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1 Groupe de Recherche sur les Maladies Infectieuses du Porc (GREMIP), Faculté de Médecine Vétérinaire, Université de Montréal, Québec, Canada
2 Canadian Research Network on Bacterial Pathogens of Swine, Faculté de Médecine Vétérinaire, Université de Montréal, Québec, Canada
3 Institute für Biochemie, Technische Hochschule Darmstadt, Petersenstrasse 22, 64287 Darmstadt, Germany
Correspondence
Marcelo Gottschalk
marcelo.gottschalk{at}umontreal.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). To date, 15 serotypes have been described using an immunodiffusion test with heat-stable antigens (Kielstein et al., 1991; Morozumi & Nicolet, 1986a; Rapp-Gabrielson & Gabrielson, 1992), although several non-typable isolates are frequently recovered from diseased animals (Rafiee & Blackall, 2000). Recently, the use of the indirect haemagglutination test has been proposed by two studies in which less than 10 % of non-typable strains were reported (Del Rio et al., 2003; Tadjine et al., 2004). However, Turni & Blackall (2005) recommended the use of an indirect haemagglutination test only as a complement of the gel diffusion test in the case of a non-typable result or cross-reaction. In North America, serotypes 4 and 5 are the most frequently isolated (Rapp-Gabrielson & Gabrielson, 1992; Tadjine et al., 2004).
Virulence factors involved in H. parasuis infection are still unknown. The association of either capsule expression or whole-cell protein profiles with virulence of H. parasuis is controversial (Oliveira & Pijoan, 2004). In addition, virulent and non-virulent strains share similar patterns of lipooligosaccharides (LOS) (Zucker et al., 1996) and the presence of fimbriae-like structures has been observed after in vivo passage (Munch et al., 1992). Finally, a neuraminidase activity has been described as a putative virulence factor (Lichtensteiger & Vimr, 1997). It is hypothesized that this enzyme, in concert with permease and aldolase, may contribute to bacterial virulence by scavenging carbohydrates from host cells. However, the actual role of this enzyme in the persistence or virulence of H. parasuis has yet to be demonstrated. More recently, differential-display RT-PCR has been used to search for genes associated with virulence (Hill et al., 2003). Although some interesting candidate genes were found, no single virulence factor was clearly identified. It remains unclear if there is a relationship between the virulence of isolates and the serotype (Kielstein et al., 1991; Morozumi & Nicolet, 1986a, b; Rapp-Gabrielson & Gabrielson, 1992). However, it is generally acceptable to use serotype as a marker of pathogenicity (Oliveira & Pijoan, 2004). In this way, serotypes 1, 2, 4, 5, 10, 12, 13, 14 and 15 are considered to be virulent while the other serotypes are thought to be avirulent (Amano et al., 1994; Kielstein & Rapp-Gabrielson, 1992).
The pathogenesis of H. parasuis infection is poorly understood. The bacteria normally colonize the upper respiratory tract of pigs, which constitute their natural reservoir (Moller & Kilian, 1990). Virulent strains of H. parasuis are able to breach the mucosal barrier and get into the bloodstream (Vahle et al., 1997). In vivo studies indicated that bacteria were not closely apposed to cilia or other cell structures (Vahle et al., 1997). However, no studies on the specific interactions between H. parsuis and host epithelial and/or endothelial cells have been performed.
Factors involved in systemic invasion by H. parasuis are unknown. To cause meningitis, H. parasuis has to reach the central nervous system (CNS). One way by which bacteria could gain access to CNS is to cross the blood–brain barrier (BBB). The BBB, an anatomically and functionally unique barrier, separates the CNS from the bloodstream and is responsible for maintaining homeostasis within the CNS. The BBB is formed, in part, by the endothelium lining the brain capillaries. This endothelium is composed of brain microvascular endothelial cells (BMEC) that form tight junctions regulating the traffic of solutes, macromolecules and cells across the BBB (Huang et al., 2000; Tuomanen, 1996). These microvessels are covered by pericytes and outgrowths of astrocytes (called astrocytic end-feet) (Goldstein & Betz, 1986; Kniesel & Wolburg, 2000). The primary site of breakdown of the BBB in most cases of bacterial meningitis appears to be the BMEC (Townsend & Scheld, 1995). However, it is not clear how circulating H. parasuis crosses the BBB.
To further understand the pathogenesis of H. parasuis-induced meningitis in swine, the objective of this study was to investigate the capacity of H. parasuis to interact with porcine brain microvascular endothelial cells (PBMEC).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and kindly provided by Dr R. F. Ross (Iowa State University), served as the reference strain. Other H. parasuis strains used in this study are listed in Table 1. Bacteria were grown for 48 h on PPLO agar plates (Difco) at 37 °C, and isolated colonies were inoculated in PPLO broth (Difco) for 8 h at 37 °C with agitation. Working cultures were obtained by inoculating 10 µl of a 10–3 dilution of these cultures in 30 ml PPLO broth for 16 h at 37 °C with agitation. Bacteria were washed twice in phosphate-buffered saline (PBS; 140 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 1·5 mM KH2PO4, pH 7·3), and were appropriately diluted in cell culture medium before infection (see Results). An accurate determination of the number of c.f.u. per ml in the final suspension was made by plating onto PPLO agar supplemented with glucose (0·1 %, w/v) and 
-nicotinamide adenine dinucleotide (-NAD; 0·004 %, w/v; Sigma). In addition, Actinobacillus pleuropneumoniae serotype 7 reference strain WF 83 was used as a negative control. A. pleuropneumoniae, which causes swine pleuropneumonia, is very rarely found in the blood circulation and is not known to cause meningitis (Gottschalk & Taylor, 2005).
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) was cultivated as previously described (Vanier et al., 2004). Briefly, cells were grown in complete IF medium, which is a mixture of 1 : 1 Iscove's modified Dulbecco's medium and Ham's F-12 (Invitrogen) supplemented with 7·5 % (v/v) heat-inactivated foetal bovine serum (hiFBS), penicillin-streptomycin (Invitrogen), sodium bicarbonate, L-glutamine, human transferrin (MP Biomedicals), N-acetylcysteine, hypoxanthine, porcine heparin, human recombinant fibroblast growth factor-basic (Sigma), and -mercaptoethanol (Bio-Rad). Flasks (Falcon; Becton Dickinson) and Primaria 24-well tissue culture plates (Falcon) were precoated with 1 % (w/v) type A gelatin from porcine skin (Sigma) to support the cells. Cells were incubated at 37 °C with 5 % CO2 in a humid atmosphere and used before the 20th passage for all experiments. Primary porcine brain microvascular endothelial cells (pPBMEC) were purchased from Cell Systems and were grown according to the supplier's recommendations. For assays, PBMEC and pPBMEC were trypsinized and diluted in their culture medium at 1x105 cells ml–1. The cell suspension was distributed in tissue culture plates and incubated to confluence. Immediately before the experiments, medium was removed from the plates and replaced by the respective medium without antibiotics.
PBMEC adhesion and invasion assays.
The adhesion assay to quantify total cell-associated (intracellular plus surface-adhered) bacteria was performed as previously described (Vanier et al., 2004), with some modifications. Briefly, bacteria were pelleted, washed twice with PBS, and resuspended in fresh cell culture medium without antibiotics at 107 c.f.u. ml–1. Confluent monolayers of PBMEC grown in 24-well plates were infected with 1 ml aliquots of bacterial suspension. The plates were incubated for different times up to 2 h at 37 °C to allow bacterial adhesion. Thereafter, cells were vigorously washed five times with PBS to eliminate non-specific bacterial attachment and incubated for 10 min at 37 °C in the presence of 200 µl 0·05 % trypsin/0·03 % EDTA. After this incubation period, 800 µl ice-cold deionized water was added, and cells were disrupted by scraping the bottom of the well and by repeated pippeting to liberate cell-associated bacteria. Serial dilutions of this cell lysate were plated onto PPLO agar and incubated for 48 h at 37 °C. Levels of adhesion are expressed as the total number of c.f.u. recovered per well.
The invasion assay to quantify intracellular bacteria was performed in a similar manner to the adhesion assay, but the plates were centrifuged at 800 g for 10 min to bring bacteria to the surface of the monolayer and incubated for different times up to 4 h. The monolayers were then washed four times with PBS; 1 ml cell culture medium containing 100 µg gentamicin ml–1 and 5 µg penicillin G ml–1 (Sigma) was added to each well, and the plates were incubated for 2 h at 37 °C to kill extracellular and surface-adhered bacteria. The monolayers were washed three times with PBS before incubation with 0·05 % trypsin/0·03 % EDTA. Controls using a bacterial concentration similar to the resulting concentration of surface-adhered bacteria were performed to ensure that the antibiotic concentrations used were sufficient to kill all extracellular cell-associated bacteria after 2 h of treatment. Adhesion and invasion assays were performed under non-toxic conditions (see below). For the intracellular survival assay, an invasion assay was performed as described above except that after a 2 h invasion period, the initial gentamicin/penicillin G treatment was lengthened for different times up to an additional 6 h. Data are expressed as the total number of recovered c.f.u. per well after antibiotic treatment.
Invasion inhibition studies.
For experiments testing the effects of inhibitors of bacterial DNA, RNA and protein synthesis, bacteria were pre-treated with ciprofloxacin (Bayer), rifampicin (Sigma) or tetracycline (Sigma), respectively. H. parasuis strain Nagasaki (107 c.f.u. ml–1) was treated with the appropriate concentration of inhibitor at 37 °C for 30 min prior to infection, as well as during the 2 h invasion period. Respective MICs (determined according to NCCLS recommendations) for strain Nagasaki were <0·125 µg ml–1 for ciprofloxacin, 0·125 µg ml–1 for rifampicin and 0·25 µg ml–1 for tetracycline. In addition to MICs, concentrations corresponding to one-half and twice the MICs were also used for each antibiotic. Results are expressed as percentage invasion relative to the invasion level without inhibitor (taken as 100 % invasion).
To study the effect of proteases affecting outer-surface proteins, H. parasuis strain Nagasaki was treated with proteinase K (Roche) at 0·1–2 mg ml–1 for 1 h at 37 °C, washed twice in PBS, resuspended in culture medium, and used to infect PBMEC for 2 h.
For experiments testing the effects of inhibitors of the formation of microfilaments and microtubules, PBMEC monolayers were preincubated with cytochalasin D or colchicine (Sigma), respectively. Different concentrations of the inhibitors were added at 37 °C for 30 min prior to the infection with strain Nagasaki, as well as during the 2 h invasion period. Cytochalasin D concentrations of 0·25, 0·5, 1·0 and 2·0 µg ml–1 and colchicine concentrations of 1·0, 5·0, 10 and 20 µg ml–1 were used. Results are expressed as percentage invasion relative to invasion level without inhibitor (taken as 100 % invasion).
PBMEC cytotoxicity assay.
The cytotoxic effect of bacteria was evaluated by lactate dehydrogenase (LDH) measurement. Briefly, bacteria were grown and diluted as described above and confluent monolayers of PBMEC grown in 24-well plates were infected with 1 ml aliquots of bacterial suspension. The plates were centrifuged at 800 g for 10 min to bring bacteria to the surface of the monolayer. The plates were incubated at 37 °C for different times up to 18 h. Non-infected cells and bacteria in IF medium without a PBMEC monolayer were used as negative controls, whereas cells lysed in 0·025 % Triton X-100 were used as a positive control (100 % toxicity). At the end of the incubation period, a 100 µl aliquot of each supernatant was transferred to a 96-well plate, which was centrifuged at 250 g for 5 min. LDH measurement was performed on 50 µl aliquots of each centrifuged supernatant using the CytoTox 96 non-radioactive cytotoxicity assay (Promega).
Electron microscopy studies.
For transmission electron microscopy (TEM), PBMEC cell monolayers were grown on 13 mm Thermanox coverslips in a 24-well culture plate. Adhesion and invasion assays were performed as described above. After two washes with PBS, the monolayers were fixed for 1 h at room temperature with 2 % (v/v) glutaraldehyde in 0·1 M cacodylate buffer pH 7·3 and then samples were postfixed in 2 % (v/v) osmium tetroxide in deionized water. Specimens were dehydrated in a graded series of ethanol solutions and embedded in Spurr resin. Thin sections were cut with a diamond knife on a Leica Ultracut ultramicrotome and post-stained with uranyl acetate and lead citrate. Samples were observed with a model 420 electron microscope (Philips Electronics). For scanning electron microscopy (SEM), samples were processed as for the TEM, except that they were not postfixed with osmium tetroxide. Samples for SEM were dehydrated in a graded series of ethanol solutions and covered with gold after critical-point drying and examined using a Hitachi S-3000N microscope.
Statistical analysis.
All data are expressed as means ± standard deviations (error bars). Data were analysed by two-tailed, unpaired t test. For comparison of adhesion and invasion capacities of several H. parasuis strains, a linear mixed model followed by Tukey–Kramer post hoc test were used. A P value of <0·05 was considered significant. All assays were repeated at least three times in independent experiments.
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RESULTS |
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H. parasuis adhesion to PBMEC
H. parasuis was able to adhere to PBMEC at high levels. This adhesion was time-dependent (Fig. 1a) and reached a plateau after 30 min incubation. The time-dependent adhesion kinetics was not due to an increase in the number of c.f.u. since H. parasuis did not grow during the 2 h incubation period used for adhesion assay (data not shown). Also, adhesion was dependent on the concentration of bacteria (Fig. 1b). A. pleuropneumoniae strain WF 83 was used as negative control for the adhesion assays and no bacteria were recovered throughout the 2 h invasion period (Fig. 1a).
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) and bacterial concentration (Fig. 2b). No significant increase in invasion was observed after 1 h incubation (P>0·05). A. pleuropneumoniae strain WF 83 was used as a negative control for the invasion assays and no bacteria were recovered throughout the 4 h invasion period (Fig. 2a).
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Confirmation of adhesion and invasion by electron microscopy
TEM and SEM were used to confirm adhesion and invasion. After 15 min incubation, bacteria were observed to adhere to PBMEC (Fig. 3a). Fig. 3(b) shows H. parasuis bacteria in close contact with a cell and within an invagination. Fig. 3(c) provides evidence confirming the intracellular invasion of PBMEC by H. parasuis.
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, after an initial 2 h invasion period and a minimal 2 h treatment with antibiotics, viable H. parasuis strain Nagasaki were found inside PBMEC up to an additional 6 h of incubation in the presence of antibiotics to exclude any extracellular source of bacteria. The results showed a gradual decrease in the number of viable intracellular bacteria, which is significant (P<0·05) after 4 h of incubation with antibiotics.
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Characterization of PBMEC invasion by H. parasuis
To evaluate the possible role of bacterial DNA, RNA and protein synthesis in invasion of PBMEC by H. parasuis, bacteria were pre-treated with ciprofloxacin, rifampicin or tetracycline. When used at their respective MICs, none of these antibiotics was able to inhibit PBMEC invasion by H. parasuis strain Nagasaki (data not shown). Similar levels of invasion were observed using concentrations corresponding to one-half and twice the MICs for all antibiotics (data not shown).
To determine whether bacterial proteins play a role in the invasion of PBMEC, bacteria were pretreated with proteinase K at a maximal concentration of 2 mg ml–1 that did not exert any effect on the viability of the bacteria (data not shown). The treatment did not affect invasion, as demonstrated by the similar level of invasion (94 %±22 %) by treated bacteria as that by non-treated bacteria (taken as 100 %). Parallel studies using an invasive strain of Streptococcus suis showed that the proteinase K treatment was effective since it significantly affected the invasion capacity of this bacterial species (data not shown).
To evaluate the possible role of microfilaments and microtubules in invasion of PBMEC by H. parasuis, cells were pre-treated with cytochalasin D or colchicine, respectively. As shown in Fig. 5, both cytochalasin D and colchicine inhibited the invasion of PBMEC by H. parasuis strain Nagasaki. A cytochalasin D concentration of 2 µg ml–1 inhibited more than 80 % of invasion (P<0·05), while 20 µg colchicine ml–1 was able to inhibit more than 70 % of invasion (P<0·05). None of the products used as eukaryotic inhibitors of PBMEC invasion was shown to be toxic to the PBMEC or bacteria at the concentrations used (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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showed that experimental inoculation of 5-day-old piglets with a live systemic strain of H. parasuis may be more effective in reducing morbidity and mortality than the colonization of piglets by nose-to-nose contact with inoculated sows. The pathogenesis of H. parasuis infection is also poorly understood. Increasing our understanding of the mechanisms by which these bacteria cause disease may help advance the development of effective tools to control the infection.
Of the many clinical diseases arising from H. parasuis infection, meningitis is one of the most frequently observed complications. To enter the CNS and cause disease, circulating H. parasuis has to first resist phagocytosis in order to remain at high concentrations in the bloodstream. In the only study published to date, Segales et al. (1998) reported that H. parasuis is able to resist phagocytosis by alveolar macrophages. In fact, the notion that a capsular polysaccharide is involved in protecting bacteria from phagocytosis is still controversial (Oliveira & Pijoan, 2004). Once in the bloodstream, bacteria must be able to cross the BBB in order to reach the CNS and cause meningitis. Since BMEC are the most important components of the BBB, in this study we investigated the interactions between H. parasuis and PBMEC.
High numbers of H. parasuis were observed to adhere to PBMEC in a time-dependent manner. This finding is in agreement with the time-dependent adhesion of Streptococcus suis, another swine meningeal pathogen, to PBMEC (Vanier et al., 2004). However, H. parasuis appears to possess a considerably higher capacity than S. suis to adhere to these cells. Indeed, adhesion to PBMEC by H. parasuis reached a plateau (at 30 min; this study) more rapidly than the adhesion by S. suis (at 4 to 6 h; Vanier et al., 2004). In contrast to the experiments with S. suis, results in this study were obtained without spinning down bacteria to facilitate contact with PBMEC. In fact, almost 100 % adhesion was obtained immediately when H. parasuis was spun down with the cells (unpublished observations). The high capacity of H. parasuis to adhere to PBMEC was also observed by electron microscopy.
In addition to adhesion, this study demonstrated that H. parasuis is also able to invade PBMEC. To the best of our knowledge, this is the first report on H. parasuis invasion of endothelial cells. Invasion of PBMEC was confirmed by SEM and TEM. Invasion of BMEC by free bacteria has also been reported for other Gram-negative meningeal pathogens, such as Haemophilus influenzae (Daines et al., 2003), Escherichia coli K1 (Badger & Kim, 1998) and Citrobacter freundii (Badger et al., 1999). Our results showed that binding to and entry of H. parasuis into PBMEC were not due to immortalization of PBMEC since H. parasuis was able to invade primary PBMEC at levels similar to those observed with immortalized cells. Interestingly, A. pleuropneumoniae, a respiratory pathogen that does not cause systemic disease, was unable to adhere to or invade either type of cells.
Results in this study suggest that active bacterial DNA, RNA and protein synthesis may not be required by H. parasuis to invade PBMEC. These findings are consistent with those reported by Virji et al. (1991) on HUVEC invasion by Haemophilus influenzae. Thus, the entry of the bacterium may be mediated by pre-existing putative invasin(s) interacting with a cell receptor (Huang & Jong, 2001; Townsend & Scheld, 1995). In addition, proteinaceous invasin(s) does not seem to play a major role in H. parasuis invasion. However, the possibility that some surface proteins that might also contribute to the invasion process could have been restored after the 2 h incubation period of invasion can not completely be ruled out. It may be hypothesized that other non-proteinaceaous surface components, such as LOS, could play an important role in H. parasuis adhesion and entry into PBMEC. LPS from A. pleuropneumoniae were shown to be involved in adherence to porcine respiratory tract cells (Belanger et al., 1990; Jacques et al., 1991). Studies on the possible role of LOS in the interactions between H. parasuis and endothelial cells are warranted.
Reorganization of host cytoskeleton components, namely actin microfilaments and microtubules, was observed to be required for entry of H. parasuis into PBMEC. In agreement with our findings, the invasion of Chang epithelial cells by H. influenzae (St Geme & Falkow, 1990) and of bovine aortic endothelial cells by Pasteurella multocida (Galdiero et al., 2001) was found to be dependent on microfilament and microtubule formation. Thus, internalization of other members of the family Pasteurellaceae may involve similar microfilament and microtubule reorganizations.
It has been suggested that many invading pathogens enter endothelial cells as a transient process (Hippenstiel & Suttorp, 2003). This is likely to be the case for H. parasuis since the intracellular bacteria survive inside PBMEC but their number decreases gradually with time. It can be hypothesized that H. parasuis undergoes exocytosis after invasion as described for other pathogens, such as P. multocida with bovine aortic cells (Galdiero et al., 2001). However, degraded H. parasuis bacteria were exceptionally observed inside PBMEC by TEM (data not shown), indicating that the decreasing number of live intracellular bacteria could also be due in part to bacterial death. In vitro BBB models should be used to confirm the proposed hypothesis.
Cell adherence and subsequent entry of the bacteria into PBMEC were not associated with pathology caused by the strains. A previous study by Rapp-Gabrielson et al. (1992) showed that following an intraperitoneal inoculation, H. parasuis strains of serotypes 2, 3 and 5 could be isolated from nearly all cultured tissues, including brain and lungs. Although no differences were observed in levels of adhesion, strains of serotypes 4 and 5 exhibited a higher level of invasion than strains of other serotypes. Interestingly, serotypes 4 and 5 are the most prevalent types isolated from diseased pigs in North America (Rapp-Gabrielson & Gabrielson, 1992). However, since only a few strains of some serotypes were used in this study, conclusions are limited. A study including a large number of strains from all known serotypes would confirm this hypothesis.
It has been previously suggested that H. parasuis is able to damage the mucosal epithelium and induce inflammation through the release of one or more toxic compounds (Vahle et al., 1997). Interestingly, H. parasuis does not posses Apx toxin genes, in contrast to other members of the family Pasteurellaceae such as A. pleuropneumoniae and Actinobacillus suis (Schaller et al., 2000). However, direct studies on the toxic effect of H. parasuis on host cells have not been performed thus far. In the present study, using a highly sensitive test, H. parasuis exerted no detectable toxic activity toward PBMEC even though it was expected that high concentrations of LOS from H. parasuis would be toxic to the cells. It has been shown that whole cells and LPS from H. influenzae are able to disrupt a monolayer of bovine BMEC (Patrick et al., 1992). However, this toxic activity appears to be specific to the origin of the cells since another study, using rat BMEC, showed an increase of albumin permeability of the BBB in the absence of cell death as assessed by LDH release (Tunkel et al., 1991). Similarly, a possible role of H. parasuis and/or its LOS in potentially increasing permeability of BBB with swine cells cannot be ruled out. In fact, a role of endotoxin in the pathogenicity of H. parasuis in acute septicaemia in pigs has been reported (Amano et al., 1997). Cell toxicity observed with A. pleuropneumoniae serotype 7 after prolonged incubation was probably due to the presence of ApxII toxin (Choi et al., 2001).
In conclusion, this study is the first to describe a high capacity of H. parasuis to adhere to and invade PBMEC. Further studies are needed to characterize the molecule(s) that mediate the adherence to and/or invasion of PBMEC. In swine, the entry of H. parasuis into endothelial cells constituting the BBB may be a key step in the pathogenesis of meningitis caused by this pathogen.
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ACKNOWLEDGEMENTS |
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Received 30 June 2005;
revised 7 September 2005;
accepted 4 October 2005.
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) or A. pleuropneumoniae (
) for the indicated times and then washed extensively to remove nonadherent bacteria. Cell lysates were plated onto PPLO agar to obtain bacterial viable plate counts as described in Methods. (b) Effect of H. parasuis concentration on level of adhesion to PBMEC after 30 min using the protocol described above.
