ISOLATION AND CHARACTERISATION OF P.MULTOCIDA ISOLATES FROM SMALL RUMINANTS AND AVIAN ORIGIN

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1 ISOLATION AND CHARACTERISATION OF P.MULTOCIDA ISOLATES FROM SMALL RUMINANTS AND AVIAN ORIGIN P. Prabhakar 1, A. Thangavelu 2, J. John Kirubaharan 3 and N. Daniel Joy Chandran 4, Dept. of Veterinary Microbiology, Madras Veterinary College, Chennai 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 548 samples were screened and twenty nine Pasteurella multocida isolates from sheep, goat, turkey, duck, emu and egret were obtained and characterized. Twenty nine isolates were confirmed 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 Pasteurella multocida, a gram-negative bacterium, is the causative agent of a wide range of disease in wild and domestic animals and in humans. It is an opportunistic pathogen as well as common commensal in the upper respiratory tract of animals. The organism causes fowl cholera in birds, haemorrhagic septicemia in cattle and buffalo, atrophic rhinitis in swine, and snuffles in rabbits (Rhoades and Rimler, 1989).Pasteurellosis is one of the most common disease of cattle, sheep and goats throughout the world. Outbreaks usually lead to high mortality and great economic loss to the ruminant industry (Links et al., 1992). P. multocida has 5 capsular serogroups (A, B, D, E, and F). Among avian strains of P.multocida, serogroup A strains cause the majority of fowl cholera cases, P.multocida in bovines is caused by serotypes B:2 and E:2 in Asia and Africa, respectively (Heddleston et al., 1972). In small ruminants, serogroup A and D are usually associated with pasteurellosis. In swine, toxigenic strains (both capsular types A and D) are most often associated with atrophic rhinitis. Pasteurellosis is serotype specific and it is necessary to monitor continuously the prevalence of various serotypes as this overall assessment can lead to development of newer and more effective vaccines to be used for prophylaxis against pasteurellosis. 1 Senior Research Fellow. Dept. of Veterinary Microbiology, Madras Veterinary College, Chennai ,3 Professor, Dept. of Veterinary Microbiology, Madras Veterinary College, Chennai Professor and Head, Dept. of Veterinary Microbiology, Madras Veterinary College, Chennai

2 Isolation MATERIALS AND METHODS A total of 548 samples were collected from dead, affected, and healthy livestock and poultry in various areas of Tamil Nadu State(Coimbatore, Chennai, Chennai-Slaughter house, Rajapalayam, Thoothukudi, Tirunelveli, Nagarkoil, Vandalur, Kancheepuram)at different time intervals for the period between as follows Cattle - 53 Buffaloes - 29 Sheep Pigeon - 6 Goat - 80 Turkey - 5 Egret - 1 Wild Birds - 10 Duck - 3 Emu - 2 Mouse bioassay The specimen were pooled flockwise of same area or village exhibiting similar symptoms and inoculated in Brain Heart Infusion(BHI) broth. After 16 hrs incubation at 37oC 0.1ml from each broth culture was injected subcutaneously into a set of Swiss Albino mice. Heart blood smears, aspirates of heart blood and impression smears of spleen, liver and lung were collected from dead mice and stained with Leishman stain. Isolation and Biochemical Characterisation Aspirated heart blood from dead mice was streaked onto 10% sheep blood agar and incubated at 37oC. The heart blood was also inoculated in BHI broth and incubated at 37oC overnight and the broth culture was streaked onto blood agar and MacConkey agar. The colonies suggestive of P.multocida were subjected to biochemical tests for identification. The biochemical tests included IMVIC, sugar fermentation test and catalase and oxidase test. Antibiotic sensitive assay Antibiotic sensitive assay was performed for the P.multocida isolates with 7 Prabhakakar et. al., antibiotics(enrofloxacin, Gentamicin, Tetracycline, Streptomycin, Ampicillin, Cephalexin and Ciprofloxacin) as per the disc diffusion method of Kirby-Bauer(Bauer et.al.,1966). Molecular Characterization and Confirmation DNA Isolation by High Salt Method DNA was extracted from the culture by high salt method as described by Senthilkumar and Ramadass (2000). Overnight 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 at 370C for 15 min in water bath. Fifty micro litre of 10% SDS and 250µl of 6 M NaCl were then added and centrifuged at 10,000 rpm for 5 min at 40C and ethanol precipitated. The pellets were resuspended in LTE(Low Tris EDTA) buffer and stored at -200C until used. P.multocida species specific PCR (PM-PCR) The species specific primers(kmtit7 and KMTISP6) designed by Townsend et.al.,(1998) were used to amplify the gene sequences in P.multocida, The PCR reaction mixture and the thermal cycle protocol were as follows. Initial denaturation at 940C for 5 min, followed by 30 cycles, each cycle consisting of 3 steps- denaturation at 950C for 1 min, annealing at 550C for 1 min, Extension at 720C for 1 min. Final Extension was carried out at 720C for 9 min. Capsular PCR typing The P.multocida capsular serogroup specific primers designed by Townsend et al. (2001) 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". 132

3 Isolation and... The thermal cycle protocol was as follows. Initial denaturation at 95 C for 5 min, followed by 30 cycles, each cycle consisting of 3 steps- denaturation at 950C for 30 sec, annealing at 490C for 30 sec, Extension at 720C for 80 sec. Final Extension was carried out at 720C for 5 min. MacConkey agar. Gram's staining of the smears revealed characteristic gram negative coccobacillary organisms The isolates were positive for indole, Nitrate reduction, oxidase and catalase. These results were same as reported by Kawamota et al., (1990)& OIE (2004). RESULTS AND DISCUSSION Pasteurellosis caused by P.multocida is an opportunistic respiratory pathogen of in tropical climate causing high morbidity and mortality. The predisposing factors include the hot tropical climate and stress induced by management practices such as docking, drenching, castration etc, as reported by Chandrasekaran et.al.,(1991) Pasteurella multocida showed variation among strains with respect to host predilection, pathogenicity, carbohydrate fermentation, colonial morphology, and antigenic specificity (Carter and Chengappa 1981). A total of 548 samples suspected for pasteurellosis were pooled area wise into groups and were subjected to biological test in mice and 29 isolates of P.multocida were obtained. All the 29 isolates were found to be lethal to mice with variation in virulence as determined by mean death time(mdt).out of the 29 isolates 23 isolates (Table-2)showed MDT between hours, 4 isolates with MDT between hours, 2 isolates with MDT between hours. The mouse bio assay findings are in agreement with the findings of Mustafa et al., (1978), Diallo et al., (1995) and Suresh Babu (2003). Heart blood smear, liver and spleen impression smears showed characteristic bipolar organisms on Leishman staining and Gram negative coccobacilli by Gram staining method in accordance with Adlam and Rutter (1989). The isolates showed typical cultural characteristics of dew drop, mucoid, non haemolytic colonies in blood agar. No growth was observed in Antibiotic sensitivity testing of bacteria has both laboratory and clinical significance. In the present study a total of 13 antibiotics (Table-1)were used. 100% sensitivity was recorded to Ciprofloxacin, 93% sensitivity to Enrofloxacin, 90% sensitivity to Gentamicin, 83% sensitivity to Tetracycline, 76% sensitivity to Streptomycin, 66% sensitivity to Spectinomycin and Cephalothin in the order of frequency. Dyer et al., (2000) reported, P.multocida isolates showed 83% sensitivity to Gentamicin and Tetracycline and less sensitivity to Spectinomycin and Erythromycin and this results correlate with our findings. Overall, the majority of Pasteurella species were susceptible to multiple antibiotics and this is most likely due to the limited exposure to antibiotics. Pasteurella multocida species specific PCR (PM - PCR) assay developed by Townsend et a.,l (1998) was used in this study to identify the subspecies of P.multocida by amplifying 460 bp DNA fragment within KMTI gene using the Primers KMTISP6 and KMTIT7. The molecular weight of the PCR products of all the isolates were found to be 460 bp(fig-1),indicating specificity for P.multocida. Capsular PCR method designed by Townsend et al (2000) was used for capsular typing(table-3). Out of 29 isolates 20 belonged 'A' serogroup, 3 to 'B' serogroup, 3 to 'D' serogroup and 3 to 'F' serogroup. These results also coincided with the serotyping results obtained from Indian Veterinary Research Institute (IVRI), U.P.(Fig-2) Ewers et al., (2006) reported that in ovine P. multocida isolates two serotypes 'A' and 'D' were detected among the isolates. Type D was found only 133

4 in diseased cases, while type A was found in diseased and healthy samples. Prevalence rate of type A was higher than type D. The results suggest that type A strain are the most common independent of disease status and this results coincides with our results. Chengmin Wang et al. (2009) reported fowl cholera in wild waterfowl in China and causative agent P. multocida capsular type 'A', when inoculated in Muskovy ducks caused disease. Therefore it is ascertained that P.multocida serotype 'A' is found to be predominant among waterfowls and associated avian species. This result coincides with our results as all our avian isolates are serotyped as capsular type 'A'. In the present study P. multocida ovine isolates revealed the presence of both capsular type 'B' and 'F' (though a small percentage)along with capsular type 'A' and 'D' So it is necessary to monitor continuously the prevalence of various serotypes as this overall assessment can lead to development of newer and more effective vaccines. 134 ACKNOWLEDGEMENTS Sincere thanks are due to ICAR, New Delhi and Dean, Madras Veterinary College, Chennai for providing necessary facilities. REFERENCES Adlam.C and Rutter.J.M, (1989), Pasteurella & Pasteurellosis. United States edition, Academic Press, Inc, San Diego: 37. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck. (1966). Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology. 45: Carter, G. R., and M. M. Chengappa. (1981). Recommendations for a standard system of designating serotypes of Pasteurella multocida. Am. Assoc.Vet. Lab. Diagn. 24: Prabhakakar et. al., Chandrasekaran.S (1991). Evaluation of combined Pasteurella vaccines in control of sheep pneumonia. British veterinary journal. 147: Chengmin Wang, Yanyun Wu, Xiaojun Xing, Guocheng Hu, Jiayin Dai, and Hongxuan. (2009), He An Outbreak of Avian Cholera in Wild Waterfowl in Ordos Wetland, Inner Mongolia, China Journal of Wildlife Diseases 45(4), Diallo, I.S., Bensink, J.C., Frost, A.J., Spradbrow, P.B., (1995). Molecular studies on avian strains of Pasteurella multocida in Australia. Veterinary. Microbiology. 46, Dyer. N.W., A.C.S. Ward, G.C. Weiser, D.G. White., (2000) Seasonal incidence and antibiotic susceptibility patterns of Pasteurellaceae isolated from American bison. The Canadian Journal of Veterinary Research.7-14 Ewers, C., Luabke-Becker. A, Bethe. A, Kieayling. S, Filter.M and Wieler. L.H., (2006). Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status. Veterinary Microbiolgy., 114: Heddleston, K. L., J. F. Gallagher and P. A. Rebers, (1972). Fowl cholera: Gel diffusion precipitation test for serotyping P. multocida from avian species. Avian Dis., 16: Kawamoto, E., T.sawada and T.Maroyama.(1990). Prevalence and characterization of Pasteurella multocida in rabbits and their environment in Japan. Journal of Veterinary Science. 52: Links I.J., Searson J.E., Godwin J., Glastonbury J.R., Philbey A.P., Mathews L.M. (1992): P. multocida and P. haemolytica infections in ruminants and pigs in Southern New South Wales. In: Patten B.E.,Spencer T.L., Johnson

5 R.B., Hoffman D., Lehane L.(eds.): Pasteurellosis in Production Animals. ACIAR Proceedings No. 43, Mustafa (1978) Carrier rate of P.multocida in a cattle herd associated with an outbreak of Haemorrhagic septicaemia in Sudan. British Veterinary Journal.,134: Office International des epizooties.(2004). Manuals of standards for diagnostic test and vaccine. 4thedition, France, 19 (2), Rhoades, K.R., Rimler, R.B., (1989). Fowl Cholera. Academic Press, London, pp Senthilkumar and Ramadass (2000). Rapid DNA isolation from leptospiral cultures using high salt method. Indian Veterinary Journal,78: Isolation and... Table - 1 Antibiotic sensitivity assay Suresh Babu, (2003) Studied on the carrier status of pasteurellosis in animals and birds M.V.Sc. thesis Submitted to TANUVAS. Townsend,K.M.,Frost.A.J.,Lee.C.W., Papadimitriou. J.M., Dawkins.H.J.S.,(1998) Development of PCR assays for Species and type specific identification of P.multocida isolates Journal of Clinical Microbiology, 36: Townsend,K.M, John D. Boyce, Jing. Y. Chung, Alan. J. Frost, and Ben Adler(2001) Genetic Organization of Pasteurella multocida cap Loci and Development of a Multiplex Capsular PCR Typing. Journal of Clinical Microbiology, 39: S. No Antibiotics Concentration No of Isolates Sensitive No of Isolates Resistance Percentage of Sensitivity 1 Amoxicillin 30 mcg Ampicillin 10 mcg Cephalothin 30 mcg Streptomycin 10 mcg Gentamicin 10 mcg Tetracycline 30 mcg Trimethoprim 10 mcg Erythromycin 15 mcg Ciprofloxacin 10 mcg Enrofloxacin 10 mcg Norfloxacin 10 mcg Cephotaxime 30 mcg Spectinomycin 100mcg

6 Prabhakakar et. al., Table - 2 Mouse bioassay S. No Mean death time No. of isolates Hours 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) Hours 4 (Sheep 3,4,17, Goat 1) Hours 2 (Sheep 21, 22) Fig-1 P.multocida species specific PCR (PM-PCR) M S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 M 460bp S1-S10 - P. multocida of sheep origin M - 100bp molecular weight marker Fig-2 Capsular PCR typing M S4 S7 S9 S1 S2 S6 S8 1044bp 850bp 760bp S1,S2,S4,S6,S7,S8,S9 - P. multocida of sheep origin M - 100bp molecular weight marker 136

7 S.No Isolation and... Table 3 P.multocida isolates Serotyped at IVRI, Izatnagar Name of host animal Capsular Serotype Area 1 Sheep 1 B 2 Sheep 2 B 3 Sheep 3 B Chennai (Slaughter house) 4 Sheep 4 A Chennai 5 Sheep 5 F 6 Sheep 6 F 7 Sheep 7 A 8 Sheep 8 F 9 Sheep 9 A 10 Sheep 10 A 11 Sheep 11 A 12 Sheep 12 A 13 Sheep 13 A Rajapalayam 14 Sheep 14 D Thoothukudi 15 Sheep 15 A 16 Sheep 16 A 17 Sheep 17 A 18 Sheep 18 A Tirunelveli 19 Sheep 19 A 20 Sheep 20 D 21 Goat 1 A Nagarkoil 22 Turkey 1 A Chennai 23 Egret 1 A Vandalur 24 Sheep 21 D 25 Sheep 22 A 26 Duck 1 A 27 Duck 2 A 28 Emu 1 A 29 Emu 2 A Mathuranthagam Chennai Chennai 137