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ISOLATION AND CHARACTERISATION OF
P.MULTOCIDA ISOLATES FROM SMALL
RUMINANTS AND AVIAN ORIGIN
P. Prabhakar1, A. Thangavelu2, J. John Kirubaharan3 and
N. Daniel Joy Chandran4,
Abstract
Pasteurella multocida is a heterogeneous species that produces septicemic or respiratory diseases in domesticated and wild animals. In the present study a total of 548samples were screened and twenty nine Pasteurella multocida isolates from sheep, goat,turkey, duck, emu and egret were obtained and characterized. Twenty nine isolates wereconfirmed as P.multocida by Pasteurella multocida Polymerase Chain Reaction (PM-PCR).
Capsular PCR typing revealed the presence of serotypes 'A','B','D',& 'F' among the isolates.
Out of 13 antibiotics used, 100% sensitivity to Ciprofloxacin, 93% sensitivity to Enrofloxacin,90% sensitivity to Gentamicin was recorded.
Key Words: Pasteurella multocida - Small Ruminants and avian origin, PM-PCR.
INTRODUCTION
(A, B, D, E, and F). Among avian strains of Pasteurella multocida, a gram-negative P.multocida, serogroup A strains cause the majority bacterium, is the causative agent of a wide range of of fowl cholera cases, P.multocida in bovines is disease in wild and domestic animals and in humans.
caused by serotypes B:2 and E:2 in Asia and Africa, It is an opportunistic pathogen as well as common respectively (Heddleston et al., 1972). In small commensal in the upper respiratory tract of animals.
ruminants, serogroup A and D are usually associated The or ganism causes fowl cholera in birds, with pasteurellosis. In swine, toxigenic strains (both haemorrhagic septicemia in cattle and buffalo, capsular types A and D) are most often associated atrophic rhinitis in swine, and snuffles in rabbits with atrophic rhinitis. Pasteurellosis is serotype (Rhoades and Rimler, 1989).Pasteurellosis is one of specific and it is necessary to monitor continuously the most common disease of cattle, sheep and goats the prevalence of various serotypes as this overall throughout the world. Outbreaks usually lead to assessment can lead to development of newer and high mortality and great economic loss to the more effective vaccines to be used for prophylaxis ruminant industry (Links et al., 1992).
1 Senior Research Fellow. Dept. of Veterinary Microbiology, Madras Veterinary College, Chennai - 600 0072,3 Professor, Dept. of Veterinary Microbiology, Madras Veterinary College, Chennai - 600 0074 Professor and Head, Dept. of Veterinary Microbiology, Madras Veterinary College, Chennai - 600 007 Tamilnadu J. Veterinary & Animal Sciences 8 (3) 131-137, May - June, 2012 MATERIALS AND METHODS
antibiotics(Enrofloxacin, Gentamicin, Tetracycline,Str eptomycin , Ampicillin, Ceph alexin an d Isolation
Ciprofloxacin) as per the disc diffusion method ofKirby-Bauer(Bauer et.al.,1966).
A total of 548 samples were collected from dead, affected, and healthy livestock and poultry in Molecular Characterization and Confirmation
various areas of Tamil Nadu State(Coimbatore,Chennai, Chennai-Slaughter house, Rajapalayam, DNA Isolation by High Salt Method
Thoothukudi, Tirunelveli, Nagarkoil, Vandalur,Kancheepuram)at different time intervals for the high salt method as described by Senthilkumar andRamadass (2000). Overn igh t cultures were centrifuged at 10,000 rpm for 20 min. The pellets were washed with PBS twice. The resulting pellets were suspended in 0.5 ml of solution I(10mM Tris HCl, 10mM KCl, 10mM MgCl2,2mM EDTA) and in 0.5ml of solution II(10mM Tris HCl, 10mM KCl,10mM MgCl2,2Mm EDTA,0.4M NaCl) and incubated Mouse bioassay
at 370C for 15 min in water bath. Fifty micro litre of 10% SDS and 250µl of 6 M NaCl were then added same area or village exhibiting similar symptoms and centrifuged at 10,000 rpm for 5 min at 40C and and inoculated in Brain Heart Infusion(BHI) broth.
ethanol precipitated. The pellets were resuspended After 16 hrs incubation at 37oC 0.1ml from each broth in LTE(Low Tris EDTA) buffer and stored at -200C culture was injected subcutaneously into a set of Swiss Albino mice. Heart blood smears, aspirates ofheart blood and impression smears of spleen, liver P.multocida species specific PCR (PM-PCR)
and lung were collected from dead mice and stainedwith Leishman stain.
Isolation and Biochemical Characterisation
KMTISP6) designed by Townsend et.al.,(1998) wereused to amplify the gene sequences in P.multocida, The PCR reaction mixture and the thermal cycle streaked onto 10% sheep blood agar and incubated protocol were as follows. Initial denaturation at at 37oC. The heart blood was also inoculated in BHI 940C for 5 min, followed by 30 cycles, each cycle broth and incubated at 37oC overnight and the broth consisting of 3 steps- denaturation at 950C for 1 cultur e was str eaked on to blood agar an d min, annealing at 550C for 1 min, Extension at 720C for 1 min. Final Extension was carried out at 720C were subjected to biochemical tests for identification.
Capsular PCR typing
The biochemical tests included IMVIC, sugarfermentation test and catalase and oxidase test.
The P.multocida capsular serogroup specific primers designed by Townsend et al. (2001) Antibiotic sensitive assay
were used for capsular PCR typing. The serogroup A specific primers hya D and hya C were used to amplify capsule biosynthetic loci of serogroup "A".
Tamilnadu J. Veterinary & Animal Sciences 8 (3) 131-137, May - June, 2012 The thermal cycle protocol was as follows. Initial MacConkey agar. Gram's staining of the smears denaturation at 95°C for 5 min, followed by 30 cycles, revealed characteristic gram negative coccobacillary each cycle consisting of 3 steps- denaturation at organisms The isolates were positive for indole, 950C for 30 sec, annealing at 490C for 30 sec, Nitrate reduction, oxidase and catalase. These Extension at 720C for 80 sec. Final Extension was results were same as reported by Kawamota et al., RESULTS AND DISCUSSION
Antibiotic sensitivity testing of bacteria has both laboratory and clinical significance. In the Pasteurellosis caused by P.multocida is an present study a total of 13 antibiotics (Table-1)were opportunistic respiratory pathogen of in tropical used. 100% sen sitivity was r ecor ded to climate causing high morbidity and mortality. The Ciprofloxacin, 93% sensitivity to Enrofloxacin, 90% predisposing factors include the hot tropical climate sensitivity to Gentamicin, 83% sensitivity to and stress induced by management practices such Tetracycline, 76% sensitivity to Streptomycin, 66% as docking, drenching, castration etc, as reported sensitivity to Spectinomycin and Cephalothin in the by Chandrasekaran et.al.,(1991) Pasteurella order of frequency. Dyer et al., (2000) reported, multocida showed variation among strains with P.multocida isolates showed 83% sensitivity to r espect to h ost pr edilection , path ogenicity, Gentamicin and Tetracycline and less sensitivity to carbohydrate fermentation, colonial morphology, Spectinomycin and Erythromycin and this results and antigenic specificity (Carter and Chengappa correlate with our findings. Overall, the majority of Pasteurella species were susceptible to multiple antibiotics and this is most likely due to the limited pasteurellosis were pooled area wise into groups and were subjected to biological test in mice and 29 Pasteurella multocida species specific isolates of P.multocida were obtained.
PCR (PM - PCR) assay developed by Townsend et All the 29 isolates were found to be lethal a.,l (1998) was used in this study to identify the to mice with variation in virulence as determined by subspecies of P.multocida by amplifying 460 bp DNA mean death time(MDT).Out of the 29 isolates 23 fragment within KMTI gene using the Primers isolates (Table-2)showed MDT between 12-18 KMTISP6 and KMTIT7. The molecular weight of hours, 4 isolates with MDT between 19-24 hours, 2 the PCR products of all the isolates were found to isolates with MDT between 24-48 hours. The mouse be 460 bp(Fig-1),indicatin g specificity for bio assay findings are in agreement with the findings of Mustafa et al., (1978), Diallo et al., (1995) and Townsend et al (2000) was used for capsular typing(Table-3). Out of 29 isolates 20 belonged 'A' impression smears showed characteristic bipolar serogroup, 3 to 'B' serogroup, 3 to 'D' serogroup organisms on Leishman staining and Gram negative and 3 to 'F' serogroup. These results also coincided coccobacilli by Gram staining method in accordance with the serotyping results obtained from Indian Veterinary Research Institute (IVRI), U.P.(Fig-2) Ewers et al., (2006) reported that in ovine characteristics of dew drop, mucoid, non haemolytic P. multocida isolates two serotypes 'A' and 'D' were colonies in blood agar. No growth was observed in detected among the isolates. Type D was found only Tamilnadu J. Veterinary & Animal Sciences 8 (3) 131-137, May - June, 2012 in diseased cases, while type A was found in diseased Chandrasekaran.S (1991). Evaluation of combined and healthy samples. Prevalence rate of type A was higher than type D. The results suggest that type A pneumonia. British veterinary journal. 147:437- strain are the most common independent of disease status and this results coincides with our results.
Chengmin Wang et al. (2009) reported fowl Guocheng Hu, Jiayin Dai, and Hongxuan.
cholera in wild waterfowl in China and causative (2009), He An Outbreak of Avian Cholera in agent P. multocida capsular type 'A', when inoculated in Muskovy ducks caused disease. Therefore it is Mongolia, China Journal of Wildlife Diseases ascertained that P.multocida serotype 'A' is found to be predominant among waterfowls and associated Diallo, I.S., Bensink, J.C., Frost, A.J., Spradbrow, avian species. This result coincides with our results P.B., (1995). Molecular studies on avian strains as all our avian isolates are serotyped as capsular of Pasteurella multocida in Austr alia.
Veterinary. Microbiology. 46,335-342.
In the present study P. multocida ovine Dyer. N.W., A.C.S. Ward, G.C. Weiser, D.G. White., isolates revealed the presence of both capsular type 'B' and 'F' (though a small percentage)along with susceptibility patterns of Pasteurellaceae capsular type 'A' and 'D' So it is necessary to monitor isolated from American bison. The Canadian continuously the prevalence of various serotypes as this overall assessment can lead to developmentof newer and more effective vaccines.
Ewers, C., Luabke-Becker. A, Bethe. A, Kieayling.
S, Filter.M and Wieler. L.H., (2006). Virulence genotype of Pasteurella multocida strainsisolated from different hosts with various Sincere thanks are due to ICAR, New Delhi disease status. Veterinary Microbiolgy., 114: and Dean, Madras Veterinary College, Chennai for Heddleston, K. L., J. F. Gallagher and P. A. Rebers, REFERENCES
(1972). Fowl cholera: Gel diffusion precipitationtest for serotyping P. multocida from avian Adlam.C and Rutter.J.M, (1989), Pasteurella & Pasteurellosis. United States edition, AcademicPress, Inc, San Diego: 37.
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Prevalence and characterization of Pasteurella Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M.
multocida in rabbits and their environment in Turck. (1966). Antibiotic susceptibility testing Japan. Journal of Veterinary Science. 52: 915- by a standardized single disk method. American Journal of Clinical Pathology. 45:493-496.
Links I.J., Searson J.E., Godwin J., Glastonbury J.R., Carter, G. R., and M. M. Chengappa. (1981).
Philbey A.P., Mathews L.M. (1992): P.
multocida and P. haemolytica infections in designating serotypes of Pasteurella multocida.
Am. Assoc.Vet. Lab. Diagn. 24:37-42.
Wales. In: Patten B.E.,Spencer T.L., Johnson Tamilnadu J. Veterinary & Animal Sciences 8 (3) 131-137, May - June, 2012 Suresh Babu, (2003) Studied on the carrier status of Pasteurellosis in Production Animals. ACIAR pasteurellosis in animals and birds M.V.Sc.
Mustafa (1978) Carrier rate of P.multocida in a cattle Townsend,K.M.,Frost.A.J.,Lee.C.W., Papadimitriou.
Haemorrhagic septicaemia in Sudan. British J.M., Dawkins.H.J.S.,(1998) Development of identification of P.multocida isolates Journal Office International des epizooties.(2004). Manuals of standards for diagnostic test and vaccine.
of Clinical Microbiology, 36:1096-1100.
4thedition, France, 19 (2), 626-637.
Townsend,K.M, John D. Boyce, Jing. Y. Chung, Alan.
Rhoades, K.R., Rimler, R.B., (1989). Fowl Cholera.
Organization of Pasteurella multocida cap Loci Senthilkumar and Ramadass (2000). Rapid DNA and Development of a Multiplex Capsular PCR isolation from leptospiral cultures using high salt Typing. Journal of Clinical Microbiology, 39: method. Indian Veterinary Journal,78:1158-1159.
Antibiotic sensitivity assay
No of Isolates
No of Isolates
Percentage
Antibiotics
Concentration
Sensitive
Resistance
of Sensitivity
Tamilnadu J. Veterinary & Animal Sciences 8 (3) 131-137, May - June, 2012 Mouse bioassay
Mean death time
No. of isolates
23(Sheep1,2,5,6,7,8,9,10,11,12,13,14,15,16,18,19,20 Duck – 1,2, Turkey –1, Emu – 1,2, Egret -1) Fig-1 P.multocida species specific PCR (PM-PCR)
M S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 M
P. multocida of sheep origin M - 100bp molecular weight marker Fig-2 Capsular PCR typing
M S4 S7 S9 S1 S2 S6 S8
Tamilnadu J. Veterinary & Animal Sciences 8 (3) 131-137, May - June, 2012 P.multocida isolates Serotyped at IVRI, Izatnagar
Name of host animal
Capsular Serotype
Tamilnadu J. Veterinary & Animal Sciences 8 (3) 131-137, May - June, 2012

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