The Formation, Persistence, and Resistance to Disinfectant of the Erysipelothrix piscisicarius Biofilm.

Erysipelothrix piscisicarius is an emergent pathogen in fish aquaculture, particularly in the ornamental fish trade. Very little is known on the biology of this pathogen; however, the recurrence of infection and disease outbreaks after removing the fish from a system and disinfecting the tank suggest its environmental persistence. Moreover, biofilm lifestyle in E. piscisicarius has been suspected but not previously shown. The purpose of this study was to investigate the formation of biofilms on an abiotic surface in Erysipelothrix spp. We used hydroxyapatite-coated plastic pegs to demonstrate the attachment, growth, and persistence of E. piscisicarius on abiotic surfaces in both fresh and marine environments and to investigate the susceptibility of this pathogen to different disinfectants that are used in the aquaculture industry. E. piscisicarius formed biofilms that persisted significantly longer than planktonic cells did in both freshwater and saltwater over a period of 120 h (P = 0.004). The biofilms were also more resistant to disinfectants than the planktonic cells were. Hydrogen peroxide was the most effective disinfectant against E. piscisicarius, and it eradicated the biofilms and planktonic cells at the recommended concentrations. In contrast, Virkon and bleach were able to eradicate only the planktonic cells. This information should be taken into consideration when developing biosecurity protocols in aquaculture systems, aquariums, and private collections.

Genome-Wide Identification of Virulence Genes in Erysipelothrix rhusiopathiae: Use of a Mutant Deficient in a tagF Homolog as a Safe Oral Vaccine against Swine Erysipelas.

Swine erysipelas is caused by the Gram-positive pathogen Erysipelothrix rhusiopathiae The swine erysipelas live vaccine in Japan, the E. rhusiopathiae Koganei 65-0.15 strain (Koganei), has been reported to cause arthritis and endocarditis. To develop a vaccine with increased safety, we used a virulent Fujisawa strain to construct transposon mutants for a total of 651 genes, which covered 38% of the coding sequence of the genome. We screened the mutants for attenuation by inoculating mice with 108 CFU of each mutant and subsequently assessed protective capability by challenging the surviving mice with 103 CFU (102 times the 50% lethal dose) of the Fujisawa strain. Of the 23 attenuated mutants obtained, 6 mutants were selected and evaluated for protective capability in pigs by comparison to that of the Koganei strain. A mutant in the ERH_0432 (tagF) gene encoding a putative CDP-glycerol glycerophosphotransferase was found to be highly attenuated and to induce humoral and cell-mediated immune responses in conventional pigs. An in-frame deletion mutant of the gene, the Δ432 mutant, was constructed, and attenuation was further confirmed in germfree piglets; three of four piglets subcutaneously inoculated with 109 CFU of the Δ432 mutant showed no apparent clinical symptoms, whereas all four of the Koganei-inoculated piglets died 3 days after inoculation. It was confirmed that conventional pigs inoculated orally or subcutaneously with the Δ432 strain were almost completely protected against lethal challenge infection. Thus, the tagF homolog mutant of E. rhusiopathiae represents a safe vaccine candidate that can be administered via the oral and subcutaneous routes.

Use of Commercial Swine Live Attenuated Vaccine to Control an Erysipelothrix rhusiopathiae Outbreak in Commercial Cage-Free Layer Chickens.

Erysipelothrix rhusiopathiae septicemia was diagnosed in three cage-free commercial layer flocks from Washington State that experienced an increase in mortality and slight drop in egg production. Erysipelothrix rhusiopathiae was isolated from multiple organs and from environmental samples. An agar gel diffusion test of several E. rhusiopathiae isolates confirmed the presence of serotype 1b, and multiplex real-time PCR of the surface protective antigen (Spa) gene confirmed presence of SpaA. Bacitracin administered via the water reduced mortality minimally and only for a short period of time. Mortality was finally controlled by vaccination with a live attenuated swine E. rhusiopathiae vaccine delivered via the drinking water. This is the first report describing the use of an attenuated vaccine to control an E. rhusiopathiae outbreak in a chicken flock.

Reporte de caso- Uso de vacunas vivas atenuadas comerciales para uso en porcinos para controlar un brote de Erysipelothrix rhusiopathiae en aves de postura libres de jaula. Septicemia por Erysipelothrix rhusiopathiae se diagnosticó en tres parvadas comerciales libres de jaula en el estado de Washington que experimentaron un aumento de la mortalidad y una leve disminución en la producción de huevos. Se aisló Erysipelothrix rhusiopathiae de múltiples órganos y de muestras ambientales. La prueba de difusión en gel de agar de varios aislamientos de E. rhusiopathiae confirmó la presencia del serotipo 1b y un método múltiple de PCR en tiempo real del gene del antígeno protector de superficie (Spa) confirmó la presencia de SpaA. La bacitracina administrada a través del agua redujo la mortalidad en forma mínima y solo durante un tiempo corto. La mortalidad finalmente se controló mediante la vacunación con una vacuna viva atenuada de E. rhusiopathiae para porcinos administrada a través del agua de bebida. Este es el primer reporte que describe el uso de una vacuna atenuada para controlar un brote de E. rhusiopathiae en una parvada de pollos.

Evaluation of the protective efficacy of four newly identified surface proteins of Erysipelothrix rhusiopathiae.

Erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and human erysipeloid. Bacterial surface proteins are promising vaccine candidates. We recently identified 3 E. rhusiopathiae surface proteins (GAPDH, HP0728, and HP1472) and characterized their roles as virulence factors. However, their efficacy as protective antigens is still unknown. The N-terminal region of a previously identified surface protein, CbpB (CbpB-N), is speculated to be a protective antigen, but this needs to be verified. The aim of this study was to evaluate the protective efficacy of GAPDH, HP0728, HP1472, and CbpB-N. Immunization with recombinant GAPDH provided complete protection in a mouse model, recombinant CbpB-N provided partial protection, while recombinant HP0728 and HP1472 provided no protection. Recombinant GAPDH also provided good protection in a pig model. GAPDH antiserum exhibited significant blood bactericidal activity against E. rhusiopathiae. In conclusion, GAPDH and CbpB-N were found to be protective antigens of E. rhusiopathiae, and GAPDH is a promising vaccine candidate.

Serotypes and Spa types of Erysipelothrix rhusiopathiae isolates from British pigs (1987 to 2015).

Erysipelothrix spp. cause a range of clinical signs in pigs and at least 28 different Erysipelothrix spp. serotypes have been identified. In this study, 128 isolates of Erysipelothrix spp. from pigs in Great Britain from 1987 to 2015 were characterised by serotyping and multiplex real time PCR assays targeting the surface protective antigen (Spa) and the main genotypes (Erysipelothrix rhusiopathiae, Erysipelothrix tonsillarum and Erysipelothrix spp. strain 2). All 128 British isolates were characterised as E. rhusiopathiae and were classified as serotypes 1a (n=21), 1b (n=17), 2 (n=75), 5 (n=2), 9 (n=2), 10 (n=2), 11 (n=4) and 15 (n=1), while four isolates were untypeable. All isolates were positive for the spa A gene. Serotypes 1a, 1b and 2 constituted 88.3% of the isolates; current serotype 2 based vaccines should protect against these isolates.

Evaluation and improvement of a single nucleotide polymorphism-based PCR assay for rapid differentiation of live attenuated vaccine strains from field isolates of Erysipelothrix rhusiopathiae.

A single nucleotide polymorphism-based PCR assay has been developed to differentiate the attenuated vaccine strain used in Japan from field isolates of Erysipelothrix rhusiopathiae found in pigs. However, this assay has been evaluated with only Japanese strains and isolates; therefore, it is unknown whether it could be used in other countries with E. rhusiopathiae strains and isolates of different genetic backgrounds. In our study, the PCR assay was evaluated using Chinese E. rhusiopathiae vaccine strains and field isolates. The PCR assay was able to differentiate the attenuated vaccine strains from the field isolates of E. rhusiopathiae in China but with a pattern different from that observed in Japan (only a single nucleotide polymorphism was detected in the Chinese vaccine strains compared with 5 in the Japanese vaccine strains). Importantly, either a DNA polymerase without 3' to 5' exonuclease activity or an exo+ polymerase with an antibody inhibiting the proofreading activity was required. In conclusion, after evaluation and improvement, this fast differentiation assay can be extended from Japan to China.

Evaluation of anti-Erysipelothrix rhusiopathiae IgG response in bottlenose dolphins Tursiops truncatus to a commercial pig vaccine.

Erysipelothrix rhusiopathiae is the causative agent of erysipeloid in humans and of erysipelas in various animals, including bottlenose dolphins Tursiops truncatus, in which an infection has the potential to cause peracute septicemia and death. The purpose of this study was to evaluate the efficacy of using an off-label porcine (ER BAC PLUS®, Zoetis) E. rhusiopathiae bactrin in a bottlenose dolphin vaccination program by determining the anti-E. rhusiopathiae antibody levels in vaccinated dolphins over a 10 yr period. Serum samples (n = 88) were analyzed using a modified fluorescent microbead immunoassay from 54 dolphins, including 3 individuals with no history of vaccination and 51 dolphins with an average of 5 vaccinations, 3 of which had previously recovered from a natural E. rhusiopathiae infection. A mean 311-fold increase in the immunoglobulin G (IgG) antibody index was measured in a subsample of 10 dolphins 14 d after the first booster vaccination. Serum IgG antibody titers were influenced by number of vaccines received (r2 = 0.47, p < 0.05) but not by age, gender, history of natural infection, adverse vaccine reaction, vaccination interval or time since last vaccination. The commercial pig bacterin was deemed effective in generating humoral immunity against E. rhusiopathiae in dolphins. However, since the probability of an adverse reaction toward the vaccine was moderately correlated (p = 0.07, r2 = 0.1) with number of vaccines administered, more research is needed to determine the optimal vaccination interval.

Fluorescent microbead-based immunoassay for anti-Erysipelothrix rhusiopathiae antibody detection in cetaceans.

A fluorescent microbead-based immunoassay (FMIA) for detection of anti-Erysipelothrix rhusiopathiae antibodies in pigs was adapted for use in cetaceans. The FMIA was validated and adjusted using serum samples from 10 vaccinated captive bottlenose dolphins Tursiops truncatus collected between 1 and 13 mo after immunization. The technique was then used to analyze specimens from 15 free-ranging cetaceans stranded alive on the Valencian Mediterranean coast between 2006 and 2014: 11 striped dolphins Stenella coeruleoalba, 3 Risso's dolphins Grampus griseus and 1 bottlenose dolphin Tursiops truncatus. One of these wild animals was confirmed to have died from E. rhusiopathiae septicemia, but no anti-E. rhusiopathiae antibodies were detected in its serum, pericardial fluid or milk samples. Another free-ranging individual, which lacked any signs or lesions that might be indicative of E. rhusiopathiae infection, showed high fluorescence intensity similar to that measured in captive dolphins at 6-13 mo after vaccination. These results suggest that this animal underwent an E. rhusiopathiae infection several months before stranding. The findings in the present study suggest that FMIA can be useful for detecting anti-E. rhusiopathiae antibodies in cetaceans, and its application to free-ranging animals is particularly interesting because of the great value of these specimens. Furthermore, the FMIA can be multiplexed to allow the determination of up to 100 analytes per sample in a single well, thereby reducing the cost, time and sample volume needed.

Safety and efficacy of a new octavalent combined Erysipelas, Parvo and Leptospira vaccine in gilts against Leptospira interrogans serovar Pomona associated disease and foetal death.

The safety and protective efficacy of a new octavalent combination vaccine containing inactivated Erysipelothrix rhusiopathiae, Parvovirus, and Leptospira interrogans (sensu lato) serogroups Canicola, Icterohaemorrhagiae, Australis (Bratislava), Grippotyphosa, Pomona and Tarassovi - Porcilis(®) Ery+Parvo+Lepto - was evaluated in laboratory studies and under field conditions. The safety (2× overdose and repeated dose) was tested in 26 gilts. In this study, neither vaccine related temperature increase nor other systemic reactions were observed after intramuscular vaccination. No local reactions were observed except for one animal that had a small local reaction (2cm diameter) that lasted for 5 days after the third vaccination. Efficacy was tested in 40 gilts. A group of 20 gilts was vaccinated at 20 and 24 weeks of age with Porcilis(®) Ery+Parvo+Lepto and a group of 20 age- and source-matched animals served as the control group. The gilts were inseminated at 41 weeks or 66 weeks of age and were challenged with serovar Pomona 10 weeks after insemination, corresponding to 6 months (n=2×10) and 12 months (n=2×10) after the last vaccination. After both the 6- and 12-month challenges the control animals developed clinical signs (fever, lethargy and anorexia) and leptospiraemia as determined by positive blood culture. In addition, both the 6- and 12-month challenges resulted in death of 21% and 27% of the total number of foetuses in the control groups, respectively. Clinical signs and leptospiraemia were statistically significantly lower in vaccinated gilts after both the 6- and 12-month challenges. In addition, foetal death was statistically significantly lower (3% and 2%, respectively) in vaccinated gilts after both the 6- and 12 month challenges. The vaccine was tested further under field conditions on a Portuguese farm with a history of an increasing abortion rate associated with a Leptospira serovar Pomona infection (confirmed by PCR and serology). This study was designed as an observational-longitudinal field study. At the start of the study, all breeding sows and replacement gilts on the farm were vaccinated twice with Porcilis(®) Ery+Parvo+Lepto at an interval of 4 weeks. Starting six months after the primary vaccination schedule, the animals were re-vaccinated during the second week of every subsequent lactation. New replacement gilts were vaccinated using the same schedule. After vaccination, the abortion rate reduced rapidly from 12.6% in winter months of 2012 (December 2011 to March 2012) to 0.5% in winter months of 2013, a statistical significant decrease of 96%. The total number of abortions on the farm decreased from 55 in 2012 to 6 in 2013. Thereafter, the abortion rate remained stable and in the period December 2013 to April 2014 was still low (0.6%). In conclusion, the present studies demonstrate that the octavalent Porcilis(®) Ery+Parvo+Lepto vaccine can be safely used in gilts and sows and induces significant protection, for the duration of at least one year, against serovar Pomona induced clinical signs, leptospiraemia and foetal death. Protection against Pomona associated reproductive failure was confirmed under field conditions where a significant reduction in abortion rate was observed.

Prevalence of IgY antibodies against Erysipelothrix rhusiopathiae in a critically endangered parrot (kakapo, Strigops habroptilus) and associated responses to vaccination.

An enzyme-linked immunosorbent assay (ELISA) was developed to estimate levels of IgY antibody against the bacterium Erysipelothrix rhusiopathiae in serum samples collected from the critically endangered kakapo (Strigops habroptilus, Psittaciformes, Aves) before and after vaccination against this bacterium. Relative IgY antibody titres in pre-vaccination serum samples (n = 71 individual kakapo) were normally distributed with the exception of four outliers which displayed low IgY levels. Notably all four low IgY samples were collected from fledglings 3 - 6 months old. Pre-vaccination serum samples from nine nestlings <3 months old, seven of which were hatched in incubators and had no contact with either adult kakapo or their natural environment (e.g. soil), were found to have relatively high IgY levels, suggesting transfer of maternal IgY molecules to fledglings via the yolk. IgY levels in pre-vaccination serum samples from seven kakapo aged 25 - 30 months were also relatively high, suggesting that most kakapo naturally acquire anti- E.rhusiopathiae IgYs within their first 2 years. There was no evidence that vaccination increased the kakapo population's mean anti-E.rhusiopathiae IgY levels. However, there was a significant negative relationship between an individual bird's pre-vaccination IgY level and any subsequent increase following vaccination, suggesting that vaccination may only raise the IgY levels of birds with relatively low pre-vaccination IgY levels. A statistical model of the relationship between 'death from erysipelas' and sex, age and transfer from one to island sanctuary to another found that only transfer was significantly associated with death from erysipelas.

Genetic variability in swine leukocyte antigen class II and Toll-like receptors affects immune responses to vaccination for bacterial infections in pigs.

The genes encoding swine leukocyte antigen (SLA) and Toll-like receptor (TLR) are highly polymorphic in pig populations, and likely have influences on infection and the effects of vaccination. We explored the associations of different genotypes of SLA class II and of the genes TLR1, TLR4, TLR5, and TLR6 with antibody responses after vaccination against Erysipelothrix rhusiopathiae (ER) and Actinobacillus pleuropneumoniae (APP) serotypes 1, 2, and 5 in 191 Duroc pigs maintained under specific pathogen-free conditions. We demonstrated close relationships between SLA class II and ER antibody response and between TLR genes other than TLR4 and APP antibody responses. Pigs with specific haplotypes in SLA class II or TLR5 showed decreased antibody response to ER vaccination or increased responses to APP2 and APP5 vaccination, respectively. It might be possible to breed for responsiveness to vaccination and to implement new vaccine development strategies unaffected by genetic backgrounds of pigs.

Effect of age and maternally-derived antibody status on humoral and cellular immune responses to vaccination of pigs against Erysipelothrix rhusiopathiae.

The effects of age and maternally-derived antibodies (MDA) on the immune response to Erysipelothrix rhusiopathiae were investigated in piglets orally vaccinated with a live E. rhusiopathiae vaccine at 6, 8 or 10 weeks of age. Seroconversion, determined by ELISA, was evident in MDA positive piglets vaccinated at 8 or 10 weeks of age and in all MDA negative vaccinates. Two weeks after vaccination in the presence of MDA, a T cell response, measured by a lymphocyte proliferation assay, was observed in 25% of piglets vaccinated at 6 weeks of age and in 100% of piglets vaccinated at 8 or 10 weeks of age. The post-vaccinal response to E. rhusiopathiae was more strongly influenced by the maternal antibody status of the piglet at the time of vaccination than the age of the piglet.

Development of an enzyme-linked immunosorbent assay for the serological detection of exposure of poultry in New Zealand to Erysipelothrix rhusiopathiae and their serological response to vaccination.

AIMS: To modify and validate an existing swine erysipelas ELISA for use with poultry serum and to assess the safety of a swine erysipelas vaccine for use in New Zealand layer birds. METHODS: An existing swine erysipelas ELISA was modified for use in domestic poultry and was validated using sera from birds injected with either 2 mL of a commercially available killed swine erysipelas vaccine (low-dose; n=12 birds), 4 mL of vaccine (high-dose; n=11 birds), or 2 mL saline (control; n=11 birds) on Day 0 and again on Day 21. Blood samples were collected on Days 0, 21, 42, and 63, and safety of the vaccine for use in layer birds was determined by assessing cloacal temperature and injection site reactions in birds at 0, 4, 24, 48, 72 and 96 h post-vaccination. RESULTS: The ELISA that was developed had a diagnostic sensitivity and specificity of 93% and 98%, respectively, after being optimised for a positive cut-off at an optical density (OD) ≥ 1.50 read at 450-nm wavelength. OD readings were higher on Days 21, 42, and 63 than Day 0 in both the low-dose and high-dose groups (p<0.05), and differed amongst the three groups on Days 21, 42, and 63 (p<0.05), suggesting that vaccination using either dose induced detectable levels of antibody, even after a single dose. In addition, the high-dose protocol induced higher levels of antibody production than the low-dose protocol. No local or systemic reactions to the vaccine were observed and cloacal temperatures remained in the normal biological range after vaccination. CONCLUSIONS: The ELISA that was developed had satisfactory diagnostic performance characteristics and the vaccine appeared to be safe for use in layer birds. However, the study design did not permit an assessment of the vaccine's efficacy to protect birds from clinical erysipelas. CLINICAL RELEVANCE: A diagnostic ELISA has been developed for determining the exposure of layer birds to E. rhusiopathiae. The test will be useful for monitoring flock-level erysipelas, response to vaccination, and in epidemiological studies designed to identify risk factors for exposure to the disease.

Immunization with truncated recombinant protein SpaC of Erysipelothrix rhusiopathiae strain 715 serovar 18 confers protective immunity against challenge with various serovars.

Previously, we showed that surface protective antigen (Spa) proteins of Erysipelothrix rhusiopathiae can be classified into three molecular species-SpaA, SpaB, and SpaC-and that SpaC is the most broadly cross-protective antigen among the three Spa proteins. In this study, we examined the ability of the α-helical domain, which comprises the N-terminal half of SpaC, to elicit cross-protective immunity in mice and pigs. Mice actively immunized with the full-length protein (rSpaC664) or the α-helical domain (rSpaC427), but not the C-terminal domain (rSpaC253), were protected against challenge with E. rhusiopathiae serovars 1a, 2, 6, 19, and 18 expressing heterologous (SpaA or SpaB) and homologous (SpaC) Spas. The α-helical domain seemed to provide better protection than rSpaC664, although the differences did not reach statistical significance. Similarly, mice passively immunized with rabbit anti-rSpaC664 or anti-rSpaC427 sera, but not anti-rSpaC253 serum, were protected from challenge with various serovars. Pigs immunized with SpaC427 also developed specific antibodies against Spa proteins and were protected from challenge with the highly virulent heterologous E. rhusiopathiae strain Fujisawa (serovar 1a). Taken together, these results demonstrate for the first time the striking protective efficacy of the α-helical domain-mediated immunization in both mice and pigs, thereby highlighting its utility as the most promising candidate for the development of a safe and effective vaccine against erysipelas.

Symposium III report: Current status of veterinary vaccines.

The 13th Annual Meeting of the Japanese Society of Vaccinology convened the symposium on current status of veterinary vaccines on September 27, 2009 in Sapporo, Japan. A broad overview of veterinary vaccines from the past to the present and future as well as the latest developments such as Erysipelothrix rhusiopathiae and Mycoplasma hyopneumoniae for a platform vector for mucosa vaccine, inactivated avian influenza vaccine prepared from reassortant virus, and inactivated feline immunodeficiency virus (FIV) vaccine were presented.

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

A collection of swine, fish, and cetacean Erysipelothrix rhusiopathiae strains representing 16 serotypes was analyzed for possession of the three currently recognized surface protective antigen (spa)-types: spaA, spaB, and spaC. Polymerase chain reaction (PCR) assays and Western blotting with a SpaA-specific monoclonal antibody demonstrated that spa-type is not confined to specific serotype groups. In particular, the spa-type of strains of aquatic origin was more variable than those of terrestrial origin, and possessed the distinct ability to express more than one spa. In a cross-protection study, mice immunized with an E. rhusiopathiae serotype 2 SpaA-type strain and challenged with various E. rhusiopathiae isolates were completely protected against strains exhibiting a single homologous spa, but variably protected against strains possessing a heterologous spa or those harboring more than one spa-type.

Etiological and biological characteristics of Erysipelothrix rhusiopathiae isolated between 1994 and 2001 from pigs with swine erysipelas in Japan.

We investigated 66 Erysipelothrix rhusiopathiae strains isolated from pigs affected with swine erysipelas in Japan from 1994 to 2001 for serotype, pathogenicity towards mice, protection in vaccinated mice and antimicrobial susceptibility. Most of the isolates (84.8%) were serotype 1 or 2. For the first time, strains belonging to serotype 21 were isolated from cases of septicemia. Fifty isolates (75.8%) were highly virulent, 12 isolates (18.2%) were weakly virulent and 4 isolates were avirulent strains. All the mice vaccinated with the Koganei 65-0.15 vaccine strain survived challenge exposure with 50 highly virulent isolates. Six isolates (9.1%) grew on TPB-T80 agar containing 0.02% of acriflavine, and this was identical to the growth of the vaccine strain. Forty-seven isolates (71.2%) were resistant to oxytetracycline. The number of strains resistant to oxytetracycline among field isolates increased rapidly each year. Tylosin-resistant strains were also isolated (6.1%). These results suggest that certain characteristics, particularly antimicrobial susceptibility of E. rhusiopathiae isolates, change yearly in the field. Therefore, further investigation of the characteristics of E. rhusiopathiae field isolates is necessary.

Genetic and antigenic diversity of the surface protective antigen proteins of Erysipelothrix rhusiopathiae.

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 ( approximately 60%). 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 endocarditis: a preventable zoonosis?

BACKGROUND: Erysipelothrix rhusiopathiae is a bacterium ubiquitous in the environment. It can cause a variety of diseases and the risk of infection is closely related to the level of occupational exposure to infected or colonised animals. AIMS: To discuss the clinical features and treatment of this zoonosis, to increase awareness of this pathogen and to emphasise the need for meticulous attention to hygienic work practices in reducing the risk of infection. METHOD: A case report of a farmer with E. rhusiopathiae endocarditis and the management of the infection. RESULTS: The patient was successfully treated with valve replacement surgery and antimicrobial therapy. CONCLUSIONS: Early identification of this microorganism is essential for appropriate treatment of endocarditis. Greater awareness and safe work practices can help reduce the risk of human infection by this microorganism.

Adhesive surface proteins of Erysipelothrix rhusiopathiae bind to polystyrene, fibronectin, and type I and IV collagens.

Erysipelothrix rhusiopathiae is a gram-positive bacterium that causes erysipelas in animals and erysipeloid in humans. We found two adhesive surface proteins of E. rhusiopathiae and determined the nucleotide sequences of the genes, which were colocalized and designated rspA and rspB. The two genes were present in all of the serovars of E. rhusiopathiae strains examined. The deduced RspA and RspB proteins contain the C-terminal anchoring motif, LPXTG, which is preceded by repeats of consensus amino acid sequences. The consensus sequences are composed of 78 to 92 amino acids and repeat 16 and 3 times in RspA and RspB, respectively. Adhesive surface proteins of other gram-positive bacteria, including Listeria monocytogenes adhesin-like protein, Streptococcus pyogenes protein F2 and F2-like protein, Streptococcus dysgalactiae FnBB, and Staphylococcus aureus Cna, share the same consensus repeats. Furthermore, the N-terminal regions of RspA and RspB showed characteristics of the collagen-binding domain that was described for Cna. RspA and RspB were expressed in Escherichia coli as histidine-tagged fusion proteins and purified. The recombinant proteins showed a high degree of capacity to bind to polystyrene and inhibited the binding of E. rhusiopathiae onto the abiotic surface in a dose dependent manner. In a solid-phase binding assay, both of the recombinant proteins bound to fibronectin, type I and IV collagens, indicating broad spectrum of their binding ability. It was suggested that both RspA and RspB were exposed on the cell surface of E. rhusiopathiae, as were the bacterial cells agglutinated by the anti-RspA immunoglobulin G (IgG) and anti-RspB IgG. RspA and RspB were present both in surface-antigen extracts and the culture supernatants of E. rhusiopathiae Fujisawa-SmR (serovar 1a) and SE-9 (serovar 2). The recombinant RspA, but not RspB, elicited protection in mice against experimental challenge. These results suggest that RspA and RspB participate in initiation of biofilm formation through their binding abilities to abiotic and biotic surfaces.