Food Protection Trends - March/April 2022 - 101

infection from eating pork, during a 40-year span of time,
is estimated at 1 in 100 when considering per person-year,
consuming the average number of servings or kilograms (12).
More recently, the Foodborne Disease Outbreak Surveillance
System (8) reported a total of 43 outbreaks, 1,539 illnesses, 206
hospitalizations, and 3 deaths associated with Salmonella from
pork between the years 2009 and 2015. In the European Union,
pork has been recognized as the third most common cause of
salmonellosis infections, behind eggs and poultry meat (2).
A variety of pork products have been associated with
Salmonella contamination; however, prevalence rates differ
between ground pork and pork chops (3). These differences
are likely due to the compilation of muscle and adipose tissue
from many animals during grinding, contamination during
grinding procedures, and incorporation of lymph nodes during
ground pork production (23). The literature suggests that
contamination of ground pork or other comminuted pork
products with Salmonella may be due to contaminated lymph
nodes (4, 23) due to the location of some peripheral lymph
nodes (also referred to as deep-tissue lymph nodes; DTLNs)
within the adipose tissue that may be incorporated into ground
pork (4). In addition to ground pork, contaminated lymph
nodes may also cross-contaminate carcasses and primal cuts if
they are cut open during slaughter (5, 7, 15) or fabrication.
The U.S. Department of Agriculture Food Safety Inspection
Service (FSIS) commenced exploratory sampling and testing
for Salmonella in raw pork cuts, both intact and nonintact, as
well as in raw comminuted pork products in fiscal year 2019
(18). Research aimed at understanding the distribution of
Salmonella in lymph nodes located throughout market hog
carcasses is necessary to support FSIS testing priorities and the
pork industry and to inform future intervention strategies (e.g.,
lymph node removal) for Salmonella control. The primary
objective of this study was to address these knowledge gaps by
mapping the Salmonella prevalence in splanchnic (mesenteric
and tracheobronchial) and somatic (subiliac and inguinal)
lymph nodes of market hog carcasses.
MATERIALS AND METHODS
Sample collection
All lymph node samples were collected post mortem at
a commercial swine processing facility in the midwestern
United States. On two different sampling days (7 and 21
October 2019), four lymph nodes were collected per carcass
from a total of 25 carcasses, resulting in approximately 100
lymph node samples collected per sampling day (4 lymph
nodes per carcass × 25 carcasses = 100 lymph nodes). A
convenience sample of 197 lymph nodes were collected
in total (three lymph node samples were unable to be
collected). Carcasses were removed from the processing
line, and four lymph node samples were collected from each
carcass, including tracheobronchial, mesenteric, subiliac, and
inguinal. Relevant lot information and general observations
were recorded for each carcass. Lymph node samples were
placed in sterile Whirl-Pak bags (Nasco, Madison, WI) and
transported to the laboratory at Kansas State University.
All samples were stored at 4°C until processing, which was
completed within 24 h after collection.
Sample processing
Lymph nodes were processed as described by Chaves et
al. (7). Briefly, fat and fascia were removed, and the lymph
nodes were weighed, surface sterilized in boiling water for
approximately 5 s, placed individually in filter Whirl-Pak bags
(Nasco), and pulverized with a rubber mallet. All samples were
then enriched with 80 ml of tryptic soy broth (TSB; BD Difco,
Franklin Lakes, NJ), stomached for 1 min (Stomacher 400
Circulator, Seward, Bohemia, NY), and incubated for 6 h at
42°C. Following incubation, samples were held at 4°C for ≤12
h until subsequent analysis.
Detection of Salmonella
Following incubation, enrichments were subjected to
automatic immunomagnetic separation as previously described
by Chaves et al. (7) using anti-Salmonella Dynabeads (Applied
Biosystems, Waltham, MA) and a KingFisher mL Purification
System (Thermo Fisher Scientific, Waltham, MA) according
to manufacturer's guidelines. Recovered anti-Salmonella
beads were transferred to 3 ml of Rappaport-Vassiliadis broth
(Oxoid, Lenexa, KS) and incubated at 35°C for 18 h. Samples
were held at 4°C for ≤12 h until subsequent analysis. Following
incubation, the Rappaport-Vassiliadis broth was streaked
on xylose lysine desoxycholate (XLD; Remel, Lenexa, KS)
and brilliant green sulfa (Remel) agar, and then incubated
for 18 to 24 h at 35°C. Following incubation, XLD plates
were considered presumptive positive if colonies appeared
black on a pink background; brilliant green sulfa plates were
considered presumptive positive if colonies appeared light
pink. Presumptive positive colonies were subjected to latex
agglutination (Wellcolex Colour Salmonella, Remel) and then
streaked on XLD to isolate individual Salmonella colonies.
XLD plates were incubated for 18 to 24 h at 37°C, and one
isolated colony from each plate was then transferred to 9 ml
of TSB. TSB tubes were incubated at 37°C for 24 h and then
frozen in 1-ml aliquots with 10% glycerol (Thermo Fisher
Scientific) at −80°C for subsequent PCR confirmation.
Salmonella confirmation
Frozen isolates were resuspended in 9 ml of TSB, incubated
at 35°C for 18 to 24 h, and then subjected to PCR to confirm
the presence of Salmonella using MicroSEQ Salmonella spp.
kit (Applied Biosystems) with RapidFinder Express software
(Applied Biosystems) and a calibrated ABI 7500 Fast RealTime
PCR machine (Applied Biosystems).
Statistical analysis
Prevalence, standard error, and confidence intervals were
calculated using GraphPad Prism 9.0 (GraphPad Software,
March/April Food Protection Trends 101
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