No beneficial effects evident for Enterococcus faecium NCIMB 10415 in weaned pigs infected with Salmonella enterica serovar Typhimurium DT104

Abstract

Salmonella enterica serovar Typhimurium DT 104 is the major pathogen for salmonellosis outbreaks in Europe. We tested if the probiotic bacterium Enterococcus faecium NCIMB 10415 can prevent or alleviate salmonellosis. Therefore, piglets of the German Landrace breed that were treated with E. faecium (n = 16) as a feed additive and untreated controls (n = 16) were challenged with S. Typhimurium 10 days after weaning. The presence of salmonellae in feces and selected organs, as well as the immune response, were investigated. Piglets treated with E. faecium gained less weight than control piglets (P = 0.05). The feeding of E. faecium had no effect on the fecal shedding of salmonellae and resulted in a higher abundance of the pathogen in tonsils of all challenged animals. The specific (anti-Salmonella IgG) and nonspecific (haptoglobin) humoral immune responses as well as the cellular immune response (T helper cells, cytotoxic T cells, regulatory T cells, γδ T cells, and B cells) in the lymph nodes, Peyer's patches of different segments of the intestine (jejunal and ileocecal), the ileal papilla, and in the blood were affected in the course of time after infection (P < 0.05) but not by the E. faecium treatment. These results led to the conclusion that E. faecium may not have beneficial effects on the performance of weaned piglets in the case of S. Typhimurium infection. Therefore, we suggest a critical discussion and reconsideration of E. faecium NCIMB 10415 administration as a probiotic for pigs.

Fig 1

Gating and selection strategies of the flow cytometry data for the analyses of lymphocyte subpopulations. The x and y axes show the intensity of the light scatter or fluorescent (FL) signal. Framed cells populations in panel A were further analyzed in panel B, those in panel B were further analyzed in panel C, and those in panel C were further analyzed in panel D. (A) Lymphocytes gated according to their forward and sideward light scatter signals. Circulating lymphocytes represent 40,000 cells. (B) Only living PI-negative cells were taken for analyses. (C) Relative cell counts of living lymphocyte subpopulations (CD8pos, cytotoxic T cells; CD4pos, CD8pos, pig-specific double-positive cells; CD4pos, T helper cells) detected with antibodies CD8β and CD4 were calculated. (D) The relative number of CD25^high cells among the CD4⁺ cells was calculated.

Fig 2

Colonization of piglets with salmonellae and the reaction of their immune systems to infection. Shedding of Salmonella Typhimurium in feces (A) and in organs after either 2 (B) or 28 days (C), as well as serum anti-salmonella IgG titers (D) and haptoglobin concentrations (E) after intragastric S. Typhimurium infection in groups of piglets that were either untreated controls (C) or were treated with E. faecium (P). CFU/g feces were log transformed to obtain normal distributions. Box-whisker plots indicate the medians (horizontal lines) and the lower and upper quartiles (lower and upper borders of the boxes); minimum and maximum values, as well as the outliers (mild, open circles; extreme, asterisks), are presented.

Fig 3

Cell counts of αβ T cells relative to the living lymphocyte population. (A) CD8β pos (cytotoxic T cells), (B) CD8β and CD4 pos, and (D) CD4 pos (T helper cells) in peripheral blood mononuclear cells (PBMCs), ileal lymph nodes (IL LN), ileal Peyer's patches (IL PP), and papilla (PAP IL) of the E. faecium-supplemented group (P) and the non-E. faecium-supplemented control group (C). (C) Fluorescence-activated cell sorting (FACS) scatter plots of one female piglet derived from P group at 2 dpi as an example for the appearance of T helper cells, cytotoxic T cells, and CD4⁺ CD8⁺ T cells in the PBMCs, IL LN, IL PP, and PAP IL. All piglets were infected with S. Typhimurium.

Fig 4

Cell counts of γδ T cells relative to the living lymphocyte population. (A) CD2- and γδ TCR-positive cells. (B) γδ TCR-positive (CD2-negative) cells in the PBMCs, IL LN, IL PP, and PAP IL of piglets from the E. faecium-supplemented group (P) and the control group (C) 2 and 28 days after challenge with S. Typhimurium.

Fig 5

Cell counts of B cells relative to the living lymphocyte population. (A) IgM-positive cells (B cells) of one piglet representative for the E. faecium-supplemented group (P) at 2 dpi in the ileal lymph node (IL LN) and one piglet representative of the control (C) at 2 dpi in IL LN. (B to D) IgM-positive cells (B cells) of 12 piglets from P group and of 12 piglets from C group at 2 (n = 6) and 28 dpi (n = 6) in mesenteric lymph nodes (LN) of jejunum (Je LN) and ileum (IL LN) (B), in PBMCs (C), and in the ileal Peyer's patch (IL PP) and papilla (PAP IL) (D). All piglets were infected with S. Typhimurium.