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1.
Vet Microbiol ; 291: 110014, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38335675

ABSTRACT

It is widely known that integrative and conjugative elements (ICEs) play an important role in the transmission of resistance genes and other exogenous genes. The present study aimed to characterize the three novel ICEs including ICEGpa76, ICEGpa44, and ICEGpa11, from Glaesserella parasuis. The ICEs from G. parasuis strains d76, Z44, and XP11 were predicted and identified by whole-genome sequencing (WGS) analysis, ICEfinder, and PCR. Characterization of G. parasuis strains carrying ICEs were determined by conjugation assay, antimicrobial susceptibility testing, WGS, phylogenetic analysis, and comparative sequence analysis.The WGS results showed that three ICEs from G. parasuis have a common genetic backbone belonging to characteristics ofthe ICEHpa1 family. The sequence comparison showed that the ICEHpa1 family has five hot spots (HSs) determined by IS6, IS110, and IS256. Moreover, two variable regions (VRs), VR1 and VR2 were determined by multidrug resistance genes and the rearrangement hotspot (rhs) family, respectively. VR1 consists of multidrug resistance genes, ISApl1s, and other accessory genes, while VR2 is composed of IS4, rhs family, transposase, and hypothetical protein genes. Conjugation experiments and MICs revealed that three ICEs could be transferred to G. parasuis strain IV52, indicating these three ICEs could be transmitted horizontally among G. parasuis strains. Additionally, the difference in resistance genes from ICEs might be due to the insertion function of the ISApl1s in VR1, and the rhs family in VR2 might evolve andthen be stably inherited in G. parasuis. These results further elucidated the transmission mechanism of exogenous genes in G. parasuis.


Subject(s)
Conjugation, Genetic , Genes, MDR , Animals , Phylogeny
2.
J Glob Antimicrob Resist ; 35: 163-171, 2023 12.
Article in English | MEDLINE | ID: mdl-37726088

ABSTRACT

OBJECTIVES: The aim of this study was to characterize the floR-carrying plasmids originating from Glaesserella parasuis and Actinobacillus indolicus isolated from pigs with respiratory disease in China. METHODS: A total of 125 G. parasuis and 28 A. indolicus strains collected between 2009 and 2022 were screened for florfenicol resistance. Characterization of floR-positive isolates and plasmids were determined by antimicrobial susceptibility testing, serotyping, multilocus sequence typing (MLST), conjugation and transformation assays, whole-genome sequencing (WGS), and phylogenetic analysis. RESULTS: One A. indolicus and six G. parasuis were identified as positive for floR. The six G. parasuis were assigned to four different serovars, including serovars 6, 7, 9, and unknown. In addition to strain XP11, six floR genes were located on plasmids. The six floR-bearing plasmids could be transformed into Pasteurella multocida and divided into two different types, including ∼5000 bp and ∼6000 bp plasmids. The ∼5000 bp plasmids consisting of rep, lysR, mobB, and floR genes, exhibited high similarity among Pasteurellaceae bacteria. Furthermore, the ∼6000 bp plasmids, consisting of rep, lysR, mobC, mobA/L, and floR genes, showed high similarity between G. parasuis and Actinobacillus Spp. Notably, WGS results showed that the floR modules of the two types of plasmids could be transferred and integrated into the diverse Pasteurellaceae- origined plasmids. CONCLUSION: This study firstly reported the characterization of floR-carrying plasmids from A. indolicus and a non-virulent serovar of G. parasuis in pigs in China and elucidated the transmission mechanism of the floR resistance gene among the Pasteurellaceae family.


Subject(s)
Actinobacillus , Anti-Bacterial Agents , Animals , Swine , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Multilocus Sequence Typing , Phylogeny , Plasmids/genetics , Actinobacillus/genetics
3.
Viruses ; 15(8)2023 08 17.
Article in English | MEDLINE | ID: mdl-37632093

ABSTRACT

Porcine sapelovirus (PSV) is a ubiquitous virus in farmed pigs that is associated with SMEDI syndrome, polioencephalomyelitis, and diarrhea. However, there are few reports on the prevalence and molecular characterization of PSV in Fujian Province, Southern China. In this study, the prevalence of PSV and a poetical combinative strain PSV2020 were characterized using real-time PCR, sequencing, and bioinformatics analysis. As a result, an overall sample prevalence of 30.8% was detected in 260 fecal samples, and a farm prevalence of 76.7% was observed in 30 Fujian pig farms, from 2020 to 2022. Noteably, a high rate of PSV was found in sucking pigs. Bioinformatics analysis showed that the full-length genome of PSV2020 was 7550 bp, and the genetic evolution of its ORF region was closest to the G1 subgroup, which was isolated from Asia and America; the similarity of nucleotides and amino acids to other PSVs was 59.5~88.7% and 51.7~97.0%, respectively. However, VP1 genetic evolution analysis showed a distinct phylogenetic topology from the ORF region; PSV2020 VP1 was closer to the DIAPD5469-10 strain isolated from Italy than strains isolated from Asia and America, which comprise the G1 subgroup based on the ORF region. Amino acid discrepancy analysis illustrated that the PSV2020 VP1 gene inserted twelve additional nucleotides, corresponding to four additional amino acids (STAE) at positions 898-902 AAs. Moreover, a potential recombination signal was observed in the 2A coding region, near the 3' end of VP1, owing to recombination analysis. Additionally, 3D genetic evolutionary analysis showed that all reference strains demonstrated, to some degree, regional conservation. These results suggested that PSV was highly prevalent in Fujian pig farms, and PSV2020, a PSV-1 genotype strain, showed gene diversity and recombination in evolutionary progress. This study also laid a scientific foundation for the investigation of PSV epidemiology, molecular genetic characteristics, and vaccine development.


Subject(s)
Amino Acids , Enteroviruses, Porcine , Swine , Animals , Prevalence , Farms , Phylogeny , China/epidemiology , Genetic Variation , Recombination, Genetic
4.
Front Microbiol ; 14: 1153740, 2023.
Article in English | MEDLINE | ID: mdl-37260682

ABSTRACT

This study aimed to characterize two novel mcr-1 variants, mcr-1.35 and mcr-1.36, which originated from Moraxella spp. that were isolated from diseased pigs in China. The Moraxella spp. carrying novel mcr-1 variants were subjected to whole-genome sequencing (WGS) and phylogenetic analysis based on the 16S rRNA gene. The mcr-1 variants mcr-1.35 and mcr-1.36 were characterized using phylogenetic analysis, a comparison of genetic environments, and protein structure prediction. The WGS indicated that two novel mcr-1 variants were located in the chromosomes of three Moraxella spp. with a genetic environment of mcr-1-pap2. In addition to the novel colistin resistance genes mcr-1.35 and mcr-1.36, the three Moraxella spp. contained other antimicrobial resistance genes, including aac(3)-IId, tet(O), sul2, floR, and blaROB-3. A functional cloning assay indicated that either the mcr-1.35 or mcr-1.36 gene could confer resistance to colistin in Escherichia coli DH5α and JM109. The nucleotide sequences of mcr-1.35 and mcr-1.36 presented 95.33 and 95.33% identities, respectively, to mcr-1.1. The phylogenetic analysis showed that mcr-1.35 and mcr-1.36 were derived from Moraxella spp. that belonged to subclades that were different from those of the mcr-1 variants (mcr-1.1 to mcr-1.34 except mcr-1.10) originating from Enterobacteriaceae. The deduced amino acid sequences of MCR-1.35 (MCR-1.36) showed 96.67% (96.49%), 82.59% (82.04%), 84.07% (83.52%), 55.52% (55.17%), 59.75% (59.57%), and 61.88% (61.69%) identity to MCR-1.10, MCR-2.2, MCR-6.1, MCR-LIN, MCR-OSL, and MCR-POR, respectively, that originated from Moraxella sp. Notably, protein structure alignment showed only a few changes in amino acid residues between MCR-1.1 and MCR-1.35, as well as between MCR-1.1 and MCR-1.36. In conclusion, this study identified Moraxella spp. carrying two novel mcr-1 variants, mcr-1.35 and mcr-1.36, conferring resistance to colistin, which were isolated from pig farms in China. In addition, mcr-like variants were observed to be located in the chromosomes of some species of Moraxella isolated from pig samples.

5.
J Med Virol ; 95(1): e28228, 2023 01.
Article in English | MEDLINE | ID: mdl-36251622

ABSTRACT

Pseudorabies virus (PRV), as a neuroherpes virus, leads to heavy economic losses in the pig industry worldwide. This study was designed to establish recombinant PRV glycoprotein B (gB), C, and D proteins as PRV diagnostic antigens. The gB/C, gC/D, and gB/C/D fusion sequences were synthesized and inserted into pET-28a+ vector to generate the recombinant plasmids. The identified positive recombinant plasmids were transformed into BL21 Escherichia coli. The results of the polymerase chain reaction and enzyme digestion showed that the gB/C, gC/D, and gB/C/D fusion proteins were successfully expressed. An indirect sandwich ELISA was developed with the gB/C, gC/D, and gB/C/D as coating antigens. The results of indirect enzyme-linked immunosorbent assay (ELISA) analysis of 184 PRV-positive porcine sera showed that the positive coincidence rates of three recombinant proteins ELISAs relative to IDEXX kit were 98.25%, 95.32%, and 98.83%, and the negative coincidence rates were 85.71%, 75% and 100%, respectively. The inter and intra batch repeatability tests showed that the coefficient of variations of our kits were all less than 5%. Especially, the gB/C/D-ELISA has the highest specificity and sensitivity among the ELISA methods developed in this study. We established a series expression system of gB/C, gC/D, and gB/C/D antigen epitope genes and Recombinant protein-based indirect ELISA, providing new ideas for PV diagnosis and vaccine development.


Subject(s)
Herpesvirus 1, Suid , Pseudorabies , Animals , Swine , Pseudorabies/diagnosis , Pseudorabies/prevention & control , Recombinant Proteins , Viral Envelope Proteins , Enzyme-Linked Immunosorbent Assay/methods , Herpesvirus 1, Suid/genetics , Epitopes/metabolism , Antibodies, Viral
6.
Curr Microbiol ; 80(1): 50, 2022 Dec 21.
Article in English | MEDLINE | ID: mdl-36542185

ABSTRACT

The type II toxin-antitoxin (T-A) HicAB system is abundant in several bacteria and archaea, such as Escherichia coli, Burkholderia Pseudomallei, Yersinia pestis, Pseudomonas aeruginosa, and Streptococcus pneumoniae. This system engages in stress response, virulence, and bacterial persistence. This study showed that the biofilm-forming ability of the hicAB deletion mutant was significantly decreased to moderate ability compared to the extra-intestinal pathogenic Escherichia coli (ExPEC) parent strain and the complemented strain, which are strong biofilm producers. Congo red assay showed that the hicAB mutant maintained the ability to form curli fimbriae. Using RNA-seq and comparative real-time quantitative RT-PCR, we observed the difference in gene expression between the hicAB mutant and the parent strain, which was associated with biofilm formation. Our data indicate that the HicAB type II T-A system has a key role in biofilm formation by ExPEC, which may be associated with outer membrane protein (OMP) gene expression. Collectively, our results indicate that the hicAB type II T-A system is involved in ExPEC biofilm formation.


Subject(s)
Escherichia coli Infections , Escherichia coli Proteins , Extraintestinal Pathogenic Escherichia coli , Toxin-Antitoxin Systems , Humans , Escherichia coli , Extraintestinal Pathogenic Escherichia coli/genetics , Extraintestinal Pathogenic Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Toxin-Antitoxin Systems/genetics , Biofilms , Escherichia coli Infections/microbiology
7.
Microorganisms ; 10(10)2022 Sep 21.
Article in English | MEDLINE | ID: mdl-36296157

ABSTRACT

An emerging pseudorabies virus (PRV) variant has been reported on Bartha-K61-vaccinated farms since 2011, causing great economic losses to China's swine-feeding industry. In this study, two vaccines, FJ-2012ΔgE/gI-GEL02 and FJ-2012ΔgE/gI-206VG, were administered to piglets for immune efficacy investigation. Humoral immunity response, clinical signs, survival rate, tissue viral load, and pathology were assessed in piglets. The results showed that both vaccines were effective against the PRV FJ-2012 challenge, the piglets all survived while developing a high level of gB-specific antibody and neutralizing antibody, the virus load in tissue was alleviated, and no clinical PR signs or pathological lesions were displayed. In the unimmunized challenged group, typical clinical signs of pseudorabies were observed, and the piglets all died at 7 days post-challenge. Compared with commercial vaccines, the Bartha-K61 vaccine group could not provide full protection, which might be due to a lower vaccine dose; the inactivated vaccine vPRV* group piglets survived, displaying mild clinical signs. The asterisk denotes inactivation. These results indicate that FJ-2012ΔgE/gI-GEL02 and FJ-2012ΔgE/gI-206VG were effective and could be promising vaccines to control or eradicate the new PRV epidemic in China.

8.
Biomed Res Int ; 2020: 5673145, 2020.
Article in English | MEDLINE | ID: mdl-32420350

ABSTRACT

This study described a TaqMan based real-time fluorescent quantitative PCR (qPCR) method to detect porcine cytomegalovirus (PCMV) infection, targeting the conserved region of the DNA polymerase (DPOL) gene. The standard curve showed a linear regression relationship with a coefficient of 0.999 and a slope of y = -3.249x + 38.958 corresponding to the amplification efficiency at 99.8%. The limit of the qPCR method was 51.9 copies/µl. The established qPCR method showed excellent specificity, with no cross-reaction observed with common porcine pathogens. The coefficient of variation for intra-assay and interassay variability ranged up to 1.51% and 2.24%, respectively. PCMV positive signals can be found in semen using this qPCR method, which suggested that we should pay more attention to PCMV contamination in semen in order to eliminate PCMV infection in artificial insemination and xenotransplantation.


Subject(s)
Cytomegalovirus/genetics , Real-Time Polymerase Chain Reaction/methods , Semen/virology , Swine Diseases , Animals , Cytomegalovirus/isolation & purification , DNA-Directed DNA Polymerase/genetics , Male , Swine , Swine Diseases/diagnosis , Swine Diseases/virology , Viral Zoonoses/diagnosis , Viral Zoonoses/virology
9.
Biomed Res Int ; 2018: 1096079, 2018.
Article in English | MEDLINE | ID: mdl-30003086

ABSTRACT

We isolated an influenza strain named A/Swine/Fujian/F1/2010 (H1N2) from a pig suspected to be infected with swine flu. The results of electron microscopy, hemagglutination (HA) assay, hemagglutination inhibition (HI) assay, and whole genome sequencing analysis suggest that it was a reassortant virus of swine (H1N1 subtype), human (H3N2 subtype), and avian influenza viruses. To further study the genetic evolution of A/Swine/Fujian/F1/2010 (H1N2), we cloned its whole genome fragments using RT-PCR and performed phylogenetic analysis on the eight genes. As a result, the nucleotide sequences of HA, NA, PB1, PA, PB2, NP, M, and NS gene are similar to those of A/Swine/Shanghai/1/2007(H1N2) with identity of 98.9%, 98.9%, 99.0%, 98.6%, 99.0%, 98.9%, 99.3%, and 99.3%, respectively. Similar to A/Swine/Shanghai/1/2007(H1N2), we inferred that the HA, NP, M, and NS gene fragments of A/Swine/Fujian/F1/2010 (H1N2) strain were derived from classical swine influenza H3N2 subtype, NA and PB1 were derived from human swine influenza H3N2 subtype, and PB2 and PA genes were derived from avian influenza virus. This further validates the role of swine as a "mixer" for influenza viruses.


Subject(s)
Genes, Viral , Influenza A Virus, H1N2 Subtype/genetics , Phylogeny , Reassortant Viruses/genetics , Animals , China , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N2 Subtype/isolation & purification , Influenza A Virus, H3N2 Subtype/genetics , Influenza, Human , Mice , Mice, Inbred BALB C , Orthomyxoviridae Infections , Swine , Swine Diseases
10.
Open Life Sci ; 13: 217-226, 2018 Jan.
Article in English | MEDLINE | ID: mdl-33817086

ABSTRACT

Haemophilus parasuis is an important bacterium affecting pigs, causing Glässer's disease. To further characterize this species, we determined the complete genomic sequence of H. parasuis CL120103, which was isolated from diseased pigs. The strain H. parasuis CL120103 was identified as serovar 2. The size of the largest scaffold is 2,326,318 bp and contains 145 large contigs, with the N50 contig being 20,573 bp in length. The complete genome of H. parasuis CL120103 is 2,305,354 bp in length with 39.97% GC content and contains 2227 protein-coding genes, 19 ribosomal rRNA operons and 60 tRNA genes. Sequence similarity of the genome of H. parasuis CL120103 to the previously sequenced genome of H. parasuis was up to 96% and query cover to 86%. Annotation of the genome of H. parasuis CL120103 identified a number of genes encoding potential virulence factors. These virulence factors are involved in metabolism, adhesion, secretion and LPS biosynthesis. These related genes pave the way to better understand mechanisms underlying metabolic capabilities. The comprehensive genetic and phylogenetic analysis shows that H. parasuis is closely related to Actinobacillus pleuropneumoniae and provides a foundation for future experimental confirmation of the virulence and pathogen-host interactions in H. parasuis.

11.
Can J Infect Dis Med Microbiol ; 2017: 9073172, 2017.
Article in English | MEDLINE | ID: mdl-29527230

ABSTRACT

The outbreaks of pseudorabies have been frequently reported in Bartha-K61-vaccinated farms in China since 2011. To study the pathogenicity and evolution of the circulating pseudorabies viruses in Fujian Province, mainland China, we isolated and sequenced the whole genome of a wild-type pseudorabies virus strain named "FJ-2012." We then conducted a few downstream bioinformatics analyses including phylogenetic analysis and pathogenic analysis and used the virus to infect 6 pseudorabies virus-free piglets. FJ-2012-infected piglets developed symptoms like high body temperature and central nervous system disorders and had high mortality rate. In addition, we identified typical micropathological changes such as multiple gross lesions in infected piglets through pathological analysis and conclude that the FJ-2012 genome is significantly different from known pseudorabies viruses, in which insertions, deletions, and substitutions are observed in multiple immune and virulence genes. In summary, this study shed lights on the molecular basis of the prevalence and pathology of the pseudorabies virus strain FJ-2012. The genome of FJ-2012 could be used as a reference to study the evolution of pseudorabies viruses, which is critical to the vaccine development of new emerging pseudorabies viruses.

12.
J Zhejiang Univ Sci B ; 14(6): 460-7, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23733422

ABSTRACT

BACKGROUND: Epithelial-mesenchymal transition (EMT) is believed to be the critical process in malignant tumor invasion and metastases, and has a great influence on improving the survival rate in non-small-cell lung cancer (NSCLC) patients. Recent studies suggested that eukaryotic initiation factor 5A-2 (eIF5A-2) might serve as an adverse prognostic marker of survival. We detected eIF5A-2 in NSCLC A549 cells, and found that the invasive capability correlates with the eIF5A-2 expression. METHODS: Transforming growth factor (TGF)-ß1 was used to induce EMT in A549 cells. Western blotting, immunofluorescence, wound healing assay, and transwell-matrigel invasion chambers were used to identify phenotype changes. Western blotting was also used to observe changes of the expression of eIF5A-2. We down-regulated the eIF5A-2 expression using an eIF5A-2 siRNA and identified the phenotype changes by western blotting and immunofluorescence. We tested the change of migration and invasion capabilities of A549 cells by the wound healing assay and transwell-matrigel invasion chambers. RESULTS: After stimulating with TGF-ß1, almost all A549 cells changed to the mesenchymal phenotype and acquired more migration and invasion capabilities. These cells also had higher eIF5A-2 protein expression. Down-regulation of eIF5A-2 expression with eIF5A-2 siRNA transfection could change the cells from mesenchymal to epithelial phenotype and decrease tumor cell migration and invasive capabilities significantly. CONCLUSIONS: The expression of eIF5A-2 was up-regulated following EMT phenotype changes in A549 cells, which correlated with enhanced tumor invasion and metastatic capabilities. Furthermore, in the A549 cell line, the process of EMT phenotype change could be reversed by eIF5A-2 siRNA, with a consequent weakening of both invasive and metastatic capabilities.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/physiopathology , Down-Regulation/genetics , Epithelial-Mesenchymal Transition/genetics , Gene Knockdown Techniques/methods , Peptide Initiation Factors/genetics , RNA-Binding Proteins/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Movement/genetics , Cell Survival/genetics , Humans , Eukaryotic Translation Initiation Factor 5A
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