Virginia Aragon

Biofilm formation in bacterial pathogens of veterinary importance

Abstract Bacterial biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that is attached to a surface. Biofilms protect and allow bacteria to survive and thrive in hostile environments. Bacteria within biofilms can withstand host immune responses, and are much less susceptible to antibiotics and disinfectants when compared with their planktonic counterparts. The ability to form biofilms is now considered a universal attribute of micro-organisms. Diseases associated with biofilms require novel methods for their prevention, diagnosis and treatment; this is largely due to the properties of biofilms. Surprisingly, biofilm formation by bacterial pathogens of veterinary importance has received relatively little attention. Here, we review the current knowledge of bacterial biofilms as well as studies performed on animal pathogens.

Differences in phagocytosis susceptibility in Haemophilus parasuis strains

Haemophilus parasuis is a colonizer of the upper respiratory tract of healthy pigs, but virulent strains can cause a systemic infection characterized by fibrinous polyserositis, commonly known as Glässer’s disease. The variability in virulence that is observed among H. parasuis strains is not completely understood, since the virulence mechanisms of H. parasuis are largely unknown. In the course of infection, H. parasuis has to survive the host pulmonary defences, which include alveolar macrophages, to produce disease. Using strains from different clinical backgrounds, we were able to detect clear differences in susceptibility to phagocytosis. Strains isolated from the nose of healthy animals were efficiently phagocytosed by porcine alveolar macrophages (PAM), while strains isolated from systemic lesions were resistant to this interaction. Phagocytosis of susceptible strains proceeded through mechanisms independent of a specific receptor, which involved actin filaments and microtubules. In all the systemic strains tested in this study, we observed a distinct capsule after interaction with PAM, indicating a role of this surface structure in phagocytosis resistance. However, additional mechanisms of resistance to phagocytosis should be explored, since we detected different effects of microtubule inhibition among systemic strains.

Advances in the quest for virulence factors of Haemophilus parasuis

Haemophilus parasuis colonises healthy pigs and is the aetiological agent of Glässers disease. The pathogenicity of H. parasuis is poorly characterised, while prevention and control of Glässers disease continues to be challenging. Understanding the pathogenicity of H. parasuis is essential for determining how this bacterium produces disease and to better distinguish between virulent and non-virulent strains. Infection by H. parasuis requires adhesion to and invasion of host cells, resistance to phagocytosis by macrophages, resistance to serum complement and induction of inflammation. Identification of virulence factors involved in these mechanisms has been limited by difficulties in producing mutants in H. parasuis. Recent advances in understanding the pathogenesis of H. parasuis are due in part to the production of deletion mutants, although most of the potential virulence factors described so far require further characterisation. Data supporting the role of lipooligosaccharide, capsule formation, porin proteins, cytolethal distending toxin and trimeric autotransporters (VtaA), among other molecules, in the virulence of H. parasuis have been described. This review provides an overview of the current knowledge of virulence factors of H. parasuis.

Correlation between clinico-pathological outcome and typing of Haemophilus parasuis field strains.

Haemophilus parasuis is the etiologic agent of Glässers disease in pigs, which is pathologically characterized by serofibrinous polyserositis and arthritis. H. parasuis include virulent and non-virulent strains and confirmation of virulence in H. parasuis is still dependent on experimental reproduction of the disease. Since the variability in virulence is supported by serotyping and genotyping (particularly, multilocus sequence typing [MLST]), we examined the relationship between the classification of 8 field strains by these methods and their capacity to cause disease in snatch-farrowed, colostrum-deprived piglets. The severity of clinical signs and lesions produced by the different strains correlated with the quantity of H. parasuis recovered from the lesions. However, the virulence of the strains in the animal model did not show a total correlation with their serovar or their classification by MLST. More studies are needed to identify a virulence marker that could substitute animal experimentation in H. parasuis. In addition, we reproduced disease in domestic pigs with a strain isolated from the nasal cavity of wild boars. This result indicates the existence of virulent strains of H. parasuis in wild suids, which could produce disease under appropriate circumstances, and suggests a possible source of infection for domestic pigs.

Immunogenicity and protection against Haemophilus parasuis infection after vaccination with recombinant virulence associated trimeric autotransporters (VtaA)

Haemophilus parasuis is the etiological agent of Glässers disease in swine, characterized by fibrinous polyserositis, polyarthritis and meningitis. The lack of a vaccine against a broad spectrum of strains has limited the control of the disease. Recently, virulence associated trimeric autotransporters (VtaA) were described as antigenic proteins of H. parasuis. In this study 6 VtaA were produced as recombinant proteins and used to immunize snatch-farrowed, colostrum-deprived piglets. Immunized animals developed specific systemic and mucosal antibodies. The protective capacity of the anti-VtaA antibodies was evaluated by the inoculation of 3 × 10(8) or 6 × 10(6) colony forming units (CFU) of the highly virulent strain Nagasaki. Vaccinated animals had a delayed course of disease and 33 or 57%, respectively, of the animals survived the lethal challenge. The partial protection achieved with the recombinant VtaA supports their potential as candidates to be included in future vaccine formulations against H. parasuis.

VtaA8 and VtaA9 from Haemophilus parasuis delay phagocytosis by alveolar macrophages

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.

Changes in Macrophage Phenotype after Infection of Pigs with Haemophilus parasuis Strains with Different Levels of Virulence

ABSTRACT Haemophilus parasuis is a colonizer of healthy piglets and the etiological agent of Glässers 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.

Identification of potentially virulent strains of Haemophilus parasuis using a multiplex PCR for virulence-associated autotransporters (vtaA)

Haemophilus parasuis is the aetiological agent of Glässers disease and is also a commensal of the upper respiratory tract of pigs. Trimeric autotransporter (vtaA) genes have been identified in H. parasuis and divided into three groups on the basis of the translocator domain sequence. In this study, group 3 vtaA genes were demonstrated by PCR in all 157 H. parasuis isolates tested. Group 1 vtaA genes were associated with virulent strains; 52/54 (96%) group 1 vtaA negative field isolates were isolated from the nasal passages of healthy animals, whereas no group 1 vtaA negative field isolates were isolated from cases of Glässers disease. There was an association between absence of group 1 vtaA, sensitivity to phagocytosis and serum and classification of isolates into nasal cluster C by multilocus sequence typing. A multiplex PCR was developed for diagnosis of H. parasuis at the species level (group 3 vtaA positive) and to differentiate putative non-virulent strains (group 1 vtaA negative). When applied to field samples, the PCR confirmed a high prevalence of H. parasuis in conventionally farmed pigs and demonstrated that almost half of the animals carried potentially virulent strains.

Distribution of genes involved in sialic acid utilization in strains of Haemophilus parasuis

Haemophilus parasuis is a porcine respiratory pathogen, well known as the aetiological agent of Glässers disease. H. parasuis comprises strains of different virulence, but the virulence factors of this bacterium are not well defined. A neuraminidase activity has been previously detected in H. parasuis, but the role of sialylation in the virulence of this bacterium has not been studied. To explore the relationship between sialic acid (Neu5Ac) and virulence, we assessed the distribution of genes involved in sialic acid metabolism in 21 H. parasuis strains from different clinical origins (including nasal and systemic isolates). The neuraminidase gene nanH, together with CMP-Neu5Ac synthetase and sialyltransferase genes neuA, siaB and lsgB, were included in the study. Neuraminidase activity was found to be common in H. parasuis isolates, and the nanH gene from 12 isolates was expressed in Escherichia coli and further characterized. Sequence analysis showed that the NanH predicted protein contained the motifs characteristic of the catalytic site of sialidases. While an association between the presence of nanH and the different origins of the strains was not detected, the lsgB gene was predominantly present in the systemic isolates, and was not amplified from any of the nasal isolates tested. Analysis of the lipooligosaccharide (LOS) from reference strains Nagasaki (virulent, lsgB(+)) and SW114 (non-virulent, lsgB(-)) showed the presence of sialic acid in the LOS from the Nagasaki strain, supporting the role of sialylation in the virulence of this bacterial pathogen. Further studies are needed to clarify the role of sialic acid in the pathogenicity of H. parasuis.

Sow vaccination modulates the colonization of piglets by Haemophilus parasuis

Haemophilus parasuis is the etiologic agent of Glässers disease in pigs and a colonizer of the upper respiratory tract of healthy pigs. A good balance between colonization and immunity is important to avoid a disease outbreak. This work studied the colonization of H. parasuis in healthy piglets coming from vaccinated and non-vaccinated sows. Piglets from vaccinated sows had higher IgG levels at early time points and subsequently were colonized later and to a lower degree than piglets from non-vaccinated ones. The variability of H. parasuis isolates was investigated by 2 genotyping methods: enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus sequence typing (MLST). A high turnover of strains was found in both groups of piglets, with few strains found on more than one sampling occasion. We found a higher number of H. parasuis strains (16 strains) within a given farm than previously thought. Overall, more H. parasuis diversity was found in piglets from non-vaccinated sows than in those from vaccinated sows. These results indicate that vaccination of sows in a farm delays the colonization of piglets and reduces the carriage and heterogeneity of H. parasuis strains.