Veterinary Microbiology 105 (2005) 291–300 www.elsevier.com/locate/vetmic Exudative epidermitis in pigs caused by toxigenic Staphylococcus chromogenes Lars Ole Andresen*, Peter Ahrens, Lise Daugaard, Vivi Bille-Hansen Danish Institute for Food and Veterinary Research, Department of Veterinary Diagnostics and Research, Bu¨lowsvej 27, DK-1790 Copenhagen V, Denmark Received 3 September 2004; received in revised form 1 December 2004; accepted 8 December 2004 Abstract Staphylococcus chromogenes is closely related to Staphylococcus hyicus, which is recognised as the causative agent of exudative epidermitis (EE) in pigs. S. chromogenes is part of the normal skin flora of pigs, cattle and poultry and has so far been considered non-pathogenic to pigs. A strain of S. chromogenes producing exfoliative toxin type B, ExhB, was identified by the use of a multiplex PCR specific for the exfoliative toxins from S. hyicus. The exfoliative toxin from S. chromogenes reacted in immunoblot analysis with polyclonal and monoclonal antibodies specific to ExhB from S. hyicus and had an apparent molecular weight of 30 kDa. Sequencing the gene encoding the exfoliative toxin from S. chromogenes revealed that the molecular weight of the toxin with the signal peptide and the mature toxin was 30,553 and 26,694 Da, respectively. Comparison of the exhB genes from S. chromogenes strain VA654 and S. hyicus strain 1289D-88 showed differences in seven base pairs of the DNA sequences and in two amino acid residues in the deduced amino acid sequences. Pigs were experimentally inoculated with S. chromogenes strain VA654. By clinical observations and histopathological evaluation of the skin alterations, all pigs revealed development of generalized exudative epidermitis. No toxin producing S. hyicus was isolated from the pigs and all ExhB-positive bacterial isolates were identified as S. chromogenes. This confirmed that the disease-causing agent was the inoculated S. chromogenes strain VA654. The results of this study show that S. chromogenes may cause exudative epidermitis in pigs. # 2004 Elsevier B.V. All rights reserved. Keywords: Staphylococcus chromogenes; Exfoliative toxin; Exudative epidermitis; Experimental infection; Pig-bacteria 1. Introduction pigs and have also been isolated from cattle, poultry (Devriese et al., 1985; Shimizu et al., 1992) and goats The bacterial species Staphylococcus chromogenes (Valle et al., 1991). Both species were previously and Staphylococcus hyicus are part of the skin flora of subspecies of the species S. hyicus, which was a new combination of Micrococcus hyicus, coagulase-posi- * Corresponding author. Tel.: +45 723 46 282; tive S. epidermidis biotype 2, and Staphylococcus fax: +45 723 46 001. aureus avian biotype 2 (Devriese et al., 1978). E-mail address: [email protected] (L.O. Andresen). Elevation of S. hyicus ssp. chromogenes to S. 0378-1135/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2004.12.006 292 L.O. Andresen et al. / Veterinary Microbiology 105 (2005) 291–300 chromogenes was suggested by Ha´jek et al. (1986) In dairy cattle, S. chromogenes is a well-known based on several criteria, including DNA–DNA pathogen associated with mastitis (Devriese et al., hybridisation studies, phenotypic characteristics, 2002) and has also been reported as the cause of skin differences in penicillin-binding proteins and phage infection in a goat (Andrews and Lamport, 1997). S. host range, which separated the two subspecies of S. chromogenes has been isolated from the skin of both hyicus from one another. Consequently both S. hyicus diseased and healthy pigs (Devriese et al., 1978; Saito ssp. hyicus and S. hyicus ssp. chromogenes were raised et al., 1996) but in those studies was not established as to species status (Annon., 1987). A few character- an ethiological agent of disease. S. chromogenes from istics, which are useful in diagnostic laboratories, can chickens and bovine udders was not pathogenic to pigs easily differentiate S. chromogenes and S. hyicus. The in inoculation experiments (Devriese and Oeding, former lacks heat-stable nuclease, hyaluronidase, and 1975), and consequently, S. chromogenes has been does not possess lipase activity that hydrolyse Tween regarded as non-pathogenic in pigs (Ha´jek et al., 1986). 80, whereas S. hyicus shows positive reactions for In the present study, we have identified and char- these enzymes. In addition, S. chromogenes most often acterised an exfoliative toxin from S. chromogenes appears with orange or creamy colonies in contrast to similar to the exfoliative toxin ExhB from S. hyicus.By the white colonies of S. hyicus. Isolation of non- experimental infection studies it was demonstrated that pigmented S. chromogenes from pigs has, however, S. chromogenes may provoke EE in pigs. been reported (Saito et al., 1996). S. hyicus is recognized as the causative agent of exudative epidermitis in pigs, which is a generalized 2. Materials and methods skin infection (Wegener and Skov-Jensen, 1999). Exfoliative toxins that are produced by certain strains 2.1. Bacterial strains and culture conditions of S. hyicus cause the characteristic histopathological signs of EE (Andresen et al., 1997). Both toxigenic S. chromogenes investigated in the present study and non-toxigenic S. hyicus can be isolated from comprised 15 from Belgium (13 porcine and 2 diseased animals (Andresen, 1998). The human bovine), all originating from different farms and 4 disease staphylococcal scalded skin syndrome (SSSS), strains from bovine mastitis isolated in Norway (see caused by exfoliative toxin-producing S. aureus Table 1). The type strains of S. chromogenes, (Ladhani et al., 1999), has several comparative aspects NCTC10530 (Ha´jek et al., 1986) and S. hyicus, to EE in pigs including histopathology, i.e., blister NCTC10350 (Devriese et al., 1978) were used for formation and exfoliation of the skin caused by comparison and as control strains in the identification splitting of the skin at the granular layer of the procedures. S. hyicus strains NCTC10350T, 1289D- epidermis. S. aureus from cases of SSSS and bullous 88, 842A-88 (Wegener et al., 1993) and A2869C impetigo in humans produces exfoliative toxins, (Amtsberg, 1979) were used as reference strains with designated ETA, ETB and ETD (Hanakawa et al., regard to the exfoliative toxins ExhA, ExhB, ExhC 2002; Yamaguchi et al., 2002). These toxins have been and ExhD, respectively, and the corresponding genes. shown to specifically cleave the desmoglein-1, which Additionally, four ExhB-positive field strains of S. is a desmosomal cadherin-like molecule involved in hyicus isolated from pigs with EE in Denmark in 1996, cell-to-cell adhesion (Hanakawa et al., 2002). 1997, 2003 and 2004, respectively, were used for Recently, it was shown that the amino acid sequences sequencing of the exhB gene. The strains were grown of the exfoliative toxins from S. hyicus and S. aureus aerobically at 37 8C for 17–24 h on Columbia agar share similarities both between and within the two base (Oxoid, Unipath Ltd., Basingstoke, UK) supple- species (Ahrens and Andresen, 2004). On the basis of mented with 5% bovine blood (C-blood agar). Liquid the DNA sequences of the exfoliative toxins and growth medium consisted of 30 g lÀ1 trypticase soy available reference strains for the exfoliative toxins broth (Becton Dickinson and Co., Cockeysville, MD) ExhA, ExhB, ExhC and ExhD from S. hyicus a supplemented with 10 g lÀ1 yeast extract (Oxoid), pH multiplex PCR was recently developed (Andresen and was adjusted to 7.2 before autoclaving the medium. Ahrens, 2004). The liquid growth medium was supplemented with L.O. Andresen et al. / Veterinary Microbiology 105 (2005) 291–300 293 Table 1 Strains of Staphylococcus chromogenes and Staphylococcus hyicus used in present study Species Strain designation Origin Year of isolation, Referencea Exfoliative country toxin produced S. chromogenes NCTC10530T –, England 1 VA103, VA104, VA110, VA401, Pig, healthy skin 1979, Belgium 2 VA404, VA650 VA654 Pig, healthy skin 1979, Belgium 2 ExhB VAC 44 Pig, healthy skin 1989, Belgium – G91 Pig, joint 1984, Belgium – G92 Pig, skin 1984, Belgium – G248, G254 Sow, endometritis 1990, Belgium – Bo59, Bo81 Cow, lesion 1977, Belgium 2 6105-4, 6042-3, 6087-2, 6128-3 Cow, mastitis 1997–1998, Norway – S. hyicus NCTC10350T Pig with EE, skin 1949, Denmark 3 ExhA 1289D-88 Pig with EE, skin 1988, Denmark 4 ExhB 842A-88 Pig with EE, skin 1988, Denmark 4 ExhC A2869C Pig with EE, skin 1970, Germany 5 ExhD 9606363-1 Pig with EE, skin 1996, Denmark – ExhB 9716142-1 Pig with EE, skin 1997, Denmark – ExhB 7513260-1 Pig with EE, skin 2003, Denmark – ExhB 7611103-1 Pig with EE, skin 2004, Denmark – ExhB a 1, Baird-Parker (1962);2,Devriese et al. (1978);3,Sompolinsky (1950);4,Wegener et al. (1993); 5, Amtsberg et al. (1979). Strains with no reference indicated (–) have not previously been published. 0.5 mM CoCl2 and 0.5 mM ZnSO4 after sterilization. multiplex PCR as previously described (Andresen and Cultures were grown in 10-ml tubes containing 5 ml Ahrens, 2004). Expression of exfoliative toxin was liquid growth medium inoculated with a single colony detected by sodium dodecyl sulfate-polyacrylamide from an overnight culture on C-blood agar. Liquid gel electrophoresis (SDS-PAGE) and immunoblotting cultures were incubated for 24 h at 37 8C with shaking as previously described (Andresen, 1999) using the at 130 rpm. monoclonal antibody MabEXH5.1 and polyclonal rabbit antibodies specific for ExhB (Andresen et al., 2.2. Identification 1997) as primary antibodies. Characters that were used for distinguishing 2.4. DNA sequencing between S. chromogenes and S. hyicus were pigmen- tation of the bacterial colonies on C-blood agar, lipase Genes encoding 16S rDNA were sequenced as activity tested for by the use of a selective and previously described (L’Abe´e-Lund et al., 2003)by indicative agar-medium previously described by PCR amplification, purification of the PCR-products Devriese (1977), hyaluronidase activity (Devriese and sequencing on an ABI 377 automatic sequencers et al., 1985) and heat-stable nuclease (Lachica et al., using the Prism BigDye terminator cycle sequencing 1971).