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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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