Seroprevalence survey of Hepatitis E Virus in Domestic Pigs in Guangdong, China.

The Hepatitis E virus (HEV) causes acute and chronic Hepatitis E and is a global public health concern. HEV genotypes 3 (HEV-3) and 4 (HEV-4) are common to humans and animals, and domestic pigs and wild boars have been identified as the main reservoirs. However, limited information is available on the status of HEV infection in pigs, particularly in the Guangdong Province, China. This study aimed to investigate the seroprevalence of HEV in pig farms within the Guangdong Province. A total of 1568 serum samples were collected from 25 farms and tested for anti-HEV IgG antibodies. Enzyme-linked immunosorbent assay (ELISA) results revealed that 57.53% (902/1568) of serum samples from 24 farms (24/25, 96%) were positive for anti-HEV IgG antibodies. Year, season, region, and age were all linked risk factors for HEV in Guangdong, with season and region showing more significant impacts. The results showing a high seroprevalence of HEV confirmed its circulation among domestic pigs in the Guangdong Province, China. The presence of this antibody indicates that HEV infection was or is present on farms, posing a risk of zoonotic transmission of HEV from pigs to exposed workers and from pork or organs to consumption.

Development and application of a TaqMan-probe-based multiplex real-time PCR assay for simultaneous detection of porcine circovirus 2, 3, and 4 in Guangdong province of China.

Porcine circoviruses disease (PCVD), caused by porcine circovirus (PCVs), is an important swine disease characterized by porcine dermatitis, nephrotic syndrome and reproductive disorders in sows. However, diseases caused by PCV2, PCV3, or PCV4 are difficult to distinguish, so a simple, rapid, accurate and high-throughput diagnostic and identification method is urgently needed to differentiate these three types. In this study, specific primers and probes were designed based on the conserved region sequences of the Rep gene of PCV2, and the Cap gene of PCV3 and PCV4. A multiplex qPCR assay was developed and optimized that the limit of detection concentration could reach as low as 3.8 copies/µL, with all correlation coefficients (R2) exceeding 0.999. Furthermore, the method showed no cross-reaction with other crucial porcine viral pathogens, and both intra-repeatability and inter-reproducibility coefficients of variation were below 2%. The assay was applied to the detection of 738 pig samples collected from 2020 to 2021 in Guangdong Province, China. This revealed positive infection rates of 65.18% for PCV2, 29.27% for PCV3, and 0% for PCV4, with a PCV2/PCV3 co-infection rate of 23.17%. Subsequently, complete genome sequences of 17 PCV2 and 4 PCV3 strains were obtained from the above positive samples and pre-preserved positive circovirus samples. Nucleotide sequence analysis revealed that the 17 PCV2 strains shared 96.7-100% complete nucleotide identity, with 6 strains being PCV2b and 11 strains being PCV2d; the 4 PCV3 strains shared 98.9-99.4% complete nucleotide identity, with 2 strains being PCV3a-1 and 2 strains being PCV3b. This research provides a reliable tool for rapid PCVs identification and detection. Molecular epidemiological investigation of PCVs in pigs in Guangdong Province will help us to understand PCV2 and PCV3 epidemiological characteristics and evolutionary trends.

Pseudorabies gD protein protects mice and piglets against lethal doses of pseudorabies virus.

Introduction: Pseudorabies (PR) is a highly contagious viral disease caused by the pseudorabies virus (PRV), which can cause disease in a wide range of domestic and wild animals. Studies have shown that new mutant strains have emerged in pig farms in many regions and that commercial inactivated and live attenuated vaccines are becoming less effective at protecting pigs. Methods: Porcine pseudorabies glycoprotein D (gD) gene (GenBank: QEY95774.1) with hexa-His tag to the C terminus for further purification processes was cloned into the lentiviral expression plasmid pLV-CMV-eGFP by restriction enzyme, the resulting plasmid was designated as pLV-CMV-gD. HEK-293T cells with robust and stable expression of recombinant gD protein was established by infection with recombinant lentivirus vector pLV-CMV-gD. We expressed porcine pseudorabies virus gD protein using HEK-293T cells. Results: We describe in this study that individual gD proteins produced by a mammalian cell expression system are well immunogenic and stimulate high levels of PRV-specific and neutralizing antibodies in mice and piglets. All mice and piglets survived lethal doses of PRV, significantly reducing the amount of PRV virus in piglets' lymph nodes, lungs, spleen, and other tissues. It also significantly reduced the time cycle and amount of viral excretion from piglets to the environment through the nasal and anal cavities. Discussion: The results suggest that PRV gD protein is expected to be a potential candidate for the preparation of genetically engineered PR vaccines for the prevention of PRV infection and the control of PR epidemics.

iTRAQ-based quantitative proteomic analysis of the antibacterial mechanism of silver nanoparticles against multidrug-resistant Streptococcus suis.

Background: The increase in antibiotic resistance of bacteria has become a major concern in clinical treatment. Silver nanoparticles (AgNPs) have significant antibacterial effects against Streptococcus suis. Therefore, this study aimed to investigate the antibacterial activity and mechanism of action of AgNPs against multidrug-resistant S. suis. Methods: The effect of AgNPs on the morphology of multidrug-resistant S. suis was observed using scanning electron microscopy (SEM). Differentially expressed proteins were analyzed by iTRAQ quantitative proteomics, and the production of reactive oxygen species (ROS) was assayed by H2DCF-DA staining. Results: SEM showed that AgNPs disrupted the normal morphology of multidrug-resistant S. suis and the integrity of the biofilm structure. Quantitative proteomic analysis revealed that a large number of cell wall synthesis-related proteins, such as penicillin-binding protein and some cell cycle proteins, such as the cell division protein FtsZ and chromosomal replication initiator protein DnaA, were downregulated after treatment with 25 µg/mL AgNPs. Significant changes were also observed in the expression of the antioxidant enzymes glutathione reductase, alkyl hydroperoxides-like protein, α/ß superfamily hydrolases/acyltransferases, and glutathione disulfide reductases. ROS production in S. suis positively correlated with AgNP concentration. Conclusion: The potential antibacterial mechanism of AgNPs may involve disrupting the normal morphology of bacteria by inhibiting the synthesis of cell wall peptidoglycans and inhibiting the growth of bacteria by inhibiting the cell division protein FtsZ and Chromosomal replication initiator protein DnaA. High oxidative stress may be a significant cause of bacterial death. The potential mechanism by which AgNPs inhibit S. suis biofilm formation may involve affecting bacterial adhesion and interfering with the quorum sensing system.

High-throughput sequencing of 16S rRNA gene analysis reveals novel taxonomic diversity among vaginal microbiota in healthy and affected sows with endometritis.

In sows afflicted with endometritis, vaginal microbiota can provide valuable information regarding bacterial community diversity. Our aim was to compare the vaginal microbiotas between endometritis and healthy sows and characterize the vaginal microbiota of endometritis sows using high-throughput sequencing of the 16S rRNA gene. Vaginal swabs were collected from healthy (Healthy_A, n = 10; Healthy_B, n = 10) and diseased (Endometritis_A, n = 10; Endometritis_B, n = 10) sows from two swine farms located in Guangdong and Yunnan province, in Southern China. The results of V3-V4 region of the 16S rRNA gene showed that Corynebacterium_1, Clostridium_sensu_stricto_1, Porphyromonas, Anaerococcus, Streptococcus, and Bacteroides comprised the core microbiota in all healthy sows. Two type of endometritis microbiota patterns were presented in two farms: the first comprised mostly of Burkholderia in farm A and the second comprised of Parvimonas in farm B. In farm A, the percentages of Burkholderia, Serratia, and Enterobacter were higher in the endometritis group, while only Parvimonas was significantly increased in the endometritis group in farm B (p < 0.05). Interestingly, the genus Burkholderia and Serratia were found only in the endometritis sows from farm A. Burkholderia was the most dominant genus found in endometritis sows and was confirmed by full-length 16S rRNA analysis using PacBio sequencing.

Proteome Analysis of Outer Membrane Vesicles From a Highly Virulent Strain of Haemophilus parasuis.

Haemophilus parasuis has emerged as an important bacterial pathogen in pig husbandry, as H. parasuis can coinfect pigs with a variety of pathogenic microorganisms and further cause an aggravation of the disease. It is crucial to investigate its pathogenetic mechanism. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs), and their potent virulence factors play prominent roles that affect the interaction between bacteria and host. Still, the pathogenesis that is associated with the bacterial OMVs has not been well-elucidated. In this study, we investigated the secretion of OMVs from a clinical H. parasuis isolate strain (H45). In addition, we further analyzed the characterization, the comprehensive proteome, and the virulence potential of OMVs. Our data demonstrated that H. parasuis could secrete OMVs into the extracellular milieu during infection. Using liquid chromatography with tandem mass spectrometry (MS/MS) identification and bio-information analysis, we identified 588 different proteins associated with OMVs. Also, we also analyzed the subcellular location and biological function of those proteins. These proteins are mainly involved in immune and iron metabolism. Moreover, we confirmed the pathogenicity of H. parasuis OMVs by observing a strong inflammatory response in J774A.1 and porcine alveolar macrophages. Taken together, our findings suggested that OMVs from H. parasuis were involved in the pathogenesis of this bacterium during infection.

Single Multiple Cross Displacement Amplification for Rapid and Real-Time Detection of Porcine Circovirus 3.

Since 2016, a novel porcine circovirus, PCV3, has been infecting pigs, causing significant economic losses to the pig industry. In recent years, the infection rate of PCV3 has been increasing, and thus rapid and accurate detection methods for PCV3 are essential. In this study, we established a novel probe-based single multiple cross displacement amplification (P-S-MCDA) method for PCV3. The method was termed as P-S-MCDA. The P-S-MCDA uses seven primers to amplify the capsid gene, and the assay can be performed at 60°C for 30 min, greatly shortening the reaction time. The results of P-S-MCDA can not only be monitored in real time through fluorescence signals but also be determined by observing the fluorescence of the reaction tubes using a smartphone-based cassette. This method demonstrated good specificity and the same sensitivity as qPCR, with a minimum detection limit of 10 copies. In 139 clinical samples, the coincidence rate with qPCR was 100%. The P-S-MCDA can be widely applied in PCV3 detection in laboratories or in rural areas.

RIPK3-Dependent Necroptosis Limits PRV Replication in PK-15 Cells.

Pigs infected by pseudorabies virus (PRV) display necrotic pathology in multiple organs. The mechanism by which PRV induces cell death is still unclear. Recently, necroptosis was identified as a programmed process dependent on the receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase-like protein (MLKL). In this study, we demonstrated that PRV induced RIPK3-dependent necroptosis in PK-15 cells. The data showed that PRV infection caused cell death with Propidium Iodide (PI)-positive staining. Transmission electron microscopy analysis indicated plasma membrane disruption in PRV-infected cells. A pan-caspase inhibitor did not prevent PRV-induced necrotic cell death. Western blot analysis indicated that caspase-3 and caspase-8 were not cleaved during PRV infection. Although the transcription of tumor necrosis factor-alpha (TNF-α) was increased by PRV infection, RIPK1 was shown to be not involved in PRV-induced necrotic cell death by use of its specific inhibitor. Further experiments indicated that the phosphorylation of RIPK3 and MLKL was upregulated in PRV-infected cells. Stable shRNA knockdown of RIPK3 or MLKL had a recovery effect on PRV-induced necrotic cell death. Meanwhile, viral titers were enhanced in RIPK3 and MLKL knockdown cells. Hence, we concluded that initiation of necroptosis in host cells plays a limiting role in PRV infection. Considering that necroptosis is an inflammatory form of programmed cell death, our data may be beneficial for understanding the necrotic pathology of pigs infected by PRV.

Comparison of Host Cytokine Response in Piglets Infected With Toxigenic and Non-toxigenic Staphylococcus hyicus.

Staphylococcus hyicus is the most common causative agent of exudative epidermitis (EE) in piglets. Staphylococcus hyicus can be grouped into toxigenic and non-toxigenic strains based on its ability to cause EE in pigs. However, the inflammatory response of piglets infected with toxigenic and non-toxigenic S. hyicus has not been elucidated. In this study, we evaluated the serum cytokine profile in piglets inoculated with toxigenic and non-toxigenic S. hyicus strains and recorded the clinical signs in piglets. Fifteen piglets were divided into three groups (n = 5) and inoculated with a toxigenic strain (ZC-4), a non-toxigenic strain (CF-1), and PBS (control), respectively. The changes in serum levels of cytokines (interleukin [IL]-1ß, IL-4, IL-6, IL-8, IL-10, IL-12, granulocyte-macrophage colony-stimulating factor, interferon-γ, transforming growth factor-ß1, and tumor necrosis factor-α) were evaluated using a cytokine array at 6, 24, 48, and 72 h post inoculation. The results showed that piglets infected with the toxigenic strain exhibited more severe clinical signs and higher mortality than those infected with the non-toxigenic strain. The serum levels of pro-inflammatory cytokine IL-1ß were significantly increased in toxigenic-and non-toxigenic-strain-infected piglets compared to those in the control group (p < 0.05), while the anti-inflammatory cytokine IL-10 was significantly up-regulated only in toxigenic group than in control group (p < 0.05). These results indicated that piglets infected with toxigenic and non-toxigenic S. hyicus showed differential infection status and inflammatory responses. Both toxigenic- and non-toxigenic- S. hyicus infection could induce a pro-inflammatory reaction in piglets. In addition, the toxigenic strain induced a strong anti-inflammatory response in piglets as indicated by the increased serum level of IL-10, which may be associated with the severe clinical signs and increased mortality and may be the key cytokine response responsible for pathogenic mechanisms of S. hyicus.

The Colorimetric Isothermal Multiple-Self-Matching-Initiated Amplification Using Cresol Red for Rapid and Sensitive Detection of Porcine Circovirus 3.

In 2016, a novel porcine circovirus (PCV), PCV3, was identified in USA. Subsequently, it was proved to be also epidemic in China, Poland, and Korea. To analyze and control the epidemic situation of PCV3, it is necessary to establish accurate and high-throughput detection methods. In this study, the colorimetric isothermal multiple-self-matching-initiated amplification (IMSA) using cresol red was developed to detect PCV3 for the first time. The reaction can be easily performed by incubating the tube at 63°C for 60 min. By the addition of pH-sensitive indicator dye cresol red, the initial color of the reaction mixture is red. When PCV3 capsid gene DNA was positive in the sample, the color of the reaction mixture changed from red to yellow after the isothermal incubation at 63°C, while the negative control maintained the red color. The colorimetric IMSA displayed good specificity in detecting PCV3, PCV2, and PCV1 and 4 porcine DNA pathogens. Moreover, it has a low and repeatable detection limit of 10 copies, which is consistent with TaqMan-based qPCR, but 10 times more sensitive than PCR. In diagnosing 128 clinical specimens, it not only showed 100% agreement with qPCR but also detected 15 positive results more than PCR. The colorimetric IMSA we offered might be a good choice for PCV3 epidemiological investigation and point-of-care testing.

Metabolomics Exploration of Pseudorabies Virus Reprogramming Metabolic Profiles of PK-15 Cells to Enhance Viral Replication.

For viral replication to occur in host cells, low-molecular-weight metabolites are necessary for virion assembly. Recently, metabolomics has shown great promise in uncovering the highly complex mechanisms associated with virus-host interactions. In this study, the metabolic networks in PK-15 cells infected with a variant virulent or classical attenuated pseudorabies virus (PRV) strains were explored using gas chromatography-mass spectrometry (GC-MS) analysis. Although total numbers of metabolites whose levels were altered by infection with the variant virulent strain or the classical attenuated strain were different at 8 and 16 h post infection (hpi), the predicted levels of differential metabolic components were shown to be associated with specific pathways, including glycolysis as well as amino acid and nucleotide metabolism. The glucose depletion and glycolysis inhibitors 2DG and oxamate could reduce the level of PRV replication in PK-15 cells. In addition, the inhibition of the pentose phosphate pathway (PPP) resulted in an obvious decline of viral titers, but the prevention of oxidative phosphorylation in the tricarboxylic acid (TCA) cycle had a minimal effect on viral replication. Glutamine starvation resulted in the decline of viral titers, which could be restored by supplemental addition in the culture media. However, inhibition of glutaminase (GLS) activity or the supplement of 2-ketoglutarate into glutamine-deleted DMEM did not alter PRV replication in PK-15 cells. The results of the current study indicate that PRV reprograms the metabolic activities of PK-15 cells. The metabolic flux from glycolysis, PPP and glutamine metabolism to nucleotide biosynthesis was essential for PRV to enhance its replication. This study will help to identify the biochemical materials utilized by PRV replication in host cells, and this knowledge can aid in developing new antiviral strategies.

Dynamic Change of Gut Microbiota During Porcine Epidemic Diarrhea Virus Infection in Suckling Piglets.

Porcine epidemic diarrhea (PED) is a disease that has a devastating effect on livestock. Currently, most studies are focused on comparing gut microbiota of healthy piglets and piglets with PED, resulting in gut microbial populations related to dynamic change in diarrheal piglets being poorly understood. The current study analyzed the characteristics of gut microbiota in porcine epidemic diarrhea virus (PEDV)-infected piglets during the suckling transition stage. Fresh fecal samples were collected from 1 to 3-week-old healthy piglets (n = 20) and PEDV infected piglets (n = 18) from the same swine farm. Total DNA was extracted from each sample and the V3-V4 hypervariable region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform. Statistically significant differences were observed in bacterial diversity and richness between the healthy and diarrheal piglets. Principal coordinates analysis (PCoA) showed structural segregation between diseased and healthy groups, as well as among 3 different age groups. The abundance of Escherichia-Shigella, Enterococcus, Fusobacterium, and Veillonella increased due to dysbiosis induced by PEDV infection. Notably, there was a remarkable age-related increase in Fusobacterium and Veillonella in diarrheal piglets. Certain SCFA-producing bacteria, such as Ruminococcaceae_UCG-002, Butyricimonas, and Alistipes, were shared by all healthy piglets, but were not identified in various age groups of diarrheal piglets. In addition, significant differences were observed between clusters of orthologous groups (COG) functional categories of healthy and PEDV-infected piglets. Our findings demonstrated that PEDV infection caused severe perturbations in porcine gut microbiota. Therefore, regulating gut microbiota in an age-related manner may be a promising method for the prevention or treatment of PEDV.

Association Between agr Type, Virulence Factors, Biofilm Formation and Antibiotic Resistance of Staphylococcus aureus Isolates From Pork Production.

Livestock-associated Staphylococcus aureus colonization and/or infections exist in pigs and people in frequent contact with pigs. In this study, a total of 130 S. aureus isolates obtained from different stages of pork production were subjected to antimicrobial susceptibility, biofilm formation, as well as PCR screening to identify virulence genes, and the accessory gene regulator alleles (agr). Among all 130 S. aureus isolates, 109 (83.8%, 109/130) isolates were positive for agr. All swine farms isolates belonged to agr IV, whereas S. aureus isolated from slaughterhouse and retail indicated diverse agr types. All isolates exhibited biofilm formation ability, and raw meat isolates (belonging to agr I) exhibited a greater ability to form strong biofilms than swine farms isolates (belonging to agr IV). agr-positive isolates were associated with more virulence genes than agr-negative isolates. Most biofilm-producing isolates were positive for microbial surface component recognizing adhesive matrix molecule (MSCRAMM), capsule type and ica group genes. The results illustrate a significant association between the prevalence rate of MSCRAMM, capsule type and ica group genes among isolates producing weak, moderate and strong biofilms. The high prevalence of resistance to ciprofloxacin, gentamicin, tetracycline, clarithromycin, clindamycin, and trimethoprim-sulfamethoxazole were mainly observed in moderate and weak biofilm producers. Our findings indicate that S. aureus isolates from pork production displayed diverse molecular ecology.

Prevalence of Enterotoxin Genes in Staphylococcus aureus Isolates from Pork Production.

In this study, 130 Staphylococcus aureus isolates from samples associated with pork production were tested for prevalence of 18 staphylococcal enterotoxin (SE) genes. Approximately 94.6% (123/130) of isolates from different stages of pork production harbored one or more SE genes forming 37 different enterotoxin gene profiles. Seb was present in 60.0% of the S. aureus isolates, the highest among the genes tested. The genes, sed, sej, seo, sep, ser, and seu, were not found. The five classical SE genes (including sea, seb, sec, sed, see) had lower prevalence than the egc gene cluster (seg, sei, sem, sen, seo, or seu). Notably, ∼6.9% (9/130) isolates harbored five SE genes. Classical SE genes were relatively higher in raw meat isolates than swine farm isolates, suggesting that raw meat isolates have a greater potential for classical staphylococcal food poisoning. Incomplete egc clusters were mainly distributed in swine farm isolates, and some of them coexisted with other classical SE genes (seb, sec), showing that swine farms could be potential sources of enterogenic S. aureus of food safety concern. Characterizing the distributions of enterotoxin genes among S. aureus may provide epidemiological information for the benefit of public health and food safety.

Comparison of Oropharyngeal Microbiota in Healthy Piglets and Piglets With Respiratory Disease.

Porcine respiratory disease (PRD) is responsible for severe economic losses in the swine industry worldwide. Our objective was to characterize the oropharyngeal microbiota of suckling piglets and compare the microbiota of healthy piglets and piglets with PRD. Oropharyngeal swabs were collected from healthy (Healthy_A, n = 6; Healthy_B, n = 4) and diseased (PRD_A, n = 18; PRD_B, n = 5) piglets at 2-3 weeks of age from two swine farms in Guangdong province, China. Total DNA was extracted from each sample and the V3-V4 hypervariable region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform. No statistically significant differences were observed in bacterial diversity and richness between the healthy and PRD groups in the two farms except for Shannon index in farm A. Principal coordinates analysis (PCoA) showed structural segregation between diseased and healthy groups and between groups of different farms. Among all samples, the phyla Firmicutes, Proteobacteria, and Bacteroidetes were predominant. At the genus level, Streptococcus, Lactobacillus, and Actinobacillus were the core genera in the oropharynx of healthy piglets from the two farms. Significant differences in bacterial taxa were found when the microbiota was compared regarding the health status. In farm A, the percentages of Moraxella and Veillonella were higher in the PRD group, while only Porphyromonas was significantly increased in the PRD group in farm B (p < 0.05). Compared to PRD groups, statistically significant predominance of Lactobacillus was observed in the healthy groups from both farms (p < 0.05). Our findings revealed that Moraxella, Veillonella, and Porphyromonas may play a potential role in PRD and Lactobacillus may have a protective role against respiratory diseases.

Rapid detection of Haemophilus parasuis using cross-priming amplification and vertical flow visualization.

Haemophilus parasuis infection is of considerable economic importance in the swine industry due to high morbidity and mortality in naive swine populations. Accurate detection and identification of the causative agent are difficult, yet necessary, for disease control. In this study, a simple and rapid method of cross-priming amplification (CPA) with a vertical flow (VF) visualization strip was established to detect H. parasuis. The reaction can specifically identify 15 serovar reference strains and 57 clinically isolated strains of H. parasuis, with a detection limit of 14CFU. The performance of the CPA-VF assay was evaluated and compared with that of species-specific PCR by testing 62 clinical culture-positive specimens of H. parasuis. The entire process, from specimen processing to analysis of the results, can be completed in 2h without a complicated apparatus. The convenience and speed of the CPA-VF assay in this study make it a suitable choice for epidemiological investigation and point-of-care testing (POCT) for H. parasuis infection.

Evaluation of immunogenicity and protective efficacy of recombinant outer membrane proteins of Haemophilus parasuis serovar 5 in a murine model.

Glässer's disease is an economically important infectious disease of pigs caused by Haemophilus parasuis. Few vaccines are currently available that could provide effective cross-protection against various serovars of H. parasuis. In this study, five OMPs (OppA, TolC, HxuC, LppC, and HAPS_0926) identified by bioinformatic approaches, were cloned and expressed as recombinant proteins. Antigenicity of the purified proteins was verified through Western blotting, and primary screening for protective potential was evaluated in vivo. Recombinant TolC (rTolC), rLppC, and rHAPS_0926 proteins showing marked protection of mice against H. parasuis infection, and were further evaluated individually or in combination. Mice treated with these three OMPs produced humoral and host cell-mediated responses, with a significant rise in antigen-specific IgG titer and lymphoproliferative response in contrast with the mock-immunized group. Significant increases were noted in CD4+, CD8+ T cells, and three cytokines (IL-2, IL-4, and IFN-γ) in vaccinated animals. The antisera against candidate antigens could efficiently impede bacterial survival in whole blood bactericidal assay against H. parasuis infection. The multi-protein vaccine induced more pronounced immune responses and offered better protection than individual vaccines. Our findings indicate that these three OMPs are promising antigens for the development of multi-component subunit vaccines against Glässer's disease.

Deletion of ssnA Attenuates the Pathogenicity of Streptococcus suis and Confers Protection against Serovar 2 Strain Challenge.

Streptococcus suis serotype 2 (SS2) is a major porcine and human pathogen which causes arthritis, meningitis, and septicemia. Streptococcus suis nuclease A (SsnA) is a recently discovered deoxyribonuclease (DNase), which has been demonstrated to contribute to escape killing in neutrophil extracellular traps (NETs). To further determine the effects of ssnA on virulence, the ssnA deletion mutant (ΔssnA) and its complemented strain (C-ΔssnA) were constructed. The ability of ΔssnA mutant to interact with human laryngeal epithelial cell (Hep-2) was evaluated and it exhibited dramatically decreased ability to adhere to and invade Hep-2 cells. This mutation was found to exhibit significant attenuation of virulence when evaluated in CD1 mice, suggesting ssnA plays a critical role in the pathogenesis of SS2. Finally, we found that immunization with the ΔssnA mutant triggered both antibody responses and cell-mediated immunity, and conferred 80% protection against virulent SS2 challenge in mice. Taken together, our results suggest that ΔssnA represents an attractive candidate for designing an attenuated live vaccine against SS2.