Haemophilus parasuis invades porcine brain microvascular endothelial cells

Abstract

Haemophilus parasuis, an important swine pathogen, is the aetiological agent of Glässer's disease. It is responsible for cases of polyserositis, meningitis and pneumonia in young pigs. To date, 15 serotypes have been described, although several non-typable isolates are frequently recovered from diseased animals. The pathogenesis of H. parasuis infection is poorly understood. To cause meningitis, H. parasuis would have to cross the blood–brain barrier (BBB), composed of brain microvascular endothelial cells (BMEC). The objective of this study was to investigate the ability of H. parasuis to interact with porcine brain microvascular endothelial cells (PBMEC). It was demonstrated that the serotype 5 reference strain of H. parasuis, Nagasaki (originally recovered from a case of meningitis), was able to adhere at very high levels to and, most importantly, invade PBMEC. These capacities were confirmed by electron microscopy. Actinobacillus pleuropnemoniae serotype 7 (strain WF 83), used as negative control, was not able to adhere to or invade PBMEC. Comparisons of the levels of adhesion and invasion by several H. parasuis field strains from different serotypes isolated from cases of either meningitis or pneumonia showed that isolates of serotypes 4 and 5 had a higher invasion capacity than isolates belonging to other serotypes. Inhibition studies demonstrated that PBMEC invasion by H. parasuis required rearrangement of actin microfilaments and microtubular cytoskeletal elements but not active bacterial DNA, RNA or protein synthesis. Characterization studies demonstrated that proteinaceous invasin(s) does not seem to play a major role in entry of H. parasuis into PBMEC. Intracellular viable H. parasuis were found in PBMEC up to 6 h after antibiotic treatment. Even at high bacterial doses, H. parasuis was not toxic to PBMEC. In swine, the invasion of endothelial cells of the BBB may play an important role in the pathogenesis of meningitis caused by H. parasuis.

Interactions ofHaemophilus parasuisand its LOS with porcine brain microvascular endothelial cells

“…The adhesion assay was performed as previously described [41]. Briefly, PBMEC were infected with H. parasuis Nagasaki strain at 1 × 10 6 CFU/mL in culture medium (1 mL per well) and incubated at 37…”

“…Field strains of H. parasuis serotypes 4 (n = 18) and 5 (n = 10) isolated from cases of meningitis, septicemia, arthritis or pneumonia in swine, as well as seven strains of serotype 4 recovered from nasal swabs of clinically healthy animals (from farms considered free from endemic disease caused by H. parasuis) were also used in this study. Bacterial strains were grown to the late logarithmic phase of growth as described previously [41]. Bacteria were appropriately diluted in cell culture medium before infection.…”

“…Alterations of endothelial cells could also be the result of bacteria-induced cytotoxic effects or cell destruction by apoptosis [35,38]. Research thus far has suggested that H. parasuis is not toxic to endothelial cells [41].…”

“…(BMEC) [14,41] and/or by increasing BBB permeability [38]. We recently showed that H. parasuis can adhere to and invade porcine BMEC [41].…”

“…We recently showed that H. parasuis can adhere to and invade porcine BMEC [41]. BMEC, the primary components of the BBB, are linked by intercellular tight junctions that control molecular exchanges between the blood and the brain [38].…”