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ThesisItem Open Access STUDIES ON EXPERIMENTAL SUPPRESSION OF BURSA AND THYMUS DEPENDENT IMMUNE SYSTEM IN DUCKS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 1992-12) PRASADU, VAKANI; UMAMAHESWARA RAO, S(MAJOR); SUBBA RAO, M.V; SURI BABU, T; SRIRAMAN, P.kThesisItem 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 EPIZOOTIOLOGICAL STUDIES OF INFECTIOUS BRONCHITIS IN CHICKEN IN RAYALASEEMA AREA(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 1979) SUDHAKAR REDDY, A; GOPALA KRISHNA MURTHY, K(MAJOR); KRISHNA SWAMY, SThesisItem Open Access IMMUNOLOGCAL STUDIES ON MAREK'S DISEASE IN CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 1979) Rama Moorthy, A; Gopala Krishnamurthy, K(MAJOR); krishna swamy, SThesisItem Open Access THE INCIDENCE OF CARRIER STATE OF SALMONELLA AMONG SLAUGHTERED PIGS IN ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 1979) SUDERSHAN SINGH; MAHENDRA NATH, D(MAJOR); KRISHNA SWAMY, S; DHANANJEYA REDDY, BThesisItem 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 virusesThesisItem Open Access ISOLATION, CHARACTERIZATION AND ANTIBIOGRAM PATTERN OF SALMONELLA AND ESCHERICHIA COLI IN ENTERIC INFECTIONS OF PIGS IN ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2008-12) RUPA SUNDARI, A; SATYANARAYANA CHETTY, M(MAJOR); SREENIVASULU, D; SRI LATHA, ChABSTRACT: Multiple serovars of Salmonella and serotypes of E. coli are responsible to cause severe infections in pigs leading to morbidity and mortality and economic loss to swine industry. These bacteria also affect human health and have public health impact. Isolation and identification of indigenous Salmonella serovars / E. coli serotypes is of prime importance in order to carryout research on development of vaccines and for the study of molecular epidemiology. Hence, an attempt was made in this direction in the present study. Among 101 faecal samples screened for salmonellosis the notable serovars Salmonella Derby, S. Typhimurium and S. Typhisuis surfaced as major incriminating agents in pigs while the E. coli serotypes identified were O1, O2, O5, O8, O10, O12, O22, O24, O60, O88, O89, O92, O123, O138, O154 and O159. The bacterial aetiology was confirmed with standard procedures and protocols of Bergey’s manual of determinative bacteriology, ninth edition. The haemagglutination pattern of Salmonella and E. coli exhibited agglutination of erythrocytes viz., cattle, sheep, goat, chicken, rabbit, guinea pig and mice indicating the importance of fimbriae in colonization of organisms in intestine and production of toxins. Anti-sensi disk patterns revealed that the drugs of choice were chloramphenicol, co-trimoxazole and gentamicin for Salmonella infection while gentamicin, amoxycillin and chloramphenicol were the suitable drugs for chemotherapy of E. coli infection. The in vivo pathogenicity studies in mice revealed that S. Typhimurium was most pathogenic followed by S. Derby and S. Typhisuis whereas the pathogenicity studies carried out in chicks with E. coli serotypes viz., O1, O2, O8, O10, O22, O88, O92, O123, O138, O138 and O159 produced characteristic lesions and mortality in affected chicks. An innovative study on effect of Salmonella serovars viz., S. Typhimurium and S. Derby and E. coli serotypes O1, O24, O60 O89, O92, O123, O138 and O159 on germination of seeds were found to be toxic causing the inhibition of germination. Cefotaxime extract of Salmonella serovars on SDS-PAGE yielded 11 polypeptide bands ranging in molecular weight from 14 to 95 kDa while fimbrial extract of E. coli revealed a single band of 18 kDa.