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.     

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.     

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.     

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.     

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 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.     

SEROLOGICAL CLASSIFICATION OF AUSTRALIAN STRAINS OF ERYSIPELOTHRIX RHUSIOPATHIAE ISOLATED FROM PIGS, SHEEP, TURKEYS AND MAN

154 strains of Erysipelothrix rhusiopathiae from pigs, sheep, turkeys and man were serotyped by using the double diffusion gel precipitation test. Ten of the 18 serotypes were detected in 151 of the strains. Three strains failed to react with any of the type specific antisera. It was found that serotype 1a shared an antigen(s) with serotype 1b, and that serotype 6 shared an antigen(s) with serotype 14. Serotype 2a and 2b were difficult to distinguish. Since serotypes 1 and 2 were isolated from cases of septicaemia in pigs, and since serotypes 1, 2, 4 and 7 were isolated from cases of arthritis, it was suggested that factors other than serotype were important in causing the various forms of swine erysipelas. The fact that the distribution of serotypes 1a, 1b and 2b between septicaemic and arthritic pigs was similar supported the conclusion that arthritis was consequent to bacteraemia. Serotypes 1a, 1b, 2b, 5, 12 and 15 were isolated from cases of arthritis in sheep, and serotypes 1a and 5 from cases of erysipelas in turkeys. Serotype 2b was isolated from a human specimen.     

Haemophilus pleuropneumoniae serotypes--cross protection experiments

Pigs vaccinated with a killed 6-hour culture of Haemophilus pleuropneumoniae serotype 2 with Freund's incomplete adjuvant were not protected against challenge with serotypes 1, 5, 6 or 8. Equivalent results were obtained when pigs were vaccinated with serotypes 4 or 5 and challenged with serotype 2. In earlier studies of immunity induced by intranasal immunization with live H. pleuropneumoniae organisms, it was clearly shown that intranasal inoculation with one serotype of H. pleuropneumoniae would induce a strong immunity to both homologous and heterologous serotypes (Nielsen 1979). The present study has shown that cross immunity is not obtained with parenteral immunization. The results strongly suggest that the immune response of the pig to parenteral vaccination is different from the response seen after natural infection, and indicate that an important part of the defence mechanism against H. pleuropneumoniae infection is a local immune-barrier which is effective in preventing the bacterium from penetrating the mucosa. In earlier vaccination experiments 90 per cent of vaccinates were protected against homologous challenge (Nielsen 1976). In the present work a vaccine containing serotypes 1 through 6 was fully protective against serotypes 2 and 3 and also against serotype 8, which shares antigenic determinants with serotypes 3 and 6. These results indicate that the protection obtained by parenteral immunization is serotype-specific. Vaccines must therefore contain the serotypes existing in the swine population.     

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.     

Lipopolysaccharides of the Heddleston serotypes of Pasteurella multocida

Lipopolysaccharides (LPS) were extracted from 13 of the 16 Heddleston serotypes of Pasteurella multocida by phenol-chloroform-petroleum ether (PCP). Serotypes 3, 9, and 13 were extracted only by phenol-water (PW). After extraction of LPS of serotype 9 by PW, an additional LPS was isolated by PCP. All LPS contained glucose, 2-keto-3-deoxyoctonate, and heptose. Two isomers of heptose, D-glycero-D-mannoheptose and L-glycero-D-mannoheptose, were found in serotypes 2 and 5. Antisera made against purified LPS of serotypes 2 and 5 reacted with both heat-stable antigens and LPS from serotypes 2 and 5 in the gel-diffusion precipitin test. Antisera against serotype 2 LPS protected turkeys against challenge with capsulated serotype 5, indicating that a structural relationship exists between LPS of strains that cause hemorrhagic septicemia and fowl cholera. Rhamnose was a component of serotype 9 LPS, and galactose was found in all LPS, except for serotype 11. The LPS of serotype 13 contained an isomer of heptose that has not been identified. The LPS had buoyant densities in CsCl of 1.40 +/- 0.0148 g/ml, and all hemagglutinated chicken and turkey, but not sheep or horse, RBC.     

Serotypes of previously unclassified isolates of Erysipelothrix rhusiopathiae from swine in the United States and Puerto Rico

Serotypes of 46 previously unclassified isolates of Erysipelothrix rhusiopathiae from porcine tissues in the United States and serotypes of 31 isolates of the organism from porcine tissues received from Puerto Rico were determined. The 46 isolates from the United States were classified in serotype 21. Four isolates (from Georgia, Minnesota, Ohio, and Oklahoma) were tested and found to be pathogenic for swine. Serotypes 1 (subtypes 1a and 1b), 2, 5, 6, and 21 were found in porcine tissues from Puerto Rico. The relative frequency of the various serotypes was similar to that previously reported in the United States.     

Biochemical and serological properties of Actinobacillus pleuropneumoniae biotype 2 strains isolated from swine

The biochemical and serological properties of 21 strains of Actinobacillus pleuropneumoniae biotype 2 isolated from haemorrhagic necrotic pleuropneumonia of swine were examined. For serologic typing, the indirect haemagglutination (IHA) and the double gel-diffusion tests were used. On the basis of their soluble surface antigens, our A. pleuropneumoniae biotype 2 isolates could be assigned to two proposed serotypes. Serotype 1 comprised 11 strains and serotype 2 comprised 10 strains. All strains contained two surface antigen components. In the strains belonging to serotype 1, one of the antigens was identical with the serotype-specific antigen of Pasteurella haemolytica T4. Both antigens of serotype 2 strains proved to be type-specific. Four strains received from Switzerland, including the holotype strain of A. pleuropneumoniae biotype 2, and three strains isolated from swine in the G.D.R. belonged to serotype 2. Both the double gel diffusion and the IHA tests detected a 2-way cross-reaction between biotype 1, serotype 2 and biotype 2, serotype 2 strains of A. pleuropneumoniae, which could be eliminated using cross-absorbed sera.     

Prevalence of various phenotypes of Streptococcus suis isolated from swine in the U.S.A. based on the presence of muraminidase-released protein and extracellular factor.

The present study describes the prevalence of muraminidase-released protein (MRP) and extracellular factor (EF) proteins associated with virulence of Streptococcus suis serotype 2 from a collection of USA strains. Sixty-six strains belonging to serotypes 1, 2, 3, 4, 7, and 10, were analyzed with a set of double antibody sandwich ELISAs and Western blots. Nineteen of 34 serotype 2 strains from cases of swine meningitis had the MRP+EF+ phenotype. Five of 7 serotype 2 strains isolated from lungs had an MRP*EF- phenotype. An MRP-EF+ phenotype was found in 4/34 strains isolated from swine meningitis. The MRP*EF- and MRP-EF+ phenotypes have not been reported previously. All strains of serotypes other than 2, including isolates from cases of meningitis, had the MRP-EF- phenotype, suggesting that these strains must have other, as yet undetected, virulence factors.     

Antimicrobial susceptibilities of Erysipelothrix rhusiopathiae isolated from pigs with swine erysipelas in Japan, 1988-1998

The susceptibility to 21 antimicrobial agents of 214 strains of Erysipelothrix rhusiopathiae isolated from pigs affected with swine erysipelas in Japan between 1988 and 1998 was determined. Ampicillin, cloxacillin, benzylpenicillin, ceftiofur, tylosin, enrofloxacin and danofloxacin were the most active agents [minimum inhibitory concentrations (MICs); < or = 0.025-0.78 microgram/ml], followed by cefazolin, virginiamycin, tiamulin, chloramphenicol, florphenicol and oxolinic acid (MICs; 0.1-25 micrograms/ml). Activity was poor or absent with kanamycin and sulfadimethoxine. Strains resistant to dihydrostreptomycin, erythromycin, clindamycin, lincomycin, oxytetracycline and doxycycline were detected. The susceptibilities to dihydrostreptomycin and oxytetracycline tended to decrease. Investigation of the differences in antimicrobial susceptibility of the 214 strains according to their serotypes, sources, isolation years and regions, showed that the strains resistant to dihydrostreptomycin were most frequently found in the strains of serotype 1a and in strains from septicaemic cases. Strains resistant to oxytetracycline were detected in all serotypes and all sources, and most of the strains resistant to erythromycin were detected in the strains of serotype 2. The frequency of strains resistant to dihydrostreptomycin gradually increased from 1988 to 1996, but then decreased between 1997 and 1998. The frequency of strains resistant to oxytetracycline was remained more than 38% from 1988 to 1998. It was suggested that the strains resistant to dihydrostreptomycin and oxytetracycline were distributed over almost all districts of Japan.     

Occurrence of different serotypes of Erysipelothrix rhusiopathiae in retail pork and fish.

Retail pork (38 samples), cod (10 samples) and herring (10 samples) were obtained from 12 stores in the area of Lund in southern Sweden during September and October 1990. Erysipelothrix rhusiopathiae was isolated from 50% of the pork samples, 60% of the cod samples and from 30% of the samples from herring. Serotype 2 dominated on retail pork as well as on fish samples constituting 53% of the pork isolates (10 strains) and 33% of the cod isolates (2 strains). All E. rhusiopathiae isolates originating from herring were serotype 2 (3 strains). Serotypes 1b, 6, and 8 were isolated from retail pork only (6, 2 and 1 strains, respectively). Serotype 5 was isolated from cod only (3 strains) and so was serotype 9 (1 strain). The public health hazards with the occurrence of virulent strains of Erysipelothrix rhusiopathiae in retail pork and fish are discussed.     

Serovar, pathogenicity and antimicrobial susceptibility of erysipelothrix rhusiopathiae isolates from farmed wild boars (Sus scrofa) affected with septicemic erysipelas in Japan

Six strains of Erysipelothrix rhusiopathiae were isolated from farmed wild boars with acute septicemic erysipelas during the period from 1983 to 1998 in Japan. All isolates belonged to serovar 1a or 2 (predominant serovars in swine). The 50 per cent lethal dose values of those isolates ranged from 10(1.3)to 10(6.2)colony forming units in mice. In swine, all isolates were virulent, capable of inducing localized or generalized urticarial lesions after intradermal inoculation. All of the isolates were resistant to oxytetracycline and/or dihydrostreptomycin. These observations suggest that E. rhusiopathiae strains isolated from wild boars may have aetiological significance in swine erysipelas.     

Grouping of Actinobacillus pleuropneumoniae strains of serotypes 1 through 12 on the basis of their virulence in mice

Variations in virulence among strains of different serotypes of Actinobacillus pleuropneumoniae were detected on intraperitoneal or intranasal inoculation in mice. In general, strains of serotypes 1, 5, 9, 10 and 11 were found to be highly virulent and those of serotypes 2, 3, 4, 6, 7, 8 and 12 to be less virulent. However, a few strains of serotype 5 caused low mortality in mice while some strains of serotype 3 and 7 were found to be highly virulent. Highly virulent strains of A. pleuropneumoniae were invasive and appeared in the blood within 3 to 6 h of intranasal inoculation. The type specific antigen as detected by the coagglutination test was distributed in lungs, liver, heart and spleen after intraperitoneal inoculation whereas it was mostly concentrated in the lungs after intranasal inoculation. Lowest concentration of boiled whole-cell suspension of A. pleuropneumoniae showing limulus amebocyte lysate activity was variable and independent of the serotype. Mortality caused by boiled whole cell suspension was also variable and serotype independent.     

Serological assay for swine erysipelas using nitrocellulose particles impregnated with an immunodominant 65 kDa antigen from Erysipelothrix rhusiopathiae.

Pigs (n = 10) that were experimentally challenged with an arthritogenic isolate of Erysipelothrix rhusiopathiae (strain VRS 229; serotype 1a) developed arthritis in at least one of twelve major limb joints. Immunoblots using sera obtained from these pigs at necropsy revealed a major band of immunoreactivity against a subunit polypeptide of apparent molecular mass 65 kDa. The usefulness of the 65 kDa immunodominant subunit as an assay reagent in an ELISA test was examined by presentation of antigen impregnated onto nitrocellulose particles (AINP). This was prepared by electro-transfer of bacterial polypeptides from SDS-PAGE gels to nitrocellulose. Protein bands were visualized by staining with amido black and a strip of nitrocellulose bearing the 65 kDa band was excised and extracted with formic acid. Nitrocellulose particles impregnated with the 65 kDa antigen (65-AINP) were precipitated from solution by neutralization with ammonium hydroxide. 65-AINP was suspended in water and the optimum dilution for ELISA assay was determined by titration to be 0.1 A650 units. Sera from all pigs challenged with VRS 229 reacted against the 65-AINP antigen in the ELISA assay while sera from control, and experimental pigs prior to challenge, failed to do so. The 65-AINP antigen could also be used efficaciously to quantify serological reactivity of pigs experimentally infected with other strains of E. rhusiopathiae representing the three major serotypes (1a, 1b and 2) that are most commonly associated with swine erysipelas infections. Mouse immunizations with 65-AINP also confirmed that nitrocellulose particles bearing the immunodominant subunit antigen will elicit murine antibodies that are monospecific against this determinant.     

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.     

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.