Streptococcus suis type 2

Transcription

1 Epiemiol. Infect. (1994), 113, Copyrigt 1994 Cambrige University Press Clonal analysis an virulence of Australian isolates of Streptococcus suis type 2 C. G. MWANIKI, I. D. ROBERTSON, D. J. TROTT, R. F. ATYEO, B. J. LEE AND D. J. HAMPSON* Scool of Veterinary Stuies, Muroc University, Muroc, Western Australia 6150, Australia (Accepte 9 May 1994) SUMMARY Multilocus enzyme electroporesis was use to ivie 124 Australian isolates of Streptococcus suts type 2 into 17 electroporetic types (ETs). Isolates in ET 1 were te most frequent cause of isease amongst Western Australian pigs, but isolates of ET 8 were more commonly associate wit isease in oter Australian states. Multiple isolates from 10 of 19 farms all belonge to te same ET, wilst isolates from te oter farms belonge to between 2 an 4 ifferent ETs. Some isolates coul be ifferentiate furter by DNA restriction enonuclease analysis, wilst oters wit te same restriction pattern were locate in ifferent, but closelyrelate ETs. Fourteen isolates were teste for teir virulence in mice. Most cause isease if given in ig numbers, but isolates in ET 1 were virulent at lower ose rates. Tis virulent clone also was istinguise by te fact tat 80% of isolates prouce extracellular factor (EF). INTRODUCTION Streptococcus suis can cause a variety of clinical problems in young pigs, especially meningitis, pneumonia, septicaemia an artritis [1-3]. Te bacterial species as been ivie into 29 capsular serotypes, esignate 1 to 28, an type 1/2 [4-6]. Of tese, serotype 2 is te most important causing isease in pigs in most parts of te worl [7, 8], altoug serotype 7 preominates in Scaninavia [6, 9]. Despite te association of tese bacteria wit isease, tey may also be recovere from te nasal cavities an tonsils of ealty pigs [10-12]. Outbreaks of isease may follow perios of stress, suc as wen weane pigs are move an mixe, or are overcrowe. Te buil-up of fumes from slurry, combine wit poor ventilation, also preisposes to isease [13]. Outbreaks also may occur following te introuction of new pigs to a er [14]. It is presume tat tese are ealty carrier animals wic bring new virulent strains into te er, an tat tese strains ten sprea to susceptible pigs. Strains of S. suis type 2 wit iffering egrees of virulence to pigs ave been escribe [14, 15]. Virulent strains prouce a muramiase-release protein (MRP) an, particularly, an extracellular factor (EF) [16, 17]. Strains tat prouce a iger molecular weigt * Corresponence an requests for reprints to: Dr D. J. Hampson.

2 322 C. G. MWANIKI AND OTHERS form of EF (EF*) were of intermeiate virulence in newborn germ-free pigs, wilst strains lacking bot proteins i not inuce any signs of isease [17]. Te species S. suis is genetically omogeneous as assesse by levels of DNA sequence omology between 13 strains [18]. Te use of multilocus enzyme electroporesis (MEE) to examine a muc larger collection of S. suis isolates of various serotypes as, owever, sown te species to be relatively iverse [19]. For instance 31 isolates of serotype 2 a a mean genetic iversity of 0-264, an were ivie into 6 electroporetic types (ETs), representing 3 clonal groups. Genetic ifferences between strains of S. suis type 2 also ave been emonstrate using DNA restriction enonuclease analysis (REA), an ribotyping wit an Escericia coli rdna probe [20, 21]. Tese tecniques ave also been use to stuy te istribution of strains of te bacteria amongst pigs uring outbreaks of S. suisassociate isease. Te purpose of tis stuy was to etermine te iversity of Western Australian (WA) isolates of S. suis type 2, an te association of specific strains wit clinical problems on piggeries. MEE was use as it is applicable to stuies of bacterial populations [22], an is a useful tecnique for analysing isolates of S. suis [19]. Results were compare wit tose previously obtaine for oter Australian strains of S. suis type 2 [19]. In aition REA was use to elp clarify te epiemiology of te infections. Te virulence of isolates in ifferent genetic groups was assaye in mice, an correlate wit te presence or absence of MRP an EF in Western blot analysis of culture supernatants, using specific monoclonal antisera. MATERIALS AND METHODS Bacterial isolates Te isolates of S. suis type 2 (n = 124) were from 4 main sources (Table 1). Source 1 comprise 67 isolates collecte uring a survey of pigs from 19 Western Australian (WA) piggeries (esignate A to S respectively). Te isolates were from te tonsils or lungs of ealty pigs following teir slaugter at local abattoirs, except for 2 isolates from farm S wic were obtaine by swabbing te nasal cavities of 2 ealty grower pigs. Between 2 an 5 isolates were examine from slaugtere pigs from eac er, wit an average of 3-4 isolates per er. Source 2 comprise 15 isolates recovere from 6 litter-mates on farm Q. Tese animals were followe as a coort from weaning to slaugter, wit nasal swabs taken from eac pig at 1, 2, 3 an 4 monts of age, an swabs from te nose, tonsils an lungs taken following slaugter at 6 monts. Source 3 comprise 11 isolates from WA pigs clinically affecte wit streptococcal meningitis. Tese were receive from N. B. Buller of te Veterinary Diagnostic Microbiology Laboratory, WA Department of Agriculture, Sout Pert, or were isolate from ea pigs submitte for iagnosis to te Muroc University Veterinary Hospital. Tese animals came from 4 farms (R to U respectively). Te 4t source was 31 strains of S. suis type 2 stuie previously [19], an inclue isolates from pigs in Sout Australia (n = 22), Tasmania (n = 3), Victoria (n = 2), an New Sout Wales (n = 2). Seventeen were from pigs wit meningitis or septicaemia, an 12 were isolate from te tonsils of ealty animals at slaugter. One isolate was recovere in Australia from te meninges of a uman, an te oter was a porcine reference strain from te State Serum Institute, Copenagen, Denmark.

3 Analysis of Streptococcus suis type 2 Table 1. Electroporetic types of Streptococcus suis type 2 isolate from A ustralian pigs Electroporetic type WA piggeries* Oter statest 1 G(3); 1(3); J(3); L(1); SA(1); SA(1)T M(2); N(3); R(3); R(6)$; S(2)$; U(1)$ 2 TAS(1)t 3 Q(3) 4 C(2) 5 S(1) 6 TAS(1)T; NSW(2)$ 7 Q(1); R(2) SA(6); SA(2)T 8 A(3); C(1); D(3); E(3); SA(4); SA(7$); F(3); H(3); L(2); P(3); TAS(1)$; VIC(2)$ 323 Q(14); T(1)T 9 M(3) 10 B(3); L(1) 11 H(1) SA(1) 12 K(2); R(1)T 13 K(1); O(1) 14 O(1) 15 S(2) 16 S(3) 17 S(2) * Twenty-one piggeries in WNestern Australia, esignate A to IU. Numbers in parenteses are te number of isolates. t SA. Sout Australia. TAS, Tasmania. NSW, New Sout Wales, VIC, Victoria. i Inicates isolates recovere from pigs wit isease. All te oter isolates are from ealty pigs. ET 1 also containe an isolate from a uman wit meningitis, an ET 2 containe a Danis porcine reference strain. Bacterial isolation an caracterization Swabs were taken from te live an te slaugtere pigs as escribe previously [23]. Tese were use to inoculate meium incorporating 3 9% (w/v) Columbia agar base (Oxoi), 1-5 % (w/v) streptococcal selective antibiotic supplement (Oxoi), an 5 % (v/v) specific seep antiserum raise against a Sout Australian reference strain of S. suis type 2 [24]. Plates were incubate aerobically for at 37 C, an ten for 24 at 4 'C. Colonies of S. suis type 2 were ientifie by te presence of brigt broa aloes of immunoprecipitation aroun tem wen viewe by inirect ligt in semi-arkness [25, 26]. Specificity of te meto was confirme by testing representatives of eac batc of plates wit S. suis isolates of serotypes 1, 3-9, 13, 15, 18, 20, an untypable isolates of S. suis [19], as well as wit Enterococcus faecalis, S. pyogenes, S. equi, S. zooepiemicus, S. bovis, an a group G streptococcus. Positive colonies were subculture to (i) Columbia agar containing 5 % (v/v) efibrinate ovine bloo (SBA plates), an incubate overnigt at 37 'C, an (ii) te selective agar, wic in tis case containe specific seep antiserum to S. suis type 1. Colonies tat reacte positively on bot antiserum plates were recore as being type 1/2, an were not investigate furter. Small alpa-aemolytic colonies of Gram positive cocci growing on te SBA plates were confirme as being S. suis by testing for absence of growt on 6-5 %

4 324 C. G. MWANIKI AND OTHERS 9M I I...I Genetic istance Fig. 1. Penogram of genetic istance (expresse as percent fixe allelic ifferences) among 17 ETs, containing 124 isolates of S. suis type 2, clustere by te unweigte pair group meto wit averages (UPGMA) strategy. NaCl agar, a negative Voges-Proskauer test, an prouction of aci in eiter trealose or salicin brots or bot [27]. Te serotype of selecte isolates from eac of te major genetic groups was subsequently confirme using te tube precipitation an/or te capsular swelling reaction tests [27]. Multilocus enzyme electroporesis (MEE) Te tecnique as been escribe [19]. Briefly, bacterial cells were grown overnigt in 500 ml of To-Hewitt brot (Oxoi), arveste by centrifugation, wase in pospate buffere saline (PBS; ph 7 2), centrifuge again, an resuspene in sonication buffer (40 mm-k2hpo4, 10 mm-l-cysteine HCl, 5 jtg/ml bovine serum albumen, 3 mm itiotrietol, ph 7 5). Tey were lyse by four cycles of sonication, clarifie by centrifugation, an te supernatant collecte. Tese lysates were subjecte to electroporesis in 11F4% orizontal starc gels, an te electroporetic mobilities of te following 16 enzymes were etermine by staining for specific enzyme activity: Leucyl-tyrosine peptiase (LT), glucose-6- pospate eyrogenase (G6P), mannose-6-pospate isomerase (MPI), leucylglycine peptiase (LGG), nucleosie posporylase-1 (NP), a-naptyl esterase (EST), leucyl-proline peptiase (LP), glutamate eyrogenase (GDH), pospoglucomutase (PGM), lactate eyrogenase (LDH), 6-pospogluconate eyrogenase (6PG), nucleosie posporylase-2 (NSP), aenylate kinase (ADK), glyceraleye-3-pospate eyrogenase (GI1), alanine eyrogenase (ALD), an pospoglucose isomerase (PGI). Te gel an electroe buffers use were triscitrate (ph 8-0). Mobility variants of te enzymes were interprete as te proucts of ifferent alleles at te corresponing enzyme locus. Groups of one of more isolates wit te same alleles at all loci were referre to as being an electroporetic type (ET).

5 Analysis of Streptococcus suis type 2 Table 2. DNA restriction enonuclease analysis (REA) patterns of 33 Australian isolates of S. suis type 2 belonging to ifferent electroporetic types (ETs) Healt REA Isolates* statust ETI pattern S9, S10, U1 1 1 R1-R3 1 2 R6-R C4-5 (SA) 8 4 F2 8 5 F1, F3 8 6 N (TAS) 2 8 St 5 9 S2, S S4-S S7, S R4, R C7-32 (SA) 7 12 BI R * TAS, Tasmania; SA, Sout Australia. All te oter isolates originate from Western Australia., isease pig;, ealty pig. t ETs in MEE analysis, sown in Fig. 1. Following igestion wit HaeIIJ. Patterns were not obtaine for 53 oter isolates examine. Genetic iversity () at eac enzyme locus was calculate as = (1 - XPi2) (n/n - 1), were Pi is te frequency of te it allele an n is te number of ETs or isolates in te sample [28]. Total genetic iversity (H) was calculate as te mean of over all loci. Genetic istances between ETs were calculate as te proportion of fixe loci at wic issimilar alleles occurre, an te unweigte pair-group meto of aritmetic averages (UPGMA) clustering fusion strategy was use to create a penogram (Fig. 1) to sow relationsips between isolates [29]. DNA restriction enonuclease analysis (REA) REA was base on tecniques escribe for S. suis [20, 21]. Attempts were mae to obtain DNA baning patterns from 83 of te WA isolates, an 3 oter Australian isolates (Table 2). Te bacteria were grown overnigt in 100 ml To-Hewitt brot, arveste by centrifugation at g, an wase twice in PBS (ph 7 2). Te pellet was resuspene in 750 jl of 250 mm-tris HCl, ph 8-0, containing 10 jug/ml fresly prepare lysozyme, an was incubate at 37 C for 1, wit occasional saking. Te tube was centrifuge at g for 3 min, te supernatant remove, an te pellet resuspene in 750 #1 of TE buffer (50 mm- Tris, 10 mm-edta, ph 8 0). Te tube was frozen at -20 C, tawe, an 150 /ul of pre-warme STEP buffer (0 5 % SDS, 50 mm-tris HCl, ph 7-5, 40 mm-edta) ae, followe by proteinase K to a concentration of 1 mg/ml. Te tube was mixe by inversion an incubate at 50 C for jul of penol-cloroform was ae an te tube repeately mixe by gentle inversion until an emulsion forme. Following centrifugation at 5000 g for 5 min, te upper aqueous layer was 12 HYG

6 326 C. G. MWANIKI AND OTHERS remove an re-extracte wit penol-cloroform. After centrifugation, te upper aqueous layer was transferre to a new tube, an 90 1ul of 3 M soium acetate an I ml of isopropanol ae. After mixing, te DNA was spoole out, rinse briefly in 70 % etanol, rie, an issolve in 250,1 of TE buffer. Te concentration of te issolve DNA was etermine by measuring absorbance at 260 nm [30]. 15,tg DNA was ten igeste wit 20 units of HaeIII (Boeringer Manneim) in 10% meium salt buffer for 6 at 37 'C. Digeste DNA was subjecte to electroporesis for 21 at 30 V in orizontal 0-7 % agarose gels using TBE electroporesis buffer (45 mm-tris borate, 1 mm-edta, ph 8-0). Page lamba DNA preigeste wit HinIII an HinIII +EcoR1 (Boeringer Manneim) was use as a molecular mass marker. Gels were staine by soaking in etiium bromie (0'5 mg/ml), an were potogrape uner u.v. ligt. Were istinct baning patterns coul not be obtaine after 2-5 attempts, isolates were recore as being not susceptible to igestion wit HaeIII. DNA baning patterns were assesse visually witout knowlege of results obtaine by oter metos. Caracteristic patterns were given a numeral, an DNAs wit similar patterns were subjecte to electroporesis on te same gel to ceck for ifferences. Virulence testing Tree-week-ol outbre female Swiss ARC Albino SPF mice (Animal Resources Centre, Pert, Western Australia) were use to test te virulence of 14 selecte isolates (te first 14 isolates in Table 3). Tis proceure was approve by te Muroc University Animals Experimentation an Etics Committee, an was monitore by te University's Animal Welfare Officer. Six of te isolates were recovere from clinical cases of S. suis type 2 infections in pigs, an 8 were recovere from ealty pigs at slaugter. Eigt of te isolates teste belonge to ET 1, 4 to ET 8, an 2 to ET 12 (Fig. 1). Te mice were ouse in 14 groups of 7, in iniviual mouse boxes. Six mice were inoculate initially wit 2 x 108 colony forming units (c.f.u.), wic a been resuspene from early log-pase culture in To-Hewitt brot into 0 1 ml of sterile PBS, by te intraperitoneal route; te 7t mouse acte as an in-contact control. One week later te surviving animals were callenge wit 4 x 108 c.f.u. of te same strain in 0-2 ml PBS, an a week later survivors were callenge wit 1 x 109 c.f.u. in a 0 5 ml volume. Surviving mice were kille 1 week later, an swabs were taken from te eart bloo an culture for S. suis type 2. Trougout te experiment te mice were observe twice aily, an any clinical signs recore. Analysis for MRP an EF prouction Te prouction of muramiase-release protein (MRP) an extracellular factor (EF) by te 14 isolates teste for virulence in mice, an anoter 17 isolates (Table 3), was assaye [16, 17]. Overnigt cultures in To-Hewitt brot were ilute 1:10 in fres brot, an incubate at 37 'C for 4. Te cultures were centrifuge at 4000 g for 15 min, an te supernatants analyse by soium oecyl sulfate-polyacrylamie gel electroporesis (SDS-PAGE) in 8% separating gels, an by Western immunoblotting, using a mini-protean II electroporesis unit an transblot cell (Bio-Ra Laboratories, Ricmon, Calif.). A control culture supernatant from a Dutc strain, 4005,wic was positive for MRP an EF, an

7 Analysis of Streptococcus suis type Table 3. Prouction of muramiase release protein (MRP) an extracellular factor (EF) by 31 isolates S. suis type 2 Isolate* Statust ETt MRP EF S9 1 S10 11 U1 R6 1 I1 1 LI 1 Ni 1 Ji D2 8 + E Q5V4 8 Ti 8 Kl 12 R12 12 C5-16 (SA)II (TAS) C5 4 DR31-V (TAS) 6 C7-25 (SA) 77 D7-42 (SA) 04 9 B3 10 C8-39 (SA) 88 C6-13 (SA) 14 (TAS) 8 C4-5 (SA) 8 Q6 13 Qi 14 DR DR27-T 16 * Te first 14 isolates (S9-R12)) were teste for virulence in mice, an S9, Ul, I1, LI an Ni cause isease at 2 x 108 c.f.u. wen aministere intraperitoneally. SA, Sout Australia; TAS, Tasmania. All oter isolates from Western Australia. t Disease status of pig from wic te isolate was recovere., isease;, ealty. t ET, electroporetic type in Fig. 1. +, protein present in te supernatant; -, protein absent; x, iger molecular mass ban present (EF*). 11 Isolate from a case of meningitis in a uman. wic was supplie by Dr Uri Vect, DLO-Central Veterinary Institute, Lelysta, Te Neterlans, was also inclue on te gels. Following electroporesis, gels were staine using a silver staining kit (Bio-Ra Laboratories). Proteins tat transferre to nitrocellulose membranes in Western blots were blocke for 1 in 5 % (w/v) non-fat milk power, an ten incubate wit a 1: 1 mixture of mouse anti-mrp monoclonal antiboy (Mab) (1 13 mg/ml) an anti-ef Mab (8-4 mg/ml), eac in a 1:200 ilution [16, 17], provie by Dr Vect. After wasing, te membranes were incubate wit a 1: 2000 ilution of a 1: 1 mixture of goat antimouse IgG an goat anti-mouse IgM, bot conjugate wit orserais peroxiase (Bio-Ra laboratories). After wasing, antiboies were localize by aing te 12-2

8 328 C. G. MWANIKI AND OTHERS substrate 3-3'-iaminobenziine tetrayroclorie iyrate (Bio-Ra Laboratories). RESULTS MEE Te enzymes LDH, ADK, GP1 an ALD were monomorpic wilst te oter 12 enzymes were polymorpic, wit 2-4 alleles. Te average number of alleles per locus was Te 124 isolates were ivie into 17 ETs, wit a mean genetic iversity per locus of 0 397, or wen te number of isolates in eac ET was consiere. Two broa genetic iversions were observe (Fig. 1). ETs 1-11 containe 111 (89-5 %) of te 124 isolates, separate from ETs at a genetic istance of Te WA isolates were istribute in 15 of te ETs, but not in ETs 2 or 6 (Table 1). Te Danis isolate an te oter Australian isolates of serotype 2, from our previous stuy [19], were locate in ETs 1, 2, 6, 7, 8 an 11. Tese ETs correspone respectively to ETs 33, 34, 50, 51, 53 an 54 in our previous stuy [19]. Overall, tere were 50 isolates in ET 8. ET 1 containe 30 isolates, ET 7 11 isolates, an te oter ETs between 1 an 4 isolates. Isolates in ET 8 were common in WA (39 % of isolates), were tey mae up 35 of te 82 (43 %) isolates from ealty pigs, but only 1 of te 11 (9%) isolates from isease animals. Isolates in tis ET also were common in te oter states (14 of 29 porcine isolates; 47 %), but ere tey were more frequently recovere from isease pigs (10 of 17; 59%) tan from ealty animals (4 of 12; 33%). In contrast, isolates in ET 1 were common in WA (29% of 93 isolates), but were muc less so in te oter states (2 of 29; 7 %). In WA tey mae up 82% (9 of 11) of te clinical isolates, but only 22% (18 of 82) of tose from ealty pigs. Isolates were collecte from ealty pigs on 19 farms, an on 11 of tese all belonge to te same ET. Five farms a isolates belonging to 2 ETs, 2-3 ETs, wilst te 8 isolates from farm S belonge to 4 ETs. In te coort stuy on farm Q, 9 isolates from 5 of 6 pigs belonge to ET 8 (as i 3 isolates collecte earlier in te abattoir survey). In te sixt pig, owever, 2 isolates were of ET 8, 3 ET 3, an 1 ET 7. Of te 11 isolates recovere from isease pigs in WA (Table 1), 9 belonge to ET 1, an 1 eac to ETs 8 an 12. Isolates belonging to tese ETs were also recovere from ealty WA pigs. Te 17 isolates from isease pigs in oter Australian states belonge to ETs 1(1), 2(1), 6(3), 7(2) an 8(10); tose from ealty pigs were of ETs 1(1), 7(6), 8(4) an 11 (1) (Table 1). REA Fourteen ifferent REA patterns (Fig. 2) were ientifie for 33 isolates, using te enzyme HaeIII (Table 2). Patterns coul not be obtaine for 53 oter isolates as tese eiter faile to lyse aequately, yiele poor quality DNA, or te DNA was refractory to HaeIII. Te 12 belonging to ET 1 in MEE analysis were ivie into tree REA patterns. Notably, 3 nasal isolates from ealty pigs on farm R a a ifferent pattern from 6 clinical isolates from isease pigs on te same property. Te latter a an aitional ban at approximately 22 kb. Five isolates belonging to ET 8 were ivie into 4 REA patterns. In contrast 7 isolates, from farm S, all a

9 Analysis of Streptococcus suis type 2 kb Fig. 2. Restriction fragment patterns of DNA from isolates of S. suis type 2. Samples igeste wit HaeJII an separate by electroporesis in 07% agarose. Fourteen patterns were recognize, an representative isolates from eac of tese are presente in ascening orer in lanes 2-8, an Lanes: 1 an 9, A DNA size markers; 2, isolate S9; 3, isolate RI; 4, isolate R6; 5, isolate C4-5 (SA); 6, isolate F2; 7, isolate F3; 8, isolate L3; 10, isolate 13 (TAS); 11, isolate SI; 12, isolate S5; 13, isolate R4; 14, isolate C7-32 (SA); 15, isolate Bi; 16, isolate R12. REA pattern No. 10, but were separate by MEE into te closely relate ETs 15, 16 an 17. Similarly, anoter 3 isolates wit REA pattern No. 14 belonge to te closely relate ETs 12, 13 an 14. Virulence testing Following te first callenge, some mice (mean 1P7 of 6) inoculate wit isolates Ut, It an LI ie, an S. suis type 2 was isolate from tese. Oter mice inoculate wit tese cultures, as well as S9 an NI, also evelope clinical signs

10 330 C. G. MWANIKI AND OTHERS MRP - EF kda kda MRP - EF 80 kda kda - Fig. 3. Western blots of cell culture supernatants of te 14 isolates of S. suis type 2 tat were use to infect mice. Membranes probe wit a 1:1 mixture of mouse anti-mrp an anti-ef Mab (supplie by U. Vect). Lane 1, positive control supernatant (MRP + ve; EF + ve), supplie by U. Vect; Lane 2, isolate S9; lane 3, isolate S10; lane 4, isolate Ul; lane 5, isolate R6; lane 6, isolate I1; lane 7; isolate LI; lane 8, isolate NI; lane 9, isolate J1; lanes 10 an 11, prestaine molecular mass markers (low range, Bio-Ra Laboratories); lane 12, isolate D; lane 13, isolate E2; lane 14, isolate Q5V4; lane 15, isolate T1; lane 16, isolate K; lane 17, isolate R 12; lane 18, positive control supernatant. (10 of 25 mice), wic inclue lack of grooming, letargy an incoorination. All five isolates belonge to ET 1, altoug only S9 an Ul originate from isease pigs. Following te secon callenge, wit a iger inoculum, only tose mice expose to isolate LI evelope isease. Two of tese 4 animals ie, an te oter 2 sowe clinical signs. After te 3r inoculation, eats occurre in 12 of te 14 groups, altoug not in mice callenge wit isolates S9 or Ki. Forty-one of 77 mice ie (53%), an 23 sowe clinical signs (30%). MRP an EF prouction Prouction of MRP an EF by te 31 isolates is recore in Table 3. Specific bans in Western blots using te Mabs are sown in Fig. 3 for te 14 isolates teste for virulence in mice. Tree penotypes were recore: MRP an EF positive;

11 Analysis of Streptococcus suis type MRP positive, EF negative; MRP an EF negative. Isolates from isease animals were more likely to prouce MRP an EF tan were tose from ealty animals. Isolate C5-16 (SA), from a uman wit meningitis, a an EF protein wit a molecular mass of approximately 150 kda (EF*; membrane not sown). DISCUSSION MEE inicate tat S. suis type 2 from ealty an isease Australian pigs originate from iverse genetic backgrouns, confirming te conclusions of oters tat serotyping cannot be relie upon for strain ientification of S. suis [20, 31]. Two broa genetic groups were ientifie. Te smaller group comprise 13 isolates (10O5 %) in 6 ETs, an inclue only 1 of te 28 isolates from isease pigs. Tis group is, terefore, probably of minor significance clinically. Despite te overall iversity amongst isolates, 40, 24 an 9% belonge to ETs 8, 1 an 7 respectively. Te geograpical istribution of ETs 1 an 8 in Australia iffere, as i teir association wit isease. ET 1 was te most common cause of isease in WA, an ET 8 in Sout Australia, Tasmania an Victoria. Isolates from bot ETs were also recovere from ealty pigs on farms witout a istory of recurrent S. suis type 2-associate isease. Some isolates in ETs 2, 6, 7 an 12 were also associate wit isease, inicating tat virulence is not restricte to te two most common ETS (1 an 8). Te istribution of strains of S. suis type 2 varie on ifferent farms, an more tan one ET was foun in 9 of 19 (47 %) WA piggeries. On te oter 10 properties all isolates were of te same ET, altoug some of tese (ETs, 1, 8, 10, 13) were foun on oter properties, inicating tat tese strains may be wiesprea. To etermine weter isolates witin an ET were likely to be ientical, attempts were mae to ifferentiate tem using REA. Tis prove possible for only a proportion of isolates an it is possible tat ifferences in te tickness or composition of te capsule coul ave accounte for variations in cell lysis, an recovery of DNA. Neverteless, using REA, it was possible to etect ifferences between certain isolates in ETs 1, 8 an 7, incluing tose from te same farms, e.g. ETs 1 an 8 on farms R an F respectively. Tose in ET 1, from farm R, were particularly interesting because isolates from ealty pigs (pattern 2) iffere from isolates from isease animals (pattern 3; Fig. 2). Te latter a an aitional DNA ban (approximately 22 kb) tat migt ave been associate wit virulence. Unfortunately tese isolates were lost following a freezer failure, an coul not be investigate furter. Oter isolates from ifferent but closely relate ETs gave te same REA pattern (patterns 10 an 14). All isolates wit pattern 10 were from te same farm, but comprise tree relate ETs (ETs 15, 16 an 17). Te significance of tese minor ifferences is uncertain. In orer to assess te relationsip between genetic group an virulence, 14 isolates, from ETs 1, 8 an 12, were teste in mice. Mice were cosen because tey are a convenient moel of S. suis type 2 infection in pigs [32, 33], even toug teir use as never been fully compare wit stanarize pig moels of te isease. Intraperitoneal injection also as been foun to be an easy an effective route for establising infection in mice [32, 34]. Most isolates belonging to ET 1 were more virulent in mice tan are tose in ETs 8 or 12, altoug most isolates were capable of inucing isease at iger ose rates. It was unclear wy isolate S9 cause

12 332 C. G. MWANIKI AND OTHERS isease at 2 x 101 c.f.u., but not following callenge wit 1 x 109 c.f.u. A protective immunity may ave evelope after te mice receive te initial oses, but a similar effect was not seen wit oter isolates. Tree of te 5 most virulent isolates were recovere from te tonsils of ealty pigs at slaugter, sowing tat ealty pigs may arbour virulent strains. Vect an colleagues [16, 17] emonstrate tat isolates of S. suis type 2, virulent to newborn germ-free pigs, release MRP an EF into te culture meium. Isolates tat prouce bot MRP an EF*, a iger molecular weigt form of EF, were of intermeiate virulence, wilst isolates not proucing eiter form i not inuce isease. In tis stuy, 8 of te isolates prouce MRP, but not EF or EF*; tis penotype as not previously been escribe. Six isolates from ET 1 teste in mice prouce bot MRP an EF, an, in concorance wit results obtaine in pigs [16, 17], 4 were virulent at relatively low oses. Neverteless isolate II, also in ETI, i not prouce MRP or EF, but was virulent at low ose rates. Oter isolates, lacking eiter or bot proteins, were virulent at iger ose rates. MRP an EF, terefore, are not essential as virulence eterminants in mice inoculate by te intraperitoneal route. Tey may be important in te early establisment of infection in natural cases of isease in pigs, owever, since 6 of 13 isolates from isease pigs prouce MRP an EF, an 4 prouce MRP, wilst only 2 of 18 isolates from ealty pigs prouce MRP an EF, an 4 prouce MRP. Altoug most isolates from ealty pigs were MRP an EF negative, some tat were potentially more virulent (MRP an EF positive) were carrie by ealty animals in certain of te WA ers tat infrequently suffer from S. suis-associate isease. Te absence of isease in tese ers may be associate wit superior management practices, suc as provision of goo ventilation an low stocking ensities. Isolate C5-16 (SA) recovere from te meninges of a uman prouce an EFlike molecule (EF*) wit a iger molecular mass tan tat prouce by our porcine isolates (approximately 150 kda, compare to 110 kda). Similar material (EF*) as been escribe by Vect an colleagues [17] in strains isolate from umans, altoug in relatively few porcine isolates. Tese results suggest tat EF* may be involve in specific aspects of te patogenesis of te infection in umans. All te isolates proucing EF belonge to te closely relate ETs 1 an 2 (8 of 10 isolates analyse from tese ETs were EF positive), an 9 of te 10 isolates prouce MRP. In contrast only 7 of te 21 (33 %) isolates from oter ETs tat were examine prouce MRP, incluing only 2 of te 8 isolates from ET 8 (4 of wic were from isease pigs). ETs 1 an 2, wic coul be istinguise by te presence of allele 1 for esterase an allele 4 for glutamate eyrogenase, appeare to be more virulent tan oter ETs. Prouction of EF an MRP by tese isolates terefore may only be a marker for tis virulent clone, rater tan te proteins temselves necessarily being eterminants of virulence. Tis possibility was suggeste by Vect an colleagues [17]. Tey pointe out tat, in orer to etermine te function of te proteins, it was necessary to obtain isogenic strains tat varie in teir prouction of te proteins, an to test tese strains for virulence in pigs. Currently, irrespective of te function of EF, screening for its prouction is a means of etecting isolates belonging to te virulent clone

13 Analysis of Streptococcus suis type represente by ETs 1 an 2. Isolates from oter clones tat o not prouce EF owever may also be capable of causing isease. ACKNOWLEDGEMENTS Tis stuy was supporte by grants from te Western Australian Pig Inustry Compensation Fun, an from te Australian Pig Researc an Development Corporation. C. G. M. was in receipt of a Postgrauate Scolarsip from te Australian International Development Assistance Bureau. Dr Uri Vect generously onate te monoclonal antiboies against MRP an EF, an provie constructive comment on te manuscript. REFERENCES 1. Lamont MH, Ewars PT, Winsor RS. Streptococcal meningitis in pigs: results of a five year survey. Vet Rec 1980; 107: Sanfor SE, Higgins R. Streptococcal iseases. In: Leman A et al., es., Diseases of swine, 7t e. Ames, USA: Iowa State University Press, 1992: Winsor RS, Elliot SD. Streptococcal infections in young pigs. IV. An outbreak of streptococcal meningitis in weane pigs. J Hyg 1975; 75: Gottscalk M, Higgins R, Jacques M, Mittal KR, Henricsen J. Description of 14 new capsular types of Streptococcus sui8. J Clin Microbiol 1989; 27: Gottscalk M, Higgins R, Jacques M, Beauoin M, Henricsen J. Caracterisation of six new capsular types (23 troug 28) of Streptococcus suis. J Clin Microbiol 1991; 29: Perc B, Peersen KB, Henricsen J. Serology of capsulate streptococci patogenic for pigs: six new serotypes of Streptococcus suis. J Clin Microbiol 1983; 17: Alexaner TJL. Streptococcus suis: an upate. Pig Vet Soc Proc 1991; 27: Touil F, Higgins R, Naeau M. Isolation of Streptococcus suis from isease pigs in Canaa. Vet Microbiol 1988; 17: Siovenen L, Kurl DN, Henricsen J. Streptococcus suis isolate from pigs in Finlan. Acta Vet Scan 1988; 29: Clifton-Haley FA, Alexaner TJL. Te carrier site an carrier rate of Streptococcus suis type 2 in pigs. Vet Rec 1980; 107: Clifton-Haley FA, Alexaner TJL, Enrigt MR, Guise J. Monitoring ers for Streptococcus suis type 2 by sampling tonsils of slaugter pigs. Vet Rec 1984; 115: Robertson ID, Blackmore DK. Prevalence of Streptococcus suis types 1 an 2 in omestic pigs in Australia an New Zealan. Vet Rec 1989; 124: Clifton-Haley FA. Streptococcal meningitis in pigs. Pig News Info 1989; 10: Clifton-Haley FA, Alexaner TJL, Enrigt MR, Linsay HJ. Monitoring ers for Streptococcus suis type 2: cross-reactions an variations in virulence. Proc Int Pig Vet Soc Cong. Barcelona, Spain, 1986: Vect U, Arens JP, van er Molen EJ, van Leengoe LAMG. Differences in virulence between two strains of Streptococcus suis type II after experimental inuce infection of newborn germ-free pigs. Am J Vet Res 1989; 50: Vect U, Wisselink HJ, Jellema MlL, Smit HE. Ientification of two proteins associate wit virulence of Streptococcus sui.s type 2. Infect Immun 1991; 59: Vect U, Wisselink HJ, van Dijk JE, Smit HE. Virulence of Streptococcus suis type 2 strains in newborn germfree pigs epens on penotype. Infect Immun 1992; 60: Klipper-Balz R. Scleifer KH. Streptococcus suits sp. nov., nom. rev. Int J Svst Bacteriol 1987: 37: Hampson DJ, Trott DJ, Clarke IL, Mwaniki CG, Robertson ID. Population structure of Australian isolates of Streptococcus suis. J Clin Microbiol 1993; 31: Beauoin M. Harel J, Higgins R. Gottscalk M, Frenette M, Maclnnes JI. Molecular analysis of isolates of Streptococcus suis capsular type 2 by restriction-enonuclease-igeste DNA separate on SDS-PAGE an by ybriization wit an rdna probe. J Gen Microbiol 1992; 138:

14 334 C. G. MWANIKI AND OTHERS 21. Mogollon JD, Pijoan C, Murtaug MP, Kaplan EL, Collins JE, Cleary PP. Caracterisation of prototype an clinically efine strains of Streptococcus suis by genomic fingerprinting. J Clin Microbiol 1990; 28: Selaner RK, Caugant DA, Ocmann H, Musser JM, Gilmour MN, Wittam TS. Metos of multilocus enzyme electroporesis for bacterial population genetics an systematics. Appl Environ Microbiol 1986; 51: Robertson ID, Blackmore DK, Hampson DJ, Fu ZF. A longituinal stuy of natural infection of piglets wit Streptococcus suis types 1 an 2. Epiemiol Infect 1991; 107: Davies PR, Ossowicz CJ. Evolution of metos use for etecting Streptococcus suis type 2 in tonsils, an investigation of te carrier state in pigs. Res Vet Sci 1991; 50: Clifton-Haley FA, Alexaner TJL, Enrigt MR. Diagnosis of Streptococcus suis type 2 infection in pigs. Pig Vet Soc Proc 1985; 14: Moreau A, Higgins R, Bigras-Poulin M, Naeau M. Rapi etection of Streptococcus suis serotype 2 in weane pigs. Am J Vet Res 1989; 50: Higgins R, Gottscalk M. An upate on Streptococcus suis ientification. J Vet Diagn Invest 1990; 2: Nei M. Estimates of average eterozygosity an genetic istance from a small number of iniviuals. Genetics 1978; 89: Sneat PHA, Sokal RR. Numerical taxonomy, San Francisco: WH Freeman, 1993: Sambrook J, Fritsc EF, Maniatis T. Molecular cloning: a laboratory manual. Col Spring Harbor: Spring Harbor Laboratory Press, Mogollon JD, Pijoan C, Murtaug MP, Collins JE, Cleary PP. Ientification of epiemic strains of Streptococcus suis by genomic fingerprinting. J Clin Microbiol 1991; 29: Robertson ID, Blackmore DK. Experimental stuies on te comparative infectivity an patogenicity of Streptococcus suis type 2. II. Porcine an uman isolates in laboratory animals. Epiemiol Infect 1990; 105: Williams AE, Blackmore WF, Alexaner TJL. A murine moel of Streptococcus suis type 2 meningitis in te pig. Res Vet Sci 1988; 45: Kebee M, Cengappa MM, Stuart JG. Isolation of caracterisation of temperaturesensitive mutants of Streptococcus suis: efficacy trial of te mutant vaccine in mice. Vet Microbiol 1990; 22: