Yogender Singh*1, KalpanaYadav1, Vinod Kumar Singh2
College of Biotechnology, NanajiDeshmukh;Pashu-ChikitsaVigyanVishwavidyalaya, Jabalpur (MP)
Division of Virology, I.V.R.I.,Izatnagr, Bareilly (U.P.)
*corresponding author: [email protected]
Introduction
Among infectious diseases of poultry, infectious bursal disease (IBD) is major threat to poultry industry because it causes immunosuppression and high mortality; generally at 3 to 6 weeks of age so early diagnosis of disease is very important to lessen the economic loss. Information about the immunology and molecular make up of IBDV is accumulating rapidly which has led to the development of more sensitive and specific diagnostic tools for the detection and differentiation of IBD viruses.Both conventional and molecular techniques are being used for the diagnosis of IBD. Conventional diagnosis involve diagnosed by signs and symptoms, postmortem lesions and histopathological findings along with isolation and identification of the virus along with virological and serological tests while the molecular techniques used for diagnosis include techniques like reverse transcription-polymerase chain reaction (RT-PCR), restriction enzyme analysis (RE analysis) and sequencing. These molecular techniques are very effective and having high sensitivity.
Virus structure and genome organization
IBDV is a nonenveloped isometric particle with a diameter of about 60–65 nm. IBDV contains two segments of linear double-stranded RNA. Segment A has 3.2 kb and contains two partially overlapping open reading frames (ORFs). The largest ORF encodes for a polyprotein that is proteolytically cleaved to form three polypeptides: VP2 and VP3 are the structural proteins, whereas VP4 is a protease. VP2 contains the major antigenic sites, responsible for eliciting neutralizing antibodies. Segment B has 2.8kb and encodes for VP1, an RNA-dependent RNA polymerase. Sequencing of the VP2 hypervariable region, which contains important neutralizing epitopes, has been important to characterize IBDV strains.The virus has two distinct serotypes based on the cross neutralization test. Serotype-1 is pathogenic to chickens and varies in its virulence and serotype 2 isolated from turkey is pathogenic to both turkey and chicken.
Diagnostic of IBD
Apart from epidemiology and prophylaxis, early diagnosis is also very important in controlling the disease and its spread and thus reducing the economic loss it will cause. Infectious bursal disease is one of the major diseases of poultry which cause large number of mortality in poultry and its early diagnosis is very important to reduce the loss and avoid spread to other birds. Both conventional and molecular techniques are being used for the diagnosis of IBD.
A. Clinical diagnosis
1.Sign and symptoms
Infectious bursal disease in flock is firstly identified by the sing and symptoms.
2.Postmortem lesions
The postmortem lesions observed are edematous, enlarged and hemorrhagic bursa which becomes turgid and turns atrophic in 7-10 days. The infected bursa also shows necrotic foci and petechial hemorrhages on the mucosal surface and distension of ureter with urates, thymicatrophy and congestion, spleenomegaly with uniformly dispersed small grey foci on serosal surface having mottled appearance, ecchymotic hemorrhages in muscles of thigh, pectoral regions, and mucosa of proventriculus.
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B. Laboratory daignosis
1. Isolation
Chorioallantoic membrane (CAM) route inoculation of 9 -11 days old embryonated chicken eggis the most sensitive route for isolation of the IBDV which could subsequently be adapted to the allantoic sac and yolk sac route of inoculation.
2.Molecular techniques
Molecular techniques are used for detection and differentiation of both serotypes and antigenic subtypes. The techniques such as reverse transcription-polymerase chain reaction (RT-PCR), restriction enzyme analysis (RE analysis) and sequencing are highly sensitive, extremely specific and versatile. These techniques not only have the potential to detect minute quantity of the infectious agent, even when pathogen has lost its infectivity but can also differentiate closely related viruses directly from clinical samples without going for virus isolation.
(a)Reverse Transcriptase Polymerase Chain Reaction and RE analysis
The potential of using RT-PCR followed by RE analysis as a method for differentiation of different IBDV strains. It was concluded that bursa and caecal tonsils were suitable organs for the routine diagnosis of IBD by RT-PCR. The RNA isolated from clinical samples is used for the amplification of structural genes, out of them VP2 gene is more suitable target.
(b)Nucleotide Sequencing
The most precise and reliable technique for the genomic characterization, taxonomic classification and molecular epidemiology of the pathogens is nucleotide sequencing. The RT-PCR amplified segments of targeted gene are sequenced and the obtained sequences can be used for analysis.
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