VETERINARSKI ARHIV 69 (6), 319-326, 1999

ISSN 1331-8055 Published in Croatia

Distribution and protein profiles of porcine Staphylococcus hyicus isolates

Branka Šeol*

Department of Microbiology and Infectious Diseases, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia

* Contact address:
Dr. Branka Šeol,
Department of Microbiology and Infectious Diseases, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia,
Phone 385 1 23 90 205; Fax 385 1 244 13 90; E-mail: seol@mavef.vef.hr

ŠEOL, B.: Distribution and protein profiles of porcine Staphylococcus hyicus isolates. Vet. arhiv 69, 319-326, 1999.

ABSTRACT

A total of 165 swabs originating from skin and various mucosa of 33 healthy sows were bacteriologically examined. From 23 (69.7%) animals, 52 Staphylococcus hyicus strains were isolated. All isolates were serotyped by slide agglutination test. Most isolated strains belonged to serovar ATCC 19226. S. hyicus was most predominantly represented on udders (13 sows), skin of snout and ears and nasal mucosa (11 sows), vaginal mucosa (nine sows) and conjunctivas (eight sows). Sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) profile patterns of whole cell samples of epidemiologically unrelated strains showed great homology among strains of the same serovar. SDS-PAGE profile patterns of whole cell samples of 29 epidemiologically unrelated strains showed great homology among strains of the same serovar. SDS-PAGE profile of sterile concentrated supernatant showed that five strains, all ATCC 19226 serovar, isolated from healthy sows belonged to separate groups. Those strains contained a band corresponding to a protein with a molecular mass of approximately 50 kDa, while that of 45 kDa was not observed.

Key words: Staphylococcus hyicus, distribution, healthy sow, protein profile

Introduction

Staphylococcus hyicus, originally described by Sompolinsky (1950), is a causative agent of exudative epidermitis, an infectious skin disease characterised by exfoliation of the skin, excessive sebaceous secretion, and formation of a brownish coat of exudate that may cover the entire body (Jones, 1956; Underdahl et al., 1965; Jubb et al., 1993). It is known that healthy pigs can carry S. hyicus on their skin and mucosa and can, therefore, be a source of infection for piglets (Amtsberg, 1978; HOLLAND and HODGES, 1981; Hajsig et al., 1985; Onet and Pommer, 1991). There are very few data regarding S. hyicus findings and distribution in sows (Amtsberg, 1978; Hajsig et al., 1985; Wegener and Skov-Jensen 1992; ŠEOL, 1996). The purpose of the present study was to investigate the distribution of S. hyicus strains on skin and some mucosa of healthy sows, serotyping of isolated strains and a comparison between strains of the same serovar isolated from the same animal, as well as between different serovars of the same bacteria based on sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) of protein profiles of whole cells and concentrated, unpurified sterile supernatants.

Materials and methods

In order to establish the frequency of S. hyicus occurrence in healthy sows, 165 swabs were collected from skin of the snout, ears, mammary glands, conjunctivae, nasal and vaginal mucosa of 33 healthy animals. Identification of S. hyicus was performed using the method described by Devriese (1977, 1981). All identified strains of S. hyicus were typed by slide agglutination. Serological typing was carried out using seven absorbed sera for the following S. hyicus serovars: ATCCC 19226, S 3588, ZH 1029, ZH 1037, Hunter A, Hunter B and A 636 / A 1743. For sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) 29 epidemiologically unrelated Staphylococcus hyicus strains isolated from sows and five referent strains (ATCCC 19226, S 3588, ZH 1029, ZH 1037, Hunter A), were inoculated in 20 ml of BHI broth (Difco, Detroit, Michigan, USA) and incubated at 37 °C for 24 hours in a shaker operated at 100 oscillations per min. After incubation, cultures were centrifuged at 10,000 × g for 30 minutes at 4 °C. Supernatants were passed through a 0.45 µm-pore-size membrane filter (Milipore; Bedford, Massachusetts, USA), lyophilised and re-suspended in 1 ml of sterile distilled water and obtained 20-times concentrated unpurified sterile supernatants. Sediment of whole bacterial cells was re-suspended in 5 ml sterile PBS (Phosphate buffered saline), centrifuged three times at 10,000 × g for 30 minutes at 4 °C. Finally, 1 ml of sterile PBS was added and samples were stored at -20 °C. Protein concentration was determined by BCA protein assay (Smith et al., 1985; Sorensen and Brodbeck, 1986) with bovine serum albumin (Sigma Chemical Co., St. Louis, USA).

SDS-PAGE was performed using the Laemmli (1970) method with a discontinuous system employing slab gels 0.75 mm thick, a 10% (w/v) resolving gel and a 3% (w/v) stacking gel. Samples were mixed 1:1 with sample buffer containing 2% SDS, 5% 2-mercaptoethanol, 10% sucrose, 0.01% bromophenol blue, and boiled for three minutes before application to gels. Molecular mass standards (pre-stained high molecular mass marker and low molecular mass marker) were also run at each gel.

Results

From 33 examined animals S. hyicus strains were found on the skin of ear and snout in eleven animals (33.3%); in thirteen sows (39.4%) S. hyicus was found on udders. Nine sows (27.3%) carried these bacteria on vaginal mucosa and eleven (33.3%) on nasal mucosa. S. hyicus was recovered from conjunctiva of eight animals (24.2%). As seen in Table 1, most strains belonged to serovar ATCC 19226 (67.3%); thirteen strains (25%) to serovar ZH 1037 and only four strains (7.7%) to serovar ZH 1029. SDS-PAGE profile patterns of whole cell samples of 29 investigated strains showed great homology among strains of the same serovar. SDS-PAGE profile of concentrated unpurified sterile supernatants showed that five strains, all ATCC 19926 serovar (strains marked as 26 A, 27 C, 27 E, 28 D and 29 D), isolated from healthy sows belong to separate group (Figs. 1-4). These strains contained a band corresponding to a protein with a molecular mass of approximately 50 kDa, while those of 45 kDa were not observed.

Table 1. Results of serological identification Staphylococcus hyicus strains isolated from healthy sows

Fig. 1. Protein profiles of sterile concentrated supernatants of S. hyicus from sows: lane 1-6; strains marked: 24 C, 26 A, 26 D, ref. serovar ATCC 19226, 25 D, ref. serovar ZH 1037. Left and right lanes: molecular mass markers.

Fig. 2. Protein profiles of sterile concentrated supernatants of S. hyicus from sows: lane 1-6; strains marked: 27 A, 27 B, 27 C, 27 D, 27 E, ref. serovar ATCC 19226. Left and right lanes: molecular mass markers.

Fig. 3. Protein profiles of sterile concentrated supernatants of S. hyicus from sows: lane 1-6; strains marked: 28 A, 28 C, 28 D, ref. serovar ATCC 19226, 29 A, 29 D. Left and right lanes: molecular mass markers.

Fig. 4. Protein profiles of sterile concentrated supernatants of S. hyicus from sows: lane 1-6; strains marked: 29 A, 29 C, 29 D, ref. serovar ATCC 19226, 29 B and ref. serovar ZH 1037. Left and right lanes: molecular mass markers.

Discussion

Considering the fact that studies of S. hyicus in healthy piglets, and especially sows, are rare (Hunter et al., 1970; Devriese, 1977; Hajsig et al., 1985) we attempted to expand our knowledge of the distribution of this bacteria in healthy sows and which can be transmitted to their litters. We examined the presence of S. hyicus on the conjunctiva and nasal mucosa, the skin of the snout and ears, as well as udders, because they appear to be the first sites of visible signs of exudative epidermitis (Jubb et al., 1993; Radostits et al., 1994). Vaginal mucosa was chosen because of reports on abortion in sow attributed to S. hyicus (ONET and POMMER, 1991). Out of 165 swabs samples taken from 23 sows, S. hyicus was isolated from the udders of 13 animals (39.4%) and from ears and snout of 11 (33.3%). S. hyicus was found on vaginal mucosa in nine sows (27.3%) and the conjunctiva of eight animals (24.2%). These findings agree with results obtained by the other authors (AMTSBERG, 1978; Hajsig et al., 1985; WEGENER and SKOV-JENSEN, 1992). The serological typing of S. hyicus showed the presence of serovars ATCC 19226, ZH 1029 and ZH 1037. Out of 52 isolates the most prevalent serovar was ATCC 19226 (35 strains) followed by ZH 1037 (13 strains) and ZH 1029 (4 strains). In 11 sows S. hyicus strains present at two or more sites belonged to the same serovar, and in six sows to a different serovar. These results contribute to the knowledge of the geographical spread of some S. hyicus serovars. While strains of serovar ATCC 19226 have been established in most countries of Europe and America, serovars ZH 1029 and ZH 1037, previously found only in Switzerland (STUKER and BERTSCHINGER, 1976), have been isolated in Croatia (HAJSIG et al., 1985). Serovars Hunter A and Hunter B were also, for a short period, found only in Great Britain (HUNTER, 1970), and in 1985 were isolated in Croatia by HAJSIG et al. (1985), although not in the current survey. None of investigated strains belonged to serovars S 3588 or A 636 / A 1743 (SIEVERDING, 1988). According to the literature there are no data regarding comparison between protein profiles of whole cells and concentrated supernatants of S. hyicus strains of the same or different serovars. Our results showed that most of the strains belonged to the same serovar, had a similar protein profile of whole bacterial cell, as well as a protein profile of sterile concentrated supernatants. The most significant differences were shown between five strains of S. hyicus, all serovar ATCC 19226, isolated from four animals. Those strains were different from all other strains, as well as from the referent strain of same serovar, in two protein bands. They possess a protein of approximate molecular mass of 50 kDa, while those of 45 kDa were absent. At the same time, they differ from strains of other serovars.

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Received: 27 October 1999
Accepted: 15 December 1999

SEOL, B.: Raspodjela i bjelancevinski profili bakterije Staphylococcus hyicus izdvojene iz svinja. Vet. arhiv 69, 319-326, 1999.

SAZETAK

Bakteriološki je pretrazeno 165 materijala što su potjecali s koze i razlicitih sluznica 33 zdravih krmaca. Bakterijska vrsta S. hyicus izdvojena je iz 23 krmace (69,7%). Ukupno su izdvojena 52 soja koja su bila serotipizirana postupkom brze aglutinacije na predmetnici. Najzastupljeniji serovar bio je ATCC 19226. Bakterija S. hyicus bila je najcešce prisutna na kozi mlijecne zlijezde, zatim kozi rila i uški te nosnoj sluznici, sluznici rodnice i ocnim spojnicama. Elektroforezom u poliakrilamidnom gelu cijelih bakterijskih stanica ustanovljena je velika ujednacenost bjelancevinskih profila obradenih izolata, a usporedbom bjelancevinskog izgleda pripravaka djelomice koncentriranih sterilnih supernatanata ustanovljeno je da pet sojeva, svi serovara ATCC 19226, pripada zasebnoj skupini. Bitna znacajka bjelancevinskog izgleda ovih sojeva bila je prisutnost bjelancevine priblizne molekularne mase 50 kDa kao i nedostatak bjelancevine molekularne mase 45 kDa.

Kljucne rijeci: Staphylococcus hyicus, zdrave krmace, distribucija, proteinski profil, bjelancevinski profil