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1996 - 19995
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Subject: Veterinary Microbiology × End date: 2000 × Start date: 1996 ×
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    ThesisItemOpen Access
    Isolation and identification of viruses from waterfowls seen in Kerala
    (Department of microbiology, College of veterinary and animal sciences, Mannuthy, 1997) Bindu, M S; KAU; Krishnan Nair, G
    In the recent years, frequent outbreaks of poultry diseases have caused significant economic losses and among the various sources of infection, freeflying migratory birds and waterfowls are reported to be an important source for the introduction of disease to domestic flocks. These freeflying pirds and waterfowls abound in the waterlogged areas of Kerala especially Alleppey, Kottayam, Thrissur and Malappuram districts. Heavy losses were reported each year due to severe outbreaks of duck plague and duck pasturellosis resulting in the wiping out of the duck population in the state. This may be due to the introduction of the disease agents by migratory waterfowls. Hence a study was undertaken to elucidate the role of waterfowls in the spread of diseases to domestic poultry and ducks. A total of 52 waterfowls were caught from different parts of Kerala of which 15 were lesser whistling teals and 37 were gargany. Postmortem examination of these birds were carried out and the required materials were collected. Impression smears from the cut surface of the liver revealed intranuclear inclusion bodies in the hepatic cells in four cases (Bird Nos. 17, 23, 27 and 49) and two haemagglutinating agents were isolated from the cloacal swabs of the bird numbers 18 and 22. The haemagglutinating agents developed lesions in the chicken embryos which died 3 to 5 days after inoculation. the allantoamniotic fluid of the affected embryos agglutinated teal and chicken erythrocytes. The lesions exhibited by the embryos infected with Ta were congestion of the CAM and embryos while in T22 the embryos were stunted, curled and at the same time congested. The liver of the embryo had yellowish brown patches. The haemagglutination activity of both the viral isolates were tested with red cells of various species like cattle, horse, human '0', duck, rabbit and pig but was negative in all cases. Both the isolates lost their infectivity and haemagglutination property at 56°C for 30 min. the infectivity and HA activity of the viral isolates T18 and T22 were retained at pH 7.2 and were completely destroyed at pH 3.2 and pH 9. Both the isolates were sensitive to treatment with chloroform, revealing both as enveloped viruses, wherein the infectivity was completely lost and HA activity was considerably reduced. In the case of Tl8 HA activity was reduced from 512 to 16 and for T22 from 128 to 8. The nucleic acid types of the viral isolates were confirmed by inoculating the isolates to chicken embryo fibroblast cultures pretreated with 100 µg and 200 µg per ml of IudR which led to the conclusion that Ta was a RNA virus and T22 a DNA virus. The ELD50 of Ta and T22 were 107 ELD50/ml and 106 ELD50/ml. The ICPI, MDT and IVPI. were calculated as for NDV. The ICPI for the isolates were 0.83 and 0.30 for T18 and T22 respectively, MDT was 120 hr for both and the IVPI was calculated to be zero in both cases, indicating that both the isolates were nonpathogenic. In cell cultures both the isolates produced CPE which affected the whole monolayer by 96 hrs. In the case of Tl8 the CPE was characterised by rounding and clumping of cells, the infected cells showing a tendency to get separated from the neighbouring cells leaving long cytoplasmic strands, syncytium formation with four or five nuclei and severe cytoplasmic vacuolation. For T22 the CPE was characterised by rounding and cytoplasmic vacuolation and karryorhexis. Intranuclear and intracytoplasmic inclusion bodies could be demonstrated. The pathogenicity studies of both the isolates were carried out in one week old and six week old ducklings and chicken, for which both were nonpathogenic and did not exhibit any clinical signs or mortality. But viral infection was established in six week old chicks by virus isolation till the 14th day of infection from the cloacal swabs for both the isolates. The sera collected from these birds revealed an antibody titre of 16 for T¬18 and 8 for T22 indicating the infection. The antigenic relationship of the isolates was examined with NDV, EDS-76 and fish viruses (FV and F6), of which T22 did not show any antigenic similarity with any of the viruses. But T1B on the contrary exhibited antigenic relationship with the fishviruses FV and F6 but no antigenic similarity with NDV and EDS-76. The antigenic similarity exhibited by T18 with the fish viruses leads to the conclusion that waterfowls may be disseminating the viruses responsible for the outbreak of epizootic ulcerative syndrome in fishes. The morphological features of T18 by electronmicroscopy revealed an enveloped virus with a size of 150-185 nm with pleomorphic forms and peplomers of a length of 18-20 nm. Except the length of the peplomers it similated a paramyxovirus. The morphology of T22 was not studied due to technical defects.
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    ThesisItemOpen Access
    Production and application of monoclonal antibodies against duck plague virus
    (Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1999) Ravindra Dattatraya, Padalkar; KAU; Jayaprakasan, V
    Monoclonal antibodies (Mabs) were raised against the vaccine strain of DPV and three strains of DPV Viz, Vaccine (DPV-V), IVRI (DPV-I) and Alleppy strain (DPV-A) were used to raise polyclonal serum in the present investigation. DPV-V was revived in 11 day old chicken embryo and the embryo death was recorded Tour to five days PI- with congestion all over the body and spleen and necrotic foci in liver. The cytopalhy in CEF cell culture observed was rounding and clumping of the cells, syncylium formation and bridge formation with extensive vacuolation in the Cytoplasm. The detachment of the cells was observed at 120 h PI. DPV-I a virulent strain was inoculated in the ducklings, death was recorded in all the inoculated birds with extensive hemorrhages on serous membranes, muscles and visceral organs. Necrotic foci on liver,' enlargement and congestion of liver, and spleen, and white hecrotic foci in the gizzard were evident. The virus was further passaged in DDE and cultivated in bulk in DEF cell culture. The DPV-V and DPV-A were titrated in CEF cell culture and the TC1D J0 was 4.7 X 105 per ml of the inoculum Tor DPV-V and 3.2 X 10"1 for DPV-A. DPV-I cultivated in DEF cell culture had a TCID50 of 1X10 3'per ml of the inoculum All (lie strains were partially purified at 100000 g for 4.5 h at 4" C in Beckman ultra centrifuge and the protein concentration oF the virus was estimated by biuret method and was found to be 11 mg, 8 mg and 7 mg for DPV-V, A and 1 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 ELISA litres more than 1:12800, one mouse showed a titre of 1:6400. The mice inoculated with DPV-A showed a titre of more than 1,12800 and those inoculated with DPV-I, 1:6400 ELISA ’Was1’ used to test the sera samples of the mice inoculated with DPV strains. The test was found to be highly sensitive, easy to perform and less time consuming. The test therefore can be recommended for routine diagnosis of DPV
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    ThesisItemOpen Access
    Secretory immunoglobulins of the duck (Anas platyrrhynchos domesticus)
    (Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1996) Hari, Kumar A V; James, P C
    The profile and functional properties of the immunoglobulins of serum, bile, mucos of trachea and intestine of ducks were studied. The immunoglobulins were separated by salting out using ammonium sulphate. The various fractions of immunoglobulines were further resolved by Sephadex G-200 gel filteration which gave two peaks for serum and tracheal immunoglobulins and a single peak each for bile and intestinal immunoglobulins. The purity of these fractions were checked by immunodiffusion and immunoelectrophoresis. The different fractions obtained were quantified by single radial immunodiffusion. The level of fraction 1 in the bile ranged between 1718 µg/ml and 1959 µg/ml and that of serum, 1718.06 µg/ml to 2442 µg/ml, in the S.typhimurium treated groups. The level of fraction 1 in the NDV treated groups ranged from 1115 µg/ml to 1597.35 µg/ml in bile, and 1597.35 µg/ml to 1959.34 µg/ml in serum. The level of fraction 2 in serum ranged from 1797 µg/ml to 2591 µg/ml in S.typhimurium treated group and 1400 µg/ml to 1797 µg/ml in the NDV treated group. Fraction 2 was not detectable in bile. The antibody response of ducks to a bacterial and viral antigen (anaculture of S.typhimurium and R2B strain of New Castle Disease virus respectively) was assessed. On conducting standard tube agglutination test, the serum, bile and oviduct washings revealed antibody tires against S.typhimurium in inoculated birds ranging between 1:20 and 1:160 in the case of serum; 1:10 and 1:80 in the case of bile and tire less than 1:10 for oviduct washings. No antibody titre could be detected for tracheal and intestinal washings and testicular extracts. The HI titre ranging from 1:32 to 1:128 could be observed for serum, 1:32 to 1:64 for bile and a titre of 1:16 was observed for oviduct washings of ducks parenterally administered with NDV. The tire was relatively low for serum when NDV was administered intranasaly. Intestinal and tracheal washings and testicular extract failed to reveal any HI antibodies.
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    ThesisItemOpen Access
    Comparative efficacy of different antigenic preparations from Pasteurella multocida for detection of antibodies by enzyme immuno assay
    (Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1997) Rinita Sing; KAU; Jayaprakasan, V
    In this study IHA, plate ELISA and DIA were employed to monitor antibody from ducks vaccinated with three different types of vaccine ( bacterin, bacterin with adjuvant and sonicate adjuvanated vaccine) prepared from P. multocida. Three different type of antigens viz., Crude capsular extract (CCE), Potassium thiocyanate extract ( KSCN) and sonicated antigen were used to coat NCM/ microtitre plate or for sensitization of SRBC. Forty ducklings of four week age were used for immunization. They were divided into four groups each group comprising of ten ducklings. Groups one, two and three were vaccinated with bacterin, bacterin with adjuvant and sonicate adjuvanated vaccine, respectively. Antibody was monitored upto 35th day post vaccination by IHA, Plate ELISA and DIA, employing CCE, KSCN and sonicated antigen. Group one ( vaccinated with bacterin) gave a higher mean titre value followed by IHA and plate ELISA, irrespective of the type of antigen employed, followed by group three ( birds vaccinated with sonicate adjuvanated vaccine) and group two ( birds vaccinated with bacterin with adjuvant ). Irrespective of the antigens employed in the tests, plate ELISA gave the highest sensitivity ( cent per cent) followed by IHA and DIA, whereas the highest specificity was observed by DIA, over IHA and plate ELISA. When the comparison was made between antigens a high mean titre value was obtained with sonicated antigen, followed by KSCN extract and CCE. Crude capsular extract antigen gave cent per cent specificity by all the tests, while the other two antigens gave a low percentage of specificity. As the immunological test with highest specificity is the one preferred, CCE was shown to be superior over KSCN extract and sonicated antigen in IHA, plate ELISA and DIA for the detection of specific antibodies against P. multocida in ducks.
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    ThesisItemOpen Access
    Cerytain plasmid-mediated characters of staphylococci isolated from bovine mastits
    (Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1996) Anil Kumar, M; KAU; Punnose, K T
    Twenty – six staphylococci were isolated from 70 cases of clinical / sub – clinical bovine mastitis. They were characterized by various biochemical tests and biotyped using coagulase production, haemolysin production, pigment production, Tween – 80 hydrolysis and casein hydrolysis. These 26 isolates comprised 19 biotypes of which, most preponderating one was biotype – B comprising of 5 isolates. The reliability of biotyping in distinguishing the isolates was found to be 73.08 per cent. The antibiogram study revealed that chloramphenicol and vancomycin were cent per cent effective. Cloxacillin, nitrofurantoin, pefloxacin and polymixin – B were also effective. Ampicillin and nalidixic acid were found to be least effective. The reliability of this method was found to be 96.15 per cent. Resistogram study revealed that maximum degree of resistance was noticed against barium chloride and potassium permanganate. All the isolates were found to be sensitive to antimony trichloride, cetrimide, copper sulphate, ferrous sulphate, iodine and potassium tellurite. The reliabiling of resistogram study was found to be 76.92 per cent. Production of haemolysin and resistance to antibiotics were found to be plasmid – mediated. Correlation between resistances to certain antibiotics and metal salts/chemical agents was also found. The plasmid profiling revealed only 16 isolates carrying plasmids. No plasmid was found common to in all the isolates. The maximum number of plasmids in an isolate was five, and this isolate carried both the largest and smallest plasmids. The reliability of plasmid profiling was 61.54 per cent. Conjugation studies revealed transfer of ampicillin, penicillin, streptomycin, erythromycin and gentamicin resistances and beta- haemolysin production to the recipient. But alpha – haemolysin was not transferred. Protoplast fusion studies revealed the expression of only ampicillin, pencillin and streptomycin resistance and alpha – haemolysin production, by the biparental strains. Determination of Numerical Index of Discrimination indicated that antibiogram typing and all its combinations were having the maximum ‘D’ value of one. Plasmid profiling was having the least value, i.e. 0.862. So it is suggested that antibiogram typing and its combinations can be used in differentiating and identifying the staphylococci causing bovine mastitis, more accurately.