Vaccine 23 (2005) 1491–1501 Protective ability of subcellular extracts from Salmonella Enteritidis and from a rough isogenic mutant against salmonellosis in mice Javier Ochoa-Rep´ araz a , Bego˜ na Garc´ ıa a,b , Cristina Solano b ,I˜ nigo Lasa b , Juan Manuel Irache c , Carlos Gamazo a,∗ a Departamento de Microbiolog´ ıa, Universidad de Navarra, 31080 Pamplona, Spain b Instituto de Agrobiotecnolog´ ıa y Recursos Naturales, UPNA-CSIC, 31006 Pamplona, Spain c Department of Pharmaceutical Technology, Universidad de Navarra, 31008 Pamplona, Spain Received 26 May 2004; accepted 7 September 2004 Available online 30 October 2004 Abstract We evaluated the efficacy of surface components enriched hot saline extracts (HE) from parental and two isogenic rough mutant strains of Salmonella Enteritidis as subcellular vaccine candidates. By a randomized mutagenesis approach from a clinical isolate of S. Enteritidis there were selected two rough mutants defective in LPS synthesis (R1 and R2 mutants). The mutations mapped to the wcaI gene and gmd gene, respectively, of the O-antigen gene cluster involved in O-antigen synthesis. BALB/c mice received intraperitoneally one single dose of 30 g of HE from parental and mutant strains, and the protection against a lethal infection with S. Enteritidis was determined. In contrast to the wild type extract, immunization with rough extracts did not induce any distress symptoms in the mice. HE extract from wild type and R1 strains induced the highest immunogenic response with respect IFN- eliciting splenic cells, in contrast with HE-R2. These results correlated with the obtained levels of protection. Thus, at day 63 post-infection, HE from parental strain rendered an 80% level of protection; HE-R1 conferred a 60% level of protection, whereas HE-R2 did not protect the mice. Any of the antigenic extracts elicited systemic IgG1 and IgG2a responses, although these antibodies did not, however, correlate with protection. These results put forward the importance of cellular immune response mediated by IFN- in protection against salmonellosis. The significantly different protective capacity between HE extracts from both rough mutants suggest that other factors independent of the O-chain, like outer membrane proteins and fimbrial antigens, may be involved in protection. In summary, the HE is a good candidate acellular extract for evaluation of its protective ability against salmonellosis following vaccination in poultry. © 2004 Elsevier Ltd. All rights reserved. Keywords: Salmonellosis; Vaccine; Rough mutant 1. Introduction Salmonella enterica serovar Enteritidis (Salmonella En- teritidis) is a major cause of human food-borne illness, the most frequently serovar detected in outbreaks of human salmonellosis [1]. Poultry products are known to be a signif- icant reservoir for Salmonella and the most important source of S. Enteritidis infection in humans [2,3]. Antibiotics, com- petitive exclusion, genetic selection of chicken strains and ∗ Corresponding author. Tel.: +34 9 48 42 56 88; fax: +34 9 48 42 56 49. E-mail address: cgamazo@unav.es (C. Gamazo). vaccines have been employed in the control of Salmonella in poultry farms [4]. Considering vaccines as the most practical approach in the control of salmonellosis, the choice of using live attenuated organisms versus inactivated ones is weighed according to the positive and negative factors of each. Live attenuated vaccines, administered parenterally, are protective and used widely. Auxotrophic [5–7] and rough mutants [8,9] from a number of Salmonella strains have pro- vided a substantial protection against infection in experimen- tal animals. The R9 S. Gallinarum vaccine, a rough attenuated strain, has been assessed extensively, in spite of the protec- tion conferred not being complete. However, mass introduc- 0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2004.09.016