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 * nkorsak@ulg.ac.be

CBL 20th edition Lille, France, 17June 2015

INTRODUCTION OBJECTIVES

MATERIALS AND METHODS Sampling

RESULTS (I)

RESULTS (II)

DISCUSSION & CONCLUSIONS

TVC & Enterobacteriaceae (log UFC / cm2) 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)

Actinobac

teria

Bacter

oidetes

Firmicu

tes

Proteo

bacter

ia0

20

40

60

80

Rel

ativ

e po

pula

tion

abun

danc

e (%

)

Statistical differences - Phylum level

*

Fusobac

teria

0

1

2

3

4

Rel

ativ

e po

pula

tion

abun

danc

e (%

) Classic_NecklineHalal_Neckline

Halal_SurfaceClassic_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

Rel

ativ

e po

pula

tion

abun

danc

e (%

)

Statistical differences - family level

Classic_NecklineHalal_Neckline

Halal_SurfaceClassic_Surface

***

**

Classic

_Nec

kline

Halal_N

eckli

ne

Classic

_Surfa

ce

Halal_S

urface

0

2

4

6

8

Bacterial diversity

non

para

met

ric s

hann

on in

dex

Classic

_Nec

kline

Halal_N

eckli

ne

Classic

_Surfa

ce

Halal_S

urface

0

500

1000

1500

2000

2500

Bacterial richness

Cha

o1 ri

chne

ss in

dex

Classic

_Nec

kline

Halal_N

eckli

ne

Classic

_Surfa

ce

Halal_S

urface

0.0

0.2

0.4

0.6

0.8

1.0

Bacterial evenness

Shan

non

even

ness

inde

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_Caryophanon

Beijerinckiaceae_unclassified

Bradyrhizobiaceae_unclassified

Caulobacteraceae_unclassified

Comamonadaceae_Rhodoferax

Lactobacillaceae_Lactobacillus

0

2

4

6

8

10

Rel

ativ

e po

pula

tion

abun

danc

e (%

)

Statistical differences - genus level

******

*** ****

***

** *******

** * **

Corynebacteriaceae_Corynebacterium

Staphylococcaceae_Macrococcus

0

10

20

30

40

50

Rel

ativ

e po

pula

tion

abun

danc

e (%

)

Classic_NecklineHalal_NecklineClassic_SurfaceHalal_Surface

***** ***

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