INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1976, p. 545-553 Vol. 26, No. 4 Copyright 0 1976 International Association of Microbiological Societies Printed in U.S.A. Streptococcus iniae sp. nov. , a Beta-Hemolytic Streptococcus Isolated from an Amazon Freshwater Dolphin, Inia geoffrens is GERALD B. PIER’ AND STEWART H. MADIN Department of Biomedical and Environmental Health Sciences, School of Public Health, University of California Berkeley, California 94720; and Steinhart Aquarium, California Academy of Science, Golden Gate Park, San Francisco, California 94122 Evidence is presented for the recognition of a new species of Streptococcus isolated from abscess foci in an Amazon freshwater dolphin, Inia geoffrensis . The organism appears to be immunologically distinct from members of the recognized Lancefield groups of streptococci. Antigens prepared by five different extraction procedures do not react with antisera to Streptococcus groups A to U, whereas antisera prepared against the new isolate react well with the extracted homologous antigens but not with antigens from groups A to U. Based on cultural, morphological, biochemical, and serological results, it is suggested that this isolate belongs to a new species for which we propose the name Streptococcus iniae. The type strain of this new species is strain PW (=ATCC 29178). The basis for the serological grouping of beta- When excised, these foci were shown to con- hemolytic streptococci, as originally defined by tain 2 to 5 ml of purulent exudate which, when Lancefield (111, rests on the demonstration of a cultured, contained few or no organisms. How- distinct immunological specificity of an antise- ever, when cultures were obtained by swabs rum to the group-specific “C” polysaccharide from the interior surface of the abscess wall and usually located in the cell wall (10). Currently streaked onto blood agar, a pure culture of a there are 19 recognized serogroups, each pre- beta-hemolytic streptococcus grew in abun- sumably having a different chemical composi- dance. tion or structure of the “C” polysaccharide (19). This original culture was sensitive to penicil- The serogroups often indicate the host specific- lin, and treatment was instituted using a mix- ity of the organism; for example, group A strep- ture of long-acting penicillin and tylocin. After tococci are primarily associated with human intensive treatment for 10 days, the animal disease, group B streptococci are mostly associ- made an uneventful recovery. ated with bovine mastitis (12), and group E The disease, known as golf ball disease be- streptococci are mostly associated with swine cause of the resemblance of the foci to golf balls, infections (1, 20). This paper describes a beta- is believed by aquarists to be fatal for this hemolytic streptococcus isolated from an Ama- particular species, as was the case with two zon freshwater dolphin, Inia geoffrensis, suffer- specimens of I. geoffrensis that had contracted ing from an acute infection termed “golf ball the disease at The Steinhart Aquarium in San disease.” Francisco, Calif. A brief review of the disease In March 1972, on&of us (S.H.M.) was asked and its pathology will be reported elsewhere. to examine a freshwater dolphin reportedly suf- In studying the streptococcus isolated from fering from golf ball disease. On inspection, the the skin lesion, we were unable to obtain a animal, a mature adult male, was found to grouping reaction with any serum against the have numerous subcutaneous abscess foci on recognized serogroups utilizing antigen pre- both sides of the thoracic and abdominal cavi- pared by the Lancefield hot HCl method (ll), ties. These abscess foci were discrete, rounded the Fuller formamide method (51, the Rantz masses, 2 to 5 cm in diameter, which were and Randall hot autoclave method (171, the raised approximately 1 to 2 cm above the skin Streptomyces albus lytic enzyme method (161, surface and which fluctuated upon palpation. or extraction with Pronase (4).The chance that There were approximately 25 such foci on the the dolphin had contracted the disease from one right side of the animal and about 15 on the left. of its handlers or from another aquarium ani- Present address: Department of Bacterial Diseases, mal was considered, so we examined the possi- Walter Reed Army Institute of Research, Washington, D.C. bility of its being a member or a variant of 20012. either group A or group C. However, the isolate 545 546 PIER AND MADIN INT.J. SYST.BACTERIOL. was resistant to the action of a group C bacte- parison of the color produced in the inoculated cul- riophage and its phage-associated murilysin, ture with that of an uninoculated control tube simi- which lyses group A streptococci (19). Our sero- larly incubated. Catalase production was deter- mined on BHIA plates. logical and biochemical findings were con- Sodium hippurate hydrolysis, gelatin hydrolysis, firmed by R. R. Facklam, Center for Disease starch hydrolysis, growth in 6.5, 4.0, and 2.5% Control (CDC), Atlanta, Ga., and R. L. Wood, NaCl, growth in 10 and 40% bile, and growth in National Animal Disease Center (NADC), 0.04% tellurite, 0.1 % tetrazolium, and bile-esculin Ames, Iowa. Further studies also indicated that media were measured by adding the appropriate test the organism is a member of a previously un- substance to THB. Reaction in litmus milk was de- described species of Streptococcus. The follow- termined from stock media, and the methylene blue ing evidence is presented for regarding this milk reaction was determined by the addition of the isolate as belonging to a new species of Strepto- dye to skim milk. Esculin agar medium was pre- coccus. pared by the method of Shuman et al. (18). All reactions were observed for 5 days. Nitrate reduc- tion was first tested for by R. R. Facklam, CDC, MATERIALS AND METHODS Atlanta, Ga., and then confirmed by ourselves. Bacterial strains. In addition to the new orga- Antibiotic susceptibility test. The qualitative an- nism, the isolation of which was described above, tibiotic test was performed by the standardized three other streptococcal strains were employed for method of Kirby and Bauer (21) on Mueller-Hinton comparison of characteristics and for testing with plates (Baltimore Biological Laboratories [BBLI) the phage murilysin. Group A strain C203 and group containing 5 % defibrinated sheep blood. Susceptibil- C strain Azgazarhad were provided by J. T. Douglas ity disks (BBL) were placed on the agar by a dis- from our department. Another group C streptococ- penser, and plates were incubated overnight at cus, one isolated from a guinea pig suffering from 37°C. Zones of inhibition were then measured and chronic streptococcal lymphadenitis, was provided susceptibilities were determined. by A. Larson, also of our department. DNA base composition. Organisms grown over- Maintenance and growth of cultures. The dol- night were harvested for isolation of their DNA by phin skin lesion was lanced, and a swab from the the method of Marmur (13), with the following mod- interior wall of the lesion was streaked onto blood ifications: 2.5 g (wet weight) of cells was suspended agar base plates containing 5% sheep red blood cells in 5 ml of saline-ethylenediaminetetraacetic acid (Microbiological Associates) for colony differentia- (EDTA) and added to 5 g of fine glass beads (Minne- tion and hemolysis testing. Single colonies of a pure sota Mining and Manufacturing). The mixture was culture of a beta-hemolytic streptococcus were then shaken in a Mickle cell disintegrator for 2 h at 4°C. transferred to Todd-Hewitt broth (THB) and incu- The entire mixture was diluted to 25 ml with saline- bated overnight at 37°C. For stock cultures, brain EDTA, 2 ml of sodium dodecyl sulfate was added, heart infusion agar (BHIA) slants were inoculated and incubation was carried out for 10 min at 60°C. and similarly incubated. New subcultures were The procedure then followed the Marmur method, made every 2 months. The other three strains were with the glass beads being removed in the first maintained on BHIA slants in a similar manner. centrifugation. For the preparation of material for vaccines, anti- The melting temperature (T,) of the DNA was gens, deoxyribonucleic acid (DNA) isolation, muri- determined by the method of Marmur and Doty (141, lysin testing, and staining, the organisms were and the moles percent guanine plus cytosine (mol% grown overnight in THB at 37°C. G+C) was calculated by the method of DeLey (2). Morphological studies. Blood agar plates (BAP) Serological study. Antigens for testing the group- were used to study colonial morphology and cellular specific polysaccharide were prepared by the follow- appearance on solid media. Todd-Hewitt broth was ing methods: (i) Lancefield hot HCl (ll), (ii) Fuller used to study growth in broth and cellular appear- hot formamide (51, (iii) Rantz and Randall hot auto- ance in liquid media. Incubation of the above was at clave (171, (iv)Streptomyces albus enzyme (16)using 37°C overnight. commercially prepared Lytase (BBL), and (v) Pro- Physiological studies. The inoculum for physio- nase B (Calbiochem)extraction at 50°C for 2 h (4). logical studies was prepared by picking single colo- Antiserum was produced in New Zealand White nies from a blood agar plate into 3 ml of THB, rabbits, weighing about 2 kg each, by repeated incubating for 3 h until there was slight visible intravenous (i.v.) injections of either a formalin- turbidity, and then pipetting 0.1 ml into the test killed preparation of the organism or a saline sus- media. pension of an overnight culture of the live organism. Acid production from carbohydrates was deter- Both preparations were adjusted to approximately mined in phenol red broth base, supplemented with lo7 colony-forming units (CFU) per ml with the aid 1%(wt/vol) of the appropriate sugar.