SEROLOGICAL CLASSIFICATION O F AUSTRALIAN STRAINS O F ERYSIPELOTHRIX RHUSIOPATHIAE ISOLATED FROM PIGS, SHEEP, TURKEYS AND MAN G. M. J .

CROSS*$

and P. D.

CLAXTONtS

SUMMARY: 154 strains of Erysipelothrix rhusiopathiae from pigs, sheep, turkeys and man were serotyped by using the double diffusion gel precipitation test. Ten of the 18 serotypes were detected in 151 of the strains. Three strains failed to react with any of the type specific antisera. It was found that serotype l a shared an antigen(s) with serotype l b , and that serotype 6 shared an antigen(s) with serotype 14. Serotype 2a and 2b were difficult to distinguish. Since serotypes 1 and 2 were isolated from cases of septicaemia in pigs, and since serotypes 1,2, 4 and 7 were isolated from cases of arthritis, it was suggested that factors other than serotype were important in causing the various forms of swine erysipelas. The fact that the distribution of serotypes l a , l b and 2b between septicaemic and arthritic pigs was similar supported the conclusion that arthritis was consequent to bacteraemia. Serotypes l a , l b , 2b, 5, 12 and 15 were isolated from cases of arthritis in sheep, and serotypes l a and 5 from cases of erysipelas in turkeys. Serotype 2b was isolated from a human specimen. Introduction

E. rhusiopathiae was considered to be antigenically homogeneous until Watts (1940) distinguished 2 serological types. Atkinson (l941), Gledhill (1945), Dedie (1949) and Szent-lvanyi (1952) confirmed the existence of antigenic differences between strains. Subsequently, more serological types were described in different parts of the world by many authors (Heuner 1958; Murase et a1 1951a; 1959b, c; Murase and Ebi 1960; Kucsera 1962, 1971; Truszczynski 1963; Castro 1969). Kucsera (1972, 1973) found that the above authors had used different designations to describe identical serotypes and the same designations to describe unrelated serotypes. He proposed that the serotypes be given Arabic numbers, and identified 18 serotypes (Kucsera 1973; 1975), keeping as much as possible to the chronological sequence of the type descriptions. N o attempt has been made to serotype E. rhusiopathiae strains isolated in Australia since Atkinson's study (1941). This paper describes the serotyping of Australian isolates of E . rhusiopathiae from pigs, sheep, turkeys and man. Materials and Methods Type Srrains Tnenty-foul- type strains of E. r/~u.tioparhiarrepresen~ing 18 serotypes \\ere obtained from I l r G. Simmons o f the Department of Primary Indu%tries,Q u e e n h n d , \\ h o had obtained

Ausrralian \'ereIiriar~.loiirnal, V o l . 5 5 , February, 1979

them trom Dr G . Kucsera of the Institute for the Control of Veterinary Serobacteriological Products, Budapest. The type strains are set out in Table 1 .

TABLE 1

Designations used for Type Strains of E. rhusiopathiae Antigen Arabic Designation la lb 2a 2b 3 4 5

5 6 6 I 8 8 9 10 10 11 12 12 13 13 14 15 16

Antigen Alphabe tic Designation

Type Strain Designation A360 EW2

B;

B2

C D L L M M H

B4 Seelachs Seechecht Heilbu tt Pecs 6 7 P-2 3iCastro Tuzok

P-3 2

0

Rotzunge Goda

0

P-92

E P

P

P G

I I J

J K R S

Kaparek 1578/1589 Pecs 100 Pecs 9

M2

Pecs 1 8 Pecs 56 Iszap4 Pecs 3596 Tanzania 111 71

Preparation of Antigen for Double Diffusion Gel Precipitation Test Each type strain was incubated at 37°C for 6 hours in 30 ml of Erysipelothrix Growth F-actor (EGF) broth. 0.5 ml of this culture was transferred t o 300 ml of E G F broth and incubated at 37°C for 16 hour\. The cultures were centrifuged at 2000 g for 30 minutes, the bacteria washed 3 times in saline a n d suspended in 30 ml of distilled water. They were then autoclaved at 121°C for one hour, preserved with 1:1O,OOO merthiolate and the autoclaved \uspensions stored at 4°C. Antigens from isolates t o be serotyped were prepared in the same manner, except that they were grown in 30 ml of EGF broth and suspended in 3 ml of distilled water. Preparation 01' Antigen tor use in the Productiori of Precipitating Aririsera Formalin-killed antigen. A 16-hour EGF broth culture was washed three times in saline and made up t o an opacity of Wellcome tube 4 with 0.2 per cent formalin-saline. Sterility was checked by incubating 1 mi of the formalin-killed antigen in 30 ml of EGF; broth for 24 hours. I f sterile, the antigen was stored at 4°C. Live antigen war prepared in a similar manner except that the washed bacteria were suspended in sterile saline to an opacity of Wellcome tube 4 and stored a t 4°C. Preparation of Anri.sera Each formalin-killed type strain antigen was administered intravenously to 2 adult N e w Zealand White rabbits on 4 occasions at 3 to 4-day intei-vals, using doses rising from I to 4 ml. Subsequently the animals were given live antigen o n the same schedule. Care ivas taken to avoid crosc-sensitisation of rabbits. The animals were e x w g u i n a t e d by cardiac puncture eight days after the last antigen administration. Antisera were preserved with I : 10,ooO merthiolate and stored at 4°C. Preparation of Erysipelothrix Growth Factor Broth (Pullal 1951)

Fresh beef liver was freed from all fat and debris, finely minced and placed in a stainless steel or glass vessel with 2 lirres of tap water for each kilogram of mince. The mixture was stirred well and allowed to soak at 4°C overnight. T h e following morning any fat was qkimmed from the top a n d the vessel was placed in a steamer and heated t o 9 5 ° C in 5 hours. The broth was allowed to cool to 70°C a n d filtered through muslin, cotton wool and glass wool. The reaction was adjusted to p H 8.0 hy the addition of ION NaOH. Difco proteose peptone, or proteose peptone No. 3, 20 gllitre, w a s lightly shaken over the surface of the infwion while i t was \till warm (50OC) and during the process of peptone solution, the following mixture was added to each litreof broth: Glucose -2g NaHCO, -2g NaCl -2g Na,H PO, f 2 H 2 0 --I& The reaction was readjusted t o p H 8.0 and the broth stored at 4°C overnight. The following morning the broth was centrifuged and the clear supernatant was poured off and filtered through either a Seitz EK pad (D9) or a membrane filter (0.22 microns). The filtered broth was distributed into sterile bottles of 300 mls capacity, and stored at 4°C. Preparation of Gel Plates Buffer c o i i i ~rra n tr NaCl Na,HPO,l 2 H 2 0 ( N a 2 H P 0 , - 14.2 g ) NaH2P0,2 H,o 80 per cent phenol Di\tilled Water

78

-

85.0 g 35.8 g

--

60.0 ml 940.0 ml

--

15.6 g

Agar gel Buffer concentrate - 100.0 ml - 900.0 ml Distilled water Ionagar No. 2 (Oxoid) - 15.0 g In order to dissolve the agar, the agar gel was autoclaved ai 110°C for 10 minutes. After cooling to 60"C, the gel was plated out in 12 tnl amounts into plastic Petri dishes (9.5 cm diameter), care being taken to ensure that the agar was of w e n depth. The plates were stored at 4 ° C in a sealed container to prevent dehydration. Technique of Precipirarion Test Serotyping was carried out by the double diffusion gel precipitation method of Oucliterlony ( 1949). Six patterns were cut on each plate. Each pattern con.sisted of a central well .surrounded by 6 wells. Each well was 6 min i n diameter and was separated from other well? by 2 mm. T o test the specificity of the type strain antisera, precipitation tests were set up in a chequerboard-like sy,tem, running all the type strain antisera against all the type strain antigens. Each isolate was then run against all the typc strain antisera. Plates were incubated at 37OC in a humid chaniber for 18 to 24 hours. Source of Isolates The 154 isolates for serofyping were obtained from the following sources: Department of Veterinary Clinical Studies, Sydney University, Camden, New South Wales - 49 Veterinary Research Station, Glenfield, New South Walec - 46 District Veterinary Laboratory, Armidale, Ne\v South Wales - 3 District Veterinary Labot-atory, Wollongbar, New South Wales -. 6 Department of Primary Industries, Queensland - 18 Department of Agriculture, Western Australia 10 Department of Veterinat-y Pathology, Sydney - 17 University Veterinary Research Institute, Victoria - 4 Royal Prince Alfred Hospital, Sydney, New South Wales 1 Information concerning the clinical condition aasociated with each isolate was obtained where possible. The isolates were recovered from the following animals: Pig - 135; sheep - 1 I ; turkey - 4; man - 1; unknown 3.

Results

The results of testing all the type strain antisera against all the type strain antigens are shown in Table 2. With the exceptions of serotypes la, 2a, 2b and 6, each specific antiserum reacted exclusively with its homologous antigen. Antiserum l a reacted with antigens l a and l b , while antiserum Ib reacted only with antigen l b . Antisera 2a and 2b each reacted with antigens 2a and 2b. Antiserum 6 reacted with antigens 6 and 14, while antiserum 14 reacted only with antigen 14. The distribution of serotypes for each clinical condition in pigs is shown in Table 3. The isolates from cases of septicaemia came from serotypes 1 and 2 (la:19; lb:20; 2b:S). Isolates from cases of arthritis had a similar distribution (la:26; lb:22; 2b: 10). Table 4 shows the serotype distribution in each animal species. Isolates from pigs were mainAustralian Veterinarv.loirrna1, Vol. 55, February, 1979

Totakeadvantageof

taxbenefitsonvGurcar, surgeryequipmentgridfur&ture we prescribe

Pioneers in Leasing. Mercantile Credits Limited. NSW: Sydney 20556; Newcastle 26 1355; Albury 21 5277; Port Macquarie 833166; Tamworth 66 1399; Wollongong 299144; Orange 62 8555. ACT: 496111. VIC: Melbourne 6902811; Shepparton 21 1322; Mildura 234231. QLD: Brisbane 2294566; Southport 32 2322; Toowoomba 32 8044. SA: Adelaide 2125641; Mount Gambier 253855. WA: Perth 322 1366: Fremantle 3358922. TAS: Hobart 348188. Australian Vererinary Journal, Vol. 5 5 , February, 1979

MC 263178

xxi

TABLE 2

Results of Double Diffusion Gel Precipitation Tests Between 5 p e Strains of E. rhusiopathiae An tigens

A360 EW2 B4 Seelachs Seechecht Heilbutt Pecs 6 7 P-2 3/Castro Tuzok P-3 2 Rotzunge Goda P-9 2 Kaparek 1578/1589 P P-100 Pecs 9 M2 Pecs 1 8 Pecs 5 6 Iszap-4 Pecs 3596 Tanzania I11

ly serotypes l a , l b and 2b (50:51:17 respectively), with a few from serotypes 2a, 4, 5, 6 and 7. Those from sheep were serotypes l a , lb, 2b, 5, 12 and 15, while those from turkeys were la and 5. The human isolate was serotype 2b. A mixture of serotypes was found in 6 isolates from pigs. These are set out in Table 5 . Three isolates did not react with any of the type strain antisera. Two of these isolates failed to react with homologous antiserum. TABLE 3 Serotypes Isolated from Different Clinical Conditions in Pigs

Discussion

Kucsera (1972) stated that E. rhusiopathiae could be reliably and reproducibiy serotyped by using autoclaved, rnerthiolate-preserved antigens and high-titre precipitating antisera in the agar gel double diffusion precipitation test. By using this method, Kucsera (1972, 1973, 1975) differentiated 18 serotypes of E. rhusiopathiae. In this study, 10 of the 18 serotypes were detected. Since antiserum l a reacted with antigens l a and lb, serotype l a must share an antigen(s) with TABLE 4 Serotypes Isolated from Different Species

Serotype

Serotype

Untyp4 5 6 7 able Total

Condition

la

l b 2a

2b

Septicaemia Arthritis Urticaria Endocarditis Tonsil Lymphnode

19 2 6 1 0 2 2

20 2 2 1 3 3 2

0 0 0 1 0 0

5 0 1 0 2 0 0 0 0 2 0 0 0

Total

50

51

1

17

0 0 0 0 3 0

0 0 0 0 1 0

0 1 0 0 0 0

2 3 1 1

1 2 0 0 0 0

45 63 2

3

129

4 11

2b

UntyP4 5 6 7 12 15 able Total

Species l a

lb

2a

50 Pig Sheep 2 Turkey2 Man 0

51 3 0 0

1 0 0 0

17 2 3 1 1 2 0 2 0 0 2 0 0 0 0 1 0 0 0 0

0 1 0 0

3 0 0 0

129 11

54 54

1

25

1 1 3

148

0 1 0 0

4 1

4

Australian Veterinary .lournal, Vol. 5 5 , February, 1979

Total

2 5

1 1

79

septicaemic pigs ( l a : 19; 1 b:20; 2b:5) and arthritic pigs (la:26; 1 b:22; 2b: 10). This is not surprising if it is accepted that chronic polyarthritis is conseClinical Number of quent to bacteraemia and is not a distinct entity Condition Sero types Occasions related to serotype. Arthritis l a , 2b 2 Murase et a1 (1959a) stated that the recovery of Arthritis la, 6 1 serotypes other than 1 and 2 from the tonsils of Septicaernia lb, 2b 1 clinically normal pigs meant that these additional Septicaernia la, 12 1 serotypes, although capable of multiplying i n the Endocarditis la, 2b 1 tonsils, were unable to extend beyond this site. I n this study, serotypes 4 and 7 were isolated from serotype 1ti. Although this antigen would be pro- cases of arthritis in pigs, and serotypes 5, 12 and duced in m a l l amounts by serotype l a , it would 15 from cases of arthritis in sheep. Murase ef a/ be sufficient to induce antibody formation when (1959a) examined only 40 strains of E. rabbits are hyper-immunised. A similar reason rhusiopathiae, so that i t is possible (as far as pigs could be advanced for the reactions obtained with are concerned) that their population sample was serotypes 6 and 14. The reactions obtained with too low to show up serotypes other than I and 2. these 2 serotypes suggest that they might be sub- The proportion of serotj'pes other than 1 and 2 types of the one serotype, and it is surprising that when compared with the total number of isolates other workers have not found this to be so. Since from cases of arthritis in the pig (5163) is much EGF broth enables E. rhusiopat/?iae to produce lower than the proportion in sheep (5111). I t excessive amounts of cell wall (Cross 1977), it is seems that these additional serotypes might be suggested that this broth is responsible for these more important as causes of arthritis in sheep. The shared antigens being detectable, for they might findings of this study d o not support the concept not be produced in significant amounts in other of an exclusive serotype-related pathology in broths. Any isolsolates which reacted with an- swine erysipelas. Some authors have isolated more than one tiserum la only were allotted to serotype la, while those reacting with antisera l a and 1 b were allot- serotype from the one pig. Szent-lvanyi (1952) ted to serotype 1 b. Serotypes 2a and 2b were dif- isolated serotype 1 from lesions of endocarditis in ficult to differentiate. They share a common an- 2 pigs, as well as serotype 2 from their spleens. He tigen(s) and although serotype 2a appeared to also suggested that the organism might undergo a have a specific antigen the difference between the change in serotype in the living animal. Alter2 sub-types may be quantitative rather than natively, the animal might be infected by a mixqualitative. 'The 3 strains which failed to react with ture of serotypes or might be suffering from a any of the type-specific antisera could have been super imposed infection, The possibility of change N-strains (Iledie, 1949). Such strains d o not pro- in serotype in the living animal has been considered by Fiege (1951), who reported that duce a heat stable type-specific antigen. Only serotypes 1 and 2 have been previously serotypes 1 and 2 became N strains after 50 reported from diseased pigs (Murase et a/ 1959a; passages in mice, and reverted to their original Norrung 1970; Sasaki 1970; Seto et a1 1972; serotypes after seven passages in pigeons. The Hashimoto er a1 1974). These last workers found passage of serotypes 1 and 2 through 20 transfers that isolates from cases of septicaemia belonged to in broth containing ten per cent homologous typeserotype 1 , from cases of arthritis o r urticaria to specific antiserum resulted in a change to N serotype 2, and of endocarditis to serotypes 1 and serotype (Kucsera 1959). Meese (1961) found that 2. These results agreed with those of Murase et a1 after several pigeon passages he induced a change (1959a) from cases of septicaemia and urticaria, of from serotype 2 to serotype 1 . The possibility of Norrung (1970) and Sasaki (1970) from cases of pre-existing or superimposed infection was not considered by these authors. On the other hand, arthriris, and of Seto et a1 (1972) from cases of septicaemia, arthritis arid endocarditis. Murase et Wellman and Thal (1967) could not induce a af (1959a) did state, however, that organisms of change in serotype after either culture in the presence of immune serum or culture on media serotype 2 could cause septicaemia in pigs. In this study, serotypes 1 and 2 were isolated containing broth culture filtrates. N o variation in from cases of septicaemia in pigs, as well as from serotype occurred a f t e r passage of E . cases of arthritis, and serotype 1 was isolated from rhusiopathiae through pigs (Shuman er a / 1965; cases of urticaria. These findings are at variance Wellman and Thal 1967). Cross (1977) found that with those of the above authors. There was little serotype remained constant f o r up to 8 months in experimentally-infected pigs. The finding o f 2 difference i n the distribution of serotypes between TABLE 5 Pig Isolates from which a Variety of Serotypes was Isolated

80

Ausrralian C'rrerirrar~/.I(liiriial, Vol. 5 5 , February. I979

serotypes in some strains (Table 5 ) in this study could mean that the pigs were infected by a mixture of serotypes, they were suffering from a superimposed infection, or that the samples were contaminated. Since a variety of serotypes was recovered from septicaemic and arthritic pigs in this study, it could be that vaccination with one serotype might not protect against infection by another serotype. Dedie (1 949) reported that killed organisms of serotype 2 protected mice against infection by serotypes 1 and 2, but that killed organisms of serotype 1 did not. White (1962) found that pigs vaccinated with killed strains of serotypes 1 and 2 protected pigs against homologous, but not against heterologous challenge. Truszczynski (1963) reported that an attenuated vaccine of serotype 2 failed to protect mice against challenge by serotype 10. Other workers have reported that there is no relation between immunising potential and serotype (Uhlig 1964; Janowski et a1 1966; .lanowski and Mierzejewska 1966). Cross (1977) found that a vaccine prepared from a n organism of serotype 2b, protected mice against challenge by 100 LD,, units of each of serotypes la, l b , 2b, 6, 7 and 15. I n addition a vaccine prepared from serotype 1 b (a highly virulent strain for ice) failed to protect mice against challenge with the homologous organism and a vaccine prepared from an organism of serotype 2b protected mice against challenge by the above organism of serotype lb. Thus it would appear that immunogenicity of a strain is more important than its serotype or pathogenicity as far as vaccine production is concerned. This study has included Australian isolates from pigs, sheep, turkeys and man. The examination of isolates from birds in zoos as well as from fish might reveal additional serotypes. Kemenes et a1 (1970) isolated three serotypes (6, 8 and 14) from birds in the one enclosure in the Budapest zoo. They considered that all E. rhusiopathiae strains isolated within a zoo were worth serotyping, since many species of wild free-living birds shared the water and food with captives, as well as the small fishes which either populated ponds or were supplied daily for feeding purposes. This study has also demonstrated the superior ability of EGF broth t o enable E . rhusiopafhiae to from 16 produce its antigens rapidly. organisms hour EGF broth cultures were used to produce antisera in rabbits of sufficient titre to use in the gel diffusion precipitation test. Overseas workers have reported that it is necessary to incubate E. rhusiopathiae for at least 48 hours to obtain adequate amounts of antigen (Murase et a1 1959a;

Ausrralian Vsterinarv Jo u r n al, Vol. 5 5 , February, 1979

Murase and Ebi 1960; Truszczynski 1963; Norrung 1970; Kucsera 1972). Acknowledgements The authors would like to acknowledge the assistance of workers in the laboratories listed above who provided isolates for serotyping. This work was supported by a grant from the Pig Industry Research Council and by the New South Wales Department of Agriculture. References Atkinson, N. (1941) - Ausr. 1.exp. Biol. med. Sci., 19: 45. Castro, A. F. (1969) - Contribuico para o estudo d o Erysipelothrix rhusiopathiae. Thesis, Sao Paulo. Cited by Kucsera (1973). Cross, G . M. 1. (1977) - A Study o f Erysipelothrix rhusiopathiae Polyarthritis in the Pig. Ph.D. Thesis, University of Sydney. Dedie, K. (1949) - M h . Vet. Med., 4: 7. Fiege, H. (1951) - Ueber die Varianten A , B and N von Erysipel0thri.u rhusiopathiae suis. Inaug. Diss. Munich. p. 31. Cited in Vet. Bull., 23: 234(1953). Gledhill, A. W. (1945) - J. P a f h . Bacr., 57: 179. Hashimoto, K., Yoshida, Y. and Sugawara, H . (1974) - Nafn. Inst. Anim. Hlth. Quart., 14: 113. Heuner, F. (1958) - Arch. exp. Vet. Med., 12: 40. Janowski, H. and Mierzejewska, M. (1966) - Bull. vet. Inst. Pulawy, 10: 106. lanowski, H., Truszczynski, M. and Wasinski, K. (1966) Bull. ve!. Inst. Pulawy, 10: 99. Kemenes, F., Benyeda, .I., Dobos-Kovacs, .I. and Fabian, L. ( 1 970) - Acra. vef. Acad. Sci, Hung., 20: 405. Kucsera, G. (1959) - Acta. vef. Acad. Sci. Hung., 9: 13. Kucsera, G. (1962) - Acta. vet. Acad. Sci. Hung., 12: 43. Kucsera, G. (1971) - Acra. vet. Acad. Sci. Hung., 21: 21 I . Kucsera, G. (1972) - Acta. ver. Acad. Sci. Hung., 22: 251. Kucsera, G . (1973) - Int. .I. Syst. Bact., 23: 184. Kucsera, G.(1975) - Personal Communication t o Mr G. Simmons, Department of Primary Industries, Queensland. Meese, M. (1961) - Arch. exp. Vet. Med., 15: 89. Murase, N., Suzuki, K.. Nakahara, T., Araumi, W. and Hashimoto, K. (l959a) - Jap. .I. vet. Sci., 21: 113. Murase, N., Suzuki, K. and Nakahara, T . (1959b) - Jap. J . vet. Sci., 21: 177. Murase, N., Suzuki, K., Isayarna, Y. and Murata, M . ( 1 9 5 9 ~ ) - Jap. .I. vet. Sci., 21 :2 1 5 , Murase, N. and Ebi, Y . (1960) - .lap. .I. vet. Sci., 22: 1. Norrung, V. (1970) - Acta. vet. scand., 11: 577. Ouchterlony, 0. (1949) - 4cta. path. microbiol. scand., 25: 507. Pullar, E. M. (1951) - Personal communication to the Director, Veterinary Research Station, Glenfield. New South Wales. Sasaki, y . (1970) - Natn. Inst. Anim. Hlth. Quart., 10: 97. Seto, K., Muramatsu, M . an d Yoshida, K . (1972) - a. rep. Natn. Vet. Assay Lab., Japan, No. 9: 31. Shuman, K. D . , Wood, R. L. and Monlux, W. S. (1965) Cornell Vel.9 55: 378. Szent-lvanyi, T. (1952) - Acra. Vet. (Budapest), 2: 109. Truszczynski, M. (1963) - Bull. vet. Inst. Pulawy, 7: 85. Uhlig, H. (1964) - Arch, exp, vet. Med,, 18: 623, Watts, P. S. (1940) - J. Path. Bacr., 50: 355. Wellmann, G. and Thai, E. (1967) - Zentbl. Bakt. Parasit.,

W;ftfg;::$;

? 2 ;4 :,;

vet. Res., 23: 752, (Accepted for publication 21 .luly 1978)

81

Serological classification of Australian strains of Erysipelothrix rhusiopathiae isolated from pigs, sheep, turkeys and man.

SEROLOGICAL CLASSIFICATION O F AUSTRALIAN STRAINS O F ERYSIPELOTHRIX RHUSIOPATHIAE ISOLATED FROM PIGS, SHEEP, TURKEYS AND MAN G. M. J . CROSS*$ and...
494KB Sizes 0 Downloads 0 Views