Detection and differentiation of infectious bursal disease virus from the outbreaks in two layer farms by PCR-RFLP in Jos , Nigeria

Aim: Characterization of Infectious bursal disease viruses (IBDV) from the two outbreaks in Jos Nigeria, using reverse transcriptase polymerase chain reaction/restriction fragment length polymorphism (RT-PCR/RFLP) technique. Materials and Methods: A total of 40 bursa samples were collected from two outbreaks in November 2011 from two farms of 6-8 weeks old pullets within Jos South Local Government Area, with mortality between 60 – 74.2 % in commercially reared layer chicken flocks experiencing signs typical of infectious bursal disease (IBD). All the samples were found to contain IBDV genome by One Step RT-PCR using VP2 gene specific primers. Result: The assay amplified a 743bp fragment from 701-1444 nucleotides. RT-PCR product was further subjected to restriction digestion using TaqI, MvaI and SacI Restriction enzymes to differentiate classical from very virulent phenotypes. The RFLP profile was found similar for all eight isolates with TaqI and MvaI enzyme but different for SacI. All eight TaqI positive Viruses were further found positive for MvaI digestion and yielded RFLP profile similar to vvIBDV in Europe whereas one isolate was SacI positive and had a RFLP profile similar to classic IBDV strains. Conclusion: The clinical history of high mortality and TaqI and MvaI restriction enzyme positivity revealed that vvIBDV strains still exist in Jos, North central Nigeria.


Introduction
protein.The polyprotein is bio-transformed into VP2 and VP3 and viral protease VP4.The VP5 plays a role Infectious bursal disease (IBD) or Gumboro is an in maturation and release of the virion while VP1 acute and highly contagious disease of immature encodes for RNA dependent RNA polymerase (RdRp) chickens.Chickens of age 3 to 6 weeks are most by B segment of the genome [2,6].susceptible to clinical infection.The causative agent is The VP2 house the region responsible for Infectious bursal disease virus (IBDV) with primary differences in phenotypic characteristics.It is also a affinity for actively mitotic B-lymphocytes within the major structural protein of the viral capsid that carries bursa of fabricius where it multiplies.In the process of highly conformational epitopes responsible for infection other organs systems are involved in its induction of protective antibodies [7,8].immunology.Viral activity leads to immune-suppre-Clinically, IBDV detection and isolation on chick ssion [1,2] thereby making chickens susceptible to other embryo fibroblast is a time consuming and laborious diseases and subsequently drops in egg production and procedure.Serologic and molecular diagnosis enables quality [3].more exact differentiation of strains.The application of Infectious bursal disease virus belongs to the molecular techniques covers detection and charactegenus Avibirnavirus and family Birnaviridae divided rization with regard to virulence factors and epidemiology into two serotypes (1 and 2).Serotype 1 is further subof the virus [9].In other to control this economically divided into six (6) subtypes that ranges from apathogenic important disease of commercial poultry, rapid identito very virulent strains for chickens while serotype 2 fication of IBDV strains is vital.have been reported to be non-pathogenic [4,5].
Reverse transcriptase-polymerase chain reaction/ The non-enveloped and bi-segmented IBDV Three bands of 425, 321, and 101 bp were After homogenizing 9 ml of PBS was added and obtained on electrophoresis gel following digestion of centrifuged in a refrigerated centrifuge at 10,000 rpm DNA fragments with TaqI which revealed the presence for 5 min to make 10% tissue suspension.The superof two restriction sites (Table -1).Four bands were also natant was decanted into a sterile tube and kept at 4°C obtained following digestion of PCR products with for RNA extraction [9] and the pellet discarded into a MvaI.Agarose gel electrophoresis bands of 424, 209, disinfectant (Virkon®).The viral RNA extraction was 172 and 119 bp were obtained indicating three carried out using QIAamp Viral RNA Mini kit restriction sites.Moreover, agarose gel electrophoresis following the manufacturer's guidelines.Extracted bands of 475 and 285 were observed after SacI RNA was kept briefly at 4°C pending RT-PCR digestion of sample 4 (BF 36) and vaccine control DNA segments from the representative samples suggesting RT-PCR/REA: Six µl extracted viral RNA was amplialso one restriction site.fied in a single tube in a one step RT-PCR using Titan one Tube RT-PCR system (Roche, Germany) using Discussion gene specific primers in a total reaction volume of 50 IBDV is a very serious economic disease of µl.The reaction was carried out in 200 µl tubes with the poultry that has persisted in Nigeria for decades.One of following conditions: Each reaction contains 6 µl of the first outbreaks was reported from the Western part RNA, 0.2 mM each dNTPs, 10 units of RNase inhibitor of Nigeria in 1973.Since then the disease has (MBI Fermentas), two units of Enzyme mix, MgSO4 continued to occur [12].Due to the endemic nature of and the 5x RT-buffer provided by the manufacturer, the disease in Nigeria, vaccination has been the major 20mM of the primer J1 (F) 5'-GGC CCA GAG TCT tool in prevention and control of the disease.Besides, ACA CCA TAA C -3') and J2 (R) 5' -CCG GAT TAT immunization failures have been suggested during GTC TTT GAA (11), DEPC Nuclease free challenge by vvIBDV [13,14].waster was added to a total volume of 50 µl.PCR Outbreaks of IBD are detected clinically and amplification was carried out as follows: Incubation at confirmed either serologically or via molecular techni-42°C for 30 min followed by Initial denaturation at ques.Molecular techniques in Nigeria have not gain 94°C for 2 min followed by 35 cycles of denaturation at popularity in the use of restriction enzyme analysis as a 94°C for 30 sec, annealing at 50°C for 1 min, extension tool to determine field circulating strains.The IBDV at 72°C for 2 min and a final extension of 72°C for 7 primers (J1/J2) developed by Jackwood and Sommer, min.In all the RT-PCR reaction sets negative and [14] amplified a 743 bp fragment from 701-1444 positive controls were included.
nucleotides of the VP2 hyper-variable region.The PCR amplicons were resolved on 1.5% agarose In the present study, IBDV was differentiated by in Tris-borate-EDTA (TBE) buffer gels stained with therefore proving that RT-PCR/RFLP is a simple and Similar patterns were observed for TaqI and MvaI sensitive method for detection of genetic variations for both field and vaccine samples.Fragments obtained among isolates that are closely related serologically, by TaqI indicated the presence of the restriction site which could not be differentiated using current serologic methods.which is found in vvIBDV strains globally like the Our findings suggest that vvIBDV is prevalent on UK661 strain reported in Europe [16] and Indian the Jos Plateau and also circulates in other parts of MH1/97 and Croatian strains reported by Kataria et al.,Nigeria [20,21].Arguably, the differences in mortality [17] and Bidin et al., [18].Fragment 101 was not found rate may also be related to the challenge studies, in some of the strains suggesting a mutation of the sample size and the presence of other exacerbating restriction site [17,19].
diseases as coccidiosis [24,25] which can occur Digesting the RT-PCR products with MvaI concurrently.Nonetheless the IBD mortality rates we (Isoschizomer of BstNI) gave a similar RFLP profile to found were similar (75%) to that reported recently in an vvIBDV reported in Europe, US, Banglandesh and experimental study with a vvIBDV in Nsukka [15].Pakistan [10,11,14].This is similar to the report of One-step RT-PCR/RFLP analysis of 8 clinical samples position 867 [23].Our results shows SacI restriction detected and submitted to TaqI, MvaI and SacI site exit is one of the field sample, the presence of point restriction analysis, which confirmed IBDV in the two mutations cannot be ruled out and this mutations can farms with a mortality of 60-74.2%recorded.Previous play a role in influencing the pathogenicity of the virus survey by Ezeibe et al., [15] reported a mortality of 0 -[21].Arguably, the loss of the SacI restriction site 75 % in Nigeria following an experimental challenge offers a rapid method for the RFLP analysis and study with a Nigerian very virulent isolate.
Vom between November and December, product was viewed under UV light for the expected 2011.They were from two different layer flocks of 6 743 bp product and documented by a UV transand 8 weeks of age, respectively.The farms were illuminator.located in two different settlements (Bukuru and Vom) south of the city of Jos, Plateau State Nigeria which isRestriction enzyme analysis: The amplified product (4 approximately 25 and 36 km from Jos city respectively.fromeachfarm,n = 8) was digested using the enzyme: Flocks originated from different breeder flocks and MvaI, TaqI and SacI according to manufacturer's kept differently.They all had a history of vaccination instructions (Roche®, Germany).A total of 25 µl of One gram of bursae from each restriction enzymes (MvaI, TaqI and SacI) and the sample was weighed and homogenized using a RFLP compared (Table-1).homogenizer or a pestle and mortar with sterile glass.

Table - 1
. RFPL patterns of representative outbreak genomes of 743 bp products generated from RT/PCR