Haemophilus parasuis serovar 5 Nagasaki strain adheres and invades PK-15 cells

7 p.Haemophilus parasuis is the agent responsible for causing Gla¨ sser’s disease, which is characterized by fibrinous polyserositis, polyarthritis and meningitis in pigs. The purpose of this study was to investigate the in vitro ability of two H. parasuis serovars of different virulence (serovar 5, Nagasaki strain, highly virulent, belonging to serovar 5, and SW114 strain, nonvirulent, belonging to serovar 3) to adhere to and invade porcine kidney epithelial cells (PK-15 line). Nagasaki strain was able to attach at high levels from 60 to 180 min of incubation irrespective of the concentrations compared (107–1010 CFU), and a substantial increase of surface projections could be seen in PK-15 cells by scanning electron microscopy. This virulent strain was also able to invade effectively these epithelial cells, and the highest invasion capacity was reached at 180 min of infection. On the contrary, nonvirulent SW114 strain hardly adhered to PK-15 cells, and it did not invade these cells, thus suggesting that adherence and invasion of porcine kidney epithelial cells could be a virulence mechanism involved in the lesions caused by H. parasuis Nagasaki strain in this organS

TbpBY167A-Based Vaccine Can Protect Pigs against Glässer’s Disease Triggered by Glaesserella parasuis SV7 Expressing TbpB Cluster I

Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).[EN] Glaesserella parasuis is the etiological agent of Glässer’s disease (GD), one of the most important diseases afflicting pigs in the nursery phase. We analyzed the genetic and immunological properties of the TbpB protein naturally expressed by 27 different clinical isolates of G. parasuis that were typed as serovar 7 and isolated from pigs suffering from GD. All the strains were classified as virulent by LS-PCR. The phylogenetic analyses demonstrated high similarity within the amino acid sequence of TbpB from 24 clinical strains all belonging to cluster III of TbpB, as does the protective antigen TbpBY167A. Three G. parasuis isolates expressed cluster I TbpBs, indicating antigenic diversity within the SV7 group of G. parasuis. The antigenic analysis demonstrated the presence of common epitopes on all variants of the TbpB protein, which could be recognized by an in vitro analysis using pig IgG induced by a TbpBY167A-based vaccine. The proof of concept of the complete cross-protection between clusters I and III was performed in SPF pigs immunized with the TbpBY167A-based vaccine (cluster III) and challenged with G. parasuis SV7, strains LM 360.18 (cluster I). Additionally, pigs immunized with a whole-cell inactivated vaccine based on G. parasuis SV5 (Nagasaki strain) did not survive the challenge performed with SV7 (strain 360.18), demonstrating the absence of cross-protection between these two serovars. Based on these results, we propose that a properly formulated TbpBY167A-based vaccine may elicit a protective antibody response against all strains of G. parasuis SV7, despite TbpB antigenic diversity, and this might be extrapolated to other serovars. This result highlights the promising use of the TbpBY167A antigen in a future commercial vaccine for GD prevention.SIThis research was funded by Secretaria de Desenvolvimento Econômico Ciência e Tecnologia do Rio Grande do Sul (SDECT, Grant 328–2500/14-0). S.R.P. was supported by Foundation of University of Passo Fundo Master fellowship. S.C. was supported by University of Calgary Eyes High Doctoral Recruitment Award.We thank AFK Imunotech for the generous gift of pigs for this stud

Characterization of a recombinant transferrin-binding proteinA (TbpA) fragment fromHaemophilus parasuis serovar 5

9 p.Haemophilus parasuis, the etiological agent of Gl¨asser’s disease in pigs, possesses iron acquisition pathways mediated by a surface receptor that specifically bind porcine transferrin. This receptor is composed of transferrin-binding protein A (TbpA) and TbpB. As it has been reported for other gram-negative organisms, H. parasuis TbpA could be useful as a candidate target for H. parasuis vaccination. In this study, a 600-bp tbpA fragment of the gene encoding TbpA from H. parasuis serovar 5, the Nagasaki strain, was amplified by PCR and cloned into a pBAD/ Thio-TOPO expression vector, generating the pBAD-Thio-TbpA-V5-His (TbpAHis) construction. Escherichia coli LMG194-competent cells were transformed with this construction, followed by the induction of protein expression with arabinose. A band (38.5 kDa) corresponding to a 200-amino acid recombinant TbpA (rTbpA) fragment was seen on the sodium dodecyl sulfate polyacrylamide gel electrophoresis and confirmed by immunoblotting. Polyclonal antibodies raised against this fragment were specific for H. parasuis and Actinobacillus pleuropneumoniae, reacted at the cell surface with H. parasuis, and a significant bactericidal activity was also detected. Therefore, this rTbpA fragment induces an immunological response and might be useful as an antigen for vaccination against Gl¨asser’s diseaseS

Comparative efficacy of several disinfectants in suspension and carrier tests against Haemophilus parasuis serovars 1 and 5

6 p.The comparative efficacy of 16 active compounds (including the most commonly used chemical groups) 19 and 10 commercial formulations against Haemophilus parasuis serovars 1 and 5 was studied. These organ- 20 isms were tested in suspension and carrier tests in the presence and absence of serum as representative 21 of organic matter. Chloramine-T and half of the formulations from commercial sources (most of them 22 including quaternary ammonium compounds) were effective in both in vitro tests, regardless of the pres- 23 ence or absence of organic load. All 26 disinfectants except for an iodophor (0.1% available iodine) 24 resulted in at least 3-log10 reduction in colony-forming units in suspension test, and most of them 25 resulted in the maximal level of detection (>6-log10 reduction). On the other hand, disinfectants were 26 not as effective in carrier test as in suspension test, and the presence of serum considerably reduced 27 the activities of most of the compounds tested, especially in carrier test. These results suggest the impor- 28 tance of selecting suitable disinfection for routine use on surfaces contaminated with H. parasuis, partic- 29 ularly when organic matter is present. Chloramine-T and formulations 2 and 7–10 are recommended for a 30 complete inactivation of H. parasuis in swine herdsS

Aspectos clínicos e patológicos de um surto de infecção por Streptococcus suis sorotipo 9 em suínos

Streptococcus suis is a Gram-positive pathogen that inhabits the upper respiratory tract and can cause severe systemic inflammatory disease in pigs, mainly during the nursery phase. Streptococcus suis is a reemergent pathogen, and outbreaks of its inducing disease represent significant economic losses for the pig industry worldwide. In this study, we described the clinical, pathological, and molecular aspects of an outbreak of S. suis infection with atypically high mortality. The outbreak occurred in nursery farms integrated into a cooperative in the state of Paraná, Brazil. Of the 30 nurseries, 10 were severely affected by the pathogen and had high economic losses. Clinical signs usually started approximately 10 days after weaning and were mainly characterized by acute nervous and locomotor disorders. The mortality of the affected batches usually ranged between 8% and 10%, but in some cases, it reached 18%. Nine piglets were submitted to post mortem examination. Macroscopically, the synovial joints were enlarged and contained fibrinous exudates. In the central nervous system, there was hyperemia of the leptomeningeal vessels associated with deposition of fibrin and purulent exudate in the leptomeninges. In three piglets, there was thickening of the choroid plexus associated with dilation of the lateral ventricles. Microscopic lesions were characterized mainly by fibrinosuppurative inflammation, which involved the synovial membranes, leptomeninges of the brain, and spinal cord. Furthermore, it also affects the choroid plexus, ependyma, nerve roots, and central canal of the spinal cord. S. suis was isolated from the cerebrospinal fluid, meningeal swabs, and/or synovial fluid of 8/9 piglets, and typified as serotype 9 by multiplex PCR.Streptococcus suis é um patógeno Gram positivo que habita o trato respiratório superior e pode causar doença inflamatória sistêmica grave em suínos, principalmente durante a fase de creche. Streptococcus suis é um patógeno reemergente e surtos representam perdas econômicas significativas a suinocultura mundial. Neste estudo descrevemos os aspectos clínicos, patológicos e moleculares de um surto de infecção por S. suis com mortalidade atipicamente alta. O surto ocorreu em creches integradas a uma cooperativa do estado do Paraná, Brasil. Das 30 creches, 10 foram severamente afetadas pelo patógeno e apresentavam elevadas perdas econômicas. Os sinais clínicos iniciavam em torno de 10 dias após o desmame e eram caracterizados principalmente por sinais clínicos nervosos e locomotores agudos. A mortalidade dos lotes afetados variava entre 8% e 10%, mas em alguns casos ultrapassava 18%. Nove leitões foram submetidos ao exame post mortem. Macroscopicamente, as articulações sinoviais estavam aumentadas e continham exsudato fibrinoso. No sistema nervoso central havia hiperemia dos vasos leptomeníngeos associada a deposição de fibrina e exsudato purulento nas leptomeninges. Em três leitões havia espessamento do plexo coroide associado a dilatação dos ventrículos laterais. As lesões microscópicas eram caracterizadas principalmente por inflamação fibrinossupurativa que envolvia as membranas sinoviais, as leptomeninges do cérebro e medula espinhal. Além disso, também afetava o plexo coroide, epêndima, raízes nervosas e canal central da medula espinhal. S. suis foi isolado do líquido cefalorraquidiano, suabe de meninge e/ou líquido sinovial de 8/9 leitões e tipificado como sorotipo 9 por PCR multiplex

In vitro efficacy of several disinfectants against Salmonella enterica serovar Enteritidis and Escherichia coli strains from poultry = Eficácia de vários desinfetantes in vitro contra cepas de Salmonella enterica serovar Enteritidis e Escherichia coli aviária

5 p.The efficacy of 28 individual or blended disinfectants against avian Salmonella enterica serovar Enteritidis and Escherichia coli strains was determined. An in vitro test in the presence and absence of serum as source of organic material was conducted. Povidone-iodine (releasing 1% available iodine), 1% potassium permanganate, 70% ethanol, 2% chlorhexidine digluconate and three commercial formulations based on quaternary ammonium compounds + formaldehyde or cresol derivates were the most effective against all strains tested and reduced bacterial counts by more than 106 times (6-log10) regardless of the presence of organic matter. These commercial compounds as well as ethanol and chlorhexidine among the individual substances tested might be helpful in the adoption of environmental control measures against these two enterobacteria in poultry industryS

Differences in Haemophilus parasuis adherence to and invasion of AOC-45 porcine aorta endothelial cells

8 p.Background: The pathogenesis of Haemophilus parasuis depends on the bacterium’s ability to interact with endothelial cells and invade adjacent tissues. In this study, we investigated the abilities of eight H. parasuis reference strains belonging to serovars 1, 2, 4, 5, 7, 9, 10 and 13 to adhere to and invade porcine aortic endothelial cells (AOC-45 cell line). Results: The strains belonging to serovars 1, 2 and 5 were able to attach at high rates between 60 and 240 min of incubation, and serovars 4, 7 and 13 had moderate attachment rates; however, the strains belonging to serovars 9 and 10 had low adherence at all time points. Strong adherence was observed by scanning electron microscopy for the strains of serovars 5 and 4, which had high and moderate numbers, respectively, of H. parasuis cells attached to AOC-45 cells after 240 min of incubation. The highest invasiveness was reached at 180 min by the serovar 4 strain, followed by the serovar 5 strain at 240 min. The invasion results differed substantially depending on the strain. Conclusion: The reference strains of H. parasuis serovars 1, 2, 4 and 5 exhibited high adhesion and invasion levels to AOC-45 porcine aorta endothelial cells, and these findings could aid to better explain the pathogenesis of the disease caused by these serovars.S

Haemophilus parasuis Subunit Vaccines Based on Native Proteins with Affinity to Porcine Transferrin Prevent the Expression of Proinflammatory Chemokines and Cytokines in Pigs

8 p.The expression of chemokines (CCL-2 and CXCL-8) and cytokines (IL-1, IL-1, IL-6, TNF-, and IL-10) was evaluated by RTqPCR in colostrum-deprived pigs vaccinated and challenged with Haemophilus parasuis serovar 5. Two vaccines containing native proteins with affinity to porcine transferrin (NPAPTim and NPAPTit) were tested, along with two control groups: one inoculated with PBS instead of antigen (challenge group (CHG)), and another one nonimmunized and noninfected (blank group). The use of NPAPTim and NPAPTit resulted in complete protection against H. parasuis (no clinical signs and/or lesions), and both vaccines were capable of avoiding the expression of the proinflammatory molecules to levels similar to physiological values in blank group. However, overexpression of all proinflammatory molecules was observed in CHG group, mainly in the target infection tissues (brain, lungs, and spleen). High expression of CCL-2, CXCL-8, IL-1, IL-1, and IL-6 can be considered one of the characteristics of H. parasuis infection by serovar 5S

Immunoproteomic analysis of the protective response obtained with subunit and commercial vaccines against Glässer’s disease in pigs

14 p.An immunoproteomic analysis of the protective response of subunit and commercial vaccines in colostrumdeprived pigs against Glässer’s disease was carried out. A mixture of proteins with affinity to porcine transferrin (PAPT) from Haemophilus parasuis Nagasaki strain (serovar 5) was inoculated intramuscularly (PAPTM) and intratracheally (PAPTCp), along with a commercial bacterin. PAPT were separated using 2 dimensional electrophoresis (2DE) gels and with them, 2DE Western blots were carried out. A total of 17 spots were identified as positive with sera of pigs from any of the three vaccinated groups, the highest number of immunoreactive proteins being detected in those having received PAPTCp. Among them, six proteins (FKBP-type peptidyl-prolyl cis-trans isomerase, neuraminidase exo- -sialidase, xanthine-guanine phosphoribosyl transferase, CMP-N-acetylneuraminic acid synthetase, phenylalanyl-tRNA synthetase and glyceraldehyde 3-phosphate dehydrogenase) were found to be novel immunogens in H. parasuis. These proteins showed a high potential as candidates in future subunit vaccines against Glässer’s disease. The three experimental groups developed specific systemic total IgG (IgGt), IgG1, IgG2 and IgM antibodies after immunizations. In addition, those receiving PAPTCp yielded a serum IgA response.S

Acute phase protein concentrations in colostrum-deprived pigs immunized with subunit and commercial vaccines against Glässer’s disease

8 p.Haemophilus parasuis is the etiological agent of Glässer’s disease, which is characterized by fibrinous polyserositis, polyarthritis and meningitis in pigs. This study was focused on the characterization of the acute-phase response after immunization and infection of colostrum-deprived pigs with H. parasuis serovar 5, by measuring serum concentrations of three positive acute-phase proteins (APPs) (pig major acute-phase protein pig, MAP; haptoglobin, HPG; C-reactive protein, CRP) and one negative APP (apolipoprotein A-I, ApoA-I). Six experimental groups were established: a non-immunized but infected control group (CTL); two groups immunized with either a recombinant transferrin-binding protein (Tbp) A or TbpB fragment from H. parasuis Nagasaki strain (rTbpA and rTbpB, respectively); two groups immunized with native outer membrane proteins with affinity to porcine transferrin (NPAPT), one of them inoculated intramuscularly (NPAPTim) and the other intratracheally (NPAPTit), and the last group receiving a commercially available bacterin (PG). The greatest concentrations of the three positive APPs and the lowest concentration of the negative APP were detected in CTL group, as well as in those animals belonging to rTbpA or rTbpB groups that died in response to challenge. Significant differences (P < 0.005) were found in these groups when comparing challenge with the following days after it. However, no significant differences were seen for the remaining vaccinated groups (NPAPTim, NPAPTit and PG), which were effectively protected against Glässer’s disease. Therefore, APPs could be used as useful biomarkers for both evaluating disease progression and determining vaccination effectivenessS