RESUMEN
Porcine edema disease (ED) is an enterotoxaemia that frequently occurs in 4-12 week-old piglets and results in high mortality. ED is caused by Shiga toxin 2e (Stx2e), produced by host-adapted Shiga toxin-producing Escherichia coli (STEC) strains. We constructed a recombinant protein in which the B subunit of Stx2e (Stx2eB) was linked to Cartilage Oligomeric Matrix Protein (COMP)'s pentameric domain to enhance antigenicity to induce neutralizing antibodies against Stx2e. We evaluated the efficacy of this antigen as a vaccine on the farm where ED had occurred. The suckling piglets were divided into two groups. The pigs in the vaccinated group were intramuscularly immunized with the vaccine containing 30 µg/head of Stx2eB-COMP at 1 and 4 weeks of age. The control pigs were injected with saline instead of the vaccine. The neutralizing antibody titer to Stx2e, mortality, clinical score, and body weight was evaluated up to 11 weeks after the first vaccination. In the vaccinated group, the Stx2e neutralizing antibody was detected 3 weeks after the first vaccination, its titer increased during the following weeks. The antibody was not detected in the control group during the test period. The STEC gene was detected in both groups during the test period, but a typical ED was observed only in control pigs; the mortality and clinical score were significantly lower in the vaccinated group than in the control group. These data indicate that the pentameric B subunit vaccine is effective for preventing ED and offers a promising tool for pig health control.
Asunto(s)
Antitoxinas , Edematosis Porcina , Infecciones por Escherichia coli , Escherichia coli Shiga-Toxigénica , Enfermedades de los Porcinos , Animales , Porcinos , Toxina Shiga II/genética , Infecciones por Escherichia coli/prevención & control , Infecciones por Escherichia coli/veterinaria , Edematosis Porcina/prevención & control , Anticuerpos Neutralizantes , Vacunas de Subunidad , Edema/prevención & control , Edema/veterinaria , Enfermedades de los Porcinos/prevención & controlRESUMEN
The development of functional protein production systems using transgenic plants as hosts has been rapidly progressing in recent years. Lettuce (Lactuca sativa L.) has been studied as one such host, and it has been reported that the biomass of lettuce per area and target protein expression level can be increased by optimizing the cultivation conditions. Therefore, we investigated methods to minimize the input light energy per target protein to reduce production costs. Herein, we examined the yield of a nontoxic B subunit of Stx2e (Stx2eB) from transgenic lettuce under various cultivation conditions. Stx2eB acts as a vaccine against swine edema disease. The effects of photon flux densities (PPFDs), photoperiod, and light source on Stx2eB production were examined and the findings suggested that 400 µmol m-2 s-1, 24 h, and white LED lamps, respectively, contributed to energy-efficient Stx2eB production. In addition, Stx2eB was produced 1.4 times more efficiently per unit area time using a high plant density (228.5 plants m-2) than a common density (30.4 plants m-2). The findings of the present study can facilitate the development of energy-efficient and low-cost production processes for vaccine protein production, considering temporal and spatial perspectives.
Asunto(s)
Edematosis Porcina , Animales , Edema , Lactuca/metabolismo , Fotoperiodo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , PorcinosRESUMEN
The comprehensive effect size of several commercial vaccines and vaccine candidates against edema disease (ED) has not been evaluated to date. To integrate the effectiveness of ED vaccines reported so far and to compare and evaluate the posterior-effect estimates of each vaccine type with network models, we identified eligible studies (n = 12) from the electronic databases using specified search strings. Data for dichotomous outcomes (i.e., mortality and clinical symptoms) and continuous outcomes (i.e., fecal shedding and average daily gain) were extracted and analyzed. Conventional meta-analysis shows that, compared with that in non-vaccinated pigs, vaccinated animals are likely to show reduced mortality (OR = 0.07) and clinical signs of ED (OR = 0.11), and increased productivity (SMD = 0.73). Although reduced fecal shedding (SMD = - 1.29) was observed in vaccinated pigs, this could not be fully determined on insufficient grounds. In contrast to mortality and clinical symptoms, fecal shedding (I2 = 88%) and average daily gain (I2 = 85%) showed immense heterogeneity, which was attributed to the small sample size and vaccination route, respectively. According to the Bayesian network meta-analysis, the plasmid-based DNA vaccine demonstrated a better effect for all outcomes compared to other types of vaccines. However, these findings should be carefully interpreted with consideration to potential mediators, insufficient data, and inconsistent network models.
Asunto(s)
Edematosis Porcina , Infecciones por Escherichia coli , Escherichia coli Shiga-Toxigénica , Animales , Teorema de Bayes , Edema , Edematosis Porcina/prevención & control , Infecciones por Escherichia coli/prevención & control , Metaanálisis en Red , Porcinos , Eficacia de las VacunasRESUMEN
To simplify the diagnosis of swine edema disease, overnight culture supernatants of swine clinical samples were assayed using immunochromatographic test strips we developed previously. Small-intestinal contents, mesenteric lymph nodes, and fecal samples were cultured in casamino acid-yeast extract broth overnight, after which supernatants were loaded onto immunochromatographic test strips to determine whether they could detect Shiga toxin 2e (Stx2e). Among 23 clinical samples in which PCR-identified stx2e-positive E. coli were isolated, samples from seven of ten small-intestinal contents, one of three mesenteric lymph nodes and six of ten fecal samples showed Stx2e-positive reactions in the protein-based immunochromatographic test. Additionally, one small-intestinal content sample, in which stx2e-positive E. coli were not isolated, showed an Stx2e-positive reaction. Furthermore, the immunochromatographic test results of the samples were associated with the toxin concentration determined by sandwich ELISA and cytotoxicity assay results on Vero cells. The toxin concentration range of the samples with positive and negative reactions were 2.1-196.2 ng/ml and 0-12.8 ng/ml, respectively. The sensitivity and specificity of this immunochromatographic test strip calculated from all clinical samples analyzed in this study were 60.9% and 94.4%, respectively. Our immunochromatographic test strip has strong potential for simple and accurate diagnosis for edema disease by detecting toxin expression, complementing the PCR method.
Asunto(s)
Edematosis Porcina , Infecciones por Escherichia coli , Enfermedades de los Porcinos , Animales , Chlorocebus aethiops , Edematosis Porcina/diagnóstico , Escherichia coli , Infecciones por Escherichia coli/veterinaria , Toxina Shiga , Toxina Shiga II , Porcinos , Células VeroRESUMEN
Porcine edema disease (ED) is a toxemia that is caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC) and is associated with high mortality. Since ED occurs most frequently during the weaning period, preweaning vaccination of newborn piglets is required. We developed stx2eB-transgenic lettuce as an oral vaccine candidate against ED and examined its protective efficacy using a piglet STEC infection model. Two serially developed Stx2eB-lettuce strains, 2BN containing ingredient Stx2eB constituting a concentration level of 0.53 mg Stx2eB/g of powdered lettuce dry weight (DW) and 2BH containing ingredient Stx2eB constituting a concentration level of 2.3 mg of Stx2eB/g of powdered lettuce DW, were evaluated in three sequential experiments. Taken the results together, oral administration of Stx2eB-lettuce vaccine was suggested to relieve the pathogenic symptoms of ED in piglets challenged with virulent STEC strain. Our data suggested that Stx2eB-lettuce is a promising first oral vaccine candidate against ED.
Asunto(s)
Animales Recién Nacidos , Vacunas Bacterianas/administración & dosificación , Edematosis Porcina/etiología , Edematosis Porcina/prevención & control , Infecciones por Escherichia coli/complicaciones , Infecciones por Escherichia coli/veterinaria , Lactuca , Toxina Shiga II/inmunología , Escherichia coli Shiga-Toxigénica , Porcinos , Destete , Administración Oral , Animales , Escherichia coli Shiga-Toxigénica/patogenicidad , VirulenciaRESUMEN
Shiga toxin (Stx)-producing Escherichia coli (STEC) and enterohemorrhagic E. coli (EHEC) as a human pathogenic subgroup of STEC are characterized by releasing Stx AB5-toxin as the major virulence factor. Worldwide disseminated EHEC strains cause sporadic infections and outbreaks in the human population and swine pathogenic STEC strains represent greatly feared pathogens in pig breeding and fattening plants. Among the various Stx subtypes, Stx1a and Stx2a are of eminent clinical importance in human infections being associated with life-threatening hemorrhagic colitis and hemolytic uremic syndrome, whereas Stx2e subtype is associated with porcine edema disease with a generalized fatal outcome for the animals. Binding toward the glycosphingolipid globotriaosylceramide (Gb3Cer) is a common feature of all Stx subtypes analyzed so far. Here, we report on the development of a matched strategy combining (i) miniaturized one-step affinity purification of native Stx subtypes from culture supernatant of bacterial wild-type strains using Gb3-functionalized magnetic beads, (ii) structural analysis and identification of Stx holotoxins by electrospray ionization ion mobility mass spectrometry (ESI MS), (iii) functional Stx-receptor real-time interaction analysis employing the surface acoustic wave (SAW) technology, and (iv) Vero cell culture assays for determining Stx-caused cytotoxic effects. Structural investigations revealed diagnostic tryptic peptide ions for purified Stx1a, Stx2a, and Stx2e, respectively, and functional analysis resulted in characteristic binding kinetics of each Stx subtype. Cytotoxicity studies revealed differing toxin-mediated cell damage ranked with Stx1a > Stx2a > Stx2e. Collectively, this matched procedure represents a promising clinical application for the characterization of life-endangering Stx subtypes at the protein level.
Asunto(s)
Edematosis Porcina/microbiología , Infecciones por Escherichia coli/microbiología , Síndrome Hemolítico-Urémico/microbiología , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/citología , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Chlorocebus aethiops , Humanos , Separación Inmunomagnética/métodos , Viabilidad Microbiana , Escherichia coli Shiga-Toxigénica/química , Sonido , Porcinos , Células VeroRESUMEN
BACKGROUND: In the pathogenicity of porcine edema disease (ED), which is caused by the Escherichia coli-producing F18 and Shiga toxin, F18+ fimbrial adhesins and Shiga toxin 2e (Stx2e) play pivotal roles in the colonization and enterotoxicity of this pathogen. OBJECTIVE: To develop a vaccine candidate against ED by combining three selected antigens of F18+ E. coli. METHODS: Genetically engineered Salmonella Typhimurium (ST) ghosts that express Stx2eB, FedF, and FedA were individually inserted in a ghost plasmid cassette, and the resultant plasmids were transformed into an attenuated ST (JOL912). The individual expression of Stx2eB, FedF, and FedA in JOL912 was validated by using an immunoblotting assay. RESULTS: Immunization of the ghosts in BALB/c mice led to a significant increase in antigen-specific secretory IgA and serum IgG. Significantly marked elevation of the CD3+CD4+ T cell subpopulation and lymphocyte proliferating activity in the primed splenocytes were also observed. Furthermore, mRNA of IL-4 and IFN-γ were highly upregulated in in vitro stimulated splenic T cells. Subsequently, the immunized mice showed significant protection efficacy against a lethal dose 50 of a virulent strain, resulting in approximately 85% and 92% survival rates in mice with a single- and double-dose immunization, respectively, compared to only 40% of the non-immunized controls. CONCLUSION: A mixture of the ghosts expressing these three antigens is a potential vaccine candidate for protection against the porcine edema disease.
Asunto(s)
Adhesinas Bacterianas/inmunología , Edematosis Porcina/prevención & control , Escherichia coli Enterotoxigénica/inmunología , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Proteínas Fimbrias/inmunología , Toxina Shiga II/inmunología , Animales , Edematosis Porcina/inmunología , Edematosis Porcina/microbiología , Femenino , Inmunidad Celular , Inmunidad Humoral , Inmunidad Mucosa , Ratones , Ratones Endogámicos BALB C , Salmonella/inmunología , Porcinos , Vacunas Sintéticas/inmunologíaRESUMEN
Porcine edema disease (ED) is a toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC). ED occurs most frequently during the weaning period and is manifested as emaciation associated with high mortality. In our experimental infection with a specific STEC strain, we failed to cause the suppression of weight gain in piglets, which is a typical symptom of ED, in two consecutive experiments. Therefore, we examined the effects of deprivation of colostrum on the sensitivity of newborn piglets to STEC infection. Neonatal pigs were categorized into two groups: one fed artificial milk instead of colostrum in the first 24 h after birth and then returned to the care of their mother, the other breastfed by a surrogate mother until weaning. The oral challenge with 1011 colony-forming units of virulent STEC strain on days 25, 26 and 27 caused suppression of weight gain and other ED symptoms in both groups, suggesting that colostrum deprivation from piglets was effective in enhancing susceptibility to STEC. Two successive STEC infection experiments using colostrum-deprived piglets reproduced this result, leading us to conclude that this improved ED piglet model is more sensitive to STEC infection than the previously established models.
Asunto(s)
Calostro/fisiología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Infecciones por Escherichia coli , Escherichia coli Shiga-Toxigénica , Animales , Edematosis Porcina/microbiología , Infecciones por Escherichia coli/microbiología , Toxina Shiga II/biosíntesis , Escherichia coli Shiga-Toxigénica/metabolismo , PorcinosRESUMEN
Porcine edema disease (ED) caused by F18+ Shiga toxin 2e-producing Escherichia coli (STEC) has imposed significant economic losses in the swine industry worldwide, resulting in sudden deaths in post-weaned piglets. The flagellin protein of F18+ STEC, a structural component of the flagellar filament, is a known virulence factor that mediates adhesion and invasion to porcine epithelial cells. In this study, Salmonella inactivated by the E lysis gene and expressing the flagellin (fliC) antigen was genetically engineered utilizing a plasmid (pMMP184) carrying an efficient heterologous antigen delivery system. The resulting strain JOL1485 producing FliC was successfully inactivated by the E lysis gene cassette. Following the lysis procedure, FliC secretion and production of JOL1485 was validated by immunoblot analysis. To evaluate protective immunogenicity elicited by the constructed strain, BALB/c mice were injected with 1×108 lysed cells via the intramuscular route. The markedly elevated titers of FliC-specific IgG, IgG1 and sIgA antibodies were observed, indicating a robust Th2-associated humoral immune response was raised in the immunized mice. The proportion of CD3+ CD4+ splenic T cells and proliferative activity were also elevated in in vivo and in vitro stimulated mice splenocytes. Further, JOL1485 successfully elicited upregulated gene expression of cytokines IL-6, IL-8, IL17, IL-21, IFN-γ and TNF-α in naïve porcine peripheral blood mononuclear cells (PBMCs). The overall immune response elicited by JOL1485 conferred a significant rise of protection against a lethal virulent F18+ STEC challenge whereas all non-immunized mice died following the challenge. Our results demonstrate that fliC efficiently expressed in the genetically inactivated Salmonella strain has immunostimulatory and protective effects against a F18+ STEC lethal challenge, and may be promising as a potential vaccine candidate against ED infection.
Asunto(s)
Portadores de Fármacos , Edematosis Porcina/prevención & control , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/inmunología , Flagelina/inmunología , Vectores Genéticos , Salmonella/genética , Animales , Anticuerpos Antibacterianos/sangre , Linfocitos T CD4-Positivos/inmunología , Proliferación Celular , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Proteínas de Escherichia coli/genética , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/genética , Femenino , Flagelina/genética , Inmunoglobulina A Secretora/sangre , Inmunoglobulina G/sangre , Inyecciones Intramusculares , Ratones Endogámicos BALB C , Análisis de Supervivencia , Porcinos , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Edema disease in piglets is caused by Shiga toxin 2e (Stx2e)-producing Escherichia coli. However, there is currently no available Stx2e-specific immunochromatographic test strip to differentiate Stx2e from other types of Shiga toxin 2. In the present study, to develop an Stx2e-specific immunochromatographic test strip, we isolated nine different monoclonal antibody-producing hybridoma clones from Stx2e toxoid-immunized mice and confirmed that six antibodies were A subunit-specific whereas three antibodies were B subunit-specific. Only one A subunit-specific monoclonal antibody (45B2) was cross-reactive with prototype Stx2 (Stx2a) at the same sensitivity, but the remaining eight monoclonal antibodies were not. In immunochromatographic tests using the highly sensitive antibodies, test strips using some combinations of gold colloid-conjugated monoclonal antibody with the B subunit-specific monoclonal antibody on the membrane detected Stx2e, but not other types of Shiga toxin 2. These test strips had the ability to detect Stx2e in the culture supernatant of clinically isolated Stx2e gene-positive strains, but not in those of Stx2e gene-negative strains. These results indicate that our test strip is practical for the specific detection of Stx2e to diagnose swine edema disease.
Asunto(s)
Cromatografía de Afinidad/métodos , Edematosis Porcina/diagnóstico , Toxina Shiga II/análisis , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Antitoxinas/inmunología , Antitoxinas/aislamiento & purificación , Ratones , Sensibilidad y Especificidad , Toxina Shiga II/inmunología , Porcinos , Factores de TiempoRESUMEN
Enterotoxigenic Escherichia coli (ETEC) is the major pathogen responsible for neonatal diarrhea, postweaning diarrhea, and edema disease in pigs. Although it can be harmless, ETEC is also present in the intestines of other animal species and humans, causing occasional diarrhea outbreaks. The evaluation of this pathogen's presence in food sources is becoming an increasingly important issue in human health. In order to determine the prevalence of ETEC in nondiarrheic pigs, 990 animals from 11 pig farms were sampled. Using end-time polymerase chain reaction (PCR), eltA, estI genes, or both, were detected in 150 (15.2%) animals. From the positive samples, 40 (26.6%) ETEC strains were isolated, showing 19 antibiotic-resistance patterns; 52.5% of these strains had multiple antibiotic resistances, and 17.5% carried the intI2 gene. The most prevalent genotypes were rfb(O157)/estII/aidA (32.5%) and estI/estII (25.0%). The estII gene was identified most frequently (97.5%), followed by estI (37.5%), astA (20.0%), and eltA (12.5%). The genes coding the fimbriae F5, F6, and F18 were detected in three single isolates. The aidA gene was detected in 20 ETEC strains associated with the estII gene. Among the isolated ETEC strains, stx(2e)/estI, stx(2e)/estI/estII, and stx(2e)/estI/estII/intI2 genotypes were identified. The ETEC belonged to 12 different serogroups; 37.5% of them belonged to serotype O157:H19. Isolates were grouped by enterobacterial repetitive intergenic consensus-PCR into 5 clusters with 100.0% similarity. In this study, we demonstrated that numerous ETEC genotypes cohabit and circulate in swine populations without clinical manifestation of neonatal diarrhea, postweaning diarrhea, or edema disease in different production stages. The information generated is important not only for diagnostic and epidemiological purposes, but also for understanding the dynamics and ecology of ETEC in pigs in different production stages that can be potentially transmitted to humans from food animals.
Asunto(s)
Antiinfecciosos/análisis , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli Enterotoxigénica/aislamiento & purificación , Genes Bacterianos , Carne Roja/microbiología , Animales , Antiinfecciosos/farmacología , ADN Bacteriano/genética , Diarrea/microbiología , Diarrea/veterinaria , Edematosis Porcina/microbiología , Escherichia coli Enterotoxigénica/efectos de los fármacos , Escherichia coli Enterotoxigénica/genética , Proteínas de Escherichia coli/genética , Contaminación de Alimentos , Microbiología de Alimentos , Técnicas de Genotipaje , Carne Roja/análisis , Porcinos , Enfermedades de los Porcinos/microbiologíaRESUMEN
Currently, no simple assays are available for routine quantitative detection of Escherichia coli-produced Shiga toxin 2e (Stx2e) that causes porcine edema disease. Here, we present a novel quantitative detection method for Stx2e based on the measurement of Stx2e binding to the specific globotetraosylceramide (Gb4) receptor by ELISA (Gb4-ELISA). No cross-reactivity was found with the other Shiga toxins Stx1 and Stx2, indicating high specificity. When the recombinant Stx2e B subunit (Stx2eB) was used, the absorbance measured by Gb4-ELISA increased linearly with Stx2eB concentration in the range of 20-2,500 ng/ml. The Gb4-ELISA method can be easily performed, suggesting that it would be a useful diagnostic tool for porcine edema disease.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/veterinaria , Globósidos/metabolismo , Receptores de Superficie Celular/metabolismo , Toxina Shiga II/análisis , Animales , Reacciones Cruzadas/inmunología , Edematosis Porcina/diagnóstico , Edematosis Porcina/inmunología , Edematosis Porcina/microbiología , Infecciones por Escherichia coli/diagnóstico , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Globósidos/inmunología , Receptores de Superficie Celular/inmunología , Toxina Shiga II/inmunología , Toxinas Shiga/inmunología , Escherichia coli Shiga-Toxigénica/inmunología , Porcinos/microbiologíaRESUMEN
The exotoxin SLT-IIeB from the Escherichia coli Ee strain was expressed in E. coli, and the recombinant protein was purified, mixed with the Ee strain, then emulsified with oil-emulsion adjuvants to obtain a mixed subunit bacterin. Groups of Kunming mice were immunized at weeks 0 and 2, and challenged intraperitoneally with the Ee strain at week 4. Antibodies were detected by ELISA and an agglutination test. After the second immunization, the antibody level increased and the rate of immune protection against the Ee strain was 70 and 91.7% in the subunit bacterin and bacterin groups, respectively. Therefore, the mixed subunit bacterin provided good protection against the homologous Ee strain, which provides a basis for further research, into high-efficacy vaccines against porcine edema disease.
Asunto(s)
Vacunas Bacterianas/administración & dosificación , Edematosis Porcina/genética , Infecciones por Escherichia coli/genética , Toxina Shiga II/genética , Animales , Vacunas Bacterianas/genética , Edematosis Porcina/tratamiento farmacológico , Edematosis Porcina/patología , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/patología , Inmunización , Ratones , Subunidades de Proteína/genética , Toxina Shiga II/administración & dosificación , Porcinos/microbiologíaRESUMEN
Chicken egg yolk immunoglobulin (IgY) against Shiga toxin 2e (Stx2e), a major cause of swine edema disease, was prepared to evaluate its possible clinical applications. The titer of Stx2e-specific IgY in egg yolk derived from three chickens that had been immunized with an Stx2e toxoid increased 2 weeks after primary immunization and remained high until 90 days after this immunization. Anti-Stx2e IgY was found to neutralize the toxicity of Stx2e by reacting with its A and B subunits, indicating that IgY is a cost-effective agent to develop for prophylactic foods or diagnosis kits for edema disease.
Asunto(s)
Yema de Huevo/química , Yema de Huevo/inmunología , Inmunoglobulinas/análisis , Toxina Shiga II/inmunología , Animales , Anticuerpos Neutralizantes/análisis , Antitoxinas/análisis , Pollos , Edematosis Porcina/prevención & control , PorcinosRESUMEN
One-step real-time PCR using one set of primers and four probes was developed for differentiation of F18 variants (F18 common, F18ab, F18ac, F18new variant) of enterotoxigenic (ETEC) and Shiga toxin-producing (STEC) Escherichia coli from piglets with diarrhoea and oedema disease. The limits of detection for F18common, F18ab, F18ac, and F18new variant were 10(7), 10(7), 10(5) and 10(7)colony forming units/g faeces, respectively. Of 94 Korean isolates of E. coli encoding F18, 70 were F18ac (43 STEC/ETEC, 4 STEC and 23 ETEC), 15 were F18ab (all STEC) and nine were F18new variant (1 STEC/ETEC, 7 STEC, 1 ETEC).
Asunto(s)
Diarrea/veterinaria , Edematosis Porcina/diagnóstico , Escherichia coli Enterotoxigénica/aislamiento & purificación , Infecciones por Escherichia coli/veterinaria , Proteínas de Escherichia coli/genética , Proteínas Fimbrias/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Enfermedades de los Porcinos/diagnóstico , Animales , Diarrea/diagnóstico , Diarrea/genética , Diarrea/microbiología , Edematosis Porcina/genética , Edematosis Porcina/microbiología , Escherichia coli Enterotoxigénica/clasificación , Escherichia coli Enterotoxigénica/genética , Escherichia coli Enterotoxigénica/metabolismo , Infecciones por Escherichia coli/diagnóstico , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Proteínas Fimbrias/metabolismo , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , República de Corea , Análisis de Secuencia de ADN/veterinaria , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/metabolismo , Porcinos , Enfermedades de los Porcinos/genética , Enfermedades de los Porcinos/microbiologíaRESUMEN
Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.
Asunto(s)
Vacunas Bacterianas/farmacología , Edematosis Porcina/inmunología , Infecciones por Escherichia coli/veterinaria , Proteínas de Escherichia coli/genética , Nicotiana/genética , Plantas Modificadas Genéticamente/genética , Escherichia coli Shiga-Toxigénica/patogenicidad , Administración Oral , Agrobacterium tumefaciens , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/efectos adversos , Edematosis Porcina/microbiología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Femenino , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Ingeniería Genética , Intestinos/inmunología , Intestinos/microbiología , Intestinos/patología , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Plantas Modificadas Genéticamente/metabolismo , Semillas/genética , Semillas/metabolismo , Toxina Shiga II/genética , Toxina Shiga II/metabolismo , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/inmunología , Porcinos , Nicotiana/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Porcine edema disease (ED) is caused by Shiga toxin 2e-producing Escherichia coli (STEC). Post-weaned piglets often suffer from ED as a result of intestinal infection with STEC, which causes impaired growth performance and high mortality. Antimicrobial therapy is a curative treatment for piglets infected with STEC, but the emergence of antimicrobial-resistant STEC has become a serious problem for Japanese pig farmers. Therefore, an alternative strategy other than antimicrobial therapy is needed for the prevention or treatment of ED. In this study, we evaluated the effect of oral administration of Bacillus subtilisâ DB9011 (DB9011) to prevent the experimental infection of STEC in weaning piglets. Eight 21-day-old piglets were divided into two groups: STEC challenge with the basal diet, and STEC challenge with DB9011 supplemented diet. The challenge was carried out when the animals were 25, 26 and 27 days old using STEC contained in capsules resistant against gastric digestion. All pigs were euthanized at 36 days of age. DB9011 improved the symptoms of ED and decreased the number of STEC in the ileal digesta and feces. Accordingly, oral administration of DB9011 in weaned piglets prevents ED through the suppression of the growth of STEC in the ileum.
Asunto(s)
Bacillus subtilis , Edematosis Porcina/prevención & control , Escherichia coli Enterotoxigénica , Infecciones por Escherichia coli/veterinaria , Probióticos/uso terapéutico , Animales , Ciego/microbiología , Infecciones por Escherichia coli/prevención & control , Femenino , Íleon/microbiología , Masculino , Porcinos , DesteteRESUMEN
Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.
Asunto(s)
Animales , Femenino , Ratones , Administración Oral , Agrobacterium tumefaciens , Antígenos Bacterianos/genética , Vacunas Bacterianas/administración & dosificación , Edematosis Porcina/inmunología , Infecciones por Escherichia coli/inmunología , Proteínas de Escherichia coli/genética , Proteínas Fimbrias/genética , Ingeniería Genética , Intestinos/inmunología , Ratones Endogámicos BALB C , Modelos Animales , Plantas Modificadas Genéticamente/genética , Semillas/genética , Toxina Shiga II/genética , Escherichia coli Shiga-Toxigénica/genética , Porcinos , Nicotiana/genética , Factores de Virulencia/genéticaRESUMEN
The purpose of this work was to characterize 47 Escherichia coli strains isolated from 32 pigs diagnosed with postweaning diarrhea and three pigs with edema disease by PCR. Forty two (95.5 %) of the strains isolated from diarrheic pigs were characterized as enterotoxigenic E. coli (ETEC) and 2 (4.5 %) as Shiga toxin-producing E. coli (STEC). Fourteen (33.3 %) ETEC strains were positive for est/estII/fedA genes. The most complex genotype was eltA/estI/faeG/aidA. Strains isolated from pigs with ED were classified as porcine STEC and were stx2e/aidA carriers. Eleven (25 %) strains carried the gene encoding adhesin protein AIDA-I. However, genes coding for F5, F6, F41, intimin and Paa were not detected. The development of vaccines generating antibodies against prevalent E. coli adhesins in Argentina could be useful for the prevention of PWD and ED.