Background

Informed regulatory oversight and improved process control through industry adoption of Pathogen Reduction/HACCP plans have dramatically reduced the human incidence of disease caused by E. coli O157. A testament to this success is that the proportion of USDA/FSIS ground beef testing positive for this pathogen has fallen by 90% in a decade. An important outcome of which is that the human incidence of E. coli O157 has reduced by almost half and beef is no longer the primary attributed vehicle for human exposure. Yet despite these impressive successes as well as improved sanitary slaughter processes, control of Salmonella in beef remains frustratingly elusive. In fact, the percentage of ground beef samples testing positive for Salmonella has not changed even during the same period of E. coli O157 success. Moreover, the human incidence of Salmonella has not decreased and if anything, it may have slightly increased. 

These conflicting results ostensibly seem at loggerheads because our paradigm of carcass contamination, i.e., hide to carcass surface, is the same for both Salmonella and E. coli O157 and, furthermore, these two pathogens appear to be equally susceptible to validated in-plant interventions. Our preliminary data help explain this paradox in that Salmonella harborage in lymph nodes of cattle appears to be the likely avenue by which Salmonella evades carcass decontamination. Yet these phenomena also provide insights into opportunities to mitigate this food-safety threat. 

To address to food safety threat posed by Salmonella, we developed a collaborative multidisciplinary, government-university team to undertake a series of research activities to fill critical knowledge gaps about sources of Salmonella in food as well as opportunities for its control. Our approach was consistent with the USDA/NIFA/NIFSI priority area 111.C. Consequently, the Beef Checkoff funds provided for this work served as co-support for a successful USDA/NIFA/NIFSI submission (USDA Contract # 2011-51110-31081). 

The stated objectives for this work were to: 

1. Objective 1. Characterize variation in regional, seasonal, and animal-type burden of Salmonella harborage in lymph nodes of cattle presented for harvest;
2. Objective 2. Identify and describe important biological and epidemiological factors associated with Salmonella in lymph nodes of cattle;
3. Objective 3. Develop and test practical solutions for control of Salmonella in the lymph nodes of cattle;
4. Objective 4. Develop and disseminate best practices to improve food safety through mitigation of Salmonella in lymph nodes of cattle. 

Methodology

A lymph node surveillance program was conducted during the winter and spring of 2012. Lymph nodes were collected from 13 packing plants and assayed for the presence and concentration of Salmonella. In brief, surrounding fat and fascia were trimmed from lymph nodes, which were then weighed, surface sterilized by submersion in a boiling water bath, placed into individual filtered sample bags, pulverized using a rubber mallet and then enriched in 80 mL of tryptic soy broth. Enrichments were subjected to immunomagnetic separation using anti-Salmonella beads. Recovered beads were transferred to 3 mL of Rappaport-Vassiliadis broth, incubated at 42C for 18 to 20 h, then streaked onto selective agar plates prior to incubation at 37C for 18 to 20 h. 

In a separate set of studies designed to establish a model of transdermal lymph node infection, initial pilot studies were conducted in which an initial syringe application was trialed but found to be ineffective. In a second study, an allergy skin testing device, each device consists of 10 testing probes fitted at the end with a stainless steel lancet tip that protrudes from the probe enough to provide intradermal, but not sub-cutaneous, administration of the Salmonella. The device was dipped into broth containing Salmonella and then applied to the lower leg of the steer. Following Salmonella treatment, the steers were euthanized and necropsied to collect peripheral lymph nodes for Salmonella culture. Subsequent to these pilot studies, an experiment was conducted to evaluate whether a commercially available vaccine might hold promise in the control of Salmonella in the lymph nodes of cattle. Sixteen Holstein crossbred steers were assigned to either a Control or Vaccine treatment and inoculated in all legs with S. Newport or S. Montevideo using the skin test device described above. Additionally, all steers were inoculated on the belly with S. Senftenberg. Three and six days following Salmonella application, one half of the steers in each treatment were euthanized and necropsied at which time lymph nodes were collected and cultured for Salmonella.

Findings

Findings Thirty four (2.9%) positive lymph nodes have been detected among 1,178 lymph nodes assayed to date. The prevalence of positive lymph nodes harvested from feedlot animals (3.9%) was approximately double that observed among cull cows (1.8%). Some regional variation was observed in that fed plants in Texas (7.7%) appeared to have a greater prevalence of Salmonella in the subiliac lymph node than plants north of Texas (0.0%). 

In terms of the transdermal challenge models, using the syringe, most of the lymph nodes examined in the Salmonella-treated steers were culture positive for the challenge strains. The serogroups of the recovered lymph node isolates matched perfectly with the serogroups of the inoculated strains of Salmonella administered to each leg containing that particular lymph node. However, swelling around the injection sites and mild to moderate lameness was observed in varying degrees in the three Salmonella treated steers and this method of application was deemed unsuitable. Inoculation of the steer using the skin-allergy testing device resulted in Salmonella positive lymph nodes. Isolates cultured from the lymph nodes all belonged to serogroup B, the same serogroup as that administered. No swelling or lameness was observed indicative of successful intradermal administration. The third pilot study, confirmed the device selected to administer the Salmonella challenge is an effective means for inoculating Salmonella transdermally and evaluating subsequent uptake by the peripheral lymph nodes. All lymph node isolates belonged to the same serogroup utilized to inoculate all legs. In the vaccine study, no treatment differences were observed in calves inoculated with Salmonella Montevideo except for a reduction in the percentage of right sub-iliac lymph nodes that were culture positive in the vaccine treatment. The vaccine treatment decreased the percentage of left superficial cervical lymph nodes that were positive compared to control steers inoculated with Salmonella Newport. Slight reductions in the percentage of Salmonella positive sub-iliac (right) and popliteal lymph nodes were also observed in the vaccine treatment, that when combined and analyzed as percentage of all peripheral lymph nodes that were Salmonella positive, tended to be lower in the vaccine treatment.

Implications

Development of effective pre-harvest interventions to reduce the prevalence (and concentration) of Salmonella in the lymph nodes of cattle requires an understanding of the target population. Further, evaluation of potential interventions is enhances when effective models are developed. Our initial lymph node surveillance data confirm the seasonal decrease in lymph node prevalence during the winter and spring time periods. Moreover, regional and animal-type variation is evident in the data in the feedlot prevalence is greater in the south and feedlot prevalence is greater than cull cow prevalence. 

Prevalence is a function of both incidence (rate of new lymph node infections per unit time) and duration of infection. Hence interventions might be developed that target one or both of these determinants of prevalence. In our challenge models, we developed a repeatable challenge model that has substantial utility to evaluate both the duration of lymph node infection as well as opportunities to reduce it. Our preliminary vaccine work indicates that a commercially available vaccine may reduce the duration of infection of Salmonella Newport in lymph nodes.