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20004
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Subject: Microbiology × End date: 2000 × Start date: 2000 × Type: Thesis × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen Access
    Seroprevalence and Restriction enzyme analysis of Egg Drop Syndrome virus
    (Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2000) Priya, P M; KAU; Krishnan Nair, G
    In the present study, seroprevalence of EDS-76 was conducted in five districts of Kerala in duck and' chicken flocks using HI and ELISA. Out of 322 chicken and 281 duck sera samples screened, an overall incidence of 14.91 per cent and 26.69 per cent respectively were recorded. The high proportion of birds showing antibodies to EDS-76 reveals that the infection is widespread in Kerala and may be the major etiological factor associated with drop in egg production in poultry. Among the two serological tests namely, HI and ELISA employed for the detection of EDS-76 viral antibody, HI was found to be simple, sensitive and reliable. It is concluded that HI test could be used for the detection of EDS-76 infection in poultry flocks. Restriction DNA fingerprinting of the three indigenous strains were carried out in conjunction with the reference strain to check for any genetic variation between the strains. Comparison of the DNA fingerprint of all the four strains digested with restriction endonucleases BamHI, EcoRI and HindIII revealed identical banding pattern thereby conforming the genetic similarity of the strains.
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    ThesisItemOpen Access
    Characterisation of virus isolates from lesser whistling teals and channa species of fish
    (Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2000) Pradeep, V; KAU; Krishnan Nair, G
    Characterisation of virus isolates (T18 and T22) from lesser whistling teals (Dendrocygna javanica) and Channa species of fish (Fe and F12) was carried out to determine the similarities if any between the isolates and to identify the role of waterfowls in dissemination of these viruses. The virus isolates. preserved in the Department of Microbiology were revived by passaging through embryonated chicken! duck eggs through allantoic route. After the third passage, all the isolates were found to produce death of the embryos and the allantoic fluid collected agglutinated one percent chicken RBC. The isolates T18, Fs and F12 produced congestion of the embryo and CAM and the embryos showed sub-occipital and interdigital haemorrhages. Isolate T 22 also produced congestion of the embryo and CAM and the embryos were stunted. Liver of the embryos had yellowish brown patches. The EID50 of isolates were 3.2x10s, 5.6 x 105 , 1.65x 107 and 3.16 x 105 respectively for the isolates T18, T22. Fs and F12. The infectivity and haemagglutinating activity of all the isolates were retained at pH 7.2, but were completely lost at pH 3.2 and 9.0 and also by treatment at 56°C for 30 min. All the isolates were sensitive to chloroform indicating their enveloped nature. Pretreatment of chicken embryo fibroblast cultures with 1 OO~g! ml of luDR did not inhibit the multiplication of any of the isolates indicating they all had RNA genomes. All the isolates were resistant to treatment with brine indicating that they were capable of survlvinp at high salt concentration. The isolates T18 and Fa produced marked CPE in chicken embryo fibroblast culture with rounding and clumping of cells and syncytia formation. Marked cytoplasmic vacuolation was also observed. Inclusion bodies could not be detected either in nucleus or cytoplasm. For isolates T22 and F12, CPE developed later only and was not as prominent as for T18 and Fa. Rounding of cells and their fusion forming syncytia was noticed by 72 h. Cytoplasmic vacuolation though present was much less marked. Inclusion bodies were absent. Large polykaryocytes were produced by the isolates T18, T 22 and F12 in BHK-21 cell line within 24h after inoculation. Between 48-72h large syncytia were formed. Intracytoplasmic inclusions could be observed by 24h after infection, which were quite prominent by 96h. The isolate Fa failed to produce any CPE in BHK-21 cell line. Pathogenecity tests in day old and six-week-old chicken and ducklings showed that all the four isolates were non-pathogenic when given by the oral Isubcutaneous route or by both. Neither clinical signs or mortality could be observed in the birds. Virus isolation was possible from the cloacal swabs of six-week-old chicken for the isolates T18 and T22 up to the 14th and th day respectively. Antigenic relationship between the isolates was tested by gel diffusion and haemagglutination inhibition tests, which showed that the isolate T18 did not have any similarity with any of the other three isolates. The isolate T 22 showed antigenic similarity by both the tests. Fa showed similarity to T18 by HI test but not by immunodiffusion test. Isolate F12 was found to be distinct from the other three by HI test, but showed some similarity with them by immunodiffusion test. By sodium dodecyl sulphate polyacrylamide gel electrophoresis on 10 percent gels, 7-11 bands could be resolved for the different isolates. Three of the bands were common for all the four isolates and were having molecular weights similar to the three major proteins HN, NP and MP of avian paramyxoviruses, suggesting that the isolates belonged to the paramyxovirus group. Monoclonal antibody typing of the isolates T18 and T 22 at the Central Veterinary Laboratory, Surrey, England confirmed that both belonged to the paramyxovirus group with T18 belonging to group C (velogenic) and T 22 to group E (B1 and LaSota) viruses. The isolates Fe and F12 need to be further typed. It was concluded from the study that all the isolates were enveloped RNA viruses with T18 and T 22 being paramyxoviruses belonging to Group I. The properties of the isolates Fs and F12 resembled the paramyxoviruses and from the similarly in protein profile with the other two viruses can also be concluded to be paramyxoviruses.
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    ThesisItemOpen Access
    Molecular characterisation of salmonellae isolated from poultry
    (Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2000) Tressa Mary, G; KAU; Punnose, K T
    Five serotypes of salmonellae from avian sources were examined for biochemical properties, serology, drug resistance, plasmids, restriction enzyme pattern of plasmid as well as genomic DNA and pathogenicity. The biochemical characters are in confirmity with the characters described for the serotypes by the earlier workers. The study of antibiogram with 20 antibiotics/chemotherapeutic agents revealed the presence of multiple drug resistance in all the five serotypes. In the plasmid analysis, S enteritidis and S. branderup were found to be plasmid free. The number of plasmids in other serotypes ranged from two to three and the size ranged from 1.75 kb to 48.32 kb. Identical low molecular weight plasmids were present in both S. gallinarum. The presence of large plasmid in one of the S. gallinarum did not confer any additional detectable resistance character. S. typhimurium contained two plasmids of sizes 13.62 kb and 4.2 kb. Restriction enzyme analysis of plasmid DNA from three salmonellae with EcoRl and Hindlll yielded three different restriction pattern with each enzyme. Ethidium bromide and exposure to elevated temperature cured the plasmids present in the salmonellae within two days and 14 days respectively. Acridine orange and sodium dodecyl sulphate were found to be ineffective in curing the plasmid DNA. The elimination of plasmids resulted in the loss of resistance to antibiotics was demonstrated in S. typhimurium. In tests to assess the differences in pathogenicity between wild and cured isolates of S. typhimurium in day old chicks, only intraperitoneal route was found to be effective when compared to oral route. A relation of plasmids to virulence was noted only in S. typhimurium. Day old chicks were refractory to infection to S. gallinarum by both the routes. Plasmids encoding both resistance and virulence were observed in S typhimurium. Plasmid negative serotypes of S. enteritidis and S. branderup were found to be equally virulent as wild strains of S. typhimurium. So a definite correlation between virulence and plasmids could not be made. Restriction enzyme analysis of chromosomal DNA yielded bands which were indistinct and so uncomparable. Hence of the tests based on the analysis of genetic content plasmid profile was found ,to be efficient in typing the isolate rather than restriction enzyme analysis of genomic DNA.
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    ThesisItemOpen Access
    Homology between corynebacterium psedotuberculosis isolates from goats and standard reference strain
    (Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2000) Mohan, P; KAU; Jayaprakasan, V
    Corynebacterium pseudotuberculosis was isolated in pure culture on blood agar from pus collected from lymph nodes of goats, which suffered from caseous lymphadenitis. Three field isolates of C pseudotuberculosis were characterized and compared with standard reference" strain based on cultural, biochemical, toxigenicity, protein profile and restriction endonuclease digest analysis of chromosomal DNA. The morphological and cultural characters of all the field isolates and reference strain were similar and characteristics to the species. Biochemical reactions employed were also similar for all the strains except isolate F3, which fermented lactose. The biochemical characters were in confirmity with the characters described by the earlier workers. Three different types of toxin viz., culture filtrate (CF), whole cell lysate (WCL) and sodium chloride extract (SCE) were tested for dermonecro toxicity in rabbit skin. Among the preparations, the whole cell lysate produced a definite necrosis at the point of intradermal injection followed by culture filtrate and sodium chloride extract. The toxin preparations of the isolates invariably produced inflammatory and necrotic changes, the degree and severity of the reactions varied between samples. When the three toxin preparations of the isolates and reference strain were tested for synergistic haemolytic activity with Rhodococcus equi, the whole cell lysate produced maximum zone of haemolysis followed by culture filtrate and sodium chloride extract. The proteins presented in the different preparations were analyzed by SDS-P AGE. The protein profiles discerned by whole cell lysate, culture filtrate and sodium chloride extract were 25,9 and 9 protein bands with ranged on masses approximately 8-200KDa, 20-200KDa and 19-191KDa respectively. The 31.6KDa and 68 KDa proteins were found to be consistent in three toxin preparations. The DNA extracted from the isolates and reference strain were subjected to restriction endonuclease digestion employing Eco RI, Barn HI, BgI II, Eco RV and Pst I. The enzyme digest of DNA varied between the enzymes employed. There are no observable differences between the field isolates and reference strain, when the DNA digested with these five restriction enzymes. The enzymes Eco RI, Barn HI and BgI I presented similar restriction pattern whereas the DNA fragments generated by Eco RV and Pst I were different from the other three enzymes.