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 orrespiratory 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, 2012MATERIALS AND METHODS
antibiotics(Enrofloxacin, Gentamicin, Tetracycline,Str eptomycin , Ampicillin, Ceph alexin an d
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
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
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(1972). Fowl cholera: Gel diffusion precipitationtest for serotyping P. multocida from avian
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Organization of Pasteurella multocida cap Loci
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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, 2012Mouse 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, 2012P.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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