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Background Previous studies have shown that the genus Moraxella is commonly present in the nasal microbiota of swine. Results In this study, 51 isolates of Moraxella were obtained from nasal swabs from 3 to 4 week old piglets, which represented 26 different fingerprintings by enterobacterial repetitive intergenic consensus (ERIC)-PCR. Whole 16S rRNA gene sequencing allowed the identification at species level of the Moraxella spp. isolates. The majority of the field strains were identified as Moraxella pluranimalium, but Moraxella porci was also detected. In addition, a cluster of 7 strains did not group with any described Moraxella species, probably representing a new species. Subsequent phenotypic characterization indicated that strains of Moraxella pluranimalium were mainly sensitive to serum complement, while the cluster representing the putative new species was highly resistant. Biofilm formation capacity was very variable among the Moraxella spp. isolates, while adherence to epithelial cell lines was similar among selected strains. Additionally, variability was also observed in the association of selected strains to porcine alveolar macrophages. Antimicrobial tests evidenced the existence of multidrug-resistance in the strains. Conclusions In summary, phenotypic characterization revealed heterogeneity among Moraxella strains from the nasal cavity of piglets. Strains with pathogenic potential were detected as well as those that may be commensal members of the nasal microbiota. However, the role of Moraxella in porcine diseases and health should be further evaluated.

Urban wild boars (Sus scrofa) from Barcelona, Spain, harbor great diversity of Streptococcus suis strains, including strains with the cps2 gene and with the same molecular profile as local human cases. The increasing trend of potential effective contacts for S. suis transmission is of public health concern. info:eu-repo/semantics/publishedVersion

Haemophilus parasuis is a bacterium from the Pasteurellaceae family that comprises strains of different degree of virulence. Non-virulent strains are considered components of the upper respiratory tract microbiota, while virulent strains can invade systemic organs and cause fibrinous polyserositis (Glässer's disease). Genomic comparison of virulent and non-virulent strains led to the identification of a family of genes differentially associated to virulence, the virulence-associated trimeric autotransporters (vtaA). Monoclonal antibody 69C6 reacted with the surface of virulent strains and has allowed now the identification of an epitope in the C terminus of the passenger domain of the VtaAs from virulent strains. Protein modelling indicated that the epitope is probably exposed, although sera from pigs vaccinated with the passenger domain of VtaA9 and from convalescent animals did not react with the 69C6 epitope. Induction of antibodies against the 69C6 epitope by vaccination would allow a response targeting specifically virulent strains of H. parasuis.

Glässer's disease is a fibrinous polyserositis and polyarthritis of swine caused by the bacterium Haemophilus parasuis. Control by vaccination has been limited for years due to lack of cross-protection among strains. However, 6 trimeric autotransporters (VtaA) of the Nagasaki strain were shown to be antigenic and gave partial protection to a lethal challenge. The antigenic relationship among the VtaAs was examined by immunizing mice with individual VtaA showing that they cross-reacted by ELISA mainly with VtaA from the same group. When sera from protected and non-protected vaccinated piglets were examined no differences in VtaA cross-reactivity profiles were found. In addition, sera from commercial pigs immunized with a single VtaA (VtaA9) showed a wider range of VtaA cross-reaction, probably due to the previous colonization by H. parasuis. These results can help the development of new vaccine formulations against H. parasuis by allowing a rational VtaA selection.

Haemophilus parasuis causes Glässer's disease and pneumonia in swine. Serotyping is often used to classify isolates but requires reagents that are costly to produce and not standardized or widely available. Sequence-based methods, such as multilocus sequence typing (MLST), offer many advantages over serotyping. An MLST scheme was previously proposed for H. parasuis but genome sequence data only recently available reveals the primers recommended, based on sequences of related bacteria, are not optimal. Here we report modifications to enhance the original method, including primer redesign to eliminate mismatches with H. parasuis sequences and to avoid regions of high sequence heterogeneity, standardization of primer T(m)s and identification of universal PCR conditions that result in robust and reproducible amplification of all targets. The modified typing method was applied to a collection of 127 isolates from North and South America, Europe and Asia. An alignment of the concatenated sequences obtained from seven target housekeeping genes identified 278 variable nucleotide sites that define 116 unique sequence types. A comparison of the original and modified methods using a subset of 86 isolates indicates little difference in overall locus diversity, discriminatory power or in the clustering of strains within Neighbor-Joining trees. Data from the optimized MLST were used to populate a newly created and publicly available H. parasuis database. An accompanying database designed to capture provenance and epidemiological information for each isolate was also created. The modified MLST scheme is highly discriminatory but more robust, reproducible and user-friendly than the original. The MLST database provides a novel resource for investigation of H. parasuis outbreaks and for tracking strain evolution.

Haemophilus parasuis is a colonizer of healthy piglets and the etiological agent of Glässer's disease. Differences in virulence among strains of H. parasuis have been widely observed. In order to explore the host-pathogen interaction, snatch-farrowed colostrum-deprived piglets were intranasally infected with 4 strains of H. parasuis : reference virulent strain Nagasaki, reference nonvirulent strain SW114, field strain IT29205 (from a systemic lesion and virulent in a previous challenge), and field strain F9 (from the nasal cavity of a healthy piglet). At different times after infection, two animals of each group were euthanized and alveolar macrophages were analyzed for the expression of CD163, CD172a, SLA I (swine histocompatibility leukocyte antigen I), SLA II, sialoadhesin (or CD169), and CD14. At 1 day postinfection (dpi), virulent strains induced reduced expression of CD163, SLA II, and CD172a on the surfaces of the macrophages, while nonvirulent strains induced increased expression of CD163, both compared to noninfected controls. At 2 dpi, the pattern switched into a strong expression of CD172a, CD163, and sialoadhesin by the virulent strains, which was followed by a steep increase in interleukin 8 (IL-8) and soluble CD163 in serum at 3 to 4 dpi. The early increase in surface expression of CD163 induced by nonvirulent strains went along with higher levels of IL-8 in serum than those induced by virulent strains in the first 2 days of infection. Alpha interferon (IFN-α) induction was observed only in animals infected with nonvirulent strains. Overall, these results are compatible with a delay in macrophage activation by virulent strains, which may be critical for disease production.

Haemophilus parasuis, a member of the family Pasteurellaceae, is a common inhabitant of the upper respiratory tract of healthy pigs and the etiological agent of Glässer’s disease. As other virulent Pasteurellaceae, H. parasuis can prevent phagocytosis, but the bacterial factors involved in this virulence mechanism are not known. In order to identify genes involved in phagocytosis resistance, we constructed a genomic library of the highly virulent reference strain Nagasaki and clones were selected by increased survival after incubation with porcine alveolar macrophages (PAM). Two clones containing two virulent-associated trimeric autotransporter (VtaA) genes, vtaA8 and vtaA9, respectively, were selected by this method. A reduction in the interaction of the two clones with the macrophages was detected by flow cytometry. Monoclonal antibodies were produced and used to demonstrate the presence of these proteins on the bacterial surface of the corresponding clone, and on the H. parasuis phagocytosis-resistant strain PC4-6P. The effect of VtaA8 and VtaA9 in the trafficking of the bacteria through the endocytic pathway was examined by fluorescence microscopy and a delay was detected in the localization of the vtaA8 and vtaA9 clones in acidic compartments. These results are compatible with a partial inhibition of the routing of the bacteria via the degradative phagosome. Finally, antibodies against a common epitope in VtaA8 and VtaA9 were opsonic and promoted phagocytosis of the phagocytosis-resistant strain PC4-6P by PAM. Taken together, these results indicate that VtaA8 and VtaA9 are surface proteins that play a role in phagocytosis resistance of H. parasuis.