A DIVA vaccine strain lacking RpoS and the secondary messenger c-di-GMP for protection against salmonellosis in pigs

International audienceAbstractSalmonellosis is the second most common food-borne zoonosis in the European Union, with pigs being a major reservoir of this pathogen. Salmonella control in pig production requires multiple measures amongst which vaccination may be used to reduce subclinical carriage and shedding of prevalent serovars, such as Salmonella enterica serovar Typhimurium. Live attenuated vaccine strains offer advantages in terms of enhancing cell mediated immunity and allowing inoculation by the oral route. However, main failures of these vaccines are the limited cross-protection achieved against heterologous serovars and interference with serological monitoring for infection. We have recently shown that an attenuated S. Enteritidis strain (ΔXIII) is protective against S. Typhimurium in a murine infection model. ΔXIII strain harbours 13 chromosomal deletions that make it unable to produce the sigma factor RpoS and synthesize cyclic-di-GMP (c-di-GMP). In this study, our objectives were to test the protective effects of ΔXIII strain in swine and to investigate if the use of ΔXIII permits the discrimination of vaccinated from infected pigs. Results show that oral vaccination of pre-weaned piglets with ΔXIII cross-protected against a challenge with S. Typhimurium by reducing faecal shedding and ileocaecal lymph nodes colonization, both at the time of weaning and slaughter. Vaccinated pigs showed neither faecal shedding nor tissue persistence of the vaccine strain at weaning, ensuring the absence of ΔXIII strain by the time of slaughter. Moreover, lack of the SEN4316 protein in ΔXIII strain allowed the development of a serological test that enabled the differentiation of infected from vaccinated animals (DIVA)

Evaluation of a Salmonella strain lacking the secondary messenger C-di-GMP and RpoS as a live oral vaccine

Salmonellosis is one of the most important bacterial zoonotic diseases transmitted through the consumption of contaminated food, with chicken and pig related products being key reservoirs of infection. Although numerous studies on animal vaccination have been performed in order to reduce Salmonella prevalence, there is still a need for an ideal vaccine. Here, with the aim of constructing a novel live attenuated Salmonella vaccine candidate, we firstly analyzed the impact of the absence of cyclic-di-GMP (c-di-GMP) in Salmonella virulence. Cdi- GMP is an intracellular second messenger that controls a wide range of bacterial processes, including biofilm formation and synthesis of virulence factors, and also modulates the host innate immune response. Our results showed that a Salmonella multiple mutant in the twelve genes encoding diguanylate cyclase proteins that, as a consequence, cannot synthesize c-di-GMP, presents a moderate attenuation in a systemic murine infection model. An additional mutation of the rpoS gene resulted in a synergic attenuating effect that led to a highly attenuated strain, referred to as ΔXIII, immunogenic enough to protect mice against a lethal oral challenge of a S. Typhimurium virulent strain. ΔXIII immunogenicity relied on activation of both antibody and cell mediated immune responses characterized by the production of opsonizing antibodies and the induction of significant levels of IFN-γ, TNF-α, IL-2, IL-17 and IL-10. ΔXIII was unable to form a biofilm and did not survive under desiccation conditions, indicating that it could be easily eliminated from the environment. Moreover, ΔXIII shows DIVA features that allow differentiation of infected and vaccinated animals. Altogether, these results show ΔXIII as a safe and effective live DIVA vaccine.SB is a predoctoral fellow from the Public University of Navarra. CG and BG are recipients of a postdoctoral contract under Grants IIM 13329.RI1 and BIO2011-30503-C02-02, respectively. This work was supported by grant IIM 13329.RI1 from the Departamento de Innovación, Empresa y Empleo, Government of Navarra and grants BIO2011-30503-C02-02 and BIO2014-53530-R from the Spanish Ministry of Economy and Competitiveness.Peer Reviewe

Análisis de una cepa avirulenta de Salmonella enteritidis para su uso como vacuna viva atenuada

Salmonella enterica subs. enterica ser. Enteritidis es un importante patógeno zoonótico con capacidad de infectar a un amplio rango de huéspedes. Actualmente representa un importante problema de salud pública dado que en el año 2010 se declararon en Europa 100.000 casos de enterocolitis por esta bacteria (de los cuales 2500 casos fueron declarados en España), asociados principalmente al consumo de carne de origen porcino. Para reducir la prevalencia de Salmonella en las granjas, la Directiva (CE) 2160/2003 de la Unión Europea propone exigir la vacunación de toda la cabaña ganadera avícola y porcina. Para responder a esta necesidad, en este estudio hemos valorado la capacidad de una cepa atenuada de S. Enteritidis para el desarrollo de una vacuna capaz de inducir una respuesta inmune que disminuya la colonización de órganos y liberación de Salmonella en heces en explotaciones intensivas de ganado porcino. La cepa de Salmonella candidata para el desarrollo de la vacuna ha sido, S. Enteritidis 3934ΔXII*, que tiene delecionado los genes que codifican las 12 proteínas de la familia GGDEF. Esta cepa es avirulenta, tiene muy disminuida su capacidad para sobrevivir en el ambiente y como no contiene DNA exógeno no estaría catalogada como OGM. Ensayos de inmunización por vía oral utilizando un modelo de infección murino, revelaron que la inmunización con una sola dosis de 107ufc de esta cepa es capaz de inducir una respuesta inmune protectora frente a una infección por Salmonella, indicando que dicha cepa puede ser un candidato idóneo para su uso como vacuna viva atenuada.Salmonella enterica subsp. Enterica ser. Enteritidis is an important zoonotic pathogen capable of infecting a wide range of hosts. Nowadays it represents an important problem of public health provided that in the year 2010 there were declared 100.000 Enterocolitis's cases caused by this bacterium in Europe (of which 2500 cases were declared in Spain), associated mainly with the consumption of meat of porcine origin. To reduce the prevalence of Salmonella, the Directive 2160/2003 of the European Union proposes to demand the vaccination of the whole poultry and porcine cattle for the year 2015. Given this requirement, in this study we have evaluated the ability of an attenuated strain of S. Enteritidis for the development of a vaccine capable of inducing an immune response that diminishes the colonization and Salmonella's liberation in feces of pigs in intensive piggeries. The Salmonella strain evaluated for vaccine development has been, S. Enteritidis 3934ΔXII*, in which the genes coding for the 12 GGDEF proteins had been deleted. This strain is avirulent, it has strongly diminished the capacity to survive in the environment and, given that it does not contain exogenous DNA, it would not be catalogued as GMO. Trials of oral immunization using a murine infection model revealed that immunization with a single dose of 107ufc of this strain is capable of inducing a protective immune response against Salmonella infection, indicating that this strain can be a suitable candidate for the development of Salmonella attenuated life vaccine.Máster Universitario en AgrobiotecnologíaUnibertsitate Masterra Agrobioteknologia

Evaluación de guantes de protección para la poda con tijeras neumáticas y eléctricas

Ingeniería Técnica AgrícolaNekazaritza Ingeniaritza Tekniko

c-di-GMP signaling in <i>Salmonella</i> is required for intestinal epithelium and organ colonization of BALBc mice but its absence does not significantly affect BALBc mice survival.

<p>(A) Comparative lethality between wild-type (black circles) and ΔXII (black squares) strains in an oral infection mouse model. Inoculum administered was 10⁷ cfu/mouse. P-value was determined by a Log-rank (Mantel-Cox) test. ns; no significant difference. (B) Competitive index (CI) analysis of wild type and ΔXII strains after performing an ileal loop coinfection experiment. Nine ileal loops were coinfected with 2x10⁷ cfu containing equal numbers of the parental and ΔXII strains. (C) CI analysis following intragastric inoculation of ten BALBc mice with a 1:1 mixture of wild type and ΔXII strains (total inoculum administered was 2 x 10⁸ cfu). Mice were sacrificed after five days and bacteria were enumerated from livers and spleens. (D) CI analysis following intraperitoneal inoculation of seven BALBc mice with a 1:1 mixture of wild type and ΔXII strains (total inoculum administered was 2 x 10⁴ cfu). Mice were sacrificed three days postinoculation and bacteria were enumerated from livers and spleens. CI was defined as the log₁₀ of the ratio of wild type strain to ΔXII strain recovered (Output) divided by the ratio of wild type strain to ΔXII strain present in the inoculum (Input). A CI > 0 indicates wild type with a colonization advantage compared to ΔXII and a CI < 0 indicates wild type with a colonization disadvantage over ΔXII. The plots display values obtained from individual samples and the median CI is represented by horizontal bars. P-values were determined by a Wilcoxon signed-rank test. *P < 0.05; ** P < 0.01; *** P < 0.001.</p

Vaccination-challenge analysis of the protection conferred by ΔXIII.

<p>Groups of seven BALBc mice were orally vaccinated with 10⁷ cfu of ΔXIII (open squares), Δ<i>rpoS</i> (black triangles) or PBS as a control (black circles). 33 days post immunization, mice were challenged with 10⁶ cfu/mouse of <i>S</i>. Typhimurium 143/09. Survival curves were plotted and P-values were determined by a Log-rank (Mantel-Cox) test.</p

ΔXIII strain is unable to form a biofilm and does not survive after twelve days of desiccation in the absence of nutrients.

<p>(A) Survival of the wild type strain, new ΔXII (absence of c-di-GMP), the single Δ<i>rpoS</i> mutant and the vaccine candidate ΔXIII after twelve days of desiccation. Surviving bacteria were enumerated by viable plate counts, and their numbers were compared with those of initial inocula, which defined 100% survival. Means and standard deviations of results from three independent experiments are shown. (B) Biofilm formation capacity of the same strains. Biofilm phenotypes were visualized after growth in LB medium conditions and on congo red agar plates.</p