—NOTE— Microplate Sugar-Fermentation Assay Distinguishes Streptococcus equi from Other Streptococci of Lancefield’s Group C Yasushi KUWAMOTO, Toru ANZAI* and Ryuichi WADA Epizootic Research Station, Equine Research Institute, Japan Racing Association, 1400–4 Shiba Kokubunji- machi, Shimotuga-gun, Tochigi 329-0412, Japan Lancefield’s group C streptococci often contaminate test samples taken from open lesions of J. Equine Sci. the equine disease strangles, and it is difficult to distinguish the causal agent, Streptococcus Vol. 12, No. 2 equi (S. equi), from these other streptococci on the isolation plate. Here we have developed pp. 47–49, 2001 a microplate sugar-fermentation assay that distinguishes S. equi from other streptococci of Lancefield’s group C. Within 18 hr of incubation, the assay distinguished between 19 strains of S. equi, 171 strains of Streptococcus zooepidemicus and 19 strains of Streptococcus equisimilis, which were isolated from clinical horse samples and identified by API 20 STREP and Western immunoblotting. Moreover, this microplate assay can simultaneously test up to 24 samples, and is therefore valuable for the diagnosis of strangles. Key words: Strangles, Streptococcus equi, sugar-fermentation assay Strangles is a highly contagious equine disease that trehalose. Lancefield’s group C streptococci that fail to causes pyrexia, nasal discharge and secretion of pus ferment any of these sugars are usually identified as S. from the lymph nodes [4]. Recently, the number of equi, those that ferment lactose and sorbitol are outbreaks of strangles increased in Hidaka district, identified as S. zooepidemicus, and those ferment which is the main racehorse-breeding area of Japan, trehalose are identified as S. equisimilis [2]. However, and therefore better control of the disease has been an atypical S. equi strain that ferments sugar has been demanded [1]. Strangles is caused by Streptococcus equi isolated from suspected cases of strangles [5]. subsp. equi (S. equi), belongs to β-hemolytic and The aim of this study was to clarify the sugar- Lancefield’s group C streptococcus, and is diagnosed fermentation characteristics of β-hemolytic following the isolation and identification of S. equi from Lancefield’s group C streptococci isolated recently abscesses and mucopurulent nasal discharge. from horses in Japan, and to establish a reliable and β-Hemolytic and Lancefield’s group C streptococci quick diagnostic method of strangles using microplate include two other bacteria, Streptococcus equi subsp. fermentation assays. zooepidemicus (S. zooepidemicus) and Streptococcus We used 209 strains of β-hemolytic streptococci dysgalactiae subsp. equisimilis (S. equisimilis). These two isolated from various clinical horse samples for this bacteria are not only isolated from various clinical study. The strains were identified using STREPT LA samples from horse, but are also detected together with (Denka Seiken, Tokyo) for Lancefield serological S. equi in strangles lesions as a secondary opportunistic grouping, and API 20 STREP (bioMerieux-Vitek Japan, pathogen; in addition, it is difficult to distinguish S. equi Tokyo) for biochemical and immunological tests. from S. zooepidemicus and S. equisimilis by analyzing only Western immunoblot analysis of M-like proteins from their colony formation. Identification of these three streptococci was performed by the method of Galan bacteria is traditionally performed on the basis of their and Timoney [3]. A microplate sugar-fermentation respective fermentation of lactose, sorbitol and assay was carried out using 96-well flat-bottom tissue- culture plates (Nalgen Nunc International, USA) and µ This article was accepted June 28, 2001. 150 l fermentation broth consisting of purple broth *Corresponding author. e-mail: [email protected] base (Difco, USA), 10% inactivated horse serum, and 48 Y. KUWAMOTO, T. ANZAI, R. WADA 1% of either trehalose, sorbitol, ribose, glucose, or H2O as a negative control. Fermentation broth was dispensed into each well and the microplates were stored at –20°C before use. The microplate sugar fermentation assay was carried out as follows. Each strain was cultured purely in Todd Hewitt broth (THB) (Difco) supplemented with 0.2% yeast extract and 10% inactivated horse serum. A drop of this culture medium was added to each sugar reaction well and incubated aerobically at 37°C. The Lancefield serological grouping test typed all isolates of β-hemolytic streptococci from horses as group C. Subsequently, 19 strains were identified as S. equi, 169 strains as S. zooepidemicus and 19 strains as S. equisimilis; two strains were not identified by API 20 STREP. These 209 isolates of β-hemolytic streptococci were also analyzed for M-like protein of S. equi by Western immunoblotting. A strong reaction between native M- like proteins and the rabbit antiserum was observed in extracts from the 19 isolates identified as S. equi by API 20 STREP (Fig. 1A). By contrast, in extracts from the 169 isolates identified as S. zooepidemicus and the 2 isolates unidentified by API 20 STREP, a strong reaction was observed between the rabbit antiserum and proteins with different molecular masses to that of the M-like protein of S. equi (Fig. 1B). In extracts from the 19 isolates identified as S. equisimilis, no proteins reacted strongly to the rabbit antiserum in Western immunoblots (Fig. 1C). Thus, these biochemical and immunological tests demonstrated that of the 209 isolates, 19 were S. equi, 171 were S. zooepidemicus and 19 were S. equisimilis. For the sugar-fermentation microplate assay, results were accepted as accurate after confirmation of a positive reaction in the positive control well and a negative reaction in the negative control well. The assay showed that of the 19 strains of S. equi, none fermented lactose, trehalose, sorbitol or Fig. 1. Western immunoblot analysis of native M-like protein. ribose (Table 1). Of the 171 strains of S. zooepidemicus, The blot was developed with anti-Streptococcus equi CF32 134 fermented lactose, sorbitol and ribose, but not rabbit serum. A: 19 strains of Streptococcus equi identified trehalose; 36 strains fermented lactose and sorbitol, but by API 20 STREP. Nine strains of Streptococcus equi are shown out of nineteen strains that gave the same result. not trehalose or ribose; and 1 strain fermented lactose B: 169 strains of Streptococcus zooepidemicus and two and ribose, but not trehalose or sorbitol. All of the 19 streptococci unidentified by API 20 STREP. Nine out of strains of S. equisimilis fermented trehalose and ribose, the 169 strains of Streptococcus zooepidemicus and the two but not lactose or sorbitol. The results of the reaction unidentified streptococci are shown. C: Western could be read within 18 hours of incubation. immunoblot analysis of native M-like protein from 19 strains of Streptococcus equisimilis identified by API 20 None of the S. equi strains identified in this study STREP. Ten out of the 19 strains of Streptococcus equisimilis fermented lactose, trehalose, sorbitol or ribose. On the are shown. M, molecular marker. one hand, these fermentation characteristics are the same as those described previously for a typical strain of S. equi [4]. On the other hand, atypical isolates of S. MICROPLATE SUGAR-FERMENTATION ASSAY FOR STREPTOCOCCUS EQUI 49 Table 1. Reaction of fermentation with 209 strains of Lancefield’s group C β- hemolytic streptococci isolated from clinical materials of horses Sugar Bacteria No. of strains Lactose Trehalose Sorbitol Ribose S. equi 19 –––– S. zooepidemicus 134 + – ++ S. zooepidemicus 36* + – + – S. zooepidemicus 1** + ––+ S. equisimilis 19 – + – + +: positive, –: negative. *Including 1 isolate unable to identify by API 20 STREP (not identified-2 in Fig. 1B). **Unable to identify by API 20 STREP (not identified- 1 in Fig. 1B). equi that ferment lactose and trehalose have been stored in the freezer and can test up to 24 samples at reported by Grant et al. [5]. These researchers the same time, it should clearly contribute to the therefore recommended that ribose should be used to diagnosis and prevention of strangles. identify S. equi, because all the S. zooepidemicus isolates in their study fermented ribose. We found here, however, that 36 of the 171 identified strains of S. zooepidemicus References did not ferment ribose. Although the reasons why the two studies gave different results are not clear, one 1. Anzai, T., Nakanishi, A., Wada, R., Higuchi, T., explanation might be that neither the S. equi strains Hagiwara, S., Takazawa, M., Oobayashi, K., and isolated by us nor the S. zooepidemicus strains described Inoue, T. 1997. Isolation of Streptococcus equi subsp. by Grant et al. involved atypical strains of either equi from Thoroughbred Horses in a Racehorse- streptococcus. This would then suggest that there are a Breeding Area of Japan. J. Vet. Med. Sci. 59: 1031– few number of atypical strains of Lancefield’s group C 1033. streptococci that cannot be identified by only a sugar- 2. Bazeley, P.L. and Battle, J. 1940. Studies with fermentation test, and that immunological or genetical equine streptococci. I. A survey of beta-haemolytic tests are needed to identify atypical strains. streptococci in equine infections. Aust. Vet. J. 16: In summary, these results suggest that the microplate 140–146. 3. Galan, J.E. and Timoney, J.F. 1988. Immunologic sugar-fermentation assay is useful for distinguishing S. and Genetic Comparison of Streptococcus equi equi from S. zooepidemicus and S. equisimilis, both of Isolation from the United States and Europe. J. which can cause opportunistic infectious disease with Clin. Microbiol. 26: 1142–1146. clinical symptoms that resemble those of strangles. In 4. Timoney, J.F. 1993. Strangles. Vet. Clin. Am: Equine addition, these bacteria may be isolated from open Pract. 9: 365–374. strangles lesions as secondary invaders. In diagnosing 5. Grant, S.T., Efstratiou, A., and Chanter, N. 1993. for strangles, therefore, it is important to analyze many Laboratory diagnosis of strangles and the isolation β isolated colonies of suspected -hemolytic streptococci of atypical Streptococcus equi. Vet. Rec. 133: 215–216.