Detection and molecular characterization of chicken infectious anaemia virus from poultry
Kumar, Asutosh (2007) Detection and molecular characterization of chicken infectious anaemia virus from poultry. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, India.
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Chicken infectious anemia virus (CIAV), a member of genus Gyrovirus of the family Circoviridae, is the causative agent of Chicken infectious anemia (CIA), an emerging infectious disease mainly of young chickens. The mortality due to CIA in poultry flock is generally low but the problem is aggravated due to its immunosuppressive effects, then the mortality can be high has secondary infections take over. The disease is characterized by severe anemia, atrophy of thymus and bursa and aplasia of bone marrow with pale liver. The present study was aimed to find out the incidence of CIA in Anand area of Gujarat as per the farm records and post mortem reports, occurrence of CIAV in the poultry flocks from clinical cases (exhibiting frank lesion of CIAV) by PCR detection, comparing the efficacy of different primers in detecting the CIAV, and study its molecular characteristics by restriction endonuclease (RE) analysis as well as sequencing of the PCR amplified fragment of VP1 gene. The post mortem reports (2005-2006) indicated that occurance of the disease was more in growers (21.83%) than chicks (1.77%) and layers (1.14%). The post mortem gross pathological lesions were characterized by purple skin with loss of feathers particulary in rump, breast, wing, head and thigh area. The skin was moist and soft with haemorrhages and necrosis. The bursa of fabricious, spleen and thymus were atrophied, while liver showed enlargement. Necrotic foci were noticed on liver and spleen in some birds. The bone marrow was pinkish to white yellow. The strength of selected individual affected flocks (n=15) varied from 3,000 to 17,500 birds with a total population of 1,33,280. Of these, 12,510 birds died due to CIA (as per the farm records of history, symptoms and postmorterm lesions) with an over all mortality of 9.38 per cent, which varied from 2.1 to 23.2 in individual farm. A total of 75 clinical samples (bone marrow, spleen, liver, thymus and bursa of fabricious, 15 from each farm) from CIA suspected birds were collected and processed for DNA extraction by using DNeasy Tissue Kit (QIAGEN Pvt. Ltd). Samples showing acceptable DNA purity and concentration were subjected to PCR amplification using four different primers, which were selected to amplify different regions of CIAV including VP1 and VP2 gene segments. All the 75 samples, turned out to be positive by PCR with at least one primer set. PCR worked well and was found to be easy to adopt for efficient detection of CIAV in field samples. Out of the four primer pairs, VP2F/VP2R and O1F/PshA1R were maximally effective in detecting CIAV from field samples as they detected 68 and 67 samples positive respectively, followed by primers O1F/O1R (59 samples); where as primer pair VP1F/VP1R was least effective and detected CIAV in only 33 samples. To detect any possible genetic variation in the targeted sequences of VP1 and VP2 gene, PCR products amplified with primer pairs VP1F/ VP1R and VP2F/VP2R were subjected to RFLP analysis. PCR products of VP1 and VP2 genes of representative samples from each 15 farms of different locations of Anand area were analyzed for RFLP, after digesting with restriction enzymes (RE) Hae III, sac I and Mbo I. Hae III digestion revealed genetic variation in VP1 gene segment of the field CIAV, as two of the 15 farms yielded different RFLP pattern. The RE analysis by Sac I indicated the absence of any cutting site in VP1 gene. The VP2 gene PCR products of all representative samples from 15 farms, digested with Hae III and Mbo I, produced similar patterns of RFLP, indicating absence of genetic variations at the corresponding sites of these two REs. PCR product (1390 bp) of CIAV genome amplified by VP1F/VP1R primer from a represenrtative field sample having good quality DNA (i.e.1.7-1.9 OD at 260/280 nm) as determined by spectrophotometry was used for sequencing by ABI PRISM® 310 Genetic Analyzer (Applied Biosystems, USA), using BigDye® Terminator v3.1 Cycle sequencing kit (Applied Biosystems, USA). The partial sequence obtained revealed 177 bp (61bp from VP1F and 116 from VP1R). The alignment of 177 bp sequence with previously published sequences of CIAV in GenBank confirmed the identity of CIAV in field samples.
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