Tea polyphenols suppress growth and virulence-related factors of Haemophilus parasuis

Abstract

The bacterium Haemophilus parasuis (H. parasuis) is the primary cause of Glässer's disease. Currently, there are no effective vaccines that can confer protection against all H. parasuis serovars. Therefore, the present study aimed to investigate the effect of tea polyphenols on growth, expression of virulence-related factors, and biofilm formation of H. parasuis, as well as to evaluate their protective effects against H. parasuis challenge. Our findings demonstrated that tea polyphenols can inhibit H. parasuis growth in a dose-dependent manner and attenuate the biofilm formation of H. parasuis. In addition, tea polyphenols exerted inhibitory effects on the expression of H. parasuis virulence-related factors. Moreover, tea polyphenols could confer protection against a lethal dose of H. parasuis and can reduce pathological tissue damage induced by H. parasuis. In summary, our findings demonstrated the promising use of tea polyphenols as a novel treatment for H. parasuis infection in pigs.

Keywords: Haemophilus parasuis; biofilm; protection; tea polyphenols; virulence-related factor.

Fig. 1.

Kinetics of the antimicrobial effects of tea polyphenols on H. parasuis. H. parasuis and tea polyphenols (0, 40, 80, 160 and 320 µg/ml) were co-cultured for 0, 1, 2, 3, 4 and 5 hr. The number of colonies was counted, and kinetics curves were constructed. Results are expressed as the mean ± SD of three independent experiments. ‘*’ indicates statistical significance at P<0.05 (vs. Control). ‘**’ indicates statistical significance at P<0.01 (vs. Control).

Fig. 2.

Effect of tea polyphenols on H. parasuis biofilm formation based on glass tube biofilm assay (A) and quantitative assay (B). A: H. parasuis was incubated with tea polyphenols at concentrations of 0, 80, 160 and 320 µg/ml, and the tubes were stained with crystal violet solution. C: Control; D: H. parasuis treated with 80 µg/ml tea polyphenols; E: H. parasuis treated with 160 µg/ml tea polyphenols; F: H. parasuis treated with 320 µg/ml tea polyphenols. Arrow indicates biofilm formation at the air-liquid interface. B: A relative value of 100% was assigned to control. Data are expressed as the mean ± SD of three independent experiments. ‘*’ indicates statistical significance at P<0.05.

Fig. 3.

Analysis of H. parasuis cellular integrity by transmission electron microscopy. (A) Control: untreated bacteria. (B) Bacteria treated with tea polyphenols (160 µg/ml). (C): High-magnification image of the indicated area.

Fig. 4.

Effect of tea polyphenols on virulence-related factors of H. parasuis. H. parasuis was treated with tea polyphenols at 1/2 MIC (160 µg/ml), and the expression of virulence-related factors (ArcA, ClpP, RfaE, RfaD, OmpP2, OmpP5, GalU, and GalE) of H. parasuis were determined. Data are expressed as the mean ± SD of three independent experiments. ‘*’ indicates significance at P<0.05.

Fig. 5.

Survival rates of mice administered with tea polyphenols (80, 160, and 320 µg/ml) via intragastrical administration following H. parasuis SH0165 challenge. ‘*’ indicates statistical significance at P<0.05 (vs. Negative control group).

Fig. 6.

Representative lung and brain sections of mice from different treatment groups. (A) and (E): negative control group. (B) and (F): mice administered with 80 µg/ml tea polyphenols. (C) and (G): mice administered with 160 µg/ml tea polyphenols. (D) and (H): mice administered with 320 µg/ml tea polyphenols. Bars=100 µm.