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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-1ß 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.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Salmonella enteritidis , Sistemas de Secreção Tipo I , Transdução de Sinais , Caspase 1/metabolismo , Interleucina-1beta/metabolismoRESUMO
This study aimed to understand the epidemiological characteristics of Salmonella in Tibetan pigs. We isolated, identified, and examined via antimicrobial susceptibility testing on Salmonella from Tibetan pigs breeder farms and slaughterhouses in Tibet, China. A genetic evolutionary tree was constructed on the basis of whole genome sequencing (WGS). A total of 81 Salmonella isolates were isolated from 987 samples. The main serovars were Salmonella Typhimurium and Salmonella London in Tibetan pigs. The isolated Salmonella Typhimurium isolates subjected to antimicrobial susceptibility testing showed varying degrees of resistance to ß-lactams, aminoglycosides, fluoroquinolones, sulfonamides, tetracyclines, and amphenicols. WGS analysis was performed on 20 Salmonella Typhimurium isolates in Tibet (n = 10), Jiangsu (n = 10), and 205 genome sequences downloaded from the Enterobase database to reveal their epidemiological and genetic characteristics. They were divided into two clusters based on core genome single-nucleotide polymorphisms: Cluster A with 112 isolates from Tibet and other regions in China and Cluster B with 113 isolates from Jiangsu and other regions. The isolates in Cluster A were further divided into two subclusters: A-1 with 40 isolates including Tibet and A-2 with 72 isolates from other regions. Virulence factors analysis revealed that all isolates from Tibet carried adeG, but this observation was not as common in Salmonella isolates from Jiangsu and other regions of China. Antibiotic resistance genes (ARGs) analysis showed that all isolates from Tibet carried blaTEM-55 and rmtB, which were absent in Salmonella isolates from Jiangsu and other regions of China. Genetic characteristic analysis and biofilm determination indicated that the biofilm formation capabilities of the isolates from Tibet were stronger than those of the isolates from Jiangsu and other regions of China. Our research revealed the epidemic patterns and genomic characteristics of Salmonella in Tibetan pigs and provided theoretical guidance for the prevention and control of local salmonellosis.
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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-1ß, 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)-ß. 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-ß and IFN-related genes (IRFs and ISGs) in bone marrow-derived dendritic cells and macrophages. These results suggested that Salmonella flagellin can activate IFN-ß-related immune responses in macrophages, which provides new insight into the immune mechanisms of flagellin adjuvant.
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Salmonella spp. is a major foodborne pathogen that is distributed among most pork production chains worldwide. This study aimed to investigate the dynamic changes in Salmonella spp. along the pig breeding process monthly from April 2018 to March 2019 in a pig farm in Shanghai, China, and identify the potential critical control points during the production. In total, 239 Salmonella spp. isolates were obtained from 1389 samples, in which Salmonella were detected from 26.3% (222/843) of fecal samples, 7.1% (17/240) of feed samples, and 0.0% (0/306) of both water and insect samples. Seven different serotypes were identified, with the predominant serotype being Salmonella Derby (21.8%), followed by Salmonella Typhimurium (18.8%), Salmonella Rissen (16.3%), Salmonella Mbandaka (12.6%), and Salmonella 1,4,[5],12:i:- (11.8%). Most probable number (MPN) analysis revealed that the load of Salmonella spp. gradually increased along the pig production chain, while the highest number of Salmonella spp. isolates was at the fattening stage (MPN value, 11-15 MPN/g). The pulsed-field gel electrophoresis showed that both Salmonella Typhimurium and Salmonella Derby isolates were grouped to six clusters. The antimicrobial resistance analyzed demonstrated that 80.0% of the isolates were of multidrug resistance and resistant to sulfamethoxazole (84.5%), lincomycin (89.4%), ampicillin (96.9%), oxytetracycline (93.8%), and tetracycline (95.1%). We further evaluated the Salmonella spp. Resistance to quaternary ammonium compounds (QACs) showed an increasing trend along with the testing period indicating that the use of QACs could induce the resistance of Salmonella spp. to QACs. Our study confirmed the dynamic changes in Salmonella spp. over time and space in this pig farm and identified feed and the fattening house as the key points for the prevention and control of Salmonella spp. contamination.
Assuntos
Resistência Microbiana a Medicamentos/genética , Salmonella/classificação , Salmonella/efeitos dos fármacos , Salmonella/genética , Salmonella/isolamento & purificação , Matadouros , Animais , Antibacterianos/farmacologia , China , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Eletroforese em Gel de Campo Pulsado , Microbiologia de Alimentos , Carne/microbiologia , Testes de Sensibilidade Microbiana , Prevalência , Salmonelose Animal/microbiologia , Sorogrupo , Suínos , Doenças dos Suínos/microbiologiaRESUMO
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-1ß 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.
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Proteínas de Bactérias/metabolismo , Inflamassomos/metabolismo , Salmonella enteritidis/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Animais , Proteínas de Bactérias/genética , Caspase 1/metabolismo , Expressão Gênica , Interleucina-1beta/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/metabolismo , Camundongos , Mutação , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Infecções por Salmonella/virologia , Salmonella enteritidis/enzimologia , Transdução de Sinais , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , TranscriptomaRESUMO
Salmonella Enteritidis (SE) is a highly adapted pathogen causing severe economic losses in the poultry industry worldwide. Chickens infected by SE are a major source of human food poisoning. Vaccination is an effective approach to control SE infections. This study evaluated the immunogenicity and protective efficacy of a SE sptP deletion mutant (C50336ΔsptP) as a live attenuated vaccine (LAV) candidate in chickens. 14 day-old specific pathogen-free (SPF) chickens were intramuscularly immunized with various doses of C50336ΔsptP. Several groups of chickens were challenged with the virulent wild-type SE strain Z-11 via the same route at 14 days post vaccination. Compared to the control group, the groups vaccinated with 1 × 106, 1 × 107 and 1 × 108 colony-forming units (CFU) of C50336ΔsptP exhibited no clinical symptoms after immunization. Only slight pathological changes occurred in the organs of the 1 × 109 CFU vaccinated group. C50336ΔsptP bacteria were cleared from the organs of immunized chickens within 14 days after vaccination. Lymphocyte proliferation and serum cytokine analyses indicated that significant cellular immune responses were induced after the vaccination of C50336ΔsptP. Compared to the control group, specific IgG antibody levels increased significantly in vaccinated chickens, and the levels increased markedly after the challenge. The 1 × 107, 1 × 108, and 1 × 109 CFU vaccinated chickens groups showed no clinical symptoms or pathological changes, and no death after the lethal challenge. Whereas severe clinical signs of disease and pathological changes were observed in the control group chickens after the challenge. These results suggest that a single dose of C50336ΔsptP could be an effective LAV candidate to against SE infection in chickens.
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Proteínas de Bactérias/genética , Imunogenicidade da Vacina , Doenças das Aves Domésticas/prevenção & controle , Salmonelose Animal/prevenção & controle , Vacinas contra Salmonella/imunologia , Deleção de Sequência , Animais , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/imunologia , Galinhas , Citocinas/sangue , Imunoglobulina G/sangue , Doenças das Aves Domésticas/microbiologia , Salmonelose Animal/imunologia , Salmonella enteritidis/genética , Salmonella enteritidis/imunologia , Organismos Livres de Patógenos Específicos , Vacinação/veterinária , Vacinas Atenuadas/imunologiaRESUMO
BACKGROUND: Salmonella enterica serovar Enteritidis (S. Enteritidis) has emerged as one of the most important food-borne pathogens for humans. Lipopolysaccharide (LPS), as a component of the outer membrane, is responsible for the virulence and smooth-to-rough transition in S. Enteritidis. In this study, we screened S. Enteritidis signature-tagged transposon mutant library using monoclonal antibody against somatic O9 antigen (O9 MAb) and O9 factor rabbit antiserum to identify novel genes that are involved in smooth-to-rough transition. RESULTS: A total of 480 mutants were screened and one mutant with transposon insertion in rfbG gene had smooth-to-rough transition phenotype. In order to verify the role of rfbG gene, an rfbG insertion or deletion mutant was constructed using λ-Red recombination system. Phenotypic and biological analysis revealed that rfbG insertion or deletion mutants were similar to the wild-type strain in growth rate and biochemical properties, but the swimming motility was reduced. SE Slide Agglutination test and ELISA test showed that rfbG mutants do not stimulate animals to produce agglutinating antibody. In addition, the half-lethal dose (LD50) of the rfbG deletion mutant strain was 106.6 -fold higher than that of the parent strain in a mouse model when injected intraperitoneally. CONCLUSIONS: These data indicate that the rfbG gene is involved in smooth-to-rough transition, swimming motility and virulence of S. Enteritidis. Furthermore, somatic O-antigen antibody-based approach to screen signature-tagged transposon mutants is feasible to clarify LPS biosynthesis and to find suitable markers in DIVA-vaccine research.
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Proteínas de Bactérias/genética , Mutagênese , Salmonella enteritidis/genética , Salmonella enteritidis/isolamento & purificação , Virulência/genética , Testes de Aglutinação/métodos , Animais , Anticorpos Monoclonais , Elementos de DNA Transponíveis/genética , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática/métodos , Genes Bacterianos , Dose Letal Mediana , Lipopolissacarídeos/biossíntese , Mutagênese Insercional , Antígenos O/genética , Antígenos O/imunologia , Fenótipo , Coelhos , Salmonelose Animal/prevenção & controle , Salmonella enteritidis/crescimento & desenvolvimento , Salmonella enteritidis/patogenicidadeRESUMO
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-1ß and MCP-3) in avian macrophage HD11 cells. In addition, HA1-2-fliC induced significant upregulation of cytokines (IL-1ß, IL-6, IL-18 and IFN-γ) and chemokines (CXCLi1, CXCLi2 and MIP-1ß) 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.
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Galinhas/imunologia , Flagelina/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/veterinária , Adjuvantes Imunológicos , Animais , Linhagem Celular , Flagelina/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Imunidade Celular , Imunidade Humoral , Leucócitos/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/imunologiaRESUMO
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.
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Proteínas de Bactérias/imunologia , Proteínas Tirosina Fosfatases/imunologia , Salmonelose Animal/prevenção & controle , Vacinas contra Salmonella/imunologia , Salmonella enteritidis/imunologia , Administração Oral , Animais , Feminino , Deleção de Genes , Imunização/veterinária , Camundongos Endogâmicos BALB C , Salmonelose Animal/imunologia , Vacinas contra Salmonella/genética , Salmonella enteritidis/genética , Salmonella enteritidis/patogenicidade , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , VirulênciaRESUMO
Newcastle disease (ND), a highly contagious, acute, and potent infectious disease caused by Newcastle disease virus (NDV), has a considerable impact on the global poultry industry. Although both live attenuated and inactivated vaccines are used to prevent and control the spread of ND among chickens, the increasing number of ND outbreaks in commercial poultry flocks worldwide indicates that routine vaccinations are insufficient to control ND. Hence, efforts are being invested into developing alternative and more effective vaccination strategies. In this study, we focus on F protein, the neutralizing and protective antigen of NDV, and flagellin (FliC), a toll-like receptor 5 (TLR5) agonist that is an effective inducer of innate immune responses. We amplified F gene from velogenic NDV strain F48E8. The recombinant histidine (His)-tagged F protein was efficiently expressed in a Pichia pastoris (P. pastoris) eukaryotic system and verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The conditions for F protein expression in P. pastoris were optimal. The immunogenicity of F protein with FliC as the adjuvant was evaluated in a C3H/HeJ mouse model. FliC was found to enhance both F-specific and NDV-specific IgG responses and F-specific cellular immune responses following intraperitoneal co-administration with F protein. Thus, the recombinant F protein expressed by P. pastoris when used with flagellin as the adjuvant has potential as a subunit vaccine candidate.
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Adjuvantes Imunológicos/farmacologia , Proteínas de Escherichia coli , Flagelina , Expressão Gênica , Vírus da Doença de Newcastle/genética , Pichia/metabolismo , Proteínas Virais de Fusão , Vacinas Virais , Animais , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/farmacologia , Flagelina/imunologia , Flagelina/farmacologia , Camundongos , Vírus da Doença de Newcastle/imunologia , Pichia/genética , Pichia/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacocinética , Proteínas Virais de Fusão/biossíntese , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/farmacocinética , Vacinas Virais/biossíntese , Vacinas Virais/genética , Vacinas Virais/imunologia , Vacinas Virais/farmacologiaRESUMO
BACKGROUND: A novel influenza virus, subtype H7N9, circulated through China in 2013-2014. Its higher rates of human infection in a wide range of locations within China and the associated increased likelihood of human-to-human transmission have caused global concern. Recombinant subunit vaccines provide safe and targeted protection against viral infections. However, the protective efficacy of recombinant subunit vaccines tends to be less potent than vaccines made from whole viruses. Studies have shown that bacterial flagellin has strong adjuvant activity and induces protective immune responses. RESULTS: In this study, we used overlap-PCR to generate an H7N9 influenza recombinant subunit vaccine that fused the globular head domain (HA1-2, aa 62-284) of the protective hemagglutinin (HA) antigen with the potent TLR5 ligand, Salmonella typhimurium flagellin (fliC). The resulting fusion protein, HA1-2-fliC, was efficiently expressed in an Escherichia coli prokaryotic expression system, and Western blotting and TLR5-stimulating activity analysis confirmed that the HA1-2-fliC moiety could be faithfully refolded to take on the native HA and fliC conformations. In a C3H/HeJ mouse model of intraperitoneal vaccination, the fusion protein elicited significant and robust HA1-2-specific serum IgG titers, maintaining high levels for at least 3 months in the vaccinated animals, and induced similar levels of HA1-2-specific IgG1 and IgG2a that were detectable 12 days after the third immunization. HA1-2-fliC was also found to be capable of triggering the production of neutralizing antibodies, as assessed by measuring hemagglutination inhibition titers. CONCLUSIONS: We conclude that immunization with HA1-2-fliC induces potent HA1-2-specific responses, offering significant promise for the development of a successful recombinant subunit vaccine for avian influenza A (H7N9).
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Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/genética , Influenza Aviária/prevenção & controle , Influenza Humana/prevenção & controle , Vacinas de Subunidades Antigênicas/genética , Vacinas Sintéticas/genética , Animais , Aves , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Flagelina/metabolismo , Células HEK293 , Hemaglutininas/metabolismo , Humanos , Camundongos , Salmonella typhimurium/metabolismoRESUMO
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.
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Galinhas , Farmacorresistência Bacteriana Múltipla , Doenças das Aves Domésticas , Salmonelose Animal , Salmonella enterica , Sequenciamento Completo do Genoma , Animais , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/epidemiologia , Salmonelose Animal/epidemiologia , Salmonelose Animal/microbiologia , China/epidemiologia , Prevalência , Salmonella enterica/genética , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/isolamento & purificação , Sequenciamento Completo do Genoma/veterinária , Feminino , Sorogrupo , Antibacterianos/farmacologiaRESUMO
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.
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African swine fever virus (ASFV) is a large double stranded DNA arbovirus that is highly contagious and seriously endangers domestic and wild pigs. In the past decade, African swine fever (ASF) has spread in many countries in the Caucasus, Russian Federation, Eastern Europe and Asia, causing significant losses to the pig industry. At present, there is a lack of effective vaccine and treatment for ASF. Therefore, the rapid and accurate detection is crucial for ASF prevention and control. In this study, we have developed a portable lateral flow strip (LFS) detection mediated by recombinase polymerase amplification (RPA) and CRISPR/LwCas13a, which is performed at 37 â and visualized by eyes without the need for complex instruments. This RPA-LwCas13a-LFS is based on the ASFV structural protein p17 gene (D117L), with a detection sensitivity up to 2 gene copies. This method is highly specific and has no cross reactivity to 7 other pig viruses. In the detection of two batches of 100 clinical samples, the p17 (D117L) RPA-LwCas13a-LFS had 100% coincidence with conventional quantitative PCR (qPCR). These findings demonstrate the potential of this simple, rapid, sensitive, and specific ASFV detection method for on-site ASFV detection.
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Vírus da Febre Suína Africana , Febre Suína Africana , Sistemas CRISPR-Cas , Animais , Febre Suína Africana/virologia , Febre Suína Africana/diagnóstico , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/veterinária , Sensibilidade e Especificidade , Suínos , Proteínas Estruturais Virais/análise , Proteínas Estruturais Virais/genéticaRESUMO
Autophagy is a crucial innate immune response that clears pathogens intracellularly. Salmonella enterica serovar Enteritidis (S.E) has emerged as one of the most important food-borne pathogens. Here, we reported that dTDP-4-dehydro-ß-Ö-rhamnose reductase (RfbD) was able to enhance bacterial colonization in vivo and in vitro by regulating autophagy. We screened the transposon mutant library of Salmonella Enteritidis strain Z11 by High-Content Analysis System, found that rfbD gene has an effect on autophagy. The Z11ΔrfbD-infected group showed greater expression of LC3-II than the Z11-infected group in HeLa, RAW264.7, and J774A.1 cells. Overall, the survival of Z11ΔrfbD in RAW264.7 cells was reduced after 8 h of infection compared to that of the Z11 wild-type strain. In addition, we observed that inhibition of autophagic flux significantly increased the survival of Z11ΔrfbD in RAW264.7 cells. Mice infection experiments revealed that Z11ΔrfbD virulence was significantly reduced, and bacterial load was reduced in the liver and cecum in mice model, and LC3-II expression was significantly increased. These findings indicate an important role of Salmonella Enteritidis protein as a strategy to suppress autophagy and provides new ideas for manipulating autophagy as a novel strategy to treat infectious diseases.
Assuntos
Salmonelose Animal , Salmonella enteritidis , Animais , Humanos , Camundongos , Autofagia/genética , Células HeLa/microbiologia , Imunidade Inata , Células RAW 264.7/microbiologia , Salmonella enteritidis/genética , Salmonelose Animal/microbiologia , Virulência/genéticaRESUMO
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.
RESUMO
Introduction: Apicomplexan 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). Methods: The 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. Results: The 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 ß-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<0.01). EnApiAP2 protein was localized in the nuclei of SZ, MZ-2 and MZ-3 of E. necatrix. The protein of EnApiAP2 was localized in the nucleus of the DF-1 cells, whereas the ΔNLS-EnApiAP2 was expressed in the cytoplasm, which further confirmed that EnApiAP2 is nucleoprotein. Discussion: EnApiAP2 protein encoded by ENH_00027130 sequence was localized in the nucleus of E. necatrix parasites, and relied on the NLS for migration to DF-1 cell nucleus. The function of EnApiAP2 need further study.
Assuntos
Eimeria , Doenças das Aves Domésticas , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Galinhas/genética , DNA Complementar/genética , Eimeria/genética , Eimeria/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Doenças das Aves Domésticas/parasitologia , Esporozoítos/metabolismoRESUMO
Eimeria necatrix is a high pathogenic pathogen, which seriously endangers the poultry industry. The surface antigens (SAGs) of Apicomplexa are a kind of membrane protein anchored on the surface of the parasites through its carboxyl terminal glycosylphosphatidylinositol (GPI) structure. However, little is known about GPI-linked surface proteins in E. necatrix. In the present work, the E. necatrix sag gene (Ensag-CAP) was amplified and cloned for expression of the recombinant protein (rEnSAG-CAP). The full length Ensag-CAP gene was 813 bp, coding 270 amino acids with a predicated molecular weight of 28.86 kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG-CAP was about 32 kDa and mainly expressed in a soluble form. Western blot analysis indicated that the rEnSAG-CAP could be recognized by anti-rEnSAG-CAP monoclonal antibody (anti-rEnSAG-CAP McAb) and the convalescent serum of chicken infected with E. necatrix. Native protein of EnSAG-CAP was detected in second-generation merozoites (MZ-2) using anti-rEnSAG-CAP polyclonal antibody (anti-rEnSAG-CAP pAb). The findings from the indirect immunofluorescence assay and enzyme digestion utilizing Bacillus cereus phosphoinositide-specific phospholipase C (PI-PLC) revealed that EnSAG-CAP predominantly localized at the surfaces of SZ and MZ-2 via a GPI anchor. It was observed that EnSAG-CAP can be cleaved from MZ-2 by PI-PLC. Real-time quantitative PCR (qPCR) analysis showed that transcript levels of Ensag-CAP in MZ-2 was significantly higher than that in SZ (P < 0.05). The anti-rEnSAG-CAP McAb in vitro could significantly inhibit the sporozoite invasion into MDBK cells (P < 0.01), which suggests that the protein might participate in sporozoite invasion into MDBK cells. rEnSAG-CAP afforded an immune protection against E. necatrix. The ACI value was 164.99 in the chickens immunized with 200 µg rEnSAG-CAP. Chickens immunized with rEnSAG-CAP had a significantly higher antigen-specific serum IgY response (P < 0.0001). The data indicates that EnSAG-CAP could serve as a potential candidate antigen for the development of a recombinant coccidiosis vaccine.
Assuntos
Coccidiose , Eimeria , Doenças das Aves Domésticas , Animais , Eimeria/fisiologia , Galinhas/parasitologia , Coccidiose/prevenção & controle , Coccidiose/veterinária , Proteínas Recombinantes/genética , Esporozoítos , Vacinas Sintéticas , Doenças das Aves Domésticas/parasitologiaRESUMO
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.
Assuntos
Doenças das Aves Domésticas , Salmonelose Animal , Vacinas contra Salmonella , Salmonella enterica , Animais , Galinhas , Doenças das Aves Domésticas/microbiologia , Salmonella/genética , Salmonelose Animal/microbiologia , Salmonella enterica/genética , Vacinas AtenuadasRESUMO
Salmonella enterica serovar Enteritidis (S. Enteritidis), one of the zoonotic pathogens, not only results in significant financial losses for the global poultry industry but also has the potential to spread to humans through poultry and poultry products. Vaccination is an effective method to prevent Salmonella infections. In this study, we constructed a live attenuated DIVA (differentiation of infected and vaccinated animals) vaccine candidate, Z11ΔrfbG, and evaluated its protective effectiveness and DIVA potential in chickens. Compared to that of the virulent wild-type strain, the 50% lethal dose (LD50) of the rfbG mutant strain increased 56-fold, confirming its attenuation. High serum levels of S. Enteritidis-specific IgG titers indicated that a significant humoral immune response was induced in the vaccinated group. After challenge, the nonvaccinated group showed serious clinical symptoms (diarrhea, depression, decreased appetite, ruffled feathers, and weight loss), pathological changes (white nodules in the liver and fatty lesions in liver cells), and death. In contrast, there were no clinical symptoms, pathological changes, or death in the 5 × 106- and 5 × 107-CFU-vaccinated groups. Z11ΔrfbG vaccination significantly reduced S. Enteritidis colonization in the spleen, liver, and cecum. In addition, the Z11ΔrfbG-vaccinated group exhibited a negative response to the serological test, whereas the virulent wild-type Z11 infection group was strongly positive for the serological test, showing a DIVA capability of Z11ΔrfbG vaccination. Overall, our findings demonstrate the viability of the rfbG mutant as a live attenuated chicken vaccine that can discriminate between animals that have been immunized and those that have been infected. IMPORTANCE S. Enteritidis is a highly adapted pathogen that causes significant economic losses in the poultry industry around the world. Vaccination is an effective method of controlling S. Enteritidis infections. Here, we demonstrated that S. Enteritidis Z11ΔrfbG has the potential to be a safe, immunogenic, and DIVA vaccine candidate for the control of Salmonella infections in chickens. Z11ΔrfbG not only provided effective protection in chickens but also distinguished between infected and vaccinated chickens by serological tests.