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ThesisItem Open Access IMMUNO INFORMATIC APPROACHES IN DESIGNING VACCINE AGAINST PATHOGENIC LEPTOSPIRA THROUGH PAN GENOME REVERSE VACCINOLOGY(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-12) SUDHEER, P; RANIPRAMEELA, D(MAJOR); VINOD KUMAR, N; JAGADEESH BABU, AABSTRACT: Leptospirosis is a globally important zoonotic disease caused by pathogenic Leptospira and it is a disease of livestock, pet animals, wildlife and humans throughout the world. The losses are due to reproductive problems in livestock and mortality in case of humans. Current existing leptospiral vaccines are unsuccessful due to their limitations .Hence there is a need to develop a novel and efficacious vaccine to control the disease. To overcome this, reverse vaccinology is the right choice. In recent trends, it is possible to target the common vaccine candidates with genomic information of the single organism. Based on this concept the present work was planned to study the Bioinformatics approaches to identify the vaccine candidates in designing a vaccine against pathogenic Leptopsira in Bovines In the present study complete proteomes of L.borgpetersenii hardjo bovis JB 197 and L550 were screened to identify common surface exposed proteins through Rxvi language scripts and codes. Later these common surface exposed proteins were subjected to DEG analysis. 49 essential proteins were identified .Further essential proteins were subjected to non-homology analysis against both host and gut microbiota to avoid autoimmunity using NCBI-BLAST.T-helper cell epitopes were predicted from non-homologous proteins through MetaMHCII and ProPred homology search against BoLA-DRB3, and evaluated using Vaxijen server. Three dimensional structures were built for T-cell epitopes and BoLA-DRB3 using Modeller9v.15. A total of twenty five models were generated. The model with high negative DOPE score was selected and validated using PROCHECK, ProQ, and ProSA in determining protein quality. The structures of T-cell epitopes and BoLA DRB3 were prepared before docking using protein preparation wizard of Schrodinger 2015-3. Docking and free energy calculations were performed with BioLuminate Module v 2.0 of Schrödinger software suite 2015-3. The changes in structural confirmation were monitored in terms of energy plot, RMSD and RMSF during 50 ns MD simulations run time using Desmond v4.3. In Silico analysis of L.borgpetersenii JB 197 and L550 retrieved three proteins namely Ton B dependent receptor containing single epitope, ABC permease protein with three epitopes and UVr ABC protein B with single epitope. L.ballum was selected instead of L.borgpetersenii due to its non-availability of the culture during the period of the study and 99% identity on BLASTp. The nucleotide sequence corresponding to ABC permease gene containing three epitopes was retrieved, primers were designed and PCR was standardized for the amplification of ABC permease gene. PCR purified product on sequencing analysis confirmed the presence of ABC permease gene. Then, the PCR purified product was cloned in to PRSET vector using E.coli DH5α cells .The recombinant plasmid was transformed in to E.coli BL21 (DE3) cells and expression was induced by addition of 1mM IPTG. Finally recombinant protein was extracted from lysate of E.coli BL21 (DE3) cells. Recombinant protein was analysed on SDS-PAGE for characterization. The SDS-PAGE analysis yielded 20KD of expected recombinant protein on staining with commassie brilliant blueThesisItem Open Access STUDIES ON ANTIBIOTIC RESISTANCE AMONG MAJOR BOVINE MASTITIS PATHOGENS IN ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-12) USHARANI, K; CHAITANYA, R.K(MAJOR); SREENIVASULU, D; PADMAJA, KABSTRACT: Mastitis remains as a major problem to the dairy industry worldwide, as it affects the quality and quantity of milk production. In the present study a total of 130 milk samples were collected from clinical (101) and subclinical cases (29) of bovine mastitis from different regions of Andhra Pradesh. Isolation of causative bacteria was carried out and a total of 105 bacterial isolates were obtained. The incidence of Staphylococcus spp. (70/105, 66.66%) was found to be high, followed by Enterobacter spp. (16/105, 15.23%), E. coli (11/105, 10.47%) and Streptococcus spp. (8/105, 7.61%). Among the 70 isolates of Staphylococcus spp., 37 were identified as coagulase positive Staphylococci (CPS) and 33 were identified as coagulase negative Staphylococci (CoNS) on tube coagulase test and in coagulase gene PCR. Multiplex PCR carried out for species identification of Staphylococcal and Streptococcal isolates, confirmed all the 37 CPS isolates as S. aureus, a CoNS isolate as S. epidermidis and six isolates of Streptococci as St. agalactiae. The in vitro antibiotic sensitivity test of Staphylococcal isolates revealed high frequency of resistance to pencilin G (48 isolates, 68.57%) followed by cefoxitin (35, 50%), oxacillin (24, 34.28%), gentamicin (6, 8.57%), ciprofloxacin (5, 7.14) and ceftriaxone (2, 2.85%). Interestingly, all the isolates were found susceptible to chloramphenicol. As cefoxitin is used as surrogate for mecA mediated oxacillin / methicillin resistance, 35 (50%) isolates that showed resistance to cefoxitin were phenotypically identified as methicillin resistant, out of which 18 were MRSA and 17 were CoNS. In PCR for mecA and mecC genes that confer methicillin resistance in Staphylococci, only ten (10/70, 14.28%) Staphylococcal isolates were found to carry mecA gene. Three of them were S. aureus and the remaining seven were coagulase negative Staphylococci. The relative frequencies of MRSA and MR-CoNS were 8.1% (3/37) and 21.2% (7/33) respectively. All these mecA positive isolates were found resistant to cefoxitin which is a surrogate for mecA mediated oxacillin/ methicillin resistance. However, six of these mecA positive isolates were found susceptible to oxacillin. None of the 70 Staphylococcal isolates carried mecC gene. Phenotypic resistance was observed in three isolates of St. agalactiae (3/8, 37.5 %), but none was found to carry resistance genes tetO and ermB in PCR. Out of 27 (11 E. coli and 16 Enterobacter spp.) isolates of coliforms, 14 (14/27, 51.85%) isolates were suspected as ESBL producers as they showed resistance to any of the 3rd generation non combination cephalosporins tested in phenotypic screening test. Among these fourteen isolates, only four (4/14, 28.57%) have shown increased diameter of inhibition zones (≥ 5 mm) with the drugs in combination with ß- lactamase inhibitors over the individual drugs and hence these four isolates were phenotypically confirmed as ESBL producers. All the 27 isolates were susceptible to ertapenem and combination drugs of 3rd generation cephalosporins with ß-lactamase inhibitors i.e. ceftriaxone + tazobactem, ceftazidime + clavulanic acid and cefotaxime + clavulanic acid. Out of 27 isolates of coliforms tested for ESBL genes, six isolates (3 E. coli and 3 Enterobacter spp.) were found carry SHV gene in m PCR-I. In m PCR-II, an isolate each of E. coli and Enterobacter spp. were found to carry CTX-M-1 gene and another isolate of E. coli was found to carry both CTX-M-1 and CTX-M-2 gene. Hence these three isolates were confirmed as ESBL producers genotypically. Among the 4 phenotypically confirmed ESBL producers, ESBL genotype was confirmed only in 3 of them (2 E. coli and 1 Enterobacter spp.) with the presence of CTX-M genes. The other ESBL isolate didnot carry any of the ESBL genes. Results of the present study indicate considerably high levels of antibiotic resistance among the major bacterial species causing mastitis in cattle and buffaloes. Hence, it is imperative to go for antibiotic susceptibility testing prior to choosing an appropriate antibiotic for treatment.ThesisItem Open Access PRODUCTION, ISOLATION AND CHARACTERIZATION OF IgY ANTIBODIES TO CANINE PARVOVIRUS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-12) ATCHUTHARAM, A; SREENIVASULU, D(MAJOR); SREEDEVI, B; ESWARAPRASAD, PABSTRACT: The overall goal of this study was to develop practical, natural, and efficient antimicrobials from egg. The existence of an IgG-like molecule in avian eggs, referred to as IgY, has been well documented, and extensive research has been carried out on its characterization, production and purification. Although it is the functional equivalent of mammalian IgG, the major serum antibody found in mammals, IgY is structurally different, and has been found to exhibit several important differences when compared to mammalian antibodies, including its physico-chemical properties and immunological capabilities. Recently, considerable research has focused on the use of IgY as an alternative to mammalian antibodies in several applications, including immune therapeutic applications, especially for the oral passive immunization against various bacteria and viruses. Much research has also been carried out on the use of IgY as a replacement for IgG in various immunodiagnostic and immune affinity purification purposes. The use of IgY offers several advantages over polyclonal antibodies produced in mammals, including providing a much more hygienic, cost efficient, convenient, humane and plentiful source of antigen-specific antibodies. Chicken immunoglobulin Y (IgY) may provide a new modality in the therapy of various infectious animal diseases. This study presents evidence of its efficacy for canine parvovirus (CPV), which is a highly infectious, fatal viral disease in dogs. Initially IgY antibody production and separation was standardized using BSA as antigen. BSA 0.2 mg/ml was used to immunize three 21weeks old white leghorn chicken. Additional booster doses were administered weekly intervals up to 6 weeks following the first injection. Sera from immunized chicken were collected on 21st day to confirm the immune response to BSA. Among three BSA immunized birds, one birds did not show any response to BSA antigen. Initially BSA specific IgY antibodies were separated from eggs laid by immunized chicken using Ammonium sulphate method. The specificity of IgY antibody raised against BSA was determined by agar gel immunodiffusion test. A clear precipitation line was observed between BSA and anti BSA IgY antibody precipitated from egg yolk, which shows specificity of immune response to BSA. Anti BSA IgY antibody levels were monitored up to 120 days by indirect ELISA and found that the titres were maintained up to 120 days of immunization with the highest titre on 75th day. The present study was conducted as a preliminary step to monitor the in vitro efficacy of IgY as a passive immunotherapeutic agent to control Canine parvovirus infection in dogs. Canine parvovirus vaccine containing 103viral particles/ml was used as antigen to immunize 21 weeks old white Leghorn chicken. The eggs from immunized chicken were collected from 1st day to 135th day. Sera from immunized chicken were also collected on 21st day to confirm the immune response to canine parvovirus. Presence of antibodies against canine parvovirus was checked using hemagglutination inhibition assay (HI). The HI titres of the serum collected from immunized hens was found to be 256 HI units. Water soluble fraction was isolated from eggs collected after the immunization and estimated the protein content and the maximum protein concentration was found (35.20 ± 1.32a mg/ml) in the water soluble fraction collected from egg yolk on 75th day of post immunization. The IgY was separated from the WSF by using Ammonium sulphate method, Sodium chloride method, Sodium sulphate method and PEG method. Among the four methods used for IgY separation, Sodium chloride method and Sodium sulphate methods were found to yield high protein content (6.70 to 6.71mg/ml) compared with Ammonium sulphate method and polyethylene glycol method. IgY was purified using DEAE cellulose column chromatography. Highest protein concentration was observed in the 4th and 5th fractions. The purity of the immunoglobulin present in the 4th and 5th fractions of DEAE cellulose column elute was checked by determining the molecular weight of the protein. A single protein band showing molecular weight 180 KDa was recorded. The titre of the purified canine parvovirus specific IgY antibody was found to be 2048 HI units. Anti canine parvovirus IgY antibody levels were monitored up to 135 days by indirect ELISA. It was found that the titres were be maintained up to 135th day with peak titre (1.186) on day 75th after immunization. Stability of canine parvovirus IgY antibody was studied by exposing to different temperatures and different pH using HI assay. The HI titre gradually decreased when the temperature increased to 50ºC and above. At 25ºC and 37ºC the IgY antibody was found to be stable when exposed to 10, 20 and 30 minutes. Purified IgY antibody when subjected to pH 7 for 8 hours its HI titre was 1024. The HI titre gradually decreased when the pH decreased below 7 and also when pH increased above 7. The stability of purified anti canine parvovirus antibody was completely lost when it is exposed to pH 3 in the presence of pepsin as reflected by complete loss of HI activity. HI titre of the IgY antibody was found to be 32 to 64 when exposed to pH 4.0 and pH 5.0 respectively in the presence of pepsin. The results indicated that the activity of immunoglobulins reduced in the presence of pepsin. Hence, there is a need to protect IgY immunoglobulins against the action of pepsin in the stomach of puppies. Present study was helpful for production of desired IgY antibodies, which can be obtained in large quantities against specific pathogen (canine parvovirus).ThesisItem Open Access STUDIES ON BETA-LACTAMASE ANTIMICROBIAL RESISTANCE IN CANINE MICROBIOTA AND SCREENING OF LACTIC ACID BACTERIA FOR PROBIOTIC ACTION(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-12) NOORBASHA MOHAMMAD SHARIF; SREEDEVI, B(MAJOR); CHAITANYA, R.K; SRILATHA, ChABSTRACT: Microbiota plays a central role in host health and disease. Alterations in gut microbiota can have major consequences, both beneficial and harmful, for host health. In view of this, rectal swab samples from healthy (92) and diarrhoeic (44) dogs as well as pus swabs from pyometra (15) and otitis (15) clinical cases were analyzed. Isolation and identification of canine microbiota was carried out by conventional cultural methods. Gut microbiota isolated include E. coli (65.2% incidence in healthy versus 75% in diarrhoeic dogs), Proteus spp. (61.9% vs. 65.9%), Enterobacter spp. (26% vs. 11.3%), Klebsiella spp. (26% vs. 20.4%) and Pseudomonas spp. (28.2% vs. 45.4%). Microbiota isolated from pus swabs include Staphylococcus spp. (73.3% incidence in pyometra vs. 100% in otitis), Pseudomonas spp. (80% vs. 53.3%), Proteus spp. (0% vs. 60%) and E. coli (46.6% vs. 0%). E. coli isolates were further confirmed by PCR targeting E16S gene and also sent for serotyping based on ‘O’ antigen. Extensive usage of antibiotics in canine practice may lead to development of antimicrobial resistance in dogs. In view of this, all the isolates obtained in the present study were screened for β-lactamase resistance both phenotypically and genotypically. Overall incidence of β-lactamase antimicrobial resistance in phenotypic screening test was found to be 35.8% (125/349), which includes 54 E. coli, 40 Pseudomonas, 22 Klebsiella and 9 Enterobacter species. Of these 125 isolates, resistance to cefotaxime was observed in 80.8%, ceftriaxone in 57.6%, ceftazidime in 52% and aztreonam in 26.4% of isolates. β-lactamase resistance was detected in 34.5 and 42.7% of gut microbiota isolated from healthy and diarrhoeic dogs, respectively; 46.6 and 12.5% of microbiota isolated from pyometra and otitis pus samples, respectively. All the Proteus and Staphylococcus spp. were found to be highly sensitive or intermediately sensitive to β-lactam antibiotics. Overall incidence of ESBL phenotype in phenotypic confirmatory test was found to be 14.6% (51/349), with highest incidence detected in E. coli (31%, 31/100) followed by Klebsiella (21.2%, 7/33) and Pseudomonas (19.6%, 13/66) species. ESBL phenotype was detected in 12.5 and 17.7% of gut microbiota of healthy and diarrhoeic dogs, respectively and 33.3% of microbiota of pyometra pus samples. Detection of β-lactamase genes in canine microbiota was carried out using a set of three multiplex PCR assays and a single uniplex PCR assay. The overall incidence of β- lactamase genes in canine microbiota was found to be 57.3% (200/349). Predominant β- lactamase genes detected in canine microbiota include blaAmpC in E. coli (87%), blaSHV in Klebsiella (84.8%) and Enterobacter (48.2%), blaOXA in Pseudomonas (66.6%) species. Majority of the isolates with confirmed ESBL phenotype carried blaCTX-M G1 gene (72.5%). The blaACC and blaMOX genes were not detected in the canine microbiota. The efficacy of antibiotics against bacterial infections is decreasing with rise in antimicrobial resistance, thus, there is a need to search for novel probiotic strains as potential alternatives to antibiotics. In India, there are no probiotics available for canine usage, as they are host specific. In view of this, rectal swabs (67) from healthy pups were analyzed and a total of 49 (73.1%) Lactobacillus isolates were identified based on morphological, biochemical characteristics and genus specific PCR. Of these 49 isolates, 23 were found to be positive for Group IV; six for Group I, four for Group II and 16 were found to be negative for Lactobacillus group specific PCR. In vitro antibacterial activity of canine Lactobacillus isolates on test pathogens like E. coli, Klebsiella and Enterobacter species have been studied using agar well diffusion assay. Out of 49 Lactobacillus isolates, the supernatants of 20 isolates showed inhibition against majority of the test pathogens examined. The inhibition zones were large and clear against E. coli and Klebsiella spp., but limited and hazy zones were observed against Enterobacter spp. Reduction in antibacterial activity was noticed after neutralization, proteinase K and heat treatment of supernatants, suggesting that the antibacterial activity might be partly due to organic acid production and partly due to heat labile antimicrobial proteins. Nucleotide sequence analysis of genus specific PCR products of 10 selected Lactobacillus isolates that showed consistently high antibacterial activity revealed maximum sequence homology with Lactobacillus fermentum strain RCM 14 (for six isolates), Lactobacillus agilis strain 76CL (for one isolate), Pediococcus acidilactici strain G4 (for 2 isolates) and Weissella confusa strain 3W (for one isolate). In conclusion, the present study revealed alarming β-lactamase resistance in microbiota of dogs in Andhra Pradesh. Therapeutic failures may likely to occur as resistance to commonly prescribed third generation cephalosporins was observed. Lactobacillus strains of dog faecal origin were found to have potent in vitro antimicrobial action. Furthermore, the present study highlighted need for in vivo studies in India to establish probiotic potential of dog faecal Lactobacillus species in the near future.ThesisItem Open Access CHARACTERIZATION OF AN INDIAN ISOLATE OF BLUETONGUE VIRUS SEROTYPE 16(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2012-03) YOGANAND, BRUNDAVANAM; NARASIMHA REDDY, Y(MAJOR); DHANALAKSHMI, K; NAGENDRA HEGDE, RABSTRACT: Bluetongue is an arthropod-borne disease, transmitting to ruminants through the bites of Culicoides. Bluetongue is listed as a ‘notifiable disease’ by the Office International des Epozooties (OIE), causing severe hemorrhagic disease with fever, lameness, coronitis, edematous lips and tongue and death. Export of animals and animal products from Bluetongue endemic countries like India are seriously being hampered. Bluetongue virus (BTV) belongs to the family Reoviridae, genus Orbivirus. Out of 24 serotypes identified worldwide, 12 serotypes have been reported by isolation and 11 serotypes by serology in India. VP2 gene is highly variable and responsible for neutralization and serotype specificity of BTV. Characterization of isolates which are definitively sero-typed is very much essential for developing suitable vaccine to control the disease in a given geographical region. This research work is focused on the characterization of an Indian isolate (VJW784) of bluetongue virus serotype 16, by RT-PCR and molecular techniques. This is the first time to document the complete sequencing of protein coding region of VP2 gene of an Indian isolate of bluetongue virus serotype 16. The VJW784 isolate was propagated on BHK-21 cell lines and harvested. dsRNA extracted from BHK-21 cells was used for testing by RT-PCR with serotype specific primers of BTV 1, 9, 10, 16, 21 and 23. Further serotype-specific primers for VP2 gene of BTV 16 were designed and used for specific amplification of segment 2. Cloning was carried out using pDK101 vector. The positive plasmid DNA and PCR amplicons were sequenced. The sequence information was aligned with DNAstar software and BLAST analysis was carried out. The sequence information provided herein may help to determine the geographic origin of VJW784isolate and defined the phylogenetic relationship of this isolate to other BTV strains.ThesisItem Open Access CHARACTERIZATION OF Staphylococcus aureus STRAINS OF BOVINE AND CANINE ORIGIN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2011-03) JYOTHI KUMARI, GANNE; DHANA LAKSHMI, K(MAJOR); NARASIMHA REDDY, Y; MADHURI, DABSTRACT : The present investigation was carried out to isolate and characterize S.aureus from bovine and canine species. Characterization was done by examination of the isolates for simultaneous coagulase and mannitol fermentation, DNase, hemolysin, gelatin liquefaction, antibiogram and biotyping. The methicillin resistant Staphylococcus aureus (MRSA) isolates were further subjected to PCR for the detection of genes namely mec A and spa. A total of 100 clinical samples collected from bovines and canines were subjected for isolation of S.aureus following standard methods and total of 51 isolates were obtained. Most of the coagulase positive strains showed thermonuclease activity in toluidine blue DNA agar. Beta haemolysis was observed in 87.1% of isolates while 64.5% isolates showed gelatin liquefaction. The antibiogram pattern of S.aureus isolates carried out by employing 25 different antibiotics revealed higher sensitivity for chloramphenicol 84%, followed by ceftriaxone 80.3%, vancomycin 76.4%, tetracyclines 72.5% and chlorotetracyclines 70.5%. The antibiotics namely streptomycin and furazolidone exhibited low sensitivity 23.5% and 7.84% respectively. Of the 51 isolates subjected to methicillin, only 10 isolates (19.6%) were found to be resistant. All the 10 MRSA isolates and two methicillin sensitive Staphylococcus aureus (MSSA) were subjected for detection of genes namely mec A and spa by PCR technique. Of these, only four of MRSA isolates exhibited the products of both mec A and spa gene whereas three MRSA isolates revealed the product of spa gene alone while the remaining three isolates did not yield any of the gene products. The size of the gene product for mec A gene was 300bp while that of spa gene were measuring either 400bp or 1000bp.Both MSSA isolates tested failed to produce the products of mec A as well as spa gene products.. The polypeptide analysis of the 5 MRSA and 1 MSSA isolates carried out by employing SDS- PAGE revealed 20 different protein bands with approximate molecular weights ranging from 16 to 88 KDa. However, there was no difference either in number of protein bands or mol. wt between MRSA and MSSA isolates of S.aureus tested.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.