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1 Veterinary Quarterly ISSN: (Print) (Online) Journal homepage: Comparison of the effect of live Newcastle disease vaccine Clone 30 in broilers administered at day 1 or at day 7 and the effect of H120 vaccination at 17 days of age: A field experiment A. C. Voeten, J. H. H. van Eck, F. G. Davelaar & B. Kouwenhoven To cite this article: A. C. Voeten, J. H. H. van Eck, F. G. Davelaar & B. Kouwenhoven (1987) Comparison of the effect of live Newcastle disease vaccine Clone 30 in broilers administered at day 1 or at day 7 and the effect of H120 vaccination at 17 days of age: A field experiment, Veterinary Quarterly, 9:1, 3848, DOI: / To link to this article: Copyright Taylor and Francis Group, LLC Published online: 01 Nov Submit your article to this journal Article views: 1201 Citing articles: 5 View citing articles Full Terms & Conditions of access and use can be found at

2 Comparison of the effect of live Newcastle disease vaccine Clone 30 in broilers administered at day 1 or at day 7 and the effect of H120 vaccination at 17 days of age: a field experiment A. C. Voeten', J. H. H. van Eck2, F. G. Davelaar2, and B. Kouwenhoven3 SUMMARY To analyse the results of a vaccination on the first day of age against Newcastle disease (ND) and on the 17th day of age against Infectious Bronchitis (IB) resp. with spray vaccines with Clone 30 and H120 vaccine. These vaccinations are compared in fieldcircumstances with other vaccination methods. A serological examination and challenge test were used to be informed about the response and protection. From the present study the following conclusions can be drawn: (1) Clear indications are obtained that following a spray vaccination against ND with Clone 30 vaccin of onedayold broilers which possessed maternal antibodies, birds received a moderately good protection against ND, in spite of very low levels of HI antibodies. (2) A spray vaccination against IB with H120 vaccine of broilers at 17 days ofage gave some protection from two weeks after vaccination, however making a good conclusion about the protection is impossible and further investigation is required. INTRODUCTION Of the lentogenic Newcastle disease (ND) virusstrains most commonly used as vaccine strains, the Hitchner B I and the LaSota are the best known. Voeten et al. (9) reported that one of the lentogenic NDvaccins was remarkable for its long elution time (8). This vaccine differed considerably from the Hitchner B1 vaccine, and to a lesser extent, from the LaSota vaccine, in the protection it induced when given at day 1. Challenge experiments showed that vaccination at day 1 induced solid immunity at 3 weeks, although haemagglutination'inhibiting (HI) antibodies were absent. Intervet International, concentrating on a search for a similar strain, selected from various 'cloned' strains of a LaSota parent strain, one clone (Clone 30) which also induced a solid immunity, although without inducing HI antibodies when administered at the first day of life to chicks with maternal antibodies. Bijnen et al. (2) investigated this Clone 30 and classified it as a lentogenic strain. 1 Gezondheidsdienst voor Dieren in NoordBrabant, Molenwijkseweg 5282 SC Boxtel, The Netherlands.. 2 Department of Poultry Diseases, State University of Utrecht, Doom, The Netherlands. 3 Poultry Health Institute, Doom, The Netherlands. 38 THE VETERINARY QUARTERLY, VOL. 9, No. 1, JANUARY 1987

3 Papparella et al. (6) showed that after vaccination with Clone 30 at 3 to 8 days 95% of the birds were protected against mortality when challenged at 21 days, and about 80% were protected when challenged at 40 days. The present study investigated the protection at 1 to 6 weeks in birds vaccinated at day 1 and in birds vaccinated at day 7 under field conditions. Effects of the vaccination were measured by challenge experiments and by weekly determination of HI titres. Also the performance data were collected. Vaccinations against Infectious Bronchitis (IB) were adapted to the ND vaccination programme: the flocks vaccinated against ND at day I were vaccinated for IB at day 17, and flocks vaccinated against ND at day 7 were vaccinated for IB at day 1 In flocks vaccinated against Infectious Bursal Disease (IBD), vaccination was performed at the age of 8 to 11 days. MATERIAL AND METHODS Experiment 1 This trial was conducted in 20 broiler farms varying in size between 10,000 and 25,000 broilers. The farms were all situated in the province of NoordBrabant in the Netherlands. Housing and management were average standard; all broilers were of the same breed and received feed from the same origin and composition. Flocks of the experimental group (Group A) were housed on 10 farms. Vaccinations were performed as follows: 1 day ND' in hen dose, automatic spray at the hatchery 8 days IBD2 1/2 hen dose, drinking water 17 days /2 hen dose, knapsack spray Flocks of the reference group (Group B), which were vaccinated according to the program normally used, were also housed on 10 farms. Vaccinations were carried out as follows: 1 day /2 hen dose, automatic spray at the hatchery 7 days ND' 1/, hen dose, knapsack spray 8 days IBD2 1/2 hen dose, drinking water For the spray vaccination at the hatchery an automatic spraying machine was used, consisting of a compressor, pressure tank and coarse nozzle (Bimex). Data collected: Blood samples of 10 birds per flock were taken weekly for determination of HI antibodies to ND virus. At the age of 6 weeks 20 birds per flock were challenged with velogenic ND virus and mortality was recorded. Two weeks after challenge, surviving birds were killed andchanges present in trachea, air sacs and proventriculus were recorded semiquantitatively (moderate to very serious). Performance results. Experiment 2 This experiment was a field trial performed in the summer and autumn of 1984 in the southern part of the Netherlands. During five months, all broilers hatched in two hatcheries were vaccinated against ND in the hatchery with '/,, hen dose per chick of Clone 30 vaccine, using an automatic spraying machine (Bimex). The hatcheries belonged to two integrations, A and B. ' ND Clone 30 vaccine (Intervet). 2 IBD vaccine (Intervet or Mycofarm) H120 (Intervet). THE VETERINARY QUARTERLY, VOL. 9, No. 1, JANUARY

4 Vaccination against IB by spraying (knapsack spray) with a half dose of H120 vaccine per chick, at 17 days of age was advised. If the parent flock was not vaccinated with an inactivated IBD vaccine, vaccinating the broilers with live IBD vaccine (1/2 dose/bird) by the drinking water at 810 days of age was recommended. Six flocks of integration A and 4 of integration B were intensively monitored. These flocks were marked El to E10. Data collected from E flocks ND and IB Blood samples were taken at one day of age and then weekly from two weeks ofage from birds of each flock for determination of HI antibodies to ND virus. Weekly, from 2 weeks of age, 5 or 10 birds per flock were examined for thepresence of ND and IB virus (IBV) antigen in the trachea by the direct immunofluorescence test (I FT) and virusisolation trials. IBV isolates were serotyped as described (5). At 2, 4 and 6 weeks of age, 15 or 20 broilers per flock were challenged with velogen ND virus and mortality was recorded in the two weeks after challenge. At ages of 3, 4 and 5 weeks, 10 birds per flock were challenged with IBV. Five days postchallenge, postmortem examination was performed and birds were checked for the presence of IBV antigen in the trachea by the IFT. Also, virus recovery attempts (egg culturing according to standard procedures) from the trachea were carried out. Other examinations. At one day of age and then weekly from two weeks of age, twenty four of thirty blood samples per flock were tested for precipitins to IBD and for agglutinins to Mycoplasma gallisepticum (M.g.) and Mycoplasma synoviae (M.s.). Data collected from flocks of integrations A and B other than the Eflocks Simultaneously with the monitoring program of the Eflocks, data of a great number (see Results) of other flocks belonging to integrations A and B were collected. Flocks vaccinated against ND at one day as well as flocks vaccinated at 712 days of age were involved. Data collected were related to performance results (integrations A and B), thepresence of viral ND antigen in the trachea (integration A) and HI antibodies to ND in the serum at slaughter age (integrations A and B). Blood tests and IFT were performed according to the standards used by the Dutch Animal Health Institutes (7). Challenge tests Challenge to ND: experiment 1: Herts 33 virus, ELD50 per bird by the intranasal route. Experiment 2: Herts 33 virus, 1065 ELD per bird by the ocular route. According to the British Standard Test (1), 103 ELD50 of the challenge virus should be equal to at least the CLD50. Following intramuscular application'of 103 ELD" of the Herts 33 virus to 20 SPF WL cocks of 10 weeks of age, all birds died within five days. So, the challenge virus used adequately meets the demands of the British Standard Test. Challenge to IB: V387 strain (virulent Massachussets strain; 10" EIDSO per bird by the ocular route. The ND and IB challenge viruses came each from one batch, were freezedried in 2 ml ampoules and stored at 20 C until use. In all challenge experiments two SPFWL birds of 6 to 10 weeks were included as controls. Statistical analysis Statistical analysis on mortality and the occurrence of alterations following ND challenge in experiment 1 was performed using the nondirectional MannWitney U test. In experiment 2 numbers of flocks in which NDantigen was demonstrated were compared with the Chi2 method and performance data with the Wilcoxon test. 40 THE VETERINARY QUARTERLY, VOL 9, No. I, JANUARY 1987

5 Table I. Mean 2log HI titres to Newcastle disease (ND) virus in broiler flocks which were vaccinated against ND at 1 day old (Group A) or at 7 days of age (Group B). 10 Blood samples per flock were examined on each occasion. Group A agc in weeks farm no. 1 day , mean Group B age in weeks farm no. 1 day , mean RESULTS Experiment 1 Results of blood tests for HI antibodies to ND virus are presented in Table I. Flocks vaccinated at 1 day old show a slow decrease of antibody titres and at 6 weeks ofage only very low levels were detected (mean 2log HI titre at 6 weeks: 4.6). In flocks vaccinated at 7 days of age, a distinct antibody rise was observed from 5 weeks of age (mean 2log HI titre at six weeks: 7.8). Results of the ND challenge experiments at 6 weeks of age are given in Table 2. Fourteen percent of the birds vaccinated at 1 day of age died following challenge, while this percentage was 4.5 in birds vaccinated at 7 days of age. This difference proved to be significant (p < 0.01). Between groups no significant differences were observed concerning alterations in tracheas, air sacs and proventriculi of surviving birds. However, air sac lesions in birds vaccinated at 7 days of age seemed to occut less frequently and to a lower degree than in birds vaccinated at one day of age. Performance results of flocks of both groups are presented in Table 3. From this table it appears that no distinct differences in technical results between the two groups of flocks existed. Experiment 2 In all flocks except three (E3, E4 and E7) IFT for ND virus antigen in the trachea was negative (see Table 6). No clinical symptoms of ND were observed in any of the flocks. ND virus was isolated from 2 of the Eflocks (flocks E7 and E5) at 5 and 6 weeks respectively (see Table 6). THE VETERINARY QUARTERLY, VOL. 9, No. I, JANUARY

6 4=, ts.) Table 2. Mortality and alterations in trachea, air sacs and proventriculus of surviving broilers following challenge 6 weeks of age. Twenty birds by the ocular route with Herts 33 virus at per flock were challenged. Flocks of Group A were vaccinated against Newcastledisease of age. at one day of age, those of Group B at 7 days GROUP A H r.. rl GROUP B ALTERATIONS TRACHEA ALTERATIONS AIR SAC ALTERATIONS PROVENTRICULUS farm no. mortal Ity very serious serious moderate absent serious moderate no. datal absent serious moderate tbsent of birds ' Total Z Mean Percentage 144' n z ALTERATIONS TRACHEA ALTERATIONS AIR SAC ALTERATIONS PROVENTRICULUS < > w.0 farm no. mortality very serious serious moderate absent no. data c serious moderate ebsent serious moderate absent of birds n w g cii c < : , > Z > a 5 1 z Total ẉ < Mean percentage , stc. co l No. of surviving birds of which post mortem data are lacking. 2. No. of birds died. 3. No. of birds with alterations. 4. Significant difference with corresponding value of group B (p< 0.01)..

7 Table 3. Performance of two groups of broiler flocks, each consisting of 10 flocks varying in size between 10,000 and 25,000 birds. Flocks of Group A were vaccinated against Newcastle disease at one day of age, those of Group B at 7 days of age. Flocks were slaughtered at approximately 45 days of age. Group A Mean slaughter weight (g) Mean growth/day (g) Feed conversion Mortality (%) E.B.I.' Group B I E.B.I. = European broiler index = mean growth per bird per day (g) x survival rate: 10 x feed conversion. Table 4. Mean 2log HI titres to Newcastle disease (ND) virus in broiler flocks vaccinated against ND at 1 day old. Age in weeks Flock no. I E E E E E E n.d.' E E E EIO Mean n.d. = not done. Table 5. Mortality in broilers following challenge by the ocular route with Herts 33 virus at different ages. At 4 weeks of age 15 birds per flock were challenged; at the other agcs 20. Flocks were vaccinated against Newcastle disease at one day old. Flock no. 2 Age in weeks at challenge 4 6 E I 0' 0 1 E E E E5 0 3 I E6 0 0 n.d.2 E7 I 5 0 E E EIO Total Mean percentage ' No. of birds died. 2 n.d. = not done. THE VETERINARY QUARTERLY, VOL 9, No. 1, JANUARY

8 Table 6. Results of 1FT to infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) and of virus isolation (VI) in the trachea with Clone of broilers vaccinated 30 vaccine. I day old and with HIM vaccine at I7daysofage. At 4weeksofage, 5 birds per flock were examined, at the other ages 10 birds per flock. Age In weeks IFT IFT 6 1FT IFT IFT IFT IFT IFT Flock no. to IBV to NDV IFT IFT VI to IBV to NDV VI to IBV to NDV VI to IBV to NDV VI to IBV to NDV VI E _ IBV 1 1 IBV 7 6 IBV* E IBV 0 0 IBV 2 0 IBV 3 0 E IBV 5 0 IBV 6 0 IBV 3 I E IBV 8 0 IBV 7 2 E NDV E IBV 4 0 IBV 0 0 IBV* n.d.3* E 74* IBV NDV 8 0 n.d. n.d. E IBV 5 0 IBV 2 0 IBV E IBV 3 0 IBV 4 0 EIO IBV IBV* 3 0 No. of positive flocks / number examined 1/10 0/10 0/10 6/10 1/10 IBV 6/10 IBV 7/10 NDV 7/10 2/10 9/10 2/10 9/9 2/9 NDV 14DV 0/10 0/10 1/10 1/9 % No. of birds positive In IFT / 2/100 0/100 14/100 1/100 21/50 2/50 48/100 9/100 43/90 3/90 no. examined IBV 8/10 NDV IBV 1/9.1* no. of positive birds. t*. No IBV or NDV Isolated. ;* n.d.. not done. 4* flock E 7 was not vaccinated against Infectious bronchitis. Isolate was serotyped.,

9 Results of HI tests to ND virus and ND challenge experiments are given in tables 4 and 5 respectively. HI antibody titres to ND virus (Table 4) decreased with age, and at 6 weeks of age mean 'log HI titres were very low and varied from 4.0 to 5.4. Only in flock E7 was the mean titre considerably higher (9.3) at 6 weeks of age. Mortality following challenge (Table 5) increased with age. Mean mortality rates were 3.0, 11.3 and 12.2 percent at 2, 4 and 6 weeks of age respectively. Results of IFT and virusisolation trials to IB and of IB challenge experiments are presented in Tables 6 and 7 respectively. From 2 weeks of age the number of flocks with positive IFT to IB increased and at the end of the fattening period all flocks were positive (Table 6). Virus isolations succeeded from 6 of the 10 flocks at 3 weeks of age, after which the isolation score increased slowly up to 5 weeks of age, but decreased at 6 weeks. Five IB isolates were serotyped (Table 6). Three of the isolates belonged to the Massachussets serotype and 2 of the isolates were serologically different. At 5 days after challenge at 3, 4 and 5 weeks of age viral antigen was demonstrated by IFT in birds of all flocks. At 3 and 4 weeks virus was also recovered from all flocks. Following challenge at 5 weeks virus was not recovered from 4 flocks (Table 7). Results of AGP tests to IBD virus are presented in Table 8. In the second half of the fattening period six flocks (E2, E5, E6, E8, E9 and E10) possessed a high proportion of birds with precipitins to IBD. Of these flocks only E9 and EIO were vaccinated. Flocks E2, E5 and E6 originated from parents which were vaccinated with inactivated IBD vaccine; flocks E8, E9 and EIO were the progeny of parents vaccinated with live IBD vaccine. Table 7. Infectious bronchitis (IB) challenge experiment: number of broilers in which IB virus antigen was demonstrated in the trachea by the IFT and IB virus recovery 5 days following ocular challenge with the V387 strain of Massachussetts IB virus. Each time, 10 birds per flock were examined. Flocks had been vaccinated with HI20 vaccine at 17 days of age. Flock no. 3 IFT Virus recovery Age in weeks 4 I FT Virus recovery El E FT _2 E E E E E7' Virus recovery E E EIO No. of positive flocks/ 10/10 10/10 10/10 10/10 10/10 6/10 no. examined No. of birds positive in IFT/ no. examined, 94/100 68/100 55/100 No. of positive birds. 2 + = virus recovered = virus not recovered. 3 Flock E7 was not vaccinated against infectious bronchitis. THE VETERINARY QUARTERLY, VOL 9, No. I. JANUARY

10 Table 8. Precipitins to infectious bursal disease (IBD) in broilers not vaccinated with IBD vaccine. At two weeks of age thirty blood samples per flock were examined; at the other ages 24 blood per flock. samples Age in weeks Flock no.' I day El E E E E E n.d.3 E E E EIO ' Flocks EIE6 originated from parent flocks, which were vaccinated with inactivated flocks E7E10 came from parents vaccinated IBD vaccine; with live IBD vaccine. Flocks E9 and vaccinated with live IBD vaccine at 10 EIO were or 11 days of age. 2 No. of positive blood samples. 3 n.d. = not done. No antibodies to Mycoplasma gallisepticum and Mycoplasma'synoviae were detected in any of the flocks. Technical results of broiler flocks which were vaccinated against ND at 1 day or 712 days of age of integrations A and B are given in Table 9. Flocks of integration A which were vaccinated against ND at 1 day old showed significantly increased growth rates, slaughter weights and broiler indices, in comparison with flocks of the same integration vaccinated at 712 days of age. In flocks of integration B no significant differences could be established in these parameters between flocks vaccinated at 1 day or 712 days of age. Mortality rates had increased significantly in flocks of both integrations, which were vaccinated at 1 day old. Mortality rates had increased predominantly in the first week of life. No significant differences in feed conversion were observed between groups of flocks of the. same integration. Table 9. Performance of broilers in integrations A and B. Flocks were vaccinated against Newcastle disease at 1 day old or at 712 days of age. Integration A A No. of flocks vaccinated at day old 62 9 at 712 days Mean slaughter weight (g) Age at slaughter (days) Mean growth/bird/day (g) 37.8' Feed conversion Mortality (%) Condemnations (%) E.B ' ,3 Significant difference (p < 0.05) with corresponding values of flocks of integration A, which were vaccinated against Newcastle disease at 712 days of age. 2 E.B.I. = European broiler index (see Table 3). 46 THE VETERINARY QUARTERLY, VOI.. 9, No. 1, JANUARY 1987

11 At slaughter age, in 24% (8 of 34 flocks) of the flocks of integration A which were vaccinated against ND at 1 day old, ND viral antigen was demonstrated in the trachea by the IFT, while this percentage was 17 (5 of 29 flocks) in flocksvaccinated at 712 days of age. This difference was not significant. In 36% (20 of 56 flocks) of flocks vaccinated against ND at 1 day old, mean 2log HI titres to ND in the sera were 6.0 at slaughter age, and in all flocks (49 of 49 flocks) vaccinated at 712 days. DISCUSSION AND CONCLUSIONS Following spray vaccination against ND with Clone 30 vaccine of 1 day old broilers which possessed maternal antibodies, HI titres decreased, and in the majority of flocks HI antibodies were absent or at a very low level at the end of the fattening period (Tables 1 and 4). By contrast, vaccination at 712 days of age resulted in increasing titres from 3 weeks of age and in mean HI antibody titres ranging from at 6 weeks of age (Table 1). Moderate to high HI titres were present in a number of flocks, vaccinated at 1 day old, at 6 weeks of age (experiment 2: flock E7 (Table 4) and in 36% of the 'other flocks' of integration A). The presence of these antibodies is undoubtedly due to circulating virus (experiment 2: virus was demonstrated at 5 weeks of age in birds of flock E7 and at slaughter age in 24% of the 'other flocks'). Whether this circulating virus is vaccine or field virus is not clear from this study. Mortality after challenge at 6 weeks of age was significantly increased in chicks vaccinated at 1 day old in comparison with chicks vaccinated at 7 days of age (Table 2: 14% versus 4.5%). Serious air sac lesions in birds which survived after challenge also occurred in a higher proportion of birds vaccinated at 1 day old (Table 2: 17.8% versus 9.5%). In a second experiment mortality rates of broilers vaccinated on the first day of life were 3.0, 11.3 and 12.2% when challenged at 2, 4 and 6 weeks of age respectively (Table 5). So, although protection at 6 weeks of age induced by vaccinationat 1 day old is lower than induced by vaccination at 7 days of age, protection is still considerable. If necessary, emergency revaccination can be carried out, which is expected to give a quick and good response. In the secons half of the fattening period, a moderately good protection against ND induced by vaccination at 1 day old was present in spite of very low levels of HI antibodies (Tables 1, 2, 4 and 5) and is very likely a consequence of specific local immunity. Circulating virus would have caused an increase in HI titres. The absence of this indicates that the protection is derived from the vaccination at one day old. Performance of flocks vaccinated against ND at 1 day old did not differ significantly from that of flocks vaccinated at 7 days of age (experiment 1; Table 3) or was significantly improved (experiment 2; Table 9). Improvement was achieved by increased growth rates (Table 9). However, mortality was increased in the first week of life in flocks vaccinated at 1 day old (Table 9). Only poor protection against IB is induced by vaccination at 17 days of age (see Table 6). This is demonstrated by: a poor effect of vaccination as shown by the low incidence of chicks bearing I B antigen as demonstrated by IFT at 3 weeks of age; the long standing IFT positive birds in most of the flocks, indicating a long period of virus circulation after vaccination; the high score of IB isolations from flocks up to 5 weeks of age. At this time 5 isolates were serotyped. The fact that 3 of the isolates belonged to the Massachussetts serotype means that the IB vaccination at 17 days did not prevent the circula THE VETERINARY QUARTERLY, VOL 9. No. I. JANUARY

12 tion of viruses of the homologous serotype at 35 days of age. This is in sharp contrast to the usually practised dayold IB vaccination of broilers, which protects the chicks against challenge with virulent Massachussettstype IB virus already at 3 weeks of age (3, 4). The challenge results confirm the findings mentioned above, showing a marked increase in IFT positive chicks after challenge at 3 and 4 weeks of age. At 5 weeks of age the number of chicks with a positive IFT is equally high in both challenged and nonchallenged birds, making conclusions about protection and/or the effect of vaccination or field infections impossible. It may be concluded that the spray vaccination at 17 days gives some protection from 23 weeks after vaccination. In half the number of flocks not vaccinated against IBDan infection with IBD virus occurred, as shown by the presence of precipitins in a hundred percent of the birds in these flocks at the end of the fattening period (experiment 2, Table 8). This infection occurred in flocks which were the progeny of parents vaccinated with live IBD vaccine as well as in flocks which came from parents vaccinated with inactivated IBD vaccine. ACKNOWLEDGEMENTS The authors would like to express their sincere thanks to drs. G. H.. H. Bergs,A. Burger, C. Raatgever, and W. van 't Veer for their assistance. REFERENCES I. Allan WH, Lancaster JE, Toth B. Newcastle disease vaccines: their production and use. FAO, Rome, Bijnen B, Spanoghe L, Devos A. Commercial Newcastle disease vaccines Identification and characteristics of 5 different vaccine viruses, type La Sota and Clone 30. Vlaams Diergeneeskundig Tijdschrift 1983; 52: Davelaar FG, Kouwenhoven B. Influence of maternal antibodies on vaccination of chicks of different ages against infectious bronchitis. Avian Pathology 1977; 6: Davelaar FG, Kouwenhoven B. Vaccination of onedayold broilers against infectious bronchitis by eyedrop application or coarse droplet spray and the effect of revaccination by spray. Avian Pathology 1980; 9: 499., 5. Davelaar FG, Kouwenhovcn B, Burger AG. Occurrence and significance of infectious bronchitis virus variant strains in egg and broiler production in the Netherlands. The Veterinary Quarterly 1984; 6: Papparella V, Di Modugno G, Maiolino R. Premiers resultats en Italie& la vaccination spray cont re la maladie de Newcastle avec virus 'Clone 30' selectionné a travers les cultures a plaques de souche Lasota. World's Poultry Congress, Brasil. IXOP 1978; Poultry diagnostics and laboratory techniques. Poultry Health Institute, Doom, the Netherlands, ' 8. Spalatin J, Hansen RP, Beard PD. The haemagglutinationelution pattern as a marker in characterising Newcastle disease virus. Avian Diseases 1970; 14: Voeten AC, Orthel FW, Jacobs G. Comparison of live Newcastle disease vaccin in a simple vaccination and challenge experiment. Res Vet Sci 1977; 22: THE VETERINARY QUARTERLY, VOL. 9, No. I, JANUARY 1987