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Chicks without specific maternal antibody to avian infectious bronchitis virus were vaccinated intranasally at two weeks old with a live H120 vaccine and a proportion revaccinated with a live H52 vaccine at 15 weeks. Groups of these birds were then challenged intratracheally with the Massachusetts M41 strain at varying intervals and their humoral antibody responses monitored by neutralisation and haemagglutination-inhibition tests. Protection was assessed by the presence of tracheal ciliostasis four days after challenge and was shown to correlate completely with histopathological findings and immunofluorescence microscopy. Chicks were protected by H120 alone up to 12 weeks old and, following revaccination with H52, up to between 30 and 50 weeks. The antibody responses following challenge became increasingly enhanced as the vaccinal antibody titres declined. The results are discussed in relation to previous reports and the response to vaccination against infectious bronchitis virus of chickens in commercial flocks.

Six strains of avian infectious bronchitis virus (IBV) were used to inoculate explants of a range of 15 chicken tissues and virus growth kinetics observed over a period of 96 hours thereafter. Similar patterns of virus production were given by all 6 strains from explants of any particular tissue such as the nasal turbinates, trachea, lung, air sacs and oviduct. Nevertheless, significant differences in behaviour between strains were noted for different tissues and in the efficiency with which certain tissues produced virus. It is suggested that the method has a potential value in determining the virulence of different strains of IBV by comparing their pathogenesis of chicken tissues in vitro.

The ability of nine strains of avian infectious bronchitis virus (IBV) to induce chicken interferon has been investigated using Semliki Forest virus for the tests. The Beaudette, H120 and Connecticut 46 strains induced interferon in the allantoic fluids of embryonated hens' eggs, the highest titre (1 : 30) being associated with Beaudette; but these as well as the Massachusetts M-41 and H52 strains failed to yield interferon in primary monolayer cultures of chick kidney cells as did all nine strains in organ cultures of chick embryo trachea. None of six strains of IBV investigated was susceptible to the inhibitory effects of chicken interferon.

Long-term organ cultures of a range of tissues collected from specific pathogen-free chickens were employed to determine their susceptibility, and their capacity for subsequent virus production, following inoculation with avian infectious bronchitis (AIB) virus. When inoculated with approximately 2-0 log10 median ciliostatic doses (CD50) of a classical highly egg-adapted vaccine strain (H120) of AIB virus, 9 of 23 tissues were shown to be susceptible, namely the nasal turbinates, trachea, air sac membranes,lungsasal turbinates, trachea, air sac membranes, lungs, proventriculus mucosa, thyroid, kidney, ovary and oviduct. When the remaining 14 tissues were inoculated with a high dose of virus (6.8 log10 CD50), the conjunctiva, caecel tonsil, testis and bursa of Fabricius were susceptible whereas the oesophagus and cloaca responded minimally. Inoculation of the same range of tissues with a high or low dose of a field strain (HVI9) of AIB virus produced similar results, except for a number of individual variations in response, due possibly to strain differences in pathogenicity. Determinations of the minimal infectious dose requirements of the susceptible tissues revealed that the efficiency of infection with the H120 strain was highest for the nasal turbinate and tracheal tissues, and thereafter, in order of decreasing efficiency, were the air sac membranes, lung, oviduct, proventriculus mucosa, conjunctiva, kidney, ovary, bursa of Fabricius, thyroid, testis, caecal tonsil, cloaca and oesophagus. The relevance of these results is discussed in connection with the early events in the pathogenesis and the clinical syndrome of AIB infection in chickens.

Avian infectious bronchitis coronavirus (IBV) inactivated by beta-propiolactone induced partial protection of the trachea in up to 40% of chickens following one intramuscular inoculation 4 to 6 weeks prior to challenge. Retention of an intact tracheal ciliated epithelium 4 days after challenge was the criterion of protection. There was no correlation between protection and serum titres of virus-neutralizing (VN) and haemagglutination-inhibiting (HI) antibody, which were maximal at about 4 weeks after inoculation. Virus from which the S1 but not the S2 (spike-anchoring) spike glycopolypeptide had been removed by urea did not induce protection or VN or HI antibody. Four intramuscular inoculations of monomeric S1 induced VN and HI antibody in two and four chickens respectively. These results indicate that VN and HI antibodies are induced primarily by S1, that intact spikes are a major requirement for the induction of protective immunity and that this property is probably associated with S1.

The minimal infectious dose of the H52 strain of infectious bronchitis virus for organ cultures of oviduct and kidney was compared in chickens of different ages. Organ cultures of oviduct were found to be highly susceptible to infection regardless of the age of chicken and no difference in susceptibility could be demonstrated between cultures of the magnum and uterus regions of the mature oviduct. Kidney organ cultures were less susceptible and resistance to infection increased significantly (P less than 0.001) with the age of the chicken from which cultures were prepared.

The antigenic relationships of 24 strains of avian infectious bronchitis virus (IBV) were investigated by serum neutralisation tests performed in chick embryo tracheal organ cultures. The serum dilution that neutralised 100 median ciliostatic doses (CD50) of virus was estimated from the linear relationship between varying concentrations of each virus strain and the neutralisation titre of homologous antiserum; this dilution defined 1 antibody unit. Antisera diluted to contain 20 antibody units were then tested by neutralisation against 1.5--2.5 log10 CD50 of each strain. Clusters of both strains and antisera in turn were established by methods of numerical taxonomy using as measures of resemblance Euclidean distance and correlation coefficient, and by analysis by principal components. These analyses identified a group of 8 similar strains; neutralisation of the remaining 16 strains was slight. Similar results were obtained by classifying antisera, except that a further group of 3 antisera was demonstrated, each having a neutralising capacity for most strains. Implications for vaccine formulation are discussed.

Commercially bred chicks with maternally derived antibody to avian infectious bronchitis virus (IBV) were hatched in isolated conditions and a number vaccinated within the first three weeks of life with live IBV strain H120. Humoral antibody responses were assayed by haemagglutination inhibition (HI) or neutralisation (SN) tests, and the degree of protection against challenge with the virulent Massachusetts M41 strain assessed on the basis of tracheal ciliary activity four days after challenge. Maternal antibody in unvaccinated chicks declined linearly with a mean half-life of five to six days based on both HI and SN tests; these chicks were protected against challenge until four weeks old. There was complete correlation between ciliary activity and histopathological findings, but little between protection and antibody titre. It was concluded that the optimum age for primary vaccination was about two weeks.

An egg-adapted vaccine strain (H120) and an organ culture-passaged field strain (HV-10) of avian infectious bronchitis (AIB) virus were propagated in tracheal organ cultures and their growth kinetics examined using nine-day-old embryonated fowl eggs and chick tracheal explants for virus assay. When the H120 strain was assayed in embryonated eggs, titres were approximately log10 2-0 ID50 (50 per cent infectious dose) per ml higher than when assays were performed in tracheal explants. The HV-10 strain, assayed in tracheal explants, yielded higher titres than did the H120 strain, but when assayed in embryonated eggs, yielded only minimal and variable virus titres.

A study has been made of the use of chicken tracheal organ cultures for the isolation and assay of avian infectious bronchitis (AIB) virus from both naturally and experimentally infected chickens. Six strains of AIB virus were investigated, 3 of which had been isolated from natural outbreaks of disease. Two of the virus isolations from the outbreaks of AIB were made directly into tracheal organ cultures without passage in embryonated eggs. Organ cultures prepared from 20-day-old embryos were used since they were found to be somewhat more sensitive in virus assay than those derived from chickens of up to 31 days of age. Ciliostasis, which was used as the marker of infectivity, was complete by 3 days after inoculation with each strain of virus examined. Virus could be isolated from both respiratory and non-respiratory tissue in tracheal organ cultures and these cultures were found to be at least as sensitive as 9-day-old embryonated eggs in detecting AIB virus either in pathological material or in serial dilutions. When virus was assayed in both systems, the titres were very similar. It is considered, therefore, that chicken embryo tracheal organ cultures offer a reliable alternative system to embryonated eggs for studying AIB virus.