Use of 16S rDNA metagenetics and classical microbiology to assess the bacterial superficial contamination patterns in bovines classically slaughtered or following the halal ritual

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 20th _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 cm2 _(“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 Re la tiv e p o p u la tio n a b u n d an ce (% )

Statistical differences - Phylum level

* Fusobacteria 0 1 2 3 4 Re la tiv e p o p u la tio n a b u n d an ce (% ) Classic_Neckline Halal_Neckline Halal_Surface Classic_Surface *** p value Symbol < 0.05 * < 0.01 ** < 0.001 *** Actinomycetales_unclassified Corynebacteriaceae Beijerinckiaceae BradyrhizobiaceaeCaulobacteraceaeLactobacillaceae Peptostreptococcaceae Planococcaceae 0 5 10 15 20 25 Re la tiv e p o p u la tio n a b u n d an ce (% )

Statistical differences - family level

Classic_Neckline Halal_Neckline Halal_Surface Classic_Surface *** ** Classic_NecklineHalal_NecklineClassic_Surface Halal_Surface 0 2 4 6 8 Bacterial diversity non pa ra m e tr ic s ha nnon inde x Classic_NecklineHalal_NecklineClassic_Surface Halal_Surface 0 500 1000 1500 2000 2500 Bacterial richness Ch ao 1 ri ch n e s s in de x Classic_NecklineHalal_NecklineClassic_Surface Halal_Surface 0.0 0.2 0.4 0.6 0.8 1.0 Bacterial evenness Sh a n n o n e v e n n es s in d e x

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 •  ì Actinobacteria & î Fusobacteria •  Family level ì in the “legal” zone for

•  Corynebacteriaceae,

Planococcaceae, Aerococcaceae, Brevibacteriaceae and Clostridiaceae

•  Family level ì in the “neck” zone for

•  Aerococcaceae and

Clostridiaceae

•  Genus level ì in all the zones for •  Brevibacterium, Clostridium,

Corynebacterium & Macrococcus

•  Genus level î 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_CaryophanonBeijerinckiaceae_unclassifiedBradyrhizobiaceae_unclassifiedCaulobacteraceae_unclassifiedComamonadaceae_RhodoferaxLactobacillaceae_Lactobacillus

0 2 4 6 8 10 Re la tiv e p o p u la tio n a b un d a nc e (%)

Statistical differences - genus level *** *** ** * **** ** * ** **** *** * * * ** Corynebacteriaceae_Corynebacterium Staphylococcaceae_Macrococcus 0 10 20 30 40 50 Re la tiv e p o pu la tio n a b u n da n c e (%) Classic_Neckline Halal_Neckline Classic_Surface Halal_Surface *** ** _*** ***