Use of 16S rDNA Metagene3cs and classical Microbiology to Assess the bacterial superficial Contamina3on PaAerns in Bovines Classically Slaughtered or following the Halal Ritual KORSAK N. *, TAMINIAU B., HUPPERTS C., DELHALLE L., NEZER C., BURTEAU S., DELCENSERIE V., FERAUCHE C., AND DAUBE G. University of Liège - FARAH, Faculty of Veterinary Medicine, Department of Food Science, Liège, Belgium * [email protected] CBL 20 th edition Lille, France, 17June 2015 INTRODUCTION OBJECTIVES MATERIALS AND METHODS Sampling RESULTS (I) RESULTS (II) DISCUSSION & CONCLUSIONS TVC & Enterobacteriaceae (log UFC / cm 2 ) Metagenetics Methods TVC and Enterobacteriaceae: • non-parametrics statistics (Mann-Whitney) Metagenetics • percentage of reads for each OTU • conversion in cfu / g (in relation with TVC results) • Newcombe-Wilson Stat (Stamp software) Statistics no significant difference observed In several European countries, two cattle slaughtering protocols exist: the classical method, that encompasses a stunning step before the sticking procedure, and the halal method, combining the stunning and the sticking in one single step. The main difference lies in the fact that, in the halal protocol, a single cut with a sharp knife is practiced directly on live cattle, instead of two cutting steps with two different knives for the sticking in the classical slaughtering technique. The unique section in the halal technique results generally in the cross section of trachea and esophagus of cattle. The aim of this study was to seek if the two slaughtering techniques were similar regarding the superficial microbial contamination of carcasses, swabbed between 2 and 4 hours after the killing step. Abattoir Belgian Cattle slaughterhouse: • east of Belgium • practicing both slaughtering methods • separated non- rotating contention box for Halal slaughtering • ligation of esophagus in both cases Period of sampling: Augustus 2013 2 days of sampling 20 swabbed carcasses (10 Classically slaughtered and 10 Halal-slaughtered) Sex of animals: 19 male and 1 Heifer Wet-cotton swabbing method 2 to 4 hours after slaughtering 2 zones: • 1,600 cm 2 (“legal” zone) • neck area (200 cm2) close to the bleeding cut Swabbing Metagenetic analysis targeting the V1-V3 region of the 16S rDNA was performed using the Roche GS junior Mesophilic Total Viable counts (TVC) at 30 °C + counting of Enterobacteriaceae (VRBG 30 °C) Actinobacteria Bacteroidetes Firmicutes Proteobacteria 0 20 40 60 80 Relative population abundance (%) Statistical differences - Phylum level * Fusobacteria 0 1 2 3 4 Relative population abundance (%) Classic_Neckline Halal_Neckline Halal_Surface Classic_Surface *** p value Symbol < 0.05 * < 0.01 ** < 0.001 *** Actinomycetales_unclassified Corynebacteriaceae Beijerinckiaceae Bradyrhizobiaceae Caulobacteraceae Lactobacillaceae Peptostreptococcaceae Planococcaceae 0 5 10 15 20 25 Relative population abundance (%) Statistical differences - family level Classic_Neckline Halal_Neckline Halal_Surface Classic_Surface *** ** Classic_Neckline Halal_Neckline Classic_Surface Halal_Surface 0 2 4 6 8 Bacterial diversity non parametric shannon index Classic_Neckline Halal_Neckline Classic_Surface Halal_Surface 0 500 1000 1500 2000 2500 Bacterial richness Chao1 richness index Classic_Neckline Halal_Neckline Classic_Surface Halal_Surface 0.0 0.2 0.4 0.6 0.8 1.0 Bacterial evenness Shannon evenness index Classical microbiology, non significant differences, but: • higher contamination level in the “legal” zone (1,600 cm2), • and lower level for the neck arae (200 cm2) in the “Halal” group compared to the “Classic” group Metagenetics – “Halal“ vs “Classic”’: • Phylum level • differences in the “legal” zone • Actinobacteria & Fusobacteria • Family level in the “legal” zone for • Corynebacteriaceae, Planococcaceae, Aerococcaceae, Brevibacteriaceae and Clostridiaceae • Family level in the “neck” zone for • Aerococcaceae and Clostridiaceae • Genus level in all the zones for • Brevibacterium, Clostridium, Corynebacterium & Macrococcus • Genus level in all the zones for • Beijerinckiaceae, Bradyrhizobiaceae, and Caulobacteriaceae genera, Rhodoferax & Lactobacillus The slaughtering method does not influence the superficial microbiological pattern in terms of specific microbiological markers of the digestive or respiratory tract. The legal zone of swabbing reflects the hygienic conditions of slaughtering. Further studies are needed to correlate the superficial contamination with hygienic practices The metagenetics reveals different patterns of contamination between swabbing areas and slaughtering techniques Actinomycetales_unclassified Brevibacteriaceae_Brevibacterium Clostridiaceae_Clostridium Dietziaceae_Dietzia Planococcaceae_Caryophanon Beijerinckiaceae_unclassified Bradyrhizobiaceae_unclassified Caulobacteraceae_unclassified Comamonadaceae_Rhodoferax Lactobacillaceae_Lactobacillus 0 2 4 6 8 10 Relative population abundance (%) Statistical differences - genus level *** *** ** * *** * ** * ** * ** *** * * * * * * Corynebacteriaceae_Corynebacterium Staphylococcaceae_Macrococcus 0 10 20 30 40 50 Relative population abundance (%) Classic_Neckline Halal_Neckline Classic_Surface Halal_Surface *** ** *** ***