ORIGINAL ARTICLES

Streptococcus suis serotypes associated with disease in weaned pigs RP GOGOLEWSKI*, RW COOKt and CJ O’CONNELLS New South Wales Agriculture and Fisheries

SUMMARY: Streptococcus suis was recovered from 9 outbreaks of septicaemia and meningitis in weaned pigs between 1979 and 1983. Fifteen isolates from 7 outbreaks were identified as S. suis type 9, and 3 isolates from 2 outbreaks as S. suis type 2. Three further isolates of S. suis type 2 and an isolate of S. suis type 3 were recovered from cases of bronchopneumonia in weaned pigs from 4 other piggeries. Aust Vet J 67: 202-204

Introduction Streptococcus suis type 2 (the “group R”, alpha-haemolytic streptococcus) causes septicaemia and meningitis in weaned pigs in many countries (Clifton-Hadley 1985), including Australia (Buddle et a1 1981). This disease syndrome is widespread in the United Kingdom (Lamont et al 1980), where S. suis type 2 is the most common cause of meningitis in pigs (CliftonHadley and Alexander 1988). In Australia, a similar syndrome in weaned pigs has also been caused by alpha-haemolytic streptococci, whose serotyping proved inconclusive (Friend and Sims 1978; Seaman and Armstrong 1979). In this paper we examine isolates of alpha-haemolytic streptococci associated with outbreaks of septicaemia and meningitis. and cases of pneumonia in weaned pigs in Australia.

Materials and Methods Source of Specimens Field isolates of alpha-haemolytic streptococci in this study were recovered at necropsy from weaned pigs from 13 piggeries, between 1979 and 1983. The piggeries housed between 30 and 2,500 sows and were located in New South Wales, Victoria or Western Australia. Cases of septicaemia and meningitis were examined from piggeries 1 to 9, and of bronchopneumonia from piggeries 10 to 13. Bacteriology Brain, heart blood, lung, liver and intestinal contents from most animals with septicaemia and meningitis were cultured aerobically on 7% citrated sheep blood agar. Joints, including those with evidence of arthritis, were also cultured. Lungs from cases of bronchopneumonia were similarly cultured. Isolates of alpha-haemolytic, Gram-positive, catalase-negative cocci were subcultured onto 7% sheep blood agar. Biochemical reactions and growth tolerance were tested by the methods of Cowan (1974). Serology Antiserums to strepococcal isolates from piggeries 1 (isolates la, l b and lc), 2, 3, 8 and 11 and to reference isolates of S. suis types 1 to 80 were prepared in rabbits as described by Elliott (1960). Antiserum to S. suis type 9 was prepared in Denmark as described by Perch et al (1974). Antiserums to streptococcal Lancefield groups A to H, K to N, and P, and to “group R” and “group S” were obtained commercially.(

Veterinary Laboratories, Glenfield, New South Wales 2167

t Regional Veterinary Laboratory, Wollongbar, New South

* Wales Regional Veterinary Laboratory, Wagga Wagga, New South Wales 2480

2650

0 Reference isolates of

S. suis types 1 to 8 were supplied by Dr J Henrichsen, The Streptococcus Department, Statens Seruminstitut, Copenhagen, Denmark, and by Dr S D Elliott, Department of Pathology, University of Cambridge, England 1 Wellcome Streptococcal Grouping Serum, W e l l c o m e Reagents Ltd, England

202

Slide agglutination test - Test organisms from colonies growing on 7% sheep blood agar for 18 h were suspended in normal saline. A drop of this suspension was mixed with a drop of undiluted antiserum on a glass slide, and macroscopic and microscopic agglutination were assessed immediately. Control preparations were examined for autoagglutination. Precipitin test - Antigens were prepared by phenol extraction (Elliott et a1 1977) from one streptococcal isolate from each piggery, except piggeries 10 and 13, and from the reference isolates of S. suis types 1, 2 and 3. Each antigen extract was dissolved in phosphate-buffered saline and a drop was layered on top of antiserum in a tapered glass tube sealed at the tip. The tube contents were examined immediately, and after 15 minutes and 10 hours incubation at room temperature. The test was considered positive if precipitate was observed at any time. Cougglutinution test - Selected streptococcal isolates were tested for streptococcal group D antigen using a commercial test kit.#

Results History and Pathological Findings

Outbreaks of septicaemia and meningitis on piggeries 1 to 9 involved weaned pigs between 4 and 8, and occasionally up to 12 weeks of age. Most pigs died without premonitory signs. Some pigs showed neurological signs including ataxia, recumbency, clonic spasms and convulsions before death. At necropsy, visceral organs were congested and the body cavities frequently contained clear to slightly turbid, yellow fluid. In some pigs with neurological signs the leptomeninges were oedematous and opaque. Some had fibrinopurulent polyarthritis. In those that died suddenly there were few microscopic changes apart from the presence of Gram-positive cocci in lumens of blood vessels. Some of these pigs had suppurative leptomeningitis, as did all pigs that died with neurological signs. Overcrowding, poor ventilation, or draughty conditions in upper compartments of weaner crates were considered to be predisposing factors in some outbreaks. The disease has persisted on most of these piggeries, with a variable response to antibacterial medication and improvement in the housing of weaned pigs. On piggeries 10, 12 and 13, coughing and weight loss were seen in pigs aged between 5 and 12 weeks of age, and on piggery 11 similar signs were seen in 2 of 20 sentinel weaners housed with 14 recently-purchased boars. Suppurative bronchopneumonia was found at necropsy of an affected pig from each of these piggeries. Bacteriology Alpha-haemolytic streptococci were usually recovered in dense, pure growth from brain, heart blood, lung, liver and #

PhadebacP Strep D Test: Pharmacia Diagnostics AB, Uppsala, Sweden Australian veterinary Journal, Vol. 67. No. 6 , June, 1990

and 3 and antiserums to “group R” and “group S”. The results were the same as those obtained in slide agglutination tests. In tests of these extracts against antiserums to Lancefield groups A to H, K to N, and P, group antigen D was detected in the isolate from piggery 11 and in reference isolates of S. suis types 1, 2 and 3; no other reactions occurred.

joints of the 21 pigs from the outbreaks of septicaemia and meningitis on piggeries 1 to 9. In some animals with neurological signs these organisms were isolated only from brain or joints. Alpha-haemolytic streptococci were recovered from the lungs in pure growth from a pig from piggery 10, with MycopIasma hyopneumoniae and PasteureIIa multocida from a sentinel pig from piggery 11, with P. multocida from a pig from piggery 12, and with Flavobacterium sp from a pig from piggery 13. After 12 to 18 h incubation on sheep blood agar, all the streptococcal isolates formed colonies Imm in diameter, surrounded by a zone of alpha haemolysis lmm wide. After 48 h this zone of haemolysis was 2mm wide, comprising an inner zone of greening and an outer zone of partial clearing of the blood agar. On further incubation there was progressive clearing of these zones but the distinctive greening of the inner zone persisted even after 72 h incubation. Results of biochemical and growth tolerance tests for the 25 isolates from piggeries 1 to 13 and the reference isolates of S. suis types 1 to 8 were similar and conformed with those described for S. suis (Perch et al 1981; Kilpper-Balz and Schleifer 1987). S. suis type 1 differed from the other isolates in its failure to ferment melibiose and raffinose.

Coagglutination test - Lancefield group D antigen was demonstrated in the isolates tested from piggeries 1 to 13 and in reference isolates of S. suis types 1 to 9. On the basis of these biochemical and serological results we identified 15 isolates of S. suis type 9 (piggeries 1 to 7), and 3 isolates of S. suis type 2 (piggeries 8 and 9) from cases of septicaemia and meningitis, and 3 isolates of S. suis type 2 (piggeries 1 0 and 11) and 1 isolate of S. suis type 3 (piggery 13) from cases of bronchopneumonia. The isolate of S. suk type 2 from piggery 9 has been reported previously (Buddle et al 1981). Discussion S. suis type 9, a recently-identified serotype of S. suis (Gottschalk et al 1989) was isolated from 7 of the 9 outbreaks of septicaemia and meningitis in weaned pigs in this study. There is strong evidence that S. suis type 9 was the cause of these 7 outbreaks; it was usually isolated in pure growth from brain, heart blood, lung, liver and joints of affected pigs, some of which had lesions of leptomeningitis and polyarthritis. S. suis type 2, the most commonly reported cause of this syndrome in weaned pigs (Windsor and Elliott 1975; CliftonHadley 1985, Clifton-Hadley and Alexander 1988) was involved in only 2 of the 9 outbreaks. The recognition of S. suis type 9 as the main cause of this disease syndrome in this study reflects recent progress in defining S. suis serotypes, which now total 22 (Gottschalk et al 1989). We believe that untypable alpha-haemolytic streptococci associated with previous outbreaks of this syndrome in weaned pigs in Australia (Friend and Sims 1978) are also likely to have been S. suis type 9.

Serology Slide agglutination test - The results of testing are shown in Table 1. The 15 isolates tested from piggeries 1 to 7 comprised a single serotype that reacted only with the 5 antiserums to isolates from piggeries 1, 2 and 3, and with the antiserum to the reference isolate of S. suis type 9. Three other isolates from piggery 6 autoagglutinated and could not be tested. The 6 isolates from piggeries 8 to 12 reacted only with the antiserums to isolates from piggeries 8 and 11, and with antiserum to the reference isolate of S. suis type 2 (and with “group R” antiserum). The isolate from piggery 13 reacted only with antiserum to the reference isolate of S. suis type 3. Precipitin test - The antigen extracts of isolates from 11 piggeries and of reference isolates of S. suis types 1, 2 and 3 were tested against antiserums to isolates from piggeries 1, 2

TABLE 1 Slide agglutination reactions of S. suis isolates Antiserum Reference isolates* s1

s4

Number of

isolates

S8

Septicaemia/meningitis 1 (NSW 2 (NSW) 3 (NSW) 4 (NSW) 5 (NSW) 6 (VIC 7 (VIC) 8 (NSW 9 (W4

~~~

s3

s5 S6 s7

“S” Piggery (location)

s2 “R”

Field isolatest

s9

la

2

3

8

11

lb

lc

+$ +§

+ + +

NT NT

-5 -

~

*Antiserums to reference isolates of S. suis (Sl-S9) reacted only with their homologous serotype. “S” and “R” are commercial antiserums to S. suis types 1 and 2 respectively. tAntiserums to isolates from piggeries 1, 2 and 3 did not react with reference isolates of S. suis types 1 to 8. Antiserums to isolates from piggeries 8 and 11, reacted with the reference isolate of S. suis type 2 and not with reference isolates of S. suis type 1 or types 3 to 8. These antiserums were not tested against the reference isolate of S. suis type 9. $Two isolates tested §One isolate tested + = positive; - = negative; NT = not tested

Australian VeterinaryJournal. Vol. 67, No. 6, June, 1990

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Field et a/ (1954) described outbreaks in Great Britain of septicaemia, meningitis and polyarthritis in sucking pigs, caused by alpha-haemolytic streptococci that could not be placed into recognised Lancefield groups. De Moor (1963) studied similar isolates from septicaemic infections in pigs in Holland and proposed 3 new Lancefield groups, “R”, “S” and “T”; isolates from the outbreaks described by Field et af (1954) belonged to “group S”. Streptococci of groups “S”, “R” and “T” were subsequently shown to possess Lancefield group D antigen, and were designated S. suis capsular type 1 (Elliott 1966), type 2 (Windsor and Elliott 1975) and type 15 (Gottschalk et al 1989) respectively. Isolates possessing both “R” and “S” capsular antigens (S. suis type 1/2) have also been recovered (de Moor 1963, Perch ef a1 1981). The syndrome of septicaemia, meningitis and polyarthritis in sucking pigs caused by S. suis type 1 (group “S”) has recently been reported in Australia (Cook er a/ 1988). S. suis type 2 (“group R”), as well as causing septicaemia and meningitis in weaned pigs, is now frequently isolated from cases of bronchopneumonia in pigs in Europe and North America (Clifton-Hadley and Alexander 1988), often in association with other bacteria including Pasreurella spp. (Sanford and Tilker 1982). Field isolates from among the more recently identified S. sub types 3 to 22, or of non-typable streptococci biochemically consistent with S. sub, have most frequently been from cases of bronchopneumonia, in Denmark (Perch ef a/ 1983), Holland (Vecht et a/ 1985), Belgium (Hommez et a/ 1986) and Finland (Sihvonen et a/ 1988). This association of an increasing number of S. mis serotypes with bronchopneumonia in pigs, and the isolation, in this study, of S. suis types 2 and 3 from cases of bronchpneumonia in 4 piggeries, suggest that the role of S. sub in the pathogenesis of pnewnonia in pigs in Australia warrants evaluation. Previous workers have had difficulty demonstrating Lancefield group D antigen in isolates now designated as S. suis (Field et al 1954; de Moor 1963; Hommez er a/ 1986), which on the basis of hybridisation studies is not closely related to other group D streptococci (Kilpper-Biilz and Schleifer 1987). Elliott et al (1977) suggested that extraction of intracellular group D antigen (lipid-bound teichoic acid) by disruption of cocci or by use of aqueous phenol should replace the traditional methods developed for extraction of cell-wall Lancefield group carbohydrates (eg. heating at pH2 or in formamide). Perch et a/ (1983) confirmed the value of physical disruption of cells for detection of group D antigen in their isolates of S. suis types 7 and 8. With the precipitin test we demonstrated group D antigen in only one field isolate, despite using aqueous phenol for antigen extraction. However, using the commercial coagglutination test, we consistently detected group D antigen in S. suis isolates. In contrast to the group D antigen, the capsular antigens of S. suis are easily demonstrated. The slide agglutination test is a simple method for determining the capsular serotype of

isolates. The occasional isolates of S. suis that autoagglutinate can be serotyped by the more time-consuming precipitin test. Our findings confirm the need to serotype all isolates of S. suis associated with disease in pigs, to improve our understanding of the epidemiology and pathogenicity of S. suis in Australia.

Acknowledgments We gratefully acknowledge the supply of streptococcal isolates from the Regional Veterinary Laboratories Benalla and Bendigo in Victoria, and from Dr JR Buddle, School of Veterinary Studies, Murdoch University in Western Australia. We thank Drs SD Elliott and J Henrichsen, for examining a selection of streptococcal isolates from piggeries in this study, and M Lawler, AE Stevenson and L Whittingham for technical assistance.

References Buddle JR, Jones JET, Pass DA and Robertson J (1981) - Aust Vet J 57: 437 Clifton-Hadley FA (1985) - In Vet. Annual, edited by CSG Grunsell et a/, 25th issue, Scientechnica, John Wright and Sons Ltd, Bristol, p161 Clifton-Hadley FA and Alexander TJL (1988) - ‘In Practice’ J Vet Postgrad Clin Study (Suppl to Vet Rec) 10: 185 Cowan ST (1974) - In Cowan and Steel’s Manual for the Identification of Medical Bacteria, 2nd edn, Cambridge University Press, Cambridge Cook RW, Jackson ARB and Ross AD (1988) - Aust Vet J 65: 64 Elliott SD (1960) - J Exp Med 111: 621 Elliott SD (1966) - J Hyg, Camb 6 4 205 Elliott SD, McCarty M and Lancefield RC (1977) - J Exp Med 145: 490

Field HI, Buntain D and Done JT (1954) - Vet Rec 66: 453 Friend SCE and Sims LD (1978) - Vic Vet Proc 36: 51 Gottschalk M, Higgins R, Jacques M, Mittal KR and Henrichsen J (1989) - J Clin Microbiol 27: 2633 Hommez J, Devriese LA, Henrichsen J and Castryck F (1986) - Vet Microbiol 11: 349 Kilpper-B5lz R and Schleifer KH (1987) - In1 J Syst Bucteriol 37: 160

Lamont MH, Edwards PT and Windsor RS (1980) - Vet Rec 107: 467 Moor CE de (1963) - Antonie Van Leeuwenhoek, J Microbiol Serol 2 9 272 Perch B, Kjems E and Ravn T (1974) - Acta Path Microbiol Scand (Sect B) 82: 357 Perch B, Kjems E, Slot P and Pedersen KB (1981) - Acta Path Microbiol Scand (Sect B) 8 9 167 Perch B, Pedersen KB and Henrichsen J (1983) - J Clin Microbiol 17: 993 Sanford SE and Tilker AME (1982) - J A m Vet Med Assoc 181: 673 Seaman JT and Armstrong KJ (1979) - Vet Notes, NSW Dept Agriculture, Div Anim Hlth 13: 74 Sihvonen L, Kurt DN and Henrichsen J (1988) - Acta Vet Scand 29: 9 Vecht U, van Leengoed LAMG and Verheijen ERM (1985) - Vet Quart 7: 315 Windsor RS and Elliott SD (1975) - J Hyg, Camb 75: 69

(Accepted for publication 28 November 1989)

CORRECTION An outbreak of listerial myelitis in sheep In the article An outbreak of listerial myelitis in sheep, by JT Seaman, MJ Carrigan, FA Cockram and GI Carter which was published in the Ausr Vet J (1990) Vol 67, page 142-143, the order of the authors was inadvertently changed. The correct order of the authors is JT Seaman, GI Carter, MJ Carrigan and FA Cockram.

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Australian Veterinary Journal, Vol. 67, No. 6, June, 1990

Streptococcus suis serotypes associated with disease in weaned pigs.

Streptococcus suis was recovered from 9 outbreaks of septicaemia and meningitis in weaned pigs between 1979 and 1983. Fifteen isolates from 7 outbreak...
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