Protective effect of chicken egg yolk immunoglobulins (IgY) against enterotoxigenic Escherichia coli K88 adhesion in weaned piglets

Abstract

Background: Enterotoxigenic Escherichia coli K88 (E. coli K88) are considered as a major cause of diarrhea and death in newly weaned piglets. Oral passive immunization with chicken egg yolk immunoglobulins (IgY) have attracted considerable attention for treatment of gastrointestinal infection due to its high specificity. In this study it was estimated the protective effect of anti-K88 fimbriae IgY against E. coli K88 adhesion to piglet intestinal mucus in vitro and to investigate the potential use of IgY for controlling E. coli-induced diarrhea in weaned piglets in vivo.

Results: E. coli K88 was incubated with IgY for 24 h, and the bacterial growth profiles showed that specific IgY with a concentration higher than 5 mg/mL was observed to significantly inhibit the growth of E. coli K88 compared to nonspecific yolk powder in a liquid medium. Moreover, pretreatment with 50 mg/mL of IgY was found to significantly decrease the adhesion ability of E. coli K88 to porcine jejunal and ileal mucus, further supported by the observations from our immunofluorescence microscopic analysis. In vivo, administration of IgY successfully protected piglets from diarrhea caused by E. coli K88 challenge. Additionally, IgY treatment efficiently alleviated E. coli-induced intestinal inflammation in piglets as the gene expression levels of inflammatory cytokines TNF-α, IL-22, IL-6 and IL-1β in IgY-treated piglets remained unchanged after E. coli K88 infection. Furthermore, IgY significantly prevented E. coli K88 adhering to the jejunal and ileal mucosa of piglets with E. coli infection and significantly decreased E. coli and enterotoxin expression in colonic contents.

Conclusion: Outcome of the study demonstrated that IgY against the fimbrial antigen K88 was able to significantly inhibit the growth of E. coli K88, block the binding of E. coli to small intestinal mucus, and protect piglets from E. coli-induced diarrhea. These results indicate that passive immunization with IgY may be useful to prevent bacterial colonization and to control enteric diseases due to E. coli infection. The study has great clinical implication to provide alternative therapy to antibiotics in E coli induced diarrhea.

Keywords: Bacteria adhesion; Diarrhea; Egg yolk immunoglobulins (IgY); Escherichia coli K88; Piglets.

Fig. 1

Growth inhibitory effect of specific IgY. E. coli K88 was incubated with 0.5 mg/mL (a), 1 mg/mL (b), 5 mg/mL (c), 12.5 mg/mL (d), 25 mg/mL (e) and 50 mg/mL (f) of yolk powder or specific IgY in LB liquid medium containing 50 μg/mL streptomycin. LB medium without yolk powder or IgY was used as blank control. Samples were taken at 0, 2, 4, 6, 8, 10, 12, 14 and 24 h of incubation and optical density at 600 nm was measured. Results were expressed as means ± SEM, n = 3, (Specific IgY versus Yolk powder; ***, P < 0.001) (Yolk powder versus Control group; ###, P < 0.001). Unless otherwise noted, significance was determined by one-way ANOVA with Tukey’s post hoc test. Results were repeated in three independent experiments. The raw data was shown in Additional file 1: Figure S1

Fig. 2

Inhibition of E. coli K88 adhesion by specific IgY. E. coli K88 was pretreated with specific IgY and the number of bacteria adhered to crude porcine jejunal (a) and ileal (b) mucus was determined by bacterial colony counts. FITC-labelled E. coli was incubated with 50 mg/mL of yolk powder or specific IgY. Bacterial adherence to jejunal (c) and ileal (d) mucus was measured by fluorescence intensity changes. Results were expressed as means ± SEM, n = 3, (*, P < 0.05; ***, P < 0.001). FITC-labelled E. coli was pre-treated with PBS (e), 50 mg/mL of yolk powder (f) or specific IgY (g). Immunofluorescence microscopy data show the adhesion of E. coli K88 (in Green) to jejunal mucus (magnification × 10). Data shown are generated from three independent experiments. The raw data was shown in Additional file 2: Figure S2

Fig. 3

Diarrhea scores of piglets after E. coli challenge. Piglets pretreated with yolk powder or specific IgY were orally inoculated with 5 mL of E. coli K88 at a dose of 10⁸ CFU/mL. Diarrhea scores were recorded: (0 = normal; 1 = soft feces; 2 = mild diarrhea; 3 = watery stool). Results were expressed as means ± SEM (n = 5–15 per group). Yolk powder (Group II) versus Control group (Group I); #, P < 0.05. The raw data was shown in Additional file 3: Figure S3

Fig. 4

Inflammatory profiles in jejunal and ileal mucosa. a to h The relative gene expressions of inflammatory cytokines TNF-α, IL-22, IL-6 and IL-1β in jejunal and ileal mucosa were detected by real-time PCR (n = 5). Results were expressed as means ± SEM. Specific IgY (Group III) versus Yolk powder (Group II); *, P < 0.05. Yolk powder (Group II) versus Control group (Group I); #, P < 0.05. NS stands for Not Significant. The raw data was shown in Additional file 4: Figure S4

Fig. 5

Counts of E. coli K88 adhering to intestinal mucosa and enterotoxin expression in luminal content. Mucosal samples were obtained at 12, 24 and 72 h post infection and numbers of E. coli that adhered to jejunal (a) and ileal (b) mucosa were counted by plating serial dilutions of mucosal homogenates onto MacConkey agar plates. Intestinal digesta samples were collected at 72 h after infection and fold changes of genes encoding E. coli (c), STa (d), STb (e), LT (f) and K88 (g) were quantified by PCR analysis (n = 5). Results were expressed as means ± SEM. Specific IgY (Group III) versus Yolk powder (Group II); *, P < 0.05, **, P < 0.01. Yolk powder (Group II) versus Control group (Group I); #, P < 0.05, ##, P < 0.01. NS stands for Not Significant. The raw data was shown in Additional file 5: Figure S5