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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.
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, V
Two 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.
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, G
This 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.
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, 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
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 C
During 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.
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 T
Escherichia 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.
Open Access
Characterisation of Pasteurella multocxda isolates from rabbits
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1992) Sheela Yohannan; KAU; Jayaprakasan, V
One 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.
Open Access
Development and evaluation of outer membrane protein vaccine against duck pasteurellosis
(Department of Veterinary Microbiology, College of Veterinary and Animal Science, Mannuthy, 2007) Ranjini, A R; KAU; Krishnan, Nair G
A research work was undertaken to prepare effective vaccines against P. multocida grown under different conditions and their immunopotency was assessed in one month old ducklings. The purity of the P. multocida A:l strain (DP1) was confirmed as per standard procedure. Pathogenicity of the isolate was assessed in six to eight weeks old mice. The isolate killed the mice with in 8 h intra peritoneally and within 24 h when injected subcutaneously. Whole cell protein of P. multocida (DP1) was extracted and they were subjected to discontinuous system of SDS- PAGE which revealed 20 to 26 visible protein bands of molecular weight ranging from 102 to 19 kDa. After growing P .multocida in iron sufficient and iron restricted medium its OMPs were extracted and they were analysed by SDS PAGE. In iron sufficient medium, 10 protein bands of MW which ranging from 91.84 to 19.02 kDa were revealed. In addition to the above a protein band with MW of 97.8 kDa were detected in iron restricted medium.The protein concentration was estimated by Modified Lowry method,and it was found to be 4mg/ml. The Median Lethal Dose (LDso) of P. multocida when determined in one, month old ducklings was found to be 10 -7.13 which contained 13 cells. Oil adjuvanted formalin inactivated vaccines were prepared from DPl grown under three different conditions viz., TSB, BHIB and BHIB supplemented with 100 u M 2,2' dipyridyl. Sterility, safety and potency test of the vaccines were done as per standard procedures. A total of 120 one month old ducklings were divided in to four groups with 30 birds in each group. The first three groups were vaccinated with ordinary bacterin, OMP vaccine prepared under iron sufficient condition and OMP vaccine prepared under iron restricted condition respectively and the fourth group served as control.The birds were vaccinated with 0.5millilitre of vaccine intramuscularly. The blood was collected from all the ducks on day 0,7, 14,21,28,45 and 60 day PV. Passive haemagglutination was done and the increase in antibody titre was observed from day 7 PV onwards for groups I,.Il and In. The highest antibody titre was obtained at 14th day PV and 21 st day PV for aMP vaccine prepared under iron restricted condition. All the vaccine groups had shown a significant difference from the control group at all stages of study. On homologous challenging OMP vaccine under iron restricted media on 42nd day PV afforded 60 per cent protection. On 60th day PV afforded 50 per cent protection. Though the aMP vaccine prepared under iron restricted condition was found to provide high antibody titre, the protection percentage afforded by it in comparison with ordinary bacterin and aMP vaccine prepared under iron sufficient media was low. Hence, the aMP vaccine prepared under iron restricted condition need to be subjected to further investigation.
Open Access
Evaluation of pathogenicity and antigenic relationship of salmonella isolates from poultry
(Department of Veterinery microbiology, College of Veterinary and Animal Science, Mannuthy, 2007) Sunil, G; KAU; Koshy, John (Guide)
In the present study the pathogenicity and the antigenic relationship of three local isolates of S. gallinarum was compared with that of a reference/known strain. The antigenic homogeneity/heterogeneity of these isolates was studied using SDS PAGE and AGID. All the four isolates when subjected to antibiogram revealed similar pattern. The only difference was with isolate X 4 which was susceptible to co-trimoxazole while all the other three were resistant. Except for the reference/known strain BGL, all the other three isolates were found pathogenic to mice. All the four isolates were pathogenic to day-old chicks. Isolates QS 1 and X 4 were most pathogenic. Major clinical signs were somnolence, weakness, inappetance and whitish diarrhea and were more prominent in IM inoculated group. All chicks that died during the experimental period gave positive Salmonella isolation from liver, spleen, heart blood, lungs, yolk and ceca. At the end of experimental period survived birds were sacrificed and they gave positive isolation only from ceca. All the four isolates were pathogenic to layers. The clinical signs observed were listlessness, inappetance, ruffled feathers, shrunken comb, greenish-yellow diarrhea with gradual weight loss and was more prominent in IV challenged group, followed by those inoculated by oral and IC routes. The most pathogenic strain was QS 1. Cloacal swabs examined for Salmonella revealed intermittent shedding in all groups. The highest re-isolation was obtained from liver. The egg production was affected in all test groups and was most severely affected in IV group. No significant difference was noticed between different routes of inoculation and among different isolates in re-isolation from egg. The re-isolates were confirmed as S. gallinarum by standard biochemical reactions. The gross and histopathological changes observed in experimental infection with all the isolates were the same. There was enlargement and necrosis of liver and spleen, with congestion of heart and lungs. Day-old chicks had omphalitis. Histopathologically there was congestion, necrosis and infiltration of inflammatory cells into the internal organs, including submucosa of intestine. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis analysis of the OMPs of all four isolates revealed 13 different polypeptide bands. These bands were of similar molecular weight in all the four isolates, indicating antigenic homogeneity. Agar gel immunodiffusion carried out using hyper immune serum raised against isolates BGL and QS 1 revealed lines of identity between the four isolates, indicating antigenic homogeneity by serology.

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