SHORT COMMUNICATIONS Suilysin production by Streptococcus suis strains isolated from diseased and healthy carrier pigs in Spain C. TARRADAS, C. BORGE, A. ARENAS, A. MALDONADO, R. ASTORGA, A. MIRANDA, 1. LUQUE Veterinary Record (200 1) 148, 183-184 C. Tarradas, DVM, C. Borge, PhD, A. Arenas, DVM, A. Maldonado, DVM, R. Astorga, DVM, A. Miranda, DVM, I. Luque, DVM, Departamento Patologia Infecciosa, Edificio Sanidad Animal, Campus Universitario de Rabanales, Ctra de Madrid, s/n, 14071- C6rdoba, Spain SINCE Streptococcus suis was first reported as a cause of disease in pigs in the Netherlands and the UK, new cases have been cited in major swine-producing countries around the world (Windsor and Elliot 1975, Sanford and Higgins 1992). S suis is responsible for a variety of pathological conditions, such as septicaemia, meningitis, arthritis, bronchopneumo- nia, and reproductive failures in pigs, with important losses in production in herds (Sanford and Higgins 1992, Luque and others 1998a). It is now generally accepted that among strains within one serotype of S suis there are differences in virulence (Vecht and others 1991, Jacobs and others 1994, Gottschalk and others 1998). Different bacterial structures or products have been considered to be virulence factors (Jacobs and others 1994, Salasia and Lammler 1994). Vecht and others (1991) described two proteins, a muramidase-released protein (MRP), which is a 136 kD cell wall-associated protein, and an extra- cellular factor (EF), a 110 kD protein, both related to the vir- ulence of the strains in pigs. Jacobs and others (1994) described a thiol-activated haemolysin called suilysin, a secreted protein, which can also be a virulence factor of S suis. The aim of this study was to determine the production of suilysin by studying both strains isolated from diseased pigs (clinical strains) and strains isolated from healthy carrier pigs (tonsillar strains) in Spain, showing any possible relation with other virulence-related proteins (MRP and EF proteins) which had already been studied (Luque and others 1998b). A total of 203 swine strains were studied, 85 of them iso- lated from pigs with different clinical conditions (septicaemia, meningitis, arthritis, reproductive failure, bronchopneumo- nia, abscess formation) and the remaining 118 isolated from healthy carrier pigs. Several processes such as isolation, biochemical and sero- logical identification, and production of MRP and EF proteins, were carried out as described by Luque and others (1998b). Production of suilysin was carried out by using a microtitre assay as described by Jacobs and others ( 1994). Colonies from Columbia agar base (Oxoid) were inoculated in 100 ml Todd- Clinical strains (0/o) Tonsillar strains (%o) Phenotype Sly+ Sly- Sly+ Sly- MRP+EF+ 31 (36-5) 19 (22-4) 9 (7-6) 15 (12-7) MRP-EF- 7 (8-2) 12 (14-1) 2 (1-7) 37 (31-4) MRP+EF- 1 (1.2) - - 2 (1-7) MRP-EF+ 1 (1.2) - 3 (2-5) 8 (6-8) MRP+EF* - 12 (14-1) 4 (3-4) 6 (5-1) MRP*EF- - 2 (2-4) 4 (3-4) 22 (18-6) ND - - 5 (4-2) 1 (0-8) Total (%) 40 (47) 45 (53) 27 (22-9) 91(77-1) ND Not determined Production of suilysin Serotype Clinical strains Tonsillar strains 1/2 2 3 4 5 6 7 8 9 10 14 15 16 21 22 23 24 26 27 28 A ND Total 1/3 3/5 27/49 0/4 0/1 0/1 0/0 2/2 4/4 1/4 0/1 2/4 0/2 0/2 0/0 0/1 0/0 0/0 0/0 0/1 0/0 0/1 0/0 40/85 2/7 1/8 15/50 0/1 0/1 0/0 0/2 0/1 0/2 1/6 0/2 2/5 5/11 0/0 1/3 0/0 0/1 0/2 0/3 0/0 0/2 0/2 0/9 27/118 Total* 3/10 4/13 42/99 0/5 0/2 0/1 0/2 2/3 4/6 2/10 0/3 4/9 5/13 0/2 1/3 0/1 0/1 0/2 0/3 0/1 0/2 0/3 0/9 67/203 * Number of positive strains/total number of strains of serotype ND Not determined Hewitt broth (Oxoid) supplemented with 0- 1 per cent L-cys- teine (Sigma Chemical) and cultured at 370C for six to nine hours (A. Jacobs, personal communication). After centrifu- gation at 10,000 g for 10 minutes, the supernatant was used. Serial two-fold dilutions (100 V1) of test samples with 10mM Tris-buffered saline (pH 7-4) as diluent were prepared in poly- styrene deep-well titre plates. Titration of haemolytic activity was performed with a 2 per cent ovine erythrocyte suspen- sion that appeared to be more stable than horse erythrocyte suspension. The plates were incubated at 370C for two hours on a rotative shaker (Esclat ES-21; Biotec), and the unlysed ery- throcytes were allowed to pelletise overnight at 40C. Finally, the supernatant was transferred to a flat-bottomed microtitre plate and measured at 450 nm with a micro-ELISA reader. To confirm the identity of suilysin, a second microtitre assay was carried out after incubation of the supernatant from each strain with cholesterol (Jacobs and others 1994). None of the strains showed haemolytic activity in the second assay. Among the S suis capsular types isolated from diseased pigs and from the tonsils from healthy carrier pigs, serotype 2 appeared to be the most prevalent, although other serotypes of S suis were also isolated (serotype 1, 1/2, 3, 4, 5, 7, 8, 9, 14, 15,16,22 and 27) (data not shown). In this study, most of the different strains (serotype 1, 1/2, 2, 7, 8, 9, 14, 15, 21) tested showed haemolytic activity due to suilysin (Table 1). These results agree with previous reports (Jacobs and others 1994, Segers and others 1998). Titres of haemolytic activity 2' to 29 were obtained. Of the 85 strains isolated from diseased pigs, 40 (47 per cent) (Table 1) produced suilysin, a lower frequency than pre- viously described both in European and Asian countries (>70 per cent) (Jacobs and others 1995, Segers and others 1998), but higher than the 41 per cent described in North America by Segers and others (1998). Twenty-seven S suis serotype 2 strains out of 49 (55 per cent), produced suilysin at this stage (Table 1), which is also low if compared with those found in other European and Asian countries (95 per cent), but much higher than those isolated in North America (7 per cent) (Segers and others 1998). These results show that the frequency of suilysin produc- tion in clinical samples from diseased pigs is approximately The Veterinary Record, February 10, 2001 .4-n-rf-T E le* .. .em l r. S 5, 6el 5 KWT. 5 5 .., D * S " 183