J Vet Diagn Invest 2:249-252 (1990)

REVIEW ARTICLE

An update on Streptococcus suis identification Robert Higgins, Marcelo Gottschalk sular type 2 isolate was recovered in pure culture from lungs and kidneys of a 4½-month-old aborted bovine fetus and its placenta. This suggests that S. suis may be pathogenic for more than 1 animal species. In Canada, it was reported that 94% of 4-8-weekold clinically healthy piglets harbored S. suis in their nasal cavities, and 79% of these isolates did not belong to the 9 capsular types.3 Recently, we demonstrated that almost 90% of these untypeable isolates belong to only 4 of the new capsular types (types 17, 18, 19, and 21, unpublished data). Other investigators have attributed the development of rhinitis4,7,22 to untypeable S. suis isolates. At this time, it is not possible to know whether any of the capsular types of S. suis belong to the normal flora of the nasal cavity or whether they represent real pathogens. Of the new capsular types, types 9 and 22 are more frequently found in diseased pigs than in healthy ones. An outbreak of S. suis type 9 infection recently occurred in swine in Canada? Although 22 of 23 capsular types of S. suis are present in North America, our studies revealed that almost 30% of isolates recovered from diseased pigs are still untypeable (unpublished data). As the number of capsular types increases, serotyping becomes more complicated, and it could soon become limited to reference laboratories. Even biochemical identification, when used alone, can be misleading. Thus, veterinary diagnostic laboratories face a difficult situation. There is an urgent need for a standardization of both the biochemical identification of S. suis and the techniques for capsular typing. The purpose of this paper is to summarize the knowledge of biochemical characterization and serotyping of S. suis and to suggest means to facilitate the proper identification of this infectious agent of growing importance. Streptococcus suis is a gram-positive coccus that occurs singly, frequently in pairs, or occasionally in short chains. The organism grows well in aerobiosis, but growth is often enhanced by microaerophilic condiFrom the Research Group in Swine Infectious Diseases, Faculty tions. The majority of strains are alpha-hemolytic on of Veterinary Medicine, University of Montreal, Saint-Hyacinthe, bovine blood agar plates after 24 hours of incubation Quebec J2S 7C6, Canada. Presented at the 32nd Annual Meeting of the AAVLD, Las Vegas, at 37 C. In the first study that included a complete scheme NV, October 28-31, 1989. Received for publication November 1, 1989. for identification of groups R, S, and RS (types 1, 2,

Streptococcus suis is a worldwide cause of a variety of porcine infections. It has been isolated from cases of meningitis, bronchopneumonia, arthritis, pericarditis, endocarditis, polyserositis, septicemia, rhinitis, and abortion.20,22,24 Different alpha-hemolytic streptococci were ascribed to Lancefield groups R, S, RS, and T in 1963.5 Other investigators working with capsulated streptococci similar to de Moor’s groups S and R realized that the polysaccharides involved in serotyping originated from the capsular material rather than from the cell wall. They considered these isolates as a new species (Streptococcus suis) within the Lancefield group D, with 2 capsular types: 1 and 2, respectively. The de Moor’s group RS was also added as S. suis type ½.26 In 1983, 6 new capsular types of S. suis originating from diseased pigs were described. 20 In 1987 the species was officially recognized, l4 but the authors demonstrated by DNA hybridization that S. suis was not closely related to group D streptococci. Despite the presence of 9 capsular types of S. suis, untypeable isolates were still frequently reported. These isolates were recovered from diseased4,10,12,13,22,24,25 and clinically healthy pigs. 2,3,21 More recently, 14 new capsular types have been described.9 Some of the reference strains originated from diseased pigs, whereas others were from the nasal cavities of clinically healthy pigs. One strain was isolated from a diseased calf (capsular type 20) and another from a human case of meningitis (capsular type 14). Isolates belonging to capsular type 14 have recently been recovered from diseased pigs in our laboratory as well as in Denmark and Belgium (Dr. J. Henrichsen, personal communication, 1989). This confirms that types other than capsular type 2 could be involved in a zoonosis.9 In addition to the bovine capsular type 20, several other S. suis isolates have been recovered from ruminants.9,12 Capsular type 16 was recently isolated in our laboratory from lungs of a calf suffering from bronchopneumonia, and a cap-

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and ½)19 the authors reported that fermentation of inulin and glycogen and resistance to optochin and arginine dihydrolase were sufficient tests to positively identify S. suis isolates. They also suggested that the production of acid from raffinose was a good test to differentiate group S from group R streptococci. Since then, several workers have studied the biochemical characteristics of different capsular types of S. suis, and it is now obvious that the exclusive use of the above mentioned tests can lead to the misidentification of many S. suis isolates. Several authors considered arginine dihydrolase and production of acid from inulin as variable characteristics for the identification of S. suis 11,13,22 It has also been shown that neither the raffinose fermentation test nor any other test can be used to differentiate one capsular type from another. Detection of group D antigen was considered another useful test for the identification of S. suis,2,21 but other 13,14,22 do not recognize this antigenic deinvestigators terminant as a specific characteristic. Among the new capsular types, other biochemical variations have been noted. For instance, S. suis was originally considered negative for mannitol fermentation. However, > 70% of new capsular types 17, 19, and 21 tested positive. Despite this variation, a minimal number of biochemical tests carried out in combination with capsular typing may allow for the definitive identification of most S. suis isolates. Four tests are used for a presumptive identification of S. suis in our laboratory: no growth in 6.5% NaCl agar, a negative Voges-Proskauer (VP) test, and production of acid in trehalose and salicin broths.10 Although some isolates are trehalose or salicin negative, very few are negative for both tests. The VP test is critical and appears to be the most reliable for differentiating S. suis from S. bovis. False negative results are possible, and the constant use of control strains is advisable. Isolates identified as S. suis according to these 4 biochemical tests can be serotyped. Untypeable isolates should be confirmed by additional biochemical tests, including positive arginine dihydrolase and production of acid in Phenol Red brotha from lactose, sucrose, and inulin. Glycerol, mannitol, and Sorbitol will generally be negative. Nevertheless, atypical results can be expected with some isolates, and only a complete serotyping system will solve this problem. Some authors have used a commercial multitests system.b 13,22,25 Our experience indicates that this system is not adequate for identification of S. suis, particularly the new capsular types. Nearly 50% of 137 strains belonging to capsular types 9-22 could not be identified as S. suis using this system (unpublished data). Certain characteristics, such as ß-glucuronidase, arginine dihydrolase, and production of acid from mannitol should be questioned. Other investigators

have reached the same conc1usion13,22 (Gottschalk M, Higgins R, Mittal KR, Jacques M: 1989, Abstr 5th Int Symp World Assoc Vet Lab Diagn #101). Only encapsulated isolates can be serotyped. These isolates will present a homogeneous growth in ToddHewitt broth,a whereas uncapsulated isolates will deposit at the bottom of the tube, leaving a very clear supernatant. 9,19 Loss of capsule has been reported among some S. suis isolates. 17,18 Data from our laboratory indicate that almost 5% of isolates recovered from diseased animals were not encapsulated (unpublished data). Because of the large number of capsular types that have recently been described, it would be unreasonable for most diagnostic laboratories to perform capsular typing for all serotypes. Preparation of reagents and culture conditions are critical. It has been demonstrated, by electron microscopy, that the amount of capsular material can vary depending on culture conditions. Reference strains used to immunize rabbits must possess a well-developed capsule or antibody titers may vary significantly. It is important to note that capsular material may degenerate very rapidly, and an interruption of cellular growth before it reaches its peak is essential. In general, several passages of a 5-6-hour subculture in Todd-Hewitt broth supplemented with 5% inactivated bovine serum increases capsule production. A quality control of formalinized immunogens should be performed by the capsular reaction test. The antigen should be stored in dense suspension at 4 C. Rabbits weighing 3 kg are given 3 injections per week of increasing concentrations of bacteria during 4 weeks as follows: first week: 2-4 x 109 streptococci, second to fourth week: 4-8 x 109 streptococci. Ten days after the last injection, blood samples are taken, and sera are evaluated by the capsular reaction test. Inoculations are continued for 1 or 2 weeks unless a clear reaction is obtained. Some capsular types cross-react, indicating the possession of common antigenic determinants. This crossreaction is probably due to similar composition of capsular polysaccharides. To date, the following crossreactions have been demonstrated: capsular type ½ with types 1 and 2 antisera, capsular type 6 with type 16 antiserum, capsular type 16 with. type 6 antiserum, and capsular type 22 with type 2 antiserum.9,19 Absorption is recommended to obtain monospecific antisera. Several laboratories, especially in North America, use only the coagglutination test for S. suis typing.2,21 Nonspecific reactions with streptococcal agglutination and coagglutination tests have been reported earlier 8,13,21 Nonetheless it has been demonstrated that nonspecific cross-reactions may occur among different capsular types of S. suis.9 Todd-Hewitt broth and other

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Review article

serum-supplemented broths should not be used for the preparation of antigen because a false positive reaction may occur with the coagglutination reagents.23 The coagglutination test should not be used without a confirmatory test if weak or multiple positive reactions occur. The capillary precipitation test or the capsular reaction test may be used. Acid extraction (0.066 N HCl) of capsular material is recommended for the cap13,20 However, it is a long proillary precipitation test. cedure and, in some cases, it is not reliable for some field isolates. The capsular reaction is the test of choice, and it should be used by all laboratories. Because aging cultures may cause a degradation of the capsular antigen, a 5- or 6-hour culture should be used. The technique of the capsular reaction is easy, rapid, and specific. Although some modifications have been added,1,5 the following procedure is recommended. A loopful of a 5- or 6-hour serum (5%) culture broth is spread over an area of 0.5 cm in diameter on a glass slide. A loopful of the antiserum is then mixed thoroughly with the culture on the slide. A coverslip is placed over the mixture and the preparation is examined under a phase contrast microscope with an oil immersion objective (1,000 x magnification). The reaction occurs between the type-specific serum and the capsular material, making the capsule visible. The streptococci appear twice as big as those of control strains, which are mixed with nonimmune rabbit serum. The increasing number of capsular types (probably more than 30 by 1990) makes it difficult for a diagnostic laboratory to serotype S. suis. The relative importance of different capsular types of S. suis is unknown and may vary with time and geographic area. In most countries, capsular type 2 is currently the cause of most S. suis infections,4,10,24-26 whereas in Denmark and Finland, capsular type 7 appears to be most preva1ent.20,22 Efforts are presently directed toward the establishment of a definitive serotyping system with the aim of making it accessible to every diagnostic laboratory. The development of this system is a prerequisite for further studies on the pathogenicity of the different capsular types, for the understanding of the physiopathology of the infection, and for the production of vaccines. Sources and manufacturers a. Difco Laboratories, PO Box 1058, Detroit, MI. b. API, Analytab Products, Division of Sherwood Medical, 200 Express Street, Plainview, NY.

References 1. Austrian R: 1976, The Quellung reaction, a neglected microbiologic technique. Mt Sinai J Med 43:699-709.

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2. Breton J, Mitchell WR, Rosendal S: 1986, Streptococcus suis in slaughter pigs and abattoir workers. Can J Vet Res 50:338341. 3. Brisebois L, Charlebois R, Higgins R, Nadeau M: 1990, Prevalence of Streptococcus suis in four to eight week-old clinically healthy piglets. Can J Vet Res 54:174-177. 4. Castryck F, Hommez L, Devriese L, Cassimon P: 1987, Streptococcus suis infections in pigs. Vlaams Diergeneeskd Tijdschr 56:231-237. 5. de Moor CE: 1963, Septicaemic infections in pigs caused by haemolytic streptococci of new Lancefield groups designated R, S and T. Antonie Leeuwenhoek J Microbiol Serol 29:272-280. 6. Elliott SD: 1966, Streptococcal infections in young pigs. I. An immunological study of the causative agent (PM Streptococcus). J Hyg (Camb) 64:205-212. 7. Freeze W: 1989, Streptococcus suis: culture and identification techniques and serological field results. Proc Annu Meet Am Assoc Swine Pract, pp. 361-364. 8. Giunta S, Sampaoli G, Galeazzi L, et al.: 1983, Inhibition of nonspecific streptococcal coagglutination reactions. J Clin Microbiol 17: 192-194. 9. Gottschalk M, Higgins R, Jacques M, et al.: 1989, Description of 14 new capsular types of Streptococcus suis. J Clin Microbiol 27:2633-2636. 10. Higgins R, Gottschalk M, Mittal KR, Beaudoin M: 1990, Streptococcus suis infection in swine. A 16-month study. Can J Vet Res 54:170-173. 11. Hoffman L, Henderson L: 1985, The significance of Streptococcus suis in swine diseases: clinical, pathologic and bacteriologic data from a two year study. Proc Annu Meet Am Assoc Vet Lab Diagn 28:201-210. 12. Hommez L, Wullepit J, Cassimon P: 1988, Streptococcus suis and other streptococcal species as a cause of extramammary infection in ruminants. Vet Rec 123:626-627. 13. Hommez L, Wullepit J, Cassimon P, et al.: 1986, Identification and characterization of Streptococcus suis. Vet Microbiol 11: 349-355. 14. Kilpper-Balz R, Schleifer KH: 1987, Streptococcus suis sp. nov. nom. rev. Int J Syst Bacteriol 37:160-162. 15. Lund E, Henrichsen J: 1978, Laboratory diagnosis, serology and epidemiology of Streptococcus pneumoniae. In: Methods in microbiology, Bergan T, Norris JR, vol. 12, pp. 241-262. Academic Press, New York, NY. 16. Orr J, Copeland S, Chirino-Trejo M: 1989, Streptococcus suis type 9 outbreak in swine. Can J Vet Res 30:680. 17. Pedersen KB, Henrichsen J, Perch B: 1984, The bacteriology of endocarditis in slaughter pigs. Acta Microbiol Immunol Scand Sect B 92:237-238. 18. Pedersen KB, Kjems E, Perch B, Slot P: 1981, Infection with RS streptococci in pigs. Acta Microbiol Immunol Scand Sect B 89:161-165. 19. Perch B, Kjems E, Slot P, Pedersen KB: 1981, Biochemical and serological properties of R, S and RS streptococci. Acta Microbiol Immunol Scand Sect B 89:167-171. 20. Perch B, Pedersen KB, Henrichsen J: 1983, Serology of capsulated streptococci pathogenic for pigs: six new serotypes of Streptococcus suis. J Clin Microbiol 17:993-996. 21. Rosendal S, Breton J, Henrichsen J, et al.: 1986, Isolation of Streptococcus suis using a selective medium. Can J Vet Res 50: 537-539. 22. Sihvonen L, Kurl DN, Henrichsen J: 1988, Streptococcus suis isolated from pigs in Finland. Acta Vet Scand 29:9-13. 23. Sorensen UB, Henrichsen J: 1987, Cross-reactions between

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An update on Streptococcus suis identification.

J Vet Diagn Invest 2:249-252 (1990) REVIEW ARTICLE An update on Streptococcus suis identification Robert Higgins, Marcelo Gottschalk sular type 2 is...
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