INTRODUCTION Along with Salmonella, Campylobacter is one of the important zoonotic foodborne bacterial pathogen that causes diarrhoeal disease in human and animals (WHO, 2020; Garcia et al., 2018). Campylobacter is one of the most frequently occurring bacterial agents of gastroenteritis. The true incidence of gastroenteritis due to Campylobacter spp. is poorly known, particularly in Low and Middle Income Country (LMIC); studies in 3 per 1000 population (WHO, 2012). The pathogen can be transmitted to human via food, water and through contact with farm animals and pets (Elbrissi et al., 2017). The Campylobacter organism is characterized by possessing Gram-negative, spiral, non-spore forming rods that form spherical or coccoid bodies in older cultures (Penner, 1988). They are between 0.2 to 0.9 microns wide and 0.5 to 5 microns long, are motile and usually move with a polar unsheathed flagellum at one or both ends, and are microaerobic with a respiratory-type metabolism, although there are some that grow aerobically or anaerobically (Fitzgerald and Nachamkin, 2011). Campylobacter is a fastidious organism generally requiring specific atmospheres and temperatures to grow, uses menaquinones as their respiratory quinones, does not ferment or oxidize carbohydrates but requires microaerophilic environment (5% O2, 10% CO2 and 85% N2) for growth and isolates causing human gastroenteritis are primarily of the thermotolerant variety which can also grow at 42°C–43°C (Vandamme and De Ley, 1991). Present study is based on the assessment of different artificial media for Campylobacter isolation and identification. MATERIALS METHODS Sample collection and culture. A total of 430 faecal samples of wild animals including mammals and birds were collected during April 2020 to March 2021 from six Zoos and National parks/Sanctuary of Uttarakhand (n=2), Uttar Pradesh (n=2) and Chhattisgarh (n=2), province of India (Table 1). All samples were processed immediately in the department of Veterinary Public Health and Epidemiology, College of Veterinary and Animal Sciences, GBPUA&T, Pantnagar, Uttarakhand, India as per ISO 10272-1:2017(E) guidelines and put for pre-enrichment in Buffered peptone water for overnight at 42°C in CO2 incubator with 5 % CO2 supply, after that enrichment of samples were done in Bolton broth (Hi-Media, Mumbai, India) supplemented with 5% sterile lysed defibrinated sheep blood and FD231 supplement (Hi-Media, Mumbai, India) to be incubated micro-aerobically in CO2 incubator at 42°C for 48 hours. After primary isolation of Campylobacter species five artificial media were assessed, which were broadly categorized into two group- blood free and blood containing media. Culture Media. Blood free media were Modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA) and Hi-chrome Campylobacter agar (HCCA) while blood based media were Columbia blood agar (CBA), Sheep blood agar (BA) and chocolate agar (CA). For mCCDA agar media, CCDA selective supplement (FD135, Hi-Media, Mumbai, India) was added to the basal agar as per the manufacturer’s instructions. Briefly, 22.74 gm of basal medium was suspended in 500 ml of distilled water and dissolve uniformly by boiling, sterilized by autoclaving at 15 lbs pressure (121°C) for 15 minutes and the media were cooled to 45-55°C, then added aseptically rehydrated contents of 1 vial of CCDA selective supplement (FD 135), mixed well and poured into sterile Petri plates (90x15 mm, Genaxy, Solan, Himachal Pradesh, India). For preparation of HCCA agar medium, 1 vial of a selective supplement FD078 (Hi-Media) was added to 29.77 gm HCCDA basal medium in 500 ml distilled water and further were processed same as mCCDA media prepared. For Columbia blood agar (CBA) media, 44.0 gm of CBA basal medium was added in 1000 ml of distilled water, dissolve properly by heating and sterilized by autoclaving, cool down the medium to 45-55°C, 5% v/v sterile defibrinated sheep blood was added, then rehydrated content of Campylobacter growth supplement (FD009) was added and poured about 25 ml into each sterile Petri plate (size 90 × 15 mm, Genaxy, Solan, H. P., India). For, Sheep blood agar (BA) media, 10 per cent defibrinated sheep blood and one vial rehydrated content of Campylobacter growth supplement (FD009) were added in 14.0 gm of Nutrient agar (Hi-media) in 500 ml distilled water. Cooling, sterilizing and pouring into sterile Petri plates were done same as in CBA media. For making Chocolate Agar media, all steps will be same as in Blood Agar media except in place of autoclaving; media were heated at 80°C for 10 minutes with constant agitation. After sterility testing of all prepared media were kept at 4° C and used within 3-4 days. Culture on media After pre-enrichment and enrichment, the obtained isolates were inoculated in mCCDA, HCA, CBA, BA and CA media and incubated in CO2 incubator (New Brunswick, Germany) which were maintained 5 per cent CO2 supply and 42°C temperatures. The incubation was done for 48-72 hrs period. Suspected and well-isolated colonies were subcultured onto the same media for purification which were checked by Gram’s staining and all presumed colonies were further identified by standard biochemical tests as oxidase test, catalase test, Hippurate hydrolysis test, Campylobacter nitrate reduction test, urease test and H2S production on TSI test methods (Atabay and Corry, 1997). Biochemically positive isolates were grown in Tryptone soya broth and aliquoted in 4.5 ml cryo-vials with 20 per cent sterile glycerol and preserved in -80°C for future use. Campylobacter speciation. On the basis of colonies appearance, staining properties and biochemical results, positive isolates were further confirmed through multiplex PCR (Shams et al., 2017) after DNA was extraction (Ertas et al., 2004). Statistical Analysis. The prevalence data of Campylobacter spp. recovered from each culture media was statistically compared by one way analysis of variance followed by least significance difference (DMRT). The analyses were carried out by using SPSS version 26 statistical programme. RESULTS AND DISCUSSIONS All the suspected Campylobacter isolates were identified on the basis of their colonies characteristics, motility test, inability to grow in aerobic condition at 36°C temperature and Gram’s staining features. Campylobacters colonies were small (1-2 mm), circular, flat to slightly raised, sticky, spreading, shiny grey coloured or water droplets on mCCDA, CBA, BA and Chocolate agar plates while in HCA plates Campylobacter species appear mauve to purple coloured colonies. The organisms appeared as pink coloured Gram-negative rod, spiral curved rods with comma shaped (S) or gull wing appearance cells. The similar colonies characteristics were also recorded by Monika (2014), and Garhia, (2017). The recovery rate of Campylobacter spp. in this study was higher (Table 2) in CBA culture media than that of mCCDA, HCA, BA and Chocolate agar culture method studied. The overall positivity of Campylobacter spp. was 13.72% (59/430) with Campylobacter jejuni (7.67 %) and Campylobacter coli (6.05 %) agreeing with findings of Acke et al. (2009). After enrichment, plating on CBA with selective supplement yielded a significantly higher (P<0.05) prevalence as 4.65% of Campylobacter species. The similar observations were also reported by Hutchinson and Bolton (1984). However, we could observe 3.72% recovery on mCCDA as reported by Roma (2019); Ansari (2021); Corry and Atabay (1997); and 3.48% on BA (Byrne et al., 2001) showing none significant differences (Table 3) and they are followed by CA (2.0%) as also reported by Aspinall et al. (1996) and on 1.16% HCCA (Humphrey et al., 2007). Multiplex PCR results confirmed its speciation as well as sensitivity of each culture methods. As the majority of Campylobacter spp. were isolated by CBA media with selective supplement. It was also found that pre-enrichment and an enrichment step reduces the transport stress and enhances the recovery of Campylobacter spp than either direct plating or filtration on to selective media. Since CBA showed higher recovery rate of Campylobacter spp (P<0.05), hence it might be more accurate blood based assessment method for the actual prevalence of Campylobacter spp. in the sampled population. In blood free method mCCDA would be comparatively better for assessment of Campylobacter spp prevalence. In both the method - CBA and mCCDA, hemin (Fe3+) and charcoal respectively act as source of energy and oxygen quenching agent, which is needed for growth and microaerophilic environment.