Serovars and SpaA Types of Erysipelothrix rhusiopathiae Isolated from Pigs in Japan from 2012 to 2019.

Erysipelothrix rhusiopathiae causes swine erysipelas (SE), which results in considerable economic loss on pig farms. During SE outbreaks that occurred sporadically from 2008 to 2011 in Japan, new E. rhusiopathiae strains were isolated with a specific surface protective antigen (Spa)A protein characterized by methionine at position 203 and isoleucine at position 257 (M203/I257 SpaA type). To determine whether strains with the M203/I257 SpaA type are still prevalent in Japan, we collected 79 strains of E. rhusiopathiae from pigs showing various SE symptoms from 2012 to 2019 and classified them based on serovar typing, spaA gene sequence analysis, and lineage typing. We found that the majority of recent E. rhusiopathiae strains (59/79) belonged to the serovar 1a strain, and that the M203/I257 SpaA type (56/59) was predominant continuing from 2008 to 2011. Furthermore, serovar 1a strains with IVb-1 and IVb-2 lineages that had been isolated in specific regions of Japan were no longer local but were found across Japan. The pathogenicity of recent isolates tested in mice was not significantly changed when compared to that of previously isolated strains. Our results suggest that recent SE outbreaks were not due to changes in the SpaA protein or to altered virulence of E. rhusiopathiae but were rather caused by the persistent presence of E. rhusiopathiae with the M203/I257 SpaA type.

The Shigella flexneri effector OspI deamidates UBC13 to dampen the inflammatory response.

Many bacterial pathogens can enter various host cells and then survive intracellularly, transiently evade humoral immunity, and further disseminate to other cells and tissues. When bacteria enter host cells and replicate intracellularly, the host cells sense the invading bacteria as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) by way of various pattern recognition receptors. As a result, the host cells induce alarm signals that activate the innate immune system. Therefore, bacteria must modulate host inflammatory signalling and dampen these alarm signals. How pathogens do this after invading epithelial cells remains unclear, however. Here we show that OspI, a Shigella flexneri effector encoded by ORF169b on the large plasmid and delivered by the type ΙΙΙ secretion system, dampens acute inflammatory responses during bacterial invasion by suppressing the tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6)-mediated signalling pathway. OspI is a glutamine deamidase that selectively deamidates the glutamine residue at position 100 in UBC13 to a glutamic acid residue. Consequently, the E2 ubiquitin-conjugating activity required for TRAF6 activation is inhibited, allowing S. flexneri OspI to modulate the diacylglycerol-CBM (CARD-BCL10-MALT1) complex-TRAF6-nuclear-factor-κB signalling pathway. We determined the 2.0 Å crystal structure of OspI, which contains a putative cysteine-histidine-aspartic acid catalytic triad. A mutational analysis showed this catalytic triad to be essential for the deamidation of UBC13. Our results suggest that S. flexneri inhibits acute inflammatory responses in the initial stage of infection by targeting the UBC13-TRAF6 complex.

Characterization of Actinobacillus pleuropneumoniae field strains antigenically related to the 3-6-8-15 group from diseased pigs in Japan and Argentina.

The objectives of this study were to determine the serovar of a collection of Actinobacillus pleuropneumoniae strains within the 3-6-8-15 cross-reacting group and to analyze their phenotypic and genetic properties. Based on the serological tests, forty-seven field strains of Actinobacillus pleuropneumoniae isolated from lungs with pleuropneumonia lesions in Japan and Argentina were found to be serovars belonging to the 3-6-8-15 cross-reacting group. By using a capsule loci-based PCR, twenty-nine (96.7%) and one (3.3%) from Japan were identified as serovars 15 and 8, respectively, whereas seventeen (100%) from Argentina were identified as serovar 8. The findings suggested that serovars 8 and 15 were prevalent within the 3-6-8-15 cross-reacting group, in Argentina and Japan, respectively. Phenotypic analyses revealed that the protein patterns observed on SDS-PAGE and the lipopolysaccharide antigen detected by immunoblotting of the reference and field strains of serovars 8 and 15 were similar to each other. Genetic (16S rDNA, apxIIA, apxIIIA, cps, cpx genes, apx and omlA patterns) analyses revealed that the apxIIA and apxIIIA genes of the field strains of serovars 8 and 15 were similar to those of the reference strains of serovars 3, 4, 6, 8 and 15. The results obtained in the present study may be useful for the development of more effective vaccines against disease caused by A. pleuropneumoniae by including the homologous antigens to the most prevalent serovars in specific geographical areas.

Genetic and antigenic characteristics of ApxIIA and ApxIIIA from Actinobacillus pleuropneumoniae serovars 2, 3, 4, 6, 8 and 15.

Apx toxins produced by Actinobacillus pleuropneumoniae are essential components of new generation vaccines. In this study, apxIIA and apxIIIA genes of serovars 2, 3, 4, 6, 8 and 15 were cloned and sequenced. Amino acid sequences of ApxIIA proteins of serovars 2, 3, 4, 6, 8 and 15 were almost identical to those of serovars 1, 5, 7, 9 and 11-13. Immunoblot analysis showed that rApxIIA from serovars 2 and 15 reacts strongly with sera from animals infected with various serovars. Sequence analysis revealed that ApxIIIA proteins has two variants, one in strains of serovar 2 and the other in strains of serovars 3, 4, 6, 8 and 15. A mouse cross-protection study showed that mice actively immunized with rApxIIIA/2 or rApxIIIA/15 are protected against challenge with A. pleuropneumoniae strains of serovars 3, 4, 6, 8, 15, and 2 expressing ApxIII/15 and ApxIII/2, respectively. Similarly, mice passively immunized with rabbit anti-rApxIIIA/2 or anti-rApxIIIA/15 sera were found to be protected against challenge with strains of serovars 2 and 15. Our study revealed antigenic and sequence similarities within ApxIIA and ApxIIIA proteins, which may help in the development of effective vaccines against disease caused by A. pleuropneumoniae.

Numerical simulation of red blood cell distributions in three-dimensional microvascular bifurcations.

We constructed three-dimensional microvascular bifurcation models using a parent vessel of diameter 10µm and investigated the flow behavior of the red blood cells (RBCs) through bifurcations. We considered symmetric and asymmetric model types. Two cases of equal daughter vessel diameter were employed for the asymmetric models, where the first was 10µm, which is the same as the parent vessel and the second was 7.94µm, which satisfies Murray's law. Simulated blood flow was computed using the lattice Boltzmann method in conjunction with the immersed boundary method for incorporating fluid-membrane interactions between the flow field and deformable RBCs. First, we investigated the flow behavior of a single RBC through microvascular bifurcations. In the case of the symmetric bifurcation, the turning point of the fractional plasma flow wherein the RBC flow changed from one daughter vessel to the other was 0.50. This turning point was however different for asymmetric bifurcations. Additionally, we varied the initial offset of RBCs from the centerline of the parent vessel. The simulation results indicated that the RBCs preferentially flow through the branch of a larger flow ratio. Next, we investigated the distribution characteristics of multiple RBCs. Simulations indicated that the results of the symmetric model were similar to those predicted by a previously published empirical model. On the other hand, results of asymmetric models deviated from those of the symmetric and empirical models. These results suggest that the distribution of RBCs varies according to the bifurcation angle and daughter vessel diameter in a microvascular bifurcation of the size considered.

Bacteria hijack integrin-linked kinase to stabilize focal adhesions and block cell detachment.

The rapid turnover and exfoliation of mucosal epithelial cells provides an innate defence system against bacterial infection. Nevertheless, many pathogenic bacteria, including Shigella, are able to surmount exfoliation and colonize the epithelium efficiently. Here we show that the Shigella flexneri effector OspE (consisting of OspE1 and OspE2 proteins), which is highly conserved among enteropathogenic Escherichia coli, enterohaemorrhagic E. coli, Citrobacter rodentium and Salmonella strains, reinforces host cell adherence to the basement membrane by interacting with integrin-linked kinase (ILK). The number of focal adhesions was augmented along with membrane fraction ILK by ILK-OspE binding. The interaction between ILK and OspE increased cell surface levels of 1 integrin and suppressed phosphorylation of focal adhesion kinase and paxillin, which are required for rapid turnover of focal adhesion in cell motility. Nocodazole-washout-induced focal adhesion disassembly was blocked by expression of OspE. Polarized epithelial cells infected with a Shigella mutant lacking the ospE gene underwent more rapid cell detachment than cells infected with wild-type Shigella. Infection of guinea pig colons with Shigella corroborated the pivotal role of the OspE-ILK interaction in suppressing epithelial detachment, increasing bacterial cell-to-cell spreading, and promoting bacterial colonization. These results indicate that Shigella sustain their infectious foothold by using special tactics to prevent detachment of infected cells.

Characterization of Actinobacillus pleuropneumoniae biovar 2 isolates reportedly reacted with the serovar 4 antiserum, and development of a multiplex PCR for O-antigen typing.

We have analyzed the capsule (CPS) and the lipopolysaccharide O-Antigen (O-Ag) biosynthesis loci of twelve Spanish field isolates of Actinobacillus pleuropneumoniae biovar 2, eleven of them previously typed serologically as serovar 4 and one non-typable (NT) (Maldonado et al., 2009, 2011). These isolates have the common core genes of the type I CPS locus, sharing >98% identity with those of serovar 2. However, the former possesses the O-Ag locus as serovar 4, and the latter possesses the O-Ag locus as serovar 7. The main difference found between the CPS loci of the 11 isolates and that of serovar 2 reference strain S1536 are two deletions, one of an 8 bp sequence upstream of the coding sequence and one of 111 bp sequence at the 5' end of the cps2G gene. The deletion mutations mentioned lead to a defect in the production of CPS in these isolates, which contributed to their previous mis-identification. In order to complement the serotyping of A. pleuropneumoniae in diagnostics and epidemiology, we have developed a multiplex PCR for the comprehensive O-Ag typing of all A. pleuropneumoniae isolates.

Characterization of nonencapsulated Actinobacillus pleuropneumoniae serovar K12:O3 isolates.

Three Actinobacillus pleuropneumoniae isolates from clinical cases of porcine pleuropneumonia were positive by capsular serovar 12-specific PCR assay, but not reactive to antiserum prepared against serovar 12 using the rapid slide agglutination (RSA) test. The isolates were positive for apxIICA, apxIIICA, apxIBD, apxIIIBD, and apxIVA in the PCR toxin gene assay, which is the profile seen in serovars 2, 4, 6, 8, and 15, and reacted with antisera against serovars 3, 6, 8, 15, and 17. Nucleotide sequence analysis revealed that genes involved in the biosynthesis of capsular polysaccharide of the 3 isolates were identical or nearly identical to those of serovar 12. However, genes involved in the biosynthesis of O-polysaccharide of the 3 isolates were highly similar to those of reference strains of serovars 3, 6, 8, 15, 17, and 19. In agreement with results from the RSA test, transmission electron microscopic analysis confirmed the absence of detectable capsular material in the 3 isolates. The existence of nonencapsulated A. pleuropneumoniae serovar K12:O3 would hamper precise serodetection.

Pulmonary lesions with asteroid bodies in a pig experimentally infected with Actinobacillus pleuropneumoniae serovar 15.

Five pigs experimentally infected with Actinobacillus pleuropneumoniae serovar 15 isolated in our previous study were pathologically examined. One pig died at 2 days post inoculation (dpi) and four pigs were euthanized at 7 dpi. Autopsy revealed fibrinohemorrhagic pleuropneumonia in all pigs. Histopathologically, the lesions were characterized by extensive hemorrhage and necrosis, fibrin deposition, and multifocal abscesses composed of numerous neutrophils including oat cells and numerous Gram-negative bacilli. In one survived pig, asteroid body formation was confirmed in the lung. The bacteria within the abscesses and asteroid bodies were immunohistochemically positive for antiserum raised against A. pleuropneumoniae serovar 15. This is the first report describing porcine pleuropneumonia with asteroid bodies in a pig experimentally infected with A. pleuropneumoniae serovar 15.

Characterization of an atypical Actinobacillus pleuropneumoniae serovar 2 isolate with a rough-type lipopolysaccharide.

We describe phenotypic and genetic characterization of an atypical Japanese Actinobacillus pleuropneumoniae isolate OT761. Nucleotide sequence analysis revealed that gene clusters involved in capsular polysaccharide and O-polysaccharide (O-PS) biosynthesis of the isolate were nearly identical to those of serovar 2 reference strain. The main difference found between the O-PS loci is the shortening of 31 amino acids from the C terminus of WcaJ in the atypical isolate due to a 93 bp deletion at the 3' end of wcaJ gene. Immunoblot analysis revealed that this isolate could not produce O-PS. Taken together, our results showed that the C-terminal domain of the A. pleuropneumoniae WcaJ plays a critical role in enzyme function of WcaJ involved in the biosynthesis of O-PS.