Porcine proliferative enteropathy(PPE),an important infectious disease in pig production caused by an obligate intracellular bacterium Lawsonia intracellularis,is commonly associated with diarrhea and reduced weight g...Porcine proliferative enteropathy(PPE),an important infectious disease in pig production caused by an obligate intracellular bacterium Lawsonia intracellularis,is commonly associated with diarrhea and reduced weight gain in growing pigs widespread.An accurate method for detecting L.intracellularis is particularly important for preventing and controlling PPE.Heat shock protein 60(Hsp60)is an immunodominant bacterial antigen found in all eukaryotic and prokaryotic organisms.Thus,the purpose of the current investigation was to produce a novel L.intracellularis Hsp60 monoclonal antibody(mAb)useful for immunodiagnostics.Three hybridomas secreted anti-Hsp60 termed 3E5,4E2,and 9G6 were generated,and the titers of ascitic fluids of 3E5,4E2,9G6 were 1:1024000,1:2048000 and 1:2048000,respectively.The Western blotting analysis demonstrated that recombinant Hsp60(rHsp60)was recognized by mAbs 3E5,4E2 and 9G6.Subsequently,analyses of specificity showed all the mAbs were highly specific to L.intracellularis while could not significantly react with other enteric bacteria commonly found in the ileum of pigs,such as Escherichia coli,Salmonella Choleraesuis,Salmonella Typhimurium,and Brachyspira hyodysenteriae.Furthermore,the mAbs were useful for detecting L.intracellularis in the infected monolayer cells and histological sections of the ileum from PPE-affected pigs.Our research will provide a foundation for the development of immunological diagnostic tests.展开更多
Streptococcus agalactiae is one of the most common pathogens that cause bovine mastitis worldwide. Identifying pathogen prevalence and virulence factors is critical for developing prevention and control approaches. He...Streptococcus agalactiae is one of the most common pathogens that cause bovine mastitis worldwide. Identifying pathogen prevalence and virulence factors is critical for developing prevention and control approaches. Herein, 1 161 milk samples from various dairy farms in China(n=558) and Pakistan(n=603) were collected between 2019–2021 and were subjected to S. agalactiae isolation. Prevalence, serotyping, virulence genes, and antibiotic-resistant genes of S. agalactiae were evaluated by PCR assay. All isolates were characterized for haemolysis, biofilm production, cytotoxicity, adhesion, and invasion on bovine mammary epithelial cells. The prevalence of S. agalactiae-induced mastitis in cattle was found to be considerably higher in Pakistan than in China. Jiangsu and Sindh provinces had the highest area-wise prevalence in China and Pakistan, respectively. Serotypes Ia and II were prevalent in both countries, whereas serotype III was found only in Pakistan. Moreover, all isolates tested positive for PI-2b gene but negative for PI-1 and PI-2a genes. All isolates harboured cfb, cylE, hylB, and fbsB virulent genes, whereas many of them lacked bibA, rib and bca. However, the absence of bac and scp genes in Chinese isolates and cspA in Pakistani isolates was noted, while spb1 and lmb were not detected in isolates of both countries. Pakistani isolates, particularly serotype Iapositive, had a considerably higher ability to produce biofilm, haemolysis, cytotoxicity, adhesion, and invasion than Chinese isolates. Most of the isolates were phenotypically resistant to tetracycline, erythromycin, and clindamycin and genotypic resistance was confirmed by the presence of ermA, ermB, tetM and tetO genes. Our study highlights the antimicrobial resistance profile and virulence-related factors contributing to the epidemiological spread of mastitis-causing S. agalactiae in China and Pakistan. The findings may facilitate future studies designed to develop improved treatment and control strategies against this pathogen.展开更多
Streptococcus equi subsp.zooepidemicus(SEZ)is an important zoonotic agent.Here,a virulence-attenuated strain M35246 derived from natural variation of wild-type SEZ ATCC35246 was found.M35246 showed a deletion of 25con...Streptococcus equi subsp.zooepidemicus(SEZ)is an important zoonotic agent.Here,a virulence-attenuated strain M35246 derived from natural variation of wild-type SEZ ATCC35246 was found.M35246 showed a deletion of 25contiguous genes as well as a loss-of-function mutation in covS.Subsequently,a 25-gene-deleted strain(ΔPI),a covS-mutant strain(Mcov S),and relevant complementary strains were constructed and investigated.M35246 and Mcov S were significantly less encapsulated and exhibited poorer anti-phagocytic capacity compared to wild-type SEZ.McovS was significantly more sensitive toβ-lactams,aminoglycosides,macrolides,and lincosamides than wild-type SEZ.M35246,McovS,andΔPI exhibited an increase in median lethal dose(LD_(50))in mice by 10~5,10~5,and 5 times when compared to wild-type SEZ,respectively.Neither M35246 nor McovS were isolated from mice 48 h after being challenged with approximately 2000 times the LD_(50)of wild-type SEZ.Transcriptome analysis showed that 668 significantly differentially expressed genes existed between McovS and wild-type SEZ.Numerous virulence factor-encoding genes and anabolicrelated genes in McovS that were involved in anti-phagocytosis,capsule formation,pathogenicity,and antibiotic resistance were downregulated significantly relative to the wild-type strain.This study revealed that the CovS plays a vital role in the establishment of SEZ virulence.展开更多
Streptococcus equi ssp. zooepidemicus(SEZ) is a pathogen associated with a wild range of animal species. Frequent outbreaks have occurred in recent years in pigs, horses, goats and dogs which is liable to infect human...Streptococcus equi ssp. zooepidemicus(SEZ) is a pathogen associated with a wild range of animal species. Frequent outbreaks have occurred in recent years in pigs, horses, goats and dogs which is liable to infect humans. There is a lack of efficient vaccines against this disease and the occurrence of antibiotic resistance may render drug therapies ineffective. In this study, gene deletion mutant(ΔSEZ) in pathogenicity islands SeseCisland_4 was constructed. The mutant ΔSEZ had a 52-fold decrease in 50% lethal dose(LD_(50)) and had less capacity to adhere epithelial cells. Importantly, immunization of mice with attenuated vaccine ΔSEZ at the dose of 10~2 colony-forming units(CFU) mL^(–1) elicited a significant humoral antibody response, with an antibody titer of 1:12 800. Therefore, 10~2 CFU mL^(–1) might be used as the appropriate immune dose for the attenuated vaccine ΔSEZ, which provided mice with efficient protection against virulent SEZ. In addition, the hyperimmune sera against 10~2 CFU m L–1 attenuated vaccine ΔSEZ could confer significant protection against virulent SEZ infection in the passive immunization experiment and exhibited efficient bactericidal activity in the whole blood assay. Meanwhile, no viable bacteria was detected in blood when mice were immunized with ΔSEZ at the dose of 10~2 CFU mL^(–1) via hypodermic injection. Thereafter, the mutant ΔSEZ at the dose of 10~2 CFU mL^(–1) could confer significant protection in mice and had less negative effects on host, which could be an effective attenuated vaccine candidate for the prevention of SEZ.展开更多
The G-H loop of the foot-and-mouth disease virus（FMDV） virion contains certain dominant immunogenic epitopes, as well as an arginine-glycine-aspartic acid（RGD） motif that is recognized by cell surface integrin rec...The G-H loop of the foot-and-mouth disease virus（FMDV） virion contains certain dominant immunogenic epitopes, as well as an arginine-glycine-aspartic acid（RGD） motif that is recognized by cell surface integrin receptors. Previous experiments indicate that it is critical to maintain virus structural integrity when inserting an exogenous epitope into the surface of an FMDV structural protein. However, it remains to be determined how factors such as different insertion positions affect interactions among the virus, cells and host immune system. In this study, one infectious c DNA clone of the swine FMDV Cathay topotype strain O/CHA/90 was constructed. Then, a FLAG marker（DYKDDDDK） was inserted upstream（–4） or downstream（＋10） of the RGD motif to generate tagged viruses vFLAG-O/CHA/90 or vO/CHA/90-FLAG, investigating the possibility of expressing foreign antigen and effect on its immunogenicity. Compared to the parental virus, both tagged viruses exhibited similar plaque phenotypes, suckling mouse pathogenicity and antigenicity. Additionally, the FLAGtag insertion position did not change the use of integrin-mediated cell entry by the tagged viruses. Interestingly, both tagged vaccines protected pigs against challenge with the parental virus O/CHA/90 and induced immune responses against FMDV in BALB/c mice and pigs, but only vaccination with vFLAG-O/CHA/90 generated anti-FLAG antibodies. Our findings demonstrated that two sites（RGD–4 and RGD＋10） tolerated the insertion of an exogenous gene in the swine FMDV O/CHA/90 strain. However, only RGD–4 was a novel and appropriate inserting site which could tolerate exogenous FLAG. The resultant tagged virus is a promising candidate for FMD vaccine which can be differentiating infected from vaccinated animals（DIVA）.展开更多
Streptococcus equi ssp. zooepidemicus （S. zooepidemicus） is a zoonotic pathogen with worldwide distribution. Lacking suitable vaccine and virulent maker is still bottleneck to control this infection. An immunoproteo...Streptococcus equi ssp. zooepidemicus （S. zooepidemicus） is a zoonotic pathogen with worldwide distribution. Lacking suitable vaccine and virulent maker is still bottleneck to control this infection. An immunoproteomic approach has been used to screen the membrane-associated and cell wall-associated proteins of S. zooepidemicus isolate in China CY to discover vaccine candidate antigens and therapeutic agents. Finally, 11 membrane-associated proteins, and 13 cell wall-associated proteins were successfully identified. BLAST （www.sanger.ac.uk） results also indicated that nucleotide sequences of majority identified proteins shared high homology （60%） with S. zooepidemicus, except for AC1-3, AC5, AC8, and AC13. Moreover, genes for 7 of the identified proteins were detected from CY; compared with ST171, 3 of them （AM1, AM8 and AC11） were only found in virulent strains （CY）. All of the proteins identified in this study remain not to be reported in S. zooepidemicus. Some of the proteins serve a vital role in the immune system and reproduction of host species according to available data, while the functions of the rest were seldom researched.展开更多
Streptococcus suis is one of the major pathogens of swine streptococcosis. Among them, the strongest virulence and highest rate of clinical isolation serotype is S. suis serotype 2（SS2）. Moreover, SS2 is also an imp...Streptococcus suis is one of the major pathogens of swine streptococcosis. Among them, the strongest virulence and highest rate of clinical isolation serotype is S. suis serotype 2（SS2）. Moreover, SS2 is also an important zoonosis pathogen, which caused severe public health issues in China. It has been reported that SS2 has several virulence factors, including muramidase released protein, extracellular factors, capsule, fibronectin-binding protein, enolase, hemolysin, small RNA, biofilm, two-component regulatory systems, STK/STP, etc., whose functions involved in adhesion, anti-phagocytosis, inflammatory pathway activation, invasion, etc. Actually, SS2 has developed a variety of ways to escape from host immune system during evolution. In particularly, capsule could resist phagocytosis through inhibiting sphingosine dependent immune cell recognition, which plays an important role in escaping host inflammation response; moreover, superoxide dismutase encoding by sod A enables SS2 escaping reactive oxygen species（ROS） in host immune cells; besides, binding complement factor h with Fhb could suppress the activation of complement alternative pathway and bactericidal effect. And SS2 could also hinder the formation of neutrophil extracellular traps（NETs） to avoid trapping by swine neutrophils, while host immune globulin could be degraded by Ig A1 hydrolase and Ig M protease. In addition, SS2 could escape host immune defense with the help of multiple transcriptional factors and micro-RNA. So far, the pathogenesis of meningitis, arthritis caused by SS2 infection, is still unclear, and the virulence regulatory mechanism of phosphorylation, micro-RNA need to be further clarified. Importantly, the study of interaction mechanism of pathogen and host contribute to further demonstration the pathogenesis of SS2.展开更多
基金supported by the National Key Research and Development Program of China(2021YFD1800400)the National Natural Science Foundation of China(31872480)+1 种基金the Jiangsu Agriculture Science and Technology Innovation Fund,China(CX(19)2020)the Priority Academic Program Development of Jiangsu Higher Education Institutions,China(PAPD).
文摘Porcine proliferative enteropathy(PPE),an important infectious disease in pig production caused by an obligate intracellular bacterium Lawsonia intracellularis,is commonly associated with diarrhea and reduced weight gain in growing pigs widespread.An accurate method for detecting L.intracellularis is particularly important for preventing and controlling PPE.Heat shock protein 60(Hsp60)is an immunodominant bacterial antigen found in all eukaryotic and prokaryotic organisms.Thus,the purpose of the current investigation was to produce a novel L.intracellularis Hsp60 monoclonal antibody(mAb)useful for immunodiagnostics.Three hybridomas secreted anti-Hsp60 termed 3E5,4E2,and 9G6 were generated,and the titers of ascitic fluids of 3E5,4E2,9G6 were 1:1024000,1:2048000 and 1:2048000,respectively.The Western blotting analysis demonstrated that recombinant Hsp60(rHsp60)was recognized by mAbs 3E5,4E2 and 9G6.Subsequently,analyses of specificity showed all the mAbs were highly specific to L.intracellularis while could not significantly react with other enteric bacteria commonly found in the ileum of pigs,such as Escherichia coli,Salmonella Choleraesuis,Salmonella Typhimurium,and Brachyspira hyodysenteriae.Furthermore,the mAbs were useful for detecting L.intracellularis in the infected monolayer cells and histological sections of the ileum from PPE-affected pigs.Our research will provide a foundation for the development of immunological diagnostic tests.
基金supported by the National Key Research and Development Program of China (2021YFD1800400)the National Natural Science Foundation of China (31872480)+1 种基金the Jiangsu Agriculture Science and Technology Innovation Fund of China (CX(19)2020)the Priority Academic Program Development of Jiangsu Higher Education Institutions, China (PAPD)。
文摘Streptococcus agalactiae is one of the most common pathogens that cause bovine mastitis worldwide. Identifying pathogen prevalence and virulence factors is critical for developing prevention and control approaches. Herein, 1 161 milk samples from various dairy farms in China(n=558) and Pakistan(n=603) were collected between 2019–2021 and were subjected to S. agalactiae isolation. Prevalence, serotyping, virulence genes, and antibiotic-resistant genes of S. agalactiae were evaluated by PCR assay. All isolates were characterized for haemolysis, biofilm production, cytotoxicity, adhesion, and invasion on bovine mammary epithelial cells. The prevalence of S. agalactiae-induced mastitis in cattle was found to be considerably higher in Pakistan than in China. Jiangsu and Sindh provinces had the highest area-wise prevalence in China and Pakistan, respectively. Serotypes Ia and II were prevalent in both countries, whereas serotype III was found only in Pakistan. Moreover, all isolates tested positive for PI-2b gene but negative for PI-1 and PI-2a genes. All isolates harboured cfb, cylE, hylB, and fbsB virulent genes, whereas many of them lacked bibA, rib and bca. However, the absence of bac and scp genes in Chinese isolates and cspA in Pakistani isolates was noted, while spb1 and lmb were not detected in isolates of both countries. Pakistani isolates, particularly serotype Iapositive, had a considerably higher ability to produce biofilm, haemolysis, cytotoxicity, adhesion, and invasion than Chinese isolates. Most of the isolates were phenotypically resistant to tetracycline, erythromycin, and clindamycin and genotypic resistance was confirmed by the presence of ermA, ermB, tetM and tetO genes. Our study highlights the antimicrobial resistance profile and virulence-related factors contributing to the epidemiological spread of mastitis-causing S. agalactiae in China and Pakistan. The findings may facilitate future studies designed to develop improved treatment and control strategies against this pathogen.
基金supported by the National Key Research and Development Program of China(2021YFD1800400)the National Natural Science Foundation of China(31872480)+1 种基金the Jiangsu Agriculture Science and Technology Innovation Fund,China(CX(19)2020)the Priority Academic Program Development of Jiangsu Higher Education Institutions,China(PAPD)。
文摘Streptococcus equi subsp.zooepidemicus(SEZ)is an important zoonotic agent.Here,a virulence-attenuated strain M35246 derived from natural variation of wild-type SEZ ATCC35246 was found.M35246 showed a deletion of 25contiguous genes as well as a loss-of-function mutation in covS.Subsequently,a 25-gene-deleted strain(ΔPI),a covS-mutant strain(Mcov S),and relevant complementary strains were constructed and investigated.M35246 and Mcov S were significantly less encapsulated and exhibited poorer anti-phagocytic capacity compared to wild-type SEZ.McovS was significantly more sensitive toβ-lactams,aminoglycosides,macrolides,and lincosamides than wild-type SEZ.M35246,McovS,andΔPI exhibited an increase in median lethal dose(LD_(50))in mice by 10~5,10~5,and 5 times when compared to wild-type SEZ,respectively.Neither M35246 nor McovS were isolated from mice 48 h after being challenged with approximately 2000 times the LD_(50)of wild-type SEZ.Transcriptome analysis showed that 668 significantly differentially expressed genes existed between McovS and wild-type SEZ.Numerous virulence factor-encoding genes and anabolicrelated genes in McovS that were involved in anti-phagocytosis,capsule formation,pathogenicity,and antibiotic resistance were downregulated significantly relative to the wild-type strain.This study revealed that the CovS plays a vital role in the establishment of SEZ virulence.
基金was supported by the National Key R&D Program of China(2017YFD0500203)the National Natural Science Foundation of China(31672574)+3 种基金the Special Fund for Agro-scientific Research in the Public Interest,China(201403054)the Primary Research&Development Plan of Jiangsu Province,China(BE2017341)the Jiangsu Agricultural Science and Technology Innovation Fund [CX(16)1028]the Priority Academic Program Development of Jiangsu Higher Education Institutions,China(PAPD)
文摘Streptococcus equi ssp. zooepidemicus(SEZ) is a pathogen associated with a wild range of animal species. Frequent outbreaks have occurred in recent years in pigs, horses, goats and dogs which is liable to infect humans. There is a lack of efficient vaccines against this disease and the occurrence of antibiotic resistance may render drug therapies ineffective. In this study, gene deletion mutant(ΔSEZ) in pathogenicity islands SeseCisland_4 was constructed. The mutant ΔSEZ had a 52-fold decrease in 50% lethal dose(LD_(50)) and had less capacity to adhere epithelial cells. Importantly, immunization of mice with attenuated vaccine ΔSEZ at the dose of 10~2 colony-forming units(CFU) mL^(–1) elicited a significant humoral antibody response, with an antibody titer of 1:12 800. Therefore, 10~2 CFU mL^(–1) might be used as the appropriate immune dose for the attenuated vaccine ΔSEZ, which provided mice with efficient protection against virulent SEZ. In addition, the hyperimmune sera against 10~2 CFU m L–1 attenuated vaccine ΔSEZ could confer significant protection against virulent SEZ infection in the passive immunization experiment and exhibited efficient bactericidal activity in the whole blood assay. Meanwhile, no viable bacteria was detected in blood when mice were immunized with ΔSEZ at the dose of 10~2 CFU mL^(–1) via hypodermic injection. Thereafter, the mutant ΔSEZ at the dose of 10~2 CFU mL^(–1) could confer significant protection in mice and had less negative effects on host, which could be an effective attenuated vaccine candidate for the prevention of SEZ.
基金supported by the National Key Research and Development Program of China(2016YFD0501500)the Special Fund for Agro-scientific Research in the Public Interest,China(201303046)
文摘The G-H loop of the foot-and-mouth disease virus（FMDV） virion contains certain dominant immunogenic epitopes, as well as an arginine-glycine-aspartic acid（RGD） motif that is recognized by cell surface integrin receptors. Previous experiments indicate that it is critical to maintain virus structural integrity when inserting an exogenous epitope into the surface of an FMDV structural protein. However, it remains to be determined how factors such as different insertion positions affect interactions among the virus, cells and host immune system. In this study, one infectious c DNA clone of the swine FMDV Cathay topotype strain O/CHA/90 was constructed. Then, a FLAG marker（DYKDDDDK） was inserted upstream（–4） or downstream（＋10） of the RGD motif to generate tagged viruses vFLAG-O/CHA/90 or vO/CHA/90-FLAG, investigating the possibility of expressing foreign antigen and effect on its immunogenicity. Compared to the parental virus, both tagged viruses exhibited similar plaque phenotypes, suckling mouse pathogenicity and antigenicity. Additionally, the FLAGtag insertion position did not change the use of integrin-mediated cell entry by the tagged viruses. Interestingly, both tagged vaccines protected pigs against challenge with the parental virus O/CHA/90 and induced immune responses against FMDV in BALB/c mice and pigs, but only vaccination with vFLAG-O/CHA/90 generated anti-FLAG antibodies. Our findings demonstrated that two sites（RGD–4 and RGD＋10） tolerated the insertion of an exogenous gene in the swine FMDV O/CHA/90 strain. However, only RGD–4 was a novel and appropriate inserting site which could tolerate exogenous FLAG. The resultant tagged virus is a promising candidate for FMD vaccine which can be differentiating infected from vaccinated animals（DIVA）.
基金supported by the Program for New Century Excellent Talents (NCET) at the University of China (NCET-08-0794)the Key Technology R&D Program of Jiansu Province, China (BE2009388)+1 种基金the Fundamental Research Funds for the Central Universities, China (KYT 201003)the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, China
文摘Streptococcus equi ssp. zooepidemicus （S. zooepidemicus） is a zoonotic pathogen with worldwide distribution. Lacking suitable vaccine and virulent maker is still bottleneck to control this infection. An immunoproteomic approach has been used to screen the membrane-associated and cell wall-associated proteins of S. zooepidemicus isolate in China CY to discover vaccine candidate antigens and therapeutic agents. Finally, 11 membrane-associated proteins, and 13 cell wall-associated proteins were successfully identified. BLAST （www.sanger.ac.uk） results also indicated that nucleotide sequences of majority identified proteins shared high homology （60%） with S. zooepidemicus, except for AC1-3, AC5, AC8, and AC13. Moreover, genes for 7 of the identified proteins were detected from CY; compared with ST171, 3 of them （AM1, AM8 and AC11） were only found in virulent strains （CY）. All of the proteins identified in this study remain not to be reported in S. zooepidemicus. Some of the proteins serve a vital role in the immune system and reproduction of host species according to available data, while the functions of the rest were seldom researched.
基金supported by the National Key R&D Program of China (2017YFD0500203)the National Transgenic Major Program of China (2014ZX0800946B)+3 种基金the National NaturalScience Foundation of China (31672574)the Special Fund for Agro-scientific Research in the Public Interest, China (201403054)the Jiangsu Agricultural Science and Technology Innovation Fund, China ([CX (16) 1028])the Priority Academic Program Development of Jiangsu Higher Education Institutions, China (PAPD)
文摘Streptococcus suis is one of the major pathogens of swine streptococcosis. Among them, the strongest virulence and highest rate of clinical isolation serotype is S. suis serotype 2（SS2）. Moreover, SS2 is also an important zoonosis pathogen, which caused severe public health issues in China. It has been reported that SS2 has several virulence factors, including muramidase released protein, extracellular factors, capsule, fibronectin-binding protein, enolase, hemolysin, small RNA, biofilm, two-component regulatory systems, STK/STP, etc., whose functions involved in adhesion, anti-phagocytosis, inflammatory pathway activation, invasion, etc. Actually, SS2 has developed a variety of ways to escape from host immune system during evolution. In particularly, capsule could resist phagocytosis through inhibiting sphingosine dependent immune cell recognition, which plays an important role in escaping host inflammation response; moreover, superoxide dismutase encoding by sod A enables SS2 escaping reactive oxygen species（ROS） in host immune cells; besides, binding complement factor h with Fhb could suppress the activation of complement alternative pathway and bactericidal effect. And SS2 could also hinder the formation of neutrophil extracellular traps（NETs） to avoid trapping by swine neutrophils, while host immune globulin could be degraded by Ig A1 hydrolase and Ig M protease. In addition, SS2 could escape host immune defense with the help of multiple transcriptional factors and micro-RNA. So far, the pathogenesis of meningitis, arthritis caused by SS2 infection, is still unclear, and the virulence regulatory mechanism of phosphorylation, micro-RNA need to be further clarified. Importantly, the study of interaction mechanism of pathogen and host contribute to further demonstration the pathogenesis of SS2.
