MATERIALS AND METHODS

Arcobacter isolates: A total of 56 isolates (A. butzleri (33), A. skirrowii (03), A. cryaerophilus (20)), recovered from different sources (chicken meat, pork, sheep faeces, goat faeces, poultry intestinal contents and human stool samples), were used in this study (Table 1). All these isolates were maintained in Division of Veterinary Public Health, Indian Veterinary Research Institute Izatnagar, Bareilly, Uttar Pradesh, India.

DNA extraction: DNA extraction from Arcobacter isolates was done using commercially available DNA extraction kit, DNeasy Blood and Tissue Kit (QIAGEN, USA) following the manufacturer’s protocol, isolated DNA were stored at -20°C till used.

RAPD-PCR profiling of Arcobacter isolates: RAPD-PCR was employed for genotyping and evaluating genetic diversity of the 56 isolates of three Arcobacter spp. (A. butzleri, A. skirrowii, A. cryaerophilus). Two primers, HLWL85: 5'-ACAACTGCTC-3' (Mazurier et al., 1992) and OPA-11: 5'-CAATCGCCGT-3' (Hernandez et al., 1995) were employed in the present study. RAPD-PCR was performed by slight modification of the protocol of Houf et al. (2002). Each PCR mixture consisted of 2.5 μL of 10x Taq buffer, 2.5 μL of 2 mM concentration of each dNTPs, 25 pmol of the primer, 1 μ of Taq polymerase, 3 μL of template DNA and nuclease-free water up to 25 μL. The amplification cycles included initial denaturation at 94°C for 5 min followed by 45 repeats of denaturation at 94°C for 1 min, annealing at 36°C for 1 min and extension at 72°C for 1 min 30 sec. Final extension was carried out at 72°C for 7 min.

Table 1:	Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii isolates used in the study
CM: Chicken meat, P: Pork, SF: Sheep faeces, GF: Goat faeces, PI: Poultry intestinal contents, HS: Human stool

Gel electrophoresis and data analysis: The PCR products were characterized by gel electrophoresis on 1.5% agarose gel for 95 min at 80 V and stained with ethidium bromide (0.5 μg mL^-1) and visualized in gel documentation system. Approximately, 10 μL of the PCR product was loaded along with 100 bp plus ladder (GeneRuler 100 bp plus DNA Ladder, Fermentas, Canada) used as the molecular marker. The interpretation of results was done by pairwise binary band matching (Tenover et al., 1995). Only the distinct bands were considered for analysis by binary scoring pattern, wherein a score of 1 for the presence and 0 for absence of a band was assigned to each isolate. The dendrogram was created using the software TREECON for Windows v1.3b, Bioinformatics and Evolutionary Genomics, Belgium (Van de Peer and de Wachter, 1994). Dendrograms were constructed and analyzed separately for the three Arcobacter species. Numerical index of discrimination was calculated by Simpson’s index of diversity (Hunter and Gaston, 1988).

RESULTS

Both the primers employed in this study yielded RAPD fragments in 54 out of 56 isolates giving a typeability of 96.4% (Table 2). Out of 33 A. butzleri isolates, primer 1 (HLWL85) yielded RAPD fragments in 31 isolates while 02 failed to show any RAPD fragments. RAPD fragments were present in all 20 A. cryaerophilus and 3 A. skirrowii isolates. The number of bands ranged between 2-8 and was compared between ~500-~3100 bp (Fig. 1-3). Distinct polymorphic bands at ~2200 bp were observed in CM 10, 12, 61, 68, 76, 79, 86, 90, 94, 104, P4, 19, 37, 17, 44, SF 12, 15, 55 Arcobacter isolates. Out of the 56 isolates, 54 typable strains and a total of 35 types were observed, giving a typeability of 96.4%. The discriminatory power of the primer was 0.9762 (Table 2). The profiles generated by RAPD-PCR using the primer 2 (OPA-11) comprised of 2-7 bands (Fig. 4-6), which were compared across the molecular weight of ~210-~2800 bp. Out of the total 56 isolates, 54 were typable, giving a typeability of 96.4%. Two isolates of A. butzleri failed to show any bands. There were a total of 29 RAPD types out of the 54 typable strains. A single largest cluster of 13 Arcobacter isolates was observed accounting for 23.21% of the isolates. The discriminatory power of this primer was 0.9336 (Table 2).

Table 2:	Summary of RAPD typing technique

Fig. 1:

The RAPD profiles obtained for Arcobacter butzleri isolates with HLWL85 primer. Lane M: 100 bp plus ladder, Lane 1: CM 10, Lane 2: CM 12, Lane 3: CM 61, Lane 4: CM 68, Lane 5: CM 76, Lane 6: CM 79, Lane 7: CM 86, Lane 8: CM 90, Lane 9: CM93, Lane 10: CM 94, Lane 11: CM 104, Lane 12: P 4, Lane 13: P19, Lane 14: P 37, Lane 15: P 18, Lane 16: P 17, Lane 17: P 44, Lane 18: SF 12, Lane 19: SF 15, Lane 20: SF 55

Fig. 2:	RAPD profiles obtained for Arcobacter cryaerophilus isolates with HLWL85 primer. Lane M: 100 bp plus ladder, Lane 1: CM 4, Lane 2: CM 6, Lane 3: CM 48, Lane 4: CM 70, Lane 5: CM 75, Lane 6: CM 83, Lane 7: CM 98, Lane 8: P 9, Lane 9: P 11, Lane 10: P 20

Fig. 3:	RAPD profiles obtained for Arcobacter skirrowii isolates with HLWL85 primer. Lane M: 100 bp plus ladder, Lane 1: CM 1, Lane 2: SF 59, Lane 3: PI 6

Dendogram showed clustering of Arcobacter isolates in same group from different sources indicative of their epidemiological relationship. A significant genetic diversity was observed from the different and same sources of Arcobacters (Fig. 7-12).

DISSCUSION

In recent years, Arcobacters are considered as potential emerging food and water-borne pathogens. They are increasingly being isolated from a wide range of food products all over the world (Houf et al., 2000; Patyal et al., 2011; Dhama et al., 2013; Ramees et al., 2014a).

Fig. 4:

RAPD profiles obtained for Arcobacter butzleri isolates with OPA-11 primer. Lane M: 100 bp plus ladder , Lane 1: CM 10, Lane 2: CM 12, Lane 3: CM 61, Lane 4: CM 65, Lane 5: CM 76, Lane 6: CM 79, Lane 7: CM 86, Lane 8: CM 90, Lane 9: CM93, Lane 10: PI 61, Lane 11: CM 104, Lane 12: P 4, Lane 13: P19, Lane 14: P 37, Lane 15: P 18, Lane 16: P 17, Lane 17: P 44, Lane 18: SF 12, Lane 19: SF 15, Lane 20: SF 55

Fig. 5:	RAPD profiles obtained for Arcobacter cryaerophilus isolates with OPA-11 primer. Lane M: 100 bp plus ladder, Lane 1: CM 4, Lane 2: CM 6, Lane 3: CM 48, Lane 4: CM 70, Lane 5: CM 75, Lane 6: CM 83, Lane 7: CM 98, Lane 8: P 9, Lane 9: P 11, Lane 10: P 20, Lane 11: P 38, Lane 12: P 48

In the last 5 years, the number of new species has risen exponentially due to the application of molecular techniques such as multiplex-PCR, 16S rRNA gene-RFLP, sequencing of the 16S rRNA gene, ERIC-PCR, AFLP and Pulsed-Field Gel Electrophoresis (PFGE) (Hume et al., 2001; On et al., 2004; Quinones et al., 2007; Collado and Figueras, 2011; Kayman et al., 2012).

Fig. 6:	RAPD profiles obtained for Arcobacter skirrowii isolates with OPA-11 primer. Lane M: 100 bp plus ladder, Lane 1: CM 1, Lane 2: SF 59, Lane 3: PI 6

Fig. 7:	Dendrogram showing RAPD types obtained for Arcobacter butzleri isolates amplified with HLWL85 primer

The present study reports the diversity and epidemiological relationship among Arcobacter spp. isolated from different sources from India using RAPD-PCR.

Fig. 8:	Dendrogram showing RAPD types obtained for Arcobacter cryaerophilus isolates amplified with HLWL85 primer

Fig. 9:	Dendrogram showing RAPD types obtained for Arcobacter skirrowii isolates amplified with HLWL85 primer

Fig. 10:	Dendrogram showing RAPD types obtained for Arcobacter butzleri isolates amplified with OPA-11 primer

Fig. 11:	Dendrogram showing RAPD types obtained for Arcobacter cryaerophilus isolates amplified with OPA-11 primer

Fig. 12:	Dendrogram showing RAPD types obtained for Arcobacter skirrowii isolates amplified with OPA-11 primer

Using primer 1 (HLWL85) RAPD-PCR, yielded bands ranging between 2-8 (500-3100 bp) and of the 56 Arcobacter isolates, 54 typable strains and a total of 35 types were observed, e with discriminatory power of 0.9762. Recently, Suelam (2012) reported 9 genotypes out of 10 Arcobacter isolates recovered from rabbit. In an earlier study by Houf et al. (2002), using a universal random primer 5'-GGTGCGGGAA-3’ high genetic diversity among Arcobacter spp. was reported. AFLP profiling of 73 isolates of A. butzleri from different sources (human infections, chickens, turkeys, ducks, sheep and poultry abbatoir effluent) distinguished 51 subtypes and the similarity level was 91%, of which 39 included single strains. The remaining 34 isolates were scattered among 12 subtypes, each of which contained strains homogeneous with respect to their respective source of isolation (On et al., 2004). In a total of 72 strains of Arcobacter subjected to AFLP profiling 62 distinct types were defined, with evidence of clonal lineages within A. butzleri, A. cryaerophilus and A. skirrowii and a new taxon identified (On et al., 2003). Among Arcobacter isolates, there was a significant level of variation reported from a Farrow-to-Finish Swine Facility (Hume et al., 2001). In dendogram analysis, the clustering pattern of some of the A. butzleri isolates (CM 68, 76, P 17 and 61) of chicken meat, pork and poultry intestinal content origin in the same group indicated possible homogeneity and their phylogenic relationship. Similarly, some of the A. cryaerophilus isolates (CM 48, 7, 75, 83, GF 41 and PI 1) of chicken meat, goat fecal sample and poultry intestinal content showed origin in the same group. A significant level of homogeneity among Arcobacter species from same sources has also been reported previously (Aydin et al., 2007). Isolate of A. butzleri from cattle of beef and dairy farms in the North West of England showed significant diversity using multilocus sequence typing (Merga et al., 2013).

A. cryaerophilus isolates from pork, sheep faeces and goat faeces did not cluster with other isolates, indicating the possibility of heterogeneity as per the source of isolates. Shah et al. (2012) reported12 different clusters consisting of 29 different PFGE patterns within the Arcobacter species using PFGE technique, which is indicative of diversity among the Arcobacter isolates. Fingerprints data generated by RAPD-PCR in the present study showed 21 genotypes of A. butzleri and 14 genotypes of A. cryaerophilus, which showed genomic diversity within the Arcobacter species. Earlier studies have also reported Arcobacers to be having high genetic diversity within and between the species (Atabay et al., 1998, 2006; Collado et al., 2010; Kayman et al., 2012). However, a study using PFGE reported homogeneity among the A. butzleri isolates, indicative of common source of contamination (Rivas et al., 2004). A number of closely related A. butzleri and A. cryaerophilus isolates were found from chicken meat samples which indicate cross contamination of common type of Arcobacter. A high genetic diversity among Arcobacter spp. and their continuous evolving nature has been reported recently from United Kingdom, using MLST technique (Merga et al., 2011). Characterization of 13 A. cryaerophilus and 10 A. butzleri isolates by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) resulted in 10 and 5 different genotypes, respectively (De Smet et al., 2010).

Primer 2 (OPA-11) yielded RAPD-PCR profiles comprising of 2 to 7 bands (210-2800 bp) and of total 56 isolates, 54 were typable, giving a typeability of 96.4%. Two isolates of A. butzleri failed to show any band. There were a total of 29 RAPD types out of the 54 typable strains. A single largest cluster of 13 Arcobacter isolates was observed accounting for 23.21% of the isolates is showing homogeneity among Arcobacter spp. The discriminatory power of this primer was 0.9336. Both the primers yielded a satisfactory typeability and discriminatory power, which indicated RAPD-PCR to be a highly desirable genotyping method. Arcobacter isolates (15) from domestic geese were evaluated for RAPD, wherein 7 A. cryaerophilus, 2 A. butzleri and 6 A. skirrowii isolates produced 6, 2 and 3 distinct profiles, respectively. These showed high heterogeneity among Arcobacter spp. supporting previous study. From the same flocks, the isolates showed same patterns (Atabay et al., 2008). A wide variation of Arcobacter isolate has been reported from poultry (Houf et al., 2002). Nine isolates of A. butzleri obtained from diarrheal patients have been reported to show different ERIC-PCR profiles (Kayman et al., 2012).

In dendogram analysis, clustering together of one of the A. butzleri isolate of sheep feces and human stool in the same group indicated similarity in phylogeny and possibility of zoonotic nature of Arcobacters. The presence of multiple parent genotypes for the three important Arcobacter spp. (A. butzleri, A. cryaerophilus and A. skirrowii) and high genetic recombinations between the progeny of parent genotypes may be reasons for huge amount of heterogeneity in Arcobacters, this is indicative of a multiple source contamination events to be happening (Houf et al., 2002; Aydin et al., 2007). Arcobacter butzleri isolates (92) from different sources gave 13 distinct DNA profiles and some of the isolates originated from different sources contributed the same DNA profiles (Aydin et al., 2007). The possible explanations for the large amount of heterogeneity include multiple sources of contamination, the presence of multiple parent genotypes for all the three species in a single animal and a high degree of genomic recombination among the progeny of parent genotypes (Houf et al., 2002; Collado and Figueras, 2011). The present study reporting for the first time the genotyping and diversity of Arcobacter spp. recovered from different sources (chicken meat, pork, poultry intestines, sheep feces, goat feces and human stools) from India adds to the heterogeneity reports among Arcobacter species worldwide, supporting diversity among same species.

CONCLUSION

The present study reports for the first time the significant rate of genetic diversity among Arcobacter spp. recovered from different sources from India using RAPD-PCR, which is a very rapid tool for such studies. The results revealed that both the primers used (HLWL85, OPA-11) yielded a satisfactory typeability and discriminatory power and the high genetic diversity was observed among the Arcobacter species. The analysis of Arcobacter isolates showed that single host may harbour not only more than one species but also multiple genotypes. Arcobacters showing close clustering between human and animal origin are indicative of zoonotic and public health concerns, for which further explorative studies are needed to reveal more information about the organism as such and to combat any adversary effect of Arcobacters in future.