinary) 1985, HMSO, London 2 Balla, L., Papocsi, L., Szurop, I. and Toth, B. Studies on the correlation between the results of the haemagglutination-inhibition test and the immunity against Newcastle disease. Acta Vet. Hung. 1976, 26, 235 3 Phillips, J.M. Vaccination against Newcastle disease: an assessment of haemagglutination-inhibition titres obtained from field samples. Vet. Rec. 1973, 93, 577 4 Roepke, W.J. The control of Newcastle disease in the Netherlands. Bull. Off. Int. Epiz. 1973, 79, 43 5 Wilson, R.A., Perrotta, C., Frey, B. and Eckroade, R.J. An enzymelinked immunosorbent assay that measures protective antibody levels to Newcastle disease virus in chickens. Avian Dis. 1984, 28, 1079 6 Charan, S., Rai, A. and Mahajan, V.M. Comparison of enzyme-linked immunosorbent assay and haemagglutination inhibition test for the detection of Newcastle disease virus antibodies in human sera. J. C/in. Pathol. 1981, 34, 90 7 Snyder, D.B., Marquardt, W.W., Mallinson, E.T. and Russek, E. Rapid serological profiling by enzyme-linked immunosorbent assay. 1. Measurement of antibody activity titer against Newcastle disease virus in a single serum dilution. Avian Dis. 1983. 27, 161 8 Miers, L.A., Bankowski, R.A. and Zee, Y.C. Optimizing the enzymelinked immunosorbent assay for evaluating immunity of chickens to Newcastle disease. Avian Dis. 1983, 27, 1112 9 Marquardt, W.W., Snyder, D.B., Savage, P.K., Kadavil, S.K. and Yancey, F.S. Antibody response to Newcastle disease virus given by two different routes as measured by ELISA and haemagglutination-inhibition test and associated tracheal immunity. Avian Dis. 1985, 29, 71 10 Allan, W.H. and Gough, R.E. A standard haemagglutination inhibition test for Newcastle disease. 1. A comparison of macro and micro methods. Vet. Rec. 1974, 95, 120
Introduction The current method for determining the potency of inactivated Newcastle disease (ND) vaccines in the UK is that prescribed by the British Pharmacopoeia (Veterinary) 1. In this test three groups of chickens are given graded doses of the vaccine under test and, together with unvaccinated controls, challenged with virulent virus 17-21 days later. The potency of the vaccine is calculated from the ratio of protected and unprotected birds within the groups after observation for 10 days. This method has a number of disadvantages: it is expensive, it requires high-security housing, it is timeconsuming and it causes stress. Whilst a non-animal test would be ideal, eliminating the challenge step from the existing test would be an improvement both in terms of economics and animal welfare. The haemagglutination inhibition (HI) test has been shown to be a reliable indicator of flock protection and of vaccine immunogenicity2-4. Wilson et al. 5 demonstrated enzyme-linked immunosorbent assay (ELISA) results that corresponded directly with survival rates of challenged birds. Charan et al. 6 and Snyder et al. 7 found a significant correlation between ELISA and HI, whereas Miers et al. 8 failed to demonstrate a direct correlation due to the wide range of ELISA results for each HI titre. Marquardt et al. 9 pointed out that, although the ELISA/HI correlation is generally good, it can vary with the vaccination and sampling regime. The work presented here examines the serological response of chickens, using the ELISA and the HI test, under the specified conditions of potency testing, to see whether a sound basis for a serology-based potency test exists.
Materials and methods Vaccines
Twenty-five batches of commercially available inactivated oil-emulsion Newcastle disease (ND) vaccines Central Veterinary Laboratory, New Haw, Weybridge, Surrey, KT15 3NB, UK. (Received 19 February 1988; revised 15 July 1988) 0264-410X/88/060530-03 $03.00 ~) (British) Crown copyright
530 Vaccine, Vol. 6, December 1988
from five different manufacturers were tested. Six were monovalent; the others contained, in addition, one or more non-ND antigens. P o t e n c y test
The method set out in the British Pharmacopoeia 1 was used, with challenge 21 days after vaccination. The suggested 1/25, 1/50 and 1/100 dose pattern was used initially, and some later batches of vaccines were tested at 1/50, 1/100 and 1/200 dose. Vaccines were tested in groups of 3 to 7 and the potency of each vaccine was calculated by probit analysis. Serology
Birds from the 1/50 dose group were bled from a wing vein 20 days after vaccination, the day before challenge. All sera were subjected to the HI test and the sera from 23 vaccine groups were tested by indirect ELISA.
Haemagglutination inhibition test The HI test was performed following the method of Allan and Gougha° but using Nunc 96-well roundbottomed microwell plates and manual dispensing and dilution using Titertek multichannel pipettes. The incubation time was 1 h at 4°C. The test was considered valid if the recommended result of 25-26 was obtained for the international reference preparation of anti-Newcastle disease serum and 22 or less with the unvaccinated controls. Indirect E L I S A
The Hitchner B1 strain of ND virus, used as coating antigen in the ELISA, was grown in the chorio-allantois of 10-day-old SPF embryonated eggs for 72 h. All subsequent steps were carried out at 4°C. After cooling the eggs overnight the infected fluid was removed and clarified by centrifugation at 1600g for 30 min. The supernatant was pelleted by centrifugation at 30 000g for 45 min. The pelleted virus was resuspended in 1/100th of the original volume in phosphate-buffered saline (PBS, pH 7.4) and was layered on top of a discontinuous gradient of 1 ml 20% and 1 ml 60% (w/w) sucrose. After centrifugation at 45 000g for 3 h, virus at the
Potency test for ND vaccines: R.D. Goddard et al. Table 1
Results obtained from groups of 25 chickens given 1/50 dose of inactivated Newcastle disease vaccines PDso per dose
Vaccinated
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
> 100 >100 >100 37 (31-46) 8 58 (47-72) >100 > 100 >100 25 (22-30) 45 (36-56) 33 (27-40) >100 > 100 >200 >200 >200 >200 > 100 >200 162 (133-219) >200 191 (141-377) >200 196 (148-364) >100
4.36 3.92 4.16 2.20 2.64 4.76 4.60 4.32 2.00 2.60 2.40 4.90 4.40 4.80 4.96 5.00 4.52 3.84 4.64 4.56 4.64 4.52 4.36 4.69 5.76
" 9 5 % confidence limits
Mean ELISA absorbance
Log2 mean HI titre
Vaccine No.
Control
Vaccinated
Control
Ratio vaccinated/control
1.40 1.40 1.40 1.57 1.57 1.57 1.57 1,57 1.90 1.90 1.90 1.90 1.90 2.00 2.00 2.00 2.00 2.00 1.35 1.35 1.35 1.35 1.35 1.35 1.35
1.24 1.13 1.16 0.50 0.82 1.05 0.98 0.97 0.33 0.69 0.40 1.10 1.04 1.23 1.37 1.10 1.12 0.91 1.01 1.05 0.89 -
0.16 0.16 0.16 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.22 0.22 0.22 0.22 0.22 0.18 0.18 0.18 -
7.75 7.06 7.25 2.78 4.56 5.83 5.44 5.39 1.83 3.83 2.22 6.11 5.78 5.59 6.23 5.00 5.09 4.14 5.61 5.83 4.94 -
- , Not done
interface was collected, dissaggregated by passing up and down a pipette several times and diluted 1/5 in PBS. Antigen was stored at -70°C until needed. Unless otherwise stated, all volumes dispensed in the ELISA were 50 ~tl. Antigen was diluted 1/400 in carbonate/bicarbonate buffer pH 9.6 and dispensed in all wells of the polyvinyl chloride microtitre plates (Becton Dickinson). The plates were stored overnight at 4°C and then washed five times in PBS containing 0.05% Tween 20. Serum samples were diluted 1/100 in PBS/ Tween and tested in duplicate. The plates were kept at 37°C for 1 h and washed as before. Rabbit anti-chicken IgG conjugated to alkaline phosphatase (Bio Yeda) was added to all wells at a dilution of 1/1000. The plates were incubated and washed as before. The substrate, p-nitrophenyl phosphate (5 mg dissolved in 5 ml diethanolamine buffer) was added to all wells and incubated for 45 min, after which time the reaction was stopped by the addition of 2 M sodium hydroxide. The absorbance was read at 410 nm using a Dynatech MR600 microplate reader. The variations from plate to plate and test to test were controlled by the use of a positive reference preparation titrated in duplicate on each plate at dilutions of 1/100, 1/500, 1/2500 and 1/12 500. The results of test sera on a single microtitre plate were considered valid if the mean slope of the standard tested on that plate was within the 95% tolerance limits set in earlier work (data not shown). The system had previously been tested to ensure that no cross-reactions occurred with sera from birds inoculated with inactivated oil-emulsion vaccines containing infectious bursal disease virus, infectious bronchitis virus or egg-drop syndrome virus, the antigens contained in combination with ND virus in the multivalent vaccines tested.
Results Results obtained from the potency test, the HI test and ELISA are given in Table 1. Of the 25 vaccines
tested for potency, four had less than the required 50 PDs0 (median protective dose) per dose. Seventeen of the vaccines had potencies too high to allow adequate statistical analysis from the dilutions tested. Regression analysis of the remainder showed correlation coefficients of 0.988 (p=0.001) between HI titres and PDs0 and 0.861 (p=0.01) between ELISA absorbance and PDs0. Mean ELISA absorbance values ranged from 0.82 to 1.37 for vaccines that clearly passed the potency test and from 0.33 to 0.69 for those that failed to reach the pass mark. The corresponding HI titres w e r e 2 2.64 to 2 5.76 and 22 to 22.6 . Statistical analysis after about onehalf of the vaccines had been tested indicated that adequate vaccines should yield mean HI titres in groups of 25 birds of not less than 2381 in 95% of tests. Although this was increased in the final analysis by the inclusion of several vaccines of very high potency, 20 of the 21 adequate vaccines (95.2%) gave a mean titre of >2381. For 11 of the vaccines, all three dose groups (1/25, 1/50 and 1/100) were subjected to HI test and mean titres of 2 4'39, 2 3.53 and 22.83 (r=0.998) indicated a clear dose-related response. Similarly, all three groups from five vaccines were subjected to the ELISA and mean absorbance values of 1.026, 0.862 and 0.767 (r=0.988) showed a clear dose-related response.
Discussion The direct correlation between both the HI titre and ELISA absorbance with the PDs0 reported here for products from five different manufacturers provides a sound basis for using serology as a measure of ND vaccine potency. Since the majority of ND vaccines produced greatly exceed the minimum potency, it is recommended to use a threshold titre of 24, making the possibility of an inadequate vaccine passing the test even less likely, whilst having little effect on the adequate ones. On this basis three vaccines (11% of those tested) which performed well when challenged would also fail the HI-based test. This closely matches the V a c c i n e , Vol. 6, December 1988
531
P o t e n c y test for N D v a c c i n e s : R.D. G o d d a r d et al.
observations of Balla et a L e on both commercially farmed and experimental chickens. A similar observation is seen with the E L I S A . From H I / E L I S A regression analysis, a HI titre of 24 was equivalent to an absorbance of 0.96. Using this figure as a cut-off point, the E L I S A , like the HI test, correctly identifies the inadequate vaccines, but also wrongly includes three vaccines which performed well after challenge. If, however, the E L I S A values are expressed as a ratio of the mean absorbance for vaccinated to unvaccinated, a ratio of 4:1 would correctly classify all the vaccines listed: those with a ratio < 4 would fail and those with more would pass. Indeed, in view of the inherent difficulties in standardizing E L I S A between different laboratories, the use of a ratio would be a more practical, more robust and, from this study, a more accurate measure of vaccine potency. From the results obtained here it is reco m m e n d e d that, for the estimation of the potency of inactivated oil-emulsion N D vaccines, serology is used as a screen to eliminate the need to challenge birds vaccinated with obviously efficacious vaccines. Only those vaccines that at 1/50 dose do not evoke the required serological response (HI titre I>24 or an E L I S A ratio t>4) would need to be challenged.
Acknowledgements The authors are grateful to Ms B. Westbury and Mr A. Ream for invaluable technical assistance.
532
V a c c i n e , Vol. 6, D e c e m b e r 1 9 8 8
References 1 British Pharmacopoeia (Veterinary) 1985, HMSO, London 2 Balla, L., Papocsi, L., Szurop, I. and Toth, B. Studies on the correlation between the results of the haemagglutination-inhibition test and the immunity against Newcastle disease. Acta Vet. Hung. 1976, 26, 235 3 Phillips, J.M. Vaccination against Newcastle disease: an assessment of haemagglutination-inhibition titres obtained from field samples. Vet. Rec. 1973, 93, 577 4 Roepke, W.J. The control of Newcastle disease in the Netherlands. Bull. Off. Int. Epiz. 1973, 79, 43 5 Wilson, R.A., Perrotta, C., Frey, B. and Eckroade, R.J. An enzymelinked immunosorbent assay that measures protective antibody levels to Newcastle disease virus in chickens. Avian Dis. 1984, 28, 1079 6 Charan, S., Rai, A. and Mahajan, V.M. Comparison of enzyme-linked immunosorbent assay and haemagglutination inhibition test for the detection of Newcastle disease virus antibodies in human sera. J. C/in. Pathol. 1981, 34, 90 7 Snyder, D.B., Marquardt, W.W., Mallinson, E.T. and Russek, E. Rapid serological profiling by enzyme-linked immunosorbent assay. 1. Measurement of antibody activity titer against Newcastle disease virus in a single serum dilution. Avian Dis. 1983. 27, 161 8 Miers, L.A., Bankowski, R.A. and Zee, Y.C. Optimizing the enzymelinked immunosorbent assay for evaluating immunity of chickens to Newcastle disease. Avian Dis. 1983, 27, 1112 9 Marquardt, W.W., Snyder, D.B., Savage, P.K., Kadavil, S.K. and Yancey, F.S. Antibody response to Newcastle disease virus given by two different routes as measured by ELISA and haemagglutination-inhibition test and associated tracheal immunity. Avian Dis. 1985, 29, 71 10 Allan, W.H. and Gough, R.E. A standard haemagglutination inhibition test for Newcastle disease. 1. A comparison of macro and micro methods. Vet. Rec. 1974, 95, 120