Phosphorylation of GntR reduces Streptococcus suis oxidative stress resistance and virulence by inhibiting NADH oxidase transcription.

GntR transcription factor of Streptococcus suis serotype 2 (SS2) is a potential substrate protein of STK, but the regulation mechanisms of GntR phosphorylation are still unclear. This study confirmed that STK phosphorylated GntR in vivo, and in vitro phosphorylation experiments showed that STK phosphorylated GntR at Ser-41. The phosphomimetic strain (GntR-S41E) had significantly reduced lethality in mice and reduced bacterial load in the blood, lung, liver, spleen, and brain of infected mice compared to wild-type (WT) SS2. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments demonstrated that the promoter of nox was bound by GntR. The phosphomimetic protein GntR-S41E cannot bind to the promoter of nox, and the nox transcription levels were significantly reduced in the GntR-S41E mutant compared to WT SS2. The virulence in mice and the ability to resist oxidative stress of the GntR-S41E strain were restored by complementing transcript levels of nox. NOX is an NADH oxidase that catalyzes the oxidation of NADH to NAD+ with the reduction of oxygen to water. We found that NADH is likely accumulated under oxidative stress in the GntR-S41E strain, and higher NADH levels resulted in increased amplified ROS killing. In total, we report GntR phosphorylation could inhibit the transcription of nox, which impaired the ability of SS2 to resist oxidative stress and virulence.

Streptococcal autolysin promotes dysfunction of swine tracheal epithelium by interacting with vimentin.

Streptococcus suis serotype 2 (SS2) is a major zoonotic pathogen resulting in manifestations as pneumonia and septic shock. The upper respiratory tract is typically thought to be the main colonization and entry site of SS2 in pigs, but the mechanism through which it penetrates the respiratory barrier is still unclear. In this study, a mutant with low invasive potential to swine tracheal epithelial cells (STECs) was screened from the TnYLB-1 transposon insertion mutant library of SS2, and the interrupted gene was identified as autolysin (atl). Compared to wild-type (WT) SS2, Δatl mutant exhibited lower ability to penetrate the tracheal epithelial barrier in a mouse model. Purified Atl also enhanced SS2 translocation across STEC monolayers in Transwell inserts. Furthermore, Atl redistributed the tight junctions (TJs) in STECs through myosin light chain kinase (MLCK) signaling, which led to increased barrier permeability. Using mass spectrometry, co-immunoprecipitation (co-IP), pull-down, bacterial two-hybrid and saturation binding experiments, we showed that Atl binds directly to vimentin. CRISPR/Cas9-targeted deletion of vimentin in STECs (VIM KO STECs) abrogated the capacity of SS2 to translocate across the monolayers, SS2-induced phosphorylation of myosin II regulatory light chain (MLC) and MLCK transcription, indicating that vimentin is indispensable for MLCK activation. Consistently, vimentin null mice were protected from SS2 infection and exhibited reduced tracheal and lung injury. Thus, MLCK-mediated epithelial barrier opening caused by the Atl-vimentin interaction is found to be likely the key mechanism by which SS2 penetrates the tracheal epithelium.

Glaesserella parasuis serotype 5 breaches the porcine respiratory epithelial barrier by inducing autophagy and blocking the cell membrane Claudin-1 replenishment.

Glaesserella parasuis (G. parasuis), the primary pathogen of Glässer's disease, colonizes the upper respiratory tract and can break through the epithelial barrier of the respiratory tract, leading to lung infection. However, the underlying mechanisms for this adverse effect remain unclear. The G. parasuis serotype 5 SQ strain (HPS5-SQ) infection decreased the integrity of piglets' lung Occludin and Claudin-1. Autophagy regulates the function of the epithelial barrier and tight junction proteins (TJs) expression. We tested the hypothesis that HPS5-SQ breaking through the porcine respiratory epithelial barrier was linked to autophagy and Claudin-1 degradation. When HPS5-SQ infected swine tracheal epithelial cells (STEC), autophagosomes encapsulated, and autolysosomes degraded oxidatively stressed mitochondria covered with Claudin-1. Furthermore, we found that autophagosomes encapsulating mitochondria resulted in cell membrane Claudin-1 being unable to be replenished after degradation and damaged the respiratory tract epithelial barrier. In conclusion, G. parasuis serotype 5 breaks through the porcine respiratory epithelial barrier by inducing autophagy and interrupting cell membrane Claudin-1 replenishment, clarifying the mechanism of the G. parasuis infection and providing a new potential target for drug design and vaccine development.

Comparative analysis of Streptococcus agalactiae serotypes Ia and II isolates from China and Pakistan in a murine model: A focus on pathogenesis and immune response.

Bovine mastitis, caused by Streptococcus agalactiae (Group B Streptococcus; GBS), poses significant economic challenges to the global dairy industry. Mouse models serves as valuable tools for assessing GBS-induced infections as an alternative to large animals. This study aimed to investigate the LD50 dose, organ bacterial load, and quantification of peritoneal leukocyte populations for GBS serotypes Ia and II isolates from China and Pakistan. Additionally, we measured indicators such as lactoferrin, albumin, and myeloperoxidase (MPO) activity. Pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-2) and anti-inflammatory cytokines (IL-10 and TGF-ß) in serum and tissue samples were evaluated using ELISA and qPCR, respectively. BALB/c mice (4 mice per group) received individual intraperitoneal injections of 100 µl containing specific bacterial inoculum concentrations (ranging from 105 to 109 CFU per mouse) of Chinese and Pakistani GBS isolates (serotypes Ia and II). Control groups received 100 µL of sterile PBS. Results revealed that the LD50 bacterial dose causing 50 % mortality in mice was 107 CFU. The highest bacterial load in all experimental groups was quantified in the peritoneum, followed by blood, mammary gland, liver, spleen, lungs, and brain. The most significant bacterial dissemination was observed in mice inoculated with Pakistani serotype Ia at 24 h, with a subsequent notable decline in bacterial counts at day 3. Notably, infection with Pakistani serotype Ia showed a trend of increased total leukocyte counts, significantly higher than Pakistani serotype II, Chinese Serotype Ia, and Chinese serotype II. A substantial influx of neutrophils and lymphocytes was observed in response to all tested serotypes, with Pakistani serotype Ia inducing a significantly higher influx compared to other groups (Pakistani serotype II, Chinese serotype Ia, and Chinese serotype II). Furthermore, TNF-α, IL-1ß, IL-2, and IL-6 expressions were significantly increased in mice one day after infection with the Pakistani serotype Ia. Compared to mice infected with the Pakistani serotype II, Chinese Serotype Ia, and Chinese serotype II, those infected with the Pakistani serotype Ia isolate exhibited the highest production of IL-10 and TGF-ß, along with significantly increased concentrations of lactoferrin, albumin, and MPO. These findings suggest that the persistence and severity of infection caused by the Pakistani serotype Ia may be linked to its ability to spread to deeper tissues. This study enhances our understanding of the clinical characteristics of bovine mastitis caused by S. agalactiae in China and Pakistan.

Ni-Co Complex Ionic Synergistically Modifying Octahedron Lattice of Cordierite Ceramics for the Application of 5G Microwave-Millimeter-wave Antennas.

In this work, phase-pure Mg1.8(Ni1-xCox)0.2Al4Si5O18 (0 ≤ x ≤ 1) ceramics were synthesized by a high-temperature solid-state method. On the basis of Rietveld refinement data of X-ray powder diffraction and Phillips-Vechten-Levine theory, the atomic ionicity, lattice energy, and bond energy of the compound were calculated to explore their influence on the microwave dielectric properties of ceramics. The Mg1.8Ni0.1Co0.1Al4Si5O18 (x = 0.5) ceramic exhibited the best microwave dielectric properties: εr = 4.44, Qf = 73 539 GHz@13 GHz, and τf = -23.9 ppm/°C. (Ni1-xCox)2+ complex ionic doping, compared with only Ni2+ or Co2+, is beneficial for improving the symmetry of [Si4Al2O18] hexagonal rings and reducing distortion. Subsequently, 8 wt % TiO2 was added to Mg1.8Ni0.1Co0.1Al4Si5O18, resulting in a near-zero τf and high Qf values for the composite ceramic, with εr = 5.22, Qf = 58 449 GHz@13 GHz, and τf = -2.06 ppm/°C. Finally, a 5G millimeter-wave antenna with a central operating frequency of 25.52 GHz was designed and fabricated using the Mg1.8Ni0.1Co0.1Al4Si5O18-8 wt % TiO2 ceramics. Operating in the 24.7-26.0 GHz range, it demonstrated favorable radiation characteristics with a simulated efficiency of 85.2% and a gain of 4.58 dBi. The antenna's performance confirms the high potential of the cordierite composite for application in 5G communication systems.

Vimentin affects inflammation and neutrophil recruitment in airway epithelium during Streptococcus suis serotype 2 infection.

Streptococcus suis serotype 2 (SS2) frequently colonizes the swine upper respiratory tract and can cause Streptococcal disease in swine with clinical manifestations of pneumonia, meningitis, and septicemia. Previously, we have shown that vimentin, a kind of intermediate filament protein, is involved in the penetration of SS2 through the tracheal epithelial barrier. The initiation of invasive disease is closely related to SS2-induced excessive local inflammation; however, the role of vimentin in airway epithelial inflammation remains unclear. Here, we show that vimentin deficient mice exhibit attenuated lung injury, diminished production of proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and the IL-8 homolog, keratinocyte-derived chemokine (KC), and substantially reduced neutrophils in the lungs following intranasal infection with SS2. We also found that swine tracheal epithelial cells (STEC) without vimentin show decreased transcription of IL-6, TNF-α, and IL-8. SS2 infection caused reassembly of vimentin in STEC, and pharmacological disruption of vimentin filaments prevented the transcription of those proinflammatory cytokines. Furthermore, deficiency of vimentin failed to increase the transcription of nucleotide oligomerization domain protein 2 (NOD2), which is known to interact with vimentin, and the phosphorylation of NF-κB protein p65. This study provides insights into how vimentin promotes excessive airway inflammation, thereby exacerbating airway injury and SS2-induced systemic infection.

The recombinant swinepox virus expressing sseB could provide piglets with strong protection against Salmonella typhimurium challenge.

Salmonella spp. poses a great threat to the livestock, food safety and public health. A recombinant swinepox virus expressing a protective antigen sseB was constructed by homologous recombination to develop a vaccine against Salmonella infection. The rSPV-sseB was verified using PCR, Western blot and indirect immunofluorescence assay. The immune responses and protective efficacy of rSPV-sseB were assessed in piglets. Forty piglets were immunized with rSPV-sseB, inactive Salmonella vaccine, wild-type SPV (wtSPV), or PBS. The results showed that the level of the sseB-specific antibody of the rSPV-sseB-vaccinated piglets was significantly higher at all time points post-vaccination than those of the inactivated Salmonella vaccine (P < 0.05), wtSPV (P < 0.001) or mock treated piglets (P < 0.001). The IL-4 and IFN-γ in the rSPV-sseB group were significantly higher than the other three groups at all post-infection time points. rSPV-sseB provided piglets with strong protection against the challenge of S. typhimurium with lethal dose. These results suggest the possibility of using recombinant swinepox virus rSPV-sseB as a promising vaccine to prevent Salmonella infection.

Evaluation of the differences between biofilm and planktonic Brucella abortus via metabolomics and proteomics.

This study analyzed the difference between biofilm and planktonic Brucella abortus using metabolomics and proteomics. Brucella abortus was cultured in different media to induce Brucella abortus biofilm formation and planktonic cells, followed by metabolomics and proteomics analyses for these two samples. Significant differential metabolites were identified, followed by KEGG pathway analysis. Differentially expressed proteins were identified, followed by subcellular localization, GO annotation, and KEGG pathway enrichment. Additionally, a correlation analysis of metabolomics and proteomics was performed. Metabolomics analysis showed 7682 positive and 4433 negative metabolites, including 188 positive and 117 negative significant differential metabolites. These differential metabolites were enriched in fatty acid/unsaturated fatty acid biosynthesis and linoleic acid metabolism. Proteomics analysis revealed 1759 proteins, including 486 differentially expressed proteins, which were enriched in various metabolic and degradation-related pathways. Subcellular localization showed that 74.3% of the differential proteins were cytoplasmic proteins. Correlation analysis showed that 1-palmitoyl-2-oleoyl-phosphatidylglycerol had the most significant correlations with proteins, followed by cytosine. Both metabolites correlated with the protein Q57EI7 (RbsB-1, ribose ABC transporter). One common pathway, fatty acid biosynthesis, was identified by both proteomics and metabolomics analyses that involved the metabolites, oleic acid, and protein Q57DK3 (biotin carboxylase). There were metabolomic and proteomic differences between Brucella abortus biofilm and planktonic cells, and these results provide novel insights into the biofilm-forming process of Brucella abortus.

A streptococcal Fic domain-containing protein disrupts blood-brain barrier integrity by activating moesin in endothelial cells.

Streptococcus equi subsp. zooepidemicus (SEZ) is a zoonotic pathogen capable of causing meningitis in humans. The mechanisms that enable pathogens to traverse the blood-brain barrier (BBB) are incompletely understood. Here, we investigated the role of a newly identified Fic domain-containing protein, BifA, in SEZ virulence. BifA was required for SEZ to cross the BBB and to cause meningitis in mice. BifA also enhanced SEZ translocation across human Brain Microvascular Endothelial Cell (hBMEC) monolayers. Purified BifA or its Fic domain-containing C-terminus alone were able to enter into hBMECs, leading to disruption of monolayer barrier integrity. A SILAC-based proteomic screen revealed that BifA binds moesin. BifA's Fic domain was required for its binding to this regulator of host cell cytoskeletal processes. BifA treatment of hBMECs led to moesin phosphorylation and downstream RhoA activation. Inhibition of moesin activation or moesin depletion in hBMEC monolayers abrogated BifA-mediated increases in barrier permeability and SEZ's capacity to translocate across monolayers. Thus, BifA activation of moesin appears to constitute a key mechanism by which SEZ disrupts endothelial monolayer integrity to penetrate the BBB.

Glaesserella parasuis serotype 4 HPS4-YC disrupts the integrity of the swine tracheal epithelial barrier and facilitates bacterial translocation.

Glaesserella parasuis (G. parasuis) is a commensal bacterium in the upper respiratory tract of pigs that can also cause the swine Glässer disease, which induces an intensive inflammatory response and results in significant economic losses to the swine industry worldwide. G. parasuis can cause disease through infection of the respiratory tract, resulting in systemic infection, but the mechanism is largely unknown. Recently we showed that Glaesserella parasuis serotype 4 (GPS4) increased swine tracheal epithelial barrier permeability, resulting in easier bacterial translocation. Tight junction proteins (TJ) play a crucial role in maintaining the integrity and impermeability of the epithelial barrier. GPS4 decreased the expression of the TJ ZO-1 and occludin in swine tracheal epithelial cells (STEC). Furthermore, the proinflammatory cytokines IL-6, IL-8 and TNF-α were significantly upregulated in GPS4-infected STEC, and both the MAPK and NF-κB signaling pathways were activated and contributed to the expression of TNF-α. We demonstrate that the production of proinflammatory cytokines, especially TNF-α, during GPS4 infection was involved in barrier dysfunction. Additionally, animal challenge experiments confirmed that GPS4 infection downregulated TJ in the lungs of piglets and induced a severe inflammatory response. In general, G. parasuis infection downregulated the expression of TJ and induced massive secretion of proinflammatory cytokines, resulting in epithelial barrier disruption and favoring bacterial infection. This study allowed us to better understand the mechanism by which G. parasuis crosses the respiratory tract of pigs.

Porcine circovirus type 2 exploits JNK-mediated disruption of tight junctions to facilitate Streptococcus suis translocation across the tracheal epithelium.

Porcine circovirus type 2 (PCV2) is considered as the primary pathogen of porcine circovirus-associated disease (PCVAD), which results in significant economic losses worldwide. Clinically, PCV2 often causes disease through coinfection with other bacterial pathogens, including Streptococcus suis (S. suis), and especially the highly prevalent S. suis serotype 2 (SS2). The present study determined that continuous PCV2 infection in piglets down-regulates tight junction proteins (TJ) ZO-1 and occludin in the lungs. Swine tracheal epithelial cells (STEC) were used to explore the mechanisms and consequences of disruption of TJ, and an in vitro tracheal epithelial barrier model was established. Our results show that PCV2 infection in STEC decreases the expression levels of ZO-1 and occludin and increases the permeability of the tracheal epithelial barrier, resulting in easier translocation of SS2. Moreover, Western blot analysis indicates that PCV2 infection activates the JNK/MAPK pathway. The disruption of TJ in SETC and increased permeability of the epithelial barrier induced by PCV2 could be alleviated by inhibition of JNK phosphorylation, which indicates that the JNK/MAPK pathway regulates the expression of ZO-1 and occludin during PCV2 infection. This study allows us to better understand the mechanisms of PCV2 coinfection with bacterial pathogens and provides new insight into controlling the occurrence of PCVAD.

Screening of Streptococcus Suis serotype 2 resistance genes with GWAS and transcriptomic microarray analysis.

BACKGROUND: Swine streptococcosis has caused great economic loss in the swine industry, and the major pathogen responsible for this disease is Streptococcus Suis serotype 2 (SS2). Disease resistance breeding is a fundamental way of resolving this problem. With the development of GWAS and transcriptomic microarray technology, we now have powerful research tools to identify SS2 resistance genes. RESULTS: In this research, we generated an F2 generation of SS2 resistant C57BL/6 and SS2 susceptive A/J mice. With the F2 generation of these two mice strains and GWAS analysis, we identified 286 significant mouse genome SNPs sites associated with the SS2 resistance trait. Gene expression profiles for C57BL/6 and A/J were analyzed under SS2 infection pressure by microarray. In total, 251 differentially expressed genes were identified between these two mouse strains during SS2 infection. After combining the GWAS and gene expression profile data, we located two genes that were significantly associated with SS2 resistance, which were the UBA domain containing 1 gene (Ubac1) and Epsin 1 gene (Epn 1). GO classification and over-representation analysis revealed nine up-regulated related to immune function, which could potentially be involved in the C57BL/6 SS2 resistance trait. CONCLUSION: This is the first study to use both SNP chip and gene express profile chip for SS2 resistance gene identification in mouse, and these results will contribute to swine SS2 resistance breeding.

Swinepox virus vector-based vaccines: attenuation and biosafety assessments following subcutaneous prick inoculation.

Swinepox virus (SPV) has several advantages as a potential clinical vector for a live vector vaccine. In this study, to obtain a safer and more efficient SPV vector, three SPV mutants, Δ003, Δ010, and ΔTK were successfully constructed. A virus replication experiment showed that these SPV mutants had lower replication abilities compared to wtSPV in 10 different host-derived cell lines. Animal experiments with mouse and rabbit models demonstrate that these three mutants and wtSPV did not cause any clinical signs of dermatitis. No fatalities were observed during a peritoneal challenge assay with these mutants and wtSPV in a mouse model. Additionally, the three mutants and wtSPV were not infectious at 60 h after vaccination in rabbit models. Furthermore, we evaluated biosafety, immunogenicity and effectiveness of the three mutants in 65 1-month-old piglets. The results show that there were no clinical signs of dermatitis in the Δ003 and ΔTK vaccination groups. However, mild signs were observed in the Δ010 vaccination groups when virus titres were high, and apparent clinical signs were observed at the sites of inoculation. Samples from all experimental pig groups were assessed by qPCR, and no SPV genomic DNA was found in five organs, faeces or blood. This suggests that the infectious abilities of wtSPV and the SPV mutants were poor and limited. In summary, this study indicates that two mutants of SPV, Δ003 and ΔTK, may be promising candidates for an attenuated viral vector in veterinary medicine.

Development and application of an indirect ELISA for the detection of antibodies to porcine epidemic diarrhea virus based on a recombinant spike protein.

BACKGROUND: As the major causative agent of swine viral diarrhea, porcine epidemic diarrhea virus (PEDV) has caused massive losses to the economies of swine raising countries. Accordingly, the serological detection of corresponding antibodies would be beneficial to diagnose PEDV indirectly to control the disease. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) based on the recombinant truncated spike (S) protein of PEDV was developed and validated. RESULTS: The reaction conditions of the developed indirect ELISA were optimized. This indirect ELISA was compared to indirect immunoinfluscent assay (IFA), and the overall coincidence rate was 96.74% based on testing 368 clinical serum samples with different PEDV antibody levels. No cross-reactivity with other common swine pathogens was detected for the developed S1 indirect ELISA. Finally, the S1 indirect ELISA was applied to detect serum antibodies of 3304 field samples collected from different pig farms in eastern China, and it presented an overall substantial agreement on the PEDV infection status. CONCLUSIONS: This established S1 indirect ELISA is capable of detecting serum antibodies against PEDV, and due to its high sensitivity and specificity, it could be applied for serological evaluation and indirect diagnosis of PEDV infection.

Detection of Salmonella Infection in Chickens by an Indirect Enzyme-Linked Immunosorbent Assay Based on Presence of PagC Antibodies in Sera.

The outcomes of infection of humans and animals with Salmonella range from a persistent asymptomatic carrier state to temporal mild gastroenteritis or severe systemic infection. A rapid and accurate diagnostic test would help formulate strategies for effective prevention of their infections in the animal population. Current sequencing data predict that the outer membrane protein, PagC, is present in all common Salmonella serovars with sequence similarities of more than 98%. PagC sequences in other bacterial species are less than 65% similarity at the amino acid level to those of Salmonella PagC. We hypothesized that PagC could be immunogenic and detection of antibodies to this protein could be an accurate indicator of Salmonella infection. The pagC gene from Salmonella enterica serovar Typhimurium CVCC542 was expressed in Escherichia coli. The purified recombinant PagC protein was immobilized in microtiter plate wells. Sera from SPF chickens infected with Salmonella or other non-Salmonella pathogens by injection were added and binding of PagC protein was detected by the horseradish peroxidase (HRP)-labeled goat anti-chicken antibody. Sera from Salmonella-infected chickens showed high specificity in contrast to the sera from chickens infected with other bacteria. When 87 Salmonella antibody-positive sera from Salmonella Pullorum orally infected SPF chicken and 93 negative sera from uninfected SPF chicken were tested, 98.3% agreement was detected. The rPagC enzyme-linked immunosorbent assay (ELISA) and agglutination had 80.6% agreement in detecting 252 clinical chicken sera samples. These results suggest that PagC antibody-based indirect ELISA can serve as a convenient and novel method for the diagnosis of Salmonella infection.

Differential Protein Analysis of IPEC-J2 Cells Infected with Porcine Epidemic Diarrhea Virus Pandemic and Classical Strains Elucidates the Pathogenesis of Infection.

Porcine epidemic diarrhea (PED) re-emerged in China in late 2010 and has now become widespread. Accumulated evidence indicates that this large-scale outbreak of diarrhea was caused by variants of the highly virulent porcine epidemic diarrhea virus (PEDV). A pandemic PEDV YC2014 strain (YC2014) was isolated from clinical samples. An iTRAQ-based comparative quantitative proteomic study of IPEC-J2 cells infected with YC2014 and a classical CV777 strain (CV777) was performed to determine the differences between pandemic and classical PEDV strain infection. Totals of 353 and 299 differentially expressed proteins were identified upon YC2014 and CV777 infection, respectively. The canonical pathways and functional networks involved in both PEDV infections were analyzed. The results indicated that the PEDV suppressed protein synthesis of IPEC-J2 cells through down-regulation of the PI3K-AKT/mTOR signaling pathways. Infection with YC2014 could activate the JAK-STAT signaling pathway and the NF-κB pathway more intensively than CV777. YC2014 could activate NF-κB pathway more intensively than CV777. On the basis of differentially expressed proteins, we propose that PEDV might disrupt apoptosis and may elicit stronger inflammatory cascades as well. This study might contribute to an understanding of the pathogenesis of PEDV infection and aid in the development of effective preventive and control vaccines.

Efficacy and immunogenicity of recombinant swinepox virus expressing the truncated S protein of a novel isolate of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) causes significant loss to the swine industry. The emergence of novel PEDV strains in recent years has decreased the effectiveness of PEDV vaccines. We have developed a live recombinant vaccine, a swinepox virus vector that expresses a truncated S protein (rSPV-St) from a recent PEDV strain, SQ2014, and evaluated its immunogenicity and effectiveness in a swine model. Vaccination of swine with rSPV-St elicited a robust antibody response specific for the homologous PEDV SQ2014. Serum IgA titers in rSPV-St-vaccinated animals were significantly higher than in those immunized with inactivated vaccines. The effectiveness of antibodies induced by the rSPV-St vaccine in protection against PEDV was tested in a passive-transfer model in which piglets were challenged with the homologous virus SQ2014 and the heterologous strain CV777. When challenged with the homologous virus, sera from rSPV-St vaccination provided complete protection. However, sera from rSPV-St vaccination did not provide any protection against the heterologous virus challenge. Amino acid sequence differences in the S proteins of the two viruses were identified within neutralizing epitopes, which might have contributed to the divergent clinical results. Our data suggest that rSPV-St is potentially an effective vaccine against infection with emerging PEDV strains.

Epidemic strain YC2014 of porcine epidemic diarrhea virus could provide piglets against homologous challenge.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is the main causative agent of porcine epidemic diarrhea (PED). Since December 2010, a large-scale outbreak of diarrhea has been observed in swine farms in China. Accumulated evidence indicates that this large-scale outbreak of diarrhea were caused by highly virulent PEDV variants. METHODS: A PEDV strain, YC2014, was isolated from intestinal samples of suckling piglets with acute diarrhea in 2014. The complete genomic sequence of YC2014 and the nucleotide sequence of S gene were aligned with sequences of published isolates using MEGA 5.1 software. The immune protective efficiency of YC2014 were determined by testing PEDV neutralizing antibodies in sera, the colostrum and the milk on 7th day after farrowing of the immunized sows. The diarrhea symptoms of piglets after challenge were also observed. RESULTS: Phylogenetic analysis of the complete genomic sequence of YC2014 and the nucleotide sequence of S gene demonstrated that the YC2014 PEDV strain was clustered with the PEDV epidemic strains, with >99 % nucleotide identity to these PEDV strains. The S gene sequence of YC2014 shared only 93.9 % ~ 94.4 % identities with classical CV777, DR13 and JS2008 strains, with 15 nucleotide insertion in three sites and three nucleotide deletion in one site. The amino acid (AA) sequence of S gene of YC2014 shared only 92.8 % ~ 93.4 % identities with classical CV777, DR13 and JS2008 strains, with 5 AA insertion in two sites and 1 AA deletion in one site. In the immune protective efficiency tests, the neutralizing antibody titers in sera, the colostrum and the milk on 7th day after farrowing of the inactivated YC2014 PEDV strain immunized group were significantly higher than the inactivated CV777 immunized group and the inactivated DR13 immunized group (P < 0.05). The traditional inactivated PEDV vaccines made from CV777 or DR13 could not protect piglets from YC2014 challenge, while inactivated YC2014 could provide piglets with 100 % protection against YC2014 challenge. CONCLUSIONS: The results showed that, great antigenicity variation had occurred to this YC2014 PEDV strain. The YC2014 PEDV strain could provide piglets against homologous challenge. It is critical for future pathogenic and antigenic studies, as well as for the development of effective preventive and control vaccines against PEDV.

Identification and characterization of a Streptococcus equi ssp. zooepidemicus immunogenic GroEL protein involved in biofilm formation.

Streptococcus equi ssp. zooepidemicus (S. equi spp. zooepidemicus) is an opportunistic pathogen that causes major economic losses in the swine industry in China and is also a threat for human health. Biofilm formation by this bacterium has been previously reported. In this study, we used an immunoproteomic approach to search for immunogenic proteins expressed by biofilm-grown S. equi spp. zooepidemicus. Seventeen immunoreactive proteins were found, of which nine common immunoreactive proteins were identified in planktonic and biofilm-grown bacteria. The immunogenicity and protective efficacy of the S. equi spp. zooepidemicus immunoreactive GroEL chaperone protein was further investigated in mice. The protein was expressed in vivo and elicited high antibody titers following S. equi spp. zooepidemicus infections of mice. An animal challenge experiment with S. equi spp. zooepidemicus showed that 75% of mice immunized with the GroEL protein were protected. Using in vitro biofilm inhibition assays, evidence was obtained that the chaperonin GroEL may represent a promising target for the prevention and treatment of persistent S. equi spp. zooepidemicus biofilm infections. In summary, our results suggest that the recombinant GroEL protein, which is involved in biofilm formation, may efficiently stimulate an immune response, which protects against S. equi spp. zooepidemicus infections. It may therefore be a candidate of interest to be included in vaccines against S. equi spp. zooepidemicus infections.

Glaesserella parasuis serotype 5 induces pyroptosis via the RIG-I/MAVS/NLRP3 pathway in swine tracheal epithelial cells.

Glaesserella parasuis (G. parasuis) is a common Gram-negative commensal bacterium in the upper respiratory tract of swine that can cause Glässer's disease under stress conditions. Pyroptosis is an important immune defence mechanism of the body that plays a crucial role in clearing pathogen infections and endogenous danger signals. This study aimed to investigate the mechanism of G. parasuis serotype 5 SQ (GPS5-SQ)-induced pyroptosis in swine tracheal epithelial cells (STECs). The results of the present study demonstrated that GPS5-SQ infection induces pyroptosis in STECs by enhancing the protein level of the N-terminal domain of gasdermin D (GSDMD-N) and activating the NOD-like receptor protein 3 (NLRP3) inflammasome. Furthermore, the levels of pyroptosis-related proteins, including GSDMD-N and cleaved caspase-1 were considerably decreased in STECs after the knockdown of retinoic acid inducible gene-I (RIG-I) and mitochondrial antiviral signaling protein (MAVS). These results indicated that GPS5-SQ might trigger pyroptosis through the activation of the RIG-I/MAVS/NLRP3 signaling pathway. More importantly, the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) repressed the activation of the RIG-I/MAVS/NLRP3 signaling and rescued the decrease in Occludin and zonula occludens-1 (ZO-1) after GPS5-SQ infection. Overall, our findings show that GPS5-SQ can activate RIG-I/MAVS/NLRP3 signaling and destroy the integrity of the epithelial barrier by inducing ROS generation in STECs, shedding new light on G. parasuis pathogenesis.