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.

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.

Vaccination of porcine circovirus type 2 (PCV2)-infected sows against porcine Parvovirus (PPV) and Erysipelas: effect on post-weaning multisystemic wasting syndrome (PMWS) and on PCV2 genome load in the offspring.

The effect of different Parvovirus+Erysipelas vaccination schemes in PCV2-infected sows on PMWS outcome in the offspring was investigated under experimental conditions. Six PCV2-free sows were first infected oro-nasally with PCV2 two months before insemination (D0; "Day 0") and then by the intra-uterine route at insemination (D62). On D21 and D42, vaccinated sows received either the two commercial monovalent vaccines, A1(PPV) and A2(Erysipelas), or the bivalent vaccine B (PPV+Erysipelas). In addition, three SPF sows (foster-sows) were synchronized for farrowing dates to enable them to foster piglets born to infected sows and removed at birth before colostrum intake. A significantly higher proportion of mummified fetuses was obtained from PCV2-infected non-vaccinated sows than from vaccinated sows. Acute myocarditis lesions were found in their piglets, together with a high PCV2 genome load. The latter was significantly higher than in those born to PCV2-infected vaccinated sows. Sentinel PCV2-negative piglets, born to SPF foster-sows, seroconverted at almost the same time as piglets without PCV2 passive immunity and born to infected sows. Sixteen of the 84 liveborn piglets born to infected sows and foster-sows were affected by a syndrome possibly related to PMWS, as judged by clinical signs and histological lesions. Most were born to PCV2-infected non-vaccinated sows and 12/16 did not receive PCV2 passive immunity. The probability of PCV2 infection and the number of PCV2 genome copies per gram of tissue were significantly increased in piglets that did not receive PCV2 passive immunity.

Efficacy of oral vaccination against swine erysipelas in growing-finishing pigs in a clinically infected Slovakian pig herd.

In a large Slovakian growing-finishing pig production unit, the effects of oral vaccination against swine erysipelas (SE) were investigated in three groups of pigs of 10 weeks of age. In group 1, the pigs were vaccinated intramuscularly at 1 and 3 weeks after arrival in the growing-finishing barn using an Erysipelothrix rhusiopathiae bacterin. Group 2 pigs were vaccinated at the same time as group 1 using an oral avirulent live SE vaccine administered through drinking water; the pigs in the third group were placebo treated. Clinical signs of acute SE, arthritic changes, average daily weight gain (ADG), feed conversion ratio, and mortality were evaluated. None of the pigs in groups 1 and 2 but 31.7% of the control animals (group 3) showed typical clinical signs of acute SE. More (P<0.01) non-vaccinated pigs had chronic arthritic changes compared with groups 1 and 2. No significant differences in mortality were recorded between the groups. Groups 1 and 2 had higher (P<0.05) ADG and lower feed conversion ratios compared with group 3 pigs. The results demonstrated that the oral avirulent live culture was efficacious in significantly reducing the clinical symptoms caused by E. rhusiopathiae infection, so enhancing the pigs' performance.

Evaluation of Erysipelothrix rhusiopathiae vaccines in pigs by intradermal challenge and immune responses.

In a vaccine trial, pigs were challenged intradermally with eight E. rhusiopathiae strains of serovars 1a, 1b or 2 given concurrently. The strains were derived from six herds affected with vaccine breakdowns in 1997-1999, one herd without vaccine breakdown and a serovar 2 reference strain. Responses to two commercial bacterins (one implicated in the vaccine breakdowns), and two experimental bacterins (based on field isolates from affected herds) showed distinct differences in protection, particularly in clinical responses measured at 72 h. Less protection was afforded against serovar 1 challenge by the vaccine implicated in the vaccine breakdowns. Antibody and cell-mediated immune (CMI) responses were significantly different between treatments, and highlighted a similar post-vaccinal antibody response was produced against serovar 2 lysate by all vaccines, but only those providing significant protection against serovar 1 [corrected] produced significantly elevated antiserovar I lysate [corrected] antibodies. Vaccination in general significantly reduced CMI responses to the mitogens concanavalin A and phytohaemagglutinin. This experimental pig challenge system was readily able to confirm suboptimal performance of a commercial bacterin that had passed potency tests in mice but was associated with vaccine failure in commercial herds. This vaccine was also the most immunosuppressive to CMI responses associated with E. rhusiopathiae-specific and non-specific stimulation. The best vaccine response was associated with the highest mean serovar 1 antibody response and the highest CMI response (by lymphoproliferation assay) to serovar 2.

Characterisation of Erysipelothrix rhusiopathiae isolates from pigs associated with vaccine breakdowns.

Swine erysipelas vaccines are routinely used to protect pigs against peracute and acute/urticarial forms of Erysipelothrix. Between 1995 and 1998, 34 swine herds across four Australian states experienced vaccine failure. Forty-four isolates of Erysipelothrix rhusiopathiae of serovars 2, 1a, 1b and 1bx21 were recovered from 15 of these 34 vaccine breakdown herds. These isolates were characterised by restriction fragment length polymorphism (RFLP) analyses using RsaI and AluI on whole cell DNA and for the presence of plasmid DNA. Results were compared with those of 20 isolates from 16 herds unaffected by vaccine breakdown and 13 isolates representing 10 reference strains. The majority of breakdown herds possessed isolates of serovar 2 (9/15 herds), followed by serovar 1a (5 herds). No geographic predominance of a single serovar was evident. The identification of 10 RsaI profiles from whole cell DNA among the 44 isolates from 15 breakdown herds indicated that a single, new clonal lineage of E. rhusiopathiae was not responsible for vaccine failure. RsaI RFLP analyses detected a further 14 distinct profiles among 20 field strains unassociated with vaccine breakdowns, and none matched profiles of the 10 serovar reference strains for serovars 1a, 1b, 2 or 21. This technique is recommended for epidemiological studies of E. rhusiopathiae strains.

Oral vaccination against swine erysipelas--field experiences in Croatia.

In order to prove the effects of mass application of oral erysipelas vaccine via drinking water, in a farrow-to-finish production unit in Croatia, the growing-finishing animals were divided into 3 groups and treated as follows:--Group 1 (n=199) was vaccinated intramuscularly against swine erysipelas at 1 week and 3 weeks after arrival in the growing-finishing facility with a swine erysipelas bacterin.--Group 2 (n=199) were vaccinated at the same time with an avirulent culture of Erysipelothrix rhusiopathiae oral vaccine through drinking water.--Group 3 (n=200) was not vaccinated. Animals with clinical signs of swine erysipelas, chronic progressive arthritis at slaughter, mortality, average daily weight gain during the growing-finishing phase were evaluated. None of the pigs in the groups 1 and 2 showed clinical signs typical for acute swine erysipelas. Twenty-four of the pigs (12 %) in group 3 had pyrexia and skin lesions typical for swine erysipelas. Fifteen pigs in group 1, 13 pigs in group 2, and 63 pigs in group 3 had chronic progressive arthritis (group 1 and 2 vs. group 3: P < 0.01). No significant differences in mortality were recorded between the groups. Group 1 and 2 had higher (P < 0.05) average daily weight gains compared with the group 3.

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.

Serological and pathogenic characterization of Erysipelothrix rhusiopathiae isolates from tonsils of slaughter pigs in Indonesia.

Erysipelothrix rhusiopathiae was isolated from tonsils of 245 (35.7%) of 687 apparently healthy slaughter pigs in Indonesia during the period of June 1987 to February 1988. A total of 150 of the 245 E. rhusiopathiae isolated could be serotyped within the 22 recognized serotypes. Serotype 2 was most prevalent with 23.7%, followed by Serotypes 11, 12, 1a, 5 and 6 representing 7.3, 5.3, 4.9, 4.9 and 4.1% of the isolates, respectively. The nine other serotypes (Serotypes 1b, 3, 4, 8, 9, 10, 13, 19 and 22) combined to make up 11.0% of the isolates. Antibiotic-resistant strains were not found. Of 86 selected isolates belonging to various serotypes, 76 (88.4%) were highly virulent for mice (LD50 less than 10(3.0) colony-forming units). In swine, 40 (51.2%) of 78 isolates induced local or generalized urticarial lesions after intradermal inoculation, and the remaining 38 isolates induced no clinical signs. Of 76 isolates used for challenge in the cross-protection study, 29 (38.2%) killed greater than 40% of mice immunized with an erysipelas bacterin marketed in Indonesia. A tendency to be refractory to the bacterin-induced immunity was observed in some isolates of various serotypes, but this characteristic was not consistent.

Cross protection of mice and swine inoculated with culture filtrate of attenuated Erysipelothrix rhusiopathiae and challenge exposed to strains of various serovars.

Mice and swine inoculated subcutaneously with culture filtrate vaccine prepared from acriflavine-fast attenuated Erysipelothrix rhusiopathiae strain Koganei 65-0.15 (serovar 2), were challenge exposed to 20 pathogenic strains of E rhusiopathiae of 18 serovars and type N. Vaccinated mice survived after challenge exposure to serovars 1b, 2, 8 (strain Goda), and type N, but mortality occurred in vaccinated mice challenge exposed to other strains: 20% to 30% mortality in mice challenge exposed to serovars 1a, 11, 12, 15, 16, or 21; 40% to 50% mortality in mice challenge exposed to serovars 4, 5, 6, 7, or 8 (strain 911); and 60% to 80% mortality in mice challenge exposed to serovars 9, 10, 18, or 19. All vaccinated mice died after challenge exposure with strain 2553 (serovar 20). Non-vaccinated control mice died after challenge exposure to all strains. Of 2 vaccinated swine challenge exposed to strain 2553, 1 developed a local urticarial lesion at the site of intradermal exposure. Vaccinated swine challenge exposed to serovars 1a, 1b, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 18, 19, 21, or type N did not have clinical signs of acute erysipelas. Nonvaccinated control swine developed acute generalized erysipelas or localized urticarial lesions at the site of intradermal exposure.

Specificity in response of vaccinated swine and mice to challenge exposure with strains of Erysipelothrix rhusiopathiae of various serotypes.

Swine and mice were vaccinated with standard erysipelas adsorbate bacterins made from Erysipelothrix rhusiopathiae of serotype 2 and were subsequently exposed to pathogenic strains of E rhusiopathiae, serotypes 1, 2, 4, 9, 10, and 11. Response to challenge of immunity in swine was determined by presence of urticarial lesions at the sites of intradermal injection of culture; response in mice was determined by the quantal (live-dead) method. After vaccination with standard bacterins, swine and mice were significantly more susceptible (P less than of equal to 0.01) to infection with strains of serotypes 9 and 10 than with strains of serotypes 1, 2, 4, or 11. An adsorbate bacterin made from the challenge strain of serotype 10 induced specific immunity to homologous challenge exposure in swine but not in mice. Bacterins made from the other challenge strains induced little or no immunity.

Acute nonfatal erysipelas in sows in a commercial farrow-to-finish operation.

Acute nonfatal erysipelas was diagnosed in 4 sows in a 1,000-sow commercial farrow-to-finish operation in Indiana. Sows were pyrexic, lethargic, lame, and had multiple, 1.3- to 7.6-cm, erythematous rhomboid skin lesions. Outbreak was attributed to failure to properly vaccinate pigs. Further morbidity and mortality were prevented by treatment of clinically affected sows and all pigs in close proximity with procaine penicillin G daily for 3 consecutive days and vaccination of all pigs with questionable vaccination status, using bacterin of killed Erysipelothrix rhusiopathiae. Periodic review of herd management protocols is important to ensure that recommended vaccination schedules are being followed and animals are receiving quality vaccinations. Human error can contribute to many production problems and should be included on the differential diagnoses list.

Polyarthritis of pigs in western Australia: the role of Erysipelothrix rhusiopathiae.

A 3-month survey of polyarthritis in pigs was carried out at 3 metropolitan abattoirs in Western Australia. The incidence of total carcass condemnations for polyarthritis was 0.46% and partial condemnation for "arthritis" 1.66% of 15,919 pigs. It was demonstrated that the majority of joint lesions found in either total or partial condemnations were identical pathological conditions, usually differing only in the number of joints involved. Erysipelothrix rhusiopathiae was isolated from the joints of 63% of pigs condemned for polyarthritis; it appears that this organism is the most significant aetiological agent of polyarthritis in Western Australia. The role of mycoplasmas has not been determined. The groww and microscopic lesions seen in affected joints were those of a non-suppurative proliferative polysynovitis. The stifle and elbow joints showed the highest incidence of the most severe lesions. Pigs condemned for polyarthritis over a 6-month period were traced back to 125 farms. Eighty per cent of pigs that had been condemned for polyarthritis had not been vaccinated against erysipelas or were of doubtful vaccination status. Only 3% of pigs had been vaccinated as recommended. The results would suggest that erysipelas vaccine is not directly implicated in the pathogenesis of polyarthritis. The majority of pigs condemned for polyarthritis originated from poorly managed small piggeries run as sideline enterprises. There was no significant pattern to the geographical distribution of polyarthritis cases in Western Australia. Likewise, no significant realtionship was established between polyarthritis condemnations and either breed, sex or bodyweight of thepigs involved.

[The development of a technology for the production of live dried vaccines against avian pasteurellosis and swine erysipelas]. / Razrabotka tekhnologii proizvodstva zhivykh sukhikh vaktsin protiv pasterelleza ptits i rozhi svinei.

The technology of the production of dried live vaccine against Pasteurella infection of fowl from Pasteur's 2nd avirulent strain, strains AB and K, has been developed. This technology includes the process of batch cultivation of Pasteurella cells, controlled in such parameters as eH, pO2 and glucose concentration, in fermenters in optimized culture medium, based on Hottinger hydrolysate and fermentative casein-yeast hydrolysate, and preservation in improved saccharose-gelatin medium prepared in potassium sulfate buffer solution. The new technology makes it possible to increase the yield of preparations with stable biological activity 5- to 13-fold in comparison with the traditional technology. Furthermore, the technology of the production of live dried vaccine against swine erysipelas from Erysipelothrix insidiosa strain BP-2 has been developed. This technology is based on maintaining the optimum conditions of the batch cultivation of E. insidiosa in meat medium based on Hottinger hydrolysate and media obtained from hydrolysate of pancreatic fermentation products of microbial biomass; the preparation thus obtained is stabilized in peptone-saccharose-gelatin medium prepared in potassium phosphate buffer solution. This increases the yield of the vaccine 8-fold in comparison with the traditional technology, while ensuring the stability of bacteria after drying and during prolonged storage.

[Swine erysipelas: from yesterday to today]. / Vlekziekte: van toen naar nu.

The history of swine erysipelas in the Netherlands is reviewed, with special reference to the significance of vaccination and the views on epidemiology. The paper of D. A. de Jong Jzn. (1891) is discussed. The activities of the National Serum Institute in Rotterdam in this regard are discussed in detail. In conclusion, it is stated that vaccinations designed to prevent swine erysipelas are no longer required under conditions prevailing in the Netherlands.

[Comparative investigations of a combined vaccine against parvovirus and erysipelas and corresponding monovaccines in different vaccination schedules. 1: Field trial]. / Vergleichende Untersuchungen über den Einsatz eines Parvovirose-Rotlauf-Kombinationsimpfstoffes sowie entsprechender Monovakzinen bei unterschiedlichen Revakzinationszeitpunkten. Teil 1: Feldversuch

In a field trial, the development of antibodies of a combined vaccine against the porcine parvovirus (PPV) as well as against swine erysipelas was compared with corresponding mono vaccines. Furthermore, these vaccines were used in different vaccination schedules. The tests were carried out on 109 gilts in three closed farms. In all gilts, a basic immunization repeated twice was carried out at the age of six months and at intervals of three weeks. The revaccination was carried out four months after the basic immunization with half of the animals, and six months after the basic immunization with the remaining gilts. Between the combined vaccine and the mono vaccine no significant differences in the development of antibodies against PPV could be found according to different vaccination schedules. The gilts having been vaccinated with the mono vaccine and boostered six months later showed significantly higher antibody titers against Erysipelothrix rhusiopathiae. Between the remaining vaccination groups no significant difference in the development of the antibodies against swine erysipelas could be found. On only one farm, a continuous decrease of antibody titers against PPV in case of altogether 238 non-vaccinated piglets until the sixth month of life could be observed. On the two other farms, an increase of antibody titers against PPV could be found at different points of time, which indicates an infection of the piglets. Between the individual vaccination groups no significant antibody titers against PPV could be measured in milk tests. With regard to the number of piglets born alive per litter, the number of piglets born dead per litter and the number of mummies, a significant difference could neither be found between the vaccination groups 1-4.

Prophylactic application of Propionibacterium avidum KP-40 in swine with acute experimental infections. II. Bacterial infections: pleuropneumonia and swine erysipelas.

The potent immunomodifier Propionibacterium avidum KP-40 (PA) demonstrated prophylactic potency in swine infected experimentally with Haemophilus (Actinobacillus) pleuropneumoniae or Erysipelothrix rhusiopathiae. Animals received PA either together with the respective vaccine or PA only; 3 resp. 4 weeks later all animals were inoculated with virulent pathogens. Eight of 10 swine immunized with inactivated pleuropneumonia vaccine developed mild-moderate forms of infection with temporary stagnation of body weight; application of the vaccine together with PA lowered the morbidity rate to 1 of 10 (p < 0.05). Also in non-vaccinated swine infected with pleuropneumonia or erysipelas PA application resulted in milder clinical symptoms, faster recovery and a larger gain of body weight.

Characterization of Erysipelothrix species isolates from clinically affected pigs, environmental samples, and vaccine strains from six recent swine erysipelas outbreaks in the United States.

The aim of this study was to characterize Erysipelothrix sp. isolates from clinically affected pigs and their environment and compare them to the Erysipelothrix sp. vaccines used at the sites. Samples were collected during swine erysipelas outbreaks in vaccinated pigs in six Midwest United States swine operations from 2007 to 2009. Pig tissue samples were collected from 1 to 3 pigs from each site. Environmental samples (manure, feed, central-line water, oral fluids, and swabs collected from walls, feed lines, air inlets, exhaust fans, and nipple drinkers) and live vaccine samples were collected following the isolation of Erysipelothrix spp. from clinically affected pigs. All Erysipelothrix sp. isolates obtained were further characterized by serotyping. Selected isolates were further characterized by PCR assays for genotype (E. rhusiopathiae, E. tonsillarum, Erysipelothrix sp. strain 1, and Erysipelothrix sp. strain 2) and surface protective antigen (spa) type (A, B1, B2, and C). All 26 isolates obtained from affected pigs were E. rhusiopathiae, specifically, serotypes 1a, 1b, 2, and 21. From environmental samples, 56 isolates were obtained and 52/56 were E. rhusiopathiae (serotypes 1a, 1b, 2, 6, 9, 12, and 21), 3/56 were Erysipelothrix sp. strain 1 (serotypes 13 and untypeable), and one was a novel species designated Erysipelothrix sp. strain 3 (serotype untypeable). Four of six vaccines used at the sites were commercially available products and contained live E. rhusiopathiae serotype 1a. Of the remaining two vaccines, one was an autogenous live vaccine and contained live E. rhusiopathiae serotype 2 and one was a commercially produced inactivated vaccine and was described by the manufacturer to contain serotype 2 antigen. All E. rhusiopathiae isolates were positive for spaA. All Erysipelothrix sp. strain 1 isolates and the novel Erysipelothrix sp. strain 3 isolate were negative for all currently known spa types (A, B1, B2, and C). These results indicate that Erysipelothrix spp. can be isolated from the environment of clinically affected pigs; however, the identified serotypes in pigs differ from those in the environment at the selected sites. At one of the six affected sites, the vaccine strain and the isolates from clinically affected pigs were of homologous serotype; however, vaccinal and clinical isolates were of heterologous serotype at the remaining five sites, suggesting that reevaluation of vaccine efficacy using recent field strains may be warranted.