Salmonella Typhimurium interferes with the humoral immune response in pigs

Foodborne salmonellosis is one of the most important bacterial zoonotic diseases worldwide. Salmonella Typhimurium is the serovar most frequently isolated from slaughter pigs in Europe. Circumvention of the host’s immune system by Salmonella might contribute to persistent infection of pigs. We found that Salmonella Typhimurium strain 112910a, which is able to persist in pigs, was capable of downregulating the expression of major histocompatibility class II (MHC II) molecules on porcine alveolar macrophages (PAM) in a Salmonella pathogenicity island 2 (SPI-2) dependent way and that MHC II downregulation was Salmonella strain dependent. The MHC II downregulation capacity was abolished when bacteria were opsonized with Salmonella-specific antibodies. Furthermore, intracellular proliferation of Salmonella Typhimurium opsonized with Salmonella positive pig serum was significantly impaired compared to that of the bacteria opsonized with negative pig serum. In a subsequent in vivo experiment, Salmonella Typhimurium strain MB2216 that did not induce MHC II downregulation in vitro, was shed less and persisted less but induced earlier seroconversion in pigs than strain 112910a. From the in vitro data, it is proposed that Salmonella Typhimurium downregulates the humoral immune response to promote intracellular survival inside porcine macrophages, contributing to long-term Salmonella persistence in pigs. The fact that the less persistent strain MB2216 induced earlier seroconversion than strain 112910a is of major interest for Salmonella-monitoring programs primarily based on serology, since this indicates that more persisting strains are more likely to escape serological detection. Introduction In European countries, Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) is the serovar most frequently isolated from slaughter pigs (Anon., 2008). In most cases, the bacterium will asymptomatically colonize pigs, resulting in a so called ‘carrier status’ (Wood et al., 1989). In the past, Salmonella infections in pig herds have traditionally been diagnosed by culturing intestinal or faecal samples. LPS-based serological surveillance is perceived as a practical and cost-effective alternative for monitoring Salmonella infection in pig herds (Proux et al., 2000) and is currently widely applied. The success of many persisting pathogens relies on their ability to interfere with the host’s immune responses, for example by interfering with major histocompatibility complex (MHC) molecule expression and antigen presentation. SPI-2 plays a role in Salmonella-mediated downregulation of MHC II in human Mel JuSo cells (Mitchell et al., 2004). No data are available on this phenomenon in porcine cells. In the present study, we hypothesized that Salmonella Typhimurium strain 112910a induced Salmonella pathogenicity island 2 (SPI-2) mediated downregulation of MHC II expression on porcine macrophages as a possible mechanism to circumvent antibody production by the pig’s immune system. We then examined the role of antibodies in intracellular survival and proliferation of Salmonella in macrophages and in the bacterium’s ability to interfere with the MHC II presentation pathway, and if this MHC II downregulating capacity was Salmonella strain dependent. Finally, we examined whether the Salmonella induced MHC II downregulation in porcine macrophages coincides with Salmonella persistence in pigs. Material and Methods Bacterial strains and manipulations Salmonella Typhimurium strain 112910a, a pig stool isolate (Boyen et al., 2009), and its isogenic deletion mutants ΔsseA and ΔssrA/B, constructed as described by Datsenko and Wanner (2000), were used. Other strains used in this study are Salmonella Typhimurium pig isolates MB2150, MB2216, MB2222, MB2223, MB2233 and MB2498 and the pigeon isolate DAB69, Salmonella serovars Brandenburg, Derby and Infantis, all isolated from pigs and a chicken isolate Salmonella serovar Enteritidis 76Sa88. For flowcytometric analysis, strains were transformed with a plasmid expressing green fluorescent protein (GFP; Valvidia and Falkow, 1996). Pig antisera against Salmonella Typhimurium Porcine serum containing Salmonella-specific antibodies was raised by injecting pigs twice intramuscularly with a bacterin consisting of formalin-inactivated Salmonella Typhimurium strain 112910a suspended in phosphate buffered saline (PBS) and Freund’s incomplete adjuvant (positive serum). Negative serum was collected from pigs, injected twice with an emulsion of Freund’s incomplete adjuvant and PBS. The effect of Salmonella Typhimurium on MHC expression on the surface of porcine macrophages Porcine alveolar macrophages (PAM) were isolated and inoculated with GFP-transformed Salmonella strains and serovars, as described by Boyen et al. (2006). Macrophages were incubated with a primary MHC class I or class II antibody to detect MHC I or II expression, respectively, and then incubated with a secondary Alexa Fluor 633 antibody. MHC expression of uninfected and infected PAM was measured using a FACScanto TM II cytometer and analysed with FACS Diva software. Impact of opsonization with antibodies on intracellular survival of Salmonella Typhimurium in porcine macrophages Intracellular survival of bacteria that were either non-opsonized or opsonized with positive or negative pig serum, was assessed a gentamicin protection assay (Boyen et al., 2006). Comparison of the in vivo behaviour of Salmonella Typhimurium strains 112910a and MB2216 after experimental inoculation of piglets Salmonella-free piglets were orally inoculated with Salmonella Typhimurium strain 112910a (n=19), inducing MHC II downregulation in vitro, or strain MB2216 (n=9), not inducing MHC II downregulation in vitro, respectively. Faeces were collected at different days post inoculation (pi) and bacteriologically analyzed. Six days before inoculation and 11, 18, 26, 33 and 40 days pi, blood was collected and serum was isolated. Serum was analysed using an LPS-based Elisa (IDEXX Labs), and an in-house whole-cell ELISA (Leyman et al., 2011). Forty days pi, all pigs were euthanized and tonsils, ileocecal lymph nodes, ileum, ileum contents, caecum, caecum contents, colon and colon contents were collected and bacteriologically analysed (Van Parys et al., 2010). Results Infection with Salmonella Typhimurium strain 112910a did not result in a decreased MHC I expression level on PAM. In contrast, the MHC II expression level on strain 112910a infected PAM was significantly decreased (Figure 1A). MHC II expression levels between PAM inoculated with strain 112910a, ΔsseA or ΔssrA/B did not significantly differ from each other, nor from the respective ratios directly after inoculation, suggesting that MHC II expression was partly restored when SPI-2 was abolished (Figure 1A). Among the 7 different Salmonella Typhimurium isolates tested, strain MB2216 did not induce MHC II downregulation on PAM, while the other strains exhibited downregulation of MHC II expression similar to strain 112910a. Furthermore, Salmonella Typhimurium strain DAB69 and the Salmonella Derby and Infantis isolates showed no MHC II downregulation, in contrast to serovar Brandenburg and Enteritidis strains (Figure 1B). PAM inoculated with Salmonella Typhimurium opsonized with negative pig serum showed a decrease in MHC II expression level, while the MHC II expression on PAM inoculated with Salmonella Typhimurium opsonized with positive pig serum remained unaffected. Furthermore, Salmonella was able to significantly proliferate intracellularly in PAM when they were not opsonized or when they were opsonized with negative pig serum, while bacteria that were opsonized with positive pig serum proliferated less. 0,00 0,50 1,00 1,50 2,00 2,50 3,0