Genetic and Antigenic Diversity of the Surface Protective Antigen Proteins of Erysipelothrix rhusiopathiae

Abstract

The surface protective antigen (Spa) protein of Erysipelothrix rhusiopathiae has been shown to be highly immunogenic and is a potential candidate for a new vaccine against erysipelas. In this study, we cloned and sequenced spa genes from all E. rhusiopathiae serovar reference strains as well as from a serovar 18 strain which was not classified as any species in the genus Erysipelothrix. Sequence analysis revealed that the Spa proteins could be classified into three molecular species, including SpaA, which was previously found in serovars 1a and 2, and the newly designated SpaB and SpaC proteins. The SpaA protein is produced by E. rhusiopathiae serovars 1a, 1b, 2, 5, 8, 9, 12, 15, 16, 17, and N, the SpaB protein is produced by E. rhusiopathiae serovars 4, 6, 11, 19, and 21, and the SpaC protein is produced only by serovar 18. The amino acid sequence similarity was high among members of each Spa type (96 to 99%) but low between different Spa types (ϳ60%). The greatest diversity in Spa proteins was found in the N-terminal half of the molecule (50 to 57% similarity), which was shown to be involved in immunoprotection. Coinciding with this, immunoblot analysis revealed that rabbit antisera specific to each Spa reacted strongly with the homologous Spa protein but weakly with heterologous Spa proteins. A mouse cross-protection study showed that the three recombinant Spa (rSpa) proteins elicited complete protection against challenge with homologous strains but that the level of protection against challenge with heterologous strains varied depending on the rSpa protein used for immunization. Our study is the first to demonstrate sequence and antigenic diversity in Spa proteins and to indicate that rSpaC may be the most promising antigen for use as a vaccine component because of its broad cross-protectiveness.Erysipelothrix rhusiopathiae is a small gram-positive rod bacterium that causes erysipelas in swine and a variety of diseases in other animals, as well as erysipeloid, a skin disease of humans (20). E. rhusiopathiae was once thought to be the only species in the genus Erysipelothrix and was classified into 25 serovars based on peptidoglycan antigens of the cell wall. At present, the genus contains at least the following two species: E. rhusiopathiae, including serovars 1a, 1b, 2, 4, 5, 6, 8, 9, 11, 12, 15, 16, 17, 19, and 21 and type N; and Erysipelothrix tonsillarum, including serovars 3, 7, 10, 14, 20, 22, and 23. Serovars 13 and 18 are unclassified but are considered to be assigned to genetically distinct groups from the above two species (15).E. rhusiopathiae serovars 1 and 2 are most frequently isolated from swine with clinical erysipelas (11, 16), but other serovars of E. rhusiopathiae are occasionally isolated from swine with septicemia, urticaria, arthritis, lymphadenitis, and endocarditis (17). Because of their high frequency of isolation, serovar 1a (Koganei 65-0.15) and serovar 2 (Tama-96) strains have been used to prepare live and killed vaccines, respectively, in Japan. Both vaccines elicit a cross-prot...

Erysipelothrix rhusiopathiae: Association of Spa-type with serotype and role in protective immunity

“…Cultures were streaked for isolation and grown on 5% bovine blood agar at 37 • C and propagated as previously documented [17]. Strains used for testing in mice were Piquet, Immiayuk, Quitz, Naluark, Kayavak, Large Herring 182, and Large Herring 911.…”

“…The following primers were synthesized (Iowa State University DNA Facility) and used in PCR assays according to previously described reports: primer 1, 5 -ATG AAA AAG AAA AAA CAC CTA-3 , and primer 2, 5 -CTA TTT TAA ACT TCC ATC GTT-3 , were used to amplify whole spaA [17]; primer 3, 5 -ATG AAA AAG AAA AAA CAC CTA TTT CCG AAA GTA-3 , and primer 4, 5 -CTA TTT TAA ACT TCC ATC GTT CTT AAA TGC ATA-3 , were used to amplify whole spaB or spaC [17]; ERY-1F, 5 -ATC GAT AAA GTG TTA TTG GTG G-3 , and ERY-2R, 5 -CGA GTG TGA ATC CGT CGT CTC-3 , were used to verify the species of Erysipelothrix strains [19].…”

“…The genus Erysipelothrix contains two accepted species, E. rhusiopathiae which include serotypes 1a, 1b, 2, 4, 5,6,8,9,11,12,15,16,17,19,21 and N; E. tonsillarum containing serotypes 3, 7, 10, 14, 20, 22 and 23 [3,4]. Two unclassified Erysipelothrix groups representing serotypes 13 and 18 are also recognized [4].…”

“…The gene encoding the protective 64-66 kDa protein was first sequenced by Makino et al [16] who named it, surface protective antigen A (spaA). Recently, two additional types of spa-related genes were detected within E. rhusiopathiae and an unclassified serotype 18 of the genus Erysipelothrix [17]. Amino acid sequence analysis determined that serotypes 1a, 1b, 2,5,8,9,12,15,16,17 and N possess spaA; serotypes 4, 6, 11, 19 and 21 possess spaB; and the unclassified serotype 18 possesses spaC [17].…”

“…The three molecular groups are distinguished by amino acid sequence and are considered antigenically distinct [17]. In active immunization experiments in mice, recombinant Spa antigens were completely protective against virulent E. rhusiopathiae strains possessing homologous spa [17,18], but variably protective against strains possessing heterologous spa-types [17].…”