Pathogenicity and Competitive Fitness of Salmonella enterica Serovar 4,[5],12:i:- Compared to Salmonella Typhimurium and Salmonella Derby in Swine

Abstract

Since 2014, Salmonella 4,[5],12:i:- has emerged as the most common serovar of Salmonella enterica identified from swine samples submitted to veterinary diagnostic laboratories in the United States. To compare the pathogenicity of S. 4,[5],12:i:- in swine to the known pathogenic Salmonella Typhimurium and lesser pathogenic Salmonella Derby, 72 pigs (20 per Salmonella serovar treatment and 12 controls) were inoculated with either S. Typhimurium, S. 4,[5],12:i:-, S. Derby, or sham-inoculated and followed for up to 28 days thereafter via rectal temperature, fecal scoring, and fecal culture. Animals were euthanized on days 2, 4, or 28 to determine the gross and histopathologic signs of disease and tissue colonization. The results clearly demonstrate that for the isolates selected, serovar 4,[5],12:i:- possesses similar ability as serovar Typhimurium to cause clinical disease, colonize the tonsils and ileocecal lymph nodes, and be shed in the feces of infected swine past resolution of clinical disease. To compare the competitive fitness of S. 4,[5],12:i:- to S. Typhimurium in swine when co-infected, 12 pigs were co-inoculated with equal concentrations of both S. Typhimurium and S. 4,[5],12:i and followed for up to 10 days thereafter. When co-inoculated, serovar 4,[5],12:i:- was consistently detected in the feces of a higher percentage of pigs and at higher concentrations than serovar Typhimurium, suggesting an increased competitive fitness of 4,[5],12:i:- relative to serovar Typhimurium when inoculated simultaneously into naïve pigs. Whole genome sequencing analysis of the isolates used in these studies revealed similar virulence factor presence in all S. 4,[5],12:i:- and S. Typhimurium isolates, but not S. Derby, providing additional evidence for similar pathogenicity potential between serovars 4,[5],12:i:- and Typhimurium. Altogether, this data strongly supports the hypothesis that S. 4,[5],12:i:- is a pathogen of swine and suggests a mechanism through increased competitive fitness for the increasing identification of Salmonella 4,[5],12:i:- in swine diagnostic samples over the past several years.

Keywords: 4,[5],12:i:-; Derby; Salmonella; Typhimurium; monophasic; pathogenesis; porcine; swine.

Figure 1

Mean rectal temperatures from animal study #1 following inoculation on DPI 0 with three separate isolates of S. 4,[5],12:i:- (A, B, C). The mean and standard error are represented by the vertical bars.

Figure 2

Mean fecal scores from animal study #1 following inoculation on DPI 0 with three separate isolates of S. 4,[5],12:i:- (A, B, C). The fecal scoring system ranged from 1 to 5, with 1–2 being considered normal and 5 being considered severe diarrhea. The mean and standard error are represented by the vertical bars.

Figure 3

Histologic scores from samples collected at necropsy on day 7 following inoculation with three separate isolates of S. 4,[5],12:i:- (A, B, C) in animal study #1. These scores depict the average histologic lesions, as determined by the ulceration, neutrophil infiltration, and crypt elongation and abscessation, and submucosal inflammation, at the time of necropsy on DPI 7. The mean of each isolate-tissue location combination is represented by the horizontal bar with the standard error represented by the vertical line.

Figure 4

Comparison of rectal temperatures of pigs inoculated with Salmonella serovars 4,[5],12:i:- (A), Typhimurium (B), and Derby (C), and non-inoculated control (D) pigs in animal study #2. Asterisks represent significant differences from the mean temperature on DPI 0 (p < 0.05). Each datum point represents the temperature in individual animal; short horizontal bar represents mean temperature within a group.

Figure 5

Comparison of fecal scores of pigs inoculated with Salmonella serovars 4,[5],12:i:-, Typhimurium, and Derby and non-inoculated control pigs in animal study #2. Symbols represent the mean, with vertical bars representing standard error of the mean. Fecal scores of 1–2 are normal, 3 is mild diarrhea, 4 is moderate diarrhea, and 5 is severe diarrhea. Days with significant differences present are represented by asterisks and are detailed in the text.

Figure 6

Comparison of quantitative culture results for Salmonella in feces collected from pigs inoculated with Salmonella serovars 4,[5],12:i:-, Typhimurium, and Derby in animal study #2. The mean CFU/mL and standard error are represented by the horizontal and vertical lines, respectively. Statistically significant differences (p < 0.05) were determined within each group compared to DPI 0 at which time no Salmonella was detectable. Days with significant differences present from DPI 0 are represented by asterisks and were as follows: S. 4,[5],12:i:- on DPI 2, 4, and 7; S. Derby on DPI 2, 4, and 7; S. Typhimurium on DPI 2, 4, 7, and 14.

Figure 7

Representative gross lesions following infection with Salmonella 4,[5],12:i:- and Salmonella Typhimurium. Spiral colon. (A) Pig inoculated with Salmonella 4,[5],12:i:-, DPI 4, severe diffuse fibrinous colitis. (B) Pig inoculated with Salmonella Typhimurium, DPI 4, severe diffuse fibrinous colitis.

Figure 8

Comparison of histologic lesion scores from pigs inoculated with Salmonella serovars 4,[5],12:i:-, Typhimurium, and Derby and non-inoculated control pigs in animal study #2. Histologic lesion scores represent a summary of the ulceration, neutrophil infiltration, crypt elongation and abscessation, and submucosal inflammation at the time of necropsy on DPI 2. Mean and standard error are represented by the symbols and vertical lines, respectively. Different letters indicate statistically significant differences between serovar groups, separated by tissue location (p < 0.05). Histologic lesion scores from pigs euthanized on DPI 4 and 28 depicted in Supplemental Figure 2.

Figure 9

Quantitative fecal culture results from animal study #3 in which pigs were co-inoculated with equal amounts of serovars of Salmonella: 4,[5],12:i:- and Typhimurium. Each data point represents the log₁₀ CFU/mL result in individual animals. The mean and standard error are represented by the horizontal and vertical lines, respectively. Asterisks represent days in which the mean amount of S. 4,[5],12:i:- was significantly different from the mean amount of S. Typhimurium (p < 0.05).

Figure 10

Quantitative culture results from samples collected upon necropsy in animal study #3 in which pigs were simultaneously co-inoculated with equal amounts of two serovars of Salmonella: 4,[5],12:i:- and Typhimurium. Each data point represents the log₁₀ CFU/mL result in individual animals. The mean and standard error are represented by the horizontal and vertical lines, respectively.

Figure 11

The competition index (CI) calculated based on the culture results from animal study #3. The CI was calculated for (A) fecal samples collected throughout the study period and (B) samples collected at the time of necropsy on DPI 4 and 10. The CI represents the fitness of Salmonella 4,[5],12:i:- relative to Salmonella Typhimurium, with a positive CI indicating that S. 4,[5],12:i:- is more fit within the host and a negative CI indicating that S. Typhimurium is more fit within the host. Values closer to 1 or −1 indicates that 4,[5],12:i:- or Typhimurium were the dominant Salmonella serovar in that set of samples, respectively.