Polymerase chain reaction combined with restriction enzyme analysis for detection and differentiation of fowl adenoviruses

Abstract

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.

Occurrence of Aviadenovirus in chickens from the poultry industry of Minas Gerais

“…No detection was obtained from the SPF chicks. Using these primers, FAdV would be detected by PCR regardless of serotype (Meulemans et al, 2001). The PCR protocol used in this study enabled the detection of the target genomic segment in the total DNA diluted up to 10 -9 on the positive FAdV Phelps strain used as control.…”

“…1), as visualized on agarose gel electrophoresis under ultraviolet light (Meulemans et al, 2001). No detection was obtained from the SPF chicks.…”

“…It is assumed that a lower percentage of chicks would be produced with vertical infection from infected breeders and a progressive horizontal transmission would occur in the farm during the egg production year. (Meulemans et al, 2001). …”

“…Cook) was used as positive control in each reaction, originally replicated in SPF chicken livers. Primers F 5'-(CAA GTT CAG GCA GAC GGT)-3' (Hexon A) and R 5'-(TAG TGA TGC CGC GAC ATC AT)-3' (Hexon B) and reaction protocols were as described previously (Meulemans et al, 2001) for amplifying the Aviadenovirus hexon protein coding gene sequence, including the L1 loop region (897 bp). The evolutionary relationships of Aviadenovirus sequences were inferred using the Neighbor-Joining method (Saitou and Nei, 1987) and a bootstrap consensus tree was inferred from 1000 replicates (Felsenstein, 1985) representing the evolutionary history of the Aviadenovirus sequences analyzed (Felsenstein, 1985).…”

“…The polymerase chain reaction (PCR) is a molecular tool that allows highly specific and sensitive detection of the FAdV genome (Xie et al, 1999;Meulemans et al, 2001;Singh et al, 2002), with cost-benefit advantages against the traditional routine diagnostic tests.…”