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ThesisItem Open Access Role of parrots in the epizootiology of newcastle disease(Department of Microbiology, College of Veterinary and Animal Sciences,Mannuthy, 1981) Vijayan, V; Sulochana, SThe incidence, susceptibility, mode of infection and duration of excretion of the new cattle disease virus in the common Indian parrots (psittacula Krameri) were studied in detail. The blood, cloacal and throat swabs of parrots, collected from different parts of the State were screened for the presence of ND antibodies and virus. Seventeen out of 103 blood samples were found to possess HI antibodies. The serum samples which gave positive HI titres belonged to parrots kept as pets in households and allowed to mingle freely with domestic poultry and those housed in Trivandrum Zoo along with pigeons. None of the 70 cloacal and 42 throat swabs were positive for the virus. Experimental infection of parrots with undiluted virulent virus by the intranasal and intraocular routes and by the subcutaneous and intranasal routes with 1:100 dilution of the same virus gave almost the same results. All of them died within a weeks’ time, after showing symptoms of inappettance, leg and wing paralysis and diarrhoea, from day two of infection. Virus could be isolated from the throat and cloacal swabs and also from the tissue of dead birds. Chicks kept along with these infected parrots did not develop symptoms of ND, eventhough they excreted the virus, for a few days, and had a low titre of HI antibodies in their sera. All the contact chicks died of ND with typical symptoms and lesions on challenge with the virulent virus. The parrots that received a mesogenic strain (Komorov) of the virus, also succumbed to the disease, but with less pronounced symptoms and with an extended incubation period. The parrots that were infected with lentogenic strain of the virus (F1) did not develop symptoms of the disease. However multiplication of the virus occurred in these birds as isolations could be made from cloacal and throat swab and a slight increase in H1 titre was noticed sera. However on challenge with a virulent strain of NDV, they showed symptoms of ND. All of them died within eight days and the virus could be isolated from them. Contact infection of parrots from infected chicks were quite effective, as the parrots died with the same symptoms described above, almost within the same time as direct infection. Virus was also isolated from the tissues of the dead parrots. The common Indian parrots were found to be highly susceptible to both velogenic and mesogenic strains of NDV, but they were resistant to the lentogenic strain. Uninfected chicks kept along with the parrots infected with virulent virus picked up the infection, and virus could be isolated from the cloacal and throat swabs of these chicks. They also showed an increase in the antibody titre. The failure to produce clinical disease in chicken might be attributed to a decrease in virulence of the virus on passage in parrots. The carrier state with the lentogenis strain of the virus could not be assessed as they were challenged after 2 weeks. Though a carrier role had been attributed to the parakeets imported from India, the parrots in this study were found to succumb to the disease within a week. This might be due to the variation in the susceptibility of various species of parrots to NDV. The chances of dissemination of the virus could be prevented by quarantining them for a period, not less than ten days.ThesisItem Open 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, GIn 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.ThesisItem Open Access Antigens of pasteurella multocida isolates from rabbit and their immunologencity(Department of microbiology, College of veterinary and animal sciences, Mannuthy, 1994) Manoharan S; KAU; Jayaprakasan, VTwo rabbit strains viz. R9 S and R23 S and a bovine vaccine strain P – 52 which were maintained in virulent form, were used for the preparation of three forms of antigen viz., heat inactivated crude extract, KSCN extract and sonicated antigen. These antigens were chemically analysed for protein and carbohydrate contents and were found to be higher in the sonicated antigen preparation irrespective of the source. In SDS – PAGE analysis, the profiles discerned by heat inactivated crude extract, KSCN extract and sonicated antigens were four, five and six protein bands with molecular weights lesser than 68 kDa while the KSCN extract and sonicated antigen presented an additional protein band with molecular weight higher than 68 kDa. Three types of antigen of P. multocida were characterized and analysed for the inter relationship and the immunogenic potential in mice. Antiserum was raised against each antigenic preparation from the three strains in rabbits and used for serological study. In AGPT and immunoelectrophoresis the serum developed multiple precipitin lines and arcs respectively when reacted against the three homologous and two heterologous antigens in which a few were identical to the heterologous antigens. The results revealed stronger serological relationship between the two rabbit strains than with the cattle strain and the heterogeneity of the sonicated antigen. The antibody titre in each antiserum was measured by IHA using the sensitized GA – SRBC/T – GA – SRBC and the titres were more in the homologous antiserum and high titre for the heterologous serum was seen with the sonicated antigen. The LD50 determined for the three strains R9 S, R23 S and P- 52 was found to be 3 x 104 , 3 x 103 and 3 x 105 bacteria. Immunogenic potential of the three antigens and an adjuvanted sonicated antigen were tested in mice by giving two doses of vaccine at 14 days interval and challenging on 21st day with homologous and heterologous strains. A higher percentage of protection was conferred by homologous strains and it was cent per cent (100%) with sonicated antigen. The percentage of protection against challenge with heterologous strains was low. An elaborated study on immunity trials with these immunogens is needed before recommending the R23 S as a candidate vaccine strain.ThesisItem Open Access Cellular and humoran immune responses to corynebacterium presudotuberculosis infection in goats(Department of microbiology, College of veterinary and animal sciences, Mannuthy, 1986) Jayaprakasan, V; KAU; Sulochana, SCaseous lymphadenitis was experimentally produced in cross-bred malabari goats aged 8 to 12 months by bilateral inoculation of 1 x 106 viable C. pseudotuberculosis (ATCC 19410) through intradermal, subcutaneous and submucosal routes. The clinical picture, immune response and pathological features were studied up to a period of 13 weeks. The development of immune response in experimentally infected goats was assessed, by comparing the data with those of the controls, with respect to total serum protein, serum protein fractions, antibody activity of the serum, leukocyte count, counts of lymphocyte subpopulations, leukocyte migration inhibition index and skin hypersensitivity reaction. Pathological features in the lymphnodes and other tissues of infected goats necropsied at 15 days interval were also studied. Initial febrile reaction which lasted for 72 to 96 h, local inflammatory changes caused at the site of inoculations during the first two to three weeks of infection and the development of lesions typical of caseous lymphadenitis in local/regional lymphnodes within 21 days post-inoculation were the main features of clinical manifestations of the disease. aS a result of infection, neutrophilic leukocytosis was maximum during the 2nd week of infection. No appreciable was maximum during the 2nd week of infection. No appreciable change in counts of other cells in terms of their absolute numbers was noted during the entire period of observation. The humoral immune response in infected goats was indicated by the rise in serum protein, antitoxic antibody and B-lymphocyte count in the peripheral blood. The serum protein concentration increased to significant levels from the 5th week onwards and it reached the peak value (11.346 g%) by the 8th week. From the 3rd week onwards haemolysis inhibition test, which detected goats and persisted till the end of the observation period. The peak antibody level was recorded by the 5th week of infection and thereafter there was gradual reduction in the titre. Significantly high percentage of B- lymphocyte was recorded in infected animals from the 2nd to 10th week, except at the 5th week. The percentage of B-cells in infected goats ranged between 12.3 + 0.85 – 17.63 + 1.2 while it was 8.56 + 0.75-12.3 + 1.09 in control goats. This was considered as an indication of stimulation of humoral immune response. The cell-mediated immune response was evidenced by the increased T- lymphocyte count in the peripheral blood, inhibition of leukocyte migration and the development of delayed skin hypersensitivity. The mean percentage of T-lymphocytes in the peripheral blood of infected goats by E-rosette assay recorded an initial reduction at the first week (18.44 + 1.4) followed by an increase which was significant during the 5th, 6th, 7th, 8th, 12th and 13th week of infection. The maximum value was recorded (35.24 + 1.58) at the 13th week. In the case of control goats the percentage values ranged from 24.55 + 3.66 to 26.74 + 1.34. The T-lymphocyte count in the peripheral blood enumerated the ANAE method did not show any significant change even after infection. In the experimentally infected goats, leukocyte migration inhibition index was less than 0.8 during post-infection period while the control goats had the index value above 0.8. Significant reduction in the migration index was noted by 45th day of infection and the maximum reduction was on the 60th day. Intradermal injection of the toxic supernatant of the culture elicited characteristic delayed skin hypersensitivity reaction in all the experimentally infected goats while there was no reaction in the controls. The positive reaction was found to be maximum by the 48th hour post-injection. The pathological changes were characterized by an initial stimulatory hyperplastic reaction in the lymphnodes and this was accompanied by necrobiotic changes typical of caseous lymphadenitis. The hyperplastic stimulatory reactions were characterized by the presence of several active follicles with well developed germinal centres in the cortex, distinct medullary cords densely lined with plasma cells and sinus histiocytosis indicating the early elicitation of humoral immune response to the bacterium or to its in vivo products. The results obtained from the present study revealed the operation of both cell-mediated and humoral immune responses in goats against C. pseudotuberculosis infection. Of the various methods employed to monitor the immune response, leukocyte migration inhibition and delayed skin hypersensitivity tests were found to be of value in assessing the cell-mediated immune response and haemolysis inhibition test for humoral immune response. Leukocyte migration inhibition test and haemolysis inhibition test could be employed for early diagnosis of C. pseudotuberculosis infection in goats. FINDINGS : The immune responses and pathological features in Corynebacterium pseudotuberculosis infection were studied by experimental infection of cross- bred Malabari goats of S-12 months of age. Single cell bacterial suspension in chilled sodium chloride bile salt solution was used for this purpose. Goats were inoculated at both sides of the body by three routes viz., intradermal, subcutaneous and submucosal, with 2 x 106 bacteria per site of injection. The experimentally infected and control goats were observed for clinical manifestations of caseous lymphadenitis for a period of 13 weeks. The development of immune response in experimentally infected goats was assessed by comparing the data with those of the controls with respect to total serum protein, serum protein fractions, antibody activity of the serum, leukocyte counts, counts of lymphocyte sub-populations, leukocyte migration inhibition index and skin hypersensitivity reaction. Gross and histopathological changes in the lymphnodes and other tissues of necropsied goats were studied at 15 days interval for a period of 90 days. All experimentally infected goats exhibited rise in temperature, general weakness, lethargy and impaired appetite which lasted for 72 to 96 h. The sites of inoculations showed varying degree of inflammatory reaction during the first two to three weeks of infection. All experimentally inoculated goats except one developed lesions typical of caseous lymphadenitis in regional/local lymphnodes within 21 days post-inoculation. Route of infection did not influence the ability to set up lesions in lymphnodes. Although massive dose of bacterial (1.2 x 107) was administered, none of the goats had fatal infection indicating that goats are relatively resistant to this infection. Majority of goats did not develop generalized form of caseous lymphadenitis as there was no lesions in visceral/deep seated lymphnodes or organs. The normal serum protein concentration of cross-bred Malabari goats was estimated to range from 7.187 to 9.750 g %. Consequent to experimental infection, serum protein concentration was increased and recorded significant rise from the 5th week onwards reaching the peak value by the 8th week – 11.346 g%. Estimation of quantitative distribution of serum protein fractions was done by agar gel electrophoresis and densitometer tracing of electrophoretogram. Though there was initial increase in globulin content in infected animal followed by a decrease, no significant alteration in the albumin-globulin ration (A:G ratio) was noted compared to the control group. C. psuedotuberculosis was cultivated in lemco proteose broth containing sheep serum and incubated aerobically at 370C for 72 h. Supernatant obtained from the above culture, having maximum haemolysin titre and dermonecrotoxicity was used as the toxin of the bacterium in the present studies. The haemolysin content of the culture supernatant was estimated by the haemolysis test using sheep red cells. A maximum titre of 1:256 was found in the culture aged 72 h. The dermonecrotoxicity of the toxic culture supernatant was tested by intradermal inoculation into the rabbit skin. The inflammatory and necrotic reactions were maximum by 48 h post-injection. Specific antibody activity against toxin of C. pseudotuberculosis in the serum was monitored by haemolysis inhibition test and the test was adjudged as a useful test for detecting humoral immune response to C. pseudotuberculosis infection in goats. In infected goat from the 3rd week of infection onwards MIT was positive while it was negative in control goats during the period of 13 weeks of observation. The peak antibody level was achieved by the 5th week of infection and thereafter the titre was found to dwindle gradually till the 11th week. Towards the end of the observation period (12th week) there was a marginal increase in the antibody titre, which would be considered as secondary immune response against the toxin of the multiplying bacteria. Infected goats showed leukocytosis during the entire period of observation and maximum leukocytosis was observed during the 2nd week of infection. The periodical fluctuation in leukocytosis indicated the recurrent flare up of bacterial invasion in the body. The absolute lymphocyte count obtained both at pre and post-infection periods with experimentally infected goats did not show any change which indicated no deleterious effect on peripheral blood lymphocytes. Throughout the period of observation infected animals showed numerically low lymphocyte percentage in differential counts and with several samples the percentage distribution was significantly low. Absolute counts of neutrophils were consistently high in experimental goats when compared to those of controls and the same was reflected in differential count also. The other blood cells were absolutely without any change in infected as well as control goats. Density gradient centrifugation using Ficoll-paque (1.077 g/ml, centrifuged at 720 x g) was found quite useful for separation of mononuclear leukocytes from the whole blood of goats. Such separated mononuclear cells were found to contain on an average 91.80% lymphocytes and 8.2% monocytes with an average viability of 91.2%. Peripheral blood B-lymphocytes of goats were successfully enumerated by EAC rosette assay employing bovine red cells. The normal percentage of B-cells was estimated to range 8.56 and 12.3. Significantly high percentage of B-cells was recorded in infected animal from the 2nd to 10th week post- infection except at the 5th week. B-cell percentage in infected goats ranged between 12.3 + 0.85-17.63 + 1.2 while it was 8.56 + 0.75 to 12.3 + 1.09 in control goats indicating the operation of humoral immune response to concurrently boost the specific antibody activity in the serum. T-lymphocytes of goats were identified and enumerated by E-rosette assay and ANAE activity. Goat lymphocytes presented several receptors to sheep red cells, as majority of rosettes presented erythrocytes at the entire periphery of lymphocytes. The mean percentage of E-rosette positive lymphocytes in the peripheral blood of control goats ranged from 24.55 + 3.66 to 26.74 + 1.34 during 13 weeks of observation. The E-rosette technique employed in the present study was assumed unaffected by unknown variables as the data recorded in the control goats remained near normal throughout the observation period. During the first two weeks of infection E-rosette positive lymphocyte count was found numerically decreased and the reduction was significant at the first week (18.44 + 1.40). From the third week onwards an increase in the E-rosette forming cells was observed and significant increase was noted during the 5th, 6th, 7th, 8th, 12th and 13th week of infection, the maximum being at the 13th week (35.24 + 1.58). T-cells were also identified and enumerated based on the demonstration on ANAE activity. Fixing of mononuclear cells in acetone-citric acid solution enabled the fixed smears to be stored in dry state without any interference to the enzymic activity for longer periods. T-lymphocytes presented one or two localized red coloured reaction product in the cytoplasm adjacent to the cell membrane. Mean percentage of ANAE positive cells in experimental goats was 28.09 + 1.51 while it was 35.80 + 4.86 for control goats when estimated before the start of the experiment. During infection, the count of ANAE positive cells in the peripheral blood did not show any change as the mean percentage ranged between 28.9 + 2.06-33.78 + 1.99 as against the corresponding values (30.83 + 3.5-36.91 + 3.61) in controls. In infected animals significant hike in E-rosette positive lymphocyte counts was recorded while such a change could not be observed with ANAE positive lymphocytes. Thus the results of T-cell estimation by E-rosette assay and ANAE demonstration indicated that estimation of total rosette forming cells could reflect better the T-cell competence. Cell mediated immune response to C. pseudotuberculosis infection in goats was demonstrated by leukocyte migration inhibition test under agarose. A population density of 1.5 x 108 leukocytes/ml was found suitable for LMIT. Toxic culture supernatant having haemolysin titre 1:16 whose pH adjusted to 7.2 could be successfully used as antigen in the test. In experimentally infected goats leukocyte migration index was less than 0.8 during post-infection period while with control goats it was above 0.8. Significant reduction in LMI index was noted by 45th day of infection through 75 days showing maximum reduction by the 60th day. Intradermal injection of toxic culture supernatant elicited characteristic delayed type skin hypersensitivity reaction in all experimentally infected goats, while a negative reaction in controls. Skin hypersensitivity reaction was found to be maximum by 48 h post-injection. Histopathology of skin biopsy taken from the site of inoculation revealed infiltration of lymphocytes, and macrophages at perifollicular and periglandular areas, congestion of blood vessels with perivascular infiltration of lymphocytes and macrophages and dermal oedema. Tuberculin failed to produce a positive skin hypersensitive reaction in C. pseudotuberculosis infected or control goats. From 15th day onwards, experimentally infected goats which were necropsied presented gross lesions typical of caseous lymphadenitis in lymphnodes. The lesions were found to confine to superficial lymphnodes adjacent to the site of inoculations. The histological changes observed in lymphnodes were basically of two types: hyper-plastic stimulatory reaction and degenerative changes. The changes were hyper-plastic reactive follicles with well distinguished germinal centre, accumulation of lymphocytes and varying degrees of sinus histiocytosis in medullary region, dense lining of medullary cords with plasma cells, depletion of lymphocytes from the cortical area; subcapsular and cortical oedema, congestion of blood vessels, haemorrahage, infiltration of mononuclear cells in lymphatics and blood vessels, accumulation of macrophages and plasma cels in the medulla, dilatation of sinusoids, fibrous tissue proliferation, degenerative and necrotic changes of lymphocytes in the cortex and medulla, fibrous tissue encapsulated focal areas of caseation of calcification surrounded by lymphocytes, macrophages and giant cells and finally conversion of parenchyma to a caseated mass enclosed in fibrous tissue capsule. In brief, the results obtained from the present study revealed the operation of both cell-mediated and humoral immune responses in goats against C. pseudotuberculosis infection. Of the various methods employed to monitor the immune response, leukocyte migration inhibition and delayed skin hypersensitivity tests were suitable for ascertaining the cell-mediated immune response and haemolysis inhibition test for humoral immune response. Leukocyte migration inhibition test and haemolysis inhibition test can be successfully employed for the early diagnosis of C. pseudotuberculosis infection in goats.ThesisItem Open Access Bacteria associated with respiratory infections in poultry(Department of Veterinery Microbiology, College of Veterinary and Animal Science, Mannuthy, 2007) Jesto, George; KAU; Krishnan Nair, GThis 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 11 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. Amoxycillin 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. coli 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 Characterization of staphylococci isolated from cases of mastitis and study of their R plasmids(Department of microbiology, College of veterinary and animal sciences, Mannuthy, 1986) Mathew, E S; KAU; Punnoose, K TThe emergence of drug resistant staphylococci causing mastitis deserve serious investigation. The work was intended to characterize staphylococci isolated from cases of bovine mastitis and to study their R plasmid transfer in vitro. The characterization was done using glucose and mannitol fermentation, catalase production, coagulase production, Iysostaphin sensitivity and bacteriophage typing. The antibiogram of the isolates was done by agar diffusion method using 14 chemotherapeutic agents (amoxicillin, ampicillin, bacitracin, chloramphenicol, cloxacillin, erythromycin, gentamicin, methicillin, neomycin, nitrofurantoin, penicillin, streptomycin, sulphamethoxazole and tetracycline) and by agar dilution method using eight antibiotics (ampicillin, chloramphenicol, rifampicin, streptomycin, tetracycline , gentamicin, erythromycin and penicillin) The in vitro transfer of R plasmids was tried using selected S.aureus and coagulase-negative staphylococcal isolates as donors and S.aureus RN 450RF and S.epifermidis 131S as recipients. From 360 milk samples collected from cases of bovine mastitis 17 strains of S.aureus and 35 strains of coagulase negative staphylococci were isolated. Lysostaphin sensitivity test was positive for 90.38% of the isolates, but this could not be used to differentiate between coagulase positive and negative staphylococci. The results of phage typing revealed a predominance of group III phages over the other groups and the possible role of human strains of staphylococci in producing mastitis in animals. None of the strains were resistant to mercuric chloride. From the antibiogram rifampicin , bacitracin, neomycin , methicillin, gentamicin, cloxacillin, nitrofurantoin and chloramphenicol were found to be the drugs of choice in the treatment of bovine mastitis caused by staphylococci. When S. aureus Rn 450RF was used as recipient six of the ten selected S.aureus isolates could transfer either one or more drug resistance markers and the mode of transfer was suspected to be by conjugation. All the four selected streptomycin resistant coagulase –negative staphylococcal donors were found to transfer the R plasmid DNA to the recipient., S.aureus RN 450RF, which was established to be through conjugation. The drug resistant S. aureus as well as coagulase negative staphylococcal strains failed t transfer their resistance to S.epidermidis 131S.ThesisItem Open 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, VMonoclonal 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 DPVThesisItem Open Access Prevalence of chlamydial agents in livestock in Kerala(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1988) Reji Francis; KAU; James, P CThe magnitude of the prevalence of chlamydial infections in the live stock in Kerala was assessed by screening the smears of various clinical materials after staining , isolation using chicken embryos and guinea pigs and serologically by passive haemagglutination test. The results obtained were discussed correlating to the managemental practises in the organised herds and agroclimatic conditions. A total of 71 bio sample comprising 17 bovine abortion materials , 15 bull semen, 6 seminal vesicular secretion , one synovial, fluid from a calf, 5 caprine abortion material, 15 caprine pneumonic lungs, 5 samples from perinatal mortality in kids , 4 ovine lung tissue and one conjunctival washings from a buffalo were utilised for screening the smears stained by Gimenez ,Macchiavellos, modified Ziehi-Neelsen and Giemsa's methods and for isolation purpose . On screening the stained smears, one bovine abortion material, two bull semen, one caprine abortion material and three caprine pneumonic lesions were found positive for developmental forms of chlamydiae which could be discerned intra and extracytoplasmically . The overall prevalence rate by this method was 9.9% and species wise prevalence rates were 7.3 % among cattle and 16 % among goats.. Attempts for isolation resulted in the recovery of chlamydiae from two of 17 bovine abortion materials , four of 15 bull semen and two of 15 caprine pneumonic lungs. The overall prevalence based on isolation rate was 11.3% and species wise prevalence rates were 14.6% and 8% respectively for cattle and goats. A total of 169 serum samples consisting of 92 from cattle , 67 from goats and 10 from sheep were screened serologically . Of these 21 from cattle , 13 from goats and one from sheep were found positive showing titres of 1:16 and above . The overall seroprevalence rate was found to be 20.7 % and species wise rates were 22.8%, 19.4% and 10% for cattle , goat and sheep .ThesisItem Open Access Assessment of immunity to duck plague virus (duck virus enteritis)(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1993) Diwakar Dattatrayrao, Kulkarni; KAU; James, P CDuring 1991, six outbreak clinically suspected to be duck plague (DP) with 33 per cent morbidity and 26 per cent mortality were investigated Duck plague virus was isolated from each outbreak. The isolates were able to produce the lesions and death of the duck embryos but failed to kill the chicken embryos during initial passages. One of the strains, named DP-S was partially attenuated by 10 passages in chicken .embryos following 20 passages in duck embryos. Though the attenuated strain did kill ducks, its pathogenicity index was reduced from 1.9 to 1,23. The isolate DP-S under transmission electron microscope revealed virions of herpes virus morphology. Two DP vaccines - commercial vaccine and lab-adapted vaccine having virus titres 0.74 and 3.5 log 10 ELD 50/ml respectively, were separately inoculated into four groups of ducklings respectively, two groups receiving single dose and two receiving double dose of corresponding vaccines at an interval of four weeks. Another group of ducklings was kept as control without vaccination. Three ducks in each group were challenged with 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 screened for the carrier status of DPV by examination of their rectal swabs for virus isolation. In an organized farm, 180 ducks were given commercial vaccine at one year of age and were screened for VN antibodies, LMI response and PHA titres before and eight weeks post -vaccination. Randomly selected two birds were challenged six weeks post-vaccination. The findings of the study are briefly listed as under: Six duck plague outbreaks were investigated, the virus isolated, and characterized. It was partially attenuated in duck and chicken embryos. The commercial, vaccine could elicit very poor immune response as compared to laboratory adapted vaccine. The immunity could not last long even upto eight weeks in single vaccination and 20 weeks in double vaccination.
