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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.

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.

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.

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.

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.

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.

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.

Staphylococcus hyicus strains with different phage types, plasmid profiles, and antibiotic resistance patterns were isolated from piglets with exudative epidermitis. The strains could be divided into virulent strains, producing exudative epidermitis, and avirulent strains, producing no dermal changes when injected in experimental piglets. The results showed that both virulent and avirulent strains were present simultaneously on diseased piglets. This constitutes a diagnostic problem. Concentrated culture supernatants from nine virulent strains injected in the skin of healthy piglets produced a crusting reaction in all piglets. Acanthosis was observed in the histopathological examination of the crustaceous skin. Concentrated culture supernatants from nine avirulent strains produced no macroscopic or microscopic skin changes. Protein profiles from all virulent strains and seven out of nine avirulent strains showed a high degree of protein band homology. An approximately 30 kDa protein present in all concentrated culture supernatants capable of producing skin changes, could not be detected in samples that did not produce skin changes. No other protein showed a similar association. It is concluded that crusting reaction of piglet skin is a suitable indicator of virulence in S. hyicus in relation to exudative epidermitis, and that virulent strains produce a 30 kDa protein, absent in concentrated culture supernatants from avirulent strains. This 30 kDa protein might be an exfoliative toxin.

Exudative epidermitis is an acute, often fatal skin disease in piglets caused by infection with Staphylococcus hyicus. Clinical and histopathological manifestations of exudative epidermitis are similar to those of a human blistering skin disease, staphylococcal scalded skin syndrome, in which cleavage of desmoglein (Dsg) 1 by exfoliative toxins produced by S. aureus results in blister formation in the upper part of the epidermis. The aim of this study was to determine whether the four types of exfoliative toxin produced by S. hyicus (ExhA, ExhB, ExhC and ExhD) are able to affect swine Dsg1. We first generated recombinant toxins of ExhA, ExhB, ExhC and ExhD by an E. coli expression system. To determine the pathogenic activity of these recombinant toxins, we injected them subcutaneously into 3- to 4-week-old piglets. After administration of all four recombinant toxins, local exfoliations developed at the inoculation sites of the piglets’ skin at 21–48 h postinjection. To further assess whether these Exhs affect swine Dsg1, we incubated cryosectioned pig skin with Exhs in vitro and subsequently stained Dsg1 and Dsg3 by immunofluorescence with human pemphigus foliaceus sera and anti-Dsg3 monoclonal antibody (5H10), respectively. The cell surface staining of Dsg1 was abolished by incubation with any of the four isoforms of Exh, whereas that of Dsg3 was not affected at all. These findings indicate that all four types of exfoliative toxin produced by S. hyicus (ExhA, ExhB, ExhC and ExhD) can cause skin exfoliation in pigs with exudative epidermitis, presumably by digestion of Dsg1. Funding: Ministry of Education, Culture, Sports, Science and Technology of Japan. [ABSTRACT FROM AUTHOR]