Duplex Shuttle PCR for Differential Diagnosis of Budgerigar Fledgling Disease and Psittacine Beak and Feather Disease

Abstract

Two common viral diseases in psittacine birds including budgerigar fledgling disease (BFD), generally called avian polyomavirus (APV) infection, and psittacine beak and feather disease (PBFD) have similar clinical manifestations characterized by feather disorders. A duplex shuttle PCR was developed for detection of APV and PBFD virus (PBFDV). Two pairs of oligonucleotide primers were designed to amplify a 298‐bp fragment of the t/T antigen region of APV genome and a 495‐bp fragment of the capsid protein region encoded by open reading frame (ORF) C1 of PBFDV genome, respectively. In the present study, APV and PBFDV were detected simultaneously in one tube by duplex shuttle PCR using these two pairs of primers. The detection limits were 2 viral copies of APV and 3 viral copies of PBFDV. In the clinical application, we detected 16 APV‐positive, 15 PBFDV‐positive, and 3 mixed infected samples in 39 samples examined. Sequences of the amplified products were read. The t/T antigen region was conserved in the APV‐positive samples as expected. ORF C1 of PBFDV genome showed diversity. Phylogenic analysis indicated that PBFDV ORF C1 consisted of 6 clusters which were related to subfamilies of psittacine birds. Our duplex shuttle PCR could be a useful method for differential diagnosis and molecular epidemiology of BFD and PBFD.

Mycobacterium genavenseand avian polyomavirus co-infection in a European Goldfinch (Carduelis carduelis)

“…Avian polyomavirus (APV) infections are well documented in some psittacine species (Krautwald et al, 1989;Gerlach, 1997;Ogawa et al, 2005). Infections have been also detected in non-psittacine pet birds (Johnston & Riddell, 1986;Sironi & Rampin, 1987;Forshaw et al, 1988;Sironi, 1991) but there has been limited characterization of the effects of these infections.…”

Relatedness between Host Species and Genotype of Beak and Feather Disease Virus Suggesting Possible Interspecies Cross Infection during Bird Trade

“…PCR for BFDV examination was performed using Primer2 (5'-AAC CCT ACA GAC GGC GAG-3') and Primer4 (5'-GTC ACA GTC CTC CTT GTA CC-3') [13], which targets a part of ORF V1, to compare with other epidemiological reports [3,7,12]. PCR was carried out by using TaKaRa Ex Taq (TaKaRa Bio., Otsu, Japan) as described previously [11,18]. In our laboratory, the detection limit of this PCR was 2.44 × 10 4 copies of viral DNA per 50 µl reactions.…”

“…DNA samples were amplified with PBFDdupF (5'-TTG GGT CCT CCT TGT AGT GGG ATC-3') and PBFDdupR (5'-CAG ACG CCG TTT CTC AAC CAA TAG-3'), which were used to amplify a 495-bp fragment corresponding to the part of ORF C1 [14], because genetic clusters were accorded to some psittacine species or subfamily in the phylogenic analysis using BFDV ORF C1 [5,6,11,13,18]. Molecular cloning and sequencing were examined as described previously [11], and at least three clones were sequenced. Sequences were edited using Genetyx-Mac version 13 computer software (Genetyx Co., Tokyo, Japan) and assembled using Genetyx-Mac/ATSQ version 4.2.4 computer software (Genetyx Co.).…”

Genetic Analysis of Beak and Feather Disease Virus Derived from a Cockatiel (Nymphicus hollandicus) in Japan

“…The genome sequence of this BFDV isolate, which was designated CO-JA, was amplified by PCR using two pairs of primers (Primer 2/PBFDdupR and PBFDdupF/Primer 4) as described previously (Table 1) [6,10]. The PCR products were directly sequenced by the primer walking method and assembled as described previously [3].…”