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ThesisItem Open Access MOLECULAR DETECTION AND CHARACTERIZATION OF CLOSTRIDIUM PERFRINGENS TOXIN GENES RESPONSIBLE FOR NECROTIC ENTERITIS IN POULTRY OF ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2008-04) PRAVEEN KUMAR, N; VINOD KUMAR, N(MAJOR); RANI PRAMEELA, D; SUJATHA, KABSTRACT: The present study was carried out to investigate the prevalence of Clostridium perfringens toxin genes responsible for necrotic enteritis in poultry of Andhra Pradesh. A total of 691 faecal samples (396 necrotic enteritis suspected and 295 apparently healthy) comprising of cloacal swabs from live birds and intestinal scrapings from dead birds were collected from different districts of Andhra Pradesh viz., Chittoor, Guntur, Nellore, Krishna, East Godavari and West Godavari. Gross pathological studies of affected birds revealed necrosis of the small intestinal mucosa and submucosa with fibrin deposition resulting in pseudo membrane formation and turkish towel appearance was noticed in the small intestine. Microscopically lumen of intestine with fibrinonecrotic material which forms a visible pseudo membrane composed of cell debris, necrotic/distorted villi, inflammatory cells and clumps of bacteria were observed. The samples were inoculated in to fluid thyoglycollate broth and incubated overnight. DNA extracted from 24 h broth cultures by boiling method were screened by multiplex PCR for simultaneous detection of α, β and β2 toxin genes. Out of 396 (broiler 282 & layer 114) necrotic enteritis suspected samples 337/396 (85.1%) were positive for α toxin gene of which 189/337 (56.08%) were β2 toxin gene positive. Out of 295 (broiler 182 & layer 113) apparently healthy samples 61/295 (20.67%) were positive for α of which 4/61 (6.55%) were β2 positive. None of the sample showed amplification of β toxin gene indicating the absence of C. perfringens type C. As some recent studies focused the involvement of NetB toxin in pathogenesis, therefore, uniplex PCR amplification of NetB gene was done from alpha toxin gene positive samples (C. perfringens type A) yielded no positives for NetB toxin gene. From chi square analysis a significant difference (p<0.01) in the prevalence of toxin genes (cpa & cpb2) between necrotic enteritis suspected and apparently healthy at 99% level of confidence with an increased number of positives from necrotic enteritis suspected group. The present research indicates C. perfringens type A along with β2 toxin gene might be responsible for causing necrotic enteritis in broilers and layers in Andhra Pradesh. The multiple sequence analysis of the amplified partial cpa and cpb2 gene sequences revealed 100% homology between the present isolates, and with selected published sequences from GenBank were found to be 98-99% and 94-99% homology respectively. The phylogenetic analysis of the cpa gene of the present C. perfringens isolate (MG600591) with the selected published sequences of cpa revealed the close segregation in distinct clade with cpa gene of broiler isolate of C. perfringens (GU581186) from Iran. The phylogenetic analysis of three cpb2 sequences of present isolates (MF471364; MF471366; KX001813) segregated into close group of poultry originated sequences with exception of MF471365 which segregated in distinct clade with noporcine originated C. perfringens sequence (AY609173) from USA. Since alpha toxin gene (cpa) is considered as signature toxin gene for C. perfringens, amplification of cpa by PCR is considered as confirmative diagnosis of C. perfringens. Hence, in the present study all the PCR positives for cpa (n=398) were isolated by culturing revealed only 221/398 (55.52%) isolates indicating PCR is more sensitive in detecting C. perfringens when compared with isolation by culturing. In the present study culture supernatant of B. subtilis isolated from healthy intestinal contents of birds successfully inhibited C. perfringens by disk diffusion method indicating its importance as a probiotic in controlling necrotic enteritis in poultry.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 CHARACTERIZATION OF Staphylococcus aureus ISOLATES OF ANIMAL ORIGIN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2003-11) ARUNA, VEDAVALLI; DHANA LAKSHMI, K(MAJOR); JANAKIRAMA SARMA, B; ANJANEYULU, YABSTRACT : The present investigation was carried out to isolate and characterize S. aureus from different animal and poultry species. Characterization was done by examination of the isolates for simultaneous coagulase and manitol fermentation, DNase, hemolysin, dermonecrotoxin production and antibiogram. The isolates were also subjected to finger printing analysis by REP and BOX-PCR. A total of 50 samples were collected from different animal species viz., buffalo, dog, goat, poultry and emu for isolation of S. aureus. The number of isolates from 14 buffalo milk samples,13 from birds, 13 from dogs and 10 from goats were 8, 6, 10 and 7, respectively. Identification of isolates by simultaneous testing of coagulase and mannitol fermentation was less time consuming and a convenient method. Most of the coagulase positive strains showed thermonuclease activity in toluidine blue DNA agar. Out of all the hemolysis positive isolates only one isolate from Emu bird exhibited dermonecrosis in rabbits. The RF for metronidazole was 100% and was more than 50% for ampicillin, penicllin-G, nitrofurantoin, amoxycillin and streptomycin. The RF of 25 – 50% was noticed for kanamycin and erythromycin. For doxycylcline, tetracycline, trimethoprim norfloxacin and cephalexin the RF was less than 25%. Out of 31 DNA samples of S. aureus analyzed by PCR technique, 14 samples produced PCR amplified products in REP and BOX and they were grouped into 6 major clusters in REP and 10 in BOX. There was extensive variability in DNA finger prints of S. aureus indicating differences in the genetic make up of S. aureus strains without any consideration for the animal source.ThesisItem Open Access ISOLATION AND CHARACTERIZATION OF INDIGENOUS INFECTIOUS BURSAL DISEASE VIRUS ISOLATES IN AND AROUND HYDERABAD(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2009-03) SOUMYA, VENGA REDDY; DHANALAKSHMI, K(MAJOR); SATYANARAYANA CHETTY, M; MADHURI, DABSTRACT : The present study was taken up with the objective of isolation and characterization of IBDV isolates. Ten pooled bursal samples (each pool consisting of five samples) were screened for IBD virus by AGID, out of which 5 pooled samples were positive for IBD virus. Chicken embryos were used for isolation of IBD virus by CAM route. Out of five samples two samples, PDP-1 and VH-1 yielded IBD virus after three serial passages in 11 day old chicken embryos. These isolates produced characteristic lesions like dwarfing of embryo, cutaneous edema, hemorrhages on thigh muscles, toes and greenish yellow discolouration of the liver in chicken embryos. These isolates could be adapted to BHK-21 cell cultures only after 5 successive passages as evidenced by stabilization of typical cytopathic effects. At 3rd passage level, CPE started with rounding of cells 72 h, PI. Large numbers of cells showed rounding and clumping, increase in cytoplasmic granularity at 96 h, of PI in 4th passage. In 5th passage, CPE started early i.e., 48 h, of PI and by 72 h, 60% of the cells were involved. There was extensive detachment of cells by 96 h, PI. Cell culture adapted isolates were characterized for physico-chemical properties. They were resistant to chloroform but inhibited by 1.0% formalin for 6 h, 0.1% formalin for 24 h. They were viable even after exposure to 560C for 90 min. They were resistant to acidic pH of 2.0 but completely inactivated at pH 12.0. Immunological characterization of cell culture adapted IBDV isolates by AGID and CIE revealed clear precipitation lines. The polypeptide analysis by SDS-PAGE revealed that two isolates contained identical viral polypeptides with similar molecular weights and band pattern. Both isolates revealed 5 poly peptides, in addition to these two faint bands. The molecular weights of the 5 polypeptides were: (91 KD) VP1, (52 KD) VPx, (42 KD) VP2, (32 KD) and VP3, (27KD) VP4 and molecular weights of two faint bands were 70 KD and 66 KD. In this study, RT-PCR was performed on cell culture fluids and corresponding bursal tissues to detect the virus. RT-PCR was performed using P1 and P2 primers specific to hyper variable region of VP2 gene pertaining to IBDV. These studies resulted in generation of specific amplification of 643 bp from samples tested. Pathogenecity studies with the cell culture adopted field isolates inoculated in 3 week old chicks revealed 50 % mortality with PDP-1, 37.5 % mortality with VH-1.The dead birds revealed characteristic lesion i.e., haemorrhages in thigh and pectoral muscles, enlarged and haemorrhagic bursae. Histopathological changes were more pronounced in bursa of Fabricius. The changes were characterized by lymphoid necrosis, follicular degeneration and proliferation of connective tissue.ThesisItem Open Access ISOLATION AND MOLECULAR CHARACTERIZATION OF INDIGENOUS GOATPOX VIRUS FROM TELANGANA REGION(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2009-01) VIJAYALAXMI, V; SATYANARAYANA CHETTY, M(MAJOR); Anjaneyulu, YABSTRACT:In view of the increasing incidence of goatpox out breaks the present study has been taken up with a view to isolate goatpox virus from field cases and characterize them. The disease surveillance reports on goatpox were collected for the years 1997-2008 and analysed. The analysis of epidemiological data revealed 176 outbreaks in Andhra Pradesh The outbreaks were recorded more during the years 2000-2003 and 2004-2007.Case fatality rates ranged from 3.7% - 49.4 % during the period. Samples were collected from two outbreaks of goatpox. Both the samples were positive for goatpox by Agar gel immunodiffusion. Attempts were made to isolate the virus by passaging in chicken embryos and then adapted to vero cell lines. Infected embryonating chicken eggs died between 48 h. and 120 h. Post Inoculation (PI) depending on the passage. CAM was edematous, thickened and congested. Slight hemorrhages were observed in 3rd passage. Embryo material after 3rd passage gave positive reaction when tested by Agar gel immunodiffusion with positive serum. EID50/ml (embryo infective dose 50) of the virus isolates ranged from 0.5x102 to 1x102 for the virus from 1st isolate while the virus from 2nd isolate had titres ranging from 0.7x102 to 0.9x102 / ml. Embryo passaged virus was then adapted to vero cells. On first passage cells revealed granulation and shrinkage of cells at 24 h – 48h post infection, syncitia at 72 h - 96 h PI while peeling of monolayer started at 120 h PI. Complete detachment of monolayer was seen in 6 days. Virus from 1st isolate had a titre of 104.9 TCID50 /ml and 2nd isolate had 105.2 TCID50/ml titre. Goatpox virus (GPV) was successfully purified by sucrose density gradient centrifugation. Two bands formed on the gradient, one at the interface of 50 and 60 percent sucrose layer and another at 40 percent sucrose layer. AGID and Immuno Electrophoresis were used to characterize the isolates. They resulted in a single precipitation line with hyperimmune serum. Physicochemical characterization of the viruses with regard to temperature, pH, ether and chloroform were carried out. The viruses were completely inactivated at 60oC in 30 min and 70oC in 10 min. Chloroform, ether and pH 3 were detrimental to goatpox viral isolates. Electron microscopy of the virus isolates revealed brick shaped virus with size of 250 x 290 nm. SDS - Polyacrylamide gel electrophoresis of the isolates revealed a total of 20 bands. Of these, five were major bands with size ranging from 22 to 67kd. Seroprevalance studies on goatpox was studied by employing AGID and Counter Immuno Electrophoresis (CIE). CIE was found more sensitive with positive percentage of 22.5 compared to 13.75 by AGID in a total of 80 serum samples. Polymerase chain reaction was used for detection of specific nucleotide sequences of GPV in samples and cell culture adapted viruses