Immunogenicity and Protection by DnaK and SpaA Recombinant Proteins Against Erysipelothrix Rhusiopathiae in a Murine Model.

BACKGROUND/AIMS: Swine erysipelas is a disease caused by Erysipelothrix rhusiopathiae, a Gram-positive bacillus, which has great economic importance because it leads to the loss of the swine herd. To control this disease, animals are immunized with a cellular vaccine of killed or attenuated E. rhusiopathiae, but even with herd vaccination, cases of swine erysipelas outbreaks have been reported in the United States, China and Japan, leading to the search for other antigenic components of the bacteria that may promote greater protection against E. rhusiopathiae. The surface protein SpaA from E. rhusiopathiae has been shown to be a candidate to constitute a subunit vaccine, since it has already been reported to induce a host immune response against the bacterium. DnaK, a hsp70 molecular chaperone, also seems to be a good candidate in the composition of a vaccine, as it has been demonstrated to be an antigenic protein of the bacteria. METHODS: This work evaluated the immunogenicity and protection induced by the E. rhusiopathiaee SpaA and DnaK recombinant proteins in a murine model, by intramuscular administration to mice with two doses of 100 µg at 21-day interval between them. The candidate proteins were tested either separately and together, compared with the commercial vaccine and the non-vaccination condition, and mice were challenged with a virulent strain of E. rhusiopathiae. Serum was collected to assess the produced antibodies and peripheral blood cells, whereas spleen and kidney tissues were assayed for E. rhusiopathiae presence by colony counting. RESULTS: A survival curve of the animals was performed, which confirmed the protection induced by the proteins. IgG antibodies increased in the animal serum inoculated with the proteins when compared to the control, and a significant delay in disease symptoms was observed. CONCLUSION: These results suggest that E. rhusiopathiae DnaK and SpaA are immunogenic in mice and interfere with the disease development.

Comparative study of the phenotype and virulence of recent serovar 1a, 1b, and 2a isolates of Erysipelothrix rhusiopathiae in Japan.

Erysipelothrix rhusiopathiae causes swine erysipelas (SE) and is classified -into 16 serovars based on cell surface antigens. Our previous study suggested that recent SE outbreaks were mostly caused by serovar 1a of E. rhusiopathiae with the surface protective antigen (Spa)A protein characterized by methionine and isoleucine at positions 203 and 257 (M203/I257 SpaA). In this study, four recent E. rhusiopathiae isolates comprising two serovar 1a with M203/I257 SpaA strains (2012 Miyazaki and 2012 Chiba), one serovar 1b strain (2015 Miyazaki), and one serovar 2a strain (2012 Nagano) were compared with each other and with the serovar 1a Fujisawa reference strain regarding in vitro phenotypes and in vivo virulence in mice and pigs. The serovar 1b and 2a strains, which are the less prevalent strains in the field in Japan, showed lower growth in liquid culture and lower virulence in animals than the serovar 1a variants. Adhesion of the serovar 2a strain to porcine endothelial cells was weaker than that of the serovar 1a and 1b strains. Several advantages of serovar 1a strains were found, but no plausible cause of the M203/I257 SpaA type variants to be selected for the most prevalent strains among serovar 1a strains was identified in this study.

Potential risk factors of swine erysipelas outbreak in Northeast Mainland China.

Swine erysipelas is a common infectious disease that affects the pig-breeding industry. The purpose of this study was to elucidate the risk factors and their role in the prevalence of swine erysipelas so that one may be able to better prevent and control the swine erysipelas outbreaks in Northeast China. Using spatial clusters, the study area was divided into two parts: South Central Mainland China (hot spots) and Northeast Mainland China (potential outbreak areas). We investigated a total of 31 environmental factors and used the lasso regression and k-fold cross-validation methods to determine the main factors involved. Seven risk factors were determined to have a major impact on swine erysipelas. Multiple logistic regression was used to examine the contribution of these seven risk factors to the outbreak from 2008 to 2018, in the two research regions. In South Central Mainland China, where swine erysipelas was most prevalent, the extreme maximum temperature [OR = 1.143 (95%CI: 1.032-1.342)], total precipitation [OR = 2.298 (95%CI: 1.410-5.112)] and precipitation ≥0.1 mm [OR = 2.396 (95%CI: 1.329-5.941)] exhibited positive effects. The maximum wind speed [OR = 0.550 (95%CI: 0.303-0.775)] and concentration of O3-8H-90 per [OR = 0.876 (95%CI: 0.747-0.980)] exhibited negative effects. Summer was the main season for the erysipelas epidemic in South Central Mainland China. In Northeast China, only the total precipitation [OR = 1.048 (95%CI: 0.900-0.989)] was positively correlated with the prevalence of swine erysipelas, whereas the other factors were not significant. There was no obvious seasonal feature for the epidemic. Through the comparison and analysis of risk factors between the two research regions, more attention should be given to the impacts of high temperature and precipitation on the swine erysipelas epidemic in Northeast China.

Serovars and SpaA Types of Erysipelothrix rhusiopathiae Isolated from Pigs in Japan from 2012 to 2019.

Erysipelothrix rhusiopathiae causes swine erysipelas (SE), which results in considerable economic loss on pig farms. During SE outbreaks that occurred sporadically from 2008 to 2011 in Japan, new E. rhusiopathiae strains were isolated with a specific surface protective antigen (Spa)A protein characterized by methionine at position 203 and isoleucine at position 257 (M203/I257 SpaA type). To determine whether strains with the M203/I257 SpaA type are still prevalent in Japan, we collected 79 strains of E. rhusiopathiae from pigs showing various SE symptoms from 2012 to 2019 and classified them based on serovar typing, spaA gene sequence analysis, and lineage typing. We found that the majority of recent E. rhusiopathiae strains (59/79) belonged to the serovar 1a strain, and that the M203/I257 SpaA type (56/59) was predominant continuing from 2008 to 2011. Furthermore, serovar 1a strains with IVb-1 and IVb-2 lineages that had been isolated in specific regions of Japan were no longer local but were found across Japan. The pathogenicity of recent isolates tested in mice was not significantly changed when compared to that of previously isolated strains. Our results suggest that recent SE outbreaks were not due to changes in the SpaA protein or to altered virulence of E. rhusiopathiae but were rather caused by the persistent presence of E. rhusiopathiae with the M203/I257 SpaA type.

Erisipela suína: relato de caso no município de Cachoeiras de Macacu-RJ / Swine erysipelas: case report in the city of Cachoeiras de Macacu-RJ

Na suinocultura perdas econômicas ainda são elevadas devido aos baixos padrões de qualidade e sanidade dos animais. Dentre as afecções que afetam a produção, a erisipela é uma doença considerada importante em função dos prejuízos econômicos que causa, e pela questão de saúde pública visto ser uma zoonose. Ela é uma enfermidade do tipo hemorrágica comumente causada pela bactéria ubíqua Erysipelotrix rhusiopathiae. O objetivo deste trabalho foi relatar um caso desta afecção em uma matriz da raça Large White, de dois anos de idade, recém desmamada, não vacinada, de uma pequena granja de ciclo completo no munícipio de Cachoeiras de Macacu, estado do Rio de Janeiro. Ela amanheceu prostrada, com dificuldade de locomoção, sem febre e com manchas avermelhadas sobre toda a superfície corporal. As lesões cutâneas, ligeiramente elevadas, apresentavam um formato losangular (diamante) característico e sugestivo de Erisipela. Após a identificação do problema, o animal foi isolado e tratado. O tratamento iniciou-se na manhã do mesmo dia, observando-se a regressão da maioria das lesões à tarde e na manhã seguinte. A suspeita clínica foi confirmada através do diagnóstico terapêutico, sendo a associação de penicilina e estreptomicina eficiente no tratamento.

In swine industry, economic losses are still high due to low standards of quality and health of animals. Among the diseases that affect production, erysipelas is a disease considered important due to the economic losses it causes, and because of the public health issue as it is a zoonosis. It is a hemorrhagic type disease commonly caused by the ubiquitous bacteria Erysipelotrix rhusiopathiae. The aim of this study was to report a case of this condition in a Large White breed sow, two years old, recently weaned, not vaccinated, from a small pig farm (farrow to finish operation) in the municipality of Cachoeiras de Macacu, state of Rio de Janeiro. The sow was prostrate and with limited mobility, without fever and with reddish spots on the entire body surface. The cutaneous lesions were elevated, with a characteristic diamond shape suggestive of erysipelas. After identifying the problem, the animal was isolated and treated. The treatment started in the morning of the same day, observing the regression of most lesions in the afternoon and the following morning. The clinical diagnosis was confirmed through therapeutic diagnosis, and the association of penicillin and streptomycin was efficient in the treatment.

Genome sequence of multidrug-resistant Erysipelothrix rhusiopathiae ZJ carrying several acquired antimicrobial resistance genes.

OBJECTIVES: This study aimed to determine the genetic environment of antimicrobial resistance genes (ARGs) in Erysipelothrix rhusiopathiae strain ZJ isolated from a pig with symptoms of swine erysipelas in China. METHODS: Illumina MiSeq (200× coverage) and PacBio RS II (100× coverage) platforms were used for genome sequencing. ARGs and prophages were identified using ResFinder 3.0 and PHASTER, respectively. A conjugation experiment, induced prophage infection and long-term passage assay were performed to determine the transferability and stability of ARGs in this strain. RESULTS: The assembled circular genome of E. rhusiopathiae ZJ was 1 945 689 bp with a GC content of 36.48%; no plasmid sequence was detected. Eleven acquired ARGs were identified in the genome. A novel integrative and conjugative element (ICE) encoding a multidrug resistance (MDR) gene cluster [aadE-apt-spw-lsa(E)-lnu(B)-aadE-sat4-aphA3] was identified in strain ZJ. A prophage Φ1605 harbouring mef(A)-msr(D) and tet(M) was also found in this strain, which can take a circular form and can be induced by mitomycin C to infect E. rhusiopathiae G4T10 for ARG transfer. CONCLUSION: To our knowledge, this is the first report of a complete genome sequence of E. rhusiopathiae carrying multiple ARGs obtained from a pig farm. This is the first identification of a novel chimeric ICE carrying a MDR gene cluster and a prophage carrying ARGs in E. rhusiopathiae, which will provide a valuable reference to understand the potential transfer mechanism of MDR gene clusters carried by ICEs and prophages in Gram-positive bacteria.

Wild boars: A potential source of Erysipelothrix rhusiopathiae infection in Japan.

The potential role of wild boars as a source of erysipelas infection was investigated. An ELISA test of wild boar serum samples from 41 prefectures in Japan revealed that proportions of the Erysipelothrix rhusiopathiae-positive samples were very high in all the prefectures, and the mean positive rate was 95.6% (1312/1372). Serovars of E. rhusiopathiae isolates from wild boars were similar to those of previously reported swine isolates, and all serovar isolates tested were found to be pathogenic to mice. These results suggest that wild boars in Japan constitute a reservoir of E. rhusiopathiae and may pose risks to other animals.

Production diseases in smallholder pig systems in rural Lao PDR.

Pigs in Lao People's Democratic Republic are important for income and food security, particularly in rural households. The majority of pigs are reared in smallholder systems, which may challenge the implementation of any disease control strategies. To investigate risk factors for pig production diseases in such farming systems in the country a serological survey was conducted during 2011. A total of 647 pigs were sampled, accounting for 294 households in Luang Prabang and 353 in Savannakhet province representing upland and lowland, respectively. The results demonstrated that pigs in Lao PDR had antibodies against erysipelas (45.2%), CSF (11.2%), PRRS (8.6%), FMD O (17.2%) and FMD Asia 1, (3.5%). Differences in the housing systems influenced disease risk, for example, penned pigs had reduced odds of FMD and CSF, compared to those in scavenger systems. Pigs owned by farms using a sanaam (a communal area where pigs are kept for some time of the year) had 3.93 (95% confidence interval (CI): 1.09-14.7) times the odds of having pigs seropositive for FMD. Farms on which sudden piglet deaths had been experienced were more likely to have pigs seropositive for FMD O and erysipelas. These diseases constrain the development of village farming and the wider livestock industry due to their impact on productivity and trade. Vaccination coverage for FMD and CSF was low and there was a lack of national funding for livestock disease control at the time of the study. Further investigation into sustainable low-cost control strategies for these pathogens is warranted.

Influence of swine leukocyte antigen haplotype on serum antibody titers against swine erysipelas vaccine and reproductive and meat production traits of SLA-defined selectively bred Duroc pigs.

We investigated possible associations of SLA class II haplotypes with serum antibody titers against a swine erysipelas vaccine, reproductive and meat production traits using a population of selective breeding Duroc pigs. In the selective breeding Duroc pigs, four SLA class II-DRB1 and -DQB1 alleles were assigned by using PCR-sequence specific primer technique. Low-resolution haplotype (Lr)-0.30 and/or Lr-0.13 were deduced from the SLA class II alleles in the population of SLA-defined Duroc pigs. SLA-homozygous piglets with the Lr-0.30 haplotype had relatively lower serum antibody titers against the vaccine compared to those with Lr-0.13. In contrast, there were no statistically significant differences in reproductive performance between the SLA-defined pigs with two SLA class II haplotypes. Weaning and rearing rates until the body weight of 105 kg was reached in homozygous piglets with Lr-0.30 were significantly lower than those in homozygous piglets with Lr-0.13. The SLA-defined pigs had lower birth and weaning weights, body weights at 60 days of age, and daily weight gains than non-selective breeding Duroc pigs. Furthermore, the SLA-defined pigs had slightly lower back fat thickness compared to the non-selective breeding pigs. The rib eye areas of homozygous or heterozygous pigs with Lr-0.13 were larger than those of homozygous pigs with Lr-0.30 and non-selective breeding pigs. These data suggested that SLA haplotypes had the potential as useful genetic markers for selective breeding in the population of SLA-defined Duroc pigs.

Clonal Lineages of Erysipelothrix rhusiopathiae Responsible for Acute Swine Erysipelas in Japan Identified by Using Genome-Wide Single-Nucleotide Polymorphism Analysis.

Erysipelothrix rhusiopathiae causes swine erysipelas, an important infectious disease in the swine industry. In Japan, the incidence of acute swine erysipelas due to E. rhusiopathiae serovar 1a has recently increased markedly. To study the genetic relatedness of the strains from the recent cases, we analyzed 34 E. rhusiopathiae serovar 1a swine isolates collected between 1990 and 2011 and further investigated the possible association of the live Koganei 65-0.15 vaccine strain (serovar 1a) with the increase in cases. Pulsed-field gel electrophoresis analysis revealed no marked variation among the isolates; however, sequencing analysis of a hypervariable region in the surface-protective antigen A gene (spaA) revealed that the strains isolated after 2007 exhibited the same spaA genotype and could be differentiated from older strains. Phylogenetic analysis based on genome-wide single-nucleotide polymorphisms (SNPs) revealed that the Japanese strains examined were closely related, showing a relatively small number of SNPs among them. The strains were classified into four major lineages, with Koganei 65-0.15 (lineage III) being phylogenetically separated from the other three lineages. The strains isolated after 2007 and the two older strains constituted one major lineage (lineage IV) with a specific spaA genotype (M203/I257-SpaA), while the recent isolates were further divided into two geographic groups. The remaining older isolates belonged to either lineage I, with the I203/L257-SpaA type, or lineage II, with the I203/I257-SpaA type. These results indicate that the recent increased incidence of acute swine erysipelas in Japan is associated with two sublineages of lineage IV, which have independently evolved in two different geographic regions.IMPORTANCE Using large-scale whole-genome sequence data from Erysipelothrix rhusiopathiae isolates from a wide range of hosts and geographic origins, a recent study clarified the existence of three distinct clades (clades 1, 2, and 3) that are found across multiple continents and host species, representing both livestock and wildlife, and an "intermediate" clade between clade 2 and the dominant clade 3 within the species. In this study, we found that the E. rhusiopathiae Japanese strains examined exhibited remarkably low levels of genetic diversity and confirmed that all of the Japanese and Chinese swine isolates examined in this study belong to clonal lineages within the intermediate clade. We report that spaA genotyping of E. rhusiopathiae strains is a practical alternative to whole-genome sequencing analysis of the E. rhusiopathiae isolates from eastern Asian countries.

Pathogenic characterization of Erysipelothrix rhusiopathiae Met-203 type SpaA strains from chronic and subacute swine erysipelas in Japan.

To characterize the Erysipelothrix rhusiopathiae Met-203 type surface protective antigen (Spa) A strains causing swine erysipelas in Japan, the nucleotide sequence of the hypervariable region of the spaA gene was determined in 80 E. rhusiopathiae (serotype 1a) isolates collected from pigs with chronic and subacute swine erysipelas in 14 prefectures in 2008-2014. In this study, 14 (17.5%) isolates were Met-203 type SpaA strains. We confirmed the pathogenicity of a Met-203 type SpaA strain in specific-pathogen-free pigs. In this experiment, the two challenged pigs displayed arthritis, urticaria and other clinical signs, but recovered within 10 days. Our results reveal the existence of the E. rhusiopathiae Met-203 type strains that have been causing chronic erysipelas in Japan.

Effects of amoxicillin, ceftiofur, doxycycline, tiamulin and tulathromycin on pig humoral immune responses induced by erysipelas vaccination.

It addition to their antimicrobial properties, antibiotics can influence the host immune system (modulation of cytokine secretion, antibody production and T-cell proliferation). In the present study, the authors studied the effects of therapeutic doses of amoxicillin (AMX), ceftiofur (CEF), doxycycline (DOXY), tiamulin (TIAM) and tulathromycin (TUL) on the postvaccinal immune response after pigs had been vaccinated against erysipelas. Because humoral immunity is considered as the most important in the protection against swine erysipelas, the present study focused on the interactions between antibiotics and postvaccinal humoral immunity. One hundred and five, eight-week-old pigs of both sexes were used. Specific antibodies to the Erysipelothrix rhusiopathiae antigen were determined using a commercial ELISA test. In pigs treated with DOXY or CEF or TIAM, a significant reduction in the number of positive pigs was observed four and six weeks after the second dose of vaccine, compared with the remaining vaccinated groups. In pigs treated with CEF, the ELISA score was significantly lower than in non-treated vaccinated pigs. While in vaccinated pigs treated with AMX or TUL, the ELISA score was significantly higher than in pigs treated with the remaining antibiotics and than in non-treated vaccinated controls. The results of the present study indicate that vaccination of pigs against erysipelas in the presence of antibiotics may result in a decrease (CEF, DOXY, TIAM) or enhancement (AMX, TUL) in the production of specific antibodies.

Osteochondrosis, but not lameness, is more frequent among free-range pigs than confined herd-mates.

BACKGROUND: Organic pig production is expanding and amongst the objectives of organic farming are enhancing animal health and welfare. However, some studies have reported a higher prevalence of lameness and joint condemnation at slaughter in free-range/organic pigs than in conventionally raised pigs. Organic slaughter pigs have free-range housing in which indoor and outdoor access is compulsory, while in conventional farming the pigs are commonly confined to indoor pens. The present study evaluated the effects of free-range and confined housing on lameness prevalence in a herd of 106 finisher pigs, and whether osteochondrosis and Erysipelothrix rhusiopathiae associated arthritis influences these effects. We also evaluated the association between clinical lameness during the rearing period and joint condemnations at slaughter. RESULTS: Seventy free-range and 36 confined housed fattener pigs were scored for their gait twice during the rearing period and 848 joints were evaluated post mortem. Osteochondrosis was more frequent among free-range than confined pigs (P < 0.05), and when present it was also more severe (P < 0.001). Pigs with more numerous and more severe osteochondral lesions had their gait affected more than did pigs with fewer such lesions (P < 0.05). Hence it was a paradox that we did not detect more lameness among the free-range pigs than the confined pigs. E. rhusiopathiae associated arthritis was not diagnosed. The association between gait remarks/clinical lameness and joint condemnations at slaughter was not significant. CONCLUSIONS: The results indicate that free-range housing may have both positive and negative effects on locomotory traits. Free-range pigs may be less clinically affected by osteochondrosis than are confined pigs. One explanation for this effect may be strengthening of joint supportive tissue and pain relief promoted by exercise. Visual gait scoring missed serious joint lesions that probably were harmful to the pigs, and should therefore not be used as a sole indicator of joint/leg health in welfare inspection of pigs. The association between gait scores and joint condemnation appeared to be poor. This study was limited to one herd, and so more and larger studies on the effects of free-range housing on lameness severity and osteochondrosis development in pigs are recommended.

Role of surface protective antigen A in the pathogenesis of Erysipelothrix rhusiopathiae strain C43065.

To clarify the role of surface protective antigen A (SpaA) in the pathogenesis of Erysipelothrix rhusiopathiae C43065 (serotype 2), the spaA deletion mutant of E. rhusiopathiae ΔspaA was constructed by homologous recombination. The virulence of the ΔspaA mutant decreased more than 76-fold compared with that of the wild-type strain C43065 in mice. The mutant strain was sensitive to the bactericidal action of swine serum, whereas the wild-type strain was resistant. The adhesion of wild-type strain to MEF cells was inhibited significantly by treatment with rabbit antiserum against recombinant SpaA (rSpaA) as compared with the treatment with normal rabbit serum, but the mutant strain was not affected. The mutant strain was readily taken up by mouse peritoneal macrophages in the normal rabbit serum, whereas the wild-type strain was resistant. Whereas the rabbit antiserum against rSpaA promoted the phagocytosis of wild-type strain by macrophages, the mutant strain was not affected. In addition, mice vaccinated with the formalin-killed mutant strain were provided 40% protection against challenge by the homologous virulent strain as compared with those with wild-type strain, NaOH-extracted antigen, or rSpaA, which provided more than 80% protection against the same infection. These suggested that SpaA has an important role in the pathogenesis of E. rhusiopathiae infection and could be a target for vaccination against swine erysipelas.

Complete genome assembly and characterization of an outbreak strain of the causative agent of swine erysipelas--Erysipelothrix rhusiopathiae SY1027.

BACKGROUND: Erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and, to a fewer occurrences, human erysipeloid. It is ubiquitous in nature and commensal in diverse species of animals, wild or domestic, from mammals and birds to reptiles and fish. Mechanisms of its virulence and pathogenicity are poorly understood. RESULTS: Making use of the complete genome sequencing of E. rhusiopathiae strain SY1027 and comparative genome analysis between the three highly pathogenic strains (SY1027, Fujisawa and ATCC19414), the genomic structure and putative functional elements, such as pathogenicity island (PAI)-like regions, potential virulence factors and horizontal transferring genes of the bacteria are identified. Strain SY1027 genome is 1,752,910 base pairs long, just 30 kilobases smaller than strain Fujisawa, with the same GC level of 36.36%. It contains 1,845 open reading frames (ORF) predicted by GLIMMER 3.02, of which 1,775 were annotated by PGAAP, 1,757 (~95.23%) were annotated by NCBI nr blast, 1,209 by COG database and 1,076 by KEGG database. 37 potential virulence factors were annotated in strain SY1027 by VFDB, while 19 (~51.35%) of them are common in the 2 strains, 7 of which are potentially related to antibiotic resistance and highly conserved (~98-100% match identity (ID)) amongst the three strains of E. rhusiopathiae and modestly homologous to other gastrointestinal tract-inhabiting Firmicutes (~40% match ID), e.g. Clostridium spp., Enterococcus spp. Genomic island- and pathogenicity island-like regions were also predicted, in which some showed association with tRNA and potential virulence factors. CONCLUSION: Complete genome sequencing of Erysipelothrix rhusiopathiae, the causative agent of animal erysipelas, was performed. Molecular identification of various genomic elements pave the way to the better understanding of mechanisms underlying metabolic capabilities, pathogenicity of swine erysipelas and prospective vaccine targets besides the widely used SpaA antigens.

Characterization and identification of a novel candidate vaccine protein through systematic analysis of extracellular proteins of Erysipelothrix rhusiopathiae.

Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, is a facultative intracellular Gram-positive bacterium. It has been shown that animals immunized with a filtrate from E. rhusiopathiae cultures are protected against lethal challenge. In this study, we identified and characterized the extracellular proteins of E. rhusiopathiae to search for novel vaccine antigens. A concentrated culture supernatant from the E. rhusiopathiae Fujisawa strain, which has been found to induce protection in mice, was analyzed using two-dimensional electrophoresis. From more than 40 confirmed protein spots, 16 major protein spots were selected and subjected to N-terminal amino acid sequence determination, and 14 protein spots were successfully identified. The identified proteins included housekeeping proteins and other metabolic enzymes. We searched for surface-localized proteins by analyzing the genomes of two E. rhusiopathiae strains: Fujisawa and ATCC 19414. Genome analysis revealed that the ATCC 19414 strain has three putative surface-exposed choline-binding proteins (CBPs): CbpA, CbpB, and CbpC. Each CBP contains a putative choline-binding domain. The CbpC gene is mutated in Fujisawa, becoming a nonfunctional pseudogene. Immunogold electron microscopy confirmed that CbpA and CbpB, as well as the majority of the metabolic enzymes examined, are associated with the cell surface of E. rhusiopathiae Fujisawa. Immunization with recombinant CbpB, but not with other recombinant CBPs or metabolic enzymes, protected mice against lethal challenge. A phagocytosis assay revealed that antiserum against CbpB promoted opsonin-mediated phagocytosis by murine macrophages in vitro. The protective capabilities of CbpB were confirmed in pigs, suggesting that CbpB could be used as a vaccine antigen.

Capsular polysaccharide of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, and its modification with phosphorylcholine.

The capsule has been implicated in the virulence of the swine pathogen Erysipelothrix rhusiopathiae, a rod-shaped, intracellular Gram-positive bacterium that has a unique phylogenetic position in the phylum Firmicutes and is a close relative of Mollicutes (mycoplasma species). In this study, we analyzed the genetic locus and composition of the capsular polysaccharide (CPS) of the Fujisawa strain of E. rhusiopathiae. Genome analysis of the Fujisawa strain revealed that the genetic locus for capsular polysaccharide synthesis (cps) is located next to an lic operon, which is involved in the incorporation and expression of phosphorylcholine (PCho). Reverse transcription-PCR analysis showed that cps and lic are transcribed as a single mRNA, indicating that the loci form an operon. Using the cell surface antigen-specific monoclonal antibody (MAb) ER21 as a probe, the capsular materials were isolated from the Fujisawa strain by hot water extraction and treatment with DNase, RNase, pronase, and N-acetylmuramidase SG, followed by anion-exchange and gel filtration chromatography. The materials were then analyzed by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The CPS of E. rhusiopathiae is heterogeneous and consists of the major monosaccharides galacturonic acid, galactose, mannose, glucose, arabinose, xylose, and N-acetylglucosamine and some minor monosaccharides containing ribose, rhamnose, and N-acetylgalactosamine. In addition, the capsule is modified by PCho, which comigrates with the capsular materials, as determined by Western immunoblotting, and colocalizes on the cell surface, as determined by immunogold electron microscopy. Virulence testing of PCho-defective mutants in mice demonstrated that PCho is critical for the virulence of this organism.

Towards in vitro potency testing of inactivated erysipelas vaccines.

Ph. Eur. Monograph 0064 "Swine erysipelas vaccine (inactivated)" currently advises mouse serology for batch potency testing. However, technological advances in vaccine production, improved quality control systems and comprehensive post marketing surveillance increasingly promote the acceptance of non-animal approaches for batch release testing. Protein and immune profiles of inactivated swine erysipelas vaccines obtained by SDS-PAGE and Western Blot might offer a convenient global and functional in vitro alternative. Characteristic and consistent protein and immune profiles could be obtained for aluminium-adjuvanted vaccines. Immunoreactivity of polyclonal sera raised in mice differs markedly from reactivity of swine sera.

Cloning, auto-induction expression, and purification of rSpaA swine erysipelas antigen.

This work reports the cloning, expression, and purification of a 42-kDa fragment of the SpaA protein from Erysipelothrix rhusiopathiae, the main antigenic candidate for a subunit vaccine against swine erysipelas. The use of an auto-induction protocol to improve heterologous protein expression in recombinant Escherichia coli cultures was also investigated. The cellular growth pattern and metabolite formation were evaluated under different induction conditions. The His-tagged protein was over-expressed as inclusion bodies, and was purified by a single chromatography step under denaturing conditions. Auto-induction conditions were shown to be an excellent process strategy, leading to a high level of rSpaA expression (about 25 % of total cellular protein content) in a short period of time.