This study aims to establish a convenient,new and visual detection method for the field diagnosis of Pasteurella multocida(Pm).With reference to the Pm kmt1 gene conserved sequence published in GenBank,PCR amplification primers were designed,the amplified kmt1 gene was cloned into pMD19-T vector,and the recombinant plasmid standard pMD19-T-kmt 1 was estab-lished and identified by PCR and sequencing.Using pMD1 9-T-kmt 1 plasmid as template and kmt1 gene as target gene,basic primers were designed and synthesized.According to the requirements of LFD,a probe(Pm-P)was designed,and the RPA-LFD method for Pm detection was established by optimizing the reaction conditions.Specificity and sensitivity tests were carried out,and 64 clini-cal samples were tested by the method.The results showed that the established Pm RPA-LFD method could be amplified at 37 ℃ for 15 min.Escherichia coli(E.coli),Salmonella enteriditis(SE),Riemerella anatipestifer(RA),Staphylococcus,goose parvovirus(GPV),duck plague virus(DPV),Muscovy duck parvovirus(MDPV)DNA was extracted as the template,and plasmid standard pMD19-T-kmt 1 was used as the positive control.All the positive controls were negative,indicating that the method had good specificity.The plasmid standard pMD1 9-T-kmt 1 was diluted with a 10-fold ratio,and the plasmid standard with a concentration of 107-100 copies/μL was used as the template.The sensitivity was 1.50×101 copies/μ,,which was 100 times higher than that of PCR.A total of 64 clinical samples with suspected RA were subjected to testing using PCR,RPA and LAMP-LFD,with a 100％compliance rate for all three detection tests.The results show that the established RPA-LFD method has the characteristics of strong specificity,high sensitivity,fast speed and visualization,and can be applied to the field detection of Pm.

The 16S rRNA sequencing,whole genome sequencing and drug sensitivity tests were used to identify the isolates molecularly and to detect and analyse their virulence genes,resistance genes and drug resistance.The results showed that the isolate was highly homologous to Klebsiella pneumoniae X4 and located on the same branch by 16S rRNA sequence analysis,and it was named as KLKp10.Whole genome sequencing results showed that the KLKp10 genome was 5 342 841 bp in length,containing 5 138 genes,346 repetitive segments,6 rRNAs and 81 tRNAs,with a GC con-tent of 57.30％.MLST analysis showed that KLKp10 belongs to the ST-4263 type.The functions of 4 097 of the genes encoding proteins were classified and annotated by COG,and there were also 382 genes with unknown functions.A total of 50 functional classifications were involved in the an-notation results based on the GO database;33 kinds of signaling pathways were covered based on the signaling pathway annotations in the KEGG database.A total of 443 virulence genes were screened in the VFDB database,of which 339 belonged to the Set A database and could encode 124 virulence factors.The 101 resistance genes were predicted by comparing with the CARD database,among which there were more resistance genes against β-lactam antibiotics.The results of drug sensitivity test showed that KLKp10 was highly sensitive to ceftazidime,gentamicin,azithro-mycin,chloramphenicol,norfloxacin,ofloxacin,and enrofloxacin;moderately sensitive to ceftriax-one,neomycin,kanamycin,and streptomycin;and resistant to ciprofloxacin,tetracycline,amoxicil-lin,and penicillin.In this study,we systematically revealed the gene-wide characterization,virulence factors and drug resistance of Klebsiella pneumoniae KLKp10 of Kole pig origin,which provides important data support for the study of Klebsiella pneumoniae at the overall level of its genome.

The eIF3i gene was amplified from lamb testicular(LT)cells by PCR and cloned into pCold vector to construct the pCold-eIF3i plasmid.Plasmid PCR,double enzyme digestion and se-quencing were used to verify the results.The recombinant eIF3i protein was induced under the op-timized expression conditions.The expression and reactogenicity of the target protein were detected by SDS-PAGE and Western blot.New Zealand white rabbits were immunized with purified recom-binant eIF3i protein combined with Freund's complete and incomplete adjuvants for three times.Se-rum samples were collected after immunization.Indirect ELISA was used to detect antiserum titer,and Western blot was used to analyze antibody specificity.Indirect immunofluorescence assay(IFA)was used to detect the application effect of antibodies.The results showed that the size of LT eIF3i gene was 978 bp.The optimal expression conditions for the eIF3i recombinant protein were as follows:IPTG concentration of 0.2 mmol/L,temperature of 37 ℃,and induction time of 8 h.The recombinant eIF3i protein was expressed as an inclusion body with a size of about 36 kDa.The titer of polyclonal antibody against eIF3i protein was 1∶51 200.Western blot and IFA showed that the prepared polyclonal antibody against eIF3i protein had good reactivity and specificity.In conclusion,we successfully prepared rabbit anti-eif3i polyclonal antibody and confirmed that it could specifically recognize endogenous eIF3i protein,which laid a foundation for further study on the biological function of eIF3i protein.

The 16S rRNA sequencing,whole genome sequencing and drug sensitivity tests were used to identify the isolates molecularly and to detect and analyse their virulence genes,resistance genes and drug resistance.The results showed that the isolate was highly homologous to Klebsiella pneumoniae X4 and located on the same branch by 16S rRNA sequence analysis,and it was named as KLKp10.Whole genome sequencing results showed that the KLKp10 genome was 5 342 841 bp in length,containing 5 138 genes,346 repetitive segments,6 rRNAs and 81 tRNAs,with a GC con-tent of 57.30％.MLST analysis showed that KLKp10 belongs to the ST-4263 type.The functions of 4 097 of the genes encoding proteins were classified and annotated by COG,and there were also 382 genes with unknown functions.A total of 50 functional classifications were involved in the an-notation results based on the GO database;33 kinds of signaling pathways were covered based on the signaling pathway annotations in the KEGG database.A total of 443 virulence genes were screened in the VFDB database,of which 339 belonged to the Set A database and could encode 124 virulence factors.The 101 resistance genes were predicted by comparing with the CARD database,among which there were more resistance genes against β-lactam antibiotics.The results of drug sensitivity test showed that KLKp10 was highly sensitive to ceftazidime,gentamicin,azithro-mycin,chloramphenicol,norfloxacin,ofloxacin,and enrofloxacin;moderately sensitive to ceftriax-one,neomycin,kanamycin,and streptomycin;and resistant to ciprofloxacin,tetracycline,amoxicil-lin,and penicillin.In this study,we systematically revealed the gene-wide characterization,virulence factors and drug resistance of Klebsiella pneumoniae KLKp10 of Kole pig origin,which provides important data support for the study of Klebsiella pneumoniae at the overall level of its genome.

This study aims to establish a convenient,new and visual detection method for the field diagnosis of Pasteurella multocida(Pm).With reference to the Pm kmt1 gene conserved sequence published in GenBank,PCR amplification primers were designed,the amplified kmt1 gene was cloned into pMD19-T vector,and the recombinant plasmid standard pMD19-T-kmt 1 was estab-lished and identified by PCR and sequencing.Using pMD1 9-T-kmt 1 plasmid as template and kmt1 gene as target gene,basic primers were designed and synthesized.According to the requirements of LFD,a probe(Pm-P)was designed,and the RPA-LFD method for Pm detection was established by optimizing the reaction conditions.Specificity and sensitivity tests were carried out,and 64 clini-cal samples were tested by the method.The results showed that the established Pm RPA-LFD method could be amplified at 37 ℃ for 15 min.Escherichia coli(E.coli),Salmonella enteriditis(SE),Riemerella anatipestifer(RA),Staphylococcus,goose parvovirus(GPV),duck plague virus(DPV),Muscovy duck parvovirus(MDPV)DNA was extracted as the template,and plasmid standard pMD19-T-kmt 1 was used as the positive control.All the positive controls were negative,indicating that the method had good specificity.The plasmid standard pMD1 9-T-kmt 1 was diluted with a 10-fold ratio,and the plasmid standard with a concentration of 107-100 copies/μL was used as the template.The sensitivity was 1.50×101 copies/μ,,which was 100 times higher than that of PCR.A total of 64 clinical samples with suspected RA were subjected to testing using PCR,RPA and LAMP-LFD,with a 100％compliance rate for all three detection tests.The results show that the established RPA-LFD method has the characteristics of strong specificity,high sensitivity,fast speed and visualization,and can be applied to the field detection of Pm.

The eIF3i gene was amplified from lamb testicular(LT)cells by PCR and cloned into pCold vector to construct the pCold-eIF3i plasmid.Plasmid PCR,double enzyme digestion and se-quencing were used to verify the results.The recombinant eIF3i protein was induced under the op-timized expression conditions.The expression and reactogenicity of the target protein were detected by SDS-PAGE and Western blot.New Zealand white rabbits were immunized with purified recom-binant eIF3i protein combined with Freund's complete and incomplete adjuvants for three times.Se-rum samples were collected after immunization.Indirect ELISA was used to detect antiserum titer,and Western blot was used to analyze antibody specificity.Indirect immunofluorescence assay(IFA)was used to detect the application effect of antibodies.The results showed that the size of LT eIF3i gene was 978 bp.The optimal expression conditions for the eIF3i recombinant protein were as follows:IPTG concentration of 0.2 mmol/L,temperature of 37 ℃,and induction time of 8 h.The recombinant eIF3i protein was expressed as an inclusion body with a size of about 36 kDa.The titer of polyclonal antibody against eIF3i protein was 1∶51 200.Western blot and IFA showed that the prepared polyclonal antibody against eIF3i protein had good reactivity and specificity.In conclusion,we successfully prepared rabbit anti-eif3i polyclonal antibody and confirmed that it could specifically recognize endogenous eIF3i protein,which laid a foundation for further study on the biological function of eIF3i protein.