Adherence of actinobacillus pleuropneumoniae serotype 1 to swine buccal epithelial cells involves fibronectin

Abstract

The swine pathogen Actinobacillus pleuropneumoniae serotype 1 was investigated for its ability to adhere to swine, rat, and human buccal epithelial cells (BEC). The highest number of bacteria adhered was to swine BEC. This binding ability was affected by heating, extreme pH, treatment with sodium dodecyl sulfate, ethylenediamine tetra-acetate, or periodate, and proteolysis, suggesting that cell-surface glycoproteins participate in adherence and that adherence is based mostly on ionic interactions. Mannose and swine fibronectin may play a direct role in this interaction. Convalescent-phase serum from naturally infected pigs inhibited the adhesion. There was a correlation between bacterial pathogenicity as well as host specificity and the capacity for adherence to swine BEC. Adhesion to swine BEC provides a convenient method to study in vitro the adherence of A. pleuropneumoniae and other pathogens of the pig respiratory tract.

Figure 1

Actinobacillus pleuropneumoniae serotype 1 adherence to buccal epithelial cells (BEC) after incubation of 5 × 104 BEC from swine, rats, and humans (B, D, and F, respectively) with 2 × 108 colony-forming units of bacteria. A, C, and E are controls from indigenous flora on swine, rat, and human BEC, respectively. Cells were visualized by light microscopy (original magnification, X400).

Figure 2

A1: Swine buccal epithelial cells (BEC) incubated with rabbit anti-swine fibronectin polyclonal antibody and then with anti-rabbit immunoglobulin (Ig)G labelled with fluorescein isothiocyanate (FITC). A2: Effect of trypsin on swine BEC surface; cells were incubated with trypsin (2.5 μg/mL) for 10 min, washed with 0.1 M Tris-HCl, pH 7.2, and treated with the same serum as in A1. B: Numbers of bacteria adhering to swine BEC and to trypsin-treated cells. Assay was performed as in Figure 1. None — indigenous flora; A. pleuro 1 — A. pleuropneumoniae serotype 1. C: Electron micrograph of A. pleuropneumoniae incubated for 5 min with fibronectin and stained with uranyl acetate (bar = 500 nm); bacteria are in close interaction with fibronectin networks, covered by the protein. D: Similar interaction of A. pleuropneumoniae with a fibronectin fibre (bar = 500 nm).

Figure 3

Concentration effect of anti-pig fibronectin antibodies (A) or mannose (B) on the adherence of A. pleuropneumonie serotype 1 to swine BEC. Assays were performed as in Figure 1.

Figure 4

Adhesion of 3 A. pleuropneumoniae isolates and related gram-negative bacteria to human, rat, and swine BEC. Assays were done as in Figure 1. None — indigenous flora; A. pleuro 1, 1a, 1b, and 1c — A. pleuropneumoniae serotype 1 reference strain S4074 and 3 isolates; A. porcinus — Actinobacillus porcinus [* adhesion value of 350 ± 13 (standard error)]; A. minor — Actinobacillus minor Amy 2B; A. suis — Actinobacillus suis; A. seminis — Actinobacillus seminis ATCC 15768; H. parasuis — Haemophilus parasuis serotype 5; M. haemo — Mannheimia (Pasteurella) haemolytica M16 (microbiota) and OV20 (pathogen) (2 isolates from field ovine); P. multocida 1 and 2 — Pasteurella multocida (pig pathogen isolates); E. coli — Escherichia coli K12 AB1157.