Cross protection of mice and swine given live-organism vaccine against challenge exposure with strains of Erysipelothrix rhusiopathiae representing ten serovars

Mice and swine vaccinated (subcutaneous inoculation) with live acriflavine-fast attenuated Erysipelothrix rhusiopathiae, strain Koganei 65-0.15 (serovar 2), were challenge exposed with 10 strains of E rhusiopathiae pathogenic for swine; the latter strains comprised serovars 9 and 10 and other previously undetermined. Vaccinated mice did not die after they were challenge exposed (subcutaneous inoculation) with serovars 4, 6, 7, 8, 9, 10, 15, 16, or N, but vaccinated mice challenge exposed with strain 2553 (serovar 20) had 30% mortality. Nonvaccinated control mice died after they were challenge exposed with all serovars tested. One of 2 vaccinated swine challenge exposed (intradermal inoculation) with each of strains 911 (serovar 8), 2179 (serovar 10), or 2553 developed localized urticarial lesion at the site of intradermal inoculation. Vaccinated swine challenge exposed with serovars 4, 6, 7, 9, 15, 16, or N did not have clinical signs of acute swine erysipelas. Nonvaccinated control swine developed localized lesions at the site of intradermal challenge inoculation.     

Cross protection in mice and swine immunized with live erysipelas vaccine to challenge exposure with strains of Erysipelothrix rhusiopathiae of various serotypes

Mice and swine immunized subcutaneously with live vaccine prepared from acriflavine-fast attenuated Erysipelothrix rhusiopathiae, strain Koganei (serotype 2), were challenge exposed to virulent strains of E rhusiopathiae of various serotypes. Vaccinated mice did not die after challenge exposure to serotypes 1a, 1b, 2, 3, 5, 6, 7, 8, 9, 11, 12, 15, 16, 18, 19, 21, or N, but 20% to 30% mortality occurred in vaccinated mice challenge exposed to serotypes 10, 14, 20, or 22. Nonvaccinated control mice died after challenge exposure to all serotypes tested. Vaccinated swine challenge exposed to strain 14B (serotype 9) or strain 2179 (serotype 10) developed localized urticarial lesions at the site of intradermal exposure. Vaccinated swine challenge exposed to serotypes 1a, 1b, 2, 5, 8, 11, 12, 18, 19, or 21 did not have clinical signs of acute erysipelas. Nonvaccinated control swine developed acute generalized erysipelas or localized lesions at the site of intradermal exposure.     

Antiserum against culture filtrate is cross-protective for various serovars of Erysipelothrix rhusiopathiae

The protective effect of porcine antiserum prepared against culture filtrate (CF) of an attenuated strain of Erysipelothrix rhusiopathiae (serovar 2) in mice to challenge with 20 virulent strains of 18 serovars and one type N was investigated. Passively immunized mice survived after challenge with serovars 1a, 1b, 2, 5, 6, 8 (strain Goda), 11, 12, 15, 16, 21 or type N, but 10-30% mortality occurred in immunized mice challenged with each strain of serovars 4, 7, 8 (strain 911), 9, 18 or 19 and 70% mortality to serovar 10 (strain 2179). All immunized mice died after challenge with serovar 20 (strain 2553). Non-treated control mice died after challenge with all serovars and the type tested.     

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.     

Mechanism of protection induced in mice against Erysipelothrix rhusiopathiae infection by treatment with porcine antiserum to the culture filtrate of an attenuated strain

The mechanism of protection induced in mice against challenge with a virulent strain of Erysipelothrix rhusiopathiae by porcine antiserum to the culture filtrate (CF) of an attenuated strain was investigated. Death and bacterial growth in the spleens of mice challenged with the virulent strain were completely prevented by treatment with the antiserum. The protective effect of the serum was markedly decreased in mice in which polymorphonuclear leucocytes (PMN) were depleted by cyclophosphamide (CY) treatment but not in mice in which macrophages were blocked selectively by carrageenan (CG). The phagocytic rate of PMN and the number of bacteria ingested by PMN were significantly higher in mice treated with the antiserum than in mice treated with normal serum. These results indicate that anti-CF serum exerts its protective effect by opsonic activity and that opsonized E. rhusiopathiae are eliminated mainly by PMN.     

Safety of a live attenuated Erysipelothrix rhusiopathiae vaccine for swine

The porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus. Virions of PRRSV contain six membrane proteins: the major proteins GP5 and M and the minor proteins GP2, GP3, GP4, and E. The GP5 is the major envelope proteins, which was involved in the formation and infectivity of PRRSV by coaction with other membrane proteins. Here, to determine the function of alone GP5 envelope protein in viral entry, we investigated the formation and infectivity of GP5-pseudotyped virus particles. By co-transfection of GP5 expression plasmids with murine leukemia virus (MuLV) based retroviral vectors (pHIT60, encoding MuLV Gag-Pol; pHIT111, encoding an MuLV genome with a β-galactosidase reporter gene) into 293 T cells and analysis of the culture medium using ultracentrifugation, Western blot, and infection assay. We observed that the GP5 envelope protein was incorporated into the MuLV retroviral vectors to generate an pseudotyped murine leukemia virus, which was infectious to PAM and Mack-145 target cells and displayed the same host range with wild-type PRRSV. The infection of the pseudotyped virus on PAM target cells is effectively neutralized by polyclonal antibodies specific for PRRSV or GP5. The results suggested that the GP5 protein may play a key role in the viral entry by interacting with the host cell receptor. The GP5-pseudotyped virus will be useful in the identification of the cellular receptor binding with GP5 protein.     

Susceptibility of vaccinated swine and mice to generalized infection with specific serotypes of Erysipelothrix rhusiopathiae

Swine were vaccinated with adsorbate bacterin made from Erysipelothrix rhusiopathiae of serotype 2 and were subsequently allotted to 4 exposure groups, each of which was exposed to 1 of the strains of E rhusiopathiae of serotypes 1, 2, 9, or 10. Mice were vaccinated with the same bacterin and were subsequently allotted to 60 exposure groups which were exposed to 60 strains of E rhusiopathiae, comprising 10 strains each of serotypes 1, 2, 4, 9, 10, and 11. Response to challenge of immunity in swine was determined by the presence of clinical signs of acute generalized erysipelas; response in mice was determined by the quantal (live-dead) method. Vaccinated swine were as susceptible to the strain of serotype 10 as were nonvaccinated control swine, whereas vaccinated swine were immune and control swine were susceptible to the strains of serotypes 1 and 2. The strain of serotype 9 was not sufficiently virulent to induce acute generalized erysipelas, even in control swine. Arthritis was not prevented by vaccination, but its frequency and severity were less in vaccinated swine exposed to strains of serotype 1 or 2 than in those exposed to strains of serotype 9 or 10. Vaccinated mice were significantly (P less than 0.05) more susceptible to the strains of serotype 10 than to those of any other serotype tested.     

Correlation between adherence of Erysipelothrix rhusiopathiae strains of serovar 1a to tissue culture cells originated from porcine kidney and their pathogenicity in mice and swine

Adherence of four virulent and four avirulent strains of Erysipelothrix rhusiopathiae, serovar 1a, to porcine kidney cell lines, PK-15 and ESK cells, was examined in an in vitro system. The virulent strains adhered well to the cells (range of means, 9.95 +/- 0.87-36.01 +/- 1.10 per cell). In contrast, the avirulent strains showed negligible adherence to the cells (range of means, 0.11 +/- 0.04-1.41 +/- 0.13 per cell). Pretreatment of bacteria with heat, trypsin, or antiserum resulted in a marked decrease in adherence. Scanning electron microscopic examination revealed that the bacteria attached directly to the microvilli of cells.     

Characterization of Erysipelothrix rhusiopathiae strains isolated from recent swine erysipelas outbreaks in Japan

The objective of the present study was to characterize Erysipelothrix sp. strains from recent erysipelas outbreaks in Japan. Eighty-three (100%) strains were identified as E. rhusiopathiae, based on serotyping and spaA PCR. Fifty (60.3%), 5 (6.0%), and 28 (33.7%) strains were isolated from animals with acute, subacute and chronic outbreaks, respectively, of which 79 (95.2%), 1 (1.2%), and 3 (3.6%) belonged to serotypes 1a, 2a, and untypeable, respectively. Fifteen strains (including 3, 2, and 10 from acute, subacute, and chronic cases, respectively) were sensitive to acriflavine, and showed high levels of virulence in mice; of which strains from acute cases, and from subacute and chronic cases killed 100%, and 80 to 100% mice, respectively at challenge doses of 10(2) CFU per mouse. Based on sequence analysis of a 432-bp hypervariable region in spaA gene, 83 strains could be divided into 3 groups: (i) group 1 (3 strains of serotype 1a) had Ala-195 and Ile-203; (ii) group 2 (76 strains of serotype 1a and 3 of untypeable) had Asp-195 and Met-203; and (iii) group 3 (one strain of serotype 2a) had Asn-195 and Ile-203. The results of the present study suggest that the serotype 1a strains belonging to the group 2 might be widespread in pig populations in Japan.     

Induction of cross-protection in mice against dolphin Erysipelothrix rhusiopathiae isolates with a swine commercial vaccine

Erysipelothrix rhusiopathiae is well known to cause disease in dolphins. This disease occurs either in an peracute way, leading to mortality even before clinical signs are observed or in a sub-acute way, characterized by rhomboidal skin lesions, that can be treated with penicillin or its derivatives. Commercial swine vaccines, containing inactivated serotype 2 strains, are currently used for vaccination but it is not known whether these vaccines induce protection against E. rhusiopathiae isolates from dolphins. In the present study, it was demonstrated in a mouse model that vaccination with a commercial swine vaccine (Eurovac Ery, Eurovet, Belgium) containing inactivated serotype 2 E. rhusiopathiae strains induced protection against challenge with three E. rhusiopathiae isolates from dolphins. The duration of the protection varied, depending on the challenging isolate, between 8 and >23 weeks. There was however no positive correlation between the amount of antibodies at the moment of challenge and the observed protection.In conclusion, vaccination trials in mice indicate that commercial serotype 2 swine Erysipelothrix vaccines induce protection against erysipelas caused by dolphin pathogenic isolates.     

Computerized comparison of the protein compositions of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum strains

Protein profiles of six Erysipelothrix rhusiopathiae strains, five Erysipelothrix tonsillarum strains and three Erysipelothrix strains of uncertain taxonomic position were studied by sodium dodecyl sulphate-polyactylamide gel electrophoresis (SDS-PAGE). In a computerized comparison of the protein patterns of the strains, the level of similarity between the strains was determined. The SDS-PAGE protein bands were divided into 14 groups based on molecular weight. The relative distribution of proteins within these groups was used to characterize the strains. These distribution patterns were analysed by computing Pearson's correlation coefficient between strains, and by cluster analysis based on Euclidean distances and the unweighted pair-group method of arithmetic averages (UPGMA). The geometric mean of the similarities calculated by Pearson's correlation coefficient was 0.980 +/- 0.018 between the E. rhusiopathiae strains and 0.979 +/- 0.013 for E. tonsillarum strains. The value was 0.932 +/- 0.036 between the strains belonging to different species. However, a threshold value applicable for identification of a given strain to a species could not be established. Of the three strains of uncertain taxonomic position, the strains designated Rotzunge and Iszap 4 had a protein composition more similar to that of E. tonsillarum than to that of the E. rhusiopathiae type strain. The strain designated Pécs 56, which may be a member of a new species according to literature data, gave inconsistent results by the two methods used. The computerized evaluation method developed here is suitable for the comparison of the protein composition of the strains and for the construction of the protein similarity tree by cluster analysis.     

Effect of cyclophosphamide and carrageenan on resistance of mice to Erysipelothrix rhusiopathiae

The immunosuppressive effect of cyclophosphamide (CY) or carrageenan (CG) treatment was investigated to clarify the mechanism of resistance of mice to Erysipelothrix rhusiopathiae infection. In mice inoculated with attenuated E. rhusiopathiae, death occurred and bacterial growth in the spleen was enhanced only by CY treatment; in CG-treated mice, no death occurred and bacterial growth in the spleen was kept at a low level for at least 23 days, similar to that of nontreated control mice. These results indicated that polymorphonuclear leucocytes rather than macrophages may play an important role in the resistance of mice to E. rhusiopathiae infection.     

Immunological characterization of protective antigens prepared by alkaline treatment of whole cells and from the culture filtrate of Erysipelothrix rhusiopathiae.

Culture filtrate and alkaline-extracted antigens from whole cells of an attenuated strain of Erysipelothrix rhusiopathiae (strain Koganei: serovar 1a) were fractionated with ammonium sulfate; both induced protective immunity in mice. Sephadex G-200 gel filtration revealed three protein fractions in the alkaline-extracted antigen and four protein fractions in the culture filtrate antigen. A fraction in the alkaline extract (NaOH P-2) and in the culture filtrate (CF P-2) induced protection in mice against challenge with a different serovar strain (strain Agata: serovar 5). Anti-NaOH P-2 and anti-CF P-2 mouse sera were protective against different serovars. Glycoprotein fraction derived from CF P-2 antigen by affinity chromatography with Con A-Sepharose 4B did not show protective activity. Western blotting between the antisera (anti-NaOH P-2, Anti-CF P-2 and anti-Koganei strain) and the antigens (NaOH P-2, and sonicated antigens of Agata, Fujisawa and Koganei strains) showed strong recognition of the same bands at 62, 42 and 41 kDa.     

红斑丹毒丝菌的分离鉴定及疫苗的免疫保护作用

自重庆地区屠宰场猪扁桃体样品分离红斑丹毒丝菌,在测定血清型、Spa基因型和致病性的基础上,研究了弱毒疫苗G_4T_(10)的免疫保护作用。结果显示,从146份样品中分离33株红斑丹毒丝菌,其中27株对小白鼠具有致病性;19株和2株分别在试管凝集试验和琼扩试验中与兔抗G_4T_(10)血清呈阳性反应;12株为SpaA型,21株为SpaB型;在弱毒疫苗G_4T_(10)的免疫保护试验中,被动或主动免疫小白鼠对11株分离细菌的攻击具有保护作用。结果表明,健康猪群红斑丹毒丝菌的带菌率高,弱毒菌苗G_4T_(10)对多数菌株缺乏免疫保护。     

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.     

Acute airsacculitis in turkeys inoculated with cell-free culture filtrate of Pasteurella multocida

Twenty-six female and 26 male turkeys, inoculated into the caudal thoracic air sacs with cell-free culture filtrate of Pasteurella multocida strain R44/6, were examined from 0 to 6 hours post-inoculation and compared with 26 female and 26 male sham-inoculated control turkeys given brain-heart-infusion broth. The air sac reacted rapidly with exudation of heterophils. Microscopically, low numbers of heterophils were present within air sac blood vessels and also perivascularly by 0.5 hour after inoculation. These became more numerous by 1.5 and 3 hours post-inoculation. By 6 hours post-inoculation, there was severe swelling of air sac epithelial and mesothelial cells and thickening of the air sac by proteinaceous fluid and heterophils. Ultrastructurally, mesothelial and air sac epithelial cells were vacuolated, and interdigitating processes of epithelial cells were separated. Microscopically, in control turkeys, rare heterophils were present perivascularly at 1.5, 3, and 6 hours after inoculation. Ultrastructurally, all features were normal. In turkeys given cell-free culture filtrate, total cell counts in air sac lavage fluids increased markedly by 3 hours post-inoculation in which heterophils predominated (greater than 97%). There were only slight increases in cell counts of air sac lavages from control turkeys. The circulating blood heterophil cell count dropped transiently at 1.5 hours post-inoculation, followed by a return to normal 3 hours after inoculation, and by heterophilia by 6 hours post-inoculation in turkeys given either cell-free culture filtrate or brain-heart-infusion broth. These results indicate cell-free culture filtrate of P. multocida induces hematologic, cytologic, and morphologic changes indistinguishable from those induced by cultures of P. multocida.     

Pathogenicity of field isolates of Erysipelothrix rhusiopathiae in mice, rats and pigs

Eight field isolates of Erysipelothrix rhusiopathiae serotypes 1 and 2, from different sources, were examined for their pathogenicities for mice and pigs. Arthritogenicity for pigs correlated with virulence for mice at the highest and lowest levels, but not with strains of intermediate virulence. The most virulent strain was also arthritogenic in rats. In pigs, after repeated intravenous challenge the number of affected joints ranged from 0 to 11 of 12 examined. For the 8 strains, the mean number of affected joints ranged from 1 to 7.7 per pig. Clinical course and pathological findings were correlated, but the onset, severity and duration of lameness was variable both within and between groups. Clinical lameness, joint swelling and urticariae were of limited use as indicators of joint changes. The more virulent strains caused lameness as early as 2 days, whereas strains of low virulence took up to 8 weeks.