Food Protection Trends - September/October 2023 - 384

facilities (n = 100 per plant) in Alberta, Canada (33). These
researchers also noted that the percentage of Salmonellapositive
carcasses was reduced to approximately 7% prior
to blast chilling and that Salmonella was not detected on the
surface of pork cuts (n = 100 per plant). Almost all carcasses
in that study were positive for Salmonella; thus, it would be
warranted to investigate the prevalence and concentration
of Salmonella in nonvisceral DTLNs because these will not
be eliminated during carcass processing. Several isolates in
the study by Sanchez-Maldonado et al. (33) also displayed
multidrug resistance, with five isolates resistant to five or
more antibiotics, including those considered to be of very
high importance in human medicine (20, 33). Studies on
the prevalence and concentration of Salmonella in DTLNs
have not been reported in pork in Canada. The lymph nodes
in hog carcasses have been mapped (8) because full removal
of lymph nodes in specific pork cuts and trimmings can be
required in certain markets. The objective of the study was
to determine the prevalence, concentration, and antibiotic
resistance profiles of Salmonella in ground pork and DTLNs
from the belly, ham, shoulder, and salivary glands of hog
carcasses at a pork processing plant over a 10-month period.
MATERIALS AND METHODS
Sample collection and processing
Samples were collected from belly, ham, shoulder, and
salivary gland DTLNs (n = 100 each) from blast-chilled
carcasses during fabrication at a pork processing plant in
Alberta, Canada, from June 2016 through February 2017
(10 samples per tissue per visit). Ground pork samples were
also collected during each visit (n = 100). At the time of
sampling, the plant processed up to 8,500 hogs per day from
multiple swine producers. The DTLNs were located and removed
using the Hog Carcass Lymph Node System Handbook
as a guide (8). Grab samples of ground pork and individual
DTLNs were aseptically placed into sterile sample bags and
placed on ice for transport to the laboratory. Each DTLN was
trimmed and surface sterilized by immersion in boiling water
for 3 s, cooled on ice, weighed, minced into small pieces with
a sterile scalpel, and placed into a sterile stomacher bag.
Minced DTLNs were stomached with 20 ml of sterile buffered
peptone water (BPW) for 2 min and 8 ml of homogenate
was incubated for 24 h at 35°C as a preenrichment step. The
remaining 12 ml of the homogenate was stored at 4°C to allow
samples that were positive for Salmonella to be enumerated by
hydrophobic grid membrane filtration. To enhance detection of
Salmonella among a large background microbiota in DTLNs, 1
ml of preenriched BPW was subjected to immunomagnetic separation
(IMS) with a BeadRetriever (Invitrogen, Thermo Fisher
Scientific, Waltham, MA) using anti-Salmonella magnetic beads
(Invitrogen, Thermo Fisher Scientific) according to standard
procedures prior to enrichment. For ground pork samples, a
25-g subsample was preenriched with 225 ml of BPW.
Detection, enumeration, isolation, and characterization
of Salmonella
Samples from DTLNs and ground pork were enriched by
transferring 1 ml of the preenriched BPW or pre-enriched
BPW+IMS into 9 ml of tetrathionate broth (Oxoid, Nepean,
ON, Canada). Simultaneously, 0.1 ml of the preenriched
BPW was transferred into 10 ml of modified Rappaport
Vassiliadis broth. Both inoculated broths were incubated at
42°C for 24 h. Salmonella isolates were recovered from presumptive
positive enrichment broths according to Health
Canada's Compendium of Analytical Methods (MFHPB-20)
as described by Aslam et al. (3). Briefly, 10 µl of presumptive
positive enrichment broth was streaked onto bismuth
sulphite agar (BD Difco, Fisher Scientific, Mississauga, ON,
Canada), brilliant green sulfa agar (Oxoid), and xylose lysine
desoxycholate agar (BD Difco) and incubated at 35°C
for 48 h. Three agar plates with well-separated, presumptive
Salmonella colonies from each sample were selected, and
one colony from each was restreaked onto MacConkey agar
(Oxoid) and incubated at 35°C for 24 h. Each presumptive
Salmonella colony was screened biochemically on triple sugar
iron, lysine iron, and urea agars (Oxoid) for 24 at 35°C.
Final confirmation was carried out using PCR with primers
targeting the internal transcribed spacer region (13) of
Salmonella.
For enumeration of DTLN samples that were presumptive
positive for Salmonella by enrichment, 250 μl of 0.2% Tween
80 (Sigma-Aldrich) was added to 12 ml of the original
homogenate, mixed by inversion, and then incubated at room
temperature for 5 min. A 10−2
dilution of the homogenate
was then prepared in 0.1% peptone water, and the undiluted
and 10−2
dilution were each filtered through hydrophobic
grid membrane filter units (Neogen) according to standard
procedures (22). After they were incubated for 24 h at 35°C
on xylose lysine desoxycholate agar plates, presumptive
colonies were counted and confirmed.
Up to three Salmonella isolates from each original
sample were selected and used for further characterization.
Serotyping and phage typing were performed by the
Reference Laboratory for Salmonellosis (Public Health
Agency of Canada, Guelph, Ontario). Antimicrobial
susceptibility testing using the Sensititre NARMS (National
Antimicrobial Resistance Monitoring System) gram-negative
CMV3AGPF plate (Trek Diagnostics, Independence,
OH) and the detection of 15 different antibiotic resistance
genes via PCR were carried out as described by SanchezMaldonado
et al. (33).
Whole genome sequencing of Salmonella isolates
Salmonella isolates were streaked from glycerol stocks onto
bismuth sulphite agar and grown overnight at 35°C for 48 h.
A black colony was selected, streaked onto MacConkey agar,
and incubated for 24 h at 35°C; next, an off-white opaque
colony was selected and inoculated into tryptic soy broth
384 Food Protection Trends September/October
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