Occurrence of thermophilic Campylobacter spp. in chicken meat breeds from an organised farm in thrissur district #

Campylobacteriosis is one of the principal causes of gastroenteritis in the world. Poultry, being the natural reservoir, can harbour large number of Campylobacter in their caeca. The current study was carried out to assess the occurrence of thermophilic Campylobacter spp. in different meat type breeds in an organised farm. Out of the 120 cloacal swabs analysed, 31.67 per cent were found to be positive for Campylobacter spp. There was no significant difference in occurrence of Campylobacter spp. between different meat breeds. On molecular confirmation, majority of the isolates (71.05 per cent) were identified as Campylobacter jejuni and the rest as Campylobacter coli (28.95 per cent). The present study confirms a substantial degree of Campylobacter contamination in meat type chicken breeds in the farm. Good hygienic practices with better biosecurity measures and periodic screening of live poultry, poultry meat and its products are necessary to reduce the foodborne transmission of this pathogen to ensure safer food for consumers.

Campylobacter is a Gram-negative, slender, non-spore forming, bipolar flagellated, spiral or helical shaped bacteria.Thermophilic Campylobacter grow best at 42°C and include C. jejuni, C. coli, C. lari and C. upsaliensis.Campylobacteriosis could be associated with some major complications like Guillain-Barre Syndrome (GBS), Reactive Arthritis (REA), Inflammatory Bowel Disease (IBD), Irritable Bowel Syndrome (IBS) and Miller Fisher Syndrome (MFS) (Vivekanandhan et al., 2022).Consumption of contaminated poultry meat and meat products can account for most cases of human campylobacteriosis.Campylobacter jejuni is usually seen in 2-3 weeks old broiler flocks, which corresponds with a fall in maternal antibody titres (Deepa et al., 2022).The intestinal tract of chicken, especially the caecum and colon, harbours a large number of Campylobacter spp.Campylobacter jejuni belongs to the normal avian microbiota and despite a huge colonisation number of upto 10 8 Colony Forming Units (CFU) per gram of intestinal content, chicken generally remain asymptomatic (Konkel et al., 2007).Campylobacter infection has a complex epidemiology with diverse animal and environmental reservoirs along with numerous risk factors making them a serious public health threat.There is limited information available in Kerala regarding the occurrence of Campylobacter spp. in different meat type breeds in organised farm conditions.Hence, the present study was undertaken to assess the presence of Campylobacter in an organised farm in Thrissur district.

Collection of samples
A total of 120 cloacal swab samples, 30 each from Gramasree, Red Cornish, Australorp and Rhode Island Red (RIR), were collected from an organised farm during a period of three months from December 2021 to February 2022.Swabs were transferred aseptically in Cary-Blair transport media under chilled conditions in thermo-cool containers to the laboratory of Department of Veterinary Public Health, College of Veterinary and Animal Sciences, Mannuthy.Samples were processed within four hours of collection to ensure that the organisms remain viable and culturable.Tenover and Fennell (1992) and followed by molecular confirmation.

Multiplex PCR for identification of Campylobacter spp
The isolates obtained were further subjected to confirmation by multiplex PCR for genus and species identification of Campylobacter spp. as per Denis et al. (1999) with slight modifications.The isolated DNA, from colonies on mCCDA plates were screened for the presence of Campylobacter spp., targeting genus-specific 16S rRNA gene.Species level identification was done by PCR for the detection of C. jejuni specific mapA gene and C. coli specific ceuE gene.The primer sequences used in the study are depicted in Table 1.
The multiplex PCR was done with a reaction mixture in a final volume of 30µL, consisting of 3 µL of 10X PCR buffer (200Mm), 2µL of MgCl 2 (25Mm), 0.75 µL of Taq DNA polymerase (5 Units/µL), 2.50µL dNTP Mix (2 mM ) and 2µL of 16S rRNA ( 1 µL each forward and reverse primers), 2µL of mapA (10pmoles/ µL) and 2µL of ceuE (20pmoles/μL) and 5μL of eluted DNA (50ng/µL) and nuclease free water.The mPCR amplification cycle included initial denaturation at 95°C for 10 min followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 51.8°C for 1 min and extension at 72°C for 1 min.The final stage was an extension cycle at 72° C for 10 min.The PCR

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Occurrence of thermophilic Campylobacter spp. in chicken meat breeds from an organised farm in Thrissur district J. Vet.Anim.Sci.2023.54 (3) : 641-646 was carried out in an automated thermal cycler (Bio-Rad T100 Thermal Cycler, USA).The final PCR products were visualised in two per cent agarose gel using SYBR safe DNA gel stain solution.

Results and discussion
After direct plating onto p-MCCDA agar and following incubation, greyish, flat, spreading type, shiny, mucoid and moistened colonies with a tendency to spread, and with or without metallic sheen was observed (Fig. 1).Out of 120 samples, 38 samples that were positive for Campylobacter on culture were  All the 38 isolates obtained were subjected to molecular confirmation by mPCR by targeting 16S rRNA gene for genus identification and mapA and ceuE for species level identification.An 816 bp amplicon was obtained upon PCR targeting 16S rRNA gene, 589 bp amplicon was obtained for mapA gene and 462 bp amplicons signaled ceuE gene (Fig. 2).The amplicons obtained consequent to PCR of species-specific mapA gene, ceuE gene were purified and were outsourced for sequencing and obtained accession number of C. jejuni-OQ164769, C. coli-OQ164770.per cent of occurrence of Campylobacter in cloacal swabs collected from broiler flocks in Denmark.A similar prevalence study was done by Alam et al. (2020) where they reported 48.4 per cent of conventional farms and 20.7 per cent of good practice broiler farms in Bangladesh was contaminated by Campylobacter.A slightly lower prevalence (28 per cent) from backyard chicken cloacal swabs from Thrissur, Kerala was reported by Jacob et al. (2017).On the contrary, a higher occurrence of Campylobacter, 50.9 in broiler cloacal swabs were observed in Malaysia by Singulingga et al. (2019).Baali et al. (2020) from Algeria recorded an incidence of 65 per cent, an incidence rate higher than that of the present study.
Among the positive 38 isolates, C. jejuni specific mapA gene was amplified in 27 isolates (71.05 per cent) and C. coli specific ceuE gene was present in 11 isolates (29.4 per cent) (Table 3).This finding was in prefect tune with the result of Alam et al. (2020) where the authors identified 68 percent of isolates as C. jejuni.According to Schets et al. (2017) from Netherlands, all the Campylobacter isolates from caecal contents of broilers in a farm were C. jejuni.However, Pillay et al. (2020) reported a higher prevalence of C. coli (60 per cent) among the isolated samples from poultry farms of South Africa.Poultry is frequently colonised with C. jejuni (65-95 per cent), less often with C. coli and rarely with other Campylobacter spp.(Newell et al., 2001).The difference in occurrence of two Campylobacter species varies with region, species, climatic conditions and other environmental factors.The relatively lesser prevalence observed in different meat type breeds in the present study could be due to very good husbandry practices and proper biosecurity measures adopted in the organised farm and also could be due to seasonal variations.For the present study, samples were collected in the months of December to February, which could be correlated to the fact that lower incidence of Campylobacter was

Campy- lobacter spp.
Chon et al. (2012)dure described byChon et al. (2012).It was then incubated under microaerophilic conditions in a CO 2 incubator with 10 per cent CO 2 at 42 o C for 48 h.Isolates were characterised using biochemical tests like gram staining, oxidase test, catalase test, aerobic growth test, hippurate hydrolysis and indoxyl acetate test described by

table 1 .
Primers used for identification of Campylobacter

table 2 .
Heuer et al. (2001)16S rRNA gene, for Campylobacter spp. was amplified in all the 38 isolates.The overall occurrence of Campylobacter spp.from cloacal swabs in the farm by culture and molecular methods was 31.67 per cent.Details of occurrence of Campylobacter spp. in cloacal swabs of different meat type breeds confirmed by molecular method is given in Table2.There was no significant difference in the occurrence of Campylobacter spp. in different meat type breeds.The prevalence of Campylobacter obtained from meat type breeds in present study was in accordance with the results ofHeuer et al. (2001)where they reported a 36.7 Occurrence of Campylobacter spp. in different chicken meat breeds Ns-nonsignificant at (p>0.05)

table 3 .
Distribution of C. jejuni and C. coli in chicken meat breeds Occurrence of thermophilic Campylobacter spp. in chicken meat breeds from an organised farm in Thrissur district J. Vet.Anim.Sci.2023.54 (3) : 641-646