RESUMEN

AIM: This study aimed to develop a sensitive and specific recombinant antigen protein indirect enzyme-linked immunosorbent assay (ELISA) kit to detect the Shiga toxin (Stx)-producing Escherichia coli (STEC) antibodies against porcine edema disease (ED). METHODS AND RESULTS: The recombinant antigen was co-expressed with the STEC-derived Stx2e A2-fragment and Stx2e B protein in E. coli BL21(DE3) pLysS cells and purified using maltose-binding protein open columns. We used a Shiga-like toxin 2 antibody to test the specificity of the recombinant antigen in an indirect ELISA, which was detected in antigen-coated wells but not in uncoated wells. We tested the indirect ELISA system using samples from the STEC-immunized pig group, the commercial swine farm group, and healthy aborted fetal pleural effusion group; five and twenty samples, respectively, were positive for STEC in the former, whereas all three samples were negative for STEC in the latter. CONCLUSIONS: This newly developed indirect ELISA may be a specific method for diagnosing STEC infections in pigs.

Asunto(s)

Infecciones por Escherichia coli , Escherichia coli Shiga-Toxigénica , Enfermedades de los Porcinos , Porcinos , Animales , Infecciones por Escherichia coli/diagnóstico , Infecciones por Escherichia coli/veterinaria , Enfermedades de los Porcinos/diagnóstico , Ensayo de Inmunoadsorción Enzimática/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Edema

RESUMEN

Salmonella enterica is a leading cause of food-borne diseases in humans worldwide, resulting in severe morbidity and mortality. They are carried asymptomatically in the intestine or gallbladder of livestock, and are transmitted predominantly from animals to humans via the fecal-oral route. Thus, the best preventive strategy is to preemptively prevent transmission to humans by vaccinating livestock. Live attenuated vaccines have been mostly favored because they elicit both cellular and humoral immunity and provide long-term protective immunity. However, developing these vaccines is a laborious and time-consuming process. Therefore, most live attenuated vaccines have been mainly used for phenotypic screening using the auxotrophic replica plate method, and new types of vaccines have not been sufficiently explored. In this study, we used Radiation-Mutation Enhancement Technology (R-MET) to introduce a wide variety of mutations and attenuate the virulence of Salmonella spp. to develop live vaccine strains. The Salmonella Typhimurium, ST454 strain (ST WT) was irradiated with Cobalt60 gamma-irradiator at 1.5 kGy for 1 h to maximize the mutation rate, and attenuated daughter colonies were screened using in vitro macrophage replication capacity and in vivo mouse infection assays. Among 30 candidates, ATOMSal-L6, with 9,961-fold lower virulence than the parent strain (ST454) in the mouse LD50 model, was chosen. This vaccine candidate was mutated at 71 sites, and in particular, lost one bacteriophage. As a vaccine, ATOMSal-L6 induced a Salmonella-specific IgG response to provide effective protective immunity upon intramuscular vaccination of mice. Furthermore, when mice and sows were orally immunized with ATOMSal-L6, we found a strong protective immune response, including multifunctional cellular immunity. These results indicate that ATOMSal-L6 is the first live vaccine candidate to be developed using R-MET, to the best of our knowledge. R-MET can be used as a fast and effective live vaccine development technology that can be used to develop vaccine strains against emerging or serotype-shifting pathogens.

Asunto(s)

Refuerzo Biomédico , Vacunas contra la Salmonella , Animales , Anticuerpos Antibacterianos/genética , Femenino , Humanos , Inmunoglobulina G/genética , Ratones , Mutación , Vacunas contra la Salmonella/genética , Salmonella typhimurium , Porcinos , Vacunas Atenuadas

RESUMEN

Salmonella enterica subsp. enterica serovar Gallinarum (SG) is a common pathogen in chickens, and causes an acute systemic disease that leads to high mortality. The live attenuated vaccine 9R is able to successfully protect chickens older than six weeks by activating a robust cell-mediated immune response, but its safety and efficacy in young chickens remains controversial. An inactivated SG vaccine is being used as an alternative, but because of its low cellular immune response, it cannot be used as a replacement for live attenuated 9R vaccine. In this study, we employed gamma irradiation instead of formalin as an inactivation method to increase the efficacy of the inactivated SG vaccine. Humoral, cellular, and protective immune responses were compared in both mouse and chicken models. The radiation-inactivated SG vaccine (r-SG) induced production of significantly higher levels of IgG2b and IgG3 antibodies than the formalin-inactivated vaccine (f-SG), and provided a homogeneous functional antibody response against group D, but not group B Salmonella. Moreover, we found that r-SG vaccination could provide a higher protective immune response than f-SG by inducing higher Th17 activation. These results indicate that r-SG can provide a protective immune response similar to the live attenuated 9R vaccine by activating a higher humoral immunity and a lower, but still protective, cellular immune response. Therefore, we expect that the radiation inactivation method might substitute for the 9R vaccine with little or no side effects in chickens younger than six weeks.

Asunto(s)

Inmunidad Celular , Inmunidad Humoral , Enfermedades de las Aves de Corral/prevención & control , Salmonelosis Animal/prevención & control , Vacunas contra la Salmonella/inmunología , Vacunas de Productos Inactivados/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Citocinas/metabolismo , Inmunización , Lipopolisacáridos/inmunología , Ratones , Vacunas contra la Salmonella/administración & dosificación , Salmonella enterica/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/efectos de la radiación

RESUMEN

A previous study highlighted that mastoparan V1 (MP-V1), a mastoparan from the venom of the social wasp Vespula vulgaris, is a potent antimicrobial peptide against Salmonella infection, which causes enteric diseases. However, there exist some limits for its practical application due to the loss of its activity in an increased bacterial density and the difficulty of its efficient production. In this study, we first modulated successfully the antimicrobial activity of synthetic MP-V1 against an increased Salmonella population using protease inhibitors, and developed an Escherichia coli secretion system efficiently producing active MP-V1. The protease inhibitors used, except pepstatin A, significantly increased the antimicrobial activity of the synthetic MP-V1 at minimum inhibitory concentrations (determined against 106 cfu/mL of population) against an increased population (108 cfu/mL) of three different Salmonella serotypes, Gallinarum, Typhimurium and Enteritidis. Meanwhile, the E. coli strain harboring OmpA SS::MP-V1 was identified to successfully secrete active MP-V1 into cell-free supernatant, whose antimicrobial activity disappeared in the increased population (108 cfu/mL) of Salmonella Typhimurium recovered by adding a protease inhibitor cocktail. Therefore, it has been concluded that our challenge using the E. coli secretion system with the protease inhibitors is an attractive strategy for practical application of peptide toxins, such as MP-V1.

Asunto(s)

Antibacterianos/farmacología , Escherichia coli/metabolismo , Péptidos/farmacología , Inhibidores de Proteasas/farmacología , Salmonella/efectos de los fármacos , Venenos de Avispas/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Escherichia coli/genética , Péptidos y Proteínas de Señalización Intercelular , Pruebas de Sensibilidad Microbiana , Péptidos/genética , Plásmidos , Salmonella/crecimiento & desarrollo , Venenos de Avispas/biosíntesis , Venenos de Avispas/genética

RESUMEN

In this study, we evaluated the immune responses of avian metapneumovirus harboring chicken Fc molecule. Stable Vero cells expressing chicken Fc chimera on its surface (Vero-cFc) were established, and we confirmed that aMPV grown in Vero-cFc incorporated host derived chimera Fc into the aMPV virions. Immunization of chicken with aMPV-cFc induced higher level of antibodies and inflammatory cytokines; (Interferon (IFN)-γ and Interleukin (IL)-1ß) compared to those of aMPV. The increased levels of antibodies and inflammatory cytokines in chicken immunized with aMPV-cFc were statistically significantly (p<0.05) to that of aMPV and control. The aMPV-cFc group also generated the highest neutralizing antibody response. After challenges, chickens immunized with aMPV-cFc showed much less pathological signs in nasal turbinates and trachea so that we could confirm aMPV-cFc induced higher protection than that of aMPV. The greater ability of aMPV harboring chicken Fc to that of aMPV presented it as a possible vaccine candidate.

Asunto(s)

Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Pollos/inmunología , Fragmentos Fc de Inmunoglobulinas/genética , Infecciones por Paramyxoviridae/veterinaria , Enfermedades de las Aves de Corral/prevención & control , Vacunas Virales/administración & dosificación , Animales , Pollos/virología , Chlorocebus aethiops , Expresión Génica , Inmunización , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-1beta/biosíntesis , Interleucina-1beta/inmunología , Metapneumovirus/efectos de los fármacos , Metapneumovirus/crecimiento & desarrollo , Metapneumovirus/inmunología , Infecciones por Paramyxoviridae/inmunología , Infecciones por Paramyxoviridae/prevención & control , Infecciones por Paramyxoviridae/virología , Plásmidos/química , Plásmidos/inmunología , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/virología , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Células Vero , Proteínas Virales/genética , Proteínas Virales/inmunología , Vacunas Virales/genética , Vacunas Virales/inmunología

RESUMEN

AIMS: We were to examine the effect of bradykinin (BK) in the regulation of glutamate transporter and its related signaling molecules in a human retinal pigment epithelial (ARPE) cells, which are important cells to support retina. MAIN METHODS: d-[2,3-(3)H]-aspartate uptake, western immunoblotting, reverse transcription polymerase chain reaction, [(3)H]-arachidonic acid release, and siRNA transfection techniques were used. KEY FINDINGS: BK stimulated glutamate uptake as well as the mRNA expression of excitatory amino acid transporter 4 (EAAT4) and excitatory amino acid carrier 1 (EAAC1), which was blocked by treatment with bradykinin 1 receptor (B1R) and bradykinin 2 receptor (B2R) siRNA, suggesting the role of B1R and B2R in this process. The BK-induced stimulation of glutamate uptake was also blocked by [des-Arg(10)]-HOE 140, a B1R antagonist, and HOE 140, a B2R antagonist, as well as by the tyrosine kinase inhibitors genistein and herbimycin A. In addition, the BK-induced stimulation of glutamate uptake was blocked by treatment with the phospholipase A(2) inhibitors mepacrine and AACOCF(3), the cyclooxygenase (COX) inhibitor indomethacin, and the COX-2 inhibitor Dup 697. Furthermore, the BK-induced increase in COX-2 expression was blocked by the PI-3 kinase inhibitors wortmannin and LY294002, Akt inhibitor, and the protein kinase C (PKC) inhibitors staurosporine and bisindolylmaleimide I, suggesting the role of PI-3 kinase and PKC in this process. BK stimulated Akt activation and the translocation of PKC activation via the activation of B1R and B2R. SIGNIFICANCE: BK stimulates glutamate uptake through a PKC-Akt-COX-2 signaling cascade in ARPE cells.

Asunto(s)

Sistema de Transporte de Aminoácidos X-AG/metabolismo , Ácido Aspártico/metabolismo , Bradiquinina/farmacología , Células Epiteliales/efectos de los fármacos , Receptor de Bradiquinina B1/biosíntesis , Receptor de Bradiquinina B2/biosíntesis , Epitelio Pigmentado de la Retina/metabolismo , Ácido Araquidónico/metabolismo , Western Blotting , Técnicas de Cultivo de Célula , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cicloheximida/farmacología , Ciclooxigenasa 2/biosíntesis , Dactinomicina/farmacología , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Humanos , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor de Bradiquinina B1/genética , Receptor de Bradiquinina B2/genética , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transfección