Introduction

Naz et al.,
The Journal of Animal and Plant Sciences, 22(3 Suppl.):
J.
2012, APnim
ag P
e: lant
242 Sc
- i,
24 522(Sup 3): 2012
ISSN: 1018-7081
ISOLATION, CHARACTERIZATION AND MONITORING OF ANTIBIOTIC
RESISTANCE IN PASTEURELLA MULTOCIDA ISOLATES FROM BUFFALO (BUBALUS
BUBALIS) HERDS AROUND LAHORE
S. Naz, A. Hanif, A. Maqbool, S. Ahmed and K. Muhammand Veterinary Research Institute, Ghazi Road, Lahore, Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan. Department of Parasitology, Faculty of Veterinary Science, University of Veterinary and Corresponding Author e-mail: ahanif@uvas.edu.pk ABSTRACT
Haemorragic septicaemia (HS) caused by Pasteurella multocida (P. multucida) is an important disease of buffaloes causing heavy economic losses in Pakistan. Samples from 20 HS suspected carcasses of buffaloes were processed and the recovered isolates were identified by cultural, morphological, biochemical and serological tests. All the isolates were also processed for mouse pathogenicity test and antibiogram assay. Sixteen isolates were identified as Pasteurella multocida. Serologically all isolates were associated with Carter΄s sero group B. Maximum number of isolates were recovered from samples of bone marrow, lungs and spleen. All the isolates were found pathogenic to mice. Antibiogram assay of all the fields isolates depicted the highest sensitivity 87.5% for ciprofloxacin, ofloxacin, enrofloxacin and gentamicin, 81.25% for norfloxacin and amikacin, 75% for kanamycin, 56.25% for tetracycline, 25% for chloramphenicol, doxycycline and vancomycin, erythromycin where as sulfadiazine (12.5%) was found least effective.
No resistance was observed for ciprofloxacin, enrofloxacin, ofloxacin and norfloxacin where as maximum resistance was observed against erythromycin and sulfadiazine (50%). It is concluded that all P. multucida isolates belonged to Carter’s group B and found sensitive to ciprofloxacin and ofloxacin.
Key words. Buffalo, Pasteurella multocida, Isolation, Antibiotic resistance.
INTRODUCTION
Pasteurella isolates varies according to the host animal species, time, geographical origin and antimicrobial Hemorrhagic septicemia is an economically pretreatment of the animals (Caprioli et al., 2000). The important bacterial disease of cattle and buffaloes aim of the present study was the isolation and (Chandrasekaran et al., 1994) and buffaloes are more identification of the pathogen from hemorrhagic susceptible than cattle (DeAlwis, 1990). The disease septicemia suspected carcasses of buffaloes, which could remains a significant obstacle to sustainable livestock be used for development of vaccine from local isolates to production in most parts of tropical Asia and Africa. It is effectively prevent the disease in future, to determine the caused by Pasteurella multocida a natural inhabitant of antimicrobial susceptibility status of field isolates of the mucosal surfaces of upper part of the respiratory tract Pasteurella multocida that will assist practitioners in the of ruminants, and under predisposing environmental or rational selection of antimicrobial agents and make the management conditions which constitute stress for the prudent use of these drugs for control of the disease.
animals such as transport (shipping fever), marketing, change of feed, climate or ventilation (Radostits et al., MATERIALS AND METHODS
2000). The disease is per acute, having a short clinical course, involving severe depression, pyrexia, sub Samples: Samples of blood, bone marrow and tissue
mandibular edema, and dyspnea, followed by specimens (pieces from liver, spleen and lung) were recumbency and death (Horadagoda et al., 2001).
collected from HS suspected carcasses of 20 buffaloes Diagnosis of the Pasteurellosis has been traditionally from different areas around Lahore during the period from July, 2005 to June, 2006. The samples were isolation/identification of the causative organism.
processed for isolation and identification of suspected Antibiotics are used to a large extent for treatment of pathogen by standard method (Carter, 1984).
hemorrhagic septicemia. However, the prolonged and indiscriminate use of antibiotics has resulted in organism Isolation: Samples were inoculated on nutrient agar,
and even multi drug resistant (MDR) forms of P. blood agar (5% sheep blood) and MacConkey’s agar. The multocida have emerged (Arora et al., 2005; smears were prepared from representative colonies and Shivachandra et al., 2004). Antimicrobial resistance of “Proceedings of 6th Asian Buffalo Congress held on 27-30 Oct. 2009 at Lahore Pakistan”
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microbes were characterized microscopically by using from all isolates revealed microscopically gram negative Biochemical tests: Biochemical tests for all the isolates
All the isolates fermented glucose, sucrose, were performed. Peptone water grown culture of each sorbitol, manitol, fructose, arabinose and maltose with isolate was inoculated in 1% glucose, sucrose, sorbitol, acid production only. A negative reaction was observed manitol, fructose, dulcitol, lactose, silicin, arabinose and for dulcitol, lactose and silicin. All the isolates were maltose, incubated aerobically at 37°C for 72 hours.
found positive for indol, oxidase, catalase production and Indol, oxidase, catalase, urease production and nitrate nitrate reduction tests. All the isolates were negative for reduction tests were carried out according to their urease production. Cultural, morphological and standard bacteriological procedure (Carter, 1984).
biochemical characteristics identified all isolates as A slide agglutination test using the antiserum Pasteurella multocida. Serologically all isolates were against the capsular type B was also performed for each Sixteen samples (80%) out of 20 samples of each of the bone marrow, lung and spleen yielded pure Pathogenicity test: Pathogenicity of each isolate was
growth of the causative agent where as 10 (50%) and tested in six weeks old Swiss albino mice. A total of six 5(25%) out of 20 samples of each of the liver and blood mice were used for each isolate. Mice were inoculated yielded pure growth on sheep blood agar.
intra-peritoneally with 0.1 ml of inoculum containing All the field isolates killed mice within 24 to 36 0.3x108 organisms per ml in sterile normal saline. Control hours post inoculation. Giemsa stained smears prepared mice were injected with 0.1 ml of sterile saline. All the from heart blood of dead mice revealed bipolar mice were kept under observation and mortality was organisms. From heart blood of mice colonies recorded. Blood smears were prepared from the heart representative of P. multocida were isolated on sheep blood of dead mice and stained with Giemsa stain. Re- isolation of P. multocida from heart blood of the dead mice was carried out on sheep blood agar (Buxton and Table I. Antibiogram assay of sixteen field isolates of
P. multocida
Antibiogram assay: Each of the isolate was tested for
sensitivity against 15 different antibiotics such as Antibiotics
Sensitive
Intermediate Resistant
doxycycline, vancomycin, erythromycin, sulfadiazine, amoxycillin, ampicillin, chloramphenicol, ciprofloxacin, norfloxacin, ofloxacin and enrofloxacin using the standard method of National Committee for Clinical Laboratory Standards (NCCLS, 1990). An eighteen hours culture of each isolate in Brain Heart Infusion broth was plated on Muller-Hinton agar medium enriched with 5% sheep blood. The culture was allowed to adsorb for 10 minutes and then the antibiotic discs (Oxoid) were placed on the plate at an appropriate distance from each other.
The plates were incubated aerobically at 37°C for 24 hours. The diameters of inhibition zones surrounding the antibiotic discs were measured and subsequently matched with the standard inhibition zone diameters of respective antibiotic discs. On the basis of size of inhibition zones of Sixteen field isolates of P. multocida showed various antibiotics, the isolates were classified as sensitivity to 15 antibiotics, as 87.5% isolates were found sensitive, intermediately sensitive or resistant.
sensitive to ciprofloxacin, ofloxacin, enrofloxacin and gentamicin followed by 81.25% isolates to norfloxacin and amikacin. Seventy five percent isolates were sensitive to kanamycin. For tetracycline sensitivity was Sixteen bacterial isolates were recovered from 56.25%. Fifty percent isolates were sensitive to the samples collected from 20 HS suspected buffalo chloramphenicol, doxycycline and vancomycin. Twenty carcasses. All the isolates exhibited smooth glistening, percent isolates were sensitive to erythromycin. The translucent colonies on nutrient agar, failed to grow on lowest sensitivity (12.5%) was observed for sulfadiazine.
MacConkey agar and produced non hemolytic dewdrop Resistance was not observed for ciprofloxacin, like colonies on sheep blood agar. Grams stained smears enrofloxacin, ofloxacin and norfloxacin. However for “Proceedings of 6th Asian Buffalo Congress held on 27-30 Oct. 2009 at Lahore Pakistan”
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erythromycin and sulfadiazine resistance was 50% (Table vancomycin were moderately effective against the bacteria. Erythromycin and sulfadiazine were not very effective. These observations are in accordance to Watts DISCUSSION
et al. (1994) who frequently encountered resistance of P. haemolytica and P. multocida of bovine origin to In clinical cases, diagnosis of hemorrhagic septicemia is made on the basis of clinical signs, gross In conclusion, all the field isolates were similar pathological lesions, herd history, morbidity and in cultural, morphological, biochemical and serological mortality pattern. However confirmation of tentative characteristics and can be used for development of HS clinical diagnosis needs isolation and identification of vaccine to control the disease. The local isolates of HS causative organism from the morbid material. In the showed increasing level of resistance to the antibiotics present study, cultural, morphological and biochemical that are extensively used in field for treatment of the characteristics of all the isolates recovered from morbid disease. It is therefore recommended to monitor the materials were in accordance with those of P. multocida antibiotic sensitivity of P. multocida from time to time in (Dutta et al., 2001; Kumar et al.2009).
future to design the effective regimen for treatment of the hemorrhagic septicemia were found to be the sample of choice for isolation of P.multocida. Isolation of P. REFERENCES
multocida from the lung and spleen samples was comparable to that of long bones. P. multocida can be Anwar, M. A. H., S. U. Rahman and R. Ahmad (2000).
isolated from wide range of organs such as lung, kidney, Sensitivity of Pasteurella multocida isolated heart, spleen, liver, brain, tonsils and lymph nodes (left from cattle and buffaloes. PJBS., 3(4):692-93.
and right retropharyngeal and pre-scapular nodes) from Arora, A. K., S. Virmani, S. K. Jand and M. S. Oberoi experimentally infected calves with P. multocida (OIE, 1992; Mark et al., 2007). From the liver and blood antibiogram of Pasteurella multocida isolates samples, recovery of the causative agents was poor.
from different animal species. Indian J. Anim.
Postmortem changes might have inactivated the causative Buxton, A. and G. Fraser, (1977). Animal Microbiology.
All the field isolates of P. multocida were found Blackwell Scientific Publications Ltd. Oxford.
pathogenic for mice and killed the mice within 6-24 hours post-infection. In the BALB/c mice, when Caprioli, A., L. Busani, J. L. Martel and R. Helmuth experimentally infected through intra-peritoneal route, an (2000). Monitoring of antibiotic resistance in overwhelming septicemia was observed within 30 hours bacteria of animal origin: epidemiological and post infection (Ramdani et al., 1990).
Sixteen pure isolates of P. multocida were tested for their sensitivity against different antibiotics available Carter, G. R. (1984), Diagnostic procedures in veterinary for the treatment of bacterial infections in animals. All of bacteriology and mycology, 4th ed.,Charles C the 16 isolates were found highly sensitive (87.5%) to Thomas Publishing Co., Springfield, IL.
ciprofloxacin, ofloxacin and enrofloxacin. Yoshimura et Catry, B., F. Haesebrouck, S, D. Vliegher, B. Feyen, M.
al. (2008) and Kumar et al. (2009) also found Vanrobaeys, G. Opsomer, S. Schwarz and A, D.
enrofloxacin the most effective antibiotic against P. Kruif, (2005). Variability in acquired resistance multocida. Similarly, Shayegh et al. (2009) reported of Pasteurella and Mannheimia isolates from the 100% sensitivity of the bacteria to ciprofloxacin and nasopharynx of calves, with particular reference enrofloxacin. In present study no resistance was observed to different herd types. Microb Drug Resist., for quinolones. Similar observations were also recorded by Anwar et al. (2000). In contrast to our finding, Carty Chandrasekaran, S., L. Kennett, P. C. Yeap, N.
et al. (2005) observed the acquired resistance of P. (1994). Characterization of immune response aminoglycosides, gentamicin was found highly effective (87.5%) against field isolates. Other authors found the efficacy of gentamicin against P. multocida as vaccines. Vet. Microbiol., 41:213–219.
controversial (Verma et al., 2004; Kumar et al., 2009).
De Alwis , M. C. L. (1990). Haemorrhagic Septicaemia.
Amikacin and Kanamycin were highly effective (81.25%), and (75%), respectively but Yoshimura et al. Horadagoda, N. U., J. C. Hodgson, G. M. Moon, T. G.
(2008) found aminoglycosides as less effective.
Wijewardana and P. D. Eckersall (2001). Role “Proceedings of 6th Asian Buffalo Congress held on 27-30 Oct. 2009 at Lahore Pakistan”
Naz et al.,
J. Anim Plant Sci, 22(Sup 3): 2012
haemorrhagic septicaemia in the buffalo.
haemorrhagic septicaemia in cattle and buffalo.
Kumar, P., V. P. Singh, R. K. Agrawal and S. Singh, Immunology and Cell Biology ,68: 57–61.
(2009). Identification of Pasteurella multocida Shayegh, J., P. Mikaili, J. D. Sharaf, and A. Rastgn isolates of ruminant origin using polymerase (2009). Antimicrobal resistance evaluation of chain reaction and their antibiogram study. Trop Iranian ovine and bovine Pasteurella multocida.
Manual of standards for diagnostic tests and vaccines, Shivachandra, S. B., A. A. Kumar, A. Biswas, M. A.
(1992). Office intrnational des epizooties.
Ramakrishnan, V. P. Singh and S. K. Srivastava Mark, P., J. Dagleish, C. Hodgson, S. Ataei, A. Finucane, (2004). Antibiotic sensitivity patterns among J. Finlayson, , J. Sales, R. Parton and J. G. Coote Indian strains of Avian P. multocida, Tropical (2007). Safety and protective efficacy of Animal Health and Production, 36:743–750.
intramuscular vaccination with a live aroa Verma, S. C., N. K. Mahajan, G. Malik and J. P. Dahiya , derivative of Pasteurella multocida B:2 against (2004). An epidermiological study on bovine experimental hemorrhagic septicemia in calves.
hemorrhagic septicemia in Haryana, Indian J.
NCCLS, (1990). Performance standards for antimicrobial Watts, J. L., R. J. Yancey, J. S. A. Salmon, and C. A.
disk susceptibility tests. 4th Ed., Villanova, PA, Case (1994). A 4-year survey of antimicrobial susceptibility trends for isolates from cattle with Radostits O. M., C. Gay, D. C. Blood and K.W.
bovine respiratory disease in North America. J.
Hinchcliff (2000). A textbook of the diseases of cattle, sheep, pigs, goats, and horses, 9th ed., Yoshimura, H., M. Ishimaru, Y. S. Endoh and A. Kojima Ramdani, H. J. S. Dawkins, R. B, Johnson, T. L. Spencer, Pasteurella multocida Isolated From Cattle and and B. Adler (1990). Pasteurella multocida Pigs. J. Vet. Med. Series B, 48 (7):555–560.
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