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ABSTRACT: Salmonellosis in poultry is detrimental to the advancement of the breeding industry and poses hazards to human health. Approximately 2,600 Salmonella varieties exist, among which S. Enteritidis, S. Pullorum, S. Typhimurium, and S. Infantis are prevalent serotypes in the poultry industry in recent years. They can also infect humans by contaminating poultry eggs and meat. Therefore, identifying these serotypes is crucial for successful preventive and control interventions. The White–Kauffmann–Le Minor scheme is time-consuming and requires expensive reagents. Whole-genome sequencing (WGS) and other molecular biology techniques require skilled technical staff. In comparison, the polymerase chain reaction (PCR) is more accurate, rapid, and inexpensive, thus proving suitable for widespread application in the poultry industry. Here, we selected 4 specific primers: lygD, mdh, ipaJ, and SIN_02055, which correspond to detecting S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Infantis, respectively. They were integrated into a 1-step multiplex PCR method. We optimized the PCR method by utilizing specificity test results to determine the optimal annealing temperature (57°C). The PCR method exhibited excellent sensitivity for genomic DNA and bacterial cultures. We used the developed method to determine 157 clinical Salmonella isolates from various stages of the poultry production chain. The results aligned with serotype data generated via WGS analysis, demonstrating the method's excellent accuracy. In conclusion, this study developed a 1-step multiplex PCR method that simultaneously identifies S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Infantis, allowing routine mass detection in the grass-root poultry industry.

ABSTRACT: Salmonella, which is widely distributed in nature, is an important zoonotic pathogen affecting humans, livestock, and other animals. Salmonella infection not only hinders the development of livestock and poultry-related industries but also poses a great threat to human health. In this study, we collected 1,537 samples including weak chicks, dead embryos, fecal samples and environmental samples from 2020 to 2023 (for a period of 1 to 2 months per year) to keep a long-term monitor the prevalence of Salmonella in an intensive laying hen farm, 105 Salmonella strains were isolated with an isolation rate of 6.83% (105/1,537). It revealed a significant decrease in prevalence rates of Salmonella over time (P < 0.001). Before 2020, the predominant serotype was S. Enteritidis. S. Kentucky was first detected in November 2020 and its proportion was gradually found to exceed that of S. Enteritidis since then. S. Kentucky isolates were distributed in various links of the four regions in the poultry farm. A total of 55 S. Kentucky strains, were assigned to ST198 based on whole genome sequencing. Among them, 54 strains were resistant to 12 to 16 antibiotics, indicating that they were extensively drug-resistant (XDR). Seventeen antimicrobial resistance genes were detected in 55 S. Kentucky isolates. For most of these isolates, antibiotic resistance phenotypes were concordant with their genotypes. All S. Kentucky strains isolated from this farm in 2020 to 2023 showed a high similarity based on their core-genome SNP-based phylogeny. The traceability analysis revealed that S. Kentucky was introduced to the farm through newly purchased flocks. The long-term existence of XDR S. Kentucky ST198 poses a substantial risk because of the multiage management and circulation of workers in this poultry farm. Thus, this study is the first to report extensively drug-resistant S. Kentucky ST198 detected in this intensive poultry farm in China.

Abstract Background Control of Tuberculosis (TB) infection is mainly the result of productive teamwork between T-cell populations and antigen presenting cells (APCs). However, APCs activation at the site of initiating cellular immune response during BCG early infection is not completely understood. Methods In this study, we injected C57BL/6 mice in intravenous (i.v) or subcutaneous (s.c) route, then splenic or inguinal lymph node (LN) DCs and MΦs were sorted, and mycobacteria uptake, cytokine production, antigen presentation activity, and cell phenotype were investigated and compared, respectively. Results Ag85A-specific T-cell immune response began at 6 days post BCG infection, when BCG was delivered in s.c route, Th17 immune response could be induced in inguinal LN. BCG could induce high level of activation phenotype in inguinal LN MΦs, while the MHC II presentation of mycobacteria-derived peptides by DCs was more efficient than MΦs. Conclusions The results showed that BCG immunized route can decide the main tissue of T-cell immune response. Compared with s.c injected route, APCs undergo more rapid cell activation in spleen post BCG i.v infection.

Abstract Exosomes, membrane vesicles released extracellularly from cells, contain nucleic acids, proteins, lipids and other components, allowing the transfer of material information between cells. Recent studies reported the role of exosomes in pathogenic microbial infection and host immune mechanisms. Brucella‐invasive bodies can survive in host cells for a long time and cause chronic infection, which causes tissue damage. Whether exosomes are involved in host anti‐Brucella congenital immune responses has not been reported. Here, we extracted and identified exosomes secreted by Brucella melitensis M5 (Exo‐M5)‐infected macrophages, and performed in vivo and in vitro studies to examine the effects of exosomes carrying antigen on the polarization of macrophages and immune activation. Exo‐M5 promoted the polarization of M1 macrophages, which induced the significant secretion of M1 cytokines (tumour necrosis factor‐α and interferon‐γ) through NF‐κB signalling pathways and inhibited the secretion of M2 cytokines (IL‐10), thereby inhibiting the intracellular survival of Brucella. Exo‐M5 activated innate immunity and promoted the release of IgG2a antibodies that protected mice from Brucella infection and reduced the parasitaemia of Brucella in the spleen. Furthermore, Exo‐M5 contained Brucella antigen components, including Omp31 and OmpA. These results demonstrated that exosomes have an important role in immune responses against Brucella, which might help elucidate the mechanisms of host immunity against Brucella infection and aid the search for Brucella biomarkers and the development of new vaccine candidates.

ABSTRACT: Bovine tuberculosis (bTB) caused by Mycobacterium bovis is an important zoonotic disease. This infection is difficult to control because of the limited ability of the tuberculin skin test (TST) and ancillary IFN-γ release assay to detect all infected animals. In this study, we aimed to develop an efficient assay based on the enzyme-linked immunospot (ELISpot) technique for the diagnosis of bTB, with IFN-γ monoclonal antibodies 3E9 and Bio-labeled 6F8 used as capture and detection antibodies, respectively. As expected, there were significantly more M. bovis-specific spot-forming units (SFU) in bTB-infected cattle than in healthy cattle when an M. bovis-specific antigen, CFP-10-ESAT-6 fusion protein (CE protein), was used. The M. bovis IFN-γ ELISpot assay demonstrated a high level of agreement (90.83%) with the BOVIGAM ELISA test (Thermo Fisher Scientific) for detecting bTB. Furthermore, 3 of 109 cattle tested negative by both the TST and the BOVIGAM ELISA tests, but positive by the ELISpot assay (TST− ELISA− ELISpot+). During subsequent long-term monitoring, these 3 cattle became TST+ ELISA+ ELISpot+. These results suggest that the M. bovis IFN-γ ELISpot assay we established could detect infected cattle earlier than the BOVIGAM ELISA test.

Inflammasome activation is an essential innate immune defense mechanism against Salmonella infections. Salmonella has developed multiple strategies to avoid or delay inflammasome activation, which may be required for long-term bacterial persistence. However, the mechanisms by which Salmonella evades host immune defenses are still not well understood. In this study, Salmonella Enteritidis (SE) random insertion transposon library was screened to identify the key factors that affect the inflammasome activation. The type I secretion system (T1SS) protein SiiD was demonstrated to repress the NLRP3 inflammasome activation during SE infection and was the first to reveal the antagonistic role of T1SS in the inflammasome pathway. SiiD was translocated into host cells and localized in the membrane fraction in a T1SS-dependent and partially T3SS-1-dependent way during SE infection. Subsequently, SiiD was demonstrated to significantly suppress the generation of mitochondrial reactive oxygen species (mtROS), thus repressing ASC oligomerization to form pyroptosomes, and impairing the NLRP3 dependent Caspase-1 activation and IL-1β secretion. Importantly, SiiD-deficient SE induced stronger gut inflammation in mice and displayed NLRP3-dependent attenuation of the virulence. SiiD-mediated inhibition of NLRP3 inflammasome activation significantly contributed to SE colonization in the infected mice. This study links bacterial T1SS regulation of mtROS-ASC signaling to NLRP3 inflammasome activation and reveals the essential role of T1SS in evading host immune responses.

Macrophages can participate in immune responses by altering their metabolism, and play important roles in controlling bacterial infections. However, Salmonella Enteritidis can survive and proliferate in macrophages. After the deletion of DNA adenine methylase (Dam), the proliferation of Salmonella Enteritidis in macrophages decreased, the molecular mechanism is still unclear. After infecting macrophages with Salmonella Enteritidis wild type and dam gene deletion strains, intracellular metabolites were extracted and detected by non-targeted metabolomics and fatty acid targeted metabolomics. We found Dam had significant effects on arachidonic acid and related metabolic pathways in macrophages. The dam gene can promote the proliferation of Salmonella Enteritidis in macrophages by inhibiting the metabolic pathway of cytosolic phospholipase A2-mediated arachidonic acid production and conversion to prostaglandin E2 in macrophages, reducing the secretion of the pro-inflammatory factors IL-1β and IL-6. In addition, inhibition of arachidonic acid-related pathways in macrophages by Arachidonyl trifluoromethyl ketone could restore the proliferation of dam gene deletion strains in macrophages. This study explored the role of Dam in the process of Salmonella Enteritidis invading host cells from the perspective of host cell metabolism, and provides new insights into the immune escape mechanism of Salmonella Enteritidis.

Abstract Background Colon adenocarcinoma (COAD) is one of the common cancers worldwide. Collagen triple helix repeat containing 1 (CTHRC1) has been reported to be involved in cell invasion, angiogenesis, and the promotion of epithelial-mesenchymal transformation by mediating multiple signaling pathways. However, the role of CTHRC1 in COAD has not yet been determined. Methods Differentially expressed genes were evaluated using gene expression data from the Oncomine and TIMER databases. Correlations between CTHRC1 gene expression and clinicopathological factors were analyzed using gene expression data from UALCAN databases. Then, we searched the GEPIA database to evaluate the association of CTHRC1 gene expression with clinical outcomes. The cBioPortal database was used to analyze CTHRC1 genetic alterations. Subsequently, the TIMER website was chosen to assess the correlation of CTHRC1 with the tumor immune cell infiltration level. The TCGA dataset was used for a gene set enrichment analysis (GSEA). Result CTHRC1 was highly expressed in COAD patients, and significantly related to poor prognosis. In addition, elevated expression of CTHRC1 was related to the clinical stage and pathological type of COAD. The GSEA analysis showed that CTHRC1 was enriched in Gα signaling, GCPR ligand binding, neutrophil degranulation, interleukin signaling, and tumor-associated pathways. In addition, CTHRC1 was significantly associated with the expression of multiple immune markers related to specific immune cells. Conclusion This study suggest that CTHRC1 expression is related to the prognosis and immune infiltration of COAD patients. Therefore, CTHRC1 may be a new candidate prognostic biomarker for determining immune infiltration levels and providing COAD prognoses.

The Chinese indigenous Shiqi (SQ) pigeon and the imported White King (WK) pigeon are two meat-type pigeon breeds of economical and nutritional importance in China. They displayed significant differences in such meat quality traits as intramuscular fat (IMF) content and fatty acid (FA) compositions in the breast muscles. In this study, we aimed to screen candidate genes that could affect fat-related meat quality traits in meat-type pigeons. We investigated the polymorphic variations at the genomic level using double-digest restriction-associated DNA (ddRAD) sequencing in 12 squabs of SQ and WK pigeons that exhibited significant inter-breed differences in IMF content as well as FA and amino acid compositions in the breast muscles, and screened candidate genes influencing fat-related traits in squabs through gene ontology analysis and pathway analysis. By focusing on 6019 SNPs, which were located in genes with correct annotations and had the potential to induce changes in the encoded proteins, we identified 19 genes (ACAA1, ACAA2, ACACB, ACADS, ACAT1, ACOX3, ACSBG1, ACSBG2, ACSL1, ACSL4, ELOVL6, FADS1, FADS2, HACD4, HADH, HADHA, HADHB, MECR, OXSM) as candidate genes that could affect fat-related traits in squabs. They were significantly enriched in the pathways of FA metabolism, degradation, and biosynthesis (p < 0.05). Results from molecular docking analysis further revealed that three non-synonymous amino acid alterations, ACAA1(S357N), ACAA2(T234I), and ACACB(H1418N), could alter the non-bonding interactions between the enzymatic proteins and their substrates. Since ACAA1, ACAA2, and ACACB encode rate-limiting enzymes in FA synthesis and degradation, alterations in the enzyme–substrate binding affinity may subsequently affect the catalytic efficiency of enzymes. We suggested that SNPs in these three genes were worthy of further investigation into their roles in explaining the disparities in fat-related traits in squabs.

ABSTRACT: Salmonella is the most important foodborne pathogen in poultry production systems and can infect humans via consumption of contaminated food. Ducks, an important waterfowl widely raised in China, are also a vehicle that transmits Salmonella through the food supply chain. In this study, 701 samples were collected from each production stage of the duck production chain. Salmonella was isolated and identified, and the isolates were tested for drug sensitivity and molecular typing based on whole genome sequencing (WGS) to explore the prevalence of Salmonella in the duck production chain. Altogether, a total of 180 Salmonella isolates (25.7%) were obtained from the duck production chain, 82 (35.7%) isolates were from hatchery samples, followed by 64 (29.2%) from market samples, 17 (23.6%) from farm samples, and 17 (9.4%) from slaughterhouse samples. All isolates were divided into 9 serotypes, among which S. Typhimurium, S. Anatum, and S. Enteritidis were the dominant serotypes. The S. Typhimurium was distributed in various production stages in the duck production chain. Among the 16 antibiotics, selected 60 isolates were only resistant to NAL, indicating that resistance of Salmonella in the duck production chain was low. WGS phylogenetic relationship results based on core-genome SNPs showed that S. Typhimurium can spread across geographic regions and along between different stages of the duck production chain, eventually reaching the market where it is a potential threat to consumer health. This study explored the prevalence of Salmonella in the duck production chain which will provide data support for proposing some interventions to control Salmonella.

ABSTRACT: Salmonella enterica serovar Pullorum (S. Pullorum) causes pullorum disease (PD), which is an acute systemic disease, in chickens, and leads to serious economic losses in many developing countries because of its high morbidity and mortality rate in young chicks. The live-attenuated vaccine is considered to be an effective measure to control the Salmonella infection. In addition, the DIVA (differentiation of infected and vaccinated animals) feature without the interference of serological monitoring of Salmonella infection is an important consideration in the development of the Salmonella vaccine. In this study, we evaluated the immunogenicity and protective efficacy of a S. Pullorum rough mutant S06004ΔspiCΔrfaH as a live attenuated DIVA vaccine candidate in chickens. The S06004ΔspiCΔrfaH exhibited a significant rough lipopolysaccharides (LPS) phenotype which was agglutinated with the acriflavine, not with the O9 mono antibody. Compared to the wild-type, 50% lethal dose (LD50) of the rough mutant increased 100-fold confirmed its attenuation. The mutant strain also showed a decreased bacterial colonization in the spleen and liver. The immunization with the mutant strain had no effect on the body weight and no tissue lesions were observed in the liver and spleen. The high level of the S. Pullorum-specific IgG titers in the serum indicated that significant humoral immune responses were induced in the immunization group. The cellular immune responses were also elicited from the analysis of lymphocyte proliferation and expression of cytokines in the spleen. In addition, the S06004ΔspiCΔrfaH immunized group exhibited a negative response for the serological test, while the wild-type S06004 infection group was strongly positive for the serological test showing a DIVA capability. The survival rates in the vaccinated chickens were 87% after intramuscular challenge with wild-type S. Pullorum, while the survival rates were 20% in the control groups. Overall, these results have demonstrated that the rough mutant S06004ΔspiCΔrfaH strain can be developed as an efficient live attenuated DIVA vaccine candidate to control the systemic S. Pullorum infection without the interference of salmonellosis monitoring program in poultry.

Salmonella spp. are important zoonotic pathogens that are responsible for severe diseases in both animals and humans. Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum) and biovar Pullorum (S. Pullorum) are typical infectious pathogens detected in the chicken industry that have caused great economic losses. To facilitate their detection and prevent contamination, we developed a rapid multiple PCR method, which can simultaneously detect Salmonella spp. and further identify the biovars S. Pullorum/Gallinarum. This PCR detection method is based on the cigR gene, which is conserved among Salmonella spp. but has a 42-bp deletion in S. Pullorum/Gallinarum. The specificity and sensitivity of the PCR assay was evaluated with 41 different strains: 34 Salmonella strains, including 5 S. Pullorum/Gallinarum strains, and 7 non-Salmonella strains. The lower limit of detection was 8.15 pg of S. Pullorum (S06004) genomic DNA and 20 cfu in PCR, which shows a great sensitivity. In addition, this method was applied to detect or identify Salmonella from processing chicken liver and egg samples, and the results corresponded to those obtained from serotype analysis using the conventional slide agglutination test. Overall, the new cigR-based PCR assay is efficient and practical for Salmonella detection and S. Pullorum/Gallinarum identification and will greatly reduce the workload of epidemiologic investigation.

Abstract Background Salmonella Enteritidis (SE) is one of the major foodborne zoonotic pathogens of worldwide importance which can induce activation of NLRC4 and NLRP3 inflammasomes during infection. Given that the inflammasomes play an essential role in resisting bacterial infection, Salmonella has evolved various strategies to regulate activation of the inflammasome, most of which largely remain unclear. Results A transposon mutant library in SE strain C50336 was screened for the identification of the potential factors that regulate inflammasome activation. We found that T3SS-associated genes invC, prgH, and spaN were required for inflammasome activation in vitro. Interestingly, C50336 strains with deletion or overexpression of Dam were both defective in activation of caspase-1, secretion of IL-1β and phosphorylation of c-Jun N-terminal kinase (Jnk). Transcriptome sequencing (RNA-seq) results showed that most of the differentially expressed genes and enriched KEGG pathways between the C50336-VS-C50336Δdam and C50336-VS-C50336::dam groups overlapped, which includes multiple signaling pathways related to the inflammasome. C50336Δdam and C50336::dam were both found to be defective in suppressing the expression of several anti-inflammasome factors. Moreover, overexpression of Dam in macrophages by lentiviral infection could specifically enhance the activation of NLRP3 inflammasome independently via promoting the Jnk pathway. Conclusions These data indicated that Dam was essential for modulating inflammasome activation during SE infection, there were complex and dynamic interplays between Dam and the inflammasome under different conditions. New insights were provided about the battle between SE and host innate immunological mechanisms.

This study investigated the prevalence of Salmonella and the molecular typing of all isolates in a goose production chain including hatchery, farm, slaughterhouse, and market. A total of 350 Salmonella isolates was detected from 1,030 samples, and 13 serotypes were recovered. The highest Salmonella contamination frequency was observed at the hatchery, which 51.8% (188/363) of samples were Salmonella positive. S. Potsdam and S. Typhimurium were the 2 most common serotypes. S. Potsdam was most frequently found in the hatchery, while S. Typhimurium was widely distributed in the goose production chain. In general, the antibiotic resistance of Salmonella isolates is low, which isolates from the market is comparatively higher than from other production links indicating a possibility of Salmonella cross-contamination in the market. By the multilocus sequence typing (MLST) analysis, 7 different ST types were identified. ST2039 was the most common ST type, which was mostly found from S. Potsdam isolates in hatchery indicating that S. Potsdam might have been long existed in hatchery. The pulsed-field gel electrophoresis (PFGE) analysis of S. Potsdam indicated that S. Potsdam could be transmitted along the production chain. The PFGE analysis of S. Typhimurium showed that PFGE pattern 29 (PF29) was distributed in hatchery, and also in farm and from humans indicating the risk of S. Typhimurium transmitting to humans by the food supply chain. Our study provided the evidence of Salmonella cross-contamination in the slaughterhouse and the retail market of goose production chain, and specific serotypes existed for a long time at a particular production link. The spread of Salmonella along the production chain, might cause harm to humans through cross-contamination. Further studies would be needed to control the Salmonella contamination in hatchery and prevent the transmission of the pathogen during the goose production.

Inflammation is a double-edged sword that can be induced by various PAMPs, resulting in the control of infection by invading pathogens or injuries. The inflammatory response requires strict and precise control and regulation. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression via translational inhibition or mRNA degradation. However, the role of miRNAs in inflammation induced by flagellin (ligand of TLR5) has yet to be fully determined. In this study, we identified differentially expressed miRNAs in murine bone marrow-derived dendritic cells (BMDCs) between flagellin treatment and medium alone using miRNA microarray. We found that flagellin stimulation downregulated miR-5112 expression in BMDCs and spleen DCs in vitro and in vivo. The overexpression of miR-5112 decreased inflammatory cytokine production, accompanied by a reduction of IKKγ in flagellin-stimulated BMDCs. We demonstrated that miR-5112 could directly target IKKγ to inhibit inflammatory cytokine production. Furthermore, miR-5112 inhibited the inflammatory response induced by flagellin or Salmonella infection in vivo. Interestingly, miR-5112 could also dampen the inflammatory response and alleviate dextran sulfate sodium (DSS)-induced colitis in C57BL/6 mice. These results suggest that miR-5112 could be a novel therapeutic target for both bacterial infection and DSS-induced colitis model.

Tuberculosis (TB), a zoonosis characterized by chronic respiratory infections, is mainly caused by Mycobacterium tuberculosis and is associated with one of the heaviest disease burdens in the world. Dendritic cells (DCs) play a key role and act as a bridge between innate and adaptive immune responses against TB. DCs are divided into distinct subsets. Currently, the response of DCs to mycobacterial infections is poorly understood. Herein, we aimed to evaluate the responses of splenic conventional DCs (cDC) and plasmacytoid DCs (pDC), subsets to Bacillus Calmette–Guérin (BCG) infection in mice. Splenic pDC had a significantly higher infection rate and intracellular bacterial count than cDC and the CD8+ and CD8− cDC subsets after BCG infection. However, the expression levels of CD40, CD80, CD86, and MHC-II molecules were significantly upregulated in splenic cDC and the CD8 cDC subsets compared to pDC during BCG infection. Splenic cDC had a higher expression of IFN-γ and IL-12p70 than pDC, whereas pDC had higher levels of TNF-α and MCP-1 than cDC in mice infected with BCG. At early stages of immunization with BCG containing the Ag85A protein, splenic cDC and pDC could present the Ag85A peptide to a specific T hybridoma; however, cDC had a stronger antigen presenting activity than pDC. In summary, splenic cDC and pDC extensively participate in mouse immune responses against BCG infection in vivo. Although pDC had a higher BCG uptake, cDC induced stronger immunological effects, including activation and maturation, cytokine production, and antigen presentation.

In this paper, Al-7%Si-0.6%Mg-X%Ce (X = 0, 0.1 and 0.2) casting alloys were prepared by vacuum induction furnace. Using optical microscopy (OM), x-ray diffractometer (XRD), scanning electron microscopy (SEM), energy spectrometry (EDS), differential scanning calorimetry (DSC) and transmission electron microscopy (TEM), the microstructures of the experimental alloys were studied, and the mechanism of refinement and modification of the alloy were discussed. Results show that for Al-Si alloy, AlCeSi _2 is formed by adding cerium element into aluminum alloy. Furthermore, with the increase of cerium addition, α - Al dendrite arms decrease, the AlCeSi _2 was formed, and the growth of iron-rich phase was hindered, respectively. Also, the areas of eutectic of α -Al + Si were decreased, due to the supercooling increasing of the alloys. In addition, the α -Al grains were refined, because existing of heterogeneous nucleation core supplied by AlCeSi _2 , and component supercooling caused by cerium adding. Moreover, the formation of AlCeSi _2 consumed to some silicon elements, which leaded to size and amount reduction of eutectic silicon. The formation of high-density stacking fault can induce silicon modified, and the formed high-density stacking fault was produced by cerium as impurity element into silicon.

Listeria monocytogenes (Lm) is a foodborne zoonotic pathogen that causes listeriosis with a mortality rate of 20–30%. Serovar 4b and 1/2b isolates account for most of listeriosis outbreaks, however, no listeriosis vaccine is available for either prophylactic or therapeutic use. Here, we developed a triple-virulence-genes deletion vaccine strain, and evaluated its safety, immunogenicity, and cross-protective efficiency. The virulence of NTSNΔactA/plcB/orfX was reduced 794-folds compared with the parental strain. Additionally, it was completely eliminated in mice at day 7 post infection and no obvious pathological changes were observed in the organs of mice after prime-boost immunization for 23 days. These results proved that the safety of the Lm vaccine strain remarkably increased. More importantly, the NTSNΔactA/plcB/orfX strain stimulated higher anti-Listeriolysin O (LLO) antibodies, induced significantly higher expression of IFN-γ, TNF-α, IL-17, and IL-6 than the control group, and afforded 100% protection against serovar 4b and 1/2b challenges. Taken together, our research demonstrates that the triple-genes-deletion vaccine has high safety, can elicit strong Th1 type immune response, and affords efficient cross-protection against two serovar Lm strains. It is a promising vaccine for prevention of listeriosis.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is an important zoonotic pathogen that can lead to diarrhea and systemic infections in humans and mortality in animals. This is a major public health issue worldwide. Safe and effective vaccines are urgently needed to control and prevent Salmonella infection. Subunit vaccines are safe and provide targeted protection against Salmonella spp. Here, we developed and evaluated an S. Enteritidis subunit vaccine candidate, the rHis-SseB adjuvant with simvastatin. We amplified the SseB gene from S. Enteritidis C50041 genomic DNA and expressed the recombinant proteins rHis-SseB and rGST-SseB using the Escherichia coli system. Western blotting confirmed the immunoreactivity of recombinant proteins rHis-SseB and rGST-SseB with antisera against Salmonella Enteritidis C50041. In a mouse model of intramuscular vaccination, co-immunization with rHis-SseB and simvastatin significantly enhanced both the SseB-specific antibody titer in serum (humoral immune response) and splenic lymphocyte proliferation (cellular immune response). Co-immunization with rHis-SseB and simvastatin provided 60% protection against subsequent challenge with the S. Enteritidis C50041 strain and decreased bacterial colonization in the liver and spleen. These findings provide a basis for the development of an S. Enteritidis subunit vaccine.

TNF receptor-associated factor 6 (TRAF6) is a signal transducer, which plays a pivotal role in triggering a variety of signalling cascades. Here, we cloned and identified the TRAF6 gene from the King pigeon. The open reading frame sequence of pigeon TRAF6 (piTRAF6) is 1638 bp long and encodes a 545 aa protein, including a low-complexity domain, RING finger, Zinc finger, coiled coil domain, and meprin and TRAF homology domain. The aa sequence of piTRAF6 shared a strong identity with that of other birds. PiTRAF6 transcripts were broadly expressed in all the tested tissues; piTRAF6 levels were the highest and lowest in the heart and stomach, respectively. Overexpression of piTRAF6 activated NF-κB in a dose-dependent manner and induced IFN-β expression. Upon piTRAF6 knockdown by small interfering RNAs, NF-κB activation was markedly inhibited in HEK293T cells. The expression of piTRAF6, as well as pro-inflammatory cytokines and antiviral molecules, were obviously increased after TLR ligand stimulation and Newcastle disease virus or Salmonella Pullorum inoculation. These results suggest that piTRAF6 may play a key immunoregulatory role in the innate immune response against viral and bacterial infections.

Autophagy is a cellular process to clear pathogens. Salmonella enterica serovar Enteritidis (S.E) has emerged as one of the most important food-borne pathogens. However, major studies still focus on Salmonella enterica serovar Typhimurium. Here, we reported that AvrA, a S. Enteritidis effector, inhibited autophagy to promote bacterial survival in the host. We found that AvrA regulates the conversion of LC3 I into LC3 II and the enrichment of lysosomes. Beclin-1, a key molecular regulator of autophagy, was decreased after AvrA expressed strain colonization. In S.E-AvrA−-infected cells, we found the increases of protein levels of p-JNK and p-c-Jun and the transcription level of AP-1. AvrA-reduction of Beclin-1 protein expression is through the JNK pathway. The JNK inhibitor abolished the AvrA-reduced Beclin-1 protein expression. Moreover, we identified that the AvrA mutation C186A abolished its regulation of Beclin-1 expression. In addition AvrA protein was found interacted with Beclin-1. In organoids and infected mice, we explored the physiologically related effects and mechanism of AvrA in reducing Beclin-1 through the JNK pathway, thus attenuating autophagic responses. This finding not only indicates an important role of S. Enteritidis effector in reducing host protein as a strategy to suppress autophagy, but also suggests manipulating autophagy as a new strategy to treat infectious diseases.

Salmonella has been known as the most important foodborne pathogen, which can infect humans via consuming contaminated food. Chicken meat has been known as an important vehicle to transmit Salmonella by the food supply chain. This study determined the prevalence, antimicrobial resistance, and genetic characteristics of Salmonella at different chicken slaughtering stages in East China. In total, 114 out of 200 (57%) samples were Salmonella positive, while Salmonella contamination was gradually increasing from the scalding and unhairing stage (17.5%) to the subdividing stage (70%) throughout the slaughtering. Whole-genome sequencing (WGS) was then performed to analyze the serotype, antimicrobial resistance gene profiles, and genetic relationship of all Salmonella isolates. The most common serotypes were S. Kentucky (51/114, 44.7%) and S. Enteritidis (37/114, 32.5%), which were distributed throughout the four slaughtering stages, and were also identified in the corresponding environments. The multilocus sequence typing (MLST) analysis revealed that seven sequence types (STs) were occupied by six different serotypes, respectively. Only S. Kentucky had two STs, ST314 was the predominant ST shared by 50 isolates, while the ST198 has 1 isolate. The antimicrobial resistance gene analysis demonstrated that most of the strains belonging to S. Kentucky (39/51, 76.5%) and S. Indiana (15, 100%) contained over five groups of antimicrobial resistance genes. Based on the core genome analysis, 50 S. Kentucky isolates were genetically identical, indicating that one S. Kentucky strain with the same genetic background was prevalent in the chicken slaughtering line. Although 37 S. Enteritidis isolates only had three different antimicrobial resistance gene profiles, the core genome sequence analysis subtyped these S. Enteritidis isolates into five different clusters, which revealed the diverse genetic background of S. Enteritidis in the slaughterhouse. The antimicrobial resistance phenotypes were consistent with the presence of the corresponding resistance genes of S. Kentucky and S. Enteritidis, including tetA, floR, blaTEM-1B, strA/B, sul1/sul2, and gyrA (D87Y). Our study observed a high prevalence of Salmonella in the chicken slaughter line and identified the slaughtering environment as a main source of causing Salmonella cross-contamination during chicken slaughtering. Further studies will be needed to limit the transmission of Salmonella in the slaughterhouse.

Abstract Background Control of Mycobacterium tuberculosis (Mtb) infection requires CD4+ T-cell responses and major histocompatibility complex class II (MHC II) presentation of Mtb antigens (Ags). Dendritic cells (DCs) are the most potent of the Ag-presenting cells and are central to the initiation of T-cell immune responses. Much research has indicated that DCs play an important role in anti-mycobacterial immune responses at early infection time points, but the kinetics of Ag presentation by these cells during these events are incompletely understood. Results In the present study, we evaluated in vivo dynamics of early Ag presentation by murine lymph-node (LN) DCs in response to Mycobacterium bovis bacillus Calmette–Guérin (BCG) Ag85A protein. Results showed that the early Ag-presenting activity of murine DCs induced by M. bovis BCG Ag85A protein in vivo was transient, appearing at 4 h and being barely detectable at 72 h. The transcription levels of CIITA, MHC II and the expression of MHC II molecule on the cell surface increased following BCG infection. Moreover, BCG was found to survive within the inguinal LN DC pool, representing a continuing source of mycobacterial Ag85A protein, with which LN DCs formed Ag85A peptide-MHCII complexes in vivo. Conclusions Our results demonstrate that a decrease in Ag85A peptide production as a result of the inhibition of Ag processing to is largely responsible for the short duration of Ag presentation by LN DCs during BCG infection in vivo.

Abstract Background Activation of inflammasome contributes to the clearance of intracellular bacteria. C-terminus of E. coli EscI protein can activate NLRC4 (NLR family, CARD domain containing-4) inflammasome in macrophages. The purpose of this study was to determine if activation of NLRC4 inflammasome by EscI can reduce the colonization of Salmonella in mice. Results A recombinant S. typhimurium strain expressing fusion protein of the N-terminal SspH2 (a Salmonella type III secretion system 2 effector) and C-terminal EscI was constructed and designated as X4550(pYA3334-SspH2-EscI). In vitro assay showed that X4550(pYA3334-SspH2-EscI) significantly enhanced IL-1β and IL-18 secretion (P

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the important antibiotic resistant pathogens causing infections in humans and animals. The increasing observation of MRSA in wildlife species has raised the concern of its impact on animal health and the potential of zoonotic transmission. This study investigated the prevalence of S. aureus in fecal samples from non-human primates in a zoo located in Jiangsu, China, in which 6 out of 31 (19.4%) fecal samples, and 2 out of 14 (14.3%) indoor room floor swab samples were S. aureus-positive. The antibiotic susceptibility tests of the eight isolates showed that the two isolates were resistant to both penicillin and cefoxitin, the three isolates were resistant only to penicillin, while three isolates were susceptible to all detected antibiotics. The two isolates resistant to cefoxitin were further identified as MRSA by the presence of mecA. Five different spa types were identified including t034 of two MRSA isolates from Trachypithecus francoisi, t189 of two methicillin-susceptible S. aureus (MSSA) isolates from Rhinopithecus roxellana, t377 of two MSSA isolates from Colobus guereza, and two novel spa types t19488 and t19499 from Papio anubis. Whole genome sequencing analysis showed that MRSA t034 isolates belonged to ST398 clustered in clonal complex 398 (CC398) and carried the type B ΦSa3 prophage. The phylogenetic analysis showed that the two MRSA t034/ST398 isolates were closely related to the human-associated MSSA in China. Moreover, two MRSA isolates contained the virulence genes relating to the cell adherence, biofilm formation, toxins, and the human-associated immune evasion cluster, which indicated the potential of bidirectional transfer of MRSA between monkeys and humans. This study is the first to report MRSA CC398 from monkey feces in China, indicating that MRSA CC398 could colonize in monkey and have the risk of transmission between humans and monkeys.

The Ag85 complex functions as the main secretory protein of Mycobacterium tuberculosis (M. tuberculosis) and BCG. This complex is composed of the proteins, Ag85A, Ag85B, and Ag85C, with Ag85A thought to play the largest role within the complex. However, the lack of commercially available monoclonal antibodies (mAbs) against Ag85A still hinders the biological and applicative research on this protein. In this study, we developed and identified anti-Ag85A mAbs, and five hybridoma cells were established. Using the indirect immunofluorescence test, we found that two anti-Ag85A mAbs did not cross-react with Ag85B and/or Ag85C. In addition, we showed that all of the mAbs tested in this study are able to react with endogenous Ag85A protein in BCG and rBCG:Ag85A using indirect ELISA and Western blot analyses. A competitive ELISA (cELISA) based on mAb 3B8 was developed, the analyses of clinic serum samples from cattle with bovine tuberculosis (TB) and healthy cattle demonstrated that the sensitivity of the cELISA was 54.2% (26/48) and the specificity was 83.5% (167/200). This study demonstrated that the mAbs against Ag85A will provide useful reagents for further investigation into the function of the Ag85 complex and can be used for serodiagnosis of bovine TB.

This study investigated the effects of teaching via virtual reality (VR) on creative design performance and creative experiential learning processes by using a pretest--post-test nonequivalent group design to sample 138 seventh graders from a junior high school in Taipei City. The main conclusions from this study are: (1) VR has significant positive effects on creative design processes, particularly in the "design and planning," "testing and revision" and "thoughts and appreciation" stages. (2) VR has significant positive effects on creative design outcomes, with high- and low-level effects on "creative design" and "functional appropriateness," respectively. (3) The learning process for VR and creative experiential learning shifts from "concrete experiences" to "reflective observations," "abstract conceptualization" and, finally, "innovative applications." Behaviors of "reflective observation" and "innovative application" are active and vital to the performance of creative processes and outcomes.

Virtual reality (VR) can promote design performance, and may generate a high cognitive load and affect creative design thinking as well. In order to examine the effect of VR application on cognitive load and engineering design creativity, this study recruited 81 eighth-grade students as participants and employed a non-equivalent-groups quasi-experimental design to examine how VR application influences creativity components and creative performance. A creativity component scale (creative motivation, creative thinking, and professional cognition), a measurement of creative performance, and a cognitive load scale were used to collect data. The study also investigated the ability of VR application to predict creativity components and creative performance, using cognitive load as the mediator. The conclusions are as follows: (1) VR application had marked effects on professional cognition and creative motivation but no effect on creative thinking skills. (2) VR application had marked effects on the novelty and usefulness aspects of creative performance, particularly with respect to usefulness. (3) VR application had marked effects on extraneous and germane cognitive load, but no influence on intrinsic cognitive load, and (4) Both VR application and cognitive load were able to predict creativity components and creative performance. Their abilities to predict creative performance approached an acceptable level. Nevertheless, VR application had no effect on general creative thinking skills; therefore, other teaching strategies are needed to strengthen creative thinking.