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Kerala Veterinary and Animal Sciences University, Wayanad
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ThesisItem Open Access BACTERIA ASSOCIATED WITH RESPIRATORY INFECTIONS IN POULTRY(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2007) JESTO GEORGE; G Krishnan NairThis study was undertaken to isolate and identify of bacteria from respiratory tract of poultry and to study the antibiogram of the isolates. Birds showing respiratory signs were sacrificed, postmortem examination was conducted and samples such as nasal, tracheal and air sac swabs and lungs were collected after taking all sterile precautions. A total of 105 samples were collected by sacrificing birds showing clinical signs. Isolation of causative bacteria was made by culturing on brain heart infusion agar, Mac Conkey agar and blood agar. For identification of isolates all the procedures were followed as described by Barrow and Feltham (1993). A total of 31 bacterial isolates were obtained from samples. A total of 12 Escherichia coli isolates were isolated and identified, 4 Pasteurella multocida isolates and 15 Staphylococcus sp. Isolates were isolated and identified biochemically. Out of 15 Staphylococcus sp. isolated and identified 1 1 isolates (73.33 per cent) were coagulase negative This result indicate that CoNS were more frequently isolated from staphylococcal infections although they do not possess the virulent coagulase activity. So importance must be given to CoNS also, as given to coagulase positive staphylococci and much study need to be diverted to find the virulence factors and role of them in producing bacterial infections in poultry. Multi drug resistance (resistance to at least three antimicrobials) was found among all E. coli isolates obtained in the study. Hence it may be concluded that the high level of resistance observed among poultry E coli isolates obtained in the study may be due to incorporation of antibiotics in feed as growth promoters. As 100 per cent sensitivity is shown to enrofloxacin and chloramphenicol by P. multocida isolates, these two drugs may be used for treating pasteurellois. Amoxyciiiin clavulanic acid (Ac) and cephalexin (Cp) was found to be the most effective antibiotic against Staphylococcus sp. in the study. The plasmid DNA content of the seven isolates of E. culi was analysed on agarose gel electrophoresis but correlation between the number of plasmids and antibiotic resistance could not be ascertained in this study. In conclusion, the results of this study provide evidence for significant antimicrobial resistance among bacterial isolates from birds. Long term prospective studies involving isolation, identification and antibiogram from more samples are required to identify novel pathogens causing respiratory disease in birds. Such studies provide data on temporal and spatial difference in antibiotic resistance patterns, which in turn helps the scientific community to design better disease control strategies.ThesisItem Open Access PRODUCTION AND APPLICATION OF MONOCLONAL ANTIBODIES AGAINST DUCK PLAGUE VIRUS(College of Veterinary and animal Science,Mannuthy, 1999) RAVINDRA DATTATRAYA PADALKAR; V. JayaprakasanMonocloiMl iinlibodies (Mabs) were raised against the vaccine strain of DPV and three strains of DPV Mz, Vaccine (OPV^V). IVRl (DPV-1) and Alleppy strain (DPV-A) were used to raise polyclonal serum in tite present investigation. DPV-V was revived in 11 day old chicken enibiyo and the enibrjo deatli was recorded four to five days PI with congestion all over the body and spleen and nccrotic foci in liver. The cytopathy in CEF cell culture observed was rounding and clumping of the cells, syncytium formation and bridge fonriation with extensive vacuolation m the Cytoplasm. The detachment of the cells was observed at 120 h PI. 1)PV-1 a virulent strain was inocnlaied in the ducklings, death was recorded in all the inoculated birds with extensive hemorrhages on serous membranes, muscles and visceral organs. Nccrotic foci on li\ei, enlargement and congestion of liver and spleen, and white necrotic foci m the gizzard were evident. The virus was further passaged in DDE and cultivated in bulk in DEP cell culture. The Dl'V-V and Dl'V-A were titrated in Clil' cell cultnie and the TCin,o was 4.7 X 10' per nd of the inoeulnm lor DPV-V and 3.2 X lO' for I)PV-A. ni'V-i cultivated in Did' cell culture had a IC 11),,i of the inoculum All the strains were partially purified at 100000 g for 4.5 h at 4" C in Heekman ultra centrifuge and the protein concentration of the virus was estimated by biuret method and was found to he 1 1 mg. 8 mg and 7 mg for DPV-V, A and I respectively. All the three strains of DPV were inoculated in mice to raise polyclonal serum. Four mice out of five inoculated with DPV-V showed FJdSA titres more than 1:12800, one mouse showed a litre of 1:6400. The mice inoculated witi* DPV-A showed a litre of more than 1:12800 and those inoculated with DPV-I, 1:6400 FddSA was used to test the sera samples of the miee inoculated with DPV strains. The test was found to be highly sensitive, easy to perlorm and less time consuming. The test therefore can be recommended for routine diagnosis of DPV I'olyclonal seiuin was used to study ll'.c cross reactivitv of the three strains oC I)PV with FIJSA. DI'V-V polyclonal scrum reacted with the liomologous virus with a titre of 1:12800. It also showed similar titer with other two heterologous strains. I'olyclonal serum raised against DI'V-A had a titre of 1:12800 with homologous and hcterologuus strains of DI'V. DPV-I reacted with Immnhigous strain at a titre uf 1:6400 Similar titrcs were observed with hertologous strains. Immiino pero.xidase test was used for the detection of the tissue antigens in DPV infected CEF monolayers and in liver and spleen sections. Polyclonal serum raised against DPV-V detected homologous virus in the CF.F monolayers and hetrologous virus in the liver and spleen sections. The staining reaction was observed as dark brown deposits at the virus localization. However background tissue was also stained brown to faint yellow in the stained preparation. The virus neutralization test was used to study the cross neutralization by employing polyclonal .serum raised against the three strains of DPV. Polyclonal serurTi raised against DPV-V showed a VN F of 64 with a VNl of 1.8 with homologous virus and a VN F 45 with VNI 1.65 with other two strains of DPV. Polyclonal serum raised against DPV-A neutralized the homologous virus at a titre of 32 with a VNl of 1.5. it also neutnrlized the vacciiic strain nf DI'V with same VNT and VNI, However it neutralized DPV-1 with VN I' 24 and VNI 1.35. Dl'V-i poiyclonal serum neutralized the homologous virus at a titre of 45 with a VNI of 1.65 . I he s.iiue neutralized DPV-V and DPV-A with a VN T 32 and VNI 1.5.ThesisItem Open Access ASSESSMENT OF IMMUNITY TO DUCK PLAGUE VIRUS (DUCK VIRUS ENTERlTlS) ON VACCINATION(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 1993) DIWAKAR DATTATRAYRAO KULKARNI; P.c. JamesDuring 1991, six outbreaks clinically duck plague (DP) with 33 per cent morbidity mortality were investigated. Duck plague from each outbreak. The isolates were able lesions and death of the duck embryos but fai chicken embryos during initial passages. suspected to be and 26 per cent was isolated to produce the led to kill the One of the strain, named DP-S was par by 10 passages in chicken embryos following duck embryos. Though the attenuated strain did pathogenicity index was reduced from 1.9 to 1. DP-S under transmission electron microscope of herpes virus morphology. Two DP vaccines - commercial vaccine vaccine having virus titres 0.74 and 3.5 respectively, were separately inoculated into ducklings respectively, two groups receiving two receiving double dose of corresponding interval of four weeks. Another group of duckl as control without vaccination. tially attenuated 20 passages in kill ducks, its 23. The isolate revealed virions and lab-adapted log 10 ELD 50/ml four groups of single dose and vaccines at an ings was kept (ii) challenged with Thre e ducks in each group were virulent DPV at four,eight and 20 weeks post-vaccination. The birds in all the five groups were screened at regular intervals for studying the immune response by virus neutralization (VN), leucocyte migration-inhibition (LMI) and passive haemagglutination (PHA) test. The challenged and survived birds were the carrier status of DPV by examination of their for virus isolation. In an organized farm, 180 ducks were gi vaccine at one year of age and were scre antibodies, LMI response and PHA titres before post-vaccination. Randomly selected two birds six weeks post-vaccination. The findings of the study are briefly l Six duck plague outbreaks were investigat isolated, and characterized. It was in duck and chicken embryos. partially The commercial vaccine could elicit very response as compared to laboratory adapted immunity could not last long even upto ei single vaccination and 20 weeks in double screened for rectal swabs ven commercial ened for VN and eight weeks were challenged isted as under: ed, the virus attenuated poor immune vaccine. The ght weeks in vaccination. (iii) * Single vaccination is not effective as c vaccination given four weeks apart. The assessment of antibody-mediated (AMI) and cell mediate d (CMI) immune responses indicated that both and CMI are involved in protection of ducks against duck plague. The vaccinated or vaccinated and infected show carrier status as attempts to isolate rectal swabs collected after vaccination were unsuccessful. The PHA has been standardized for diagnosis of duck plague.ThesisItem Open Access MOLECULAR CHARACTERIZATION OF Pasteurella multocida ISOLATE^ FROM DUCKS IN KERALA(College of Veterinary and Animal Sciences, Mannuthy., 2004) P. X. ANTONY; Dr.J G. Krishnan NairTwenty-five isolates from ducks and one from fow P. multocida using standard bacteriological procedures. strain (LKO) obtained from IVR1 was used for comparisor were found to be pathogenic for mice. Variation in fe dulcitol, mannitol, sorbitol and trehalose allowed the 27 into ten biovars. Two biotypes P. multocida subsp. septlca subsp. multocida were observed among the avian examined uniformly sensitive to enrofloxacin, chloramphenicol isolates from ducks and one from fowl have been sero specific PCR assay was used to confirm the identity of PCR on template DNA prepared from blood smears was fo| rapid method of diagnosis for pasteurella. Dtyped the could N o polymorphism within the KMT1 gene restriction analysis of PM-PCR product with Hae III analysis of genomic DNA of all the avian isolates w revealed three profiles each. The fowl isolates had a majority of the duck isolates. prof le were characterized as A reference chicken . All the avian isolates •mentation patterns of isolates to be grouped , and P. multocida All the isolates were ind pefloxacin. Eight as A:l. A species isolates. Performing und to be an extremely be demonstrated by Restriction endonucleases itjh Hpa II and Hha I that was common to Eighty-eight per cent of the isolates carried p profiles were observed among the isolates examined. showed a single REP-PCR profile indicating a high level them. asmids. Two plasmid All the avian isolates of homogeneity among avi£.n Th e outer membrane protein profiles of all the Two protein fractions with molecular weights of 37.1f identified as the major OMPs by SDS-PAGE. isolates were similar. and 26.36 kDa were A 37.15-kDa protein was identified the major of avian isolates as of P. multocida by Western blotting a molecular mass of 31.33 and 26.36 kDa were also this technique. antige Tw found gene The OmpH gene of all the avian isolates as well were amplified using primers designed based on sequence of a chicken isolate of P. multocida. The am with restriction endonucleases Dra I and Hinf I fragments respectively. The similarity of the profiles for; P. multocida, suggested a high degree of homogeneity region. Restriction analysis of the OmpH-PCR products and B:2 with Dra I generated profiles which were distinct three serotypes. This technique can be helpful for serotypes of P. multocida. Studies on the OmpH gene( cholera vaccine revealed similarity with the amplified re isolate of P. multocida. Since this gene codes for the maj P. multocida the vaccine can be expected to confer immu against pasteurellosis. serotypes A:3 and B:2 blished OmpH gene plified product digested •ated four and three the avian isolates of amongst the amplified The sequence the OmpH-PCR product has GenBank and has been assigned the accession No showed 98 per cent identity with P. multocida strain X membrane protein (OmpH) gene. lie fraction of OMPs o other proteins with to be antigenic using )f serotypes A:l, A:3 rom each other for the erentiation of various s) of inactivated fowl non of the local duck sr antigenic fraction of -lily to ducks in Kerala bjeen submitted to the AY 506823. The sequence 73 (serotype A:l) outerThesisItem Open Access CHARACTERISATION OF Parteurella multocida ISOLATES FROM RABBITS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES Mannuthy - Trichur, 1992-09-04) SHEELA, YOHANNAN; Jayaprakasan, VThe prevalence of Pasteurella multocida in rabbits in and around Thrissur was probed by cultural isolation and confirmed by the elucidation of the identity of the isolates by their physiological, biological and serological characteristics. A total of 112 rabbits comprising 76 apparently healthy and 36 ailing/dead animals of various age groups and breed were subjected to cultural screening. This attempt fructified in the isolation of P. multocida from five rabbits of New Zealand White breed and one of the Grey Giant breed which died of respiratory infection. Post mortem examination of these animals revealed typical haemorrhages in the trachea, haemorrhages and abscessation in lungs and necrotic foci in liver. The present study suggests the prevalence of P* tQ'-iba as one of the causative agent of respiratory tract infection of rabbits kept at laboratory animals facilities. The biological and biochemical characters of all the six isoj.ates were similar to those characters of rabbit of multocida which were reported by earlier workers. These isolates were pathogenic to mice when intraperitoneally inoculated as all the mice were killed within 72 h. In rabbits, these isolates could not establish clinical infection on experimental intranasal inoculation, while at necropsy the animals revealed macroscopic and microscopic lesions suggestive of pasteurellosis. The antibiogram of the isolates were studied and all the isolates were sensitive to several of the chemotherapeutic agents but uniformly resistant to erythromycin. Serologically/ a common somatic antigen in all the three rabbit isolates tested and vaccine strain P^2 could be established by gel diffusion precipitin test. However, additional somatic antigen for one isolate and the vaccine strain P^2 could also be detected when they were reacted against their homologous antisera.