Assay of protein A in Staphylococcus hyicus subsp. hyicus by ELISA and immunoelectron microscopy

The presence and quantity of protein A in Staphylococcus hyicus subsp. hyicus isolates were examined by an enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy. Cell-bound protein A was demonstrated in 45 (94%) of 48 isolates from diseased pigs and in 113 (86%) of 132 isolates from healthy pigs by ELISA using peroxidase-conjugated rabbit antibody, but was not found in isolates from chickens and cows. Most of these swine isolates contained about 100 to 300 ng of cell-bound protein A/ml. Extracellular protein A was not detected in any isolates from pigs, chickens or cows. In the immunoelectron microscopy assay, swine isolates were labeled with goat anti-mouse IgG conjugated to colloidal gold particles, but chicken and cow isolates were not labeled.     

Proteolytic zymograms of Staphylococcus hyicus subsp. hyicus isolated from pigs, chickens and cows

The extracellular proteases of Staphylococcus hyicus subsp. hyicus were assayed by a zymogram showing caseinolysis and gelatinolysis. Four bands were associated with caseinolysis or with gelatinolysis. The patterns shown by strains isolated from pigs, chickens and cows were compared; isolates from pigs differed from those isolated from chickens or cows but strains isolated from diseased and healthy pigs could not be differentiated.     

Bacteriophage typing of Staphylococcus hyicus subsp hyicus isolated from pigs

Four phages were isolated and used for typing Staphylococcus hyicus subsp hyicus isolated from pigs with or without exudative epidermatitis (EE) in Japan. Sixty-four (85.3%) of the 75 isolates examined were typeable at either routine test dilution (RTD) or 100 X RTD. Two or more kinds of phage patterns were present in the isolates from each pig with EE. All isolates from healthy pigs showed a single-phage pattern. Fourteen (32.6%) of 43 isolates and 7 (87.5%) of 8 isolates from pigs with EE in Belgium and Czechoslovakia, respectively, were typeable with the 4 phages. None of 180 isolates of S aureus, 7 (6.4%) of 110 isolates of S intermedius, and 2 (2.3%) of 86 isolates of S epidermidis were typeable.     

Susceptibility of various animals and cultured cells to exfoliative toxin produced by Staphylococcus hyicus subsp. hyicus

In piglets inoculated with partially purified exfoliative toxin (pp-shET) produced by Staphylococcus hyicus subsp. hyicus, exfoliation was observed at 12 h after injection. Chickens inoculated with the same dose of pp-shET also showed exfoliation within 30 min of injection. However, exfoliation was not demonstrated in mouse, rat, guinea pig, hamster, dog or cat inoculated with pp-shET until 24 h after injection. In cultured cell lines, especially L-929 and Hep-2, the rounding effect occurred after incubation with pp-shET for 1 h. The rounding effect was also seen in five other cultured cells (NCTC 2544, HeLa/S3, HmLu-1, CHO and BHK-21) 6-24 h after exposure to pp-shET. These round cells survived for 72 h after inoculation and formed a monolayer 24 h after changeover to a toxin-free medium. The rounding effect was observed in cells after the formation of the monolayer, but not before. It was suggested that the rounding effect was not caused by the increase in cyclic AMP in cells inoculated with pp-shET but by the cleavage of intracellular contacts.     

Isolation of exfoliative toxin from Staphylococcus hyicus subsp. hyicus and its exfoliative activity in the piglet

Exfoliative toxin was isolated from the sterile cell-free filtrate of 24 h culture of Staphylococcus hyicus subsp. hyicus strain P-1. The partial purification of exfoliative toxin produced by S. hyicus (shET) was performed by precipitation with 50-80% saturated ammonium sulfate, gel filtration on a Sephadex G-75 column and column chromatography on DEAE-cellulose. Partially purified shET (pp-shET) caused exfoliation in piglets at 8 to 12 h after intradermal or subcutaneous injection. However, heat-treated pp-shET did not cause exfoliation in piglets for up to 24 h after injection. On histopathological examination of the skin at 12 h after injection of pp-shET, an intraepidermal cleavage plane was shown between the stratum corneum and stratum granulosum and at the stratum granulosum.     

Staphylococcus hyicus in cattle

Staphylococcus hyicus (subsp hyicus) was found to be a frequently occurring inhabitant of the skin of cattle. Significant numbers of this bacterium were found to be present in mange lesions of cattle between one and three years old. Experimental inoculations revealed that S hyicus had a pathogenic effect on the superficially scarified skin of young cattle.     

Surface hydrophobicity of Staphylococcus intermedius and Staphylococcus hyicus.

Surface hydrophobicity of 90 Staphylococcus intermedius and 55 S hyicus isolates was evaluated using the hexadecane adherence assay and the ammonium sulphate salt aggregation test. A strongly positive hydrocarbon adherence in the hexadecane adherence assay was demonstrated in 11 per cent of the S intermedius isolates and 7 per cent of the S hyicus isolates. Bacterial aggregation in 1.6 M, or less, ammonium sulphate was observed in 28 per cent of the S intermedius isolates and 37 per cent of the S hyicus isolates. There was no statistical correlation between the two assays. The adherence of both bacterial species to hexadecane was eliminated when the cells were first treated with pronase and trypsin, while it was mildly enhanced by prior heat treatment (60 degrees C and 95 degrees C for up to three hours). In contrast, aggregation of S intermedius in ammonium sulphate was not influenced by trypsin pretreatment, and aggregation of both bacterial species was diminished, or eliminated, with pronase or prior 95 degrees C heat treatment. Surface hydrophobicity, as measured in both assays, appeared to have no relationship with growth patterns in serum soft agar or production of slime. Similarly, the presence or absence of substantial surface receptor activity to fibrinogen, fibronectin or IgG did not appear to be related to surface hydrophobicity.     

Cytotoxic activity of Staphylococcus hyicus

Culture supernatants from a number of Staphylococcus hyicus strains caused toxic effects to both murine fibroblast and porcine keratinocyte cells in culture. The extent of cytotoxicity was shown to differ between strains and may provide an indication of strain virulence. Purification of cytotoxic activity produced by S. hyicus (strain P119) using preparative isoelectric-focussing demonstrated it to be cytolytic, haemolytic and non-proteolytic. The cytotoxin demonstrates certain properties in common with the delta haemolysin of Staphylococcus aureus.     

Staphylococcus hyicus virulence in relation to exudative epidermitis in pigs.

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.     

Plasmid-encoded antibiotic resistance in Staphylococcus hyicus

A total of 33 Staphylococcus hyicus-cultures from piglets with exudative epidermatitis were analyzed for the presence of antibiotic resistance plasmids. Four small plasmids encoding resistances to chloramphenicol, macrolide-lincosamide-antibiotics, streptomycin or tetracyclines could be identified in plasmid-curing and plasmid-transformation experiments. For further characterization these plasmids were digested with restriction endonucleases. This led to the construction of a specific restriction map for each of the 4 plasmids. On the basis of their restriction maps, these 4 antibiotic resistance plasmids from "porcine" S. hyicus-cultures were compared with the respective resistance plasmids of other staphylococcal species from infections of humans and animals.     

Staphylococcus hyicus abortion in a sow

Five aborted fetuses from a mature sixth-parity sow with severe greasy pig disease as a gilt, were submitted for diagnostic evaluation. Necropsy of the fetuses revealed serogelatinous edema in the SC connective tissue of the ventral abdominal region (especially around the umbilicus), exaggerated amounts of serohemorrhagic fluid in the abdominal, pleural, and pericardial cavities, and hemorrhagic kidneys, with diminished consistency. Staphylococcus hyicus was isolated in pure culture from liver, lung, kidney, and brain tissues and from abdominal, pleural, pericardial, and gastric fluids.     

猪葡萄球菌脱落毒素研究进展

猪渗出性皮炎是一种由猪葡萄球菌引起的哺乳或断奶仔猪的急性传染病.猪葡萄球菌产生的脱落毒素被认为是引起猪渗出性皮炎的主要因子之一,不同毒株可以产生不同的毒素,分别被命名为ExhA、ExhB、ExhC和ExhD 4种.论文就近年来对猪葡萄球菌脱落毒素的结构特征、生物学功能以及在诊断和免疫学方面的研究进行综述.     

Staphylococcus hyicus, the cause of exudative epidermitis of swine. Review

Staphylococcus hyicus, the cause of porcine exudative epidermitis, could be clearly differentiated from S. aureus, S. epidermidis and from other staphylococcal species. This was based on cultural, biochemical and serological properties. A positive coagulase reaction in porcine plasma, the detection of protein A like IgG Fc-receptors and the specific reaction of the cell wall teichoic acid allowed further characterization of this species. S. hyicus additionally produced enzymes with bacteriolytic properties.     

Studies on the Virulence of Staphylococcus hyicus

Abstract— Culture supernatants from six Staphylococcus hyicus isolates were concentrated by ultrafiltration and were injected intradermally into the abdominal skin of 2-week-old piglets. Two distinct types of reaction were observed; (1) a focal erythema and (2) an exfoliative reaction with crusting. The severity of these reactions and the number of animals affected differed between isolates. The exfoliative reaction observed in these skin tests may be a good indicator of virulence. Résumé— Les surnageants de six souches de Staphylococcus hyicus ont été concentrés par ultrafiltration et furent injectés par voie intradermique dans la peau de l'abdomen de porcelets ägés de deux semaines. Deux types distincts de réactions furent observés: (1) un éryhème focal et (2) une réaction d'exfoliation avec apparition de croûtes. L'intensité de ces réactions et le nombre d'animaux atteints différaient selon les souches. La réaction d'exfoliation observée lors de ces tests pourrait être un bon indicateur de virulence. Zusammenfassung— Überstände der Kultur von sechs Staphylococcus hyicus-Isolaten wurden durch Ultra-zentrifugieren konzentriert und bei 2 Wochen alten Ferkeln intradermal in die Abdominalhaut injiziert. Es konnten zwei deutlich unterscheidbare Reaktionen beobachtet werden: (1) ein fokales Erythem und (2) eine exfoliative Reaktion mit Krustenbildung. Die Schwere dieser Reaktionen und die Zahl der betroffenen Tiere war je nach Isolat unterscheidlich. Die exfoliative Reaktion bei diesen Hauttests könnte ein guter Indikator für die Virulenz sein. Resumen Los sobrenadantes de 6 cultivos diferentes de Staphylococcus hyicus se concentraron mediante ultrafiltración y se inyectaron via intradérmica en la piel abdominal de lechones de dos semanas de edad. Se observaron dos tipos de reacción: (1) un eritem focal y (2) una reacción exfoliativa y costrosa. La gravedad de las reacciones y el nümero de animales afectados variaba en functión del cultivo utilizado. La reacción exfoliativa que se observó en estos tests intradérmicos puede ser un buen indicador de virulencia.