Comparison of the exotoxins of four strains of Corynebacterium pseudotuberculosis

Research in Veterinary Science 1989, 47. 190-194

Comparison of the exotoxins of four strains of Corynebacterium pseudotuberculosis S, S. SUTHERLAND, E. J. SPEIJERS, B, ANDRES, Department of Agriculture, Baron Hay Court,

South Perth, Western Australia 6151

The activity of exotoxins produced by four strains of Corynebacterium pseudotuberculosis were compared by their ability to kill white mice, their haemolytic activity, staphylococcal haemolysin-inhibiting effect and activity in an enzyme-linked immunosorbent assay (ELISA). Exotoxins with a haemolytic titre of ] in 256 or more killed all mice and had the most inhibitory effect (l in 64 or more) on staphylococcal haemolysin. The haemolytic test and staphylococcal haemolysin inhibitory test of exotoxin activities were highly correlated (P
CORYNEBA CTER1UM pseudotuberculosis pro-

duces a powerful exotoxin which has been shown to playa role in the pathogenesis of caseous lymphadenitis in sheep (Jolly 1965, Hsu et al 1985). The exotoxins produced by different strains and biotypes of C pseudotuberculosis are reported to be antigenically similar (Doty et al 1964, Muckle and Gyles 1983). There is, however, some variation in the ability of the different strains of C pseudotuberculosis to produce exotoxin and this has been associated with the numbers of lesions and severity of infection in sheep (Burrell 1978). The exotoxin is a phosphatidylcholine phosphatidohydrolase, commonly known as phospholipase D (Soucek et aI1971). It is responsible for the lethal effect in mice (Jolly 1965, Lovell and Zaki 1966a), the staphylococcal haemolysin-inhibiting effect (Lovell and Zaki 1966b), and the haemolytic and haemagglutination effects (Burrell 1979, I980a). In mice, no correlation between exotoxin activity (as measured by a radioassay for phospholipase D and a (3-haemolysin inhibition test) and virulence, as determined by the numbers and size of abscesses, appears to exist (Muckle and Gyles 1983). Many tests have been devised to detect and quantitate the exotoxin and these have been documented in a recent review by Brown and Olander (1987). There is, however, some speculation as to how these tests relate to one another and whether they measure the same activity of the exotoxin (Lovell and Zaki 1966b, Burrell 1979).

This study compares several in vivo and in vitro measurements of exotoxin activity produced by four strains of C pseudotuberculosis. The effects of fractionating each of the exotoxins with ammonium sulphate is compared by measuring the haemolytic activity, staphylococcal haemolysin-inhibiting effect and activity in an enzyme-linked immunosorbent assay (ELISA). The value of each test in detecting the C pseudotuberculosis exotoxin is also discussed. Materials and methods

Bacteria Toxigenic C pseudotuberculosis strain 231 (231) and a non-toxigenic strain 137E (137E), both serotype I strains (Barakat et al 1984), have been described previously (Burrell 1979). C pseudotuberculosis strain 1030 (1030) (serotype I) isolated from a caseous lymphadenitis lesion in a sheep in Western Australia was described by Robertson (1980). A nitrate positive serotype II C pseudotuberculosis strain (Theka) isolated from a horse in Kenya was kindly provided by Dr R. Batey (Department of Primary Industry, Perth, Western Australia).

Culture media and preparation of supernatant Each of the four C pseudotuberculosis strains was inoculated into 2 x 150 ml protease peptone meat extract broths containing glucose (BurrellI980b); one of the broths contained 0·1 per cent sorbitan monooleate (Tween 80) (Brogden et al 1984). The method of inoculation and incubation was as described by Burrell (1980b). After 30 hours growth, when the pH of the culture was between 5· 5 and 6,0, the supernatant (approximately 150 ml) of each of the eight broth cultures was collected following centrifugation at 2000 g for 20 minutes, passed through a 0·22 J.lm Millipore filter and stored at - 20°C.

Ammonium sulphate precipitation To 30 ml of each of the eight broth culture supernatants ammonium sulphate was added to 10 per cent

190

Corynebacterium pseudotuberculosis exotoxins 0)

000000

442000

000000

2000 u 1600 ~* o Ql 1200 E.;:; Ql800 I'" 400

100 80 60

Ul


::;-

wo

'2

3·0 2·4

c

.2. :a~ ..r:._ - .~ E

1000 800

600

0·6 oh--';:~~­ 30 24

400

18 12

200

6

o

I

1·2

o-+--,--,--,--,--f--

......

Neat 10 20 30 40 50

430000

Ql

'"

20

3 ?O+--r-T-~=-fJ"""''­ 1·8

0·6

400000

QlQl

...J

~g 100

2·4

1·8 1·2

--

rot)

.r:.

250 u 200 ~. o Ql 150

40

o"'*-,-+f::+-....-

Vl:J

01

(ii)

(a)

443400

50

o-+--.-+=f-.......

o-i;--,--,...-,...-,...--r

c

.2. c

.....-

O-+-=~~~ Neat 10 20 30 40 50

Ammonium sulphate saturation (%) "Reciprocal end-point dilution

(bl

(ii)

cot)

;~

...J

250 200

Ql

000000 1·0

0·8 .!!l

« 'c 0·6 Vl :J ::; 0·4 w 0 '2 0·2

2?5+-,...--.---.---r---r

2'0 .!!l 1·6 ~ '§ 1·2 ~ 00·8 '2 0·4

~g 150

....

191

c

2·0

:~"Ql 1·5 ~·S 1·0 E

0'5

0-+-......................_..,.

Neat 10 20 30 40 50 Ammonium sulphate saturation (%1 "Reciprocal end· point dilution

100 50

o-+--r--P..............

Neat 10 20 30 40 50 Ammonium sulphate saturation (%) "Reciprocal end-point dilution

FIG 1: The lethal effect of each 'toxin' at 0 to 50 per cent saturation with ammonium sulphate. Actual values for haemolytic titre, staphylococcal haemolysin-inhibition titre and ELISA (00 units) versus percentage ammonium sulphate saturation for (i) 1030(.) and 231 (e); (ii) 137E (.); and Theka (e) and fitted regression lines for 1030 (--),231 (- - -I, 137E ( - - ) and Theka (- - -) (a) with Tween and (b) without Tween. 0 Indicates the toxic index for each strain. The scales on the abscissa for each test vary in order to illustrate best how each 'toxin' responds to fractionation

saturation. A 5 ml sample (SI) of the supernatant was removed from each of the eight cultures after centrifugation at 2000 g for 20 minutes, ammonium sulphate was added to the remaining supernatant to give 20 per cent saturation, and a second 5 ml sample (Sz) of the supernatant removed after centrifugation. The procedure was repeated a further three times increasing the ammonium sulphate saturation in the supernatant each time by 10 per cent until the supernatant was saturated to 50 per cent ammonium sulphate. The fractions collected from each of the eight cultures were dialysed separately for eight hours against saline. The dialysis procedure was repeated a further two times and the fractions stored at - 20°C.

Assay systems Mouse test, White mice of 20 to 25 g bodyweight were used in all experiments and all inoculations were given by the intraperitoneal route. Four mice were each inoculated with O·5 ml of one of the eight broth culture supernatants or their respective five fractions. The number of deaths in each group of four mice was recorded after four days. Haemolytic activity. The haemolytic activity of each broth culture supernatant and their respective fractions was assessed using washed sheep red blood

cells (Burrell 1979). Titres were expressed as the reciprocal of the end-point dilution.

Inhibitory action of 'toxin' preparation on staphylococcal haemolysin. The inhibitory activity of the supernatants and fractions was assessed as the highest dilution of the supernatant or fraction to inhibit the lytic action of staphylococcal haemolysin on bovine red blood cells (Zaki 1968). Titres were expressed as the reciprocal of the end-point dilution. ELISA. Polystyrene flat-bottomed microtitre plates (Disposable Products, Adelaide, South Australia) were labelled at 37°C with each supernatant and fraction diluted I in 50 in carbonate bicarbonate bu ffer pH 9· 6. The test was performed as described by Sutherland et al (1987) using a standard positive control serum. The results were standardised from plate to plate by inclusion of duplicate I: 100 to 1:3200 dilutions of the positive control serum into wells labelled with a standard toxin antigen preparation as described by Sutherland et al (1987). The optical density (on) of the solution in each well was recorded.

Statistical analysis Lethal effects on mice. Logit regressions were used to describe the relationships between the percentage

S. S. Sutherland, E. J. Speijers, B. Andres

192 ta)

*

100 "C

'" Q)

"C

#.

80

*-*:....-~-*

*

60

40 20 0

lb)

1

2

4

8

16

32 64 128 256 512 10242048

Reciprocal end-point dilution

each supernatant in both broths with respect to each assay system was defined as the test value for 0 saturation estimated from the regression line.

Comparisons between tests. Correlation coefficients were calculated between each of the tests using results from all supernatants and their fractions. Results

Lethal effects on mice

The lethal effects on mice of the 'toxin' preparations from four strains of C pseudotuberculosis * ** grown in the presence and absence of Tween and the 80 corresponding ELISA, haemolytic and staphylococcal haemolytic-inhibitory activities are shown in Fig I. 60 Most of the mice that died did so in the first 48 hours 40 after inoculation. Supernatant broth cultures and ammonium 20 sulphate fractions showing a haemolytic titre of 256 o-1--fIILl~~!!Y'-+-...,...-~L,--r---r----r­ or more (Fig 2), killed all inoculated mice and had the o 0·2 0'40'60'8 1·0 1·2 1·4 1·61'8 2·02·2 most inhibitory effect (64 or more) on staphylococcal Optical density haemolysin. In contrast, supernatants and ammonium sulphate fractions which did not haemolyse red blood cells were not lethal for mice and 100 did not inhibit staphylococcal haemolysin. * The regression coefficient for all three logit 80 regressions were significantly different from zero 60 (P
(c)

"C

'" Q)

"C

#.

FIG 2: The percentage of mice dead versus the actual test values (") and fitted regression lines for (a) haemolytic titre, (b) ELISA and (c) staphylococcal haemolysin-inhibition test

of mice dying and the logarithms of haemolytic activity, staphylococcal haemolytic inhibiting effect and ELISA activity. Log likelihood statistics were used to test whether the regression coefficient for each assay system was equal to zero and to indicate whether deviations from the regression were significant. The LDSOs and their 95 per cent confidence limits were estimated from the regressions.

Toxicity ofsupernatant and fractions. The toxicity of the culture supernatants for both broths and the change with increasing ammonium sulphate saturation was examined by fitting linear regressions between a log transformation of the test result and the saturation level (0, 10,20,30,40,50 per cent) for each supernatant and each assay system. The toxic index of

Toxicity ofsupernatants and fractions Regressions between test results for haemolytic activity, ELISA activity and the inhibition effect on staphylococcal haemolysin-inhibition and ammonium sulphate saturation level for each of the four strains of C pseudotuberculosis grown in the presence or absence of Tween are shown in Fig I. When tested by the haemolytic test and staphylo-

TABLE 1: LD60a and thair uppar and lowar 95 per cent confidence limits for aach assay system Assay system Haemolysin" Haemolysin inhibition" EllS At

LD50

121 53

1·014

" Model good fit. Deviance not significant t Model poor fit. Deviance P
95% confidence limit

160·9 66·9

1·173

97·7 38·9

0·875

Corynebacterium pseudotuberculosis exotoxins coccal haemolysin-inhibition test the toxic indices of strains 231 and 1030 grown in the presence or absence of Tween were significantly higher (P<0·05) than those of the non-toxigenic 137E strain. For both tests, the toxic indices of strain Theka grown in the presence of Tween was significantly higher (P
Comparison between tests The results ~ f all assay systems used to detect the toxicity in the 'supernatants and fractions were highly correlated (P
Many C pseudotuberculosis strains may resemble one another, for example, in the antigenic specificity of their exotoxins (Shigidi 1975). However, differences in the toxicity of the exotoxins and in the ability of C pseudotuberculosis strains to reduce nitrates have been reported (Burrell 1979, Barakat et al 1984). The four C pseudotuberculosis strains used in this study were selected for their ability to produce exotoxins which would demonstrate strain differences. It was found that two of the four strains of C pseudotuberculosis (strains 231 and 1030) produced an exotoxin which was lethal for mice usually within 48 hours of inoculation. In contrast, the exotoxin

193

from the nitrate positive Theka strain, which proved more toxic than the non-toxigenic strain 137E when assessed by the haemolytic and haemolysin-inhibition tests, failed to kill mice. In guinea pigs however nitrate positive strains are reported to be more lethal than nitrate negative strains (Barakat et aI1984). Not only was the exotoxin from the Theka strain significantly less toxic than those from strains 231 and 1030, but the toxic component behaved quite differently as the test results decreased very much more rapidly at higher ammonium sulphate saturation levels indicating a different solubility pattern (Heide and Schwick 1979). Fractionation of the two toxigenic strains (1030 and 231) provided no additional information about the exotoxins produced by different C pseudotuberculosis strains. This result suggests that the differentiation of exotoxins produced by such strains is best achieved by directly testing the culture supernatant. All strains of C pseudotuberculosis used in this study were grown under optimal conditions for haemolytic activity of C pseudotuberculosis (Burrell 1979, 1980b). The haemolytic titres of the most toxigenic strains (strains 231 and 1030) used in this study were one to three twofold dilutions higher than the haemolytic titres reported for similar strains grown in the presence of serum (Burrell 1979, 1980a). In contrast the titres were considerably lower than the 16,384 reported for a toxigenic C pseudotuberculosis grown in the absence of serum (Burrell I980b). Such a large discrepancy in the haemolytic activity is difficult to explain as the media, conditions of incubation and subsequent changes in the pH of the culture after 18 hours were as described by Burrell (I980b). The addition of O· I per cent sorbitan mono-oleate (Tween 80) to the culture media did, however, increase the haemolytic activity of the toxigenic strain when compared to the same strains grown in the absence of Tween 80 but the titres obtained were still not comparable to those previously reported (Burrell 1980b). A low but non-specific haemolytic titre was recorded for the non-toxigenic strain 137E grown in the presence of Tween 80 but the toxic index for this test was still significantly lower than the toxic index of the two most toxigenic strains (1030 and 231) and for the Theka strain. A highly significant relationship between the number of mice dying and the haemolytic and staphylococcal haemolysin-inhibition results, was evident (Fig 2, regression curve) and consistent with reports on the value of each of these tests in detecting active toxin (Lovell and Zaki 1966a, Burrell 1979). The results in this study suggested that both tests measure, the same activity of the exotoxin or two different highly correlated activities. The high statistical correlation between these two tests varied from other studies which suggest that the staphylo-

194

S. S. Sutherland, E. J. Speijers, B. Andres

coccal haemolysin-inhibition test may detect both active and inactive exotoxin whereas the haemolytic assay (Burrell 1983) and radioassay measuring phospholipase D activity (Muckle and Gyles 1983) detect enzymatically active exotoxin. Nevertheless, in the latter study even though the results of the 25 C pseudotuberculosis isolates showed poor correlation between the staphylococcal haemolysin-inhibition test and radio assay, all isolates were positive for exotoxin in both tests. The ELISA results in this study on the other hand, showed little correlation with the haemolytic and staphylococcal haemolysin-inhibition results. This, in conjunction with the fact that some samples with high ELISA results (particularly the exotoxin produced by the strain Theka) were not lethal for mice suggest that the ELISA is of little or no value for the measurement of exotoxin activity. References BARAKAT, A. A., SELlM, S. A., ATEF, A., SABER, M. S., NAFIE, E. K. & EL-EBEEDY, A. A. (1984) Revue Scientifique Office International des Epizooties 3,151-163 BROGDEN, K. A., CUTLIP, R. C. & LEHMKUHL, H. D. (1984) American Journal of Veterinary Research 45, 2393-2395 BROWN, C. C. & OLANDER, H. J. (1987) Veterinary Bulletin 57,1-12 BURRELL, D. H. (1978) Proceedings of the 55th Annual Conference of the Australian Veterinary Association. pp 79-81 BURRELL, D. H. (1979) Research in Veterinary Science 26, 333-338

BURRELL, D. H. (I 980a) Research in Veterinary Science 28,51-54 BURRELL, D. H. (1980b) Research in Veterinary Science 28, 234-237 BURRELL, D. H. (1983) New South Wales Veterinary Proceedings. pp 53-57 DOTY, R. B., DUNNE, H. W., HOKANSON, J. F. & REID, J. J. (1964) American Journal of Veterinary Research 25, 1679-1685 HEIDE, K. & SCHWICK, H. G. (1979) Handbook of Experimental Immunology. Ed D. M. Weir. Oxford, Blackwell Scientific Publications. pp 7.1-7.11 HSU, T. Y., RENSHAW, H. W., LIVINGSTONE, C. W., AUGUSTINE, J. L., ZINK, D. L. & GAUER, B. B. (1985) American Journal of Veterinary Research 46, 1206-1211 JOLL Y, R. D. (1965) Journal of Comparative Pathology 75, 415-431 LOVELL, R. & ZAKI, M. M. (l966a) Research in Veterinary Science 7, 302-306 LOVELL, R. & ZAKI, M. M. (I 966b) Research in Veterinary Science 7, 307-311 MUCKLE, C. A. & GYLES, C. L. (1983) American Journal of Veterinary Research 44,1149-1153 ROBERTSON, J. P. (1980) MSc thesis, Murdoch, Western Australia SHIGIDI, M. T. A. (1975) Bulletin of Epizootic Diseases of Africa 22,263-269 SOUCEK, A., MICHALEC, C. & SOUCKOVA, A. (1971) Biochimica et Biophysica Acta 227,116-128 SUTHERLAND, S. S., ELLIS, T. M., MERCY, A. R., PATON, M. & MIDDLETON, H. (1987) Australian Veterinary Journal 64, 263-266 ZAKI, M. M. (1968) Research in Veterinary Science 9, 489-493

Received August /6, /988 Accepted November II, /988