Investigation of pathogenicity, competitive fitness, and novel methods for rapid diagnosis of Salmonella 4,[5],12:i:- in swine

Salmonella enterica subsp. enterica serovar 4,[5],12:i:- is widely accepted to be a monophasic variant of Salmonella Typhimurium. Salmonella 4,[5],12:i:- has been increasing significantly in prevalence worldwide over the past decade while S. Typhimurium has been subsequently decreasing. Both Salmonella serovars 4,[5],12:i:- and Typhimurium are contained in Salmonella serogroup B, which is currently the predominant serogroup in swine in the United States. However, this serogroup contains serovars of a wide range of pathogenic potential, with Typhimurium generally being associated with enterocolitis and Agona and Derby generally being considered to be of lesser pathogenicity. The pathogenic potential for S. 4,[5],12:i:- in swine is largely unknown but is hypothesized to be similar to S. Typhimurium based on genetic similarities. Current diagnostic procedures for the detection and identification of Salmonella have a wide range of sensitivities, and culture-based protocols have a prolonged turn-around time to serovar-level identification due to the complex serotyping process. To facilitate more rapid detection and serovar-level identification, the work conducted within this thesis validated a multiplex real-time PCR capable of detecting Salmonella spp. in general and differentiating S. 4,[5],12:i:- and S. Typhimurium from other lesser-pathogenic serovars. Further work was focused on determining the level of clinical disease, colonization of tissues, and persistence of infections to be expected with the S. 4,[5],12:i:- in swine. For this study, pigs were experimentally infected with S. 4,[5],12:i:-, S. Typhimurium, or S. Derby to compare S. 4,[5],12:i:- to the pathogenic S. Typhimurium and less-pathogenic S. Derby. This work demonstrated that S. 4,[5],12:i:- induces similar levels of clinical disease, tissue colonization, and persistent infections to that of S. Typhimurium, both of which were more severe than that of S. Derby. Simultaneous infection of swine with equal amounts of S. 4,[5],12:i:- and S. Typhimurium revealed S. 4,[5],12:i:- is detected in higher levels in the feces, tonsils and ileocecal lymph nodes than S. Typhimurium, indicating a potential increased competitive fitness of the monophasic variant. The collective results of these studies provide improved diagnostics and needed insight into the disease causing ability of an increasingly prevalent serovar of Salmonella that is capable of infecting swine, humans, and other species

\u3ci\u3eSalmonella enterica\u3c/i\u3e induces biogeography-specific changes in the gut microbiome of pigs

Swine are a major reservoir of an array of zoonotic Salmonella enterica subsp. enterica lineage I serovars including Derby, Typhimurium, and 4,[5],12:i:- (a.k.a. Monophasic Typhimurium). In this study, we assessed the gastrointestinal (GI) microbiome composition of pigs in different intestinal compartments and the feces following infection with specific zoonotic serovars of S. enterica (S. Derby, S. Monophasic, and S. Typhimurium). 16S rRNA based microbiome analysis was performed to assess for GI microbiome changes in terms of diversity (alpha and beta), community structure and volatility, and specific taxa alterations across GI biogeography (small and large intestine, feces) and days post-infection (DPI) 2, 4, and 28; these results were compared to disease phenotypes measured as histopathological changes. As previously reported, only S. Monophasic and S. Typhimurium induced morphological alterations that marked an inflammatory milieu restricted to the large intestine in this experimental model. S. Typhimurium alone induced significant changes at the alpha- (Simpson’s and Shannon’s indexes) and beta-diversity levels, specifically at the peak of inflammation in the large intestine and feces. Increased community dispersion and volatility in colonic apex and fecal microbiomes were also noted for S. Typhimurium. All three Salmonella serovars altered community structure as measured by co-occurrence networks; this was most prominent at DPI 2 and 4 in colonic apex samples. At the genus taxonomic level, a diverse array of putative short-chain fatty acid (SCFA) producing bacteria were altered and often decreased during the peak of inflammation at DPI 2 and 4 within colonic apex and fecal samples. Among all putative SCFA producing bacteria, Prevotella showed a broad pattern of negative correlation with disease scores at the peak of inflammation. In addition, Prevotella 9 was found to be significantly reduced in all Salmonella infected groups compared to the control at DPI 4 in the colonic apex. In conclusion, this work further elucidates that distinct swine-related zoonotic serovars of S. enterica can induce both shared (high resilience) and unique (altered resistance) alterations in gut microbiome biogeography, which helps inform future investigations of dietary modifications aimed at increasing colonization resistance against Salmonella through GI microbiome alterations

Salmonella enterica induces biogeography-specific changes in the gut microbiome of pigs

Swine are a major reservoir of an array of zoonotic Salmonella enterica subsp. enterica lineage I serovars including Derby, Typhimurium, and 4,[5],12:i:- (a.k.a. Monophasic Typhimurium). In this study, we assessed the gastrointestinal (GI) microbiome composition of pigs in different intestinal compartments and the feces following infection with specific zoonotic serovars of S. enterica (S. Derby, S. Monophasic, and S. Typhimurium). 16S rRNA based microbiome analysis was performed to assess for GI microbiome changes in terms of diversity (alpha and beta), community structure and volatility, and specific taxa alterations across GI biogeography (small and large intestine, feces) and days post-infection (DPI) 2, 4, and 28; these results were compared to disease phenotypes measured as histopathological changes. As previously reported, only S. Monophasic and S. Typhimurium induced morphological alterations that marked an inflammatory milieu restricted to the large intestine in this experimental model. S. Typhimurium alone induced significant changes at the alpha- (Simpson’s and Shannon’s indexes) and beta-diversity levels, specifically at the peak of inflammation in the large intestine and feces. Increased community dispersion and volatility in colonic apex and fecal microbiomes were also noted for S. Typhimurium. All three Salmonella serovars altered community structure as measured by co-occurrence networks; this was most prominent at DPI 2 and 4 in colonic apex samples. At the genus taxonomic level, a diverse array of putative short-chain fatty acid (SCFA) producing bacteria were altered and often decreased during the peak of inflammation at DPI 2 and 4 within colonic apex and fecal samples. Among all putative SCFA producing bacteria, Prevotella showed a broad pattern of negative correlation with disease scores at the peak of inflammation. In addition, Prevotella 9 was found to be significantly reduced in all Salmonella infected groups compared to the control at DPI 4 in the colonic apex. In conclusion, this work further elucidates that distinct swine-related zoonotic serovars of S. enterica can induce both shared (high resilience) and unique (altered resistance) alterations in gut microbiome biogeography, which helps inform future investigations of dietary modifications aimed at increasing colonization resistance against Salmonella through GI microbiome alterations

Investigation of pathogenicity, competitive fitness, and novel methods for rapid diagnosis of Salmonella 4,[5],12:i:- in swine

Salmonella enterica subsp. enterica serovar 4,[5],12:i:- is widely accepted to be a monophasic variant of Salmonella Typhimurium. Salmonella 4,[5],12:i:- has been increasing significantly in prevalence worldwide over the past decade while S. Typhimurium has been subsequently decreasing. Both Salmonella serovars 4,[5],12:i:- and Typhimurium are contained in Salmonella serogroup B, which is currently the predominant serogroup in swine in the United States. However, this serogroup contains serovars of a wide range of pathogenic potential, with Typhimurium generally being associated with enterocolitis and Agona and Derby generally being considered to be of lesser pathogenicity. The pathogenic potential for S. 4,[5],12:i:- in swine is largely unknown but is hypothesized to be similar to S. Typhimurium based on genetic similarities. Current diagnostic procedures for the detection and identification of Salmonella have a wide range of sensitivities, and culture-based protocols have a prolonged turn-around time to serovar-level identification due to the complex serotyping process. To facilitate more rapid detection and serovar-level identification, the work conducted within this thesis validated a multiplex real-time PCR capable of detecting Salmonella spp. in general and differentiating S. 4,[5],12:i:- and S. Typhimurium from other lesser-pathogenic serovars. Further work was focused on determining the level of clinical disease, colonization of tissues, and persistence of infections to be expected with the S. 4,[5],12:i:- in swine. For this study, pigs were experimentally infected with S. 4,[5],12:i:-, S. Typhimurium, or S. Derby to compare S. 4,[5],12:i:- to the pathogenic S. Typhimurium and less-pathogenic S. Derby. This work demonstrated that S. 4,[5],12:i:- induces similar levels of clinical disease, tissue colonization, and persistent infections to that of S. Typhimurium, both of which were more severe than that of S. Derby. Simultaneous infection of swine with equal amounts of S. 4,[5],12:i:- and S. Typhimurium revealed S. 4,[5],12:i:- is detected in higher levels in the feces, tonsils and ileocecal lymph nodes than S. Typhimurium, indicating a potential increased competitive fitness of the monophasic variant. The collective results of these studies provide improved diagnostics and needed insight into the disease causing ability of an increasingly prevalent serovar of Salmonella that is capable of infecting swine, humans, and other species.</p

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

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.This article is published as Naberhaus, Samantha A., Adam C. Krull, Bailey L. Arruda, Paulo Arruda, Orhan Sahin, Kent J. Schwartz, Eric R. Burrough et al. "Pathogenicity and competitive fitness of Salmonella enterica serovar 4,[5], 12: i:-compared to Salmonella Typhimurium and Salmonella Derby in swine." Frontiers in Veterinary Science 6 (2020): 502. DOI: 10.3389/fvets.2019.00502. Copyright 2020 Naberhaus, Krull, Arruda, Arruda, Sahin, Schwartz, Burrough, Magstadt, Matias Ferreyra, Gatto, Meiroz de Souza Almeida, Wang, and Kreuder. Attribution 4.0 International (CC BY 4.0). Posted with permission