A polymerase chain reaction combined with restriction enzyme analysis was developed for detection and differentiation of all 12 fowl adenovirus (FAdV) serotypes representing the five fowl adenovirus (A to E) species. For primer design, the published sequences of the hexon proteins of FAdV1, FAdV8 and FAdV9 were aligned and conserved regions in the two pedestal regions adjacent to the L1 loop region were determined. A primer pair (hexon A/hexon B) was constructed and was shown to amplify approximately 900 bp of the hexon gene including the L1 loop region. An amplification product was detected using supernatant of infected cell cultures from all FAdV1 to FAdV12 reference strains used in our study. The sequence and the restriction patterns of the hexon A/B fragments of the 12 FAdV strains were determined and compared. The successive use of four different endonucleases allowed the complete differentiation of the reference FAdV strains. Twenty-six fowl adenoviruses isolated during our routine virological diagnosis activities could all be amplified using hexon A/hexon B primers. Restriction analysis results showed that 8/26 adenovirus strains contained two different FAdV types. FAdV4, FAdV12, FAdV1, FAdV5 and FAdV6 were the most frequently isolated.
The sequences of the L1 loop of the hexon protein from representative fowl adenovirus (FAdV) strains of the different European and American collections were determined and compared. This study highlighted the lack of consensus in the numbering of the individual serotypes between the American and the European classifications. An identification system is proposed based on restriction fragment length polymorphism of the hexonA/hexonB polymerase chain reaction product. In addition, new insights into the relationships among FAdV strains are presented and discussed on the basis of phylogenetic analysis of the L1 loops sequences. Six clusters of strains that are supported by high bootstrap values were identified. Three of them are clearly independent, forming groups A, B and C, whereas the three others are clustered in a single 'supergroup', denominated D. Interestingly, the Japanese strain TR22 that is presently classified as European type 5 (species B) could not be assigned to any of the aforementioned clusters and might therefore constitute the sole representative of a seventh cluster.
Infectious bronchitis virus (IBV) was isolated from each of 236 broiler flocks that had respiratory infection (86%), impaired growth, enteritis and/or nephritis (14%), over a 10-year period from 1986 to 1995 in Belgium. Among them, 65% of the investigated flocks had not been vaccinated against infectious bronchitis. Type-specific reverse transcriptase polymerase chain reactions (RT-PCRs) were used after propagation of the isolates in embryonated eggs in order to detect and differentiate Massachusetts, D274, B1648 and 793/B types. The incidence of these types was approximately 50, 38, 11 and 1%, respectively. In 16% of cases, two or three types of IBV were detected, representing mostly combinations of Massachusetts and D274. The majority of the Massachusetts and D274 isolates (68 and 69%, respectively) were recovered from non-vaccinated flocks, confirming that such flocks are at greatest risk of infection by these types of IBV. Interestingly, the B1648 type was isolated from more vaccinated flocks (14%) than non-vaccinated flocks (7.6%). Most surprising was the very low incidence (1%) of the 793/B type, which was the dominant type in some neighbouring countries, during the period of investigation. The DNA derived by RT-PCR from 24 of the Massachusetts-type isolates from 12 vaccinated and 12 non-vaccinated flocks was sequenced and compared with the sequence of Massachusetts vaccines used in Belgium. This revealed that the sequence of four of the isolates (two from vaccinated and two from non-vaccinated flocks) was identical to that of a Massachusetts vaccine strain. Similar results were obtained for D274 isolates when compared with the sequence of D274 vaccines. These sequencing results demonstrate a co-circulation of vaccine and wild-type infectious bronchitis viruses in broilers, and are further justification for permanent monitoring of circulating strains in order to rationally modify vaccination strategies to make them appropriate to the field situation.
Specific-pathogen-free White Leghorn chickens and commercial broilers were inoculated orally at 1 day of age with different intestinal preparations containing a chicken parvovirus, an entero-like virus associated with a reovirus from field materials, or the entero-like viruses and reovirus alone. Despite viral multiplication in inoculated birds, no clinical signs or growth retardation were observed in SPF and broiler chickens infected with the reo or parvoviruses. Abnormal faeces and reduction in weight gains were observed after infection with the field materials and the entero-like viruses. Some easily sedimentable particles could be involved with the entero-like virus in the aetiology of runting syndrome. Proventriculitis was present in chickens inoculated with one of the field materials and with the entero-like virus isolated from that material. Specific-pathogen-free White Leghorn chickens were as susceptible as commercial broiler chickens to weight gain depression after oral inoculation with crude homogenates at 1 day of age.
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