*While the full article for this executive summary is currently under peer review, these initial findings are being made available on BeefResearch.org to enable the industry to act on the research, inform the scientific community of ongoing work, and help prevent duplication of research efforts. Once peer review is complete, a link to the published article will be added to this summary.
KEY TAKEAWAYS
- This project was exploratory to determine appropriate sampling methodology and if synovial fluids (SF) and/or bone marrow (BM) are considerations for potential harbors of Salmonella in a carcass.
- This research demonstrated that Salmonella can survive, hidden from antimicrobial interventions put in place during harvest and processing in alternative lymphatic areas of a beef carcass, though more research is needed to determine the contribution of these locations to the overall load of Salmonella.
- Overall, findings of this study support that Salmonella can be found in both synovial fluid and bone marrow of naturally exposed feedlot cattle, identifying both mediums as potential vectors of the bacteria.
- At this time, positive samples were not able to be enumerated through further serological testing.
BACKGROUND
As of 2024, the Centers for Disease Control and Prevention (CDC) estimates that foodborne nontyphoidal
Salmonella causes an estimated 1.35 million salmonellosis infections annually. Foodborne salmonellosis is an infection that presents most commonly gastroenteritis in humans and can be directly related to the consumption of undercooked beef. Cattle digestive tracts are a known reservoir for
Salmonella, and consequently the consumption of undercooked beef products has historically led to the incidence of salmonellosis. Ground beef has been linked to outbreaks of salmonellosis, and from 2012 to 2019, was linked to 87% of the
Salmonella illnesses implicated from beef. The nature of ground beef processing contributes to its susceptibility for presence of
Salmonella. Beef trim used in ground beef can come from different locations on the carcass, and from various carcasses. An identified reservoir in beef for
Salmonella is lymphatic tissue, more specifically, lymph nodes. As lymph nodes are known to harbor Salmonella, it is important to assess all areas of the lymphatic system that could potentially lead to
Salmonella contamination. In human cases of 3 salmonellosis,
Salmonella has been identified in both joints and bone marrow (5). Synovial fluid and bone marrow interact with the lymphatic system and are susceptible to infections from bacteria, including
Salmonella. Synovial fluid is found in joints and is commonly exposed during the fabrication process of beef carcasses. It is vital to better understand how and where
Salmonella is harbored in beef carcasses, specifically alternative areas of infection. No prior research has specifically evaluated the presence of
Salmonella within synovial fluid and bone marrow of commercial feedlot cattle. The objectives of this study were to evaluate the prevalence of
Salmonella found in beef synovial fluid exposed during beef harvest and fabrication processes and evaluate the presence of
Salmonella found in beef bone marrow.
Methodology
Synovial fluid (SF) and bone marrow (BM) samples were collected from a commercial beef processing facility in Texas. SF was collected using sterile pipettes from the stifle joint of beef carcasses immediately after joint exposure. This anatomical location was selected because it represents an area of the carcass where increased lean area is exposed to SF upon joint exposure and potential cross-contamination through the fabrication process. After collection, all samples were dispensed into sterile 15mL sample collection tubes and kept on ice for transport to the Texas A&M University Food Microbiology Laboratory. Frozen, center-cut femurs (n = 190) were purchased from the same commercial beef processing facility and transported on ice to Texas A&M University Food Microbiology Laboratory. Prior to marrow extraction, femurs were thawed in a 3 °C cooler then flame sterilized using a 70% ethanol solution. A sterile spatula was then used to scoop 25 g of marrow from within the diaphysis of the center cut femur. BM samples were labeled, placed in sterile whirl pack bags, and immediately prepared for further analysis.
For collection, SF samples (n = 190) were split into two collection periods to gather a more diverse sample population. One half of samples (n = 95) were collected during an afternoon shift, and the second half (n = 95) were collected during the following morning shift, surveying different sources of cattle. After transport, samples were enriched in a 1:1 ratio of Tryptic Soy Broth (TSB) and incubated at 37 °C for 48 hours. After the initial incubation period, samples were concentrated via centrifuge at 5000 rpm for 30 minutes, the supernatant of each sample was disposed of, and the remaining pellets were re-enriched with 1 mL of TSB. Pellets were thoroughly vortexed into the media and then incubated for an additional 48 hours at 37 °C. After incubation, the samples were analyzed using the Hygenia BAX Q7 and prepared following the Hygenia protocol for Salmonella Real-Time PCR Assay with both a positive and negative control.
More collections followed the same protocol, with alterations for each sample (n = 190) collected consisting of SF from both stifle joints of the carcass compositely. After transport, approximately 4 mL of synovial fluid from each sample was frozen (-65 °C) and held for further quantification if a presumptive positive was obtained, while the remainder of each sample was concentrated for PCR pre-screening. Samples that resulted in a presumptive positive via PCR detection were thawed for further analysis.
After thawing, presumptive positive samples were diluted and pre-enriched in TSB following most probable number (MPN) guidelines. Samples were then incubated and transferred to selective broths and agars to isolate Salmonella. Colonies that looked positive were selected and tested further to confirm the presence of Salmonella using PCR. Bone marrow samples were also enriched, incubated and tested for the presence of Salmonella utilizing similar methods.
After detecting no positives from the first collection, changes were made in the analysis preparation methodology to confirm this outcome. Collection 1 of SF was prepared for PCR analysis following a different methodology than Collections 2 and 3. The samples from Collections 2 and 3 were prepared in a manner to better revive, propagate, and concentrate bacteria that may have been present, providing the best opportunity to be detected 20 via PCR analysis. In bi-phasic SF study in swine, a
Salmonella prevalence of 0 and 2.63% was reported (2). Additionally, in a study of Holstein calves, 3.3% of SF samples were positive for
Salmonella (9). In both mentioned studies, the swine and calves were inoculated with
Salmonella prior to samples being taken, as opposed to the current study where we surveyed the inherent presence post-harvest in commercial feedlot cattle. In our study, the condition and history of the sampled carcasses prior to harvest was unknown. Cattle lots can vary in the presence of bacteria like
Salmonella. A study done in 2011 revealed that the prevalence of
Salmonella found in bovine lymph nodes can vary between feedyards (4). Though the origin locations of the cattle the current synovial samples were taken from were unknown, this could potentially be one factor impacting the difference of prevalence in the two collections. Nickelson et al. found that the prevalence of
Salmonella in beef lymph nodes was different between cool and warm seasons, with more positives being found in the warm season (7). A study by Belk et al. also revealed that the prevalence of
Salmonella differed between earlier and later feeding stages, as well as reaffirmed findings from Haneklaus et al. that the prevalence of
Salmonella positive lymph nodes varies between feedyards (1, 4).
Collection 3 resulted in four
Salmonella presumptive positive samples of SF. Following MLG 4.14 for quantification, of the 4 presumptive positive samples, no plates for the dilution series of these samples displayed viable colonies for further biochemical confirmation. Therefore, no
Salmonella positives were confirmed following the MPN methodology. Using the MPN index and tables (8), all four samples were determined to have less than 0.3 MPN/mL of
Salmonella. As the Hygenia BAX Q7 detects the presence of
Salmonella, this low level of detection on plates could indicate that the bacteria present were either dead or viable nonculturable. Out of the 190 BM samples collected, two tested positive (1.05%) for
Salmonella through PCR analysis, but were not further analyzed for enumeration. As BM is part of the lymphatic and circulatory systems, it would make sense to conclude that this matrix could harbor the bacterium. In human models, a study by Castro-Equiluz et al. concluded that
Salmonella can survive in lymphatic B cells, formed in BM (3). It is interesting to note that BM may shelter
Salmonella, as B cells are not able to leave the BM until they are fully mature (6). Yet a study in 1983 displayed that inoculated beef BM had a bacteriostatic effect on
Salmonella Anatum (8). This suggests the low bacteria counts found in BM in the current study from commercial feedlot cattle may be associated with this effect, but further research is warranted.
Implications
Findings support the importance of continued research focusing on
Salmonella and its presence/pathology in the lymphatic system of feedlot cattle. Identifying additional mediums within the beef matrix that may harbor
Salmonella could lead to development of additional or alternative food safety control measures, thereby lessening the risk of infection and burden on public health while also decreasing the likelihood of a recall that would have economic impact.