Background 

Recent research suggests that the peripheral lymph nodes (PLN) in cattle may be a contributor to the contamination of ground beef with Salmonella. The source of Salmonella in the PLN has not been clearly defined. The gastrointestinal tract (GIT) in cattle is often asymptomatically colonized by Salmonella, and Salmonella has been reported in the mesenteric lymph nodes of seemingly healthy cattle, therefore conventional wisdom supports systemic distribution via an oral route from the GIT to the mesenteric lymph nodes, and subsequently to the PLN. Research utilizing an experimental model of infection, demonstrated Salmonella recovery from the PLN in cattle following oral Salmonella administration. However, significant challenge dose(s) were required, suggesting other routes of infection are involved. 

One such route may involve the mucous membranes. Some fly species, such as the house fly (Musca domestica) are common pests on cattle production facilities, and often frequent the nose and eyes of cattle. These flies lay their eggs and develop in rotting and decomposing substrates, including manure, and may acquire bacteria internally and externally. Mechanical transmission of bacteria has been reported for house flies. Bacteria replication occurs within house fly mouthparts and these bacteria may be stored in the crop (facilitating recovery in the vomitus), or distributed throughout the GIT and subsequently shed in the feces. Taken together, the ability of house flies to transfer viable bacteria in their vomitus, feces, or via direct contact (external contamination) suggest the potential role of flies and the mucous membranes in dissemination of Salmonella to the PLN.   

The objective of this study was to determine if Salmonella will reach the PLN of cattle following application of Salmonella to the mucous membranes. Initial proof‐of‐concept studies will apply Salmonella directly to the mucous membranes of the nose. If successful, follow‐up studies will utilize externally‐contaminated flies as the transmission vehicle to the membranes. 

Methodology   

Holstein steers (~BW = 100 kg) were housed in individual indoor pens (one steer per pen). For treatment administration, cattle were haltered and tied securely to restrict movement within the pen. A foam tipped swab was used to administer Salmonella Cerro (log phase; avg. concentration 12.1 x 108 CFU/ml) to the inside of each nostril of three steers designated for the treatment. Two control steers were treated similarly with trypitic soy broth only. Treatment administration was done in the afternoon on three (study 1) and four (study 2) successive days. Fecal samples were collected daily, and on day 5 all steers were euthanized, necropsied and peripheral and mesenteric lymph nodes were collected for bacterial culture.   

A third experiment was completed. In study 3, Holstein steers (~BW = 100 kg) were housed in individual indoor pens (one steer per pen). For treatment administration, cattle were haltered and tied securely to restrict movement within the pen. A foam tipped swab was used to administer Salmonella Cerro (log phase; avg. concentration 8.5 x 108 CFU/ml) to the inside nostril of each of the three steers designated for the treatment group. The two control steers were treated similarly with trypitic soy broth only. In study 1 and 2, the steers were observed to lick their nostrils after halter removal, but typically did not exhibit this behavior when the halter remained on. Therefore, all steers remained haltered for 30 minutes following treatment. Treatements were administered in the afternoon on each of eight consecutive days. Fecal samples were collected on four occasions the week prior to Salmonella inoculation and daily during the 8 day treatment period. The day following the last treatment administration, all steers were euthanized and necropsied. The right and left sub‐iliac, popliteal, superficial cervical, parotic, retropharangeal, mandibular lymph nodes and mesenteric lymph node were collected for bacterial culture. 

Findings   

The inoculated strain of Salmonella Cerro, as indicated by serogrouping of the isolates, was not recovered from any of the peripheral lymph nodes in study 1 or study 2. All isolates from one Salmonella‐treated animal (Study 2) recovered following quantitative and qualitative culture of the mesenteric lymph node, were identified as serogroup K, indicative that it was the challenge strain Cerro. The majority of lymph node isolates belonged to serogroups C1 (69.9%) and C2 (26.5%) with one isolate identified as serogroup E1. In study 3, the increase in number of inoculation events resulted in the successful recovery of the challenge strain, Salmonella Cerro (serogroup K) in the lymph nodes. Only a few PLN contained quantifiable concentrations of Salmonella, while many (44%) were positive following enrichment for the challenge strain. One of the Salmonella‐treated steers had only one PLN culture positive for serogroup K. The mesenteric lymph node contained quantifiable concentrations of the challenge strain in all three treated steers. Lymph nodes from the head (parotid, retropharangeal and mandibular) were mostly positive for the challenge strain of Salmonella, in the exception of the parotid, which was positive only in one steer. Of the two steers that served as unchallenged controls in study 3, one steer had PLN test positive for Salmonella in the sub‐iliac nodes. The right node tested positive for serogroups K and the left node tested positive for serogroup C₂.   

Most steers in study 1 were shedding Salmonella in their feces daily. However, Salmonella was detected at very low levels and detected only following enrichment of the samples. Steers inoculated with Salmonella were all culture negative the first day, but started shedding primarily the challenge strain (serogroup K) on the second and third days, and continued to shed serogroup K throughout the experimental period. Fecal shedding results from study 2 were similar. The results of fecal samples in study 3 showed that all inoculated steers shed the challenge strain in their feces on multiple occasions throughout the experiment, with two of the three steers shedding serogroup K 24 hours post‐inoculation. In study 3, the unchallenged controls also tested positive for Salmonella in the feces, with the identified serotypes being C₁ and C₂ with one steer shedding serotype K on days 6 and 7. 

It is unclear what route(s) were used that resulted in Salmonella ultimately reaching the PLN. It would appear that the mucous membranes functioned as expected and the challenge strain of Salmonella was captured in the mucous, swallowed and deposited into the gastrointestinal tract, and subsequently shed in the feces. It has been suggested that Salmonella in the PLN of cattle may originate from the gastrointestinal tract and transport to other PLN is facilitated by the mesenteric lymph nodes.   

Seeking other viable explanations, the lymph nodes of the head were examined and the challenge strain of Salmonella was recovered by the majority (78%) of them. All three nodes examined receive afferent vessels from nasal cavity and muzzle, therefore, it was not surprising that the challenge strain was recovered in these nodes. It is hypothesized that the added inoculations in study 3 may have exceeded the ability of these nodes in the head to effectively handle all of the Salmonella, thus resulting in its culture from the PLN. Detection of the challenge strain of Salmonella in the control animals further confounds interpretation of the results. Cross‐contamination, exposure through aerosolized Salmonella, or any number of other routes of infection may be responsible for these results. 

Implications 

Results of the third experiment, in which the number of inoculation events was doubled and resulted in uptake of the challenge strain of Salmonella by the PLN following exposure the mucous membranes suggests that a threshold was met and exceeded that allowed for the acquisition of Salmonella by the PLN. However, a threshold is hard to explain when looking at the ease in which the control animal became contaminated with the challenge strain in this experiment. It appears from this research and other projects conducted, that the PLN rapidly and easily acquire Salmonella when exposure is “natural” versus laboratory strains inoculated directly into the animal.   

Results of this research demonstrated uptake of Salmonella by the PLN of cattle following application of Salmonella to the mucous membranes of the nose. Therefore, while it is feasible that filth flies may serve as a vector for Salmonella transmission to the PLN via contaminating mucous membranes, the dose required in this research would suggest that a substantial number of fly‐related inoculation events would be required. Unless, as supported by this and other research, “natural” Salmonella strains are easily acquired and far more invasive than laboratory strains. The route in which Salmonella is transported to the PLN remains somewhat of a mystery.