Molecular identification of Campylobacter jejuni and coli from chicken, calves and dogs to determine its potential threat on human being

Aim: Campylobacter is an emerging zoonotic pathogen and one of the leading cause of foodborne infection worldwide and it has been isolated from a variety of animal species. The aim of this study was to identify Campylobacter jejuni and Campylobacter coli from dogs, calves, and poultry using polymerase chain reaction (PCR). Methodology: A total of 104 number of samples comprising cloacal swab from poultry (38), a rectal swab from dogs (40), and calves (26) were collected for the isolation of thermophilic Campylobacters using conventional culture method. PCR was used for identification of mapA gene for C.jejuni and ceuE for C.coli. Results: The overall presence of Campylobacter was found to be 67(64.42%) from the samples, out of which 6 isolates belongs to C. jejuni species, were 5(18.51%) from chicken and 1(4.17%) from dog was recorded and about 17 isolates belongs to C. coli species were 9(33.33%), 6 (25%), and 1(9.09%) from chicken, dog and calves was recorded. Conclusion: Results suggested that Campylobacter reservoirs chicken, calves and pet dogs can play a role as the source of infection to human beings and PCR can be an ideal tool for molecular confirmation at the species level.


Introduction
The word "Campylobacter" is derived from the Greek word which denotes to its morphological shape which is curved rod, spiral or "S" shaped morphology under the microscope. The thermophilic Campylobacter spp. like Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsalensis are able to grow at 42-43°C under microaerophilic conditions (5% O 2 , 10% CO 2 , and 85% N 2 ).
Campylobacter is one of the leading causes of foodborne diarrheal illness worldwide [1,2]. It is one of the emerging zoonotic pathogen and is responsible for more gastroenteritis cases than any other reported bacterial species in many countries [3].
Natural reservoirs of the bacteria are the gastrointestinal tract of farm and wild animals. Direct contact with carrier's animals is found to be a possible source of infection [4]. It is frequently isolated from a variety of animal species such as poultry, cattle, pigs, sheep, pets, wild birds, and rodents [5]. It was reported that about 70.9% of the human cases were attributed to chickens, 19.3% to cattle and 8.6% to dogs [6]. Handling or consumption of undercooked or contaminated meat is considered as the significant source of human Campylobacter spp. infection but also other risk factors responsible for its transmission are ingestion of contaminated dairy products, water, foreign travel, and swimming in natural sources of water [7]. Large outbreaks of campylobacteriosis are rare as most cases of human illness appear to be sporadic. Difficulties in identifying the source of sporadic infections are compounded due to the widespread occurrence of these pathogens in the environment [8,9].
Molecular methods have facilitated the development of nucleic acid-based detection methods which are more rapid, sensitive and specific. Polymerase chain reaction (PCR) has been used for diagnosis which has proven to be a fast, highly discriminative and relatively simple method [10]. Virulence factors in C. jejuni and C.coli are a useful tool to assess the potential risk of poultry as a source of Campylobacter infection [11]. Adopting the specific gene target in routine diagnosis will help in the improved understanding of the prevalence and the epidemiology of this emerging infection.

Ethical approval
Prior consent of the owners was taken before collection of a rectal swab from dogs, calves and cloacal swabs from chicken. Proper ethical considerations related to handling and not to cause any injury during sampling was taken. Available at www.veterinaryworld.org/Vol.8/December-2015/10.pdf

Collection of samples
A total of 104 number of samples comprising cloacal swab from poultry (38), the rectal swab from dogs (40), and calves (26). The samples were collected from Department of Clinics and Post-graduate Research Institute of Animal science, Kattupakam using sterile cotton swabs (Himedia, India) and transported in an icebox to a laboratory for processing and microbiological analysis.

Processing of samples
The samples were collected by using sterile cotton swabs (Himedia, India) and brought immediately to the laboratory for processing. The samples were put in Blood free Campylobacter broth base (M1318, HiMedia Pvt. Ltd., Mumbai, India) with Campylobacter growth supplement (HiMedia Pvt. Ltd., Mumbai, India) and incubated under microaerophilic conditions at 42°C for 24 h. A loopful of inoculum from broth was streaked into blood free Campylobacter selectivity agar base (M887, HiMedia Pvt. Ltd., Mumbai, India) plates and incubated for 48 h at 42°C under micro-aerophilic conditions by using internal gas generation system. This was accomplished by using equal quantity of citric acid, sodium bicarbonate and sodium borohydride which fills the jar environment with 85% N 2 , 10% CO 2 , and 5%O 2 [12].

Extraction of DNA
Grey color, spreading type colonies with sticky nature were suspected for Campylobacter. Based on colony morphology suspected colonies were picked up. The DNA of Campylobacter spp. isolates was prepared by taking loopful of 48 h test culture in 100 μl of sterilized DNAse and RNAse free milliQ water in micro centrifuge. The samples were vortexed and heated at 95°C for 10min, cell debris was removed by centrifugation, and 3 μl of the supernatant was used as a DNA template in PCR reaction mixture.

Oligonucleotide primers
Stock solutions of the primers were made in nuclease-free water and stored at −20°C. The working solutions were made to 10pmol/μl after suitable dilution. The isolates were identified at genus level by PCR targeting the mapA and ceuE gene of Genus Campylobacter. The primer used in the present study is taken from already published article [10] and the details of the primer pair used are given in (Table-1). All PCR amplifications were performed in a mixture (25 μl) containing nuclease free water, primers, Taq polymerase, and template DNA. The amplification was carried out in a thermal cycler with the following cycling conditions as shown in (Table-2).

Prevalence of Campylobacter
In the present study, the overall prevalence of Campylobacter was found to be 67 (64.42%) from the samples based on colony morphology and microscopic examination, out of which species wise prevalence was 24(60%) from dog, 27(71.05 %) from poultry and 16(61.54%) from calves was recorded.

Molecular identification of the isolates
All the Campylobacter isolates recovered from conventional culture method was tested by PCR targeting mapA gene for C.jejuni and ceuE for C.coli. Out of the 67 Campylobacter isolates 6 isolates belong to C.jejuni species, were 5 (18.51%) from poultry and 1 (4.17%) from dog and 17 to C.coli species were 9 (33.33%) from poultry, 6 (25%) from dog and 1 from calves were identified as zoonotic thermophilic Campylobacter ( Figures-1 and -2).

Discussion
Thermophilic Campylobacters are major foodborne pathogens of animal origin and leading cause of gastrointestinal infections. The importance of this disease is compounded by the fact that very low doses of Campylobacter in the food samples are capable of causing infections unlike other foodborne infection. In India, the prevalence of Campylobacter is underestimated which may be due to its way of cooking and food habit.
The advent of molecular biology techniques such as PCR has largely replaced the conventional isolation procedure by virtue of its speed, sensitivity and discriminating power. The conventional isolation procedure although time-consuming and cumbersome still remains the gold standard.
The finding of the present study is comparable with a report of Kassa et al. [13] were the prevalence of Campylobacter spp. in chickens observed was (68.1%). In one study, the frequency of isolation of Campylobacter spp. in dogs varied from 17% (Brazil,  MDmapA1FP  5'-CTA TTT TAT TTT  TGA GTG CTT GTG-3'   589 bp   MDmapA2RP  5'-GCT TTA TTT GCC  ATT TGT TTT ATT A-3'  COL3 F  5'-AAT TGA AAA TTG  CTC CAA CTA TG-3'   462 bp   MDCOL2 R  5'-TGA TTT TAT TAT  TTG TAG CAG CG-3' C. jejuni=Campylobacter jejuni, C. coli=Campylobacter coli, PCR=Polymerase chain reaction  [4,14]. However, there are several reports of higher isolation rates of Campylobacter from cattle, ranging from 5% to 89.4% [15]. Workman et al. [16] also reported the prevalence of Campylobacter spp. from rectal swabs of dogs to be 46.9% and from cloacal swabs of broiler chicks 94.2%. Amar et al. [17] reported the prevalence of Campylobacter in chicken flocks (33-44%), cattle (15%) and in healthy dogs (6%) in Switzerland. Molecular characterization of two different gene targets for C. jejuni and C. coli was attempted; the targeted gene was mapA and ceuE. In one study, Sandberg et al. [18] reported that about 7% of the isolates from dog were positive for C.coli and 6% for C.jejuni. A Dutch study reported the prevalence ranging from 20% to 31% in poultry were C. jejuni was (5.38%) and C.coli (2.3%) [19,20]. Awadallah et al. [21] also reported C.coli(7.4%) and C. jejuni(3.7%) from chicken cloacal swabs.
Prevalence of Campylobacter varies between countries depending on the level of hygienic measures followed. This variation in getting a low number of isolates positive for mapA and ceuE gene may be due to the primers used, the prevalence of inhibitors, laboratory condition, seasonal variations as well as geographic diversity in the distribution of C.jejuni and C.coli isolates.

Conclusion
Our findings suggest that chicken, calves and pet dogs can play a role as the reservoir of potentially pathogenic Campylobacter strains for humans. Hence, proper public health protection measures should be taken to control it and prevent its transmission from the reservoir. PCR can be used an ideal tool for molecular confirmation at the species level.

MS and
LG have designed the study project as well as corrected the manuscript, SB has done the research work, data compiling and manuscript preparation. MG, GS, PASM, and AK helped in doing the research work. All authors read and approved the final manuscript.