The porcine acute phase protein response to acute clinical and subclinical experimental infection with Streptococcus suis.

The pig acute phase protein (APP) response to experimental Streptococcus suis (S. suis) infection was mapped by the measurement of the positive APPs C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp) and major acute phase protein (pig-MAP) and the negative APPs albumin and apolipoprotein (Apo) A-I. The aim was to elucidate the differences in the acute phase behaviour of the individual APPs during a typical bacterial septicaemic infection. Pigs were inoculated subcutaneously with live S. suis serotype 2 and blood was sampled before and on various days post inoculation (p.i.), until the pigs were killed and autopsied on day 14 p.i. Clinical signs (fever and lameness) were observed in four of the five inoculated pigs from day 2 p.i., and these pigs also had arthritic lesions at autopsy. CRP and SAA showed fast increases in serum concentrations, CRP being elevated from days 1 to 12 p.i. and peaking at 10 times the day 0-levels on day 1 p.i. SAA rose quickly to peak levels of 30-40 times the day 0-level on days 1-2 and returned to pre-inoculation level on day 5 p.i. Hp and pig-MAP showed slightly slower responses, both peaking around 5 days p.i. Hp was increased throughout the experiment with maximum levels around 10 times the day 0-levels, and pig-MAP was elevated on days 1-12 p.i. with peak levels of around seven times the day 0-levels. Apo A-I was decreased from days 1 to 8 and showed minimum levels of about 40% of day 0-levels around 1-2 days p.i. No clear pattern of changes in albumin levels could be identified. One pig, showing clinical signs on day 2 only, also showed an APP response, although of a relatively short duration, whereas three pigs presenting clinical signs for several days had a more protracted acute phase response. Remarkably, the one pig showing no clinical signs and no arthritic lesions showed an APP response comparable to that of the other, clinically affected pigs. Thus, both acute clinical and subclinical S. suis infection could be revealed by the measurement of one or more of the APPs CRP, SAA, Hp, pig-MAP and Apo A-I. The combined measurement of two or three APPs, including proteins with slow and fast kinetics, should be used to achieve the highest sensitivity for the detection of ongoing S. suis infection during a prolonged time period. A diagnostic tool based on such APP-measurements could considerably improve strategic control procedures for this important infection.

Production of exfoliative toxin by isolates of Staphylococcus hyicus from different countries.

A total of 218 isolates of Staphylococcus hyicus from pigs in eight countries (Belgium, Croatia, Germany, Japan, Korea, Slovenia, the uk and the usa) and 44 isolates from other animals in Belgium, India, Japan and the usa were examined for the genes encoding the exfoliative toxins ExhA, ExhB, ExhC and ExhD by multiplex pcr. The expression of the toxins was confirmed by immunoblot analysis, using monoclonal or polyclonal antibodies specific for each of the toxins. The porcine isolates were from pigs with exudative epidermitis, pigs with other lesions and from healthy pigs, and one or more of the toxins could be found among the isolates from the pigs in all the countries. Toxigenic strains of S hyicus were isolated from both healthy and diseased pigs, but the chance of isolating toxigenic strains from pigs with exudative epidermitis was greater than from pigs with other lesions or healthy pigs. Of the 44 isolates from other animal species, only one isolate, from a hare from Belgium, produced ExhB, and one isolate, from a cow with mastitis from Japan, produced ExhA.

A multiplex PCR for detection of genes encoding exfoliative toxins from Staphylococcus hyicus.

AIMS: To develop a multiplex PCR for detection of genes encoding the exfoliative toxins ExhA, ExhB, ExhC and ExhD from Staphylococcus hyicus and to estimate the prevalence of exfoliative toxins among Staph. hyicus isolates from Danish pig herds with exudative epidermitis (EE). METHODS AND RESULTS: A multiplex PCR employing specific primers for each of the genes encoding four different exfoliative toxins was developed and evaluated using a collection of Staph. hyicus with known toxin type and a number of other staphylococcal species. A total of 314 Staph. hyicus isolates from pigs with EE were screened by multiplex PCR and the combined results of the present and previous investigations showed that ExhA, ExhB, ExhC and ExhD was found in 20, 33, 18 and 22%, respectively, of 60 cases of EE investigated. CONCLUSIONS: This study has provided a new tool for detection of toxigenic Staph. hyicus and a more comprehensive picture of the prevalence of the Staph. hyicus exfoliative toxins in Danish pig herds. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex PCR can be used in studies on the prevalence of toxigenic Staph. hyicus elucidating the epidemiology of EE in pigs. The multiplex PCR is currently being used for selection of Staph. hyicus isolates for production of autogenous vaccine.

Passive immunization of pigs against experimental infection with Streptococcus suis serotype 2.

The safety and protective efficacy of a horse antiserum raised against inactivated whole cell preparations of Streptococcus suis serotype 2 was investigated in pigs by experimental challenge. The antiserum was evaluated in two similar experiments each comprising 12 4-week-old pigs treated with 6 ml of antiserum the day before challenge and four pigs used as challenge controls. Pigs were infected by subcutaneous injection with approximately 10(11) colony forming units of S. suis serotype 2. Clinical disease in the pigs that could be attributed to infection with S. suis was reduced from 88 to 35% (P = 0.015). The percentage of pigs with lesions that could be associated with S. suis was reduced from 88 to 22% (P = 0.002) and isolation of S. suis serotype 2 was reduced from five (63%) out of eight pigs in the combined challenge control groups to 3 (13%) out of 23 pigs in the combined treatment groups. These results indicate that passive immunization of pigs may be a way to reduce or control S. suis serotype 2 infections in pigs.

The transferrin receptor of Actinobacillus pleuropneumoniae: quantitation of expression and structural characterization using a peptide-specific monoclonal antibody.

When Actinobacillus pleuropneumoniae (A. pp) is grown under iron-restricted conditions in vitro, transferrin binding proteins (Tbps) are induced. The functional transferrin receptor of A. pp is composed of two outer membrane proteins (Tbp1 and Tbp2) and shows an exquisite specificity for porcine transferrin. This complex was studied using a monoclonal antibody (Mab 1.48) raised against a synthetic peptide corresponding to a hydrophilic domain of Tbp2 common to several A. pp serotypes. The antibody reacted specifically with a 60-70kDa Tbp2-antigen found in all serotypes of A. pp obtained from iron-restricted culture. It was found that Tbp2 was not expressed in iron replete medium by any serotype except serotypes 5a, 5b and 6 where a weak expression was seen. There was a weak expression of related antigens in Actinobacillus indolicus and Actinobacillus suis under iron-depleted conditions while no similar antigens were detected with the Mab in iron-starved Actinobacillus lignieresii, Actinobacillus porcinus, Actinobacillus minor, Haemophilus influenzae, and Haemophilus parasuis. Using an enzyme-linked immunosorbent assay (ELISA) based on the Mab 1.48, Tbp2 could be detected in both recombinant E. coli expressing Tbp2 and in wild type A. pp grown under iron restricted conditions. The subcellular location of Tbp2 in A. pp was studied by immunoelectron microscopy using the Mab 1.48. Interestingly, all antibody binding was found inside the A. pp cells, while Tbp2 expressed in recombinant E. coli was found both in the cytosol and on the outer membrane. These results indicate that the Mab 1.48-reactive epitope of Tbp2 is surface exposed when it is expressed without Tbp1 in E. coli while the inaccessibility of this epitope of Tbp2 in A. pp could be due to shading by the association between Tbp2 and Tbp1.

Blocking enzyme-linked immunosorbent assay for detection of antibodies against Actinobacillus pleuropneumoniae serotype 6 in pig serum.

A blocking enzyme-linked immunosorbent assay (ELISA) detecting antibodies against Actinobacillus pleuropneumoniae (Ap) serotype 6 was developed. The blocking ELISA was based on the inhibition of a polyclonal antibody raised against Ap serotype 6. Purified lipopolysaccharide from Ap serotype 6 was used as antigen. The blocking ELISA was tested against sera from pigs experimentally infected with the 12 serotypes of Ap biotype 1. Cross-reaction with serotypes 3 and 8 but not with other serotypes was observed. The sensitivity and specificity of the test on a herd level were evaluated with sera from herds naturally infected with serotypes 2, 6, 8 or 12 and with sera from herds free of infection with any Ap serotype. The blocking ELISA showed a high herd sensitivity (1.00 (0.79-1.00)) and specificity (0.97 (0.93-0.99)).

New technology for regiospecific covalent coupling of polysaccharide antigens in ELISA for serological detection.

In this study we demonstrate a new UV irradiation technique for covalent coupling of bacterial polysaccharides derived from lipopolysaccharides to microtiter plates and the use of such plates in an enzyme linked immunosorbent assay (ELISA). Lipopolysaccharides were cleaved by mild acid hydrolysis into the lipid A part and the polysaccharide part. The polysaccharide was conjugated regiospecifically to a photochemically active compound, anthraquinone, resulting in a polysaccharide-anthraquinone conjugate. Anthraquinones forms active radicals when exposed to soft UV irradiation (350 nm) permitting the formation of stable covalent bonds to polymers e.g. microtiter plates. By this technique the polysaccharides are bound through the anthraquinone part of the polysaccharide-anthraquinone conjugates to the microtiter plates. This minimizes denaturation of O-antigen epitopes during binding to the microtiter plates and avoids cross-reactivity due to conserved domains in the lipid A. Furthermore, the covalent binding of the polysaccharide antigens are compatible with harsh assay conditions, such as extensive washing procedures and buffers with high salt concentrations with no risk of antigen leakage. Here we describe the use of this technique for the immobilization of lipopolysaccharide derived polysaccharides from Salmonella Typhimurium and Salmonella Choleraesuis lipopolysaccharides, representing the O-antigens 1, 4, 5, 6, 7, and 12. The functional polysaccharide surface gave similar ELISA results to plates coated passively with the corresponding unmodified lipopolysaccharide antigens. The plates were highly reproducible, showed very low inter- and intra-plate variation and were stable at room temperature for more than 8 months.

Detection of Streptococcus suis by in situ hybridization, indirect immunofluorescence, and peroxidase-antiperoxidase assays in formalin-fixed, paraffin-embedded tissue sections from pigs.

Streptococcus suis is an important pathogen in pigs and is considered a zoonotic agent. To aid diagnosis of infection caused by S. suis, a species-specific probe targeting 16S ribosomal RNA was designed and used for fluorescent in situ hybridization. Two additional immunohistochemical detection methods, an indirect immunofluorescence assay and a peroxidase-antiperoxidase method, using polyclonal antibodies also were developed. The specificity of the oligonucleotide probe was examined by whole-cell and dot-blot hybridization against reference strains of the 35 serotypes of S. suis and other closely related streptococci and other bacteria commonly isolated from pigs. The probe was specific for S. suis serotypes 1-31. The specificity of the polyclonal antibodies, which has previously been evaluated for use in diagnostic bacteriology for typing of serotype 2, was further evaluated in experimentally infected murine tissue with pure culture of different serotypes of S. suis, related streptococci, and other bacteria commonly found in pigs. The polyclonal antibodies against S. suis serotype 2 cross-reacted with serotypes 1 and 1/2 in these assays. The in situ hybridization and the immunohistochemical methods were used for detection of S. suis in formalin-fixed, paraffin-embedded tissue sections of brain, endocardium, and lung from pigs infected with S. suis. The methods developed were able to detect single cells of S. suis in situ in the respective samples, whereas no signal was observed from control tissue sections that contained organisms other than S. suis. These techniques are suitable for determining the in vivo localization of S. suis for research and diagnostic purposes.

Evaluation of a novel enzyme-linked immunosorbent assay for detection of antibodies against Salmonella, employing a stable coating of lipopolysaccharide-derived antigens covalently attached to polystyrene microwells.

Polysaccharides derived from Salmonella typhimurium lipopolysaccharide (LPS) representing the O-antigen factors 1, 4, 5, and 12 and the O-antigen factors 6 and 7 from Salmonella choleraesuis LPS were derivatized with the photoreactive compound anthraquinone and subsequently covalently coupled to microtiter polystyrene plates by ultraviolet irradiation. Both polysaccharide antigens could be coupled simultaneously to the same microtiter plate. The coated surface was used in indirect ELISA for the determination of serum antibodies from pigs infected with bacteria of the two Salmonella groups and from uninfected pigs. This ELISA proved itself by having a good long-term durability and a high degree of reproducibility, including low day-to-day variations and low interplate variations. Furthermore, the ELISA showed good specificity and sensitivity when data were compared with the optical density levels of a panel of pig sera as determined by a conventional ELISA on the basis of passive coating of the two Salmonella LPS antigens (the mix-ELISA). The covalent anthraquinone mix-ELISA shows promise as a stable and durable alternative to the existing conventional ELISA for serological surveillance of Salmonella infections in pigs.

Prevention of edema disease in pigs by passive immunization.

The effect of treatment with verotoxin 2e (VT2e) specific antiserum was evaluated in 3 Danish pig herds with edema disease (ED). The antiserum was prepared by immunizing horses with a VT2e toxoid. The study was performed as a randomized blind field trial with parallel treatment and control groups. There were approximately 50 piglets in each group in each of the 3 herds and 741 piglets were included in the study (244 from herd A, 249 from herd B, and 247 from herd C). Treatment groups received 2, 4, or 6 mL anti-VT2e serum intramuscularly the day before weaning. Control groups were treated with 6 mL normal horse serum or 6 mL RPMI 1640 medium as placebo. All pigs that died in the trial period (1 d before weaning to 44 d after weaning) were examined pathologically and microbiologically. Mortality due to ED, mortality due to other causes, and adverse effects due to treatment were recorded. As there was no mortality due to ED, herd B was excluded from statistical calculations on mortality. The content of horse antibodies specific to VT2e in serum from pigs was analyzed in an indirect ELISA. A higher dose of anti-VT2e serum was reflected in higher optical density values in the indirect ELISA. Transient adverse reactions, seen as vomiting, ataxia, and cyanosis, occurred shortly after the injection of horse serum in 1.5% of the pigs, and one pig died. There were no statistically significant differences in mortality due to other causes among the 3 treatment groups in herds A and C. Only pigs from which F18+, VT2e+, ST-, LT- hemolytic E. coli (0139 or O-rough) was isolated were diagnosed as dead due to ED. Deaths due to ED in the control groups were 8.1% and 12.0% in herds A and C, respectively, compared with 0% and 0.7% in the corresponding serum groups. The difference between treatment and control groups was statistically significant (P<0.0001). It was not possible to establish an effect of dose (2, 4, or 6 mL) of anti-VT2e serum, because only one pig died of ED in the treatment groups. It was concluded that passive immunization by intramuscular injection of a VT2e-specific antiserum can be used for protecting piglets against ED.

Development and evaluation of an indirect ELISA for detection of exfoliative toxin ExhA, ExhB or ExhC produced by Staphylococcus hyicus.

Immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) confirmed previous reports that the Staphylococcus hyicus exfoliative toxins ExhA and ExhB are metalloproteins, and further indicated that ExhC is also a metalloprotein. An indirect ELISA was developed for the detection of toxigenic strains as an alternative method to the use of phage typing for selection of S. hyicus isolates to be used in autogenous vaccine against exudative epidermitis in pigs. The indirect ELISA was evaluated by investigating the presence of toxin among a total of 655 S. hyicus isolates from 69 pig skin samples, one from each of the 69 pig herds with outbreak of exudative epidermitis. Toxigenic S. hyicus were detected in 74% of the cases by ELISA. From each of the five cases, in which initially no toxigenic S. hyicus were found, a further 40 S. hyicus-like colonies were tested in ELISA. Testing of this number of colonies has a >99% probability of disclosing toxigenic S. hyicus. Toxin-producing isolates were found in only two of the five cases investigated. This may indicate the existence of one or more variants of the exfoliative toxin of S. hyicus that are not detected in the indirect ELISA or that S. hyicus may be displaced from lesions of exudative epidermitis.

Studies on the effect of divalent metal ions on exfoliative toxins from Staphylococcus hyicus: indications of ExhA and ExhB being metalloproteins.

The exfoliative toxins ExhA and ExhB produced by Staphylococcus hyicus strains NCTC10350 and 1289D-88, respectively, were investigated with regard to the effect of divalent metal ions on toxin production as measured in indirect enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies. Data were obtained as endpoint titer values and used as semiquantitative measures for the amount of exfoliative toxin detected in culture supernatants. It was shown that the endpoint titers of ExhA in supernatants from cultures of strain NCTC10350 grown in the presence of 0.5 mM CaCl2, Cu(NO3)2 or ZnSO4 were higher compared to titers obtained by growth in medium supplemented with a number of other divalent metal salts. The titer of ExhB as determined in the indirect ELISA was increased by addition of 0.5 mM CoCl2, Cu(NO3)2 or CuSO4 to the growth medium. When ExhA or ExhB, prepared without addition of metal salt to the liquid growth medium, was subsequently incubated with 25 mM of Co2+, Cu2+ or Zn2+, the endpoint titers of the toxins were increased. Dialysis of ExhA and ExhB prepared with Zn2+ and Co2+, respectively, against certain metal chelators, resulted in a reduction of the titer determined in ELISA. Other metal chelators had varied effect in the detection of the toxins in ELISA. It was, however, not possible to restore the recognition of toxins by the monoclonal antibodies by incubation of EDDHA-dialyzed toxin preparations with Co2+, Cu2+ or Zn2+. The results of this study suggest that ExhA and ExhB are metalloproteins.

16S rDNA sequence variations of some Streptococcus suis serotypes.

Streptococcus suis 16S rDNA from selected serotypes has been sequenced and compared with the 16S rDNA sequences from serotypes 1 and 2 present in Genbank. After alignment the sequenced serotypes show clusters of variation. Based on these clusters, a limited phylogenetic tree showing the relationships of all of the serotypes was constructed.

Differentiation and distribution of three types of exfoliative toxin produced by Staphylococcus hyicus from pigs with exudative epidermitis.

Exfoliative toxins of approximately 30 kDa produced by Staphylococcus hyicus strains NCTC 10350, 1289D-88 and 842A-88 were purified and specific polyclonal antisera were raised against each of the toxins. It was shown by immunoblot analysis and ELISA that three exfoliative toxins from S. hyicus were antigenically distinct. The three toxins were designated ExhA, ExhB and ExhC. From 60 diseased pigs, each representing an outbreak of exudative epidermitis, a total of 584 isolates of S. hyicus were phage typed and tested for production of exfoliative toxin. ExhA-, ExhB- and ExhC-producing S. hyicus isolates were found in 12 (20%), 20 (33%) and 11 (18%), respectively, of the 60 pig herds investigated. Production of the different types of exfoliative toxin was predominantly associated with certain phage groups. However, toxin production was found in all of the six phage groups defined by the phage typing system. Some changes in the distribution of isolates between phage groups were observed when the results of this study were compared to previous investigations. In this study two new antigenically distinct exfoliative toxins were isolated and tools for in vitro detection of toxin producing S. hyicus isolates and for further studies on the exfoliative toxins from S. hyicus have been provided.

Prevention of edema disease in pigs by vaccination with verotoxin 2e toxoid.

Pigs in 2 herds with persistent problems with post weaning edema disease caused by infection with verotoxin-2e (VT2e)-producing Escherichia coli O139 were treated with a VT2e-toxoid vaccine. Treatment was performed as a randomized blind field trial with parallel treatment and non-vaccinated control groups. In 1 herd, a group of pigs was injected with adjuvant alone. Pigs were vaccinated at 1 and 3 wk of age and weaned at 4 wk of age. The effect of vaccination was measured by average daily weight gain (ADG), mortality due to edema disease within the 1st 4 wk after weaning, and weight at 3-6 mo of age. Pathological and microbiological examinations were performed on all pigs that died during the 1st 4 wk post weaning. Only pigs from which VT2e+, F18+ E. coli O139 was isolated were categorized as "death due to edema disease." The serological response to vaccination was evaluated by an indirect ELISA. Vaccination had a statistically significant effect on the level of antibodies specific for VT2e in both herds. Vaccination resulted in a statistically significant increase in ADG in the nursery period but not in the grower-finishing period. Vaccination had a statistically significant effect on mortality due to edema disease with an odds ratio of 0.039, indicating that there was almost total elimination of mortality due to the disease in the vaccine groups.

Staphylococcus hyicus exfoliative toxin: purification and demonstration of antigenic diversity among toxins from virulent strains.

The exfoliative toxin produced by Staphylococcus hyicus strain 1289D-88 was purified as a single protein of approximately 30 kDa. Extracellular proteins of S. hyicus grown under small scale fermentation conditions were precipitated with ammonium sulfate. Separation of proteins was performed by hydrophobic interaction chromatography and successively anion exchange chromatography. The purified toxin was tested in a piglet skin assay. Weak epidermal lesions were macroscopically and microscopically similar to lesions caused by (NH4)2SO4-precipitated culture supernatant from the same strain. Addition of 0.5 mM CuSo4 to the purified toxin resulted in more intense skin alterations comparable to lesions caused by precipitated culture supernatant diluted 1:10. These results indicated that the activity of the exfoliative toxin was dependent on the presence of Cu2+. Polyclonal and monoclonal antibodies were prepared against the exfoliative toxin from strain 1289D-88. The in vivo activity of the exfoliative toxin could be neutralized by antibodies. It was shown that polyclonal as well as monoclonal antibodies only reacted with the toxin produced by two of nine well-defined virulent strains of S. hyicus. These results showed antigenic diversity among exfoliative toxins produced by different strains of S. hyicus.

Serological characterization of Actinobacillus pleuropneumoniae biotype 2 strains isolated from pigs in two Danish herds.

Eight Actinobacillus pleuropneumoniae biotype 2 strains were isolated in pure culture from lungs of pigs originating from two Danish herds with growing and finishing pigs. The antigenic properties were studied by indirect haemagglutination (IHA) and immunodiffusion (ID) tests using soluble surface antigens and by enzyme-linked immunosorbent assays (ELISA) using capsular enriched fractions and LPS. In all tests the strains proved antigenically homogeneous and serologically distinct from the known biotype 1 and 2 serotypes. Thus, the strains represent a new serotype which is provisionally proposed as biotype 2 serotype 14.

Experimental vaccination of pigs with an Actinobacillus pleuropneumoniae serotype 5b capsular polysaccharide-tetanus toxoid conjugate.

The protective efficacy of an Actinobacillus pleuropneumoniae serotype 5b capsular polysaccharide-tetanus toxoid conjugate (Ap5bCP-TT) against homologous challenge of pigs was investigated. Four pigs were non-vaccinated controls (group A), 4 pigs were injected with adjuvant without antigen (group B) and 8 pigs were vaccinated with Ap5bCP-TT and adjuvant (group C). Pigs vaccinated with Ap5bCP-TT developed antibody responses to the capsular polysaccharide from A. pleuropneumoniae serotype 5b (Ap5bCP). After challenge, all pigs in groups A and B had severe clinical signs of disease and were euthanized. In group C, 3 out of 8 pigs showed severe symptoms and were euthanized. Five pigs in group C survived throughout the study. The post challenge observation period was 72 h. All pigs were subject to necropsy and results from gross pathological findings and microbiological examination are described. Pigs vaccinated with Ap5bCP-TT had statistically significant reduced values of the mass ratio of affected to unaffected lung tissue compared to pigs in groups A and B (p = 0.01 and p = 0.007, respectively). The results showed that Ap5bCP-TT-vaccination had considerable protective efficacy against lethality and pulmonary lesions caused by experimental infection with A. pleuropneumoniae serotype 5b.

Serological characterization of Actinobacillus pleuropneumoniae biotype 1 strains antigenically related to both serotypes 2 and 7.

Nine Danish Actinobacillus pleuropneumoniae biotype 1 isolates were shown by latex agglutination and indirect haemagglutination to possess capsular polysaccharide epitopes identical to those of serotype 2 strain 1536 (reference strain of serotype 2) and strain 4226 (Danish serotype 2 strain). Immunodiffusion confirmed the antigenic relationship with serotype 2 and further demonstrated an antigenic relationship with strain WF83 (reference strain of serotype 7). SDS-PAGE with LPS from strains 1536, 4226, WF83 and strain 7317 (representative of the 9 isolates examined) showed that strains WF83 and 7317 had an identical smooth ladder pattern whereas LPS from strains 1536 and 4226 showed a distinctly different pattern. The antigenic similarities of the LPS of strains WF83 and 7317 were confirmed by immunoblots using rabbit or pig antisera prepared against the 3 strains. No antigenic similarities in the LPS of strains 1536 and 7317 were revealed. Since an antigenic determinant specific for the 9 isolates could not be demonstrated with the methods used, the strains are proposed to be designated K2:O7.

Staphylococcus hyicus-skin reactions in piglets caused by crude extracellular products and by partially purified exfoliative toxin.

Staphylococcus hyicus may cause a spontaneous generalized exudative epidermitis in piglets. The progression and regression of macroscopical and histopathological lesions in piglet skin after subcutaneous injection of sterile concentrated culture supernatant (CCS) from one virulent and one avirulent strain of S. hyicus was studied every 24 h until 144 h post-injection. CCS from the virulent strain caused local alterations of the epidermis comparable to those of spontaneous exudative epidermitis: exfoliation, crust formation, exocytosis, formation of vesicles, and pustules and acanthosis. CCS from the avirulent strain only caused a transient erythema of the skin and no histopathological alterations of the epidermis. Additionally, proteins in CCS from the virulent strain were fractionated by column chromatography. Skin reactions similar to those caused by CCS from the virulent strain were induced by one fraction of proteins that contained eight protein bands in SDS-PAGE analysis. Two of these protein bands, with molecular weights of approximately 27 kDa and 30 kDa, were unique to the virulent strain compared to the avirulent strain. The results of this study indicate that one of these two proteins or both is a heat-labile exfoliative toxin, and that the toxin is a significant factor in the pathogenesis of exudative epidermitis in piglets.