title: Direct detection of infectious bursal disease virus from clinical samples by in situ reverse transcriptase-linked polymerase chain reaction.
abstract: The presence of the very virulent (vv) Brazilian strain of infectious bursal disease virus (IBDV) was determined in the bursa of Fabricius, thymus and liver of 2-week-old broilers from a flock with a higher than expected mortality. For this purpose, a direct in situ reverse transcriptase (RT)-linked polymerase chain reaction (PCR) method was developed using specific primers for vvIBDV. Unlabelled forward and reverse biotinylated oligonucleotides were used for RT-PCR in a one-step method and the respective products were revealed by a direct enzymatic reaction. The results were compared with those obtained by standard RT-PCR using general primers for IBDV and virus isolation. The virus isolation, RT-PCR and in situ RT-PCR revealed positive results on the bursa of Fabricius in 86%, 80% and 100%, respectively. The in situ RT-PCR detected vvIBDV in all tested thymus and liver samples, whereas the standard RT-PCR detected virus in 80% and 90% of the samples, respectively. After three consecutive passages on chicken embryonated eggs, IBDV was isolated from 64% of the thymus samples and 30% of the liver samples. In the present study, no classical or antigenic variants of IBDV were detected. The developed in situ RT-PCR assay was able to detect the very virulent strain of IBDV with a higher sensitivity than the conventional RT-PCR and virus isolation.
authors.affiliation: São Paulo State University, UNESP-Laboratório de Virologia, DAPSA, Brasil. tcardoso@fmva.unesp.br
authors.forename: Tereza C
authors.initials: TC
authors.lastname: Cardoso
authors.forename: Ana C G
authors.initials: AC
authors.lastname: Rosa
authors.forename: Rafael D
authors.initials: RD
authors.lastname: Astolphi
authors.forename: Rafael M
authors.initials: RM
authors.lastname: Vincente
authors.forename: Juliana B
authors.initials: JB
authors.lastname: Novais
authors.forename: Karina Y
authors.initials: KY
authors.lastname: Hirata
authors.forename: Maria Cecilia R
authors.initials: MC
authors.lastname: Luvizotto
mesh_terms: D000818:Animals; D018175:Birnaviridae Infections / Q000453:epidemiology / Q000662:veterinary* / Q000821:virology; D001938:Brazil / Q000453:epidemiology; D002060:Bursa of Fabricius / Q000821:virology; D002645:Chickens; D007243:Infectious bursal disease virus / Q000302:isolation & purification*; D008099:Liver / Q000821:virology; D011201:Poultry Diseases / Q000453:epidemiology / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013047:Specific Pathogen-Free Organisms; D013950:Thymus Gland / Q000821:virology; D014776:Virus Cultivation