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2003 - 20072
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Author: LAKSHMI KAVITHA, K ×
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    ThesisItemOpen Access
    PREPARATION AND APPLICATION OF A DNA PROBE FOR CHICKEN EGG DROP SYNDROME-76 VIRUS
    (SRI VENKATESWARA VETERINARY UNIVERSITY , TIRUPATI – 517 502 (A.P) INDIA, 2003-08) LAKSHMI KAVITHA, K; RAO, V.D.P (Major); SREENIVASULU, D; SRILATHA, Ch; SURYANARAYANA, V.V.S
    ABSTRACT : In the present investigation, an attempt was made to develop a radiolabelled Egg Drop Syndrome-76 (EDS-76) viral DNA probe by using local isolate which enables accurate diagnosis of EDS virus infection in poultry especially in breeder stocks because of its vertical transmission. In this study, VN 1 isolate of EDS-76 virus was cultivated in duck embryos and purified by using 10-40 percent sucrose density gradients. The isolate was serologically similar to EDS-76 strain 127 by Haemagglutination Inhibition (HI), Agar gel immunodiffusion test (AGID), Counter immuno electrophoresis (CIE) with standard EDS-127 strain virus antiserum. From the purified virus, DNA was extracted by proteinase K-phenol: chloroform method and the results provided evidence to the fact that the isolate consisted of DNA of molecular weight of 34.2kb. Restriction endonuclease pattern using Bam HI resulted into four fragments viz., 16kb,11kb, 4kb and 2kb. Of these the 2kb fragment was found to be specific for EDSV by nucleotide sequence analysis. The 2kb fragment was cloned into pBluescript II KS+ vector and the transformation efficiency was 2x104 /μg of plasmid. The nucleotide sequence of 2kb insert revealed that the 2kb fragment codes for two genes, the fibre protein and pVIII protein genes. The homology with the published sequence was found to be 96 per cent. The fragment was labeled with -33P ATP and the total incorporation was 2.4x107dpm/20ng DNA. The labeled DNA when used as a probe, detected EDSV specific genome with a sensitivity level of 1.25 pg genomic DNA. Proteinase K treated, phenol:chloroform extracted DNA when denatured by alkali enabled quick diagnosis of faecal samples. However the hybridization efficiency was found to be less in case of alkali denatured proteinase K treated samples, which showed positive signals after long time of exposure. No signals could be obtained from direct denatured samples. On comparison with HI, AGID and CIE, the dot blot hybridization using labeled probe, proved to be more sensitive, specific and able to detect virus in faecal samples of infected birds as early as 24hr post inoculation .
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    ThesisItemOpen Access
    ISOLATION AND MOLECULAR CHARACTERIZATION OF INDIGENOUS GOATPOX VIRUS
    (SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI – 517 502, A.P, 2007-02) LAKSHMI KAVITHA, K; SATYANARAYANA CHETTY, M (Major); SREENIVASULU, D; SRI LATHA, Ch; ALAHA SINGARI, N
    ABSTRACT : Goat populations that are contributing to the greater extent to the economy of poor and marginal farmers of India are being threatened by a major devastating disease like Goatpox. The review of literature revealed meagre information on Goatpox and prophylactic vaccines to contain the disease. Hence, a modest attempt has been made to study on Goatpox with regards to epidemiology, isolation, characterization, diagnosis of viral agent and development of candidate vaccine strain. The seroepidemiological investigations revealed 72 per cent animals were positive for Goatpox. The animals co-habitated with Goatpox affected goats were uninfected. The disease was observed throughout Andhra Pradesh irrespective of breed, age and sex with casefatality rate of 13.3 per cent. The proviral clinical materials of infected goats were propagated in chicken embryos and VERO cell lines which yielded a titre of 0.5 x 102 to 1.4 x 103 EID50/ml and 4.8 to 6 TCID50/ml respectively. The isolates were purified by ultracentrifugation in 40-60 per cent sucrose density gradients. The isolates were confirmed by presence of lines of identity and a VNI of 3.2 to 3.6 against specific serum by subjecting to AGID and VNT respectively. The Goatpox virus was further confirmed by reproducing infection in experimental goats. Physico-chemical characterization of virus isolates revealed a temperature of 55°C for 60, 60°C for 30, 70°C for 10 min and the pH 3 and 5 were detrimental for viral infectivity. Further chloroform and ether completely inactivated the virus. The TEM observations at 39000 x magnification with TPT isolate revealed the morphology as brick shaped with 250±10 x 290±20 nm in size, while, IEM against specific antibody yielded aggregates of viral particles. The molecular studies of Goatpox virus with SDS-PAGE yielded 17 comigrating polypeptide profiles of which five were major and 12 minor, whereas RFLP studies on viral genome revealed 95 per cent homology among the four isolates. Among the conventional tests for the diagnosis I-ELISA was more sensitive and accurate with 72 per cent positives than AGID and CIE. The molecular diagnosis with PCR yielded a 470bp product which on sequence analysis exhibited 98 per cent homology with complete Goatpox viral genome available at database. Among vaccinates that were vaccinated with attenuated and inactivated vaccines yielded satisfactory immune responses and could withstand challenge.