Detection of Infectious Bursal Disease Virus in Different Lymphoid Organs by Single-Step Reverse Transcription Polymerase Chain Reaction and Microplate Hybridization Assay

Abstract

Abstract.A rapid and sensitive method for the detection of infectious bursal disease virus (IBDV) RNA in different chicken lymphoid organs was developed. The method is based on a single-step reverse transcription polymerase chain reaction (RT-PCR) procedure and the enzyme-linked immunosorbent assay (ELISA) detection method of amplified products. Vaccinal IBDV strain and field isolates were used for the optimization of RT-PCR and for the determination of conditions for microplate hybridization and colorimetric detection of the amplicons. With this method, viral RNA could be detected in various stages of infection in samples of different lymphoid organs. Bursas and cecal tonsils were suitable organs for viral RNA detection at different times during IBDV infection. The RT-PCR/ELISA method can be applied for IBDV detection in routine diagnostic tests, which are not usually carried out because of the difficulties involved in isolating the virus.Infectious bursal disease (IBD) is a highly contagious viral disease of young chickens that causes significant economic losses in the poultry industry worldwide. 15,16 The clinical disease occurs between 3 and 6 weeks of age. Severe outbreaks are characterized by sudden onset of depression in susceptible flocks. On postmortem examination of birds that died during the acute phase, the bursa of Fabricius is the principal diagnostic organ; it is turgid, edematous, and sometimes hemorrhagic and turns atrophic within 7-10 days. 21 Dehydration and nephrosis with swollen kidneys are common, and hemorrhages in the muscle and mucosa of the proventriculus are observed in the majority of affected birds. 22 The subclinical form of the disease results in immunosupression and increases susceptibility of chickens to other infections. 1 The etiologic agent of IBD is a nonenveloped virus (IBDV) that belongs to the Birnaviridae. 6 Its genome consists of 2 segments of double-stranded RNA. The smaller RNA segment (B) encodes a single 90-kD polypeptide, designated VP1, and the larger RNA segment (A) encodes a viral polyprotein that is processed to form 3 mature proteins, VP2, VP3, and VP4, 2,10 and a small protein, VP5, of unknown function. 17 The published nucleotide sequences of genome segments A and B of various IBDV strains indicate that sequences of the major host-protective immunogen VP2 are highly conserved except in the central hypervariable region, which displays the greatest amount of amino acid sequence variation among strains. 4,7,24 Various methods have been developed for the diagnosis of IBD, such as virus isolation in cell culture, embryonated chicken eggs, or young specific-pathogen-free (SPF) chickens and localization of the virus in infected tissues by electron microscopy, fluorescence assay, agar immunodiffusion, antigene-capture enzyme-linked immunosorbent assay (ELISA), or immunohistochemistry. All these methods have disadvantages, such as being time consuming, labor intensive, expensive, or nonspecific. These methods lack the ability to detect low levels of IBDV antigens in t...

New oligonucleotide microarray for rapid diagnosis of avian viral diseases

“…All these methods have disadvantages, such as being time consuming, labor intensive, expensive, or nonspecific. These methods lack the ability to detect low levels of antigens in tissues [36][37][38][39][40].…”

Diagnosis of Clinical Cases of Infectious Bursal Disease Using a Modified Rapid Taq Man-MGB Real-Time RT-PCR Assay

“…Various methods have been developed for the diagnosis of IBD, such as virus isolation in cell culture, embryonated chicken eggs, or young specific-pathogen-free (SPF) chickens and localization of the virus in infected tissues by electron microscopy, fluorescence assay, agar immunodiffusion, antigene-capture enzyme-linked immunosorbent assay (ELISA), or immunohistochemistry [11]. However, all these methods have disadvantages, such as being time consuming, labor intensive, expensive, or non-specific [12]. Recently, various diagnostic methods targeting viral nucleic acids have been elaborated such as conventional reverse transcription-polymerase chain reaction (RT-PCR), real time RT-PCR (rRT-PCR) and loop-mediated isothermal amplification (LAMP) [13].…”

Analisis Fragmen Gen VP-2 Virus Infectious Bursal Diseases yang Diisolasi dari Peternakan Ayam Komersial

“…Pada umumnya identifikasi virus IBD banyak difokuskan pada amplifikasi gen VP2, meskipun Lin et al (1994) IBD (Becht et al, 1988). Teknis diagnosis molekuler tersebut dewasa ini, juga menjadi pilihan karena kemampuan deteksi sangat akurat, sensitif, cepat, dan dapat digunakan menggantikan diagnosis mi krobiologi in vivo dan in vitro (Lin et al,1994;Barlic Maganja et al, 2002). Di samping itu, teknis mole kuler juga dapat diaplikasikan untuk mendeteksi virus IBD yang diekstraksi dari berbagai organ dan leukosit oleh BarlicMaganja et al (2002).…”