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

(2020) 51:3 Gil et al. Vet Res https://doi.org/10.1186/s13567-019-0730-3 RESEARCH ARTICLE Open Access A DIVA vaccine strain lacking RpoS and the secondary messenger c‑di‑GMP for protection against salmonellosis in pigs Carmen Gil1†, Cristina Latasa2†, Enrique García‑Ona1, Isidro Lázaro3, Javier Labairu3, Maite Echeverz1, Saioa Burgui1, Begoña García1, Iñigo Lasa1* and Cristina Solano1* Abstract Salmonellosis 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 vac‑ cination 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). Introduction Salmonellosis remains the second most common zoonosis in humans in the European Union (EU), with 91 662 confirmed cases in 2017. Despite national control programmes, in recent years, the declining trend of salmonellosis cases has levelled off and the number of reported cases in the EU has not shown any statistically significant decrease. Moreover, Salmonella is still responsible for the *Correspondence: ; † Carmen Gil and Cristina Latasa contributed equally to this work. 1 Laboratory of Microbial Pathogenesis, Navarrabiomed-Universidad Pública de Navarra (UPNA)-Complejo Hospitalario de Navarra (CHN), IdiSNA, Irunlarrea 3, 31008 Pamplona, Navarra, Spain Full list of author information is available at the end of the article highest number of food-borne outbreaks in the EU, with eggs, pig meat and derived products being main sources of this pathogen [1]. The European Commission set the criteria to control Salmonella infections within the poultry sector, resulting in a correlated reduction in the human cases associated with the consumption of eggs [2, 3]. On the contrary, proposals concerning the monitoring and control of Salmonella in pigs have been dropped [4], but still, regulations in the swine sector should follow to tackle Salmonella infection in pigs [5]. The most common serovar at EU level causing human food-borne infections from pork is Salmonella enterica serovar Typhimurium (S. Typhimurium), being widely prevalent along the entire pig chain [1]. Therefore, it is assumed © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gil et al. Vet Res (2020) 51:3 that control measures should be based on actions taken throughout the production chain, including a combination of measures aimed at preventing horizontal and vertical transmission, with the final objective of producing Salmonella-free animals. At the pre-harvest level, measures can be addressed to the prevention of introduction of Salmonella into the herd; the prevention of in-herd transmission; and the increase of resistance to infection [6, 7]. In this regard, Salmonella vaccines are currently regarded as an adjunct to other on-farm control measures [7–10], by helping to prevent Salmonella colonization and the development of a carrier state, characteristic of pigs colonized by non-adapted serovars, such as S. Enteritidis and S. Typhimurium [7, 11]. In recent years, increasing numbers of live Salmonella attenuated vaccines have been developed for pigs vaccination [7–9, 12–17], although most of them are not yet authorized. While this type of vaccines are claimed as the most effective means of immunoprophylaxis against Salmonella [11], there are major drawbacks that must be taken into account. First, vaccines are usually serovar specific, providing limited protection against infections with Salmonella belonging to other serovars; second, vaccination may interfere with established serological monitoring programs, making it difficult to differentiate between vaccinated and infected animals; and third, attenuated vaccine strains may reverse to virulent, unsafe forms [9]. We recently published a report detailing the analysis of an attenuated S. Enteritidis vaccine strain, referred to as ΔXIII, that protected mice against a lethal oral challenge of a S. Typhimurium virulent strain [18]. ΔXIII strain is a multiple mutant in rpoS, the gene encoding the master sigma factor during stationary phase and under a variety of stress conditions [19] and also in the 12 genes encoding diguanylate cyclase proteins responsible for the synthesis of the second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) [20, 21]. Absence of c-di-GMP in ΔXIII leads to a moderate attenuation [18] whilst the additional mutation in rpoS results in a highly attenuated strain [18, 22]. Moreover, the secondary messenger c-di-GMP is a key molecule in the (...truncated)