Prevalence and Resistance to Antimicrobial Agents of Campylobacter sp. Isolated from Dogs in India

INTRODUCTION

Enteropathogenic bacteria are distributed worldwide and well to live as commensal in the gastrointestinal tract of man as well as animals. The zoonotic potential of these bacteria viz., Salmonella (Verma et al., 2007, 2008, 2011a, b; Sachan et al., 2013), E. coli (Malik et al., 2013; Kumar et al., 2013) and Campylobacter (Kumar et al., 2012a, b) was very well established. Among these Campylobacters are emerging food borne pathogens with increasing incidences and leads to severe gastroenteritis and other complications like Guillain-Barre Syndrome, reactive arthritis, haemolytic uraemic syndrome, meningitis, carditis, pancreatitis, septic arthritis, etc., (Stern and Line, 2000; Tenkate and Stafford, 2001; Hannu et al., 2002; Skirrow, 2006; Levin, 2007; Lindmark et al., 2009; Kirkpatrick and Tribble, 2011; Man, 2011; Rajendran et al., 2012; Bouwman et al., 2013; Hauri et al., 2013). In animals, these Campylobacters may cause diarrhoea and other reproductive disorders like abortion, early embryonic mortality and many other complications (Humphrey et al., 2007; Kumar et al., 2012a, b).

Various studies have reported the association of Campylobacter species with diarrhea in dogs (Steinhauserova et al., 2000; Misawa et al., 2002; Kumar et al., 2012a) however, their real role in canine enteritis is not clear (Koene et al., 2004). Generally, children are more susceptible to this infection (Wolfs et al., 2001; Chattopadhyay et al., 2001). Although, there is various reports from developed countries regarding prevalence of Campylobacters in dogs but there is little published information about Campylobacter infections and their drug resistance pattern in dogs of developing countries including India. Therefore, the present study was undertaken to determine the occurence of thermotolerant Campylobacter and their antibiogram in dogs of Mathura city and nearby areas, Uttar Pradesh, India.

MATERIALS AND METHODS

Study design, area and sample collection: This cross-sectional study was conducted during the period of almost one year in 2012-2013 in Mathura city and nearby areas, Uttar Pradesh India (Fig. 1). Geographically, the city is situated at the latitude 27°30’N and longitude 77°40’E with an elevation of 174 metre above sea level. A total of 330 rectal swabs for bacteriological culture were collected from dogs presented to Teaching Veterinary Clinical Complex, Mathura with the epidemiological information viz., breed, age, sex and health status.

Thermophilic Campylobacter isolation and identification: The samples in the Campylobacter enrichment HiVeg TM broth base with addition of polymixin B sulphate, rifampicin, trimethoprim and cycloheximide were incubated at 42-43°C under microaerophilic conditions in the microaerophilic jars with a lighting candle (Fig. 2). After incubation, the enriched samples were properly shaken and sub-cultured onto Campylobacter selective agar (HiMedia, Mumbai) supplemented with 10% defibrinated sheep blood and addition of Polymixin B, vancomycin, trimethoprim and cephalothin for primary isolation ofc thermophilic Campylobacter.


Fig. 1:	Map of Uttar Pradesh showing Mathura as study area


Fig. 2:	Microaerophilic jar with a lighting candle

The inoculated petridishes were incubated at 42°C for 48 h under microaerophilic conditions. Suspected thermophilic Campylobacter colonies that were Gram negative, curved, or spiral rods and showed corkscrew-like motion, were confirmed biochemically (Skirrow and Benzamin, 1980; Garcia et al., 1985).

Antibiogram: All the Campylobacter isolates were assessed for their antimicrobial susceptibility testing using disc-diffusion method (Bauer et al., 1966) following the NCCLS guidelines (NCCLS 2002). A total of 19 antimicrobial discs (Hi-Media, Mumbai) of commonly used antibacterial drugs viz., amikacin (30 μg), ampicillin (10 μg), amoxycillin (20 μg), Aztreonam (30 μg), Cefaclor (30 μg), Cefotaxim (30 μg), ciprofloxacin (30 μg), chloramphenicol (30 μg), enrofolxacin (10 μg), gentamicin (10 μg), Lincomycin (10 μg), Nitrofurazone (100 μg), Norfloxacin (10 μg), Ofloxacin (5 μg), Oxytetracyclin (30 μg), Peefloxacin (5 μg), Penicillin (10 units), streptomycin (10 μg) and tetracyclin (30 μg) were used to assess the drug resistance pattern of Campylobacter isolates.

RESULTS AND DISCUSSION

A total of 330 faecal samples were collected from dogs with and without clinical signs of gastroenteritis and isolation of bacteria was attempted in all the samples. Based on isolation, cultural and biochemical characterization of bacteria, the prevalence of Campylobacter spp. was 34.24% (Table 1). The prevalence of Campylobacter spp. in faecal samples of dogs is within the range (17-59%) as reported in various studies (Sandberg et al., 2002; Engvall et al., 2003; Koene et al., 2004; Rossi et al., 2008; Parsons et al., 2010; Kumar et al., 2012a, b).

Table 1:	Occurrence of Campylobacter infection in dogs influenced by age, sex, breed and health status

The variation between these studies might be either due to different study population or methods used for detecting the bacteria (Guest et al., 2007; Rossi et al., 2008; Acke et al., 2009; Parsons et al., 2010; Kumar et al., 2012a, b).

Younger dogs (less than 1 year of age) were more likely to carry Campylobacter spp. (Table 1). High prevalence of Campylobacter spp. supports the hypothesis that dogs especially the puppies (less than 1 year of age) may be an important source of Campylobacter infection for man. Our findings were similar to previous studies (Engvall et al., 2003; Acke et al., 2006, 2009; Guest et al., 2007). But contrary to our findings, a small number of studies conducted by Wieland et al. (2005) and Tsai et al. (2007) have suggested that age is not a predisposing factor for Campylobacter infection. Breed-wise prevalence showed that non-descript dogs (45.97%) were more likely to carry Campylobacter infections (Table 1). This might be due to the way of living of non-descript dogs. As they roam outside the home freely in comparison to other breeds of dog leading to more exposure and chances of getting infection from stray dogs or animals in and around areas (Kumar et al. 2012b).

Dogs showing clinical signs of gastroenteritis were showing higher prevalence (47.21%) in comparison to that of animals without gastro-intestinal disorders (15.04%) (Table 1). Similar to our study, various researchers (Guest et al., 2007) reported the association between Campylobacter infection and clinical signs, especially in younger dogs (Fox et al., 1983; Nair et al., 1985; Burnens et al., 1992).

Table 2:	Drug susceptibility pattern of all the Campylobacter isolates (n = 113) from dogs

However, contrary to our findings, there are various reports suggesting no relationship between diarrhoea and Campylobacter spp. infection status (Engvall et al., 2003; Acke et al., 2006; Rossi et al., 2008; Parsons et al., 2010; Kumar et al., 2012b), suggesting that the organism is a commensal (Engvall et al., 2003). Campylobacter infection were detected in 15.04% of the dogs either apprarantly healthy or having some problem other than gastroenteritis (Table 1) showing that these animals can be carriers of Campylobacter species and may a source of infection for other pets and human beings (Acke et al., 2006; 2009; Kumar et al., 2012b).

All the Campylobacter isolates were tested for drug sensitivity using 19 antibacterial drugs and the results were shown in Table 2.

Out of 113 Campylobacter isolates of canine origin, two isolates were resistant to all the nineteen antibiotics used in the study, while all the isolates were resistant to Streptomycin, Ampicillin, Amoxycillin, Aztreonam, Lincomycin, Tetracycline, Oxytetracycline and Penicillin. A high rate of resistance was observed to Cefotaxim (97.35%), Peefloxacin (91.15%), Chloramphenicol (90.27%), Ofloxacin (84.07%), Ciprofloxacin (83.18%), Cefaclor (80.53%), Nitrofurazone (76.11%), Norfloxacin (74.33%), Gentamicin (42.48%), Amikacin (40.71%) and Enrofloxacin (36.28%). Antibiotic sensitivity studies on Campylobacter isolates were conducted by various researchers in different countries and reported varying degree of resistance to same drug (Little et al., 2008; Moran et al., 2009; Kumar et al., 2012a). High resistance to ampi-cloxacillin, tetracycline, ofloxacin, ciprofloxacin etc were previous reported by various researchers like Little et al. (2008) and Miflin et al. (2007). Quinolones were considered as suitable drug for treatment of Campylobacteriosis (Uaboi-Egbenni et al., 2011) but higher resistance to ciprofloxacin and ofloxacin was seen, which might be either due to genetic mutations interfering with bacterial DNA gyrase (Greene and Watson, 2003) or selection pressure due to injudicious use of antibiotics (Norma et al., 2007; Biasi et al., 2011). Present results indicated Amikacin and Gentamicin as drugs suitable for the treatment of canine campylobacteriosis. This also opens up therapeutic possibilities for these drugs in human medicine.

This cross-sectional study has the some limitations in its design; nevertheless, it is performed in only little veterinary practice involving small number of dogs and few related determinants. Therefore, there is a requirement of detailed study considering larger population in order to establish a better understanding of the epidemiology of Campylobacter infection in dogs of developing countries like India and ultimately help in making strategies to control or reduce the risk of this infection in man. The use of antibiotics as threauptic and/or prophylaxis for man as well as animals should be monitored because acquisition of antibiotic resistant strains of Campylobacters by man has serious public health implications.

CONCLUSION

From the present study, it can be concluded that Campylobacter infection was prevalent in dogs of study area suggesting their possible role in transmission to human beings. Younger dogs (less than 1 year of age) were more likely to carry Campylobacter spp. it is worth highlighting that dogs, particularly puppies may be an important source of Campylobacter infection for humans. High levels of resistance to antibiotics commonly used for prophylaxis and therapeutic is of public health significance. Present results indicated Amikacin and Gentamicin as drugs suitable for the treatment of canine campylobacteriosis. However, the results emphasize the use of antibiotic sensitivity test be conducted before prescribing the antibiotics.

ACKNOWLEDGMENTS

This study was supported b y Uttar Pradesh Pandit Deen Dayal Upadhayay Pashu Chikitsa Vigyan Vishvidhyalaya Evum Go-Anusandhan Sansthan (DUVASU), Mathura, India under university funding scheme. The authors are highly thankful to Head, Department of Veterinary Microbiology and Immunology; Department of Veterinary Epidemiology and Preventive Medicine, Director, Teaching Veterinary Clinical Complex, Dean, College of Veterinary Sciences and Animal Husbandry, Director Research and Hon’ble Vice Chancellor, Uttar Pradesh Pandit Deen Dayal Upadhayay Pashu Chikitsa Vigyan Vishvidhyalaya Evum Go-Anusandhan Sansthan (DUVASU), Mathura, India; for providing all the necessary support and facilities for conducting this study. We wish to thank staff of Department of Veterinary Microbiology and Immunology especially Smt. Mamta for her technical assistance. The authors are highly thankful to dog owners, who allowed their dogs to participate in the study.