Experimental Salmonella Typhimurium infection in pigs

Abstract

The genus Salmonella, particularly Salmonella enterica subsp. enterica serovars Choleraesuis and Typhimurium, poses significant challenges to swine production and leads to economic losses from conditions such as septicemia and enterocolitis. We evaluated the effects of experimental infection with Salmonella Typhimurium on clinical signs and anatomopathologic outcomes in pigs. Twenty 90-d-old pigs were divided into 2 groups: G1 received an oral inoculum of 10⁸ cfu of Salmonella Typhimurium; G2 served as a control. Pigs were monitored clinically for 30 d; postmortem examinations and microbiologic analyses were conducted. No significant differences were found in rectal temperature or weight between groups; however, diarrhea episodes were noted in the challenged group starting on day 5 post-inoculation. Isolates of Salmonella Typhimurium were detected intermittently in the challenged group; all positive samples came from pigs without diarrhea. Macroscopic lesions in G1 pigs included button-shaped ulcers in the ileocecal region, enlarged or hemorrhagic mesenteric lymph nodes, and hyperplasia of lymphoid tissue in the colon.

The genus Salmonella is known for its ability to infect a wide variety of hosts, with particular relevance to swine production. Salmonella infection is significant for 2 main reasons: it causes clinical diseases in pigs—primarily due to Salmonella enterica subsp. enterica serovars Choleraesuis and Typhimurium—leading to substantial economic losses from conditions such as septicemia and enterocolitis. Additionally, pigs can serve as a source of Salmonella infection in humans who consume contaminated meat.^9,10

Salmonella bacteria cause salmonellosis in swine, particularly in the growing and finishing phases. Common clinical signs include diarrhea, lethargy, fever, and anorexia, with high morbidity but low mortality, leading to financial losses due to weight loss.^7,12 Most infected animals become persistent carriers and remain clinically healthy throughout their lives. ¹²

The clinical manifestations of salmonellosis, including the severity of clinical signs and macroscopic lesions, are influenced by various factors, such as the route and dose of infection, the serovar involved, the virulence of the strain, and the host’s natural and acquired immunity. ¹ Host-adapted serovars, such as Salmonella Choleraesuis, a swine-adapted serovar, typically cause severe clinical signs post-infection and are not isolated from other species. In contrast, non-adapted serovars, such as Salmonella Typhimurium and Salmonella Enteritidis, result in subclinical infections that can affect multiple animal species and are the primary causes of human salmonellosis.^2,11,12

To better understand the mechanisms of infection, clinical signs, and lesions, we conducted physical evaluations, postmortem examinations, and isolation of Salmonella Typhimurium from feces and organ samples of experimentally infected pigs in a standardized experimental model. All procedures were approved by the Ethics Committee for Animal Use (protocol 3309/21) at the School of Agricultural and Veterinary Sciences (FCAV/UNESP; Jaboticabal, Brazil). Twenty 90-d-old pigs (Landrace × Large White) from a biosecurity-compliant farm were housed in group stalls in a laboratory environment, and received a nutritionally appropriate diet and ad libitum water.

After a 7-d adaptation period, pigs were randomly assigned to 2 experimental groups of 10 animals each. An inoculum was prepared following established guidelines using a Salmonella Typhimurium strain isolated from swine feces (field isolate) that was naturally resistant to nalidixic acid.^8,13 G1 pigs each received an oral inoculum of 10⁸ cfu of Salmonella Typhimurium in 10 mL of brain-heart infusion (BHI; Oxoid) broth; G2 pigs served as controls, and each received a sham inoculation of 10 mL of BHI broth without the bacteria. Pigs were monitored for 30 d post-inoculation (dpi). Laboratory analyses were conducted at the Research Support Laboratory of the Department of Veterinary Clinic and Surgery at FCAV/UNESP.

We used Mueller–Hinton agar and commercial discs for antimicrobial susceptibility testing of the inoculum strain. Rectal swabs were collected before inoculation and subsequently every 5 d until 30 dpi ( Suppl. Fig. 1 ). Fecal consistency was assessed, rectal temperature measured, and animals were weighed at each collection point. Rectal swabs were enriched in selenite cystine broth and subsequently cultured on modified brilliant green agar, followed by biochemical testing to confirm the presence of Salmonella. Every 10 dpi, at least 3 pigs from each group were euthanized for autopsy, during which samples were collected from ileum, cecum, and mesenteric and ileocolic lymph nodes using sterile techniques. Ileum samples from all pigs were preserved in 10% neutral-buffered formalin for histologic evaluation.

Rectal temperatures and weights were analyzed using ANOVA and student t-tests; fecal consistency and isolation results were evaluated using the chi-square test and Fisher exact test as appropriate. No significant differences in rectal temperature were observed between the challenged group and the control group ( Suppl. Table 1 ). The highest rectal temperature was recorded in the control group on day 0. Similarly, there were no significant differences between the 2 groups in the percentage of pigs with diarrhea at any evaluated time ( Table 1 ). Although diarrhea was more common in pigs in the control group on day 0, only the challenged group had episodes of diarrhea after bacterial inoculation. Throughout the experimental period, both groups had 3 episodes of diarrhea; all cases in the challenged group varied in severity; cases in the control group were only mild. No significant weight differences were observed between the 2 experimental groups at any time during the evaluation period ( Suppl. Table 2 ). Salmonella Typhimurium was isolated only from rectal swabs from G1 pigs ( Suppl. Table 3 ).

Table 1.

Number of pigs with diarrhea in the group of animals challenged with Salmonella Typhimurium (G1) and non-challenged animals (G2), before oral inoculation with 10⁸ cfu of Salmonella Typhimurium (day 0) and at 5, 10, 15, 20, 25, and 30 d post-inoculation (dpi).

Dpi	Group
	G1	G2
0	0/10	3/10
5	2/10	0/10
10	0/10	0/10
15	0/6	0/6
20	0/6	0/6
25	1/3	0/3
30	0/3	0/3
Total	3/40	3/40

No significant differences were present at any time between the groups.

Macroscopic lesions in the challenged group included button-shaped ulcers in the ileocecal region ( Suppl. Fig. 2A ) and enlarged or hemorrhagic mesenteric lymph nodes ( Suppl. Fig. 2B ), along with hyperplasia of lymphoid tissue in the colon ( Suppl. Fig. 2C ). Microscopically, diffuse inflammatory infiltrates were in the intestinal lamina propria and various degrees of follicular reactivity in lymph nodes ( Suppl. Fig. 3 ). In contrast, the control group had intact superficial intestinal epithelium with only mild inflammatory infiltrates and follicular reactivity. Infections caused by Salmonella Typhimurium are less invasive than infections caused by Salmonella Choleraesuis, a swine-adapted serovar, and generally cause fibrinonecrotic enterocolitis. ⁷

Episodes of diarrhea in pigs from G1 (challenged animals) began on day 5 (Table 1); rectal temperatures (Suppl. Table 1) remained within the RI. Notably, all positive samples were from G1 pigs that did not have diarrhea (i.e., were likely subclinical carriers; Suppl. Table 4 ). Salmonella Typhimurium was isolated only from G1 organ samples ( Suppl. Tables 5, 6 ), which also had the same antimicrobial susceptibility pattern as the inoculated strain, and were susceptible to florfenicol, cefotaxime, ceftriaxone, polymyxin B, chloramphenicol, sulfamethoxazole–trimethoprim, and resistant to gentamicin, cephalothin, ciprofloxacin, tetracycline, streptomycin, nalidixic acid, ampicillin, and novobiocin.

In a comparative study of experimental infection of 7-wk-old pigs with 10⁸ cfu/mL of Salmonella Derby, Salmonella Typhimurium, and a monophasic variant of Salmonella Typhimurium, transitory diarrhea was noted at 7 and 10 dpi in pigs challenged with 10⁸ cfu/mL of Salmonella Typhimurium. ⁵ Rectal temperatures were normal in pigs experimentally infected with the monophasic variant of Salmonella Typhimurium for the 49 d of the experiment. Salmonella Typhimurium was isolated from rectal swabs of the challenged pigs on all sampling days, except for 1 sample at 14 dpi and 1 sample at 45 dpi; no isolations were made from the control group. ⁵

In another study of experimental infection of weaned pigs with 10⁷ cfu/mL of Salmonella Typhimurium, constant shedding of Salmonella Typhimurium was observed from 1 to 30 dpi in most pigs, with fecal consistency varying from soft-to-watery diarrhea; rectal temperatures >40°C were frequent within the first days after infection. ³ In another experiment, constant bacterial shedding was observed in all weaned pigs challenged from 1–14 dpi, peaking at 5 dpi. ⁶

Our study and the 3 studies cited above used a field strain for the animal challenge, the same experimental oral infection route, and similar inoculum doses (10⁷ or 10⁸ cfu/mL). The observed differences in clinical signs and shedding pattern likely can be explained by other strain (storage time, virulence) and host (age, natural and acquired resistance) characteristics.^4,7

Limitations of our study include: 1) the use of a single serovar of Salmonella Typhimurium (which does not reflect the genetic diversity and virulence of isolates found in the field); 2) a single standardized inoculum dose (exposure to Salmonella in the field occurs variably and, often, at lower doses or intermittently); 3) animals from a single source, which does not reproduce the genetic and environmental variability of different farms; 4) animals of a fixed age, which does not allow extrapolation of results to animals of other age groups and with different immunologic statuses; 5) housing and biosecurity conditions, which do not reflect the conditions typical of many commercial systems; and 6) the short observation period, which may have been insufficient to assess late effects on health and productivity.