Vol. 1, No. 1 Printed in U.S.A.


Copyright 0 1975 American Society for Microbiology

Identification of Actinomyces viscosus from Canine Infections' A. A. DAVENPORT, G. R. CARTER, AND M. J. PATTERSON Department of Microbiology and Public Health, and College of Veterinary Medicine, Michigan State University, East Lansing, Michigan 48824

Received for publication 12 August 1974

Actinomyces viscosus is a gram-positive, non-acid-fast, facultative, catalasepositive, filamentous, or diphtheroidal microorganism. It was isolated from six canine infections during a period of 1.5 years. The organism was cultured from exudate and flaky granules aspirated from infectious granulomas and empyemas. All cultures grew well aerobically and anaerobically with the addition of 10% carbon dioxide. They fermented lactose, produced catalase and acetylmethylcarbinol, reduced nitrates. hydrolyzed aesculin, and did not produce gelatinase or urease. These physiological characteristics distinguish A. viscosus from other morphologically similar organisms. differentiation of this species from other filamentous or diphtheroidal microorganisms. The purpose of this study was to delineate a minimal number of physiological tests considered necessary for definitive identification.

Howell (11) and Howell and Jordan (12) isolated a gram-positive filamentous "hamster organism" from periodontal lesions in hamsters. The "hamster strain" was later named Odontomyces viscosus (13). It was excluded from the genera Nocardia and Actinomyces because it produced catalase and was non-acid-fast. Gruter in 1932 (18) and Brion in 1939 (2) reported the isolation of similar filamentous organisms from human gingiva and canine lesions. Unfortunately, physiological studies on these strains were too incomplete to be definitive and the strains were not preserved for future examination. The Subgroup on Taxonomy of the Microaerophilic Actinomycetes (6) recommended that the genus Actinomyces include catalase-positive organisms, thus renaming the "hamster strain" Actinomyces viscosus. A. viscosus and other Actinomycetes will not grow on Sabouraud agar (Difco) but Nocardia species grow well (7). A. viscosus is a member of human and hamster periodontal plaque flora (7). Recently, human and canine infections due to this organism have been reported (1, 5, 16). The chronic, suppurative granulatomous lesions resembled those seen in actinomycosis and nocardiosis. Six canine infections with A. viscosus were seen during the last 18 months at the Michigan State University Veterinary Clinic. They were: two empyemas, one flank abscess, one temporal abscess, and two cervical abscesses. A. viscosus was isolated and considered the etiologic agent in each of these cases. The variability of colonial and cellular morphology of A. viscosus makes for difficulty in the

MATERIALS AND METHODS Organisms. Cultures of A. viscosus strains 295, 470, and 99 were obtained from H. A. McAllister who isolated them from canine clinical specimens. Strain 1097 was cultured from granules obtained from lung tissue, strain 1078 was grown from thoracic fluid from the same animal, and strain 194 was cultured from exudate aspirated from a cervical granuloma. Granules were washed in saline and crushed before culturing. All strains were initially plated on Trypticase soy agar (BBL) with 5% bovine blood. Isolated colonies were then transferred to other media for further examination. Morphology. Direct smears of granules and exudate were stained by the Gram-stain method and by the modified Kinyoun acid-fast method (4). All strains were transferred from the Trypticase blood agar to 5% bovine blood in brain heart infusion (BHI) agar (Difco), BHI agar, and BHI broth. These media were supplemented with vitamin K hemin solution and yeast extract (9). The strains were incubated with 10% carbon dioxide under aerobic and anaerobic conditions. Anaerobically prepared agar and broth media were made on an Anaerobic Culture Media Apparatus (Bellco Inc.) using the procedures described by Holdeman and Moore (9). Both 15-ml test tubes with 5 ml of broth and 100-ml flat-bottomed prescription bottles with 15 ml of agar media were prepared under an atmosphere of 90% nitrogen and 10% carbon dioxide. They were sealed with rubber stoppers and inoculation of media was done with this same apparatus. Aerobically prepared media had the same composition as the anaerobically prepared media except that resazurin and cysteine hydrochloride were omitted. These media were made without

I Michigan Agricultural Experiment Station journal article no. 6869.




the use of' an Anaerobic Culture Media Apparatus and were stoppered with Dispo plugs (Scientific Products). Cultures on aerobically prepared medium were incubated in Torbal jars which were flushed with carbon dioxide and air. All cultures were incubated at 37 C and were examined after 24 and 48 h, and 7 days. Gram stains were done on cultures from 48-h broth and supplemented BHI blood agar. and Kinyoun acid-fast stains were made from 4-day aerobic cultures and supplemented BHI blood agar. Biochemical tests. Catalase production determined by the H202 slide test (9) was examined with colonies taken from 4-day cultures on supplemented BHI blood agar. Other biochemical test media and reagents were prepared as recommended by Holdeman and Moore (9). A basal medium of peptone yeast extract (Difco) with the addition of' aesculin, lactose, gelatin, or glucose was prepared. Urea and nitrate media were made without this basal medium (9). Inoculum for all tests were made from 2-day cultures grown aerobically with CO2 on supplemented BHI blood agar. A heavy suspension of' the organism was prepared by suspending colonies in physiological saline. Four drops were added to each tube of medium with a Pasteur pipette. All inoculated test media were incubated in a candle jar for 10 days at 37 C. Test reactions were read according to the directions of Holdeman and Moore (9).


tening, mucoid, or soft colony composed of diphtheroidal forms occasionally forming Y, X, and V forms (Fig. 1). This form produced ropy growth in broth media. The other form showed rough, irregular, heaped, granular, slightly dry colonies on agar and demonstrated flocculent growth in broth. Gram stains of this type showed branching, pleomorphic filaments (Fig. 2). Both colonial types were capable of producing knobs or clubbed swellings. Some strains reversibly converted from one colonial type to another during successive subcultures and in-

RESULTS Morphology. All strains were gram positive, filamentous, or diphtheroidal and were nonacid-fast. Little or no preference for the addition of 5% bovine blood to supplemented BHI agar was evident but growth on these media was heavier than that on Trypticase blood agar. Cultures grew equally well under aerobic and anaerobic conditions when CO2 was added. Two FIG. 1. Diphtheroidal form of A. viscosus associdistinct morphological types were noted on media. One was a smooth entire, convex, glis- ated with smooth colonies (x500). ;.7.


FIG. 2. Filamentous form of A. viscosus associated with rough colonies (x500).

VOL. 1, 1975



diphtheroidal form in lesions, it is important that the microbiologist attempt to identify diphtheroids recovered from chronic granulomatous lesions and suppurative processes. The rough colonial form and related cellular morphology of A. viscosus suggest that it belongs in the family Actinomycetaceae. There is considerable information (6, 7) on the separation of A. viscosus from other members of this family. The smooth form of A. viscosus has been isolated from purulent material (1). Cultures with this colonial type and the biochemical reactions of A. viscosus resemble rather closely some species of Corynebacterium. Some nonhemolytic aerobic Corynebacterium species differ from A. viscosus only by the inability to hydrolyze aesculin when the scheme of identification suggested in Table 1 is used. The rela-

termediate forms were also seen (Fig. 1-3). Biochemicals. All strains of A. viscosus were catalase positive, reduced nitrates, were urease and gelatinase negative, hydrolyzed aesculin, did not produce acetylmethylcarbinol, and fermented lactose. These reactions were adequate to differentiate A. viscosus from other microorganisms shown in Table 1. DISCUSSION A. viscosus is a slow-growing fastidious microorganism which has been found recently in human and canine infections. The organism can not be separated from Nocardia or other filamentous or diphtheroidal organisms unless a minimal number of biochemical tests such as those recommended in this report are performed. Because A. viscosus may occur in the

FIG. 3. Reversion from rough to smooth colonial morphology (x2). TABLE 1. Expected reactions of various gram-positive filamentous or diphtheroidal microorganismsa Organisms

Acid-fast Catalase





Bacterionema Arachnia Corynebacterium Erysipelothrix Lactobacillus Rothia Actinomyces (not A. viscosus) A. viscosus





- (W) -



+ _ + _ +(-) _ _ + _ +

Urease V _ _ _ V _ _ _ _

Tests" Nitrate Gelatinase reduction

V _ _ V _ _ _ _

_ _ + + V _ V ND V +(-)

Lactose Aesculin Vogesferment hydrolysis Proskauer


+(-) _ + + _ + +

ND ND + _

V +

-(D) ND ND + + +



See references 3, 8, 9, 10, 14, 15, 17. ND, Not determined; +, positive reaction; -, negative reaction; W, weak; D, delayed; V, varies; ( ),

reaction of occasional strain.



tionship of this genus to A. viscosus has not been generally emphasized. Isolation of pure cultures of diphtheroids and filamentous microorganisms, or multiple isolation of these from inflammatory lesions, indicates the necessity for proper speciation. Because antibiotic therapy of infections caused by these species varies. proper identification is of paramount importance. ACKNOWLEDGMENTS We are indebted to the staff of the Department of Small Animal Surgery and Medicine, Michigan State University. to E. S. Beneke for aid in planning the study, and to H. A. McAllister for supplying the strains of A. Liscosus. LITERATURE CITED 1. Adeniyi-Jones, C.. J. A. Minielly, and W. R. Matthews. 1973. Actinomyces uiscosus in a branchial cyst. Amer. J. Clin. Pathol. 60:711-713. 2. Brion, A. 1939. L'Actinomycose du chien et du chat. Rev.

3. 4.




Med. Vet. 91:121-159. Carter, G. R. 1973. In Diagnostic procedures in veterinary microbiology, 2nd ed., p. 109-114, 147-156. C. C Thomas, Publisher, Springfield. Georg, L. K., L. Ajello, C. McDurmont, and T. S. Hosty. 1961. The identification of Nocardia asteroides and Nocardia brasiliensis. Amer. Rev. Resp. Dis. 84:337-347. Georg, L. K., J. M. Brown, H. J. Baker, and G. H. Cassell. 1972. Actinomyces viscosus as an agent of actinomycosis in the dog. Amer. J. Vet. Res. 33:1457-1470. Georg, L. K., L. Pine, and M. A. Gerencser. 1969. Actinomyces viscosus, comb. nov., a catalase positive, facultative member of the genus Actinomyces. Int. J. Syst. Bacteriol. 19:291-293. Gerencser, M. A., and J. M. Slack. 1969. Identification of








15. 16. 17.



human strains of Actinomyces Lliscosus. Appl. Microbiol. 18:80-87. Gledhill, W. E., and L. E. Casida. 1969. Predominant catalase-negative soil bacteria. II. Occurrence and characterization of Actinom, ces humiferus, sp. N. Appl. Microbiol. 18:114-121. Holdeman, L. V., and W. E. C. Moore (ed.). 1972. Anaerobe laboratory manual. Virginia Polytechnic Institute and State University Anaerobe Laboratory. Blacksburg. Holmberg. K., and H. 0. Hollander. 1972. Numerical taxonomy and laboratory identification of Bacterionema matruchotti, Rothia dentocariosa, Actinom,ces naeslundii, and some related bacteria. J. Gen. Microbiol. 76:4:3-6:3. Howell, A., Jr. 1963. A filamentous microorganism isolated from periodontal plaque in hamsters. I. Isolation, morphology, and general cultural characteristics. Sabouraudia 3:81-92. Howell, A., Jr., and H. V. Jordan. 1963. A filamentous microorganism isolated from periodontal plaque in hamsters. II. Physiological and biochemical characteristics. Sabouraudia 3:93-103. Howell, A., Jr.. H. V. Jordan, L. K. George, and L. Pine. 1965. Odontomyxces viscosus gen. nov., spec. nov., a filamentous microorganism isolated from periodontal plaque in hamsters. Sabouraudia 4:65-68. Johnson, W. D., and D. Kaye. 1970. Serious infections caused by diptheroids. Ann. N.Y. Acad. Sci. 174(2):568-576. Laskin, A. I. and H. A. Lechavalier (ed.). 1973. In Handbook of microbiology. p. 199-220. CRC Press. Cleveland. Lewis, R., and S. L. Gorbach. 1972. Actinom, ces Ciscosus in man. Lancet 1:641-642. McAllister, H. A. 1970. Procedures for the identification of microorganisms from higher animals, p. 36. Lucas Brothers Publishers, Los Angeles. Negroni. P. 1938. Estudio micologica del ActinomYces discotuliatus. Gruter. 1932, senaldo par primera rez en la Argintena. Mvcopathologica 1:81-87.

Identification of Actinomyces viscosus from canine infections.

Actinomyces viscosus is a gram-positive, non-acid-fact, facultative, catalase-positive, filamentous, or diphtheroidal microorganism. It was isolated f...
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