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1427 J. gen. Virol. (1986), 67, 1427-1434. Printed in Great Britain Key words: IBV immunity/Escherichia coli/mixed inJbctions Infectious Bronchitis Immunity: Its Study in Chickens Experimentally Infected with Mixtures of Infectious Bronchitis Virus and Escherichia coli By J A N E K. A. COOK,* H. WILLIAMS SMITH AND M. B, H U G G I N S Houghton Poultry Research Station, Houghton, Huntingdon, Cambridgeshire, PE17 2DA, U.K. (Accepted 20 March 1986) SUMMARY The live infectious bronchitis (IB) vaccine, H120, protected chickens against intranasal challenge with a mixture of Escherichia coli strains (E. coli Pool) and IB virus (IBV) strains of the same (Massachusetts) serotype as H120; it usually also protected against challenge with the E. coli Pool and IBV strains of other serological types. W h e n these challenge strains were themselves used as vaccines they usually protected against challenge with a mixture of the E. coli Pool and an IBV strain of the Massachusetts serotype (VF69-149) or an IBV strain not of the Massachusetts serotype (HVI-116). Poor protection, when observed, was most c o m m o n in those experiments involving a minority of the IBV strains that had been incriminated in recent outbreaks of disease in vaccinated flocks of chickens. Much lower concentrations of IBV strain VF69-149 and E. coli O18 were found in the nose, trachea and spleen o f H120-vaccinated chickens killed at different times after they were given a mixture of these organisms than were found in these sites in similarly challenged unvaccinated chickens. Some protection against challenge with IBV and the E. coli Pool was also observed in chickens vaccinated with an inactivated IBV strain; it was much less effective than that obtained following vaccination with the corresponding live IBV strain. INTRODUCTION Research into immunity against infectious bronchitis (IB) has been seriously h a m p e r e d by failure to produce consistently a quantifiable disease in chickens inoculated with IB viruses (IBV). The need for a suitable experimental model has never been greater than at present because of the recent incrimination of new IBV serotypes as a cause of disease in vaccinated chicken flocks in The Netherlands (Davelaar et al., 1983) and in Britain (Cook, 1984). Smith et al. (1985) attempted to satisfy this need by studying the effect of inoculating chickens intranasally with pools of strains of IBV and Escherichia coli that are commonly involved in natural outbreaks of the disease. A n experimental model was finally developed which closely resembled the natural disease and in which a high mortality rate consistently occurred. This p a p e r describes the application of the model to study vaccinal immunity against IB. METHODS Bacterial and viral strains. The E. coli Pool comprised an 078, an 02 : K 1 and an untypable strain, selected by Smith et al. (1985) as being the most virulent representative of many strains of these three categories isolated from natural outbreaks of IB/E. coli disease. Serogroups 078 and 02 :K 1 are the two most commonly incriminated in the natural disease. An O18ac : K 1 : H7 strain, used as a nalidixic acid-resistantmutant and referred to hereafter as O18, was included in the Pool because this serotype is sometimes associated with outbreaks of natural disease and because the pathogenic characteristics of the particular strain used have been studied closely (Smith & Huggins, 1980). The origin and relevant information concerning the IBV strains used is summarized in Table 1. The 10 Massachusetts-type strains which composed the IBV Pool (Smith et al., 1985) included M41, VF69-149 and the commercial vaccine strain, H120. The method of propagating and maintaining the bacterial and viral strains (except IBV-Beaudette which was propagated in embryonated chicken eggs) and the method of performing bacterial and viral assays on cultures and on tissues of experimental animals have all been described previously (Smith et al., 1985). 0000-7040 © 1986 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 05:24:26 1428 J. K. A. COOK, H. WILLIAMS SMITH AND M. B. HUGGINS Table 1. I B V strains used IBV strain Beaudette H120 M41 VF69-149 Iowa 97 Connecticut Holte T 918 HVI-II6 D43 D41 Doorn 207 Doorn 3896 Doom 3128 2 3 4 7 8 9 10 12 Year of isolation 1937 1960 1941 1969 1947 1951 1954 1962 1965 1967 1976 1976 1980 1980 1980 1982 1982 t 1982 1982 1982 1983 1983 1984 Country of origin Source U.S.A. Laboratory strain Netherlands Vaccine strain U.S.A. -~ U.K. (N. Ireland)[ Respiratory disease U.S.A. Enteritis Australia Nephrosis ~ Respiratory U.K. (G.B.) [ disease Serotype* ) Reference to serological typing M ~ Darbyshire et aL (1979) Nephrosis Netherlands } Aberrant egg production }Respiratory t disease NM .Cook (1984) U.K. (G.B.) Aberrant egg production ) Cook & Huggins (1986) *M, Massachusetts-type; NM, non-Massachusetts-type. Experimental chickens and inoculationprocedures. Unsexed chickens from the Institute's specified pathogen-free Rhode Island Red flock were used throughout. The pathogens of which they are known to be free include IBV and Mycoplasma gallisepticum and M. synoviae. They were housed in strict isolation and fed ad libitum on a crumbed commercial diet. Unless otherwise stated, live virus vaccination was carried out when chickens were 4 days old by instilling into their anterior nares (i.n.) 0.1 ml of tracheal organ culture fluid containing, in the case of the H 120 strain approximately, log10, 4.0 median ciliostatic doses (CDs0) of virus, in the case of Beaudette approximately, log10, 7.4 median egg infectious doses of virus and in the case of all other viral strains approximately, log~o, 5.0 CDs0 of virus. The same virus dose was given when vaccination was by the subcutaneous (s.c.) route. Unless otherwise stated, all chickens were challenged at 25 days of age by i.n. inoculation of the same dose of virus as had been used for vaccination, but to which had been added approximately, logto, 8.3 viable organisms of the four strains that composed the E. coil Pool. Chicl~ens were observed daily for 14 days after challenge when the experiments were terminated. Preparation oJinactivated IBV vaccine. Nine-day-old chicken embryos were inoculated via the allantoic cavity with approximately, logt 0, 3.0 CDs0 of the virus strain VF69-149. After 36 h incubation at 37 °C the embryos were chilled, the allantoic fluid harvested and clarified by centrifugation at 8000 g for 30 rain. If required in concentrated form, the clarified allantoic fluid was re-centrifuged at 30000 g for 40 min and the resultant pellet resuspended in citrate buffer pH 6.4. The virus titres of the VF69-149 allantoic fluid and of the resuspended pellet were, loglo, 6.5 and, loglo, 7.7 CDso/ml respectively, a concentration of approximately tenfold. Before use as a vaccine each virus sample was inactivated by the addition of ~-propiolactone (BPL) to a final concentration of 0.05% and emulsified in an equal volume of Freund's complete adjuvant. The inactivated vaccine was administered (0.5 ml) into the left gastrocnemius muscle (i.m.). RESULTS Protection provided by live I B V H I 2 0 against challenge with viruses o f the same serological type and the E. coli Pool I n t r a n a s a l v a c c i n a t i o n with live IBV H120 p r o v i d e d good protection against challenge 21 days later with a m i x t u r e of the E. eoli Pool a n d the IBV Pool, two of its c o m p o n e n t strains or H120 itself, all of the Massachusetts serotype (Table 2). N o t only did the v a c c i n a t e d c h i c k e n s survive the infection, b u t clinical respiratory infection was n o t observed, n o r were lesions o f pericarditis or peritonitis seen in a n y of t h e m at the t e r m i n a t i o n of the e x p e r i m e n t (Fig. 1). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 05:24:26 Immunity against IBV/E. coli infection 1429 Fig. 1. Chicken (left) vaccinated intranasally with IBV strain H 120 21 days before intranasal challenge with a mixture of the E. eoliPool and IBV strain VF69-149.The other chicken had not been vaccinated; note the pericarditis. Magnification x 1. Table 2. Effect of vaccinating groups of chickens with live IBV strain H120 and challenging them with a mixture of l B V strains of the same serotype as H120 and the E. coli Pool* IBV challenge strain H120 IBV Pool M41 VF69-!49 of 22 chicks that died following challenge c z Vaccinated Not vaccinated 0 14 0 77 0 73 5 55 * The chickens were vaccinated when they were 4 days old and challenged 21 days later. Time taken for the live IBV H120 vaccination to produce immunity Two experiments were performed in which groups of chickens were vaccinated with live H120 virus either i.n. or s.c. and challenged at various times afterwards with the E. coli Pool and the Massachusetts-type strain VF69-149. I n one experiment all groups were vaccinated when 4 days old and in the second all groups were challenged when 25 days old (Table 3). Good protection was demonstrated in all groups in which the interval between vaccination and challenge was 11 days or more. In the groups challenged when 25 days old, i.n. vaccination was superior to s.c. vaccination. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 05:24:26 J. K. A. COOK, H. WILLIAMS SMITH AND M. B. H U G G I N S 1430 T a b l e 3. Effect of varying the interval between vaccination with live IBV strain H120 and challenge with IBV strain VF69-149 and the E. coli Pool Interval between vaccination and challenge (days) 0 2 4 7 11 14 18 of 30 chicks that died after they had been ~ , Vaccinated at 4 days old and challenged Vaccinated at different times before at different times afterwards challenge at 25 days old • ~ ~• A , i.n. s.c. Not vaccinated i.n. s.c. Not vaccinated 77 57 97 83 93 "] 40 50 73 87 97 57 -* 90 70 90 43 40 90 27 60 80 7 0 70 17 30 7 0 53 7 20 • 0 7 53 0 7 * , No observation. T a b l e 4. Effect of vaccinating groups of chickens with live IBV strain H120 and challenging them with IB V strains of other serological types and the E. coli Pool IBV challenge strain* % of 22 chicks that died following challenge r z • Vaccinated Not vaccinated HVI-116 D41 918 T D43 Iowa 97 Holte Doorn 207 2 0 0 0 0 9 0 0 0 0 8 0 Doorn 3128 Doorn 3896 7 4 10 3 9 0 5 5 9 14 14 23 82 64 55 36 36 18 14 23 18 18 18 14 14 23 41 14 23 * The last 10 strains were isolated from flocks vaccinated against IB, those with the prefix Doom were isolated in Holland, the others in the U.K. For other details, see Table 2. Protection provided by live IBV H120 against challenge with viruses of other serological types and the E. coli Pool V a c c i n a t i o n w i t h live H 1 2 0 v i r u s p r o t e c t e d efficiently a g a i n s t c h a l l e n g e w i t h t h e E. coli P o o l a n d I B V s t r a i n s H o l t e or I o w a 97 o f A m e r i c a n o r i g i n , t h e A u s t r a l i a n T s t r a i n , t h e B r i t i s h isolates H V I - 116, D41 or 918 or t h e t h r e e D u t c h isolates r e c e n t l y i n c r i m i n a t e d as c a u s i n g i n f e c t i o n i n IBv a c c i n a t e d flocks ( T a b l e 4). It p r o t e c t e d less well a g a i n s t c h a l l e n g e w i t h t h e E. coli P o o l a n d s o m e o f t h e I B V s t r a i n s r e c e n t l y i s o l a t e d in B r i t a i n f r o m d i s e a s e o u t b r e a k s i n I B - v a c c i n a t e d flocks. T h i s a p p e a r e d to b e p a r t i c u l a r l y so w i t h isolates 3 a n d 9, a l t h o u g h , as i n t h e c a s e o f s o m e o f t h e o t h e r viruses, t h e low m o r t a l i t y in t h e c o n t r o l g r o u p s m a d e c o m p a r i s o n o f a s s e s s m e n t o f p r o t e c t i o n difficult. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 05:24:26 Immunity against IB V/E. coli infection 1431 Table 5. Effect of vaccinating chickens with live IB V strains and challenging them with the E. coli Pool and either IBV strains VF69-149 or HVI-116" of 30 chicks that died following challenge with the E. coli Pool and IBV strain IB vaccine c " strain VF69-149 HVI-116 None 70 67 VF69-149 0 3 HVI-116 0 3 Iowa 97 7 0 Beaudette 20 10 Connecticut 30 10 2 t7 7 3 17 7 9 13 7 12 7 27 * For other details, see Tables 2 and 4. Table 6. Effect of vaccinating chickens intramuscularly once or twice with inactivated I B V strain VF69-149 and challenging them with strain VF69-149 and the E. coli Pool VF69-149 vaccine One dose Ten doses One dose Ten doses One dose Ten doses One dose Ten doses One dose Ten doses Freund's complete adjuvant None Interval between vaccination and challenge (days) r 21 and 14 21 and 14 3 0 0 10 0 0 3 3 0 0 0 0 - 0 0 7 14 21 of 30 chicks that had died by the following days after challenge x 5 6 7 8 10 10 17 27 33 37 0 7 10 30 33 13 27 33 37 37 23 27 27 43 53 0 0 0 3 10 3 3 3 3 7 7 7 7 10 13 10 13 13 17 20 0 7 7 10 10 0 3 3 7 7 0 7 20 27 30 14 43 53 40 60 10 10 20 23 17 13 37 10 40 17 20 33 33 Protection produced by vaccination with live IB viruses of different serotypes against challenge with the E. coli Pool and I B V strains VF69-149 and HVI-116 All of nine IBV strains gave some protection against subsequent challenge with the E. coli Pool and IBV strain VF69-149 of the Massachusetts serotype or HVI-116, a non-Massachusettstype strain (Table 5). Of the four British strains which had been isolated from vaccinated flocks, the two (3 and 9), against which H120 did not protect in the previous experiment (see Table 4), provided only incomplete protection against challenge in this experiment. In a similar experiment in which groups of 30 chickens were challenged with the E. eoli Pool and IB VF69149, the mortality rate was 5 7 ~ in the control group. I n the groups vaccinated with six nonMassachusetts strains not referred to in Table 5 and which had recently been isolated in Britain, France, Israel and Morocco, the mortality rates following challenge were 3 ~ , 13 ~ , 13 ~ , 17 ~ , 20 ~ and 33 ~ . Protection produced by inactivated I B V A reduced mortality rate was observed in groups of chickens vaccinated i.m. with inactivated IBV strain VF69-149 at 21, 14 or 21 and 14 days before challenge with that strain and the E. coli Pool. No reduction in mortality was noted in groups of chickens vaccinated 7 days before challenge or at the time of challenge (Table 6). Increasing the vaccine dose tenfold had little additional protective effect. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 05:24:26 1432 J.K.A. C O O K , H. W I L L I A M S SMITH AND M. B. H U G G I N S T a b l e 7. Amount of I B V in the nose, trachea and spleen of H120-vaccinated chickens at different times after they were challenged intranasally with I B V strain VF69-149 and E. coli 018 logt0 Amount of IBV strain VF69-149 per g tissue* r" Time after challenge ~s) A Nose .~ Trachea Spleen ~( Unvaccinated chickens 1/12 Vaccinated chickens <2"0t <2.0 5.8 3.3 5.5 3.0 5.6 2.3 4.7 <2.0 4.6 <2.0 5.5 <2.0,<2.0,2.0 4.7 <2.0 4.2 <2.0,<2.0,2.3 4.5 <2.0 <2.0,<2.0,2.5 <2.0 2.2 <2-0 2.8 <2.0 1 2 3 4 5 6 7 8 9 10 12 14 "it" `i Unvaccinated chickens Vaccinated chickens Unvaccinated chickens Vaccinated chickens <2.0 7.2 5.7 7.5 5.2 4.4 5.3 5.0 3.4 4.4 3.8 2.4 2.4 <2.0 2-5 2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2.0 <2-0 <2.0 <2.0 2.4 <2.0,<2.0,2.0 3.2 <2.0 2.4 <2.0 2.5 <2.0 <2.0 <2,0 2.0 <2,0 <2.0,<2.0,2.2 <2.0 2.3 <2,0 2.2 <2.0 * Three vaccinated and three unvaccinated chickens were killed at each of the stated times after challenge and the amount of IBV VF69-149 in the three organs determined; the medians for each set of three determinations are shown, except where only one of the three components yielded a positive result, when all three are shown. t Results are expressed as CDso; the lowest detectable concentration was, log10, 2.0 CDso per g. T a b l e 8. Amount ofE. coli 018 in the nose, trachea and spleen of Hl20-vaccinated chickens at different times after they were challenged intranasally with IBV strain VF69-149 and E . coli 018 log10 Amount of E. coli O18 per g tissue* & ( Time after challenge (days) 1/12 1 2 3 4 5 6 7 8 9 10 12 14 ~, Nose Trachea Spleen e x ~r ~ `i r x Unvaccinated Vaccinated Unvaccinated Vaccinated Unvaccinated Vaccinated chickens chickens chickens chickens chickens chickens 4.5 4.6 6.6 7.3 7.5 8.2 8.0 8.0 7.8 6.5 7-3 4-7 5-2 4.5 < 1.5, < 1.5, 1.5 <1.5, < 1.5, 3.9 <1.5 <1.5 4.2 < 1.5 < 1.5 < 1.5 < 1.5 4.9 3.5 < 1.5, < 1.5, 2.3 < 1.5, < 1.5, 2.2 < 1.5 3.7 3.5 < 1.5, < 1.5, 1.5 2.0 < 1.5, < 1-5, 2.0 3.0 6.5 < 1-5 2.4 < 1.5 3.0 6.8 < 1-5, < 1.5, 2.6 < 1-5, < 1.5, 2.7 < 1.5 < 1.5 7.0 < 1,5 2.4 < 1.5 < 1.5, < 1.5, 4.8 6.3 < 1-5, < 1-5, 4.8 3.3 < 1-5 2.3 6.6 < 1.5, < 1.5, 2.3 2.7 < 1.5, < 1.5, 1.5 3.7 6.6 < 1.5 3.6 < 1.5 < 1-5 4.3 < 1.5 2.4 < 1.5 < 1-5 3.9 < 1-5 < l-5 < 1-5 < 1-5 2.7 < 1-5 < 1-5 < 1-5 * Results are expressed as no. of viable organisms. The lowest detectable concentration was, loglo, 1.5 per g. For other details see footnotes to Table 7. Fate of l B V strain VF69-149 and E. coli 018 given to chickens that had been vaccinated with live I B V H120 T h e c o n c e n t r a t i o n s o f I B V V F 6 9 - 1 4 9 a n d E. coli O 1 8 w e r e e s t i m a t e d in t h e t i s s u e s o f H 1 2 0 vaccinated or unvaccinated chickens killed at different times after challenge with a mixture of these two organisms. High concentrations of both were found in the nose and trachea of the u n v a c c i n a t e d c h i c k e n s f r o m a b o u t t h e 1st to t h e 9 t h d a y a f t e r c h a l l e n g e ( T a b l e s 7 a n d 8). B o t h o r g a n i s m s w e r e f o u n d in t h e s p l e e n o f t h e m a j o r i t y o f t h e s e c h i c k e n s . N o n e , o r m u c h l o w e r Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 05:24:26 Immunity against IBV/E. coli infection 1433 concentrations of both organisms, were found in the nose and trachea of the vaccinated chickens. IBV was found in only one and E. coli in only two spleens examined from the vaccinated chickens. DISCUSSION Previous attempts to study IBV immunity in the chicken have depended on virus recovery from the respiratory tract (Winterfield et al., 1972) or on assessing the presence or absence of ciliary activity in the trachea (Darbyshire, 1980) following IBV challenge. Our experimental model resembles the natural disease produced by IBV and E. coli. In it the degree of protection is assessed by the fate of the intact animal following challenge. Consequently, it is a more realistic approach to studying IBV immunity than the earlier methods. The fact that H120, whose vaccinal efficiency in the field is well established, provided excellent protection against challenge with IB viruses of the same serotype as H120 and the E. coli Pool supports this view. The much poorer protection provided by inactivated IB vaccine is also in keeping with field experience. It might, however, in part be a reflection of the reduced immunological competence of young chickens. The use of young chickens in the present studies was considered necessary because the mortality rate in the model infection is considerably reduced in chickens over 5 weeks of age (Smith et al., 1985). The finding in H120-vaccinated chickens of no, or very low, concentrations of IBV strain VF69-149 and E. coli strain O18 in the nose and trachea following challenge with a mixture of these organisms, their apparent inability to invade the tissues and the contrasting observations in the challenged non-vaccinated chickens is in keeping with the hypothesis formed after studies on the pathogenesis of the experimental disease (Smith et al., 1985). This hypothesis is that the development of generalized E. coli infection, a common consequence of IB infection in the field, results from prior damage to the respiratory tract by IBV. This damage permits pathogenic E. coli to proliferate there and then invade the tissues. The virtual absence of IBV in the nose, trachea and spleen of the challenged, vaccinated chickens confirms the comprehensive nature of the protection provided by live H120 vaccination. Plaque reduction tests in chick kidney cell culture (Hopkins, 1974) and cross-neutralization tests in tracheal organ cultures (Johnson & Marquardt, 1975; Darbyshire et al., 1979; Cook, 1984) have demonstrated the serological diversity among IBV strains. The present studies, however, confirm reports (Hitchner et al., 1964; Raggi & Lee, 1965; Winterfield & Fadly, 1972) that the results of serological tests do not correlate with those of immunological studies and that protection against IBV infection in the chicken is more widely based than the results of in vitro serological tests indicate. For example, they reveal that live H 120 vaccination protected against challenge with a variety of IBV serotypes, specially chosen to assess the breadth of its immunological ability. These serotypes had a worldwide geographical distribution and included ones against which H 120 vaccine has been reported to give incomplete protection (Davelaar et al., 1984). Because many of them did not cause high mortality when given alone to experimental chickens, they could be used as live vaccines. They, too, usually gave good protection against challenge with the E. coli Pool and IB viruses of both the Massachusetts serotype (VF69-149) and a non-Massachusetts serotype (HVI-116), viruses selected because they had caused high mortality when given to chickens with the E. coli Pool (Smith et al., 1985). As was observed following live H120 vaccination, the protection in a few instances was poor. It was perhaps significant that this was so in the experiments involving a minority of the IBV strains currently believed to be causing outbreaks of disease in vaccinated flocks in the field (Cook, 1984; Cook & Huggins, 1986). The results reported here, however, suggest that, in general, inadequacy of the currently used vaccines cannot be held responsible for the majority of these outbreaks. We are grateful to Mrs M. M. Ellis and Mrs T. Berrill for their capable technical assistance. We are also indebted to Professor P. M. Biggs, Mrs S. Pilcher and Mr B. Wells and his staff for helping in a variety of ways. REFERENCES COOK, J. K. A. (1984). The classification of new serotypes of infectious bronchitis virus isolated from poultry flocks in Britain between 1981 and 1983. Avian Pathology 13, 733-741. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 05:24:26 1434 J.K.A. C O O K , H. W I L L I A M S S M I T H A N D M. B. H U G G I N S COOK, J. K. A. & HUGGINS, M. B. (1986). Newly isolated serotypes of infectious bronchitis virus: their role in disease. Avian Pathology 15, 129-138. DARBYSHIRE, J. H. (1980). Assessment of cross-immunity in chickens to strains of avian infectious bronchitis virus using tracheal organ cultures. Avian Pathology 9, 179-184. DARBYSHIRE, J. H., ROWELL, J. G., COOK, J. K. A. & PETERS, R. W. (1979). Taxonomic studies on strains of avian infectious bronchitis virus using neutralisation tests in tracheal organ cultures. Archives of Virology 6 1 , 2 2 7 238. DAVELAAR, F. G., KOUWENHOVEN, B. & BURGER, A. G. (1983). Experience with vaccination against infectious bronchitis in broilers and significance of and vaccination against infectious bronchitis variant viruses in breeders and layers in The Netherlands. Clinica veterinaria 106, 7-11. DAVELAAR,F. G., KOUWENHOVEN,B. & BURGER, A. G. (1984). Occurrence and significance of infectious bronchitis virus variant strains in egg and broiler production in The Netherlands. Veterinary Quarterly 6, 114 120. HITCHNER, S. B., APPLETON, G. S. & WINTERFIELD, R. W. (1964). Evaluation of the immunity response to infectious bronchitis virus. Avian Diseases 8, 153 162. HOPKINS, S. R. (1974). Serological comparisons of strains of infectious bronchitis virus using plaque-purified isolants. Avian Diseases 18, 231-239. JOHNSON, R. B. & MARQUARDT,W. W. (1975). The neutralizing characteristics of strains of infectious bronchitis virus as measured by the constant-virus variable-serum method in chicken tracheal cultures. Avian Diseases 19, 82 90. RAGGI, L. G. & LEE, G. G. 0965). Lack of correlation between infectivity, serologic response and challenge results in immunization with an avian infectious bronchitis vaccine. Journal of Immunology 94, 538-543. SMITH, H. WILLIAMS& HUGG1NS, M. B. (1980). The association of the O 18, K 1 and H7 antigens and the Col V plasmid of a strain ofEscherichia coli with its virulence and immunogenicity. JournalofGeneral Microbiology 121,387 400. SMITH, H. WILLIAMS,COOK, J. K.A. & PARSELL, Z. E. (1985). The experimental infection of chickens with mixtures of infectious bronchitis virus and Escherichia coll. Journal of General Virology 66, 777-786. WINTERFIELD, R. W. & FADLY, A. M. (1972). Some characteristics of isolates of infectious bronchitis virus from commercial vaccines. Avian Diseases 16, 746-755. WINTERFIELD, R. W., FADLY, A. M. & BICKFORD, A. A. (1972). The i m m u n e response to infectious bronchitis virus determined by respiratory signs, virus infection and histopathological lesions. Avian Diseases 16, 260-269. (Received 18 January 1986) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 05:24:26