Search

Summary: Toxoplasma gondii (T. gondii) is an intracellular parasite, and its regulation of host cell apoptosis directly affects its parasitism. Studies link T. gondii-induced apoptosis to abnormal expression of mammalian STE20-like protein kinase 2 (MST2), but its precise role remains unclear. In this study, the regulatory roles in apoptosis and pathogenicity of T. gondii infection were identified in vitro and in vivo. Simultaneously, MST2 and Hippo signaling pathway activation induced by T. gondii were evaluated. MST2 overexpression and knockout were used to assess its regulatory role in apoptosis and Hippo signaling pathway. Results showed that T. gondii induced apoptosis and lung damage, with Hippo signaling pathway activation via MST2 phosphorylation. MST2 was demonstrated to regulate apoptosis and Hippo signaling pathway. Notably, MST2 knockout hindered the T. gondii-induced apoptosis and weakened Hippo signaling pathway activation. MST2 is an important target for T. gondii to control host cell fate and modulate immune responses.

Abstract Poly(heptazine imide) (PHI), a semicrystalline version of carbon nitride photocatalyst based on heptazine units, has gained significant attention for solar H2 production benefiting from its advantages including molecular synthetic versatility, excellent physicochemical stability and suitable energy band structure to capture visible photons. Typically, PHI is obtained in salt‐melt synthesis in the presence of alkali metal chlorides. Herein, we examined the role of binary alkali metal bromides (LiBr/NaBr) with diverse compositions and melting points to rationally modulate the polymerization process, structure, and properties of PHI. Solid characterizations revealed that semicrystalline PHI with a condensed π‐conjugated system and rapid charge separation rates were obtained in the presence of LiBr/NaBr. Accordingly, the apparent quantum yield of hydrogen using the optimized PHI reaches up to 62.3% at 420 nm. The density functional theory calculation shows that the dehydrogenation of the ethylene glycol has a lower energy barrier than the dehydrogenation of the other alcohols from the thermodynamic point of view. This study holds great promise for rational modulation of the structure and properties of conjugated polymeric materials.

Salmonella is a common foodborne pathogen found in poultry production systems. Contaminated poultry products are a major source of human salmonellosis. Understanding the conditions of contamination and the genetic relationships of Salmonella in poultry production is necessary to develop effective interventions measures for controlling Salmonella transmission. Through epidemiological investigation and whole-genome sequence (WGS) analysis, this study revealed the epidemic law, drug resistance, and genetic characteristics of Salmonella in the production chain. In total, 130 (10.77 %) Salmonella strains were isolated from the collected samples, with Salmonella Enteritidis (S. Enteritidis) and S. Infantis being the predominant serotypes. WGS analysis revealed that the Sequence Type (ST) of all the 22 strains of S. Enteritidis were ST11, and 13 strains of S. Infantis were ST32. Antimicrobial resistance gene analysis showed that 95.45 % of S. Enteritidis carried strA/strB, sul, and blaTEM-1B; 77.3 % contained tetA genes; and 100 % of S. Infantis carried aac(6′)-Iaa and mdf(A), which was consistent with the drug resistance phenotype. The phylogenetic tree showed that S. Enteritidis strains with different links were distributed in the same branch which displayed the very close genetic relationship. Combined with epidemiological investigations, it was found that S. Enteritidis infection begins at the chicken breeding stage and spreads vertically and horizontally along the production chain. In addition, phylogenetic tree analysis of S. Infantis showed that the genomes of 13 strains from egg products and egg collection center were very similar and belonged to the same clone cluster. It is speculated that the S. Infantis transmitted in the industrial chain is the same strain; it can spread along the industrial chain, and cross-contamination may occur. This study indicates that Salmonella contamination in the layered industrial chain is more serious and that a cloning relationship exists among the strains of different links. Therefore, more stringent measures should be taken to control Salmonella during the chicken breeding stage, and the importance of implementing good hygiene practices at every level of production should be emphasized.

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.

The Chinese pangolin (Manis pentadactyla) is on the verge of extinction. Overuse and habitat loss have decimated the pangolin population across China. Understanding the habitat status of pangolins is a prerequisite for conservation, but this understanding is lacking due to difficulties in surveying and monitoring this species. This study used the MaxEnt model to analyze the current habitat distribution of Chinese pangolins by summarizing distribution records for China from 2010–2022. Moderate- and high-quality habitat of Chinese pangolins covers about 554,025 km2, mainly in 10 provinces in East and South China, together with a small area in Yunnan and Tibet. The majority of pangolin habitats (89.32%) were distributed in the forests of low mountains and hills; only 8.92% of habitats overlap with protected areas. Prey abundance and climate are key factors affecting pangolin distribution. Land-use changes leading to habitat encroachment and/or affecting prey abundance, and climate change may underlie their shrinking distribution. Considering that the current protected area coverage of Chinese pangolin habitat is low, we recommend adjusting or expanding the boundaries of protected areas to improve coverage. The focus should be on strengthening the population and habitat management of Chinese pangolin outside protected areas. Within pangolin ranges, conservation can be achieved through improving the education of community residents, implementing stronger community economic development policies, replacing lost logging income from residents’ private forest land with economic compensation, and strictly regulating the location of infrastructure and residential area construction. The goal of these four key actions is to increase the public’s awareness and enthusiasm for pangolins, reduce the willingness of community residents to hunt pangolin, and prevent the destruction of and encroachment into existing pangolin habitat.

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.

We study the fermion-boson coupled system in d=3−ϵ space dimensions near the quantum phase transition; infinite many boson modes locating on a sphere become critical simultaneously, which is dubbed “critical boson surface” (CBS). The fermions on the Fermi surface can be scattered to nearby points locating on a boson ring in the low-energy limit. The large number of the boson scattering channels N renders the well-known Landau damping effect largely suppressed. We propose an effective theory for a single point on the Fermi surface and the associated critical boson ring induced by the boson scattering channels. Based on the effective model, one-loop renormalization group analysis is performed with asymptotic ϵ-expansion. The fermion self-energy and Yukawa interaction vertex are dressed with ϵ poles and are largely enhanced due to the presence of critical boson ring. The imaginary part of the self-energy exhibits a linear-in frequency dependence and the real part gives a vanishing quasiparticle weight in the low-energy limit, which signatures the celebrated “marginal Fermi liquid” behavior. We find a novel non-Fermi liquid fixed point, at which the critical properties show features associated with the CBS.

IntroductionApicomplexan AP2 family of proteins (ApiAP2) are transcription factors (TFs) that regulate parasite growth and development, but little is known about the ApiAP2 TFs in Eimeria spp. ENH_00027130 sequence is predicted to encode a Eimeria necatrix ApiAP2 protein (EnApiAP2).MethodsThe cDNAs encoding full-length and truncated EnApiAP2 protein were cloned and sequenced, respectively. Then, the two cDNAs were cloned into the pET28a(+) expression vector and expressed expressed in Escherichia coli BL21. The mouse polyclonal antibody (pAb) and monoclonal antibody (mAb) against recombinant EnApiAP2 (rEnApiAP2) and EnApiAP2tr (rEnApiAP2tr) were prepared and used to localize the native EnApiAP2 protein in E. necatrix, respectively. Finally, the recombinant pEGFP-C1-ΔNLS-EnApiAP2s (knockout of a nuclear localization sequence, NLS) and pEGFP-C1-EnApiAP2 plasmid were constructed and transfected into DF-1 cells, respectively, to further observe subcellular localization of EnApiAP2 protein.ResultsThe EnApiAP2 gene had a size of 5019 bp and encoded 1672 amino acids, containing a conserved AP2 domain with a secondary structure consisting of an α-helix and three antiparallel β-strands. The rEnApiAP2 and rEnApiAP2tr were predominantly expressed in the form of inclusion bodies, and could be recognized by the 6×His tag mAb and the serum of convalescent chickens after infection with E. necatrix, respectively. The native EnApiAP2 protein was detected in sporozoites (SZ) and second generation merozoites (MZ-2) extracts, with a size of approximately 210 kDa. A quantitative real-time PCR (qPCR) analysis showed that the transcription level of EnApiAP2 was significantly higher in SZ than in MZ-2, third generation merozoites (MZ-3) and gametocytes (P

ABSTRACT The development of safe and efficient vaccines against coronavirus disease 2019 (COVID-19) is necessary for global public health. The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major target for the COVID-19 vaccines. Among the available vaccine types, DNA vaccines are a promising alternative to conventional vaccines. Here, we developed a novel DNA vaccine containing the SARS-CoV-2 S1 subunit and an optimized flagellin adjuvant (FliCΔD2D3) from Salmonella typhimurium. The S1 gene and S1-fliCΔD2D3 fusion gene were codon optimized and then cloned into the antibiotic-resistance-gene-free vector asd-pVAX1, producing asd-pVAX1-S1 and asd-pVAX1-S1-fliCΔD2D3, respectively. The expression of S1 and S1-FliCΔD2D3 proteins was confirmed after transient expression in HEK293T cells. After in vitro characterization of the vaccine candidates, we evaluated the humoral and cellular immune responses induced in BALB/c mice. The DNA vaccine induced a long-lasting humoral response for approximately 4 months, and the elicited serum antibodies efficiently blocked the binding of the SARS-CoV-2 S1 receptor-binding domain to its entry receptor (angiotensin-converting enzyme 2). Moreover, the DNA vaccine induced strong Th1/Th2-mixed immune responses, as demonstrated by the similar levels of S1-specific immunoglobulin (Ig) G1 and IgG2a in the serum and the significantly elevated Th1-type (IFN-γ and TNF-α) and Th2-type (IL-4 and IL-6) cytokine expression from splenic lymphocytes. Importantly, asd-pVAX1-S1-fliCΔD2D3 immunization in mice could significantly enhance the levels of immune responses compared to asd-pVAX1-S1. This study provides crucial information regarding the selection of a safer DNA vector and a new adjuvant for the vaccine development of SARS-CoV-2 and other infectious diseases. IMPORTANCE The development of safe and effective vaccines is needed to control the transmission of coronavirus disease 2019 (COVID-19). Synthetic DNA vaccines represent a promising platform in response to such outbreaks. Here, DNA vaccine candidates were developed using an optimized antibiotic-resistance gene-free asd-pVAX1 vector. An optimized flagellin (FliC) adjuvant was designed by fusion expression to increase the immunogenicity of the S1 antigen. S1 and S1-FliCΔD2D3 proteins were strongly expressed in mammalian cells. The FliCΔD2D3-adjuvanted DNA vaccine induced Th1/Th2-mixed immune responses and high titers of neutralizing antibodies. This study provides crucial information regarding the selection of a safer DNA vector and adjuvant for vaccine development. Our FliCΔD2D3-adjuvanted S1 DNA vaccine is more potent at inducing both humoral and cellular immune responses than S1 alone. This finding provides a new idea for the development of novel DNA vaccines against COVID-19 and could be further applied for the development of other vaccines.

The flagellin (FliC) of Salmonella typhimurium is a potential vaccine adjuvant as it can activate innate immunity and promote acquired immune responses. Macrophages are an important component of the innate immune system. The mechanism of flagellin’s adjuvant activity has been shown to be related to its ability to activate macrophages. However, few studies have comprehensively investigated the effects of Salmonella flagellin in macrophages using transcriptome sequencing. In this study, RNA-Seq was used to analyze the expression patterns of RAW264.7 macrophages induced by FliC to identify novel transcriptomic signatures in macrophages. A total of 2204 differentially expressed genes were found in the FliC-treated group compared with the control. Gene ontology and KEGG pathway analyses identified the top significantly regulated functional classification and canonical pathways, which were mainly related to immune responses and regulation. Inflammatory cytokines (IL-6, IL-1β, TNF-α, etc.) and chemokines (CXCL2, CXCL10, CCL2, etc.) were highly expressed in RAW264.7 cells following stimulation. Notably, flagellin significantly increased the expression of interferon (IFN)-β. In addition, previously unidentified IFN regulatory factors (IRFs) and IFN-stimulated genes (ISGs) were also significantly upregulated. The results of RNA-Seq were verified, and furthermore, we demonstrated that flagellin increased the expression of IFN-β and IFN-related genes (IRFs and ISGs) in bone marrow-derived dendritic cells and macrophages. These results suggested that Salmonella flagellin can activate IFN-β-related immune responses in macrophages, which provides new insight into the immune mechanisms of flagellin adjuvant.

Most cases of chicken salmonellosis are caused by Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum, which lead to a significant morbidity and fatality rate. Although the conventional Kaufmann-White scheme is the reliable method for the serotyping of Salmonella, it does not distinguish between closely related biotypes like S. Pullorum and S. Gallinarum. Herein, we conducted a single one-step multiplex PCR assay that can identify and distinguish between S. Pullorum and S. Gallinarum in an accurate manner. This PCR method was based on three genes, including torT for S. Pullorum identification, I137_14430 for S. Gallinarum identification, and stn as the genus-level reference gene for Salmonella. By comparing S. Pullorum to S. Gallinarum and other serovars of Salmonella, in silico study revealed that only the former has a deletion of 126 bp-region in the carboxyl terminus of torT. The I137_14430 gene does not exist in S. Gallinarum. However, it is present in all other Salmonella serotypes. The multiplex PCR approach utilizes unique sets of primers that are intended to specifically target these three different genes. The established PCR method was capable of distinguishing between the biovars Pullorum and Gallinarum from the 29 distinct Salmonella serotypes as well as the 50 distinct pathogens that are not Salmonella, showing excellent specificity and exclusivity. The minimal amount of bacterial cells required for PCR detection was 100 CFU, while the lowest level of genomic DNA required was 27.5 pg/μL for both S. Pullorum and S. Gallinarum. After being implemented on the clinical Salmonella isolates collected from a poultry farm, the PCR test was capable of distinguishing the two biovars Pullorum and Gallinarum from the other Salmonella strains. The findings of the PCR assay were in line with those of the traditional serotyping and biochemical identification methods. This new multiplex PCR could be used as a novel tool to reinforce the clinical diagnosis and differentiation of S. Pullorum and S. Gallinarum, particularly in high-throughput screening situations, providing the opportunity for early screening of infections and, as a result, more effective management of the illness among flocks.

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.

Salmonella Enteritidis is an important intracellular pathogen, which can cause gastroenteritis in humans and animals and threaten life and health. S. Enteritidis proliferates in host macrophages to establish systemic infection. In this study, we evaluated the effects of Salmonella pathogenicity island-1 (SPI-1) and SPI-2 to S. Enteritidis virulence in vitro and in vivo, as well as the host inflammatory pathways affected by SPI-1 and SPI-2. Our results show that S. Enteritidis SPI-1 and SPI-2 contributed to bacterial invasion and proliferation in RAW264.7 macrophages, and induced cytotoxicity and cellular apoptosis of these cells. S. Enteritidis infection induced multiple inflammatory responses, including mitogen-activated protein kinase (ERK-mediated) and Janus kinase-signal transducer and activator of transcript (STAT) (STAT2-mediated) pathways. Both SPI-1 and SPI-2 were necessary to induce robust inflammatory responses and ERK/STAT2 phosphorylation in macrophages. In a mouse infection model, both SPIs, especially SPI-2, resulted in significant production of inflammatory cytokines and various interferon-stimulated genes in the liver and spleen. Activation of the ERK- and STAT2-mediated cytokine storm was largely affected by SPI-2. S. Enteritidis ΔSPI-1-infected mice displayed moderate histopathological damage and drastically reduced bacterial loads in tissues, whereas only slight damage and no bacteria were observed in ΔSPI-2- and ΔSPI-1/SPI-2-infected mice. A survival assay showed that ΔSPI-1 mutant mice maintained a medium level of virulence, while SPI-2 plays a decisive role in bacterial virulence. Collectively, our findings indicate that both SPIs, especially SPI-2, profoundly contributed to S. Enteritidis intracellular localization and virulence by activating multiple inflammatory pathways.

Abstract Background Toxoplasma gondii is an obligate intracellular protozoan parasite that can cause a geographically widespread zoonosis. Our previous splenocyte microRNA profile analyses of pig infected with T. gondii revealed that the coordination of a large number of miRNAs regulates the host immune response during infection. However, the functions of other miRNAs involved in the immune regulation during T. gondii infection are not yet known. Methods Clustering analysis was performed by K-means, self-organizing map (SOM), and hierarchical clustering to obtain miRNA groups with the similar expression patterns. Then, the target genes of the miRNA group in each subcluster were further analyzed for functional enrichment by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway to recognize the key signaling molecules and the regulatory signatures of the innate and adaptive immune responses of the host during T. gondii infection. Results A total of 252 miRNAs were successfully divided into 22 subclusters by K-means clustering (designated as K1–K22), 29 subclusters by SOM clustering (designated as SOM1–SOM29), and six subclusters by hierarchical clustering (designated as H1–H6) based on their dynamic expression levels in the different infection stages. A total of 634, 660, and 477 GO terms, 15, 26, and 14 KEGG pathways, and 16, 15, and 7 Reactome pathways were significantly enriched by K-means, SOM, and hierarchical clustering, respectively. Of note, up to 22 miRNAs mainly showing downregulated expression at 50 days post-infection (dpi) were grouped into one subcluster (namely subcluster H3-K17-SOM1) through the three algorithms. Functional analysis revealed that a large group of immunomodulatory signaling molecules were controlled by the different miRNA groups to regulate multiple immune processes, for instance, IL-1-mediated cellular response and Th1/Th2 cell differentiation partly depending on Notch signaling transduction for subclusters K1 and K2, innate immune response involved in neutrophil degranulation and TLR4 cascade signaling for subcluster K15, B cell activation for subclusters SOM17, SOM1, and SOM25, leukocyte migration, and chemokine activity for subcluster SOM9, cytokine–cytokine receptor interaction for subcluster H2, and interleukin production, chemotaxis of immune cells, chemokine signaling pathway, and C-type lectin receptor signaling pathway for subcluster H3-K17-SOM1. Conclusions Cluster analysis of splenocyte microRNAs in the pig revealed key regulatory properties of subcluster miRNA molecules and important features in the immune regulation induced by acute and chronic T. gondii infection. These results contribute new insight into the identification of physiological immune responses and maintenance of tolerance in pig spleen tissues during T. gondii infection. Graphical Abstract

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 Salmonella Derby is one of the most predominant Salmonella serotypes that seriously threatens food safety. This bacterium can be further differentiated to sub-populations with different population sizes; however, whether and how the S. Derby–gut microbiota interactions affect epidemic patterns of S. Derby sub-populations remain largely unknown. Results We selected two representative strains, 14T and 14C, which represent rarely distributed and prevalent sub-populations of the S. Derby ST40 group, respectively, to address this question using a mouse model. Effects of oral administration of both strains was monitored for 14 days. Alpha diversity of gut microbiota at early stages of infection (4 h post infection) was higher in 14C-treated mice and lower in 14T-treated mice compared with controls. Strain 14T triggered stronger inflammation responses but with lower pathogen titer in spleen compared with strain 14C at 14 days post infection. Certain known probiotic bacteria that can hinder colonization of Salmonella, such as Bifidobacteriaceae and Akkermansiaceae, exhibited increased relative abundance in 14T-treated mice compared with 14C-treated mice. Our results also demonstrated that Ligilactobacillus strains isolated from gut microbiota showed stronger antagonistic activity against strain 14T compared with strain 14C. Conclusions We identified how S. Derby infection affected gut microbiota composition, and found that the 14T strain, which represented a rarely distributed S. Derby sub-population, triggered stronger host inflammation responses and gut microbiota disturbance compared with the 14C strain, which represented a prevalent S. Derby sub-population. This study provides novel insights on the impacts of gut microbiota on the epidemic patterns of Salmonella populations.

Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum cause severe chicken salmonellosis, a disease associated with high mortality and morbidity among chickens worldwide. The conventional serotyping and biochemical reactions have been used to identify Salmonella serovars. However, the conventional methods are complicated, time-consuming, laborious, and expensive. Furthermore, it is challenging to distinguish S. Gallinarum and S. Pullorum via biochemical assays and serotyping because of their antigenic similarity. Although various PCR methods were established, a PCR protocol to detect and discriminate S. Gallinarum and S. Pullorum simultaneously is lacking. Herein, a one-step multiplex PCR method was established for the accurate identification and discrimination of S. Pullorum and S. Gallinarum. Three specific genes were used for the multiplex PCR method, with the I137_14445 and ybgL genes being the key targets to identify and differentiate S. Gallinarum and S. Pullorum, and stn being included as a reference gene for the Salmonella genus. In silico analysis showed that the I137_14445 gene is present in all Salmonella serovars, except for S. Gallinarum, and could therefore be used for the identification of S. Gallinarum. A 68-bp sequence deficiency in ybgL was found only in S. Pullorum compared to other Salmonella serovars, and this could therefore be used for the specific identification of S. Pullorum. The developed PCR assay was able to distinguish S. Gallinarum and S. Pullorum among 75 various Salmonella strains and 43 various non-Salmonella pathogens with excellent specificity. The detection limit for the genomic DNA of S. Gallinarum and S. Pullorum was 21.4 pg./μL, and the detectable limit for bacterial cells was 100 CFU. The developed PCR method was used for the analysis of Salmonella isolates in a chicken farm. This PCR system successfully discriminated S. Gallinarum and S. Pullorum from other different Salmonella serovars. The PCR results were confirmed by the conventional serotyping method. The newly established multiplex PCR is a simple, accurate, and cost-effective method for the timely identification and differentiation of S. Pullorum and S. Gallinarum.

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.

Cervical cancer is a leading cause of high mortality in women in developing countries and has a serious impact on women’s health. Human papilloma virus (HPV) prophylactic vaccines have been produced and may hold promise for reducing the incidence of cervical cancer. However, the limitations of current HPV vaccine strategies make the development of HPV therapeutic vaccines particularly important for the treatment of HPV related lesions. Our previous work has demonstrated that LM4Δhly::E7 was safe and effective in inducing antitumor effect by antigen-specific cellular immune responses and direct killing of tumor cell on a cervical cancer model. In this study, the codon usage effect of a novel Listeria-based cervical cancer vaccine LM4Δhly::E7-1, was evaluated for effects of codon-optimized E7 expression, cellular immune response and therapeutic efficacy in a tumor-bearing murine model. Our data demonstrated that up-regulated expression of E7 was strikingly elevated by codon usage optimization, and thus induced significantly higher Th1-biased immunity, lymphocyte proliferation, and strong specific CTL activity ex-vivo compared with LM4Δhly::E7-treated mice. Furthermore, LM4Δhly::E7-1 enhanced a remarkable therapeutic effect in establishing tumors. Taken together, our results suggest that codon usage optimization is an important consideration in constructing live bacterial-vectored vaccines and is required for promoting effective T cell responses.

Abstract Salmonella, as a zoonotic pathogen, has attracted widespread attention worldwide, especially in the transmission between household pets and humans. Therefore, we investigated the epidemic distribution of dog Salmonella from pet hospitals and breeding base in Xuzhou, Jiangsu Province, China, and used multilocus sequence typing (MLST) and clustered regularly interspaced short palindromic repeats (CRISPRs) to subtype Salmonella isolates. From April 2018 to November 2019, a total of 469 samples were collected from pet hospitals and breeding base, including 339 dog samples and 60 cat samples. S. Kentucky (40.74%) was the most prevalent serotype, but other, such as S. Typhimurium (18.52%) and S. Indiana (18.52%), were also widespread. Eight different sequence type (ST) patterns were identified by MLST and ST198 was the highest proportion of these isolates. CRISPRs analysis showed that 9 different Kentucky CRISPR types (KCTs) was identified from ST198. 48 spacers including 29 (6 News) for CRISPR1 and 19 (4 News) for CRISPR2 that proved the polymorphic of Salmonella genes in samples from different sources. The analysis demonstrated that the common serotypes were widely present in pet hosts in the same area. This analysis shows that CRISPR genes have better recognition ability in the same serotype, which has a positive effect on the traceability of Salmonella and the prevention and treatment of salmonellosis.

Abstract Fimbriae-mediated initial adherence is the initial and critical step required for enterotoxigenic Escherichia coli (ETEC) infection. Therefore, vaccine candidates have been developed that target these fimbriae and induce specific anti-fimbriae antibodies to block initial ETEC attachment. While this vaccine effectively protects against ETEC-associated post-weaning diarrhea (PWD), developing a broadly effective vaccine against initial ETEC attachment remains a challenging problem, owing to the immunological heterogeneity among these antigens. Here, we applied multi-epitope fusion antigen (MEFA) technology to construct a FaeG–FedF–FanC–FasA–Fim41a MEFA using the adhesive subunits of predominant fimbriae K88 and F18 as the backbone, which also integrated epitopes from adhesive subunits of the rare fimbriae K99, 987P, and F41; we then generated a MEFA computational model and tested the immunogenicity of this MEFA protein in immunized mice. We next evaluated the potential of the fimbriae-targeted MEFA as a vaccine candidate to effectively prevent PWD using in vitro assessment of its anti-fimbriae, antibody-directed inhibition of bacterial adherence. Computational modeling showed that all relevant epitopes were exposed on the MEFA surface and mice subcutaneously immunized with the MEFA protein developed IgG antibodies to all five fimbriae. Moreover, anti-fimbriae antibodies induced by the MEFA protein significantly inhibited the adhesion of K88+, F18+, K99+, 987P+, and F41+ ETEC strains to piglet small intestinal IPEC-1 and IPEC-J2 cell lines. Taken together, these results indicate that FaeG–FedF–FanC–FasA–Fim41a MEFA protein induced specific anti-fimbriae neutralizing antibodies against the five targeted fimbriae. Critically, these results show the potential of fimbriae-targeted MEFA and indicate their promise as a broad, effective vaccine against PWD.

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.

PurposeTo explore the value of partial pressure of carbon dioxide (PaCO2) levels in arterial blood for predicting immediate seizures (ISs) in patients with primary intracerebral hemorrhage (ICH).MethodsDemographic information and clinical data from patients with primary ICH were prospectively collected, including arterial blood gas analysis. Immediate seizures (ISs) were determined as seizures in the first 24 h after admission. Univariate and multivariate analyses were performed to assess the association of PaCO2 levels with ISs. Propensity-score matching (PSM) analyses were adopted to reduce the baseline difference between ISs and non-ISs groups.ResultsA total of 596 patients with primary ICH were initially screened in this clinical study, 368 of whom fulfilled all the inclusion criteria [mean age, (60.46 ±12.78) years; 57.9% female patients]. ISs occurred in 30 of the 368 (8.15%) patients with primary ICH of this cohort. Patients with ISs had significantly lower PaCO2 levels [34.35(32.38–37.53) vs. 39.45(35.90–43.43), mmHg, p < 0.001] and were younger than those without ISs [(54.57±12.15 vs. 60.99 ±12.72) years, p = 0.008]. Multivariate analysis showed that lower initial PaCO2 (≤37.2 mmHg) level was a significant independent predictor of ISs [odds ratios (OR) 0.141, 95% confidence interval (CI) 0.057–0.351, p < 0.001], as well as younger age (OR 0.961, 95% CI 0.928–0.995, p = 0.023) and hematoma expansion (OR 0.340, 95% CI 0.134–0.863, p = 0.023). Receiver operating characteristic curve (ROC) analysis demonstrated that the optimal cutoff value of PaCO2 level for predicting ISs was 37.20 mmHg in patients with primary ICH (the area under the curve (AUC) was 0.760 with a corresponding sensitivity of 76.67% and specificity of 67.46%, 95%CI = 0.713–0.802, p < 0.001). After PSM, the matched ISs group had significantly lower PaCO2 levels compared with the matched non-ISs group [34.45(32.43–38.18) vs. 41.75(35.85–43.98) mmHg, p < 0.05] in the univariate analysis. The lower initial PaCO2 level was still independent of ISs following primary ICH.ConclusionsThe lower initial PaCO2 level was associated with an increased risk of ISs in patients with primary ICH.

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.

Toxoplasma gondii (T. gondii) is an obligate intracellular parasitic protozoan that can cause toxoplasmosis in humans and other endotherms. T. gondii can manipulate the host gene expression profile by interfering with miRNA expression, which is closely associated with the molecular mechanisms of T. gondii-induced brain injury. However, it is unclear how T. gondii manipulates the gene expression of central nervous system (CNS) cells through modulation of miRNA expression in vivo during acute and chronic infection. Therefore, high-throughput sequencing was used to investigate expression profiles of brain miRNAs at 10, 25, and 50 days post-infection (DPI) in pigs infected with the Chinese I genotype T. gondii strain in this study. Compared with the control group 87, 68, and 135 differentially expressed miRNAs (DEMs) were identified in the infected porcine brains at 10, 25, and 50 DPI, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that a large number significantly enriched GO terms and KEGG pathways were found, and were mostly associated with stimulus or immune response, signal transduction, cell death or apoptosis, metabolic processes, immune system or diseases, and cancers. miRNA–gene network analysis revealed that the crucial connecting nodes, including DEMs and their target genes, might have key roles in the interactions between porcine brain and T. gondii. These results suggest that the regulatory strategies of T. gondii are involved in the modulation of a variety of host cell signaling pathways and cellular processes, containing unfolded protein response (UPR), oxidative stress (OS), autophagy, apoptosis, tumorigenesis, and inflammatory responses, by interfering with the global miRNA expression profile of CNS cells, allowing parasites to persist in the host CNS cells and contribute to pathological damage of porcine brain. To our knowledge, this is the first report on miRNA expression profile in porcine brains during acute and chronic T. gondii infection in vivo. Our results provide new insights into the mechanisms underlying T. gondii-induced brain injury during different infection stages and novel targets for developing therapeutic agents against T. gondii.

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.

The internalin family proteins, which carry the leucine repeat region structural motif, play diverse roles in Listeria monocytogenes (Lm) infection and pathogenesis. Although Internalin F, encoded by inlF, was identified more than 20 years ago, its role in the Lm anti-inflammatory response remains unknown. Lm serotype 4b isolates are associated with the majority of listeriosis outbreaks, but the function of InlF in these strains is not fully understood. In this study, we aimed to elucidate the role of inlF in modulating the inflammatory response and pathogenesis of the 4b strain Lm NTSN. Strikingly, although inlF was highly expressed at the transcriptional level during infection of five non-phagocytic cell types, it was not involved in adherence or invasion. Conversely, inlF did contributed to Lm adhesion and invasion of macrophages, and dramatically suppressed the expression of pro-inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF-α). Consistent with the in vitro results, during Lm infection mice, inlF significantly inhibited the expression of IL-1β and IL-6 in the spleen, as well as IL-1β, IL-6, and TNF-α in the liver. More importantly, inlF contributed to Lm colonization in the spleen, liver, and ileum during the early stage of mouse infection via intragastric administration, inducing severe inflammatory injury and histopathologic changes in the late stage. To our knowledge, this is the first report to demonstrate that inlF mediates the inhibition of the pro-inflammatory response and contributes to the colonization and survival of Lm during the early stage of infection in mice. Our research partly explains the high pathogenicity of serovar 4b strains and will lead to new insights into the pathogenesis and immune evasion of Lm.

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.

Infection with the avian leukosis virus subgroup J (ALV-J) impairs host genes and facilitates the establishment of chronic infection and the viral life cycle. However, the involvement of long noncoding RNAs (lncRNAs) in ALV-J infection remains largely unknown. In this study, we identified a novel chicken lncRNA derived from LTR5B of the ERV-L family (namely lnc-LTR5B), which is significantly downregulated in ALV-J infected cells. lnc-LTR5B was localized in the cytoplasm and was relatively high expressed in the chicken lung and liver. Notably, the replication of ALV-J was inhibited by the overexpression of lnc-LTR5B but enhanced when lnc-LTR5B expression was knocked down. We further confirmed that lnc-LTR5B could bind to the binding immunoglobulin protein (BiP), a master regulator of endoplasmic reticulum (ER) function. Mechanistically, lnc-LTR5B serves as a competing endogenous RNA for BiP, restricting its physical availability. Upon ALV-J infection, the reduction of lnc-LTR5B released BiP, which facilitated its translocation to the cell surface. This is crucial for ALV-J entry as well as pro-survival signaling. In conclusion, we identified an endogenous retroviral LTR-activated lnc-LTR5B that is involved in regulating the cell surface translocation of BiP, and such regulatory machinery can be exploited by ALV-J to complete its life cycle and propagate.

Monitoring of the quality change of cherry tomatoes during storage is very important for the quality control of cherry tomatoes. In this study, the soluble solids content (SSC), reducing sugars (RSs), titratable acids (TAs), ascorbic acid (AA) and lycopene of cherry tomatoes during storage at 0, 4, 10 or 25 °C were measured, and the kinetic models were established. The results showed that the zero-order reaction combined with the Arrhenius kinetic model could be used for the prediction of changes in SS, RS and AA content. The first-order reaction combined with the Arrhenius kinetic model could be used for the prediction of changes in the TA and lycopene content. The volatile compounds of cherry tomatoes were simultaneously determined by the gas chromatography–mass spectrometry (GC–MS) and electronic nose (E-nose). A total of 104 volatile compounds were identified by GC–MS. Orthogonal partial least squares discriminant analysis (OPLS-DA) showed that there were 13 different metabolites among cherry tomatoes with different freshness. The accuracies of Fisher’s models based on E-nose for discriminating freshness of cherry tomatoes stored at 0, 4, 10 and 25 °C were 96%, 100%, 92% and 90%, respectively. This study provides a theoretical basis for the quality control of cherry tomatoes during storage.

ABSTRACTCRISPR-based typing was performed to subtype isolates of S. Typhimurium and its monophasic variant Salmonella 4,[5],12:i:- from humans and animals between 2009 and 2017 in China. CRISPR typing classified all isolates into two lineages and four sub-lineages. All isolates from Lineage II and Lineage IB-1 were Salmonella Typhimurium. All of Salmonella 4,[5],12:i: – isolates were distributed in Lineage IA and Lineage IB-2, which all belonged to ST34 by MLST typing. Only Lineage IB-2 contained ST34 isolates from both Salmonella Typhimurium and Salmonella 4,[5],12:i:-. Among the isolates of ST34, TST4 was identified as the most common CRISPR type representing 86.5% of Salmonella 4,[5],12:i:- and 14.5 % of Salmonella Typhimurium mainly from pigs and humans. This study demonstrated that TST4-ST34 isolates were predominant in Salmonella 4,[5],12:i:-, and pig was the main reservoir for Salmonella 4,[5],12:i:- in China, which might have the potential to transmit to humans by pig production.

Listeria monocytogenes isolates are highly heterogeneous and exhibit different levels of virulence. Here, the authors identify hypervirulent isolates that represent a hybrid sub-lineage of the major lineage II harbouring virulence factors from Listeria ivanovii and wall teichoic acids found in major lineage I.

Salmonella Enteritidis (SE) are important zoonotic pathogens, and can be easily transferred to humans by contaminated animal products. Epidemic surveys of SE are necessary in current modern large-scale chicken farms. In this study, Salmonella strains were isolated from possibly infected samples collected at 3 independent farms, and their serotype, drug resistances, virulence genes, and genetic similarity were analyzed by molecular genetic analysis technologies including multilocus sequence typing (MLST), clustered regularly interspaced short palindromic repeats (CRISPR), pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). A total of 346 Salmonella strains were isolated from 3,598 samples (9.61%); 329 isolates were identified as SE (95.09%) and 308 isolates were multidrug resistant (93.62%). Virulotyping based on 6 virulence genes showed high similarity in SE isolates of each farm, with the exception of 2 isolates. All SE isolates were found to be the same ST11 type by MLST, and 22 strains of 150 SE isolates selected at random were found to belong to 1 cluster by PFGE and the same SET1 type by CRISPR. WGS results further revealed that these isolates belonged to the same clonal cluster, with high genetic similarity of 99.80 to 100.00%. All these results indicated that these SE isolates were overwhelmingly dominant and demonstrated high genetic similarity, which revealed that the same SE clone might be transmitted in these farms.

Pullorum is a chicken-specific systemic disease caused by Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum). This study was carried out to provide basic data for understanding the trends of S. Pullorum. A total of 652 S. Pullorum isolates collected in China during 1962–2019 were examined. Overall, 525 (80.5%) isolates were resistant to at least one antibiotic; 280 (42.9%) isolates resisted 3 or more classes of antibiotics and showed an increasing trend until 2015 and then decreased significantly. The most common multidrug-resistant pattern was ampicillin–tetracycline–nalidixic acid (13.6%). After 2008, 6 classes of antibiotic-resistant strains began to appear, and they have been prevalent ever since. In 2014, a strain resistant to 7 antibiotics (ampicillin–cefazolin–streptomycin–tetracycline–sulphonamides–nalidixic acid–nitrofurantoin) was isolated. The highest antimicrobial resistance was observed for nalidixic acid (71.9%), and the lowest was found for cefotaxime, meropenem, amikacin, gentamicin, fosfomycin, and polymyxin (0%). Our findings monitored the prevalence of the resistance of S. Pullorum during the past half-century in China. Continued surveillance of antimicrobial resistance and the rational use of antimicrobials is necessary and important to control the rapid increase in antimicrobial resistance in S. Pullorum.

Avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, is known to cause immunosuppression and various types of cancer in chickens. Recent reports have shown that epigenetic changes in DNA and chromatin are widely implicated in the life cycle of diverse viruses, and reversal of these changes in host cells can lead to alterations in the propagation of viruses. In the present study, we found that disruptor of telomeric silencing 1-like (DOT1L), a histone H3 lysine79 (H3K79) methyltransferase, was upregulated during ALV-J infection in chicken macrophage HD11 cells. Subsequently, we show that targeting DOT1L with a specific inhibitor can significantly decrease the ALV-J replication and viral production. By generating of DOT1L-knockout (KO) HD11 cells using the CRISPR/Cas9 system, we show that deletion of the DOT1L led to an increase in the induction of IFNβ and interferon-stimulated genes (ISGs) in HD11 cells in response to ALV-J infection. Importantly, we confirmed that ALV-J infection impaired the activation of the melanoma differentiation-associated protein 5 (MDA5)-mediated-IFN pathway by suppressing the MDA5 expression, and knockout DOT1L rescued the expression of MDA5 and signal transducer and activator of transcription 1 (STAT1), both of which tightly control the antiviral innate immunity. Collectively, it can be deduced from the current data that blocking DOT1L activity or deletion of DOT1L can lead to ALV-J replication inhibition and restoration of the virally suppressed host innate immunity. Thus, we suggest that DOT1L might be a potential drug target for modulating host innate immune responses to combat ALV-J infection.

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.

In this study, we characterised the single exon TLR5 gene of the Chinese rural dog. Sequence analysis revealed a 2577 nucleotide-long open reading frame of canine TLR5, encoding an 858 amino acid-long protein. The putative amino acid sequence of canine TLR5 consisted of a signal peptide sequence, 15 LRR domains, a LRR C-terminal domain, a transmembrane domain and an intracellular Toll-IL-1 receptor domain. The amino acid sequence of the canine TLR5 protein shared 95.4% identity with vulpine, 72.2% with feline and 64.7% with human TLR5. Plasmids expressing canine TLR5 and NF-κB-luciferase were constructed and transfected into HEK293T cells. Expression was confirmed by indirect immunofluorescence assay. These HEK293T cells transfected with the canine TLR5- and NF-κB-luciferase plasmids significantly responded to flagellin from Salmonella enteritidis serovar Typhimurium, indicating that it is a functional TLR5 homolog. In response to stimulation with Salmonella enteritidis, the level of TLR5 mRNA significantly increased over the control in PBMCs at 4 h. The levels of IL-8, IL-6 and IL-1β also increased after exposure. The highest levels of TLR5, IL-8 and IL-1β expression were detected at 8, 4 and 12 h after stimulation, respectively. These results imply that the expression of canine TLR5 may participate in the immune response against bacterial pathogens.

Salmonella enteritidis and Salmonella pullorum belonging to Group O9Salmonella are major causative agents of infectious diseases in chicken. O9 antigen as a part of lipopolysaccharide (LPS) is a predominant detected target for Salmonella infection. To identify the infection, an anti-O9 monoclonal antibody (McAb)-based direct competitive enzyme-linked assay (O9 Dc-ELISA) was developed after constraints were optimized; the establishment and application of O9 Dc-ELISA, compared to two commercial kits and plate agglutination test (PAT), showed that O9 Dc-ELISA could screen out more positive samples than the PAT method could and produce the same agreement rates with commercial kits in terms of sensitivity in addition to strong specificity to clinical serum samples.

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.

Microbial infection can trigger the assembly of inflammasomes and promote secretion of cytokines, such as IL-1β and IL-18. It is well-known that Salmonella modulates the activation of NLRC4 (NLR family CARD domain-containing protein 4) and NLRP3 (NLR family pyrin domain-containing 3) inflammasomes, however the mechanisms whereby Salmonella avoids or delays inflammasome activation remain largely unknown. Therefore, we used Salmonella Enteritidis C50336ΔfliC transposon library to screen for genes involved in modulating inflammasomes activation. The screen revealed the galactose metabolism-related gene galE to be essential for inflammasome activation. Here, we found that inflammasome activation was significantly increased in J774A.1 cells or wild-type bone marrow-derived macrophages (BMDMs) during infection by ΔfliCΔgalE compared to cells infected with ΔfliC. Importantly, we found that secretion of IL-1β was Caspase-1-dependent, consistent with canonical NLRP3 inflammasome activation. Furthermore, the virulence of ΔfliCΔgalE was significantly decreased compared to ΔfliC in a mouse model. Finally, RNA-seq analysis showed that multiple signaling pathways related to the inflammasome were subject to regulation by GalE. Taken together, our results suggest that GalE plays an important role in the regulatory network of Salmonella evasion of inflammasome activation.

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.

Salmonella enteritidis is a major causative agent of foodborne illnesses worldwide. As the traditional serotyping and quantification methods are labor-intensive, time-consuming, and expensive, faster and more convenient molecular diagnostic methods are needed. In this study, we developed and validated a rapid duplex TaqMan real-time polymerase chain reaction (PCR) for the accurate identification and quantification of S. enteritidis. The primers and TaqMan probes were designed based on the S. enteritidis-specific gene lygD and the Salmonella genus-specific gene invA. The melt curve and gel electrophoresis analysis showed that the designed primers had potent specificity for the amplification of lygD and invA. The duplex real-time PCR specifically identified S. enteritidis from a panel of 40 Salmonella strains that represented 29 serovars and 12 non-Salmonella organisms. The duplex real-time PCR assay detected four copies of S. enteritidis DNA per reaction. The intra- and inter- assays indicated a high degree of reproducibility. The real-time PCR could accurately detect and quantify S. enteritidis in chicken organs after Salmonella infection. Furthermore, the assay identified 100% of the S. enteritidis and Salmonella genus isolates from chicken egg samples with superior sensitivity after 6 h of pre-enrichment compared to the traditional culture method. Additionally, the most-probable-number (MPN) combined with qPCR and a shortened incubation time (MPN-qPCR-SIT) method was developed for the population determination of S. enteritidis and compared with various enumeration methods. Thus, we have established and validated a new duplex real-time PCR assay and MPN-qPCR-SIT method for the accurate detection and quantification of S. enteritidis, which could contribute to meeting the need for fast detection and identification in prevention and control measures for food safety.

Abstract Background Enhancing caspase-1 activation in macrophages is helpful for the clearance of intracellular bacteria in mice. Our previous studies have shown that EscI, an inner rod protein of type III system in E. coli can enhance caspase-1 activation. The purpose of this study was to further analyze the prospect of EscI in the vaccine design. Results A recombinant Salmonella expressing SspH2-EscI fusion protein using the promotor of Salmonella effector SspH2, X4550(pYA3334-P-SspH2-EscI), was constructed. A control recombinant Salmonella expressing SspH2 only X4550(pYA3334-P-SspH2) was also constructed. In the early stage of in vitro infection of mouse peritoneal macrophages, X4550(pYA3334-P-SspH2-EscI) could significantly (P

In the past decades, as an emerging omic, metabolomics has been widely used in meat science research, showing promise in meat quality analysis and meat authentication. This review first provides a brief overview of the concept, analytical techniques, and analysis workflow of metabolomics. Additionally, the metabolomics research in quality analysis and authentication of meat is comprehensively described. Finally, the limitations, challenges, and future trends of metabolomics application in meat quality analysis and meat authentication are critically discussed. We hope to provide valuable insights for further research in meat quality.

Abstract Background Sudden increases in the number of human A (H7N9) cases reported during December and January have been observed in previous years. Most reported infection cases are due to prior exposure to live poultry or potentially contaminated environments. Low pathogenicity of influenza A (H7N9) virus in avian species complicates timely discovery of infected birds. Therefore, there is a pressing need to develop safe and effective anti-H7N9 vaccines for poultry to reduce the risk of human infection and prevent the emergence of novel mutated strains. In addition to a good antigen, an effective vaccine also requires an appropriate adjuvant to enhance its immunogenicity. Previously, we generated an H7N9 influenza recombinant subunit vaccine (HA1–2-fliC), in which haemagglutinin globular head domain (HA1–2) was fused with flagellin (fliC), a potent TLR5 ligand, and demonstrated that HA1–2-fliC elicited effective HA1–2-specific immune responses in mice. Results In this study, we determined flagellin-induced expression profiles of cytokines and chemokines in different types of avian immune cells in vitro and ex vivo. We found that flagellin significantly increased the expression levels of CXCL inflammatory chemokines (CXCLi1 and CXCLi2) and CCL chemokines (MIP-1β and MCP-3) in avian macrophage HD11 cells. In addition, HA1–2-fliC induced significant upregulation of cytokines (IL-1β, IL-6, IL-18 and IFN-γ) and chemokines (CXCLi1, CXCLi2 and MIP-1β) in ex vivo splenic lymphocytes and peripheral blood mononuclear cells (PBMCs), suggesting that flagellin promoted immune responses of avian cells in vitro. We also evaluated specific humoural and cellular immune responses induced by HA1–2-fliC and found that chickens immunised intramuscularly with HA1–2-fliC showed significantly higher HA1–2-specific immunoglobulin (Ig)G titers in serum. Furthermore, HA1–2-fliC potentiated cellular immune responses, as reflected by an increase in CD4+ and CD8+ T cells and proliferation of PBMCs. Significantly higher levels of IFN-γ and IL-4 in PBMCs from chickens vaccinated with HA1–2-fliC further indicated that HA1–2-fliC promoted a balanced Th1/Th2 immune response. Conclusions We demonstrated that the use of the flagellin as an adjuvant potentiated immunogenicity of influenza subunit vaccine HA1–2 in vitro and in vivo. These findings provide a basis for the development of H7N9 influenza HA1–2 subunit vaccines for chickens.

Abstract Background Salmonella enterica serovar Enteritidis (S. Enteritidis) is a highly adaptive pathogen in both humans and animals. As a Salmonella Type III secretion system (T3SS) effector, Salmonella protein tyrosine phosphatase (SptP) is critical for virulence in this genus. To investigate the feasibility of using C50336ΔsptP as a live attenuated oral vaccine in mice, we generated the sptP gene deletion mutant C50336ΔsptP in S. Enteritidis strain C50336 by λ-Red mediated recombination and evaluated the protective ability of the S. Enteritidis sptP mutant strain C50336ΔsptP against mice salmonellosis. Results We found that C50336ΔsptP was a highly immunogenic, effective, and safe vaccine in mice. Compared to wild-type C50336, C50336ΔsptP showed reduced virulence as confirmed by the 50% lethal dose (LD50) in orally infected mice. C50336ΔsptP also showed decreased bacterial colonization both in vivo and in vitro. Immunization with C50336ΔsptP had no significant effect on body weight and did not result in obvious clinical symptoms relative to control animals treated with phosphate-buffered saline (PBS), but induced humoral and cellular immune responses at 12 and 26 days post inoculation. Immunization with 1 × 108 colony-forming units (CFU) C50336ΔsptP per mouse provided 100% protection against subsequent challenge with the wild-type C50336 strain, and immunized mice showed mild and temporary clinical symptoms as compared to those of control group. Conclusions These results demonstrate that C50336ΔsptP can be a live attenuated oral vaccine for salmonellosis.

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