The objective of this study was to develop a rapid and precise detection technique for por-cine Senecavirus A(SVA)employing reverse transcription recombinase polymerase amplification(RT-RAA)in conjunction with CRISPR Cas13a technology.Additionally,the study aimed to opti-mize the assay's reaction conditions to enhance amplification efficiency.Eight RT-RAA primer sets were designed based on the conserved gene sequence of porcine SVA,and a series of reaction condi-tions were evaluated to refine the RT-RAA reaction system.Subsequently,CRISPR-derived RNA(crRNA)sequences were developed and selected to construct the RT-RAA-CRISPR reaction sys-tem.The method's specificity was determined by examining six prevalent porcine pathogenic nucleic acids,while its sensitivity was assessed using SVA cRNA standards quantified by digital PCR.The method's stability and the consistency of clinical sample analysis were also evaluated.The findings revealed that the optimized RT-RAA and CRISPR reaction systems exhibited the highest amplifi-cation efficiency at a reaction temperature of 37 ℃.Among the eight crRNAs,five were identified as exhibiting the strongest detection signals.The formulated RT-RAA-CRISPR Cas13a method demonstrated exceptional specificity,showing no cross-reactivity with other common porcine disea-ses,including ASFV,PRRSV,PEDV,PCV2,CSFV,and PRV.The method achieved high sensitivi-ty,detecting as low as 0.86 copies/μL of SVA,and exhibited stable fluorescence output,robust re-producibility,and the ability to complete clinical sample analysis within 50 minutes.Consistency e-valuation with six positive and 58 negative samples indicated 100％agreement in outcomes.These results substantiate that the study successfully established a rapid and specific RT-RAA-CRISPR Cas13a detection method for the on-site identification of porcine Senecavirus A,demonstrating high specificity and sensitivity,and holds promise for application in SVA monitoring and control initia-tives.

Enterotoxigenic Escherichia coli(ETEC)is the main cause of diarrhea in piglets,which seriously threatens the development of animal husbandry.In order to effectively prevent and con-trol piglet diarrhea,the neutralizing epitopes of the screened K88(FTDYEGASVELRKPDGGT-NK),K99(NVGNGSGGANIN),987P(LAAPAENNTSQAN),F41(VMAADWTEGQPGDII)and F18(PPNAQTYPLSSGDLK)ETEC adhesins were assembled to construct the multi-epitope fusion antigen(MEFA),whose immunogenicity was verified by in silico simulations and in vitro.The computer simulation results showed that MEFA has excellent physicochemical properties and reasonable prediction results of secondary and tertiary structures and exhibits strong interaction force when docking with toll-like receptor 3,and the kinetic simulation shows that it can be stably presented.In vitro validation results showed that anti-K88,K99,987P,F18 and F41 IgY could spe-cifically recognize MEFA proteins.After immunization of laying hens with MEFA,the titers of IgY antibodies against the five ETEC were higher than those of non-specific IgY.In addition,specific anti-MEFA IgY significantly inhibited the adhesion of K88,K99,987P,F41 and F18 ETEC strains to piglet intestinal IPEC-J2 cell lines.These results indicate that MEFA has strong immunogenicity and can be used as an ideal vaccine candidate for the prevention of K88,K99,987P,F41 and F18 ETEC infection,and provide reference and technical support for the development of multivalent broad-spectrum ETEC vaccine candidates.

The objective of this study was to develop a rapid and precise detection technique for por-cine Senecavirus A(SVA)employing reverse transcription recombinase polymerase amplification(RT-RAA)in conjunction with CRISPR Cas13a technology.Additionally,the study aimed to opti-mize the assay's reaction conditions to enhance amplification efficiency.Eight RT-RAA primer sets were designed based on the conserved gene sequence of porcine SVA,and a series of reaction condi-tions were evaluated to refine the RT-RAA reaction system.Subsequently,CRISPR-derived RNA(crRNA)sequences were developed and selected to construct the RT-RAA-CRISPR reaction sys-tem.The method's specificity was determined by examining six prevalent porcine pathogenic nucleic acids,while its sensitivity was assessed using SVA cRNA standards quantified by digital PCR.The method's stability and the consistency of clinical sample analysis were also evaluated.The findings revealed that the optimized RT-RAA and CRISPR reaction systems exhibited the highest amplifi-cation efficiency at a reaction temperature of 37 ℃.Among the eight crRNAs,five were identified as exhibiting the strongest detection signals.The formulated RT-RAA-CRISPR Cas13a method demonstrated exceptional specificity,showing no cross-reactivity with other common porcine disea-ses,including ASFV,PRRSV,PEDV,PCV2,CSFV,and PRV.The method achieved high sensitivi-ty,detecting as low as 0.86 copies/μL of SVA,and exhibited stable fluorescence output,robust re-producibility,and the ability to complete clinical sample analysis within 50 minutes.Consistency e-valuation with six positive and 58 negative samples indicated 100％agreement in outcomes.These results substantiate that the study successfully established a rapid and specific RT-RAA-CRISPR Cas13a detection method for the on-site identification of porcine Senecavirus A,demonstrating high specificity and sensitivity,and holds promise for application in SVA monitoring and control initia-tives.

Enterotoxigenic Escherichia coli(ETEC)is the main cause of diarrhea in piglets,which seriously threatens the development of animal husbandry.In order to effectively prevent and con-trol piglet diarrhea,the neutralizing epitopes of the screened K88(FTDYEGASVELRKPDGGT-NK),K99(NVGNGSGGANIN),987P(LAAPAENNTSQAN),F41(VMAADWTEGQPGDII)and F18(PPNAQTYPLSSGDLK)ETEC adhesins were assembled to construct the multi-epitope fusion antigen(MEFA),whose immunogenicity was verified by in silico simulations and in vitro.The computer simulation results showed that MEFA has excellent physicochemical properties and reasonable prediction results of secondary and tertiary structures and exhibits strong interaction force when docking with toll-like receptor 3,and the kinetic simulation shows that it can be stably presented.In vitro validation results showed that anti-K88,K99,987P,F18 and F41 IgY could spe-cifically recognize MEFA proteins.After immunization of laying hens with MEFA,the titers of IgY antibodies against the five ETEC were higher than those of non-specific IgY.In addition,specific anti-MEFA IgY significantly inhibited the adhesion of K88,K99,987P,F41 and F18 ETEC strains to piglet intestinal IPEC-J2 cell lines.These results indicate that MEFA has strong immunogenicity and can be used as an ideal vaccine candidate for the prevention of K88,K99,987P,F41 and F18 ETEC infection,and provide reference and technical support for the development of multivalent broad-spectrum ETEC vaccine candidates.

OBJECTIVE To investigate present conditions about the fungal infection and analyze its risk factors and the measure of prevention. METHODS Before statistics and analysis,clinical data and culture results of 175 cases from Jan 2002 to Dec 2004 were collected. RESULTS The respiratory tract occupied the majority of the fungus infection(40.00%),the gastrointestinal tract was the second(23.43%),and the urinary tract was the third(19.43%).In infection strains,Candida albicans occupied the first place(68.57%),C.tropicalis and(C.glabrata) were the second and third(6.86% and 5.71%). CONCLUSIONS The fungal infection is relevant to some risk factors,such as using antibiotics,underlying disease and aging.Therefore,using antibiotics reasonably and improving the immunity of organism are the main measures of preventing the fungal infection.