Interaction of Salmonella enterica with Bovine Epithelial Cells Demonstrates Serovar-Specific Association and Invasion Patterns

S almonella enterica is a leading cause of infection-related morbidity among humans and other animals worldwide. Dairy animals are known reservoirs of S. enterica and serovars such as Dublin, Newport, and Typhimurium are generally associated with salmonellosis in calves and adult cows, causing mild to severe illnesses. However, asymptomatic carriage and fecal shedding of Salmonella serovars such as Cerro, Kentucky, Meleagridis, Mbandaka, and Montevideo have been well documented in dairy animals (Van Kessel et al., 2012). Asymptomatic carriage may not impact dairy production, but herds with a large S. enterica burden may pose an increased public health risk through contamination of the environment of production and finally milk and meat.

Although there is ample evidence of differential interaction patterns between Salmonella strains of different serovars with human cell lines or in murine and avian models, the interactions between dairy commensal (or persistent) and transient Salmonella serovars with bovine infection models have not been described. Morgan et al. (2004) showed that salmonellae used different gene repertoires when colonizing/infecting chickens versus cows, indicating that salmonellae employ diverse strategies to interact with host cells. The aim of this study was to investigate the interactions between Salmonella strains from different serovars with bovine epithelial cells to further understand why some serovars cause severe or fatal infections, whereas others persist as commensal members within the bovine gut community.

Twenty-six Salmonella strains of bovine origins were selected for the study (two strains per serovar: Anatum, Cerro, Dublin, Give, Kentucky, Mbandaka, Meleagridis, Montevideo, Muenster, Newport, Oranienburg, Senftenberg, and Typhimurium). The Salmonella Kentucky strains were isolated from raw meat, and all other strains were from bulk tank milk or milk filters. Bovine mammary epithelial (MAC-T) cells were used as the infection model. For each strain, six independent replications of association–invasion assays were conducted following a protocol described by Federman et al. (2016) with the following modifications: bacterial inoculum (18 h broth culture of each strain), multiplicity of infection (10:1), duration of infection (2 h), washing agent of infected MAC-T monolayer (phosphate-buffered saline), and concentration of gentamicin for invasion assay (250 mg/L). A Salmonella motility assay was conducted following the methodology described by Fang et al. (2017). Statistical analyses (multiple mean comparison with Tukey's honest significance test, Pearson product-moment correlation, and Spearman rank-order correlation) were done in JMP Pro 13 (SAS Institute, Inc., Cary, NC).

Significant differences were observed between Salmonella strains from different serovars in their association capacity (attachment and invasion combined) and invasiveness to bovine epithelial cells (Fig. 1A). Salmonella Dublin strains were more significantly associated with and invaded into MAC-T cells compared with strains belonging to the other 12 serovars (p < 0.05). The least invasive strains belonged to serovars Mbandaka, Kentucky, Cerro, and Give (p < 0.05). Invasive phenotype is an important virulence property of Salmonella because, in most cases, the establishment of infection initiates with penetration of the intestinal mucosa (Hurley et al., 2014). A higher level of invasiveness was reported among Salmonella strains isolated from the organs of calves suffering from salmonellosis compared with strains isolated from healthy animal feces (Dinjus and Hänel, 1997). A study by Berndt et al. (2007) demonstrated serovar-associated invasiveness differences in a chicken model, indicating that there is a correlation between invasiveness and host cell cytokine production. It is unclear whether serovar-associated differences in invasiveness are related with bovine immune responses and whether such an association has a role in the differential carriage of serovars in the bovine gut.

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FIG. 1.

Association and invasiveness of Salmonella enterica strains from 13 serovars (two strains per serovar) with a cultured bovine (mammary epithelial) cell line (A). Normalized interactions represent the ratios of log₁₀ transformed associated or invaded Salmonella cells and the initial inoculum size. Each strain was tested with six independent repetitions. Error bars represent one standard deviation from the mean. (B) Displays motility of S. enterica strains from different serovars on semisolid medium. Each strain was tested with three independent repetitions. (C) The degrees of relationship between motility of S. enterica strains from different serovars (except Salmonella Dublin) on semisolid medium and their association with bovine cells using Pearson product-moment correlation coefficient (r) and nonparametric Spearman rank-order correlation coefficient (ρ). Means without common letters are significantly different at p < 0.05.

Salmonella Senftenberg strains showed significantly less association (combined attachment and invasion) with MAC-T cells compared with strains from the other serovars; however, almost 100% of the attached Salmonella Senftenberg cells also invaded the MAC-T cells, indicating that Salmonella Senftenberg tended to invade once they formed stable attachments to the bovine cells. Potential factors impacting the observed phenomenon may include motility and chemotaxis; strains lacking functional flagella or chemotactic machinery may have reduced capacity to approach the host cell monolayer during the early phase of infection (Stecher et al., 2004). Motility assays on semisolid agar medium demonstrated that Salmonella Seftenberg strains were less motile than strains from most of the other serovars (Fig. 1B); a significant positive correlation was observed between serovar-specific motility on semisolid medium and association with bovine cells (Fig. 1C, Spearman's ρ 0.55, p < 0.05). Salmonella Dublin strains associated more readily with bovine epithelial cells compared with the other serovars and were less motile than most of the serovars. Yim et al. (2014) reported that the aflagellate phenotype is widely distributed in Salmonella Dublin from cattle, and flagella may be nonessential in the infection cycle of this serovar. On the contrary, flagella are required by serovar Typhimurium for maximum fluid secretion in a calf enterocolitis model (Schmitt et al., 2001), indicating that the repertoires of Salmonella virulence factors that contribute to bovine infection may be serovar specific. An inter-serovar whole transcriptome comparison may help identify serovar-specific factors of Salmonella that are associated with bovine colonization and/or infection.

In this study, significantly different invasiveness was observed between strains from serovars Anatum, Kentucky, Oranienburg, Meleagridis, Montevideo, and Senftenberg (data not shown), which indicated the possibility of within-serovar variability in the colonization/infection of dairy animals by Salmonella; some strains within a serovar may serve as transient colonizers, whereas others may be more prone to establish as commensals. This observation is further supported by our previous finding in a longitudinal study where a strain of Salmonella Kentucky was supplanted by another strain in a single dairy herd (Van Kessel et al., 2012). The observed variation in strain invasiveness within a serovar demonstrates an underappreciated level of phenotypic diversity among strains of the same Salmonella serovar.

This study demonstrated significant variability of Salmonella strains within and across serovars in their capacity to attach and invade cultured bovine epithelial cells. Future studies will focus on relating observed differences to differences in gene expression. Understanding of factors responsible for the persistence of commensal-type Salmonella serovars in dairy animals is needed to develop strategies to reduce fecal shedding of these pathogens and bovine salmonellosis.