The study was aimed at establishing an indirect ELISA for epidemiological investigation of hepatitis E viral(HEV) infection in pigs from the major pig-producing regions of Zhejiang Province. The gene fragment covering...The study was aimed at establishing an indirect ELISA for epidemiological investigation of hepatitis E viral(HEV) infection in pigs from the major pig-producing regions of Zhejiang Province. The gene fragment covering the immunogenic epitopes of HEV ORF2 was synthesized and expressed in Escherichia coli.Western blot analysis revealed that the purified protein reacted to HEV positive sera,but not to positive sera from some other common viruses that infect pigs.An indirect ELISA system was then developed using truncated HEV ORF2 protein as the coating antigen and had diagnostic accuracy of 91.5% as compared with a diagnostic kit for HEV antibodies.A total of 1 330 serum samples were collected from 46 pig farms in Zhejiang Province from 2005-2008 and tested for sero-prevalence of HEV using the indirect ELISA.The average HEV-positive rate was 55.7%(741/1 330).The positive rate of 2005 and 2006 was 62.7%(175/279) and 61.4%(301/490) respectively,much higher than those of 2007(43.0%,59/137) and 2008(48.6%,206/424).Pigs aged 66-100 days had a statistically higher positive rate(58.2%,110/189) when compared to that of pigs aged 30-65 days(39.7%,73/184) or 101-160 days(44.1%,83/188).Only three herds in the study were HEV antibody-negative,indicating high sero-prevalence of HEV in the pig populations in Zhejiang Province.展开更多
Nanoparticles are significant for veterinary vaccine development because they are safer and more effective than conventional formulations.One promising area of research involves self-assembled protein nanoparticles(SA...Nanoparticles are significant for veterinary vaccine development because they are safer and more effective than conventional formulations.One promising area of research involves self-assembled protein nanoparticles(SAPNs),which have shown potential for enhancing antigen-presenting cell uptake,B-cell activation,and lymph node trafficking.Numerous nanovaccines have been utilized in veterinary medicine,including natural self-assembled protein nanoparticles,rationally designed self-assembled protein nanoparticles,animal virus-derived nanoparticles,bacteriophagederived nanoparticles,and plant-derived nanoparticles,which will be discussed in this review.SAPN vaccines can produce robust cellular and humoral immune responses and have been shown to protect against various animal infectious diseases.This article attempts to summarize these diverse nanovaccine types and their recent research progress in the field of veterinary medicine.Furthermore,this paper highlights their disadvantages and methods for improving their immunogenicity.展开更多
Specific immunoglobulin (IgY) from egg yolk against Aeromonas hydrophila was produced by immunization of White Leghorn hens with formalin-killed whole cells of A. hydrophila. ELISA test using A. hydrophila as the coat...Specific immunoglobulin (IgY) from egg yolk against Aeromonas hydrophila was produced by immunization of White Leghorn hens with formalin-killed whole cells of A. hydrophila. ELISA test using A. hydrophila as the coating antigen revealed that the specific antibody titer started to increase in the egg yolk at the 13th day post-immunization (P/N=2.18), reached the peak at the 56th day (P/N=13.82), and remained at high level until day 133 (P/N=7.03). The antibody was purified by saturated ammonium sulphate with a recovery rate of 63.5%. The specific IgY inhibited the growth of A. hydrophila at a concentration of 1.0 mg/ml during the 18 h incubation. Pre-treatment of polyploid gibel carps Carassius auratus Gibelio with specific IgY had a protection rate of 60% (6/10) against challenge with A. hydrophila, while none of the fishes in the control groups receiving sterile phosphate buffered saline (PBS) or non-specific IgY survived the challenge. Treatment of fishes with the specific IgY 4 h after the challenge also had lower mortality (70%, 7/10), a 30% reduction against the control PBS or non-specific IgY groups (10/10). These results indicate that specific IgY antibodies could be obtained easily from hens immunized with an inactivated A. hydrophila and could provide a novel alternative approach to control of diseases in fishes caused by this organism.展开更多
Chinese soft-shelled turtles （Trionyx sinens） in culture farms using an artificial warming system in Zhejiang, China, often show typical signs of white-spot disease such as white spots on their bodies, skin lesions,...Chinese soft-shelled turtles （Trionyx sinens） in culture farms using an artificial warming system in Zhejiang, China, often show typical signs of white-spot disease such as white spots on their bodies, skin lesions, anorexia and eventually death. The sick turtles were mostly 5-80 g in weight. A suspected fungal pathogen was isolated from the sick turtles and verified as Paecilomyces lilacinus by sequence analysis of the internal transcribed spacer （ITS） of its ribosomal DNA （rDNA）. Detailed morphological examinations were also conducted to confirm the white-spot disease.展开更多
Salmonella enterica has a wide diversity,with numerous serovars belonging to six different subspecies with dynamic animal-host tropism.The FimH protein is the adhesin mediating binding to various cells,and slight amin...Salmonella enterica has a wide diversity,with numerous serovars belonging to six different subspecies with dynamic animal-host tropism.The FimH protein is the adhesin mediating binding to various cells,and slight amino acid discrepancy significantly affects the adherence capacities.To date,the general function of FimH variability across dif-ferent subspecies of Salmonella enterica has not been addressed.To investigate the biological functions of FimH among the six Salmonella enterica subspecies,the present study performed several assays to determine biofilm for-mation,Caenorhabditis elegans killing,and intestinal porcine enterocyte cell IPEC-J2 adhesion by using various FimH allele mutants.In general,allelic mutations in both the lectin and pilin domains of FimH could cause changes in bind-ing affnity,such as the N79S mutation.We also observed that the N79S variation in Salmonella Dublin increased the adhesive ability of IPEC-J2 cells.Moreover,a new amino acid substitution,T260M,within the pilin domain in one subspecies llb strain beneficial to binding to cells was highlighted in this study,even though the biofilm-forming and Caenorhabditis elegans-killing abilities exhibited no significant differences in variants.Combined with point muta-tions being a natural tendency due to positive selection in harsh environments,we speculate that allelic variation T26oM probably contributes to pathoadaptive evolution in Salmonella enterica subspecies llb.展开更多
A one-step dual flow immunochromatographic assay （DICGA）, based on a competitive format, was de- veloped for simultaneous quantification of ochratoxin A （OTA） and zearalenone （ZEN） in corn, wheat, and feed sam- ...A one-step dual flow immunochromatographic assay （DICGA）, based on a competitive format, was de- veloped for simultaneous quantification of ochratoxin A （OTA） and zearalenone （ZEN） in corn, wheat, and feed sam- ples. The limit of detection for OTA was 0.32 ng/ml with a detection range of 0.53-12.16 ng/ml, while for ZEN it was 0.58 ng/ml with a detection range of 1.06-39.72 ng/ml. The recovery rates in corn, wheat, and feed samples ranged from 77.3% to 106.3% with the coefficient of variation lower than 15%. Naturally contaminated corn, wheat, and feed samples were analyzed using both DICGA and liquid chromatography-tandem mass spectrometry （LC-MS/MS） and the correlation between the two methods was evaluated using a regression analysis. The DICGA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of OTA and ZEN in food safety control.展开更多
Porcine circovirus 3(PCV3)has been detected in major pig-producing countries around the world since its first report in the US in 2016.Most current studies have focused on epidemiological investigations and detection ...Porcine circovirus 3(PCV3)has been detected in major pig-producing countries around the world since its first report in the US in 2016.Most current studies have focused on epidemiological investigations and detection methods of PCV3 because of lack of live virus strains for research on its pathogenesis in porcine cells or even in pigs.We constructed a recombinant plasmid pCMV-Cap carrying the PCV3 orf2 gene to investigate the effects of capsid(Cap)protein expression on autophagic response in human embryonic kidney cell line 293 T(HEK293 T).We demonstrate that PCV3 Cap protein induced complete autophagy shown as formation of autophagosomes and autophagosome-like vesicles as well as LC3-II conversion from LC3-I via inhibiting phosphorylation of the mammalian target of rapamycin(mTOR)in HEK293 T cells.The ubiquitin–proteasome pathway is also involved in the autophagy process.These findings provide insight for further exploration of PCV3 pathogenetic mechanisms in porcine cells.展开更多
The heat shock cognate protein 70 (Hsc70) is a member of a 70-kDa heat shock protein (HSP70) family that functions as molecular chaperones.In this study,a novel Hsc70 gene from Chinese soft-shelled turtle (Pelodiscus ...The heat shock cognate protein 70 (Hsc70) is a member of a 70-kDa heat shock protein (HSP70) family that functions as molecular chaperones.In this study,a novel Hsc70 gene from Chinese soft-shelled turtle (Pelodiscus sinensis) (tHsc70) was identified.The tHsc70 full-length complementary DNA (cDNA) is 2 272 bp long with a 1 941-bp open reading frame (ORF) encoding 646 amino acids.Three characteristic signature regions of the HSP70 family,two major domains of an adenosine triphosphate (ATP)/guanosine triphosphate (GTP) binding domain (ABD),and a substrate-binding domain (SBD) were present in the predicted tHsc70 amino acid sequence.The tHsc70 gene was expressed in Escherichia coli BL21 and the expression product reacted with the anti-Hsc70 mouse monoclonal antibody by Western blotting.Homology analysis revealed that tHsc70 shared identity from 53.9% to 87.7% at the nucleotide level,and 49.1% to 99.5% at the amino acid level with the known Hsc70s.Phylogenetic analysis showed that tHsc70 was clustered together with the Hsc70 gene of another reptile species (Alligator mississippiensis).The tHsc70 was expressed in the liver,lung,heart,and skeletal muscle.The expression patterns of tHsc70 messenger RNA (mRNA) differed among different tissues under different durations of heat stress at 40 °C.Adaptation at 25 °C for 1 h after heat stress was also different among tissues and length of heat stress.Irrespective of different profiles of expression under heat stress,tHsc70 may play roles in protecting turtles from thermal stress.展开更多
Listeria monocytogenes is an important zoonotic foodborne pathogen that can tolerate a number of environmental stresses. Rsb R, an upstream regulator of the sigma B(Sig B) factor, is thought to sense environmental cha...Listeria monocytogenes is an important zoonotic foodborne pathogen that can tolerate a number of environmental stresses. Rsb R, an upstream regulator of the sigma B(Sig B) factor, is thought to sense environmental challenges and trigger the SigB pathway. In Bacillus subtilis, two phosphorylation sites in RsbR are involved in activating the SigB pathway and a feedback mechanism, respectively. In this study, the role of RsbR in L. monocytogenes under mild and severe stresses was investigated. Strains with genetic deletion(Δrsb R), complementation(C-Δrsb R), and phosphorylation site mutations in the rsbR(RsbR-T175 A, RsbR-T209 A, and RsbR-T175 A-T209 A) were constructed to evaluate the roles of these RsbR sequences in listerial growth and survival. SigB was examined at the transcriptional and translational levels. Deletion of rsb R reduced listerial growxth and survival in response to acidic stress. Substitution of the phosphorylation residue RsbR-T175 A disabled RsbR complementation, while RsbR-T209 A significantly upregulated SigB expression and listerial survival. Our results provide clear evidence that two phosphorylation sites of Rsb R are functional in L. monocytogenes under acidic conditions, similar to the situation in B. subtilis.展开更多
基金supported by Technological Project of Zhejiang Emtry-Exit Inspection and Quarantine Bureau(ZK200611)Technology Innovation Service Platform of Inspection and Quarantine for Safe Frontier (2006C1704)
文摘The study was aimed at establishing an indirect ELISA for epidemiological investigation of hepatitis E viral(HEV) infection in pigs from the major pig-producing regions of Zhejiang Province. The gene fragment covering the immunogenic epitopes of HEV ORF2 was synthesized and expressed in Escherichia coli.Western blot analysis revealed that the purified protein reacted to HEV positive sera,but not to positive sera from some other common viruses that infect pigs.An indirect ELISA system was then developed using truncated HEV ORF2 protein as the coating antigen and had diagnostic accuracy of 91.5% as compared with a diagnostic kit for HEV antibodies.A total of 1 330 serum samples were collected from 46 pig farms in Zhejiang Province from 2005-2008 and tested for sero-prevalence of HEV using the indirect ELISA.The average HEV-positive rate was 55.7%(741/1 330).The positive rate of 2005 and 2006 was 62.7%(175/279) and 61.4%(301/490) respectively,much higher than those of 2007(43.0%,59/137) and 2008(48.6%,206/424).Pigs aged 66-100 days had a statistically higher positive rate(58.2%,110/189) when compared to that of pigs aged 30-65 days(39.7%,73/184) or 101-160 days(44.1%,83/188).Only three herds in the study were HEV antibody-negative,indicating high sero-prevalence of HEV in the pig populations in Zhejiang Province.
文摘Nanoparticles are significant for veterinary vaccine development because they are safer and more effective than conventional formulations.One promising area of research involves self-assembled protein nanoparticles(SAPNs),which have shown potential for enhancing antigen-presenting cell uptake,B-cell activation,and lymph node trafficking.Numerous nanovaccines have been utilized in veterinary medicine,including natural self-assembled protein nanoparticles,rationally designed self-assembled protein nanoparticles,animal virus-derived nanoparticles,bacteriophagederived nanoparticles,and plant-derived nanoparticles,which will be discussed in this review.SAPN vaccines can produce robust cellular and humoral immune responses and have been shown to protect against various animal infectious diseases.This article attempts to summarize these diverse nanovaccine types and their recent research progress in the field of veterinary medicine.Furthermore,this paper highlights their disadvantages and methods for improving their immunogenicity.
基金Project (No. 2004C26026) supported by the Science and Technology Department of Zhejiang Province, China
文摘Specific immunoglobulin (IgY) from egg yolk against Aeromonas hydrophila was produced by immunization of White Leghorn hens with formalin-killed whole cells of A. hydrophila. ELISA test using A. hydrophila as the coating antigen revealed that the specific antibody titer started to increase in the egg yolk at the 13th day post-immunization (P/N=2.18), reached the peak at the 56th day (P/N=13.82), and remained at high level until day 133 (P/N=7.03). The antibody was purified by saturated ammonium sulphate with a recovery rate of 63.5%. The specific IgY inhibited the growth of A. hydrophila at a concentration of 1.0 mg/ml during the 18 h incubation. Pre-treatment of polyploid gibel carps Carassius auratus Gibelio with specific IgY had a protection rate of 60% (6/10) against challenge with A. hydrophila, while none of the fishes in the control groups receiving sterile phosphate buffered saline (PBS) or non-specific IgY survived the challenge. Treatment of fishes with the specific IgY 4 h after the challenge also had lower mortality (70%, 7/10), a 30% reduction against the control PBS or non-specific IgY groups (10/10). These results indicate that specific IgY antibodies could be obtained easily from hens immunized with an inactivated A. hydrophila and could provide a novel alternative approach to control of diseases in fishes caused by this organism.
基金the Science and Technology Department of Zhejiang Province, China (No. 2004C26026)the Science and Technology Department of Hangzhou City, China (No. 20051322 B33)
文摘Chinese soft-shelled turtles （Trionyx sinens） in culture farms using an artificial warming system in Zhejiang, China, often show typical signs of white-spot disease such as white spots on their bodies, skin lesions, anorexia and eventually death. The sick turtles were mostly 5-80 g in weight. A suspected fungal pathogen was isolated from the sick turtles and verified as Paecilomyces lilacinus by sequence analysis of the internal transcribed spacer （ITS） of its ribosomal DNA （rDNA）. Detailed morphological examinations were also conducted to confirm the white-spot disease.
基金supported by the National Program on Key Research Project of China(2022YFC2604201)well as the European Union's Horizon 2020 Research and Innovation Programme under Grant Agreement No.861917-SAFFl,Zhejiang Provincial Key R&D Program of China(2023C03045)+2 种基金Hainan Provincial Joint Project of Sanya Yazhou Bay Science and Technology City(2021JJLH0083)Key Research and Development Program of Hangzhou(202203A08)District-level project for high-level innovative and entrepreneurial talents of"Zijinshan Talents Gaochun Plan"(202100677).
文摘Salmonella enterica has a wide diversity,with numerous serovars belonging to six different subspecies with dynamic animal-host tropism.The FimH protein is the adhesin mediating binding to various cells,and slight amino acid discrepancy significantly affects the adherence capacities.To date,the general function of FimH variability across dif-ferent subspecies of Salmonella enterica has not been addressed.To investigate the biological functions of FimH among the six Salmonella enterica subspecies,the present study performed several assays to determine biofilm for-mation,Caenorhabditis elegans killing,and intestinal porcine enterocyte cell IPEC-J2 adhesion by using various FimH allele mutants.In general,allelic mutations in both the lectin and pilin domains of FimH could cause changes in bind-ing affnity,such as the N79S mutation.We also observed that the N79S variation in Salmonella Dublin increased the adhesive ability of IPEC-J2 cells.Moreover,a new amino acid substitution,T260M,within the pilin domain in one subspecies llb strain beneficial to binding to cells was highlighted in this study,even though the biofilm-forming and Caenorhabditis elegans-killing abilities exhibited no significant differences in variants.Combined with point muta-tions being a natural tendency due to positive selection in harsh environments,we speculate that allelic variation T26oM probably contributes to pathoadaptive evolution in Salmonella enterica subspecies llb.
基金Project supported by the Natural Science Foundation of Zhejiang Province(No.LQ17C170002)the Talent-Start Project of Zhejiang A&F University(No.2016FR025)+1 种基金the Key Research and Development Project Funds of Zhejiang Provincial Science and Technology Department(No.2018C02041)and the National High-Tech R&D Program(863)of China(No.2012AA101602)
文摘A one-step dual flow immunochromatographic assay （DICGA）, based on a competitive format, was de- veloped for simultaneous quantification of ochratoxin A （OTA） and zearalenone （ZEN） in corn, wheat, and feed sam- ples. The limit of detection for OTA was 0.32 ng/ml with a detection range of 0.53-12.16 ng/ml, while for ZEN it was 0.58 ng/ml with a detection range of 1.06-39.72 ng/ml. The recovery rates in corn, wheat, and feed samples ranged from 77.3% to 106.3% with the coefficient of variation lower than 15%. Naturally contaminated corn, wheat, and feed samples were analyzed using both DICGA and liquid chromatography-tandem mass spectrometry （LC-MS/MS） and the correlation between the two methods was evaluated using a regression analysis. The DICGA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of OTA and ZEN in food safety control.
基金supported by the Zhejiang Provincial Natural Science Foundation of China(No.LQ14C180001)the Fundamental Research Funds for the Central Universities(No.2014QNA6026),China
基金Project supported by the Zhejiang Provincial Department of Science and Technology(No.2018C02028),China。
文摘Porcine circovirus 3(PCV3)has been detected in major pig-producing countries around the world since its first report in the US in 2016.Most current studies have focused on epidemiological investigations and detection methods of PCV3 because of lack of live virus strains for research on its pathogenesis in porcine cells or even in pigs.We constructed a recombinant plasmid pCMV-Cap carrying the PCV3 orf2 gene to investigate the effects of capsid(Cap)protein expression on autophagic response in human embryonic kidney cell line 293 T(HEK293 T).We demonstrate that PCV3 Cap protein induced complete autophagy shown as formation of autophagosomes and autophagosome-like vesicles as well as LC3-II conversion from LC3-I via inhibiting phosphorylation of the mammalian target of rapamycin(mTOR)in HEK293 T cells.The ubiquitin–proteasome pathway is also involved in the autophagy process.These findings provide insight for further exploration of PCV3 pathogenetic mechanisms in porcine cells.
基金Project (No.20082332Y03) supported by the Science and Technology Department of Hangzhou City,China
文摘The heat shock cognate protein 70 (Hsc70) is a member of a 70-kDa heat shock protein (HSP70) family that functions as molecular chaperones.In this study,a novel Hsc70 gene from Chinese soft-shelled turtle (Pelodiscus sinensis) (tHsc70) was identified.The tHsc70 full-length complementary DNA (cDNA) is 2 272 bp long with a 1 941-bp open reading frame (ORF) encoding 646 amino acids.Three characteristic signature regions of the HSP70 family,two major domains of an adenosine triphosphate (ATP)/guanosine triphosphate (GTP) binding domain (ABD),and a substrate-binding domain (SBD) were present in the predicted tHsc70 amino acid sequence.The tHsc70 gene was expressed in Escherichia coli BL21 and the expression product reacted with the anti-Hsc70 mouse monoclonal antibody by Western blotting.Homology analysis revealed that tHsc70 shared identity from 53.9% to 87.7% at the nucleotide level,and 49.1% to 99.5% at the amino acid level with the known Hsc70s.Phylogenetic analysis showed that tHsc70 was clustered together with the Hsc70 gene of another reptile species (Alligator mississippiensis).The tHsc70 was expressed in the liver,lung,heart,and skeletal muscle.The expression patterns of tHsc70 messenger RNA (mRNA) differed among different tissues under different durations of heat stress at 40 °C.Adaptation at 25 °C for 1 h after heat stress was also different among tissues and length of heat stress.Irrespective of different profiles of expression under heat stress,tHsc70 may play roles in protecting turtles from thermal stress.
基金Project supported by the National Natural Science Foundation of China(Nos.31702250 and 31600292)the Open Fund Project by Key Laboratory of Animal Preventive Medicine of Zhejiang Province(No.ZPKLPVM2017KFKT001),China
文摘Listeria monocytogenes is an important zoonotic foodborne pathogen that can tolerate a number of environmental stresses. Rsb R, an upstream regulator of the sigma B(Sig B) factor, is thought to sense environmental challenges and trigger the SigB pathway. In Bacillus subtilis, two phosphorylation sites in RsbR are involved in activating the SigB pathway and a feedback mechanism, respectively. In this study, the role of RsbR in L. monocytogenes under mild and severe stresses was investigated. Strains with genetic deletion(Δrsb R), complementation(C-Δrsb R), and phosphorylation site mutations in the rsbR(RsbR-T175 A, RsbR-T209 A, and RsbR-T175 A-T209 A) were constructed to evaluate the roles of these RsbR sequences in listerial growth and survival. SigB was examined at the transcriptional and translational levels. Deletion of rsb R reduced listerial growxth and survival in response to acidic stress. Substitution of the phosphorylation residue RsbR-T175 A disabled RsbR complementation, while RsbR-T209 A significantly upregulated SigB expression and listerial survival. Our results provide clear evidence that two phosphorylation sites of Rsb R are functional in L. monocytogenes under acidic conditions, similar to the situation in B. subtilis.
