Single-cell transcriptional profiling reveals cell type-specific responses to duck reovirus infection in the Bursa of Fabricius of Cairna moschata.

Duck reovirus (DRV) is a universal waterfowl virus that causes significant economic losses in the duck industry. However, the role of the host innate immune response of the Bursa of Fabricius to DRV infection is largely unknown. In the present study, we constructed a single-cell resolution transcriptomic atlas of the Bursa of Fabricius of Cairna moschata after infection with HN10 (a novel DRV). Ten cell-type marker genes were used to annotate the cell type, indicating a high degree of cell heterogeneity in the Bursa of Fabricius. Most of the innate and adaptive immune system-related genes were highly expressed in T cells, B cells, neutrophils, macrophages, and DCs. In the Bursa of Fabricius, the proportions of DCs and macrophages were largely increased by HN10 infection at 14 d, suggesting that DCs and macrophages play important roles in the long-term viral response. Notably, a number of innate and adaptive immune system-related genes were highly expressed at 24 h after HN10 infection, indicating that the Bursa of Fabricius has a very strong immune function even in the early developmental stage. In the immune system, the NOD-like receptor signaling pathway and RIG-I-like receptor signaling pathway were significantly activated at the early stage of HN10 infection, while the Toll-like receptor signaling pathway was significantly activated at the late stage. Enrichment analysis suggested that different immune signaling pathways play roles in specific developmental stages. Our data provide an opportunity to reveal the immune response to DRV infection at the single-cell level.

Development of a multienzyme isothermal and lateral flow dipstick combination assay for the rapid detection of goose astrovirus II.

Introduction: Goose astrovirus (GAstV) is a newly emerging pathogen that is currently widespread among geese, causing visceral gout and leading to substantial gosling mortalities, posing a severe threat to the waterfowl industry. GAstV II is the predominant epidemic strain, characterized by its high morbidity and mortality rate. Consequently, there is an urgent necessity to develop an effective diagnostic approach to control the dissemination of GAstV II, particularly in clinical farms with limited laboratory resources. Methods: In this study, a novel multi-enzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) combined assay was developed. Different primers designed specific targeting a highly conserved region within the viral RdRp gene for the detection of GAstV II. Primers optimized and MIRA-LFD assay analyzed its performance regarding limits of detection, specificity, and efficiency of detection. Results: The developed MIRA amplification is conducted at a constant temperature and accomplished within 10 minutes. Subsequent naked-eye observation of the LFD strips merely takes 5 minutes. The established MIRA-LFD method exhibits high specificity, with no cross-reaction with other pathogens and attains a detection sensitivity of 1 copy/µl, which is consistent with the reverse transcription quantitative PCR (RT-qPCR) assay. Further evaluation with clinical samples indicates that the accuracy of this MIRA-LFD method correlates well with RT-qPCR for the detection of GAstV II. Conclusion: In summary, the convenience, sensitivity, and rapidity of this newly developed detection method offer a significant advantage for on-site diagnosis of GAstV II.

Study on the Protective Immunity Induced by Pseudotyped Baculovirus Expressing the E Protein of Tembusu Virus in Ducklings.

The Duck Tembusu virus (DTMUV), a pathogenic flavivirus, has been causing significant economic losses in the Chinese poultry industry since 2010. This virus can severely decrease egg production and inhibit the growth of laying ducks and ducklings. While many vaccines have been developed to prevent DTMUV infection, fresh outbreaks continue to occur, as few effective vaccines are available. The E glycoprotein of DTMUV is the primary target for inducing protective immunity in the natural host. Therefore, we conducted an investigation and successfully developed a recombinant baculovirus containing the DTMUV E gene. Ducklings were then vaccinated with the purified protein derived from this virus as a potential vaccine candidate. Our findings demonstrated that the E glycoprotein of DTMUV was highly expressed in Sf9 cells. The vaccination of ducklings with the recombinant baculovirus Bac-E resulted in the induction of strong humoral and cellular immune responses. Most significantly, we observed that the vaccine provided 100% protective immunity against lethal challenges with the DTMUV YY5 strain.

High expression of the classical swine fever virus (CSFV) envelope protein E2 by a single amino acid mutation and its embedded in the pseudorabies virus (PRV) vector for immunization.

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases in pigs. Most pig farms in China are vaccinated against these two diseases. Gene-deleted pseudorabies virus (PRV) can be used to develop promising and economical multivalent live attenuated viral vector vaccines. It has been reported that recombinant PRV can express a truncated E2 protein (1-338 aa), but it has not been reported that recombinant PRV can express a full-length E2 protein. We constructed nine groups of E2 proteins with different expression forms and found that the E2 protein could be expressed in vitro only when the transmembrane region of E2 was removed and the signal peptide was added. Analysis of the transmembrane region of E2 revealed that the high hydrophobicity of the E2 transmembrane region was the main reason for its inability to express. By mutating an amino acid to reduce the hydrophobicity of the transmembrane region, it was found that the full-length mutant of E2 (E2FL-muta3 or E2FL-muta4) could be expressed. The expressed full-length mutant E2 could also localize to the cell membrane. Mice immunized with a PRV vector vaccine expressing E2FL-muta3 or E2FL-muta4 developed specific cellular immunity to the E2 protein and stimulated higher levels of E2 antibody than mice immunized with a PRV vector expressing truncated E2. After immunizing the rabbits, the lethal challenge by PRV-ZJ2013 and the febrile response elicited by CSFV were simultaneously prevented. These results suggest that rPRV-dTK/gE-E2FL-muta4 is a promising bivalent vaccine against CSFV and PRV infections.

Inactivation Performance of Pseudorabies Virus as African Swine Fever Virus Surrogate by Four Commercialized Disinfectants.

This study was based on similar physicochemical characteristics of pseudorabies virus (PRV) and African swine fever virus (ASFV). A cellular model for evaluation of disinfectants was established with PRV as an alternative marker strain. In the present study, we evaluated the disinfection performance of commonly used commercialized disinfectants on PRV to provide a reference for the selection of good ASFV disinfectants. In addition, the disinfection (anti-virus) performances for four disinfectants were investigated based on the minimum effective concentration, onset time, action time, and operating temperature. Our results demonstrated that glutaraldehyde decamethylammonium bromide solution, peracetic acid solution, sodium dichloroisocyanurate, and povidone-iodine solution effectively inactivated PRV at concentrations 0.1, 0.5, 0.5, and 2.5 g/L on different time points 30, 5, 10, and 10 min, respectively. Specifically, peracetic acid exhibits optimized overall performance. Glutaraldehyde decamethylammonium bromide is cost effective but requires a long action time and the disinfectant activity is severely affected by low temperatures. Furthermore, povidone-iodine rapidly inactivates the virus and is not affected by environmental temperature, but its application is limited by a poor dilution ratio such as for local disinfection of the skin. This study provides a reference for the selection of disinfectants for ASFV.

Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection.

Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the leading causes of bloodstream infections in a broad spectrum of birds and mammals, thus poses a great threat to public health, while its underlying mechanism causing sepsis is not fully understood. Here we reported a high virulent ExPEC strain PU-1, which has a robust ability to colonize within host bloodstream, while induced a low level of leukocytic activation. Two serine protease autotransporters of Enterobacteriaceae (SPATEs), VatPU-1 and TshPU-1, were found to play critical roles for the urgent blood infection of strain PU-1. Although the Vat and Tsh homologues have been identified as virulence factors of ExPEC, their contributions to bloodstream infection are still unclear. In this study, VatPU-1 and TshPU-1 were verified to interact with the hemoglobin (a well-known mucin-like glycoprotein in red blood cell), degrade the mucins of host respiratory tract, and cleave the CD43 (a major cell surface component sharing similar O-glycosylated modifications with other glycoprotein expressed on leukocytes), suggesting that these two SPATEs have the common activity to cleave a broad array of mucin-like O-glycoproteins. These cleavages significantly impaired the chemotaxis and transmigration of leukocytes, and then inhibited the activation of diverse immune responses coordinately, especially downregulated the leukocytic and inflammatory activation during bloodstream infection, thus might mediate the evasion of ExPEC from immune clearance of blood leukocytes. Taken together, these two SPATEs play critical roles to cause a heavy bacterial load within bloodstream via immunomodulation of leukocytes, which provides a more comprehensive understanding how ExPEC colonize within host bloodstream and cause severe sepsis.

Recombinant pseudorabies virus (PRV) expressing stabilized E2 of classical swine fever virus (CSFV) protects against both PRV and CSFV.

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases of pigs. Most pig farms in China are immunized against these two diseases. Here, we describe a stabilized E2 protein as an immunogen inserted into the PRV genome as a bivalent live virus-vectored vaccine. The E2 protein has 48 variant sites, there are 2-5 candidate amino acids per variant site, and the relative energy contribution of each amino acid to E2 energy was calculated. Combined substitutions of amino acids at the neighbor variant site (neighbor substitution) were performed to obtain the E2 protein sequence with the lowest energy (stabilized E2). Multiple amino acid substitutions at 48 variant sites were performed, and the results were consistent with neighbor substitutions. The stabilized E2 sequence was obtained, and its energy decreased by 22 Rosetta Energy Units (REUs) compared with the original sequence. After the recombinant PRV expressing stabilized E2 of CSFV was constructed, the secretion efficiency of stabilized E2 was increased by 2.97 times, and the thermal stability was increased by 10.5 times. Immunization of mice resulted in a 2-fold increase in antibody production, and a balanced antibody level against subtype 1.1 and subtype 2.1d E2 was achieved. In rabbits immunized, the lethal challenge of PRV-ZJ and the fever response induced by CSFV could be prevented simultaneously. These findings suggest that rPRV-muta/287aaE2 is a promising bivalent vaccine against CSFV and PRV infections.

Isolation and Pathogenicity of a Novel Goose Astrovirus from Overfed Adult Landaise Geese in China.

Goose astrovirus (GAstV) is an important pathogen causing visceral gout and high mortality in goslings, which has broken out and spread across China. In 2021, a disease characterized by urate deposition on the visceral surface and 30% mortality occurred in commercial adult Landaise geese in Zhejiang Province, China. A systematic study identified an infecting astrovirus, designated ZJCX, that was efficiently isolated from a diseased goose with a chicken hepatocellular carcinoma cell line (LMH). In contrast to other GAstVs originating from goslings, ZJCX caused cytopathogenic effects in LMH cells, and the crystalline arrangement of viral particles was observed through transmission electron microscopy. Indeed, phylogenetic analysis and nucleotide homology comparison revealed that ZJCX isolate belongs to the genotype II cluster of GAstVs and displays 97.8-98.4% identity with other GAstV II strains. However, several specific mutations occurred in the polyprotein and capsid protein regions. Moreover, a pathogenicity assessment of ZJCX with a gosling model was conducted, and typical visceral gout was reproduced and led to 18% mortality. The viral loads of ZJCX in the blood, kidney, and liver were detected with specific primers after inoculation, which demonstrated that the kidney and liver presented viral loads peaking at seven days post-inoculation (dpi). Biochemical parameter examination showed that AST, ALT, γ-GT, UA, and BUN levels were significantly increased by GAstV, whereas body weight was reduced. Overall, this study indicated that the GAstV isolate could infect adult geese, and the results regarding the viral loads and biochemical parameters induced by ZJCX provide insight into GAstV pathogenicity.

Development and Evaluation of a Monoclonal Antibody-Based Blocking Enzyme-Linked Immunosorbent Assay for the Detection of Antibodies against Novel Duck Reovirus in Waterfowl Species.

The novel duck reovirus (NDRV) is an emerging pathogen that causes disease in various waterfowl species. Since the outbreak, it has caused huge economic losses to the duck industry in China. A rapid, reliable, and high-throughput method is required for epidemiological investigation and evaluation of vaccine immunogenicity. A good first step would be establishing an enzyme-linked immunosorbent assay (ELISA) that could detect NDRV antibodies in different breeds of ducks and geese from the serum and egg yolk. This study used a recombinant NDRV σB protein and a corresponding horseradish peroxidase (HRP)-labeled monoclonal antibody to develop a blocking ELISA (B-ELISA). The cutoff value of the B-ELISA was 37.01%. A total of 212 serum samples were tested by the B-ELISA, and the virus neutralization test (VNT) was the gold standard test. The sensitivity and specificity of the B-ELISA were 92.17% (106/115) and 97.94% (95/97), respectively. The agreement rates between the B-ELISA and VNT were 94.81% (kappa value, 0.896). The B-ELISA could specifically recognize anti-NDRV sera without cross-reacting with other positive serums for other major diseases in ducks and geese. The inter- and intra-assay coefficients of variation (CVs) of the B-ELISA and VNT assays were acceptable. In conclusion, the novel B-ELISA could be a rapid, simple, safe, and economically attractive alternative to the VNT in assessing duck flocks' immunity status and in epidemiological surveillance in multiple waterfowl species. IMPORTANCE NDRV disease is a new epidemic disease in waterfowl that first appeared in China. Compared with the classical DRV (CDRV), NDRV is associated with more severe symptoms, a higher mortality rate, and a broader host range. NDRV has become the prevalent genotype in China. At present, there are no commercially available diagnostic products for the NDRV disease. VNT, as the gold standard serologic test, is not only time-consuming and laborious, but also has high requirements for facilities and equipment, which is not suitable for clinical application. Conventional ELISA requires specific antispecies conjugates that are not currently available. B-ELISA not only has the advantage of higher analysis specificity, but also can be used to test specific antibodies against different waterfowl species, because no species-specific conjugates are required in such detection. Therefore, it is necessary to establish a B-ELISA for the detection of antibodies against NDRV in waterfowl species.

Characterization and epidemiological analysis of Vibrio parahaemolyticus isolated from different marine products in East China.

Vibrio parahaemolyticus is a major foodborne pathogen with a wide distribution in the world that causes economic and public health problems. Here, we isolated 152 V. parahaemolyticus strains from shellfish, shrimp, crab, and snails from 5 provinces in East China, and analyzed the genetic diversity, population structure, and virulence profiles of these isolates. Our results showed that the 152 V. parahaemolyticus strains belong to 84 different sequence types (STs), of which 69 (82.14 %) STs and 60 alleles were newly identified. The thermostable direct hemolysin (TDH) and the TDH-related hemolysin (TRH) were present in 4 V. parahaemolyticus isolates (2.63 %) respectively, while toxRS/new, a distinctive toxRS sequence that was associated with pandemic V. parahaemolyticus strains, is present in 52 isolates (34.21 %). Thereinto, both the ZJ11 and ZJ12 strains measure up to the standard of toxRS/new+, tdh+, and trh-, which was widely used marker for the rapid screening of pandemic strains, and thus these strains may have the risk of infectious outbreaks. In addition, we observed that all the 152 V. parahaemolyticus isolates encode type III secretion systems 1 and type VI secretion system (T6SS) 2, while 119 isolates (78.29 %) of them also contain T6SS1. The genetic relatedness of our isolates to the human V. parahaemolyticus collection was explored, which showed that ST6, ST79, ST162, ST1060, and ST1061 were all identified in both human isolates (7 isolates) uploaded in the PubMLST database and our marine products isolates (7 isolates). Our findings expand the views of the genetic diversity of V. parahaemolyticus and will contribute to understanding the potential risk of the transboundary spread of this bacterium.

Airborne bacterial communities in the poultry farm and their relevance with environmental factors and antibiotic resistance genes.

The accelerating occurrence and environmental dissemination of bacteria, gas pollutants and antibiotic resistance genes (ARGs) in aerosols of poultry farms have become emerging environmental issues due to their potential threat to animals, workers, and the communities located near such farms. Here, aerosol samples were gathered from inside and outside of the chicken house in winter with a transportable high-flow bioaerosol sampler. Then, 16S rRNA gene amplicon sequencing was used to categorize the bacteria in air samples, and the abundance of 12 ARG subtypes was researched via the real-time quantitative polymerase chain reaction (qPCR). Results indicated that the bacterial richness and diversity and total absolute abundance of ARGs were similar in the bioaerosols from indoor and downwind site of the poultry farm. The zoonotic pathogens, Staphylococcus and Corynebacterium, were detected both inside and outside of the chicken house, and the four most abundant target genes were blaTEM, tetQ, ermB and sul1 in aerosols. Moreover, the correlation between the bacterial communities and environmental factors, such as NH3 and H2S concentrations, wind speed, temperature and relative humidity, was analyzed. The result revealed that the indoor bacteria community was positively associated with temperature and concentrations of air pollutants (NH3 and H2S), and could spread from confinement buildings to the ambient atmosphere through wind. In addition, the network analysis result showed that the airborne bacteria might significantly contribute in shaping the ARGs' profiles in bioaerosol from inside and outside of the poultry house. Overall, our results revealed the airborne bacterial communities and their associated influencing factors in the micro-environment (inside of the chicken house and nearby the boundary of the farm), and brought a new perspective for studying the gas pollutants and bioaerosol from poultry farms in winter.

Identification of the RNA-binding domain-containing protein RbpA that acts as a global regulator of the pathogenicity of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2), an emerging zoonotic pathogen, causes swine diseases and human cases of streptococcal toxic shock syndrome. RNA-binding proteins (RBPs) can modulate gene expression through post-transcriptional regulation. In this study, we identified an RBP harbouring an S1 domain, named RbpA, which facilitated SS2 adhesion to host epithelial cells and contributed to bacterial pathogenicity. Comparative proteomic analysis identified 145 proteins that were expressed differentially between ΔrbpA strain and wild-type strain, including several virulence-associated factors, such as the extracellular protein factor (EF), SrtF pilus, IgA1 protease, SBP2 pilus, and peptidoglycan-binding LysM' proteins. The mechanisms underlying the regulatory effects of RbpA on their encoding genes were explored, and it was found that RbpA regulates gene expression through diverse mechanisms, including post-transcriptional regulation, and thus acts as a global regulator. These results partly reveal the pathogenic mechanism mediated by RbpA, improving our understanding of the regulatory systems of S. suis and providing new insights into bacterial pathogenicity.

Testability of high-dimensional linear models with nonsparse structures.

Understanding statistical inference under possibly non-sparse high-dimensional models has gained much interest recently. For a given component of the regression coefficient, we show that the difficulty of the problem depends on the sparsity of the corresponding row of the precision matrix of the covariates, not the sparsity of the regression coefficients. We develop new concepts of uniform and essentially uniform non-testability that allow the study of limitations of tests across a broad set of alternatives. Uniform non-testability identifies a collection of alternatives such that the power of any test, against any alternative in the group, is asymptotically at most equal to the nominal size. Implications of the new constructions include new minimax testability results that, in sharp contrast to the current results, do not depend on the sparsity of the regression parameters. We identify new tradeoffs between testability and feature correlation. In particular, we show that, in models with weak feature correlations, minimax lower bound can be attained by a test whose power has the n rate, regardless of the size of the model sparsity.

Intergrated Transcriptomic and Proteomic Analysis Revealed the Differential Responses to Novel Duck Reovirus Infection in the Bursa of Fabricius of Cairna moschata.

The bursa of Fabricius is an immunologically organ against the invasion of duck reovirus (DRV), which is a fatal bird virus belonging to the Reoviridae family. However, responses of the bursa of Fabricius of Cairna moschata to novel DRV (NDRV) infection are largely unknown. Transcriptomes and proteomes of the samples from control and two NDRV strain (HN10 and JDm10) with different virulence were analyzed. Differentially expressed genes and differential accumulated proteins were enriched in the serine protease system and innate immune response clusters. Most of the immune-related genes were up-regulated under both JDm10/HN10 infections. However, the immune-related proteins were only accumulated under HN10 infection. For the serine protease system, coagulation factor IX, three chains of fibrinogen, and complements C8, C5, and C2s were significantly up-regulated by the HN10 infection, suggesting that the serine protease-mediated immune system might be involved in the resistance to NDRV infection. For the innate and adaptive immune system, RIG-I, MDA5, MAPK20, and IRF3 were significantly up-regulated, indicating their important roles against invaded virus. TLR-3 and IKBKB were only up-regulated in the liver cells, MAPK20 was only up-regulated in the bursa of Fabricius cells, and IRAK2 was only up-regulated in the spleen samples. Coagulation factor IX was increased in the bursa of Fabricius, not in the liver and spleen samples. The data provides a detailed resource for studying the proteins participating in the resistances of the bursa of Fabricius of duck to NDRV infections.

Genomic and epidemiological characteristics provide insights into the phylogeographic spread of goose astrovirus in China.

Goose astrovirus (GAstV) is an emerging pathogen with a wide distribution in China that causes visceral gout and leads to significant economic losses in the goose industry. Here, 10 GAstV strains were isolated from different farms in southeast China. We performed an integrated analysis of the full-genome sequences of these new strains alongside comprehensive epidemiological surveillance information from the database. Interestingly, the results showed two distinct genotypes of GAstV, which were evolutionarily distant from each other. Group I GAstVs were closely related to DAstV IV, and group II strains were classified with duck astrovirus (DAstV) II and turkey astrovirus (TAstV) II. Further investigation showed that among the GAstV I strains, ZJC14 and AHDY differed from FLX. Comparative analysis of 58 available genomes clustered the GAstV II strains into two subgroups. We identified two major mutation sites, 456 (E/D) and 540 (L/Q), in the capsid protein, which were related to distinct subgroups according to evolution. GAstV II subgroup 1a strains are the predominant strains in the current prevalent epidemiology. Phylogeographic analysis based on 90 reported cases from 13 provinces revealed the complexity and severity of GAstV epidemics in China, within which Henan, Anhui and Jiangsu provinces have suffered great impacts. According to these phylogeographic investigations, following the initial introduction of GAstV from Hunan Province, the dispersal of GAstV with different subgenotypes on a nationwide scale may be explained by the live gosling trade. Our findings have important implications for the evolution and dispersal of GAstV and will contribute to understanding the potential risk of GAstV.

Distinct Whole Transcriptomic Profiles of the Bursa of Fabricius in Muscovy Ducklings Infected by Novel Duck Reovirus with Different Virulence.

Novel duck reovirus (NDRV) is a newly identified reovirus that brings about more severe damage on multiple organs and mortality in various species of waterfowl. We previously characterized the transcriptomic profiles responding to NDRV in the bursa of Fabricius of Muscovy ducklings, which is a major immunological organ against virus infection. However, the molecular mechanisms of variant cell responses in the bursa of Fabricius to NDRV with different virulence is unclear. Here, we conducted a whole transcriptomic analysis to study the effects of two strains, HN10 (virulent NDRV) and JDm10 (artificially attenuated NDRV), on the bursa of Fabricius of Muscovy ducklings. We harvested a large number of differentially expressed genes (DEGs) of the bursa of Fabricius specially induced by HN10 and JDm10, and we found that HN10 induced DEGs enriched in differentiation and development in multiple organs beyond JDm10. Moreover, the ceRNA regulatory network also indicated the different connections among mRNA, lncRNA and miRNA. Interestingly, we further noticed that a population of differential expressed miRNA could particularly target to transcripts of HN10 and JDm10. We took miR-24 as an example and observed that miR-24 could reduce the transcription of GLI family zinc finger 3 (Gli3) and membrane-associated guanylate kinase, WW and PDZ domain containing 1 (Magi1) via recognition 3' UTR of these two genes by a dual luciferase reporter gene assay in vitro. However, this effect could be compromised by HN10 infection or the ectopic over-expression of the putative miR-24 targeting regions in L1 and L3 fragments of HN10. Taken together, we examined and proposed a novel regulatory competitive mechanism between transcripts of NDRV and Muscovy ducklings for miRNA. These findings may advance the understanding of the molecular pathogenesis of NDRV in Muscovy ducklings, and help provide the potential targets for vaccine and drug development against NDRV.

Distributional conformal prediction.

We propose a robust method for constructing conditionally valid prediction intervals based on models for conditional distributions such as quantile and distribution regression. Our approach can be applied to important prediction problems, including cross-sectional prediction, k-step-ahead forecasts, synthetic controls and counterfactual prediction, and individual treatment effects prediction. Our method exploits the probability integral transform and relies on permuting estimated ranks. Unlike regression residuals, ranks are independent of the predictors, allowing us to construct conditionally valid prediction intervals under heteroskedasticity. We establish approximate conditional validity under consistent estimation and provide approximate unconditional validity under model misspecification, under overfitting, and with time series data. We also propose a simple "shape" adjustment of our baseline method that yields optimal prediction intervals.

An Auto-Regulating Type II Toxin-Antitoxin System Modulates Drug Resistance and Virulence in Streptococcus suis.

Toxin-antitoxin (TA) systems are ubiquitous genetic elements that play an essential role in multidrug tolerance and virulence of bacteria. So far, little is known about the TA systems in Streptococcus suis. In this study, the Xress-MNTss TA system, composed of the MNTss toxin in the periplasmic space and its interacting Xress antitoxin, was identified in S. suis. ß-galactosidase activity and electrophoretic mobility shift assay (EMSA) revealed that Xress and the Xress-MNTss complex could bind directly to the Xress-MNTss promoter as well as downregulate streptomycin adenylyltransferase ZY05719_RS04610. Interestingly, the Xress deletion mutant was less pathogenic in vivo following a challenge in mice. Transmission electron microscopy and adhesion assays pointed to a significantly thinner capsule but greater biofilm-formation capacity in ΔXress than in the wild-type strain. These results indicate that Xress-MNTss, a new type II TA system, plays an important role in antibiotic resistance and pathogenicity in S. suis.

Comparative genetic analyses provide clues about capsule switching in Streptococcus suis 2 strains with different virulence levels and genetic backgrounds.

Streptococcus suis (S. suis) is a major bacterial pathogen in the swine industry and an emerging zoonotic agent. S. suis produces an important extracellular component, capsular polysaccharide (CPS), based on which dozens of serotypes have been identified. Through virulence genotyping, we revealed the relatedness between subpopulations of S. suis serotype 2 (SS2), S. suis serotype 3 (SS3) and S. suis serotype 7 (SS7) strains despite their serotype differences. Multilocus sequence typing (MLST) was used to characterize the whole S. suis population and revealed capsule switching between S. suis strains. Importantly, capsule switching occurred in the SS2, SS3 and SS7 strains belonging to CC28 and CC29, which are phylogenetically distinct from the main CC1 SS2 lineage. To further explore capsule switching in S. suis, comparative genomic analyses were performed using available complete S. suis genomes. Phylogenetic analyses suggested that the SS2 strains could be divided into two clades (1 and 2), and those classified into clade 2 colocalized with SS3 and SS7 strains, in accordance with the above virulence genotyping and MLST analyses. Clade 2 SS2 strains presented high genetic similarity to SS3 and SS7 and shared common competence and defensive elements with them but were significantly different from Clade 1 SS2 strains. Notably, although the cps loci shared by Clade 1 and 2 SS2 strains were almost identical, a specific region of the cps locus of strain NSUI002 (Clade 2 SS2) could be found in the SS3 cps locus but not in the Clade 1 SS2 strain. These data indicated that the SS2 strains in CC28 and CC29 might have acquired the cps locus through capsule switching, which could explain the distinct genetic lineages within the SS2 population.