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.

Immune responses upon experimental Erysipelothrix rhusiopathiae infection of naïve and vaccinated chickens.

Erysipelas, a disease caused by Erysipelothrix rhusiopathiae (ER), is an increasing problem in laying hens housed in cage-free systems. This study aimed to monitor immune responses during ER infection of naïve chickens and chickens vaccinated intra muscularly with a commercial inactivated ER vaccine. Chickens were infected intra muscularly with ER at 30 days of age and blood leukocyte counts, serum levels of mannose binding lectin (MBL) and ER-specific IgY were monitored until the experiment was terminated at day 15 after infection. ER was detected in blood from more chickens and at higher bacterial counts in the naïve group (day 1: 1 of 7 chickens; day 3: 6 of 6 chickens) than in the vaccinated group (day 1: 0 of 7 chickens; day 3: 1 of 6 chickens). During the acute phase of infection transient increases in circulating heterophil numbers and serum MBL levels were detected in all ER infected chickens but these responses were prolonged in chickens from the naïve group compared to vaccinated chickens. Before infection IgY titers to ER in vaccinated chickens did not differ significantly from those of naïve chickens but vaccinated chickens showed significantly increased IgY titers to ER earlier after infection compared to chickens in the naïve group. In conclusion, the ER infection elicited prompt acute innate responses in all chickens. Vaccinated chickens did not have high IgY titers to ER prior to infection but did however show lower levels of bacteraemia and their acute immune responses were of shorter duration.

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.

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.

Identification of serovar 1a, 1b, 2, and 5 strains of Erysipelothrix rhusiopathiae by a conventional gel-based PCR.

Among the four species of the genus Erysipelothrix, Erysipelothrix rhusiopathiae is the main species that causes disease in swine and poultry and has also been isolated from human patients. Recently, E. rhusiopathiae infections in domesticated animals have increased in many countries and are also the cause of emerging wildlife disease in arctic and boreal ecosystems. Historically, E. rhusiopathiae has been differentiated from other Erysipelothrix species by their serovars, which are determined based on cell wall antigens. Serotyping of Erysipelothrix is important, as specific E. rhusiopathiae serovars (1a, 1b, and 2) are associated with disease in pigs, poultry, and humans. However, serotyping is laborious and time-consuming and requires a full set of serovar reference strains and strain-specific antiserum. In this study, to develop a conventional gel-based PCR assay that can detect the main disease-associated serovars of E. rhusiopathiae, the draft genome sequences of E. rhusiopathiae strains of serovars 1a, 1b, 2, and 5, the last of which is often isolated from wild animals, were analyzed. Primers were designed based on the serovar-specific sequences of the strains and tested for field strains isolated from extensive origins. Among two hundred and ninety-seven isolates of various serovar strains of E. rhusiopathiae and other Erysipelothrix species, the PCR assay identified serovar 1a, 1b, 2, and 5 strains of E. rhusiopathiae. This conventional gel-based PCR assay should be useful for serovar surveillance of E. rhusiopathiae isolates in domesticated and wild animals as well as in humans.

Generation of murine monoclonal antibodies with specificity against conventional camelid IgG1 and heavy-chain only IgG2/3.

Camelids possess antibodies with a conventional four-chain structure consisting of two heavy and two light chains (of subclass IgG1) but further they also generate heavy-chain only antibodies (of subclass IgG2 and 3) which are fully functional in antigen binding. In this study subclass-specific murine monoclonal antibodies specific to conventional camelid IgG1 and heavy-chain only IgG2/3 were generated and validated for the use as potent secondary detection reagents. The monoclonal antibodies are able to differentiate between all camelid IgGs, conventional four-chain camelid antibodies (of subclass IgG1) and exclusively heavy chain-only antibodies (of subclasses IgG2 and IgG3). Further these antibodies were used to detect specific immune responses after vaccination of Camelids against bovine corona- and rotavirus strains and different E.coli and Clostridia - antigens and to identify Erysipelothrix rhusiopathiae infected animals within a herd. The described antibodies are suitable as new secondary agents for the detection of different camelid subclasses and the validation of camelid immune reactions.

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.

Reproduction of post-weaning multi-systemic wasting syndrome in an animal disease model as a tool for vaccine testing under controlled conditions.

Snatch farrowed, colostrum deprived piglets were inoculated with different combinations of porcine circovirus 2, porcine parvovirus and Erysipelothrix rhusiopathiae candidate vaccines. 10 piglets were mock-vaccinated. Following virus challenge with a combined porcine circovirus 2/porcine parvovirus inoculum, all animals were monitored and samples taken for serology, immunohistochemistry and qPCR. At 24 dpc all non-vaccinated animals remaining were exhibiting signs of post-weaning multi-systemic wasting syndrome which was confirmed by laboratory analysis. Details of the study, analysis of samples and performance of the candidate vaccines are described.

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.

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.

Cutaneous Erysipelothrix rhusiopathiae (erysipeloid) infection in an immunocompromised child.

Erysipeloid, a cutaneous infection with the gram-positive bacillus Erysipelothrix rhusiopathiae, is typically an occupational dermatosis seen in persons working with livestock or involved in commercial fishing (fishmongers). Other more-generalized forms of infection with this organism also exist, including a septic form usually associated with endocarditis. Many infections may be self-limited. They have rarely been reported in children or in immunocompromised patients. This microbe is sensitive to many mainstream antibiotic agents.

Erysipelothrix rhusiopathiae isolates recovered from fish, a harbour seal (Phoca vitulina) and the marine environment are capable of inducing characteristic cutaneous lesions in pigs.

In order to determine the diversity and pathogenicity of Erysipelothrix spp. isolates recovered from marine fish, a harbour seal (Phoca vitulina) and the marine environment, 14 isolates were characterized by genotyping, serotyping, determination of the surface protective antigen (spa) gene type and assessment of virulence in a pig bioassay. All 14 isolates were Erysipelothrix rhusiopathiae. Isolates were determined to be of serotypes 2 (n = 3), 3 (n = 1), 4 (n = 1), 12 (n = 1), 15 (n = 1) or 21 (n = 6), and one isolate cross-reacted with serotypes 5 and 21. The spa gene analysis determined that 64.3% (n = 9) were spaA and 35.7% (n = 5) were spaB1. In pigs, 10/14 isolates induced small plaques to diamond-shaped cutaneous lesions consistent with Erysipelothrix spp. infection. The results of this study indicate that the marine E. rhusiopathiae isolates have greater genetic and antigenic diversity than pig isolates and are capable of inducing classical skin lesions in pigs.

Development of a novel fluorescent microbead-based immunoassay and comparison with three enzyme-linked immunoassays for detection of anti-Erysipelothrix spp. IgG antibodies in pigs with known and unknown exposure.

A novel fluorescent microbead immunoassay (FMIA) using the recombinant polypeptide SpaA415 was developed for detection of anti-Erysipelothrix spp. IgG in pig sera. The diagnostic performance of the FMIA was evaluated on samples from pigs with known and unknown Erysipelothrix spp. exposure and compared to an in-house enzyme-linked immunosorbent assay (ELISA-1) based on the same capture antigen, and two commercially available ELISAs (ELISA-2 and ELISA-3). Sera from pigs experimentally infected with Erysipelothrix rhusiopathiae serotype 1a (n=60) or 19 (n=12), sera from pigs vaccinated with a commercial attenuated-live vaccine based on serotype 1a (n=12) or a commercial bacterin based on serotype 2 (n=12), and 90 field samples were utilized. The sensitivity on 22 true positive samples collected in the later stages of infection/post-vaccination was 100% for the FMIA and ELISA-1, 63.6% for ELISA-2 and 81.8% for ELISA-3. The earliest antibody response was detected 7days post inoculation with the FMIA (77.8%) and ELISA-1 (11.1%), and at 14days post-vaccination (dpv) with FMIA (50%) and ELISA-1 (50%). On field samples, a higher seroprevalence was found in pigs older than 21days with all four assays. Kappa analysis indicated that the FMIA and ELISA-1 had almost complete agreement whereas the agreement was slight with ELISA-2 and fair with ELISA-3. The sensitivity of both immunoassays based on the rSpaA415 antigen was higher compared to that of the two commercial ELISAs. The rSpaA415 FMIA has great potential as an inexpensive ELISA alternative for detection of antibodies against E. rhusiopathiae in the future.

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.

Evaluation on the pathogenicity of Erysipelothrix tonsillarum for pigs by immunosuppression with cyclophosphamide or dexamethasone.

The pathogenicity of Erysipelothrix tonsillarum was evaluated in pigs immunosuppressed with cyclophosphamide (CY) or dexamethasone (DM). Animals were treated with 15 mg/kg CY (n=8, five injections), 1 mg/kg DM (n=8, nine injections) or left untreated (n=8). On the fifth day after the beginning of drug treatments, swine were inoculated with one of two E. tonsillarum serovar 7 strains (approximately 10(6) CFU per pig). In the CY-treated group, both circulating neutrophil and lymphocyte counts decreased, whereas in the DM-treated group, lymphocyte counts decreased but neutrophil counts increased. During the observation period, none of the CY- or DM-treated pigs developed clinical signs or gross lesions, as well as non-treated pigs. Growth agglutination antibody titres in all pigs remained unchanged. Our findings indicate that E. tonsillarum strains are avirulent for swine, regardless of immune status.

[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.

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.

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.