Zbl. Bakt. Hyg. A 261, 407-410 (1986)

Coagulase Typing of Staphylococcus aureus Isolated fromAnimals

JUNICHI KAWANO, AKIRA SHIMIZU, and SHIGE KIMURA

Received September 7, 1985 . Accepted January 22, 1986

Abstract

Coagulase typing was performed on Staphylococcus aureus isolated from various ani­mals. Of the 383 strains examined, 209 (54.6%) strains were typable and could be differen­tiated into 8 coagulase types. The remaining 174 strains were non-typable with antiseraagainst the 8 known types of coagulases. This suggests that many strains of S. aureus ofanimal origin may possess unknown types of coagulases.

Zusammenfassung

Eine Koagulase-Typisierungwurde an Staphylococcus aureus-Isolaten aus verschiedenenTierarten durchgefiihrt. Von den 383 untersuchten Stammen waren 209 (54,6%) typisier­bar und konnten nach 8 Koagulasetypen differenziert werden. Die iibrigen 174 Starnmelief~en sich nicht mit Hilfe von Antiseren gegen die 8 bekannten Koagulasetype typisieren.Die deutet darauf hin, dag viele S. aureus-Stamme tierischen Ursprungs unbekannte Koa­gulasetype aufweisen diirften.

Introduction

Coagulase typing has been developed for classifying S. aureus isolated from clinicalspecimens of human diseases, such as food poisoning, staphylococcal scalded-skinsyndrome, abscesses, and impetigo contagiosa (8, 9, 13, 14). This method is based onthe 8 antigenic types (types I-VIII) of coagulases. Strains of S. aureus isolated fromhuman sources have all been typed into anyone of these coagulase types. However,there is little information on the antigenic properties of S. aureus coagulases isolatedfrom animals. This paper deals with the coagulase typing of S. aureus isolated fromvarious animals to verify the distribution of coagulase types and to determine whetherunknown types of coagulase are present in S. aureus of animal origin.

28 Zbl. Bakt. Hyg. A 261/4

408 ]. Kawano, A.Shimizu, and S.Kimura

Materials and Methods

Bacterial strains: A total of 383 strains of S. aureus were used (Table 1): human strainswere isolated from food poisoning incidents; bovine strains were isolated from the milk ofcows with mastitis; chicken strains were isolated from chickens with dermatitis, septicemia,arthritis or healthy animals; the other strains were isolated from the skins or nares ofapparently healthy animals. All strains produced yellow pigments, hemolysins, acetyl­methylcarbinol, and fermented mannitol.

Coagulase typing: Coagulase typing was performed according to Ushioda et al. (9) withthe exception of the preparation of coagulase antigen. Coagulase antigen was preparedaccording to the original method described by Zen-Yoii et al. (12) with some modification.Briefly, a test strain was cultured in 2 ml of heart infusion broth in a 12 mm x 105 mm testtube for 3-5 days at 37°C. The culture broth was then centrifuged for 10 min at 1,000 x g.The supernatant was used as the coagulase antigen. Preliminary tests were made to detectthe suitable conditions for coagulase production. Incubation in different media, such asbrain heart infusion broth and yeast extract broth containing casamino acid (11), in conicalflasks, and with shaking were not remarkably effective. Production of coagulase dependedmainly on the duration of cultivation. Some strains did not produce sufficient coagulase toclot the rabbit plasma within 1 or 2 days of incubation. A most satisfactory result wasobtained under the conditions described above. Coagulase-type-specificrabbit antisera werepurchased from Denka Seiken Co. Ltd.,Tokyo. All of the antisera were examined for theirreactions to the reference strains of coagulase types (9). The reference strains were kindlysupplied by Dr. T. Terayama, of the Tokyo Metropolitan Research Laboratory of PublicHealth.

Results

A total of 383 strains of S. aureus isolated from animals were subjected to coagulasetyping (Table 1). Of the 383 strains, 209 (54.6%) were typed and differentiated into 8

Table 1. Coagulase typing of S. aureus isolated from animals

No. of No . ofOrigin strains typable Coagulase type

tested strains ("10 ) II III IV V VI VII VIII NT'

Human 15 15 (100) 0 7 3 2 2 0 1 0 0Cattle 157 58 (36.9) 0 7 6 1 8 25 11 0 99Swine 18 15 (83.3) 0 7 4 1 0 1 0 2 3Horse 8 2 (25.0) 1 0 1 0 0 0 0 0 6Dog 2 1 (50.0) 0 0 1 0 0 0 0 0 1Rat :

Rattus nor-uegicus 61 41 (67.2) 4 3 9 9 0 0 12 4 20Rattus rattus 19 13(68.4) 1 6 0 0 0 0 4 2 6

Mouse 44 21 (47.7) 1 0 0 2 0 4 8 6 23Rabbit 1 1 (100) 1 0 0 0 0 0 0 0 0Ferret 3 2 (66.7) 0 2 0 0 0 0 0 0 1Chicken 44 33 (75.0) 0 13 0 0 4 1 14 1 11Pigeon 10 6 (60.0) 0 0 6 0 0 0 0 0 4Quail 1 1 (100) 1 0 0 0 0 0 0 0 0

Total 383 209 (54.6) 9 45 30 15 14 31 50 15 174

• Non-rypable.

CoagulaseTyping of Staphylococcus aureus 409

coagulase types. The remammg 174 (45.4%) strains were non-typable with the 8typing antisera. All of 15 human, 1 rabbit and 1 quail strains were typable. Thetypabilities of the other animal strains ranged from 25.0% to 83.3%. The incidences ofcoagulase types VII and II appeared to be higher than the other types among the animalstrains. However, coagulase type VI was predominant in the bovine strains.

Discussion

The immunological properties of S. aureus coagulase have been studied for manyyears (1, 2, 3, 5, 11). Upon the basis of antigenic differences among staphylococcalcoagulases, Zen- Yoji et al. (10, 12, 13) established coagulase typing. Application ofcoagulase typing appeared to be limited to human staphylococcal diseases, and has notbeen examined in S. aureus isolated from animals, with the exception of chicken andbovine strains. Sato et al. (6) reported that 194 (85.8%) of 226 chicken strains ex­amined were coagulase typable. Takeshige et al. (7) typed 86 (98.8%) of 87 bovinestrains examined. In the present study, S. aureus strains isolated from various animalswere subjected to coagulase typing. A number of non-typable strains were presentamong the animal strains, in contrast to the human strains which were all typable. Thissuggests that some coagulase types different from the known types may be present in S.aureus of animal origin.

In the present study, coagulase type VI was predominant among the bovine strainswhich were isolated from the milk of cows with mastitis. Takeshige et al. (7) alsoshowed the same result with bovine strains isolated from mastitic udders. Bovine S.aureus of coagulase type VI may be a common causative agent of mastitis in Japan. Onthe other hand, many (63.1%) of the bovine strains used in the present study remainnon-rypable, This fact probably means that the strains are "true bovine" types and notof human origin. Human strains could be introduced through contaminated milkingmachines, and so on. Therefore, it is necessary that the coagulase types of such non­typable strains should be determined for the study of the epidemiology of bovinemastitis.

With regard to chicken S. aureus, Sato et al. (6) found that coagulase types III and IVwere not detected among 194 typable strains. Neither were these two types found inthe chicken strains used in the present study. Coagulase types III and IV may thereforebe rare or completely absent in chicken S. aureus.

It has been reported that coagulase types II, III, VI and VII are predominant in S.aureus isolated from food poisoning incidents (8,9, 13, 14). Takeshige et al. (7) foundthat most of the enterotoxin-producing strains of bovine S. aureus were of thesecoagulase types, which were also found in many of the strains isolated from variousanimals in the present study. Indeed, enterotoxigenic S. aureus strains have been iso­lated from various animals (4), and it is therefore considered necessary to study therelation between coagulase types and enterotoxin production in these organisms.

Coagulase typing may be one of the most useful methods for subdividing S. aureus.However, this' method has not been employed outside Japan. Information on theantigenic variation of coagulase is inadequate internationally. It is anticipated thatcoagulase typing will be widely employed in epidemiological studies for tracing zoono­tic staphylococcal infections.

Acknowledgements. We wish to thank Dr. T. Terayama, of the Tokyo MetropolitanResearch Laboratory of Public Health, for the supply of the reference strains of coagulasetypes.

410 J. Kawano, A. Shimizu, and S.Kimura

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Dr. J. Kawano, Department of Animal Hygiene, Faculty of Agriculture, Kobe University,Kobe 657, Japan