Quantification of IgY to Erysipelothrix rhusiopathiae in serum from Swedish laying hens.

BACKGROUND: Erysipelas, caused by Erysipelothrix rhusiopathiae (ER), is an important emerging disease in free-range and organic egg-production. The aim of the present study was to assess if quantification of ER specific IgY titers may aid the understanding of erysipelas in commercial laying hens. The methodology was validated with sequentially collected sera from experimentally ER infected SPF-chickens and subsequently applied on sera from Swedish commercial laying hens collected during and after outbreaks of erysipelas or collected at slaughter from healthy hens housed in furnished cages, barn production or in organic production (with outdoor access). RESULTS: In experimentally infected SPF-chickens, titers to ER were significantly increased approximately one week after infection while IgY to ER in uninfected age-matched controls remained low. Also chickens infected with low doses of ER, not displaying clinical signs of disease and with low recovery of ER in blood samples showed high titers of IgY to ER. For laying hens during and after erysipelas outbreaks the majority of samples were considered positive for antibodies to ER with a large variation in levels of IgY titers to ER between individuals. For healthy laying hens at slaughter all samples were deemed positive for antibodies to ER. An influence of flock on levels of IgY titers to ER was observed for both healthy hens and hens during erysipelas outbreaks. For healthy laying hens at slaughter no influence of the housing systems included in the study, history of erysipelas outbreaks at the farm or vaccination on levels of IgY titers to ER was noticed. CONCLUSIONS: Taken together, these results show that high numbers of commercial laying hens showed high IgY titers to ER, comparable to those elicited by experimental ER infection, indicating that ER or bacteria that raises antibodies that cross-react with ER are common in this environment.

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.

Identification of the Chromosomal Region Essential for Serovar-Specific Antigen and Virulence of Serovar 1 and 2 Strains of Erysipelothrix rhusiopathiae.

Erysipelothrix rhusiopathiae causes swine erysipelas, an infection characterized by acute septicemia or chronic endocarditis and polyarthritis. Among 17 E. rhusiopathiae serovars, determined based on heat-stable peptidoglycan antigens, serovars 1 and 2 are most commonly associated with the disease; however, the molecular basis for the association between these serovars and virulence is unknown. To search for the genetic region defining serovar 1a (Fujisawa) strain antigenicity, we examined the 15-kb chromosomal region encompassing a putative pathway for polysaccharide biosynthesis, which was previously identified in the E. rhusiopathiae Fujisawa strain. Six transposon mutants of Fujisawa strain possessing a mutation in this region lost antigenic reactivity with serovar 1a-specific rabbit serum. Sequence analysis of this region in wild-type strains of serovars 1a, 1b, and 2 and serovar N, which lacks serovar-specific antigens, revealed that gene organization was similar among the strains and that serovar 2 strains showed variation. Serovar N strains displayed the same gene organization as the serovar 1a, 1b, or 2 strain and possessed certain mutations in this region. In two of the analyzed serovar N strains, restoration of the mutations via complementation with sequences derived from serovar 1a and 2 strains recovered antigenic reactivity with 1a- and 2-specific rabbit serum, respectively. Several gene mutations in this region resulted in altered capsule expression and attenuation of virulence in mice. These results indicate a functional connection between the biosynthetic pathways for the capsular polysaccharide and peptidoglycan antigens used for serotyping, which may explain variation in virulence among strains of different serovars.

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.

Serological surveillance for antibodies against Erysipelothrix species in wild boar and deer in Japan.

We investigated the seroprevalence of antibodies against Erysipelothrix in wild animals in Japan. Serum samples were collected from 48 wild boar, 26 Yezo deer and 26 Japanese deer in Japan. Growth agglutination (GA) test was performed to estimate antibody titers. As a result, positive results were obtained from 32 (66.7%), 1 (3.6%) and 6 (23.1%) samples from wild boar, Yezo deer and Japanese deer, respectively. Our findings suggest that wild animals may be an important reservoir of Erysipelothrix.

[Identifying immunogenic proteins of Erysipelothrix rhusiopathiae C43065 by MALDI-TOF mass spectrometry and cloning their encoding genes].

OBJECTIVE: To identify immunogenic proteins of Erysipelothrix rhusiopathiae C43065. METHODS: Antigens were extracted from E. rhusiopathiae C43065 by the alkaline extraction method. Proteins in the NaOH-extracted antigen were separated by SDS-PAGE and transferred to nitrocellulose membranes, and then Western blotting was performed with rabbit antiserum against the NaOH-extracted antigens. The immunogenic protein bands were identified by MALDI-TOF mass spectrometry. The genes encoding 5 major immunogenic proteins was amplified by PCR from the genomic DNA of E. rhusiopathiae C43065, and inserted into the pMD18-T vector and then sequenced. RESULTS: A total of 9 immunogenic surface proteins in the NaOH-extracted antigen from E. rhusiopathiae C43065 were successfully identified by MALDI-TOF mass spectrometry. Four of the proteins were putative virulence-associated proteins: enolase, ATP-binding cassette transporter, glyceraldehyde-3 -phosphate dehydrogenase and fructose-bisphosphate aldolase class-II. The genes encoding the chaperone protein GroEL, enolase, ATP-binding cassette transporter, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase class-II were 1614, 1296, 1260, 1005 and 867 bp in length, and the nucleotide sequences homologies of the genes between the C43065 strain and the previously reported E. rhusiopathiae Fujisawa strain was more than 98%. CONCLUSION: Several putative virulence-associated proteins in the NaOH-extracted antigen of E. rhusiopathiae C43065 will be useful for elucidating the roles of these proteins in the pathogenesis of the organism.

Development of an indirect immunofluorescence technique for the evaluation of generated antibody titers against Erysipelothrix rhusiopathiae in captive bottlenose dolphins (Tursiops truncatus).

Erysipelothrix rhusiopathiae is the causative agent of erysipelas, a disease of many mammalian and avian species, mainly swine and turkeys. In cetaceans, erysipelas is considered to be the most common infection in juvenile individuals, which have not been vaccinated. Moreover, the disease manifest in both forms, the dermatologic and the acute septicemic forms, has been reported in various species of dolphins and whales. It is difficult to diagnose erysipelas by currently available approaches. Moreover, it is mainly based on culture methods and also PCR methods, which are currently being developed. At the present stage, prophylactic approaches are based on antibiotic therapy and vaccination mostly with porcine erysipelas vaccines. In the present study, an Indirect Immuno Fluorescence method for the detection of dolphin antibodies levels against E. rhusiopathiae was developed and applied in two different groups of captive bottlenose dolphins (Tursiops truncatus) from Loro Parque (Tenerife, Canary Islands, Spain) and L'Oceanogràfic de Valencia (Valencia, Spain) in order to check the tittering levels of antibodies after application of porcine erysipelas vaccines in the studied dolphins.

Prevalence of Met-203 type spaA variant in Erysipelothrix rhusiopathiae isolates and the efficacy of swine erysipelas vaccines in Japan.

Since 2009, erysipelas infection among pigs in Japan has been increasing. This study investigated the prevalence, and characteristics of Erysipelothrix rhusiopathiae isolates in Japan from 2008 to 2010 and assessed the efficacy of current commercial erysipelas vaccines. Based on polymorphisms in a 432-bp hypervariable region in the surface protective antigen A (spaA) gene, 34 isolates were classified into three groups: (i) Group 1 with methionine at position 203 (Met-203) and isoleucine at position 257 (Ile-257) (18 isolates of serotype 1a and one untypable isolate). (ii) Group 2 with Ile-257 (12 isolates of serotypes 1a, 1b, 2, 10 and 11), and (iii) Group 3 with alanine at position 195 (Ala-195) and Ile-257 (three isolates of serotype 1a). Isolates with Met-203 were highly pathogenic in mice and pigs, causing death in the pig and LD50 values of 0.45-1.45 CFU per mouse. One live and three inactivated commercial E. rhusiopathiae vaccines were evaluated for efficacy against a Met-203 isolate. Almost all mice and pigs that received vaccine survived, while non-vaccinated controls all died within 5 days of the challenge. This indicates that swine erysipelas vaccines might be still effective in protecting animals against the recently prevalent Met-203 isolates in Japan.

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.

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.

Identification of surface protective antigen (spa) types in Erysipelothrix reference strains and diagnostic samples by spa multiplex real-time and conventional PCR assays.

AIM: To develop spa multiplex real-time and conventional PCR assays to detect and differentiate between spaA, spaB and spaC genes within Erysipelothrix spp. METHODS AND RESULTS: For evaluation of the assays, 28 Erysipelothrix spp. reference strains, 25 tissues from pigs inoculated with reference strains of serotypes 1, 2, 5, 10 or 18, and 15 diagnostic samples were used. SpaA was found to be present in Erysipelothrix rhusiopathiae serotypes 1a, 1b, 2, 5, 9, 12, 15, 16, 17, 23 and N; spaB was detected in E. rhusiopathiae serotypes 4, 6, 8, 11, 19 and 21 and spaC was detected in E. sp. strain 2 serotype 18. Spa-related genes were not detected in E. tonsillarum strains (serotypes 3, 7, 10, 14, 20, 22, 24, 25, 26) or E. sp. strain 1 (serotype 13). With the spa multiplex real-time PCR assay, it was also possible to further differentiate spaB into spaB1 (serotypes 4, 6, 8, 19 and 21) and spaB2 (serotype 11). Overall, spaA was detected in seven experimental tissue samples and six diagnostic tissue samples, and spaC in two experimental tissue samples. The detection limits were determined to be five colony-forming units (CFU) per reaction for the spa multiplex real-time PCR assay and 4000 CFU per reaction for the conventional PCR assay. CONCLUSIONS: Both spa PCR assays were specific and reproducible in the identification of spa types in Erysipelothrix spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The described spa PCR assays may be useful tools for investigating spa prevalence among strains isolated from field tissues and to determine the role of the Spa proteins in vaccine protection and pathogenesis.

Development and validation of an immunohistochemical method for rapid diagnosis of swine erysipelas in formalin-fixed, paraffin-embedded tissue samples.

The objective of the study was to develop an immunohistochemical (IHC) assay for rapid detection of Erysipelothrix rhusiopathiae. Serotypes 1a, 1b, and 2 are most frequently associated with clinical disease in pigs. Antiserum against serotypes 1a, 1b, and 2 was produced in rabbits, pooled, and applied to formalin-fixed, paraffin-embedded tissue sections of pigs (lungs, heart, spleen, and skin). The results obtained with the IHC assay were compared with direct culture on tissue samples from experimentally inoculated pigs either treated (n = 6) with antibiotics or untreated (n = 8) as well as on samples from field cases (n = 170) submitted to the Veterinary Diagnostic Laboratory at Iowa State University. The agreement between direct culture and IHC staining was found to be substantial. The results of the present study indicate that the IHC assay is highly sensitive and specific in detecting E. rhusiopathiae antigen in formalin-fixed, paraffin-embedded tissues. Results indicated that the IHC is particularly useful in cases in which pigs had been treated with antibiotics prior to submission and in which direct cultures of organs were negative. In addition, the IHC was found to be useful for detection of E. rhusiopathiae antigen in skin lesions, which are often culture negative.

[Immunity of native SpaA and recombinant SpaA-N against Erysipelothrix rhusiopathiae infection in mice].

OBJECTIVE: We evaluated the effects of surface protective antigen A (SpaA) and its N-teminal protective domain (rSpaA-N) against Erysipelothrix rhusiopathiae infection in mice. METHODS: The SpaA was purified by electroelution from NaOH-extracted antigen of Erysipelothrix rhusiopathiae strain C43311. The rSpaA-N was expressed in E. coli BL21 as a soluble protein by IPTG inducing, and purified with GST affinity chromatography. Mice of each group were subcutaneously immunized three times with 50 microg or 100 microg of native SpaA, rSpaA-N or NaOH-extracted antigen with in complete or incomplete Freund adjuvant at 2-week intervals. Five mice of each group were challenged with 100 LD50 of Erysipelothrix rhusiopathiae virulent strain C43065 two weeks after the third immunization, and the specific antibody responses for SpaA was determined by indirect ELISA. RESULTS: Mice immunized with 50 microg or 100 microg of native SpaA, rSpaA-N, or NaOH-extracted antigen were protected completely against the challenge with strain C43065. There was no significant difference (P < 0.05) between the antibody responses observed for native SpaA, rSpaA-N and NaOH-extracted antigen at any dosages. Western blot results indicated that the native SpaA and rSpaA-N were recognized specifically by an antiserum against the native SpaA of strain C43311. CONCLUSION: These results demonstrated that the rSpaA-N is a protective antigen of Erysipelothrix rhusiopathiae serotype 2 strains, and might be a useful vaccine candidate against swine erysipelas.

Safety of a live attenuated Erysipelothrix rhusiopathiae vaccine for swine.

Infection with Erysipelothrix rhusiopathiae has a significant economic impact on pig production systems worldwide. Both inactivated and attenuated vaccines are available to prevent development of clinical signs of swine erysipelas. The ability of a live attenuated E. rhusiopathiae strain to become persistently established in pigs after intranasal exposure and its potential to cause clinical signs consistent with swine erysipelas after being administered directly into the nasopharynx of healthy pigs was evaluated. Five, E. rhusiopathiae-negative pigs were vaccinated by deep intranasal inoculation then followed for 14 days. Nasal swabs were collected daily for 5 days and clinical observations were made daily for 14 days post-vaccination. Nasal swabs were cultured for E. rhusiopathiae with the intent of back-passaging any recovered organisms into subsequent replicates. No organism was recovered from nasal swabs in the first vaccination replicate. A second replicate including 10 pigs was initiated and followed in an identical manner to that described above. Again, no E. rhusiopathiae was recovered from any pigs. No pigs in either replicate showed any signs of clinical swine erysipelas. The live attenuated E. rhusiopathiae strain evaluated in this study did not appear to become persistently established in pigs post-vaccination, did not cause any local or systemic signs consistent with swine erysipelas, and was therefore unlikely to revert to a virulent state when used in a field setting.

Changes of immunophysiological characteristics in neonatal calves experimentally challenged with mixture of live bacteria and virus.

The aim of the present study was to define efficient immunophysiological parameters in neonatal Holstein calves with an experimentally induced microbial infection. Calves (n = 15) were challenged with classical swine fever virus (LOM strain) and Erysipelothrix insidiosa live vaccine by intravenous injection at 3 wk of age except for control calves (n = 4). The level of total serum IgA was significantly increased at 14 and 19 d post-experimental challenge (DPEC) compared with that in calves at -2 DPEC. At 5 DPEC, relative amounts of bacterial- and viral-specific IgA increased significantly and were sustained until 26 DPEC. In the hematology assay, the neutrophil:lymphocyte ratio (%) in whole blood was significantly decreased at 14 DPEC because of a significant increase in lymphocytes and a coincident decrease in neutrophils. The percentages of CD4+ and CD25+ T cells were significantly decreased at 14 DPEC and returned to initial levels at 19 DPEC. It is intriguing to note that the level of serum lactoferrin was significantly decreased by the microbial challenge within 1 d. The concentration of haptoglobin was increased within 3 d and gradually decreased in calves after microbial challenge. Our results suggest that 1) bovine serum lactoferrin plays an important role in the innate immune response against microbial infection at an early stage and 2) experimentally induced microbial challenge using porcine live bacterial and viral vaccine in calves could be a good experimental model to evaluate the effect of diet or stress induced by environmental change on the immune responses against microbial infection.

Effect of blood sample handling post-collection on Erysipelothrix rhusiopathiae antibody titres.

A study was conducted to determine the effect of blood sample mishandling on the performance of an enzyme-linked immunosorbent assay for the detection of antibodies against Erysipelothrix rhusiopathiae. Eleven sample maltreatments (storage at -10 degrees C, storage at 4 degrees C, heat treatment of clotted blood, haemolysis, repetitive freeze-thaw cycling, and substitution of plasma in place of serum) were simulated in a laboratory environment and then run concurrently against a gold standard sample (storage at -80 degrees C). The mishandling treatment groups that simulated high levels of haemolysis had significantly lower optical density (OD) readings when compared to the gold standard. However, the magnitude of the effects was relatively small and only samples with OD values close to the cut-off changed state from positive to negative. Heat treatment had a minor, but non-significant, effect on OD values. Findings from this study suggested that immunoglobulin G antibody was stable in the face of most common sample mishandling events.

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.

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.

Characterization of protective antigen CbpB as an adhesin and a plasminogen-binding protein of Erysipelothrix rhusiopathiae.

Erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and human erysipeloid. E. rhusiopathiae CbpB has been reported to be a protective antigen, but its pathogenic roles are not known. The aim of this study was to evaluate the ability of CbpB to act as an adhesin in E. rhusiopathiae adhesion to porcine endothelial cells as well as a host plasminogen- and fibronectin- binding protein. Recombinant CbpB (rCbpB) was successfully obtained, and it was found that E. rhusiopathiae CbpB was located on the cell surface of E. rhusiopathiae. Moreover, CbpB exhibited binding activity to porcine endothelial cells. Recombinant CbpB successfully bound to host plasminogen but was unable to bind to fibronectin. In conclusion, our work suggested that CbpB is a virulence factor of E. rhusiopathiae.