Abstract: Twenty five isolates of Pasteurella multocida from ducks were assayed for the presence of plasmids in seeking to determine whether any correlation exists between the presence of plasmids, pathogenicity and sensitivity to antimicrobial agents. Twenty two isolates carried plasmids. The plasmids ranged in size from 46.2 to 0.8 kbp. Two plasmids profiles were detected. No correlation was found between the presence of the plasmids and pathogenicity and resistance to antimicrobial agents. The drug of choice for the treatment of duck pasteurellosis in the geographical location is Enrofloxacin, pefloxacin or chloramphenicol.
INTRODUCTION
Plasmids are covalently closed circular DNA stably inherited in an extrachromosomal state. They harbour the genes responsible for transmissible drug resistance, colicin, haemolysin production, symbiosis and nitrogen fixation by bacteria (Stanisich, 1988).
Several workers have undertaken studies to determine the presence of plasmids in numerous strains of P. multocida isolated from several species of animals and birds and to investigate the correlation between antibiotic resistance profiles and the presence of plasmids (Berman and Hirsh, 1978; Hirsh et al., 1981; Haghour et al., 1987; Price et al., 1993; Shivshankara et al., 2000). Very little information is available about the plasmid profiles of P. multocida isolated from ducks and so the present investigation was taken up with this objective.
MATERIALS AND METHODS
Pasteurella Strains
Twenty-five isolates of P. multocida (DP1 to DP 25) obtained from
ducks from different regions of Kerala, India, were used in the study. A reference
isolate of P. multocida from fowl obtained from the Division of Bacteriology
and Mycology, Indian Veterinary Research Institute, Izatnagar, India was used
for comparison.
The bacterial isolates were identified based on morphology, cultural characters and biochemical reactions as described by Barrow and Feltham (1993). The isolates were also confirmed as P. multocida by amplification of KMT1 gene (Townsend et al., 1998).
Pathogenicity Testing of Isolates
Swiss albino mice, six to eight weeks of age were used for pathogenicity
testing. Each group of mice were inoculated intra-peritoneally with 0.1 mL of
inoculum containing 0.3x108 organisms per mL in sterile normal saline.
Control mice were injected with 0.1 mL of sterile saline. A total of six mice
were used for each isolate. All the animals were observed for signs of infection.
Blood smears were prepared from the dead mice and stained with Leishman’s
stain. Re-isolation of P. multocida from heart blood, lung, liver and
spleen of the dead mice was carried out on sheep blood agar.
Antibiogram
The antibacterial sensitivity patterns to 15 different antibacterial was
done as per the standard single disc diffusion method of Bauer et al.
(1966).
Isolation of Plasmid DNA
Plasmid DNA was isolated as per the method of Sambrook et al. (1989)
with minor modifications. Pure culture of P. multocida was inoculated
in 10 mL of Luria Bertani broth and incubated at 37°C for 8 h. The broth
was centrifuged at 8000 x g for 15 min. Resuspended the pellet in 100 μL
of TEG buffer containing lysozyme at a concentration of 10 mg mL-1
and was kept on ice for 15 min. To this mixture 200 μL SDS-NaOH was added
and gently mixed until the solution became translucent. The tube was further
incubated on ice for 15 min. To this 150 μL of 3M sodium acetate was added
and kept on ice for another 15 min. The mixture was centrifuged at 12000 x g
for 30 min at 4°C. The supernatant was carefully transferred into a fresh
Eppendorf tube and equal volume of phenol: chloroform: isoamyl alcohol (25:24:1)
was added and mixed by gentle inversion for 10 min and centrifuged at 10,000
x g for 5 min. The aqueous phase was transferred to a fresh tube and equal volume
of chloroform: isoamyl alcohol (24:1) was added and mixed gently by inversion
for 10 min and centrifuged at 10,000 x g for 5 min. The aqueous phase was transferred
into a fresh Eppendorf tube, added double the volume of ice-cold ethanol and
allowed the plasmid DNA to precipitate at -70°C overnight. The tube was
then thawed and centrifuged at 10,000 x g for 15 min. The DNA pellet was washed
in 70% ethanol and the final pellet was resuspended in 20 μL of TE buffer.
Ribonuclease A (10 mg mL-1) 2 μL was added and incubated at
37°C for half an hour and then stored at -20°C. Plasmid DNA from Escherichia
coli V517 maintained in the Department of Microbiology was prepared in a
similar manner.
Electrophoresis
The isolated plasmid DNA was analysed in 0.8% agarose gels. Escherichia
coli V517 plasmid DNA was used to ascertain the size of the plasmids. Electrophoresis
was carried out at 40V till the dye reached near the bottom of the gel. The
DNA fragments were viewed on a transilluminator and photographed using a gel
documentation system (Bio-Rad, USA).
RESULTS
Pathogenicity Testing
All the 25 isolates of P. multocida as well as the reference fowl
isolate were able to kill weaned mice. A concentration of 0.3x108
organisms per 1 mL was able to kill mice within 24 h when injected by intra-peritoneal
route.
The gross lesions observed in the internal organs of the dead mice were petechiae in the pericardium and congestion of lung, liver and spleen. Blood smear and impression smears from spleen and liver collected from dead mice, on staining with Leishman’s stain, revealed the presence of bipolar shaped organisms.
Antibiogram of P. multocida isolates indicating the susceptibility and
resistance to various antibiotics/antibacterial agents is presented in Table
1.
| Table 1: | Antibiogram of isolates |
| Table 1: | Continued |
| S = Susceptible, R = Resistant | |
| Fig. 1: | Plasmid profiles of P. multocide, Lane 1 E. coli V517, Lane 2 profile I, Lane 3 profile II |
Plasmid Profile Analysis
The twenty-five duck isolates could be placed into two plasmid profiles
(Fig. 1). Profile II consisted of a single isolate, DP6, which
carried four plasmids with molecular sizes of 46.2, 10.9, 6.5 and 0.8 kbp. Of
the remaining twenty-four duck isolates three (DP 8, DP 12 and DP 18) did not
harbour any plasmids, while the remaining 21 carried a single plasmid each of
46.2 kbp size (Profile I). The reference fowl isolate did not harbour any plasmid.
DISCUSSION
All the field isolates as well as the reference isolates were pathogenic for mice. Sambyal et al. (1988) Jayakumar (1998) observed that P. multocida of duck origin killed mice within 12 to 24 h post inoculation.
Collins and Woolcock (1976) observed an overwhelming increase in the number of P. multocida in visceral organs associated with death in mice, following experimental infection. Similar observations have also been made by Balakrishnan (1998) and Rajalakshmi (2001).
All isolates were sensitive to Enrofloxacin, Pefloxacin and Chloramphenicol. Seven isolates representing 25.92% of the isolates tested were found to be resistant to co-trimoxazole. Moderate sensitivity to co-trimoxazole has been reported by Sambyal et al. (1988) and Bhaumik and Dutta (1995). This present study indicates a shift in sensitivity pattern of drugs once considered to be the drug of choice for avian pasteurellosis. Hence it is important to check the indiscriminate use of antibiotics in the treatment of pasteurellosis. Furazolidine and streptomycin showed a sensitivity of only 22.2% while all the isolates were resistant to metronidazole. From this it is evident that the drug of choice for the treatment of duck pasteurellosis in this geographical location is Enrofloxacin, pefloxacin or chloramphenicol.
Of the twenty-five duck isolates, twenty-two (88%) carried plasmids that ranged from 46.2 to 0.8 kbp. Strains of P. multocida have been shown to harbour plasmids from 1.3 kbp. (Diallo et al., 1995) to approximately 100 kbp (Hirsh et al., 1989) in size.
Gunther et al. (1991) demonstrated that among 28 isolates of P. multocida from rabbits, 92% carried plasmids. However, two other studies showed greater variation in the occurrence of plasmids in avian isolates and it varied from 24% (Price et al., 1993) to 70.7% (Hirsh et al., 1985).
Diallo et al. (1995) observed that of the 45 avian strains of P. multocida studied, twenty strains yielded no plasmids. The remaining twenty-five were placed into two plasmid profiles. Profile I consisted of seven isolates that carried a single plasmid each of 1.3 kbp while the remaining 18 were grouped into profile II that showed two plasmids each of 2.4 and 7.5 kbp.
Similar results have also been reported by Shivshankara et al. (2000). They identified that 10 out of 12 isolates of P. multocida contained plasmids and they were placed into two groups, group one containing seven isolates, carrying a single plasmid and remaining isolates forming group II harbouring two plasmids each.
The isolates DP 8, DP 12 and DP 18 did not harbour any plasmids. However, they were found to be resistant to five, one and four antimicrobial agents respectively out of the 15 antimicrobials tested. Thus correlation between the presence of plasmids and antibiotic resistance could not be ascertained in the present study. Similar observations have been made by Diallo et al. (1995) who found that although only 55% of the 45 avian isolates of P. multocida carried plasmids, all of them were uniformly resistant to streptomycin, trimethoprim and lincomycin.
Pathogenicity tests conducted in mice revealed that all the isolates, even those which lacked plasmids were pathogenic. Thus this study could not establish any correlation between the presence of plasmids and virulence. These results are in accordance with the observations made by Diallo et al. (1995), Balakrishnan (1998) and Shivshankara et al. (2000).
ACKNOWLEDGMENTS
The authors are grateful to the Indian Council of Agricultural Research, New Delhi, India for providing financial support under the All India Network Programme on Haemorrhagic Septicaemia and the Dean, College of Veterinary and Animal Sciences, Mannuthy, Kerala, India for providing facilities to conduct this study. The first author wishes to thank the Dean Rajiv Gandhi College of Veterinary and Animal Sciences, Pondicherry, India for having deputed him for doctoral programme and also for his keen interest in the research work.