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Open 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.
Open 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.
Open Access
Enzyme immuno assay based seromonitoring and detection of canine rabies antigen
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1999) Biju, K G; KAU; Krishnan Nair, G
In the present study an attempt was made to measure the serum neutralizing antibodies against rabies virus in the sera of both vaccinated and unvaccinated dogs, employing ELISA. A long with this special emphasis was given to compare the results of diagnostic techniques used for yaJ.i.es namely Seller's staining and IP test. Four hundred and thirty-four sera samples from dogs of different age groups were screened. Enzyme immuno assay of sera of 83 unvaccinated pups revealed that under the age group of 0-3 months 59.1 percent antibody titre of 1: 50 or above indicating the presence of maternal antibodies. Out of 116 sera samples from primarly vaccinated group only eight had antibody titre below 1:50. Among the booster vaccinated 94 sera samples tested only four samples showed antibody titre below 1:50 .. The assay revealed that during diseases the immune systems of the dogs will be suppressed. It was found that the age of first vaccination could be decided as third month. The present study revealed that primary vaccination might not produces higher antibody titres in dogs. The study showed advantages of booster vaccination over primary vaccination. All the rabies suspected cases were subjected as two diagnostic tests namely Seller's staining and IPT. Out of 20 rabies suspected cases, there were detected by demonstration of Negri bodies and four were by IPT. The present study showed plate ELISA is a sensitive method for titration of rabies virus neutralizing antibodies and IPT can be successfully used for the detection of rabies antigen in impression smears taken from hippocampus.
Open 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.
Open 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.
Open Access
Prevalence of yeast and yeast like fungi in bovine mastitis and their in vitro drug sensitivity
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1996) Sukumar, K; KAU; James, P C
A total of 200 milk samples from clinical cases of bovine mastitis were culturally screened during a period of six months. Pathogenic fungal organisms could be isolated only from 26 samples. Out of this 26 positive samples, yeast and yeast like fungal organisms were isolated from 20 samples and mould from six cases. The major pathogen isolated were candida spp namely C. tropicalis, C. parapsilosis and C. guillermondi. The other organisms were Geotrichum candidum, Trichosporon cutaneum, Sacharomyces cerevisiae, Torulopsis spp and Rhodotorula rubra. The filamentous fungi isolated were Sepedonium spp, Aspergillus ochraceous group, Cladosporium carrionii, Penicillium spp and Trichophyton verrucosum. In majority of the cases yeast and yeast like fungi produced chronic mastitis in which hardness of udder and reduction in milk yield with watery milk containing flakes were noticed. In cases of mastitis where in mould was involved, chronic mastitis characterized by hardness of udder and reduction in milk yield with straw yellow coloured milk, viscid in consistency. Sensitivity pattern of the fungal isolates to the commonly employed antifungal chemotherapeutic agents like Amphotericin B, Clotrimazole, Fluconazole, Griseofuivin, Itraconazole, Ketocanazole, Nystin and Pimaricin (Natamycin) was elucidated. Among the above agents Clotrimazole and Itraconazole exhibited maximum inhibitory activity. All the isolates were found to be resistant to Griseofulvin. In vitro drug sensitivity pattern of fungal isolates employing the discs impregnated with essential oils of cinnamon, clove and lemon grass and alksloids of Cassia alata was studied. Cinnamon leaf oil possessed maximum antifungal activity and the extracts of Cassia alata failed to evince the ability to inhibit the growth of fungal isolates. The antifungal activity of plant extracts were compared with the commonly antifungal chemotherapeutic agents.
Open Access
Comparative efficacy of different vaccines against pasteurellosis in ducks
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1998) Jayakumar, P S; KAU; Jayaprakasan, V
Two isolates of Pasteurella mul tocida from ducks viz. DP1 and DP5 isolated from ailing/dead ducks from Kuttanad area and maintained in the virulent form were used separately for the preparation of different vaccines viz., bacterin, bacterin with oil adjuvant and sonicated antigen with oil adjuvant. The biological and biochemical characters of both the isolates were compared and are in confirmity with the characters described for the species by earlier workers. Both the isolates were pathogenic to ducks and mice as all the inoculated ducks and mice were killed with in 72 h. The LDso for the DP1 isolate in one month old ducklings and six month old ducks were determined to be 23 and 32 cells respectively whereas the LDso of DP5 isolate for one month old ducklings was determined to be 17 and for adult ducks LDso was 21 cells. Immunogenic potential of different vaccines prepared using DP1 and DP5 isolate were tested in ducks by giving two doses of vaccine. The first dose was given at four weeks of age and second dose was given 80 days after the first dose. The birds were challenged with 0.1 ml of culture containing 100 LDso of fully encapsulated virulent form of bacteria at 20 days interval till 80th day and then at 90th and 120th day after the first dose of vaccine. A higher percentage of protection was conferred by oil adjuvant vaccines prepared using DP1 and DP5 isolate and sonicated antigen with oil adjuvant prepared using DP1 isolate. The serum samples were collected from vaccinated birds at regular intervals of 0 I 7 I 20 I 40 I 60 I 80 I 90 and 120 for indirect haemagglutination test and the titres obtained were ranging from 8 to 256. More evaluation and elaborated field trials are required before advocating bacterin with oil adjuvant for field use.
Open Access
Plasmid profile of avian strains of Pasteurella multocida
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1998) Balakrishnan, G; KAU; Mini, M
Four isolates of P. multocida (DCl, DC2, FCl and FC2) from avian species (chicken and ducks) were subj ected to various typing methods like biotyping, antibiogram and plasmid profiling. The pathogenicity of the isolates was also ascertained in mice, rabbits chicken and ducks. By biotyping, the isolates were found to belong to two subspecies P. mul tocida subsp. mul tocida and P. mul t.oc i cie subsp. septica. All the isolates fermented xylose and none of them fermented dulcitol. Antibiogram of the isolates was carried out and they wer~ found to be resistant to furazolidone, metronidazole anC nalidixic acid. Some of the isolates are also resistant tc sulpha and trimethoprim. All the isolates were pathogenic to mice on rip injection. Duck isolates (DCl and DC2) were found to be pathogenic to rabbits killing them on fourth and fifth day respectively. The isolates DCl and DC2 were lethal to orally infected chicks within 48 h of administration and P. multocida could e reisolated from these birds. The same isolates killed the SiC injected ducks within 24 and 48 h. 11 The chicken isolates obtained from apparently healthy birds were non pathogenic to rabbits, chicks and ducks. Plasmid profiling revealed the presence of plasmids in three of the four isolates screened. One isolate was plasmid less and another isolate contained five plasmids. The molecular size of the plasmids ranged from 2.69 kb to 7.07 kb. One isolate did not contain plasmids at all. An isolate (FC2) which was not pathogenic to rabbits, chicks and ducks contained plasmids. Isolate FCl which was resistant to six antibiotics did not possess plasmids. Hence plasmids obtained in this study may not be associated with either antibiotic resistance or pathogenicity in experimental animals. A combination of two parameters such as plasmid profile and one of the typing methods, biotyping or antibiogram made it possible to differentiate the isolates. Thus, the plasmid profile analysis with anyone of the typing methods could be used as an epidemiological tool in the differentiation of P. multocida strains of avian origin.
Open Access
Influence of adaptation of the vaccine strain of duck plague virus in chicken embryo fibroblast on its immunogenicity
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1997) Senthil Kumar, K; KAU; Ponnoose, K T
A chicken embryo adapted vaccine strain of duck plague virus was serially passaged in chicken embryo fibroblast cell cultures and its immunogenicity was evaluated at different passage levels. The vaccine strain of DPV received from VBI, Pal ode was revived in 11 day old chicken embryos by CAM route. The infected embryos died in 70 to 120 hr PI with lesions of congestion on the embryo and CAM and enlargement of liver and spleen. This embryo passaged virus was propagated in CEF cell culture, prepared from 12 day old embryonated chicken eggs. The virus produced CPE, characterised by rounding and clumping of cells, syncytium formation, vacuolation of cytoplasm and eosinophilic intranuclear inclusion bodies. The virus was adapted in CEF cultures by serial passage. It was passaged for ten times and the various characters of the fifth and 10th passaged viruses were studied. There was no change in the CPE but the time required for the appearance of CPE and total detachment of the cells decreased as the passages increased. The CPE appeared at 48 hr, 30 hr and 24 hr for first, fifth and 10th passages respectively. Similarly the time required for total detachment of cells also reduced from 120 hr at first passage to 90 hr at fifth passage and 80 hr at 10th passage. The rapid onset of CPE and desquamation of cells indicated the adaptation of the virus in CEF cell culture. The titres of fifth and 10th passage viruses in chicken embryos were 104.75 and 105.77 ELD50/ml respectively. The titres in CEF cultures were slightly higher. The values were 105.67 and 106.77 TCID50/ml respectively for the fifth and 10th passaged samples. The immunogenicity of the fifth and 10th passage viruses were studied by vaccinating six weeks old ducklings. Each duckling received 3.5 log10 TCID50 of either fifth or 10th passaged virus intramuscularly. The birds remained normal till the 20th day and when challenged with virulent virus. Birds that received the fifth passaged virus showed mean antibody titres of 64 and 32 by SNT and PHA respectively. All the birds withstood challenge indicating the effectiveness of fifth CEF passaged virus as a vaccine. In birds that received the 10th passaged virus, the antibody titres were low both by the SNT (1:54) and PHA (1:22). However all the ducks survived without manifesting any clinical signs. All the control ducks developed clinical signs of DP and died in seven to nine days time. The fifth and 10th CEF passaged viruses were sensitive to pH 3 and 11, but stable at pH 7.2. They were completely inactivated at 56°C in 30 min. These indicated that there was no change in the above physical characters of the virus though it was passaged in CEF cultures incubated at 38.5°C. Though the efficacy of the 10th passage virus was slightly lower as it was evident from the low antibody level, a detailed study is required to establish the present findings that an increase in the number of passages would result in decreased immunogenicity of the DPV vaccine strain. However from the results obtained during this study, it is evident that cell culture adapted DP vaccine strain could be recommended for production of vaccine against DP.

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