Salmonella enterica subspecies enterica (here after referred to as Salmonella) is an important group of foodborne pathogens resulting in an estimated 1.2 million illnesses, more than 23,000 hospitalizations, and 450 deaths in the United States (US) each year (Scallan et al., 2011). Financial losses resulting from Salmonella infections are considerable; the United States Department of Agriculture (USDA) Economic Research Service (ERS) reports that the total annual cost of foodborne illness attributed to Salmonella is $3,666,600,031 in medical expenses, loss of productivity, and cost of premature death (Economic Research Service (ERS), 2014). Common vehicles of exposure—many of which are presumably attributable to vertical transmission, fecal contamination and poor food hygiene—include eggs, raw milk and dairy products, poultry, produce, and beef (Guo et al., 2011).

Ground beef is an important vehicle for human exposure to foodborne pathogens (including Salmonella), and was implicated in three outbreaks of salmonellosis between 2010 and 2015 (McLaughlin et al., 2006; Centers for Disease Control and Prevention (CDC), 2012b, 2013). Authors have proposed that the carriage of Salmonella by cattle may contribute to the overall prevalence within ground beef products (Bosilevac et al., 2009). It is a common belief that much of the contamination of beef products results from fecal contamination of hides that in turn contaminate the carcass surface during carcass dressing. Consequently, comprehensive food safety systems have been developed and implemented into slaughter processes to mitigate risks associated with surface contamination. In laboratory settings, the same control strategies that have been implemented generally mitigate both E. coli O157:H7 and Salmonella (Wheeler et al., 2014). These interventions appear to have effectively reduced the burden of foodborne illness attributed to E. coli O157:H7 (Centers for Disease Control and Prevention (CDC), 2015); in 2001, the USDA Food Safety Inspection Service (FSIS) recovered E. coli O157:H7 from 0.9% of ground beef samples, whereas in 2014 the recovery was just 0.04%. However, the same successful outcomes have not been observed for Salmonella (Centers for Disease Control and Prevention (CDC), 2015). Despite the implementation of interventions, the prevalence of Salmonella in ground beef products has remained relatively constant and ranges between 1.6 and 4.2% depending on the size of the sample and the analysis methods employed (Bosilevac et al., 2009; Food Safety Inspection Service, 2011).

Because current in-plant intervention strategies primarily targeting surface contamination have not been successful in decreasing the burden of Salmonella—at least to the same extent as E. coli O157, alternative potential routes of contamination other than sanitation carcass dressing procedures have been investigated (Arthur et al., 2008; Haneklaus et al., 2012; Gragg et al., 2013a). Authors proposed that pathogen contamination of ground beef might also occur via incorporation of contaminated lymph nodes, particularly certain peripheral lymph nodes (PLN) (Arthur et al., 2008; Haneklaus et al., 2012; Gragg et al., 2013a; Li et al., 2015). Harborage within the PLN provides Salmonella protection against surface-oriented mitigation approaches, and this source of contamination would explain the greater prevalence of Salmonella observed in ground beef relative to beef trim destined for ground beef. In prior exploratory work, researchers found that recovery of Salmonella from bovine PLN is not uncommon and that likelihood of recovery might be associated with factors such as season (Gragg et al., 2013a). Given the importance of (a) Salmonella as a food-borne pathogen, and (b) the need to target areas of greatest burden, we set out to more fully characterize and describe the burden of Salmonella in PLN in healthy cattle at slaughter by season, region of the country, and production source. This information will be useful in informing risk assessors and targeted risk abatement if and where it is warranted.

Subiliac PLN were collected from carcasses of cull and feedlot-fattened (FF) cattle presented for slaughter at commercial abattoirs. The subiliac was selected for it's relatively large size, the convenience with which it can be collected without affecting normal operations in commercial abattoirs, and because it has been in the PLN of choice in most prior studies. Samples were collected over approximately a 1-year period. Samples collected in spring (February through May, 2012) and winter (November and December 2012) were designated as being collected in “cooler seasons,” and those sampled in summer/fall (June through October, 2012) were designated as being collected in “warmer seasons.” The convenience sample collection included 12 commercial processing abattoirs, including: seven abattoirs that almost exclusively slaughtered cattle fattened in feedlots, three abattoirs that almost exclusively slaughtered cattle removed (or culled) from breeding herds and dairy cattle (i.e., cull cattle), and two abattoirs that slaughtered a mix of FF and cull cattle. Participating abattoirs were categorized regionally (Figure 1) as regions A, B, and C. Three times in each season, a convenience sample of 75 PLN were collected from each of the abattoirs. Following collection, PLN were shipped to either Texas Tech University or the U.S. Meat Animal Research Center for microbiological analyses. All samples were collected from carcasses of animals after post-mortem veterinary inspection at federally inspected commercial abattoirs. As such, the research described herein did not use live animals.

Previous exploratory studies established that cattle PLN can harbor Salmonella (Arthur et al., 2008; Haneklaus et al., 2012; Gragg et al., 2013a). As such, Salmonella may have the potential to circumvent in-plant carcass surface interventions and ultimately represents a human public health burden through PLN inclusion in ground beef product (Koohmaraie et al., 2012; Li et al., 2015). In the study reported here, we were able to more fully characterize and describe the burden across multiple variables. In particular, it is clear that the highest burden (and possible risk) associated with Salmonella harborage in PLN appears to be limited to particular seasons of the year, regions of the country, and cattle production sources. These data show the prevalence of Salmonella in PLN from FF cattle from Region B during the warmer season (31.1%) is responsible for driving the prevalence statistics for FF cattle higher. In fact, FF cattle PLN sampled in Regions A (1.1%) and C (4.8%) remained relatively low with values similar to those observed for cull cattle PLN. Moreover, examination of the serotypes isolated from PLN of FF cattle, especially from Region B, in comparison with serotypes most commonly isolated from ground beef (Table 2), illustrates the degree to which bovine PLN are likely a source of ground beef contaminated by Salmonella. With Salmonella concentrations ranging from 3.0 to 4.9 log₁₀ CFU/PLN in 17.6% of positive PLN in this study—and the possibility of Salmonella harborage in multiple PLN in a given carcass (Gragg et al., 2013b)—the potential for this source of Salmonella to contaminate ground beef is substantial (Li et al., 2015).

An important consideration in the assessment of risk for human illness with regard to Salmonella infection is serotype and antimicrobial resistance. Some Salmonella serotypes are more likely than others to cause severe infection in humans (Jones et al., 2008; Suez et al., 2013). A diverse set of serotypes was isolated in this study; however, the dominant serotypes identified are either infrequently implicated in laboratory-confirmed cases of human salmonellosis (as in the case of Anatum, Lille, and Cerro), or when they are (as in the case of Montevideo) the sources identified are often produce, spices, cheese, or poultry products, as opposed to beef. Despite their documented presence in ground beef, serotypes such as Montevideo and Anatum may pose less of a risk to human health when present in this commodity than serotypes Typhimurium and Newport. Salmonella serotypes Typhimurium and Newport are observed less frequently in bovine PLN surveys, as reported here and in previous studies (Gragg et al., 2013a,b). However, the increased risk of the presence of these serotypes in ground beef resulting in human illness is evidenced by a number of ground beef related outbreaks attributed to serotypes Typhimurium and Newport (Schneider et al., 2011; Centers for Disease Control and Prevention (CDC), 2012b, 2013). In addition, FoodNet data implicate S. Typhimurium and S. Newport as two of the three serotypes responsible for the majority of laboratory confirmed human salmonellosis cases across 10 sites in the US (Centers for Disease Control and Prevention (CDC), 2012a).

When human salmonellosis does occur, it is often self-resolving and only requires care to prevent dehydration. Nevertheless, in approximately 5% of cases—mostly in immune-compromised individuals—life threatening extra-gastrointestinal infections by Salmonella may occur and antimicrobial treatment is then required (Acheson and Hohmann, 2001). Recommended treatment options for salmonellosis include beta-lactams (i.e., penicillin or third generation cephalosporins) or fluoroquinolones (not recommended for children)—and alternative treatment recommendations include sulfamethoxazole-trimethoprim or azithromycin. A majority of our unique strains were pansusceptible; however, 14.3% were resistant to at least one of the antimicrobial classes recommended for treatment of life threatening salmonellosis. Also worth noting, 1.7% of the isolates were resistant to both beta-lactams and fluoroquinolones—which is certainly of concern.

In keeping with this line of thought, the observation here that S. Dublin was detected at enumerable concentrations in PLN of both FF and cull cattle at harvest in Region C (Figure 2 and Table 2)—and was often multi-drug resistant—was unexpected and warrants further investigation. Salmonella Dublin is a cattle-adapted serotype that is commonly detected in ground beef testing programs (Doerscher et al., 2015; Food Safety and Inspection Service (FSIS), 2015). A recent report brought to light the incidence rate of reported S. Dublin infections in humans has been steadily rising in the US since the 1960s, and that these are often cases of bloodstream infections and hospitalization (Harvey et al., 2017). These observations highlight the need for an increased understanding of genes that contribute to Salmonella's success as a pathogen in the human host.

Caution should be taken in interpretation of seasonal-, regional-, and production source-differences observed in the data presented herein, particularly with respect to inferring causal relationships. It is possible that a seasonal association might have been a causal relationship. Alternatively, the association might have been confounded by an unmeasured variable, such as an atypical regional weather event in, for example, Region B. Production source also reflects many unmeasured variables—for example, age of animal at slaughter; in the US, a vast majority of FF cattle are slaughtered at less than 2 years of age, whereas animals culled from breeding herds are typically older with a much wider variation in age. Further complicating the inference on causal relationships is that, in general, animals can only be sampled once, as PLN collection occurs post mortem except in controlled experimental situations. Despite these limitations, consideration of data reported elsewhere infers there are indeed likely causal associations with season, region, and production sources. Gragg et al. (2013a) reported similar associations of Salmonella burden in PLN to those observed here, and Salmonella was recovered from a substantial proportion of PLN collected from FF cattle presented for slaughter in Texas or Veracruz, Mexico (Sofos et al., 1999; Haneklaus et al., 2012; Gragg et al., 2013b; Brown et al., 2015; Cernicchiaro et al., 2016). Moreover, Salmonella is routinely recovered from the feces of cattle in the southern portion of the US (Kunze et al., 2008; Rodriguez-Rivera et al., 2016), yet remarkably, recovery of Salmonella from cattle feces decreases to the north (Wells et al., 2001; Sorensen et al., 2002; Rao et al., 2010; Morley et al., 2011). While these other reports do not preclude confounding of season, region, or production source in the data reported herein, taken together, they do add support that there are indeed predictable differences in Salmonella burden across regions and seasons, at least within North America.

The Salmonella distribution reported herein can aid in identifying production sources that are more likely contributors of Salmonella that may be more important to human health. Targeted removal of large PLN (i.e., the subiliac, superficial cervical, and popliteal) from cattle at harvest is a possible candidate for control; however, removing PLN in the slaughter process is a difficult and imprecise task. Given the limitations of PLN removal in the dressing process, as well as the sporadic nature of PLN harborage, the need for a more precise and efficient mitigation scheme is evident, and yet such methods remain to be defined. Post-harvest strategies, such as irradiation, could decrease Salmonella prevalence in ground beef products, though many consumers have not yet accepted the concept of using methods such as these. Therefore, pre-harvest interventions may be a more practical. Notably, preliminary research testing of a commercially available Salmonella vaccine suggests this form of intervention may decrease the duration of infection in PLN for specific serotypes, in this case Newport (Edrington et al., 2013b). Further research is needed to evaluate the efficacy of these interventions for decreasing the incidence or duration of infection in bovine PLN.

Ultimately, the key to understanding the variation observed in Salmonella harborage in bovine PLN may lay in defining the implications of production source differences in pre-harvest practices. It is unclear by which route Salmonella enters the animal and then is captured within the PLN. One possible explanation is that it escapes the gastrointestinal tract and mesenteric lymph nodes, and then disseminates systemically. Hanson et al. (2016) reported evidence for vertical transmission from the dam to her fetus. Further, serotypes recovered from PLN can vary within an animal, but more closely resembles serotypes recovered from the hides of animals, whereas serotypes recovered from mesenteric lymph nodes more closely resemble those recovered from the feces (Gragg et al., 2013b). It is conceivable, therefore, that a transdermal route of infection could result in accumulation of Salmonella within PLN that receive lymph from the integument. Abrasions or external parasites such as biting insects might, therefore, contribute to the observed burden of Salmonella in PLN (Edrington et al., 2013a,b; Olafson et al., 2016). It is also possible that all of these proposed routes of infection contribute to the burden of Salmonella in PLN; the data presented here illustrate the differences observed in serotype, prevalence, and concentration among FF and cull cattle; these data make it tempting to suggest that harborage of Salmonella in PLN of cull cattle may be a “remnant” of a systemic infection from which the animal recovered—as evidenced by low Salmonella concentrations in PLN with serotypes Typhimurium and Newport. Further, that observed with FF cattle may be more so the result of recent transdermal infection via abrasions or external parasites. Factors such as management (i.e., pest control, animal health best practices, etc.), age of animal at slaughter, and nutrition are all possible drivers of the illustrated differences. Knowledge gaps in the implications of pre-harvest practices ought to be addressed, as they will be instrumental in developing effective and practical solutions to mitigate the food safety risks associated with harborage of Salmonella in bovine lymph nodes.

Conceived and designed the experiments: GL, TE, and DB-H. Performed the experiments: HW, TB, DB-H, TA, JB, NK, JS, RW, SS, and TW. Analyzed the data and wrote the paper: HW, DB-H, and GL. Critical revision of the paper for important intellectual content: all authors.