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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 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.ThesisItem Open Access Characterization of plasmids of Escherichia coli isolated from mastitis(Department of Veterinary Microbiology, College of Veterinary and Animal Science, Mannuthy, 1993) Avinash Ganpatrao, Karpe; KAU; Punnoose, K TEscherichia coli. were isolated in 15.33 per cent cases of mastitis. Of the 46 E. coli isolated 43 were resistant to one to nine antibiotics and three were sensitive to all the 13 antibiotics tested. The organisms were resistant to rifampicin (78.26%) followed by oxytetracycline (50%), tetracycline (37.78%), nalidixic acid (19.56%), co-trimoxasole (8.69%) and gentamicin (6.52%). All the organisms were susceptible to kamamycin and norfloxacin. Among the .multiple drug resistance oxytetracycline - rifampicin (OR) resistance was noticed in 76.2% cases. Twenty-six different patterns of antibiotic resistance were noticed among 43 E. coli isolates giving a reliability of 60.46 per cent in differentiating the isolates. Hence, antibiogram could only be used as an adjunct to plasmid profiling in epidemiological studies. The resistograms revealed cent per cent resistance to lead, followed by antimony (32.6%), copper (30.43%), silver(19.56%) and cetrimide (2.17%). All the isolates were sensitive to cadmium and mercury. Among the 46 E. coli isolates, 9 different resistogram patterns were obtained giving reliability of 19.56 per cent in differentiating the strains. A correlation between the antibiotics and heavy metal ac; lead an+-imcny and copper, was observed inresistance such as leaa, descending order. of the forty-six E. coli isolates three (6.52%) were hemolytic on sheep blood agar. Two of the three hemolytic strains were also enterotoxigenic. Thirteen of the 46 (28.26%) E. coli isolates were enterotoxigenic, when tested by rabbit ligated ileal loop assay. Two of the thirteen (15.38%) enterotoxigenic isolates were also hemolytic. Fourteen of the 24 (58.33%) drug resistant E. coli transferred drug resistance against one or more antibiotics to the recipient organism. In none of the cases the furazolidone resistance was transferred. All the three hemolytic E. coli isolates transferred the hemolytic character by conjugation indicating the plasmid borne nature of hemolysin production. None of the enterotoxin producing E. coli could transfer the character to recipient by conjugation.ThesisItem Open Access Characterisation of Pasteurella multocxda isolates from rabbits(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1992) Sheela Yohannan; KAU; Jayaprakasan, VOne hundred and twelve rabbits were examined during this study which included 76 apparently healthy and 36 ailing/ dead rabbits. The live animals comprised 20 young and 56 adult animals, of which, 32 were of New Zealand White, 16 Grey Giant, seven Soviet Chinchilla" and 21 cross bred. This included both healthy and sick animals. Of the 36 ailxny/dead rabbits, 26 of them were of Nev/ Zealand White, six of Soviet Chinchilla and four of Grey Giant. This included 20 young and 16 adult animals. Pasteurella multocida could be isolated from five adult New Zealand White and one adult Grey Giant which died of respiratory infection. Gross pathological lesions observed in post mortem examination were typical haemorrhages in the trachea, haemorrhages and abscessation in lung and necrotic foci in liver. All the six isolates were gram negative coccobacilli, non motile and produced catalase. Two isolates were oxidase negative and four oxidase positive, grew anaerobically, utilised glucose fermentatively and none produced hemolysis of sheep red blood cells. The isolates were positive for nitrate, indole and potassium cyanide except for one isolate which was nitrate negative and two were indole negative. All were negative for hydrogen sulphide production, urease and gelatin hydrolysis. Only one isolate was positive for growth on ONPG. Majority of the sugars were fermented by these isolates. These isolates were tested for their pathogenicity in mice and rabbits. Intra peritoneal injection of one millig litre of an overnight culture containing 10 bacteria/ml, killed mice between 24-72 h post inoculation and the organism could be re-isolated from the dead animals. When rabbits were intra-nasally inoculated, with 0.5 ml of overnight culture containing 3.2 x 10^ bacteria, none of the isolates could establish clinical infection. Though the inoculated rabbits were apparently normal, one isolate colonised within the nares of the rabbit and was shedder for a period of seven days, while the other rabbits inoculated with the remaining five isolates were shedders only upto 48 h.ThesisItem Open 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, VIn 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.ThesisItem Open 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 TTwenty – 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.ThesisItem Open Access Structural analysis of Infectious bursal disease virus isolates from clinical cases in vaccinated and unvaccinated birds(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1995) Vengadabady, N; KAU; Sulochana, SField cases with history suggestive of infectious bursal disease (IBD) were screened for confirmation by Agar gel diffusion test (AGDT), using reference antigen and antisera received from Madras Veterinary College. From the positive cases, 4 isolates, two each from unvaccinated (PKD, EKM) and vaccinated (THR, KAN) flocks and an avirulent vaccine strain (VAC) were used for structural analyses and antigenic relationship studies. The percentage of mortality of the embryos infected by these strains ranged between 50 – 100 per cent, during the third and fifth day of inoculation, in the fourth passage. The lesions produced were cutaneous haemorrhages all over the body, congestion and thickening of CAM. Enlarged bursa and typical yellowish green discolouration of liver with brown patches were also noticed. All the five isolates were propagated in chicken embryo fibroblast culture, in which cytopathic changes characterised by rounding and subsequent detachment was seen from the third passage onwards. The chicks infected with field isolates revealed mildclinical symptoms. The lesions noticed after sacrificing them on the third day were moderately swollen gelatinous bursa with slight haemorrhage in some of them. Chicks that received vaccine strain revealed only mild lesion. The viral strains by SDS – PAGE revealed that all the field isolates contained nine identical polypeptides with molecular weights of 86 KD (VP2), 77 KD (VP4), 73 KD (VP5), 62 KD (VP6), 52 KD (VP7), 47 KD (VP8), 39 KD (VP10), 36 KD (VP11) and 32 KD (VP12). The vaccine strain resolved 11 peptides of whichthree, namely VP1 (93 KD), VP3 (80 KD) and VP9 (43 KD), were absent in the field isolates, but it lacked VP7 (52 KD). Mild difference in the molecular weights of VP6 and VP12 were also noticed between the field isolates and vaccine strain. Nucleic acid analyses in agarose gel showed two bands for all the five isolates without any difference in their migration pattern. Antigenic relationship of the IBDV isolates was studied by AGDT, CIE and IE. All the four isolates produced only one precipitation line against the antiserum and this precipitation line was identical to the one produced by the vaccine strain. From the observations made, the possible reasons for breakdown of immunity and a schedule of vaccination to overcome this situation have been discussed.
