Proposal of serovars 17 and 18 of Actinobacillus pleuropneumoniae based on serological and genotypic analysis

The aim of this study was to investigate isolates of Actinobacillus pleuropneumoniae previously designated serologically either as NT or as ‘K2:07’, which did not produce serovar-specific amplicons in PCR assays. We used whole genome sequencing to identify the capsule (CPS) loci of six previously designated biovar 1 non-typable (NT) and two biovar 1 ‘K2:O7’ isolates of A. pleuropneumoniae from Denmark, as well as a recent biovar 2 NT isolate from Canada. All of the NT isolates have the same six-gene type I CPS locus, sharing common cpsABC genes with serovars 2, 3, 6, 7, 8, 9, 11 and 13. The two ‘K2:O7’ isolates contain a unique three-gene type II CPS locus, having a cpsA gene similar to that of serovars 1, 4, 12, 14 and 15. The previously NT isolates share the same O-antigen genes, found between erpA and rpsU, as serovars 3, 6, 8, and 15. Whereas the ‘K2:O7’ isolates, have the same O-antigen genes as serovar 7, which likely contributed to their previous mis-identification. All of the NT and ‘K2:O7’ isolates have only the genes required for production of ApxII (apxIICA structural genes, and apxIBD export genes). Rabbit polyclonal antisera raised against representative isolates with these new CPS loci demonstrated distinct reactivity compared to the 16 known serovars. The serological and genomic results indicate that the isolates constitute new serovars 17 (previously NT) and 18 (previously ‘K2:O7’). Primers designed for amplification of specific serovar 17 and 18 sequences for molecular diagnostics will facilitate epidemiological tracking of these two new serovars of A. pleuropneumoniae

Cytokine expression by CD163+ monocytes in healthy and Actinobacillus pleuropneumoniae-infected pigs

Distinct monocyte subpopulations have been previously described in healthy pigs and pigs experimentally infected with Actinobacillus pleuropneumoniae (APP). The CD163+ subpopulation of bone marrow (BM), peripheral blood (PB) and lung monocytes was found to play an important role in the inflammatory process. The inflammation is accompanied by elevation of inflammatory cytokines. The aim of the study was to evaluate the contribution of CD163+ monocytes and macrophages to cytokine production during APP-induced lung inflammation. Cytokine production was assessed by flow cytometry (FC) and quantitative PCR (qPCR) in CD163+ monocytes and by qPCR, immunohistochemistry/fluorescence in lungs and tracheobronchial lymph nodes (TBLN). Despite the systemic inflammatory response after APP infection, BM and PB CD163+ monocytes did not express elevated levels of a wide range of cytokines compared to control pigs. In contrast, significant amounts of IL-1β, IL-6, IL-8 and TNF-α were produced in lung lesions and IL-1β in the TBLN. At the protein level, TNF-α was expressed by both CD163+ monocytes and macrophages in lung lesions, whereas IL-1β, IL-6 and IL-8 expression was found only in CD163+ monocytes; no CD163+ macrophages were found to produce these cytokines. Furthermore, the quantification of CD163+ monocytes expressing the two cytokines IL-1β and IL-8 that were most elevated was performed. In lung lesions, 36.5% IL-1β positive CD163+ monocytes but only 18.3% IL-8 positive CD163+ monocytes were found. In conclusion, PB and BM CD163+ monocytes do not appear to contribute to the elevated cytokine levels in plasma. On the other hand, CD163+ monocytes contribute to inflammatory cytokine expression, especially IL-1β at the site of inflammation during the inflammatory process.Peer reviewe

Comparative sequence analysis of the capsular polysaccharide loci of Actinobacillus pleuropneumoniae serovars 1-18, and development of two multiplex PCRs for comprehensive capsule typing

Problems with serological cross-reactivity have led to development of a number of PCRs (individual and multiplex) for molecular typing of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. Most of these assays were developed for detection of specific amplicons within capsule biosynthetic genes before the availability of complete sequences for the different serovars. Here we describe comparative analysis of the complete capsular loci for all 18 serovars of A. pleuropneumoniae, and development of two multiplex PCRs for comprehensive capsule typing of this important pig pathogen

Structural Basis of Ca2+-Dependent Self-Processing Activity of Repeat-in-Toxin Proteins

The Ca2+-dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca2+-dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. Here, we report the solution structures of the Ca2+-loaded SPM (Ca-SPM) defining the mechanism of the autocatalytic cleavage of the Asp414-Pro415 peptide bond of the Neisseria meningitidis FrpC exoprotein. Moreover, deletion of the SPM domain in the ApxIVA protein, the FrpC homolog of Actinobacillus pleuropneumoniae, resulted in attenuation of virulence of the bacterium in a pig infection model, indicating that the Ca2+-dependent clip-and-link activity plays a role in the virulence of Gram-negative pathogens.The posttranslational Ca2+-dependent “clip-and-link” activity of large repeat-in-toxin (RTX) proteins starts by Ca2+-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free ε-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neisseria meningitidis. The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a “twisted-amide” activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca2+-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface

Multilocus Sequence Genotype Heterogeneity in Streptococcus uberis Isolated from Bovine Mastitis in the Czech Republic

The ubiquitous occurrence and high heterogeneity of Streptococcus uberis strains cause difficulties in the development and implementation of effective control strategies in dairy herds. In this study, S. uberis strains from 74 farms, obtained predominantly from subclinical, acute, and chronic recurrent mastitis, as well as from udder surface swabs and milk from healthy udders, were analysed for their genetic diversity using multilocus sequence typing (MLST). Isolates were tested for the presence of the genes encoding the virulence factors using polymerase chain reaction. Antibiotic susceptibility testing was performed using a microdilution assay including 14 antimicrobials. The virulence profiles and antimicrobial (AMR) profiles of the isolates were assembled and the overall heterogeneity was evaluated. Among the 124 isolates, 89 MLST genotypes, 7 different virulence profiles, and 12 AMR profiles were identified. The large number of different MLST allelic profiles in this study points to the high heterogeneity of strains in dairy herds in the Czech Republic. Isolates of a certain MLST genotype may possess a different set of virulence factor genes. We detected up to three different resistance profiles within a single MLST genotype. The results of our study showed that fully susceptible isolates coexisted with resistant or even multiresistant isolates in the same herd. Multiple genotypes within a herd were detected on many farms (up to seven MLST genotypes and four AMR profiles in one herd). This heterogenic population structure might suggest that environmental transmission is the predominant route of infection in herds in the Czech Republic

Resistance of <i>Streptococcus suis</i> Isolates from the Czech Republic during 2018–2022

A determination of susceptibility/resistance to antimicrobials via serotype was carried out in 506 field isolates of Streptococcus suis, originating from pig farms in the Czech Republic in the period 2018–2022. A very high level of susceptibility of S. suis isolates was found to amoxicillin, in combination with clavulanic acid and sulfamethoxazole potentiated with trimethoprim. None of the tested isolates were resistant to these antimicrobial substances. Only two isolates were found to be intermediately resistant to enrofloxacin in 2020. With regard to ceftiofur, one isolate was intermediately resistant in 2020 and 2022, and two isolates were intermediately resistant in 2018 and 2021. A low level of resistance was detected to ampicillin (0.6% in 2021) and to florfenicol (1.15% in 2019; 1.3% in 2022). With regard to penicillin, a medium level of resistance was detected in 2018 (10.6%), but a low level of resistance was found in the following years (7.0% in 2019; 3.1% in 2020; 3.3% in 2021; 3.9% in 2022). On the contrary, a high or very high level of resistance was found to tetracycline (66.0% in 2018; 65.1% in 2019; 44.35% in 2020; 46.4% in 2021; 54.0% in 2022). Using molecular and serological methods, serotype 7 (16.4%) was determined to be predominant among S. suis isolates, followed by serotypes 1/2, 2, 9, 4, 3, 1, 29, 16, and 31 (10.7%; 8.5%; 5.7%; 5.5%; 4.5%; 4.3%; 3.6%; 3.4%; 3.4%, respectively). Other serotypes were identified among the investigated strains either rarely (up to 10 cases) or not at all. A relatively high percentage of isolates were detected as non-typeable (79 isolates; 15.6%). Dependence of resistance upon serotype assignment could not be proven in all but serotype 31, wherein all isolates (n = 17) were resistant or intermediately resistant to clindamycin, tilmycosin, tulathromycin, and tetracycline. The resistance to clindamycin and tetracycline may be related to the high consumption of these antibiotics on pig farms at present or in previous years. Macrolides (tilmicosin and tulathromycin) and tiamulin are not suitable for the treatment of streptococcal infections, but are used on pig farms to treat respiratory infections caused by gram-negative bacteria, so they were included in the study

Vaccine against Streptococcus suis Infection in Pig Based on Alternative Carrier Protein Conjugate

Streptococcus suis is a serious pathogen in the pig industry with zoonotic potential. With respect to the current effort to reduce antibiotic use in animals, a prophylactic measure is needed to control the disease burden. Unfortunately, immunization against streptococcal pathogens is challenging due to nature of the interaction between the pathogen and the host immune system, but vaccines based on conjugates of capsular polysaccharide (CPS) and carrier protein were proved to be efficient. The main obstacle of these vaccines is manufacturing cost, limiting their use in animals. In this work, we tested an experimental vaccine against Streptococcus suis serotype 2 based on capsular polysaccharide conjugated to chicken ovalbumin (OVA) and compared its immunogenicity and protectivity with a vaccine based on CRM197 conjugate. Ovalbumin was selected as a cheap alternative to recombinant carrier proteins widely used in vaccines for human use. We found that the ovalbumin-based experimental vaccine successfully induced immune response in pigs, and the IgG antibody response was even higher than after immunization with capsular polysaccharide-CRM197 conjugate. Protectivity of vaccination against infection was evaluated in the challenge experiment and was found promising for both conjugates