RESUMEN
OBJECTIVES: The aim of this study was to characterise the whole genome sequence of multidrug-resistant Streptococcus pluranimalium strain SP21-2 of swine origin in China. METHODS: Illumina Miseq (200X coverage) and Nanopore PromethION platform (100X coverage) were used for genome sequencing. Rapid Annotation using Subsystem Technology (RAST) was used to annotate the genome of SP21-2. The antimicrobial resistance genes (ARGs) were identified using ResFinder-4.1. RESULTS: The assembled circular genome of S. pluranimalium SP21-2 was 1,987,058 bp in length with a GC content of 39.54%, and no plasmid sequence was detected. A total of 2086 coding sequences were predicted by RAST. Oxazolidinone-phenicol resistance gene, optrA, and pleuromutilin-lincosamide-streptogramin A resistance gene, lsa(E), are both located on chromosomes, associated with IS1216 and ISS1S, respectively. In addition, SP21-2 harbours lnu(B) (lincosamide), ant (6)-Ia and aac(6')-aph(2") (aminoglycoside), erm(B) (macrolide), and tet(O) (tetracycline). CONCLUSION: We firstly report the oxazolidinone-phenicol gene, optrA, and pleuromutilin-lincosamide-streptogramin A resistance gene, lsa(E), in S. pluranimalium. In this strain, we firstly identified ISS1S and IS1216 carrying ARGs in S. pluranimalium, which will provide a valuable reference to understanding potential transfer mechanisms of ARGs in S. pluranimalium.
Asunto(s)
Antiinfecciosos , Oxazolidinonas , Animales , Porcinos , Estreptogramina A , Antibacterianos/farmacología , Lincosamidas , Cromosomas , PleuromutilinasRESUMEN
Antimicrobial resistance is a rapidly evolving and extremely complex issue, particularly due to the use of various types of antimicrobials within human, animal, and environmental sectors. Pleuromutilin antibiotics are used to prevent and control respiratory diseases in the rearing stage of hen chicks, but the current status of pleuromutilin resistance in the laying hen breeding process is unclear. ATP-binding cassette transporters encoded by lsa(A), lsa(E), lsa(C), and vga(D) can be transferred by plasmids and transposons, thereby posing a potential dissemination risk. To investigate pleuromutilin resistance genes in the laying hen production chain in China, 95 samples from five environmental types were collected in four breeding stages to determine the abundances of the main resistance genes by qPCR, i.e. lsa(A), lsa(E), lsa(C), and vga(D). The abundance (5.16 log10GC/g) and detection rate (100%) of lsa(E) was highest in all of the samples, thereby suggesting high contamination with the lsa(E) gene across the large-scale laying hen breeding environment and feces. The lsa(A) (6.02 log10GC/g) and lsa(E) (6.18 log10GC/g) genes were most abundant in flies, and the abundance of vga(D) (4.50 log10GC/g) was highest in dust (P < .05). In addition to feces, flies and dust were important sources of contamination with pleuromutilin resistance along the laying hen production chain. In summary, we determined the abundances of four pleuromutilin resistance genes in the laying hen production chain and provided direct evidence of pleuromutilin resistance transmission and environmental contamination. In particular, the chicken breeding stage needs further attention.
Asunto(s)
Antibacterianos , Pollos , Animales , Femenino , Humanos , Antibacterianos/farmacología , Prevalencia , Farmacorresistencia Bacteriana Múltiple/genética , Polvo , PleuromutilinasRESUMEN
Shared bicycles are prevailing in China but the extent to which they contribute to maintaining and transmitting pathogens and antibiotic-resistant bacteria remain largely unknown. To fill the knowledge gap, herein, swab samples (n = 963) were collected from handlebars of shared bicycles in areas of hospital, school, metro station (n = 887) and riders (n = 76) in Chengdu, China. Staphylococci (n = 241) and Enterococci (n = 69) were widely distributed across sampling locations at a frequency of 2.3%-12.9%, and 0.08%-5.5%, respectively. Bicycle or rider-borne Gram-positive bacteria were frequently resistant to clinically important antibiotics including linezolid, fosfomycin, and vancomycin, and a significant portion of these isolates (3.4%-16.6% for Staphylococci and 0.1%-13.8% for Enterococci) indicated multidrug resistance. Nineteen Staphylococcus aureus isolates were identified in this collection and 52.6% of which were considered as methicillin-resistant S. aureus. Whole genome sequencing further characterized 26 antimicrobial resistance genes (ARGs) including fosB, fusB, and lnu(G) in S. aureus and 21 ARGs including optrA in Enterococci. Leveraging a complementary approach with conventional MLST, whole genome SNP and MLST analyses, we present that genetically closely-related bacteria were found in bicycles and riders across geographical-distinct locations suggesting bacterial transmission. Further, five new ST types 5697-5701 were firstly characterized in S. aureus. ST 942 and ST 1640 are new ST types observed in E. faecalis, and E. faecium, respectively. Our results highlighted the risk of shared bicycle system in disseminating pathogens and antibiotic resistance which warrants effective disinfections.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Staphylococcus , Antibacterianos , Ciclismo , China , Enterococcus , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Prevalencia , Staphylococcus aureusRESUMEN
OBJECTIVES: This study aimed to determine the genetic environment of antimicrobial resistance genes (ARGs) in Erysipelothrix rhusiopathiae strain ZJ isolated from a pig with symptoms of swine erysipelas in China. METHODS: Illumina MiSeq (200× coverage) and PacBio RS II (100× coverage) platforms were used for genome sequencing. ARGs and prophages were identified using ResFinder 3.0 and PHASTER, respectively. A conjugation experiment, induced prophage infection and long-term passage assay were performed to determine the transferability and stability of ARGs in this strain. RESULTS: The assembled circular genome of E. rhusiopathiae ZJ was 1 945 689 bp with a GC content of 36.48%; no plasmid sequence was detected. Eleven acquired ARGs were identified in the genome. A novel integrative and conjugative element (ICE) encoding a multidrug resistance (MDR) gene cluster [aadE-apt-spw-lsa(E)-lnu(B)-aadE-sat4-aphA3] was identified in strain ZJ. A prophage Φ1605 harbouring mef(A)-msr(D) and tet(M) was also found in this strain, which can take a circular form and can be induced by mitomycin C to infect E. rhusiopathiae G4T10 for ARG transfer. CONCLUSION: To our knowledge, this is the first report of a complete genome sequence of E. rhusiopathiae carrying multiple ARGs obtained from a pig farm. This is the first identification of a novel chimeric ICE carrying a MDR gene cluster and a prophage carrying ARGs in E. rhusiopathiae, which will provide a valuable reference to understand the potential transfer mechanism of MDR gene clusters carried by ICEs and prophages in Gram-positive bacteria.
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Erysipelothrix , Erisipela Porcina , Animales , Antibacterianos/farmacología , China , Farmacorresistencia Bacteriana , Erysipelothrix/genética , PorcinosRESUMEN
Human salmonellosis caused by the consumption of eggs and chicken meat contaminated with Salmonella Enteritidis has become a continuing public health concern worldwide. In this study we adopted whole genome sequencing (WGS) to determine the genetic relationship and antimicrobial resistance of S. enterica strains isolated from a poultry breeding enterprise that consists of one breeding chicken farm, one egg hatchery and one commercial chicken farm. A total of 148 S. enterica including 147 S. Enteritidis strains were isolated from 2100 fecal swab samples, with 16 (5.3 %, 16/300) from breeding chicken farm, 38 (4.2 %, 38/900) from egg hatchery and 94 (10.4 %, 94/900) from commercial chicken farm. WGS revealed that all 147 S. Enteritidis strains belonged to ST11, and further divided into 4 different ribosomal STs and 64 core genome STs. Single nucleotide polymorphism typing suggested the presence of the vertical transmission of S. Enteritidis from breeding chicken to commercial chicken. Three different antimicrobial-resistant plasmids including one blaCTX-M-14-carrying plasmid and two virulence-resistance plasmids were characterized, resulting in the heterogeneous antimicrobial resistance of clonally related S. Enteritidis strains. Routine surveillance in breeding chicken farms is conducive to the control of S. Enteritidis from farm to fork.
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Farmacorresistencia Bacteriana/genética , Transmisión Vertical de Enfermedad Infecciosa/veterinaria , Enfermedades de las Aves de Corral/microbiología , Aves de Corral/microbiología , Salmonella enteritidis/efectos de los fármacos , Animales , Antibacterianos/farmacología , Cruzamiento , Pollos/microbiología , China , Granjas , Femenino , Masculino , Óvulo/microbiología , Plásmidos/genética , Polimorfismo de Nucleótido Simple , Enfermedades de las Aves de Corral/transmisión , Salmonelosis Animal/epidemiología , Salmonelosis Animal/transmisión , Salmonella enteritidis/genética , Virulencia/genética , Secuenciación Completa del GenomaRESUMEN
Salmonella enterica serovar Indiana (S. Indiana) was the most frequently reported foodborne pathogen, which has a broad host range including poultry, swine, and humans. Traditional methods used for the detection of S. Indiana from contaminated food products are time-consuming and labor-intensive. Therefore, rapid detection methods with high sensitivity and specificity are vitally important to prevent the spread of S. Indiana. In this study, we developed a nearly instrument-free, simple molecular method which incorporates cross-priming amplification (CPA) combined with a nucleic acid detection strip (NADS) for sensitive detection of S. Indiana. A set of CPA primers was designed based on S. Indiana specific nucleotide sequences and the specificity of CPA-NADS was tested against 42 bacterial strains. The results showed that this method was highly specific for detection of S. Indiana. The sensitivity of CPA-NADS was evaluated and compared with that of the serovar-specific PCR method and the real-time PCR method. The limit of detection of the CPA method was 8.997â¯fg/µL for genomic DNA and 6.2â¯×â¯101â¯CFU/mL for bacteria in pure culture. An application of the CPA assay was conducted with 90 inoculated specimens by S. Indiana. The accuracy of CPA-NADS was consistent with the results of the traditional culture-based methods in inoculated specimens. This method showed a higher sensitivity than the serovar-specific PCR method did and was more convenient to perform. In conclusion, we demonstrated that the CPA-NADS system offers high specificity, sensitivity, rapidity, and a simple detection tool for screening S. Indiana.
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ADN Bacteriano/aislamiento & purificación , Análisis de los Alimentos/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Salmonella enterica/aislamiento & purificación , Serogrupo , Cartilla de ADN/genética , Contaminación de Alimentos/análisis , Microbiología de Alimentos , Límite de Detección , Tiras Reactivas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
A total of 108 meropenem-resistant Enterobacteriaceae isolates were obtained from 1,658 rectal swabs collected from 15 unrelated commercial chicken farms in China between 2014 and 2016. These samples yielded 16 Escherichia coli and 2 Klebsiella pneumoniae isolates of diverse sequence types carrying a blaNDM-5-bearing IncX3 plasmid. K. pneumoniae strain sequence type 709 (ST709) has two blaNDM-5-carrying plasmids that were transferred together to E.coli.
Asunto(s)
Antibacterianos/farmacología , Enterobacteriaceae/efectos de los fármacos , Animales , Pollos , China , Granjas , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Pruebas de Sensibilidad Microbiana , Plásmidos/genéticaRESUMEN
Objectives: To identify a novel plasmid-mediated colistin resistance gene in Klebsiella pneumoniae isolated from chickens in China. Methods: WGS was used to identify a novel colistin resistance gene. The transferability of plasmids carrying mcr-7.1 was investigated by conjugation experiments. The expression of the mcr-7.1 gene was examined using an expression vector. Results: A novel plasmid-mediated colistin resistance gene mcr-7.1, sharing 70% amino acid identity with the mcr-3 gene, was identified in three K. pneumoniae strains isolated from chickens in China. The mcr-7.1 gene was found in an IncI2-type plasmid (pSC20141012) that co-harboured the blaCTX-M-55 gene in one isolate. pSC20141012 can be transferred from K. pneumoniae SC20141012 to Escherichia coli J53Azr, exhibiting a ≥8-fold increase in colistin MIC compared with the recipient E. coli J53Azr. Conclusions: We identified a novel plasmid-mediated colistin resistance gene named mcr-7.1 in K. pneumoniae in China. The prevalence of mcr-7.1 in various species of human and animal origin needs to be investigated immediately.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Colistina/farmacología , Farmacorresistencia Bacteriana/genética , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Animales , Proteínas Bacterianas/aislamiento & purificación , Pollos/microbiología , China , Genoma Bacteriano , Klebsiella pneumoniae/enzimología , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Secuenciación Completa del Genoma , beta-Lactamasas/genéticaRESUMEN
A colistin-resistant Escherichia coli isolate from a commercial poultry farm in China carried two colistin resistance genes, mcr-1 and variant of mcr-3, in an IncP plasmid. The variant of the mcr-3 gene, named mcr-3.11, encoded two amino acid substitutions compared with the mcr-3 gene. A novel genetic structure, ISKpn40-mcr-3-dgkA-ISKpn40, might be the key element mediating the translocation of mcr-3 through the formation of a circular form. The mcr-1 and mcr-3 genes, which are colocated on a plasmid, might pose a huge threat to public health.
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Colistina/farmacología , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Plásmidos/genética , Polimixinas/farmacología , Animales , Antibacterianos , Pollos , Farmacorresistencia Bacteriana , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Granjas , Pruebas de Sensibilidad Microbiana , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
A novel 61,578-bp genomic island named Proteus genomic island 2 (PGI2) was characterized in Proteus mirabilis of swine origin in China. The 23.85-kb backbone of PGI2 is related to those of Salmonella genomic island 1 and Acinetobacter genomic island 1. The multidrug resistance (MDR) region of PGI2 is a complex class 1 integron containing 14 different resistance genes. PGI2 was conjugally mobilized in trans to Escherichia coli in the presence of a conjugative IncC helper plasmid.
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Islas Genómicas/genética , Proteus mirabilis/efectos de los fármacos , Proteus mirabilis/genética , Animales , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Integrones/genética , Salmonella/efectos de los fármacos , Salmonella/genética , PorcinosRESUMEN
A novel 65.8-kb multidrug resistance transposon, designated Tn6450, was characterized in a Proteus mirabilis isolate from chicken in China. Tn6450 contains 18 different antimicrobial resistance genes, including cephalosporinase gene blaDHA-1 and fluoroquinolone resistance genes qnrA1 and aac(6')-Ib-cr It carries a class 1/2 hybrid integron composed of intI2 and a 3' conserved segment of the class 1 integron. Tn6450 is derived from Tn7 via acquisition of new mobile elements and resistance genes.
Asunto(s)
Elementos Transponibles de ADN/genética , Farmacorresistencia Bacteriana Múltiple/genética , Proteus mirabilis/genética , Animales , Antibacterianos/farmacología , Pollos , China , ADN Bacteriano/genética , Fluoroquinolonas/farmacología , Integrones/genética , Proteus mirabilis/efectos de los fármacosRESUMEN
The novel 63,558-bp plasmid pSA-01, which harbors nine antibiotic resistance genes, including cfr, erm(C), tet(L), erm(T), aadD, fosD, fexB, aacA-aphD, and erm(B), was characterized in Staphylococcus arlettae strain SA-01, isolated from a chicken farm in China. The colocation of cfr and fosD genes was detected for the first time in an S. arlettae plasmid. The detection of two IS431-mediated circular forms containing resistance genes in SA-01 suggested that IS431 may facilitate dissemination of antibiotic resistance genes.
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Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Fosfomicina/farmacología , Plásmidos/química , Infecciones Estafilocócicas/veterinaria , Staphylococcus/genética , Animales , Proteínas Bacterianas/metabolismo , Pollos , China/epidemiología , Granjas , Secuenciación de Nucleótidos de Alto Rendimiento , Pruebas de Sensibilidad Microbiana , Plásmidos/metabolismo , Enfermedades de las Aves de Corral/epidemiología , Enfermedades de las Aves de Corral/microbiología , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología , Staphylococcus/efectos de los fármacos , Staphylococcus/aislamiento & purificación , Staphylococcus/metabolismoRESUMEN
IncC plasmids are of great concern as vehicles of broad-spectrum cephalosporins and carbapenems resistance genes blaCMY and blaNDM. The aim of this study was to sequence and characterize a multidrug resistance (MDR) IncC plasmid (pPm14C18) recovered from Proteus mirabilis. pPm14C18 was identified in a CMY-2-producing P. mirabilis isolate from chicken in China in 2014, and could be transferred to Escherichia coli conferring an MDR phenotype. Whole genome sequencing confirmed pPm14C18 was a novel type 1/2 hybrid IncC plasmid 165,992bp in size, containing fifteen antimicrobial resistance genes. It harboured a novel MDR mosaic region comprised of a hybrid Tn21tnp-pDUmer, in which blaCTX-M-65, dfrA32 and ereA were firstly reported in IncC plasmid. Phylogenetic relationship reconstruction based on the nucleotide sequences of the 52 IncC backbones showed all type 1 IncC plasmids were clustered into one clade, and then merged with pPm14C18 and finally with the type 2 IncC plasmids and another type 1/2 hybrid IncC plasmid pYR1. The MDR IncC plasmids in P. mirabilis of animal origin might threaten public health, which should be drawn more attention.
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Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Genes Bacterianos/genética , Plásmidos/genética , Proteus mirabilis/genética , Animales , Pollos/microbiología , China , Escherichia coli/genética , FilogeniaRESUMEN
Recently, a novel variant of the CTX-M enzyme, CTX-M-98, was detected in Escherichia coli isolates from food animals. However, few plasmids carrying blaCTX-M-98 have been fully characterized. In this study, we sequenced the complete pHeBE7 plasmid, an 86,015-bp plasmid that contains the blaCTX-M-98b, blaTEM-1, rmtB, and traT genes, using whole-genome sequencing. The backbone of pHeBE7 shows a high similarity (>99%) to pMC-NDM, which carries the blaNDM-1 gene, however its mosaic regions remain relatively unique among sequenced plasmids. We discovered that a typical ISEcp1-blaCTX-M-IS903 element in the mosaic region harbors the blaCTX-M-98b gene. Conjugation and growth competition assays indicate that pHeBE7 can be easily transmitted and that it confers a limited fitness cost to the recipient cell. The genetic characterization of pHeBE7 may improve our knowledge of how antibiotic resistance disseminates in enterobacteria.
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Escherichia coli/genética , Plásmidos/genética , Animales , Antibacterianos/farmacología , Pollos/microbiología , Conjugación Genética , Escherichia coli/aislamiento & purificación , Genes Bacterianos , Hígado/microbiología , Pruebas de Sensibilidad Microbiana , Enfermedades de las Aves de Corral/microbiología , Virulencia/genética , Resistencia betalactámica/genética , beta-Lactamasas/genética , beta-Lactamas/farmacologíaRESUMEN
The mcr-1 gene was detected in 5.11% (58/1136) of Escherichia coli isolates of chicken origin from 13 provinces in China. A novel mcr-1 variant, named mcr-1.3, encoding an Ile-to-Val functional variant of MCR-1 was identified in a sequence type 155 (ST155) strain. An mcr-1.3-containing IncI2 plasmid, pHeN867 (60,757 bp), was identified. The transfer of pHeN867 led to a 32-fold increase in the MIC of colistin in the recipient, exhibiting an effect on colistin resistance that was similar to that of mcr-1.
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Antibacterianos/farmacología , Pollos/microbiología , Colistina/farmacología , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Enfermedades de las Aves de Corral/microbiología , Animales , Secuencia de Bases , China , Farmacorresistencia Bacteriana/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/veterinaria , Transferencia de Gen Horizontal , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Análisis de Secuencia de ADN , beta-Lactamasas/genéticaRESUMEN
SXT/R391 integrative and conjugative elements (ICEs) were detected in 8 out of 125 Proteus mirabilis isolates from food-producing animals in China. Whole-genome sequencing revealed that seven ICEs were identical to ICEPmiJpn1, carrying the cephalosporinase gene blaCMY-2. Another one, designated ICEPmiChn1, carried five resistance genes. All eight ICEs could be transferred to Escherichia coli via conjugation. The results highlight the idea that animal farms are important reservoir of the SXT/R391 ICE-containing P. mirabilis.
Asunto(s)
Elementos Transponibles de ADN/genética , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/genética , Proteus mirabilis/genética , Proteus mirabilis/aislamiento & purificación , beta-Lactamasas/genética , Animales , Proteínas Bacterianas/genética , Secuencia de Bases , Pollos , China , Conjugación Genética/genética , ADN Bacteriano/genética , Escherichia coli/efectos de los fármacos , Granjas , Genoma , Pruebas de Sensibilidad Microbiana , Enfermedades de las Aves de Corral/microbiología , Análisis de Secuencia de ADN , Porcinos , Enfermedades de los Porcinos/microbiologíaRESUMEN
OBJECTIVES: To develop a cost-effective ELISA for detection of antibodies against infectious bronchitis virus (IBV) by using a multi-fragment protein as coating antigen. RESULTS: A multi-fragment antigen, termed BE, which was composed of eight antigenic fragments selected from the three major proteins (S, M, and N) of IBV, was expressed in Escherichia coli. The entire protein had a molecular weight of 61.5 kDa. In addition to it, a smaller truncated protein was also produced; both could react with IBV-positive serum. Next, an indirect ELISA (BE-ELISA) was developed. Coefficients of variation of this assay were lower than 10 %, and no cross-reactivity between the coated antigen BE and antiserum against newcastle disease virus, avian influenza virus, or infectious bursal disease virus was observed. The performance of BE-ELISA was evaluated, and showed 95.4 % coincidence ratio with the whole virus based-ELISA (IDEXX). CONCLUSIONS: The multi-fragment antigen (BE) may represent a promising alternative to the whole virus without safety problems, and this newly established ELISA provides an effective method for anti-IBV antibody detection.
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Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Infecciones por Coronavirus/veterinaria , Virus de la Bronquitis Infecciosa/inmunología , Enfermedades de las Aves de Corral/diagnóstico , Pruebas Serológicas/métodos , Animales , Antígenos Virales/genética , Infecciones por Coronavirus/diagnóstico , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática/métodos , Escherichia coli/genética , Expresión Génica , Virus de la Bronquitis Infecciosa/genética , Peso Molecular , Enfermedades de las Aves de Corral/virología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Four different Salmonella genomic island 1 (SGI1) variants, including two novel variants, were characterized in one Salmonella enterica serovar Rissen sequence type ST1917 isolate and three Proteus mirabilis isolates from swine farms in China. One novel variant was derived from SGI1-B with the backbone gene S021 disrupted by a 12.72-kb IS26 composite transposon containing the dfrA17-aadA5 cassettes and macrolide inactivation gene cluster mphA-mrx-mphR. The other one was an integron-free SGI1 and contained a 183-bp truncated S025 next to IS6100 and S044.
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Islas Genómicas/genética , Proteus mirabilis/genética , Salmonella/genética , Animales , China , Elementos Transponibles de ADN/genética , Variación Genética , Datos de Secuencia Molecular , Familia de Multigenes/efectos de los fármacos , Salmonella enterica/efectos de los fármacos , Salmonella enterica/genética , Porcinos/microbiologíaRESUMEN
An indirect enzyme-linked immunosorbent assay (ELISA) method based on a novel multi-epitope antigen of S protein (SE) was developed for antibodies detection against infectious bronchitis virus (IBV). The multi-epitope antigen SE protein was designed by arranging three S gene fragments (166-247 aa, S1 gene; 501-515 aa, S1 gene; 8-30 aa, S2 gene) in tandem. It was identified to be approximately 32 kDa as a His-tagged fusion protein and can bind IBV positive serum by western blot analysis. The conditions of the SE-ELISA method were optimized. The optimal concentration of the coating antigen SE was 3.689 µg/mL and the dilution of the primary antibodies was identified as 1:1000 using a checkerboard titration. The cut-off OD450 value was established at 0.332. The relative sensitivity and specificity between the SE-ELISA and IDEXX ELISA kit were 92.38 and 89.83%, respectively, with an accuracy of 91.46%. This assay is sensitive and specific for detection of antibodies against IBV.