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20105
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Subject: Veterinary Microbiology × End date: 2010 × Start date: 2010 × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen Access
    ISOLATION AND CHARACTERIZATION OF AVIAN LEUKOSIS VIRUS FROM BREEDER FLOCKS OF CHICKEN
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2010-10) GOPALA, LUNAVAT; NARASIMHA REDDY, Y(MAJOR); DHANA LAKSHMI, K; ANAND KUMAR, A; REDDY, M.R
    ABSTRACT: The present study was takcn up with a view to isolate a\.ian Icukc,sis virus (ALV) from aft'ected hrecdcr flocks of chickens and characterize the isolatc(s)with rcgard ttr group specitic ac-ELISA, growth in cell culturc titration. serum neutralization, polymerase chain reaction multiplc scqucnce alignment and phylogenetic allalysis in diagnosis of avian leucc~sisv irus infection. 276 cloacal swab sarnples wcrc collected fi-on1 hrccder Ilocks 01' chicken suspcctccl li>r avian lcukosis viral inl'ections. The breedcr flocks of chickcn cxhib~trd sympto~nsli ke tumours in livcr, splccn and heart. Thc sa~nplcs\s Jcr-ct cstcd tbr ALV by group spccilic antigen capture El-ISA ;is a prcliniinnry test hcliirc attcnipts t o isolate the virus. A total of47 sa~nplesw crc positive of'270 samples by cn~pioying p27 ac-ELISA kit. DNA from 16 blood samples (buffy coat) and RNA from 25 cloacal swabs obtained from ALV gs antigen positive flocks were tested for ALV specific sequences by PCR Attempts were made to isolate avian leukosis virus from these cloacal swab samples by passaging in CEF cells. The samples were passaged five times in cell lines. The presence of virus was demonstrated at different passage levels by ac-ELISA and Polymerase chain reaction (PCR). The RT PCR using H5 and AD1 was found negative for the SVVU-I01 isolate where as RT-PCR using primers H5 and H7b was positive with expected product size of 544 bp, which indicate that SVVU-I01 belongs to ALV subgroup-.I. Virus neutralization results indicate that the homologous antiserum efficiently neutralized ALV (SVVU-I 01) isolated in this study Thc nucleotide sequence of gp85 and gp37 was determined for tht: field isolate SVVU- 10 I and compared with publishcd sequences of' ALV subgroups A. B. C. D, E and J and seven strains of' ALV subgroup-.I. The result of prrsent study showed that the SVVU- I0 I belongs to ALV subgroup-J.
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    ThesisItemOpen Access
    CHARACTERIZATION OF CANINE PARVOVIRUS
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2010-08) DEEPIKA KUMARI, GEDDADA; DHANALAKSHMI, K(MAJOR); NARASIMHA REDDY, Y; MADHURI, D; REDDY, M.R
    ABSTRACT: The present study was taken up with a view to isolate canine parvovirus (CPV) from clinical cases and characterize them with regard to growth in cell culture, protein analysis and nucleic acid analysis. Further, haemagglutination of swine RBC, polymerase chain reaction and immunochromatography tests were compared for their efficacy in diagnosis of canine parvovirus infection. Ten faecal samples were collected from dogs suspected for canine parvovirus infections. The dogs exhibited symptoms like haemorrhagic enteritis, fever and vomition. The samples were tested for CPV antigen by haemagglutination of swine RBC as a preliminary test before attempts to isolate the virus. All the samples were positive with HA titres ranging from 32-1024. Attempts were made to isolate canine parvovirus from these faecal samples by passaging in CRFK and MDCK cells. Each of the sample was passaged ten times in both cell lines. There was no cytopathic effect in CRFK and MDCK cell lines at 10th passage. The presence of virus was demonstrated at different passage levels in CRFK but not in MDCK. However, the known isolate (IIL) caused focal rounding and aggregation of cells in CRFK but not in MDCK. With a view to characterize canine parvovirus, one isolate of canine parvovirus was purified employing sucrose density gradient centrifugation method. This method revealed purified virus as single light scattering band at 20% sucrose layer of the gradient. The purified virus gave one single precipitation line with hyperimmune serum in agar gel immunodiffusion test, confirming the isolate. The polypeptide analysis of the virus by SDS-PAGE revealed two polypeptide bands with molecular weights of 65kda and 62 kda. All the ten samples were positive for CPV by PCR employing the primer CPV-2ab which amplifies both 2a and 2b strains. However only one of these samples could be amplified by the primer CPV-2b specific for 2b strains of CPV. The known isolate (IIL) belonged to 2a strain. Three tests: Haemagglutination, PCR and rapid immunochromatographic tests (Rapigen kit test) were compared for their efficacy in the diagnosis of CPV. All ten samples were positive by haemagglutination test and PCR while only four samples were positive by rapigen kit indicating that rapigen kit can detect CPV only in faecal sample with HA titre of 512 and above. Further studies are required on more number of samples for studies on efficacy of diagnostic tests for canine parvovirus infection
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    ThesisItemOpen Access
    TYPING OF BLUETONGUE VIRUS ISOLATES BY SEROLOGICAL AND GENETIC METHODS
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2010-07) SIVA RAMAKRISHNA, GOLLAPALLI; NARASIMHA REDDY, Y(MAJOR); DHANALAKSHMI, K; RAMAKOTI REDDY, M
    ABSTRACT: Bluetongue is an arthropod-borne viral disease of cattle, sheep and other ruminants which causes huge economic loses. The BTV belongs to Reoviridae family under the genus Orbivirus is transmitted by the vector Culicoides species. This disease was placed in List 'A' diseases by Office International des Epizooties (OIE). The BTV genome consists of ds RNA with 10 segments that code for 7 structural proteins and 3 non-structural proteins. Among all these L2 segment codes for VP2 proteins which is one of the major outer capsid proteins that elicits virus neutralizing antibodies in infected animals. In addition, this determines the serotype specificity. Targeting this gene and its protein function, various typing techniques were standardized for identifying the BTV isolates up to the serotype level and for molecular characterization studies. The present study deals with the standardization of the serological and genotyping methods for typing of the BTV isolates. The hyper immune sera raised in sheep and used in serum neutralization test which specifically typed the Tirupati, MBN, N15, KMN07 and BTV-16 isolates as BTV-2, 9, 10, 21 and 16 serotypes respectively. In addition, in cross neutralization studies the SNT is able to determines the serological relationship between the serotypes 1 & 2 and between 16 & 21 serotypes. The genotyping was standardized using the RT-PCR assays. With the type specific primers the isolates Tirupati, K8, K3 and KMN07 isolates are typed as BTV-2, 9, 10 and 21 respectively. The N15 isolate which is previously typed as BTV-15, was retyped by this assay as BTV-10. On analyzing the sequence of N15 isolate VP2 gene, it showed 98% homology with that of BTV-10 USA serotype. But the homology is only 89% with that of BTV-10 South Africa reference strain. This signifies the origin of BTV-9 from USA. Both the techniques significantly typed various isolates of BTV to serotype level. In addition these techniques succeeded in identifying the serological relationships and highlighting the importance of topology of BTV serotypes in genotyping.
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    ThesisItemOpen Access
    MOLECULAR CHARACTERIZATION OF BLUETONGUE SEROTYPES 2 AND 15
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2010-01) SONALI MENAMVAR; NARASIMHA REDDY, Y(MAJOR); Anjaneyulu, Y
    ABSTRACT : The present study was taken with a view to characterize two BTV strains employed for trial vaccine. The titration of the two isolates M11 (BTV 2) and N12 (BTV 15) in BHK-21 cells was done for 5 passages. Titres were 104.55, 104.68, 106.12, 106.72 and 107.22 TCID50/ml for M11 and 102.69, 103.58, 105.64, 107.55 and 107.56 TCID50/ml for N12. Further one step growth curve experiments were conducted for the isolates in BHK-21 cells. Both the viruses had maximum titres at 48h. The inoculum size had no significant effect on the harvest in the dilutions tested. RT-PCR was standardized for detection of VP7 and NS1 genes BTV. For VP7 gene cDNA synthesis at 450C for 50 min then initial denaturation at 950C for 3 min, 30 cycles of denaturation at 950C for 20s, annealing at 390C for 60s and extension at 700C for 2 min, final extension cycle of 7 min at 700C were found to be suitable. For NS1 cDNA synthesis at 420C for 60 min then initial denaturation at 950C for 3 min, 30 cycles of denaturation at 950C for 25s, annealing at 580C for 20s and extension at 720C for 30s min, final extension cycle of 5 min at 720C were found to be suitable. Molecular characterization of the isolates was taken up by the sequencing of NS1 (M6 segment) and VP7 (segment 7). The sequences were compared to the available sequences in genbank. All NS1 nucleotide sequences segregated into 6 phylogenetic clades. Further analysis revealed that NS1 gene sequence of BTV-15 (N12) is closely related to BTV-15 (N15), BTV-15 (DQ399835). BT-2 (M11) and BTV-15 (N12) clustered together with BTV-9 BTV TPT and BTV KMTAI. All VP7 nucleotide sequences segregated into 7 phylogenetic clades. Further analysis indicated that BTV-2 (M11) was closely related to BTV-12 Brazil. BTV-15 (N12) was more closely related to BTV-15 (N15), BTV-15 (DQ399835), BTV-15 China than to BTV-15 Australia and BTV-9 (MBN).
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    ThesisItemOpen Access
    MOLECULAR CHARACTERIZATION OF BLUETONGUE VIRUS SEROTYPE 9 ISOLATES
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2010-01) KARUNASREE, NADARGI; DHANALAKSHMI, K(MAJOR); NARASIMHA REDDY, Y; MADHURI, D
    ABSTRACT : The present study was taken with a view to characterize three isolates of BTV9. The titration of the three isolates MBN,K8 and W20 of BTV9 in BHK-21 cells was done for 5 passages in BHK-21. Titres were 103.57, 103.8, 104.6, 105.33 and 107.55 TCID50/ml for MBN, 102.57, 103.4, 103.5, 104.25 and 105 TCID50/ml for K8 and 102.69, 102.99, 103.58, 104.68 and 105.64TCID50/ml for W20 isolate. Further, one step growth curve experiments were conducted for the isolates in BHK-21 cells. All three isolates had maximum titres at 48h. The inoculum size had no appreciable effect on the harvest of the virus in the dilutions tested. RT-PCR was standardized for detection of VP7, NS1 and VP2 genes of BTV. For VP7 gene : cDNA synthesis at 420C for 45 min then initial denaturation at 950C for 3 min, 30 cycles of denaturation at 950C for 20s, annealing at 390C for 60s and extension at 700C for 2 min, final extension cycle of 7 min at 700C were found to be suitable. For NS1: cDNA synthesis at 420C for 60 min then initial denaturation at 950C for 3 min, 30 cycles of denaturation at 950C for 25s, annealing at 580C for 20s and extension at 720C for 30s , final extension cycle of 4 min at 720C were found to be suitable.For VP2 gene: cDNA synthesis at 420C for 45 min then initial denaturation at 940C for 3 min, 35 cycles of denaturation at 940C for 30s, annealing at 550C for 30s and extension at 680C for 2 min, final extension cycle of 10 min at 680C were found to be suitable. Molecular characterization of the K8 isolate was taken up by the sequencing of NS1 (M6 segment), VP7 (segment 7) and VP2 (L2 segment) genes. The sequences were compared to the available sequences in genbank. NS1 nucleotide sequence analysis revealed that, NS1 gene sequence of BTV 9 (K8) is closely related to BTV 9 (MBN), BTV 2 and BTV 15.Further, NS1 derived amino acid analysis indicated cent percent homology between BTV9 K8 and BTV9 MBN isolates and close identity with BTV2 and BTV15. VP7 nucleotide sequence and derived amino acid analysis revealed that, BTV 9 (K8) is closely related to BTV 9 (MBN), BTV 2 and BTV 15. VP2 nucleotide sequences, responsible for serotype specificity, segregated into 10 distinct lineages with greatest sequence similarities between serologically related serotypes BTV 9 and BTV 5.Further analysis indicated that BTV 9 (K8) was closely related to MBN, Afandou isolates of same serotype BTV 9 and BTV5 but more distantly related to other 22 serotypes of BTV.