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ThesisItem Open 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.RABSTRACT: 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.ThesisItem Open 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.RABSTRACT: 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 infectionThesisItem Open 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, MABSTRACT: 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.ThesisItem Open 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, YABSTRACT : 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).ThesisItem Open 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, DABSTRACT : 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.ThesisItem Open Access STANDARDIZATION OF REAL-TIME RT-PCR FOR RAPID DETECTION OF BLUETONGUE VIRUS(Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2010-12) SUBHADRA, SUBHRA; SREENIVASULU, D (Major); SRILATHA, Ch; ESWARA PRASAD, PABSTRACT : The present study was taken up to standardize real time reverse transcription polymerase chain reaction (rRT-PCR) for rapid detection and relative quantification of bluetongue virus. Bluetongue virus serotypes 2, 9, 10 and 23 were propagated in BHK21 cell lines, double stranded RNA was extracted and studied the migration pattern of nucleic acid in agarose gel. BTV nucleic acid resolved into nine bands with similar migration pattern in ethidium bromide stained 1% agarose gel. Primers targeting NS3 gene of BTV were designed for standardization of real-time PCR. Conventional RT-PCR was used to check the specificity of the primers before use in real-time PCR. 35 amplification cycles polymerase chain reaction with annealing temperature of 60 0C for 30 seconds at 1.5 mM concentration of MgCl2 and primer extension at 72 0C for 30 seconds were found to be optimum for amplification of NS3 gene sequences of BTV. RT-PCR yielded amplified product of 247 bp which was the expected size of amplification from NS3 gene as template. RT-PCR was applied to various ten-fold dilutions of TCID50/ ml of BTV-2 and detected 3.16×102 TCID50 of virus / ml of tissue culture fluid. RT-PCR was also employed to screen blood samples collected from suspected outbreaks of bluetongue. Out of 32 blood samples screened, 17 samples were found to be positive for the presence of BTV by NS3 specific RT-PCR. Primers targeting NS3 gene of BTV found to be specific for detecting BTV were used for standardization of real-time PCR using SYBR green. A 25 μl real-time PCR reaction mixture containing 2 μl of cDNA, 20 p.moles of primers and annealing temperature of 60 0C was found to be optimum and yielded desired specific amplification of NS3 gene of BTV without any non-specific amplification. Different dilutions of TCID50/ ml of BTV-2 were used as standards. The CT values of the standard samples ranged from 22.1 to 39.8. Lowest CT value of 22.1 was obtained for the standard sample with highest virus titer i.e. 3.16×104 TCID50/ml whereas a CT value of 39.8 was obtained for the standard sample having lowest titer value of 3.16×10-4 TCID50/ml. No template control (NTC) yielded no CT value and remained as undetermined by the test. A standard curve generated by measuring the crossing point of each standard and plotting it against the logarithmic value of titer of the virus was linear and ranged from 3.16×104 to 3.16×10-4 with a linear regression value of - 0.99. The melting curve analysis of the standards indicated the absence of non-specific amplification. Real-time PCR was employed to screen the blood samples collected from sheep during suspected outbreaks of bluetongue, already subjected to conventional PCR. Out of 32 samples screened, the test detected 24 samples to be positive for the presence of BTV. Seven samples detected as negative by conventional PCR were detected as positive by real-time PCR. Real time PCR was also used to quantify the BTV present in the blood samples. The titer of the virus in the clinical samples was calculated using the standard curve and ranged from 1.1×10-2 to 7.7×104 TCID50/ml. In conclusion real-time PCR was found to be specific and more sensitive in detecting BTV than the conventional PCR. Further the test could also be used for relative quantification of BTV in the clinical samples which is not possible by the conventional PCR.ThesisItem Open Access STANDARDIZATION OF PCR FOR DETECTION AND SEROGROUPING OF Dichelobacter nodosus(Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2010-08) ANUPAMA, R; SREENIVASULU, D (Major); SRILATHA, Ch; SREEDEVI, BABSTRACT : Traditional methods used for diagnosis of footrot were based on smear examination, isolation and characterization of D. nodosus from clinical samples. This is time consuming and difficult because the D. nodosus organisms are fastidious in nature and require anaerobic conditions in addition to enrichment media. To overcome these problems it is proposed to standardize PCR for detection and serogrouping of D. nodosus from the clinical samples. Dichelobacter nodosus JKS – 05 B cultures obtained from Srinagar was revived and maintained in the laboratory. The cultures were used for standardization of PCR techniques. The colony of D. nodosus JKS – 05 B appeared as flat concentric zones with finely granulated surface texture. Morphology of D. nodosus was found to be Gram negative, large or slightly curved rods with terminal swollen ends. Gelatin gel test showed hydrolysis of gelatin around the wells containing broth heated to 68˚C for 16 minutes, which indicated the presence of thermostable enzyme which is a characteristic feature of virulent strain. The PCR for targeting 16SrRNA gene was standardized for direct detection of D. nodosus. A 30 cycles PCR reaction with annealing temperature of 60˚C (for 5 cycles) and 58˚C (for 25 cycles) with 25 mM MgCl2 were found optimum for amplification of 783 bp product of 16SrRNA gene. Multiplex PCR targeting fimA gene was also standardized for identification of D. nodosus serogroups using the similar cyclic conditions. Analysis of epidemiological data revealed that footrot is a seasonal disease noticed mostly during North-East monsoon period in Andhra Pradesh. The disease occurrence was related to the rainfall in the area. A total of 778 outbreaks of footrot were recorded during the period of 10 years from 1998 to 2008. The maximum number of outbreaks was recorded in Mahaboobnagar district. A total of 15 outbreaks were attended in selected villages of Chittoor and Nellore districts of Andhra Pradesh during the period of November 2009 to July 2010 and collected the materials for isolation and serogrouping of D. nodosus. Of the 371 samples 76 were found positive for the presence of D.nodosus using 16SrRNA gene targeting PCR. When the samples were further subjected to multiplex PCR targeting fimA gene revealed the presence of D.nodosus serogroups A (19), B (24), C (6). Serogroups both A and B (21) were found infecting the sheep. The study revealed that multiple serogroups were found infecting the sheep and causing footrot in Chittoor and Nellore districts of AndhraThesisItem Open Access STANDARDIZATION OF ELISA FOR DETECTION OF LEPTOSPIRAL ANTIBODIES IN BOVINES(Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2010-08) SWATHI REDDY, C; SREENIVASULU, D (Major); SRILATHA, Ch; SREEDEVI, BABSTRACT: Leptospirosis is an emerging zoonosis caused by pathogenic spirochetes belonging to the genus Leptospira. In the present study, we used the humoral immune response to detect leptospiral protein antigens expressed during infection. The hyperimmune serum raised against whole cell antigen and bovine serum positive for Leptospira were used for western blotting. Whole cell, sonicate antigen and outer membrane proteins extracted by Triton X-114 method were characterized by SDS-PAGE and western blotting immunoanalysis. SDS-PAGE analysis of the whole cell and sonicated antigens revealed ten protein bands with molecular weights ranging from 18 to 67 KDa i.e. 18, 22, 32, 36, 41, 43, 45, 63, 65 and 67 KDa where as outer membrane proteins extracted by Triton X-114 revealed protein bands of 22, 32, 36, 43, 45 and 63 KDa. Westernblot analysis of outer membrane proteins using hyperimmune rabbit serum revealed protein bands of 22, 32, 41, 43 and 63 KDa as major immunogens, where as analysis using positive bovine serum of L.grippotyphosa revealed protein bands of 25, 32 and 45 KDa as major immunogens. A total of 1100 serum samples available at Leptospira research centre C.V.Sc., Tirupati, were screened against different leptospiral serovars using MAT. The MAT titre value ≥ 1:80 dilution is considered as positive as standardized in the research centre. A total of 157(14.27%) samples were found positive for Leptospira. The MAT titres varied between 1:80 to 1:640. Indirect ELISA was standardized using heat extracted antigen and evaluated for measuring the antibody titres in bovine sera. The specificity, sensitivity and efficacy of the ELISA relative to the MAT were found to be 89.8%, 92.9%, 92.1% respectively. 19 Once ELISA was standardized, a random of 200 field serum samples available at research centre were screened by ELISA. Out of 200 serum samples, 32 (16%) were found positive by ELISA. The ELISA used in the present study was easily standardized and was found to be sensitive, rapid, specific, easy to perform, semi- automated and used a non-hazardous antigen which can be routinely prepared in large amounts.ThesisItem Open Access SEROEPIDEMIOLOGY AND MOLECULAR CHARACTERIZATION OF LEPTOSPIROSIS IN ANDHRA PRADESH(Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2010-11) RANI PRAMEELA, D; SREENIVASULU, D (Major); UMAMAHESWARA RAO, S; ESWAR PRASAD, P; SRILATHA, ChABSTRACT: Leptospirosis the world wide zoonosis is considered as reemerging disease. Besides economic losses caused by leptospira to animal production, its zoonotic character makes it an important public health problem. Due to the endemicity of the disease in Tamil Nadu, Kerala and Karnataka the adjoining states of Andhra Pradesh and absence of detailed information on leptospirosis in the state, the present work was planed to study seroepidemiology, isolation, characterization of leptospira and development of inactivated adjuvanted vaccine and to asses immune response in rabbits. The seroepidemiological study conducted using MAT on 2320 serum samples collected from apparently healthy cattle, sheep, goat, dogs and pigs revealed 20.9 percent positivity. Similarly 33.37 percent positivity was recorded from clinically suspected cases of cattle, sheep, pigs, dogs and humans. High seroprevalence in coastal region (23.09 percent) followed by Rayalaseema (17.49 percent) and Telangana (16.30 percent) was observed.. High seropositivity was recorded during north east monsoon (28.29 percent) followed by south west monsoon (21.45 percent) and lowest in summer (7.26 percent). Biochemical analysis of serum samples from cattle positive for MAT showed elevated levels of total bilirubin, SGPT and SGOT. Clinical samples collected from cattle (26) sheep (42) dogs (13), Pigs (15), Humans (53), and stagnated water in rice fields (10) were inoculated in EMJH Liquid medium with tween 80, antibiotics and 5- Flurouracil. A total of 17 isolates were recovered from sheep (5), rat (5), pigs (4), Humans (2) and rice field (1) were purified and maintained in EMJH liquid medium and semi solid medium. Physiochemical characterization of isolates at 130C, growth in the presence of 8-Azaguanine and lipase activity revealed the pathogencity of the isolates. PCR detected 12 isolates, of 17 isolates tested. RAPD DNA analysis was found to be simple and rapid test for identifying serovars of leptospira. The test identified the leptopsiral isolates as L. hardjo, L. autumnalis and L.pomona. 16S rRNA PCR sequence analysis of leptospiral isolates recovered from sheep were identified as Leptonema illini, L. hardjo and L.inadai, from rats were identified as Leptonema illini, L. noghuchi and from pigs identified as L. Pomona. A trivalent inactivated vaccine was prepared with three commonly circulating serovars namely L. grippotyposa, L. hardjo and L. autumnalis. The vaccine was adjuvanted with alumminium hydroxide (Vaccine-I) and Montanide (Vaccine-II) and the immune response in rabbits was studied. There is no significant difference between the vaccines. Both the adjuvanted vaccines yielded satisfactory immune response up to 150 days post vaccination.