Studies on clostridium oedematiens

J. CCJMP.

PAW.

1970.

9

80.

VOL.

STLJDIES

ON

CLOSTRIDIUM

OEDEMATIENS”

B)

K.

S.

ROBERTS,

LINDPIFA

S.

G~~vF:N

and E. E.

0 Sheep Diseases Research Laboratories,

WORRALL

Pendik,

Turkq

INTRODUCTION

The three pathogenic types of Cl. oedematiens are the classical type A associated with gas gangrene infections, type B, the black diseasebacillus or Bacillus gigas, the causal organism of bacillary haemogloand type D, or Cl. haemolyticum, binuria of cattle. The historical background of the group was reviewed by Oakley and Warrack (1959) who give the following account of the disposition of soluble antigens : alpha, a lethal toxin, in types A and B; main haemolysins, gamma in type A and beta in type B, and the same beta haemolysin in type D; minor antigens, delta, a labile haemolysin, and epsilon, an opalescing agent, in type A. zeta, a haemolysin, in type B, and theta, an opalescing factor and eta, a tropomyosinase, in type D. To establish procedures in Turkey for the diagnosis and control of black disease in sheep and bacillary haemoglobinuria in cattle, it was necessary to identify, type and estimate the virulence of field strains of clostridia, and to decide on the antigenic composition of strains used in the preparation and the testing of vaccines. Certain problems arose, because there appeared to be many uncertainties regarding the significance of the antigens. It was especially difficult to explain why the beta haemolysin of Cl. oedematiens type B had been ignored and its acceptance as the main lethal antigen of Cl. haemolyticum. In order to throw light on the problem material from an investigation of black diseasein Turkey was obtained (lyigiiren Durkan and Kiliso$u, 1966) and strains from this source and from other workers, 28 in all, were studied. MATERIALS

ANT,

METHODS

Liquid medium. Peptone, 3 per cent. (Evans or Oxoid) or papain digest of lean meat adjusted to O-45per cent. N, 0.2 per cent. Na2HP04, and 0.5 per cent. NaCl, pH 7.5, were sterilised at 115OC. for 30 min. or for 2 hrs. according to the strain to be grown. Solid media. Agar, 1.5 per cent., in liquid medium was similarly sterilised; 10 per

cent. defibrinated horse blood was added when blood agar was required. Meat broth. Liquid medium had one-third its volume of lean beef added before sterilisation. R.B.C.‘s. A suspension of 4 per cent. in physiological saline solution. Lecithin. A 12-egg-yolk/litre solution in physiological saline solution was used at a dilution of 4 per cent. Diluting fluid for haemolysin and lecithinase assays. Calcium chloride, 0.005 M, in physiological saline. * This work was carried out by the Sheep and Goat Special Fund Project established through United Nations.

a UNDP

Diseases Research Laboratories, Pendik, the Food and Agriculture Organisation

Turkey, of the

10

STUDIES

ON

Clostridium oedematiens

Toxins. Toxic filtrates were obtained from 4-day cultures in liquid medium. Glucose was added as required and the pH was adjusted to 7.5 three or four times during active growth. The filtrates were preserved either by addition of glycerin or by precipitation with 33 per cent. ammonium sulphate to obtain dry powders. It was ascertained by experiment that the use of sugar-free media did not produce more 1938). Precipitated toxins were used toxic filtrates (cf. Walbum and Reymann, as 1 per cent. solutions these had approximately the except where stated otherwise; same toxicity as the original filtrates. Strains. Al and Bl were classical strains widely used in the preparation of vaccine and serum, A2 and A3 were strains 1494 and 1495 respectively in the Wellcome Research Laboratories collection, A4 and B2 were a sheep strain and the Tongala strain respectively from the Commonwealth Serum Laboratories, Melbourne, B3 to B15 were isolated in black disease outbreaks in Turkey, B16 and B17 and Dl to D6 were from bacillary haemoglobinuria and D7 was from diagnosis material. Haemolysin and lecithin assays. Red blood cells or lecithin solution were incubated at 38OC. with serial doubling dilutions of filtrate starting at l/20. Haemolysin tests were left at 38OC. for two hours and at room temperature for a further 16 hours and lecithin tests were left at 38OC. for 18 hours. For serum neutralisation, similar dilutions were prepared and serum added to each tube. The mixtures were incubated at 38OC. for 30 min., 1 ml. of a r.b.c. suspension or lecithin solution added, and the tests incubated and read as before. The end points were slight haemolysis or opacity respectively. The appropriate quantity of haemolysin or lecithinase and the equivalent quantities of antiserum are referred to as units. Antisera. The Al, Bl, B16 and Dl antisera bvere prepared by concentration of the plasma of hyperimmunised Merino ram lambs (Erdogon, Worrall, Roberts and Giiven, 1968). Other antisera were the bulked sera from 12 guinea pigs, bled 7 days after _2 subcutaneous injections separated by an interval of 21 days, of alum treated Sensitivity to air. Agar medium was dispensed to give 7 cm. columns in 15 mm. bore test-tubes and these were autoclaved for 2 hours at 115OC. They were removed from the autoclave while still hot and immersed in water at 50°C. As soon as the temperature of the agar had settled at this level two tubes were each inoculated with 1 drop of a meat broth culture, rolled between the hands and incubated at 38OC. for 18 to 24 hours. The zones of clear agar above the culture were measured. Four Cl. oedematirns type A cultures were grown to provide a standard, and the mean depth of the clear zone in an unknown culture was expressed in terms of the mean length of the clear zone in the type A cultures. Fermentation. The pH change in a 3-day culture in meat broth, with 1 per cent. of the appropriate carbohydrate, was compared with that in a control culture. i\ difference of 0.2 in each of two tests was regarded as positive. There was no indication of fermentable matter in the meat broth.

RESULTS

Cultural, Morphological

and Biochemical Characters

All strains conformed with the known characters of the group (Wilson and Miles, 1964), and when they were grown on blood agar or in deep agar, no type differences were found. It is generally thought that type D strains are more difficult to grow on surface media than type B and, in general, this was the experience in this work, but neither susceptibility to air nor failure rate in surface cultures proved this conclusively. Colonies which grew overnight in deep agar cultures in tubes had ‘
et

R. S. ROBERTS

11

al.

of cultivation is not commonly used. It is true that the colonies are not so recognisably characteristic as the surface ones, but cultivation is considerably easier. Strains of all types combined with type ,4 fluorescein antibody conjugate (Batty and Walker, 1964; Van Kampen and Kennedy, 1968). The latter authors, however, reported failure to stain type B with CZ. haemolyticum antibody conjugate. The type characters which were noted were as follows. Only type A strains brought about a O-5 change in pH in glycerin fermentation : this could be said, with less certainty, about maltose fermentation (Table 1). Type A could be distinguished from other types by measurement of air sensitivity (Table 1). Type A T*BLE STRAINS

USED,

THEIR

BIOCHEMICAL INTRAMUSCULAR

1

CHARACTERS INJECTION

AN,, LQ, IN MICE

OF FILTRATES

*Fermentation Tvpe and No. A 1 A2

B

1

Has t

Origin

HWnan ..

U.K. ,.

She:p

AI&.

Sheep

Amt.

.lfaltose

GlUC0.W

GJvcerin .~

t -

t-! im ~.

-t

. ~- T_~

I.

::: R

4

E B

B B B B B B B B

7

10 11 12 13 14 15 16 17

ix D7

. .

T&k.

..

1.

1.

3’

..

9%

1.

>f

7,

,,

.,

3,

..

,>

33

,,

1,

.1

.a

3,

,1

3,

1,

U;A 9,

c&k

Sh:ep

T;;k.

i-

+ -1~ .+ ++ .+i+.+-

-t T +

4

ON

‘f&mitiuity to air

LD,,

ml.

1.2 I .2 I .o 0.8

100.000 1 .ooo 100.000 5 .OOO

2.2 2.2 2.2 1.6 I +I 1.8 I .8 2.0 1.8 2.2

10.000 10.000 10.000 10.000 .500 3.000 10 2.000 5.000 ,500 5.000 200 500 5.000 5.000 10.000 5.000

;:; 2.0 1.8 1.8 2.0 2.0 2.2

0

2.2

:

;:: 1.8 3.8

0 0 0

* r = pH fall, 0.2 to 0.4; + + = pH fall, O-5 to 0.7; + + -t = pH fall 0.8 or more Tubes with no added carbohydrate and those with lactose, salicin or sucrose fell 0.1 or less. t Clear zone in deep agar; mean of 4 type A control cultures = 1.

organisms were small, but type B organisms had the pleomorphic batons of Zeissler and Rassfeld (1929) : in CZ. haemolyticum cultures the individual cells were large, with chains difficult to distinguish from batons. Cross Protection

between

Representive

alpha-Toxin

Producing

Strains

Two classical type A strains (Al, A3) one type A from a sheep (A4), an Australian type B (Bl), a Turkish type B (B3) and a type B strain received as CZ. haemolyticum (B16) were investigated by cross protection assays (Table 2).

12

STUDIES

ON

Clostridium oedematiens TABLE

2

CROSS PROTECTION

Serum dilution protecting

Toxin

A 1 A4 B 1 B 3 A 1 B 1 A3 B 1 x 1 B 16 ,4 3 B 16 illi

A A

1 I

i A A A3 .4 A A A A3 B B

f I I 3 1 1 3 1 1

800 20: 20 800 50 60 800 800 100 60 200 25 50

Toxin

2: B B

3 2

:: B B B B B B B B

f 1 1 16 16 16 16 16 16

Serunz dilution protecting

3200 32 ‘00 20 400 25 16 200 800 100 1:: 50 100

Results of 7 cross protection tests are shown. Each test consisted of two simultaneous titrations of each of two antisera, using 4 serial x 2 dilutions, one series against the homologous and the other against the heterologous toxin. In the first test, Al and A4 antisera (col. 1) were titrated against toxin Al (col. 2) and Al antiserum was 100 times the stronger (col. 3) ; they were also each titrated against A4 toxin (col. 4), and again Al antiserum was 100 times stronger (col. 5). Reading horizontally, in line 1, toxin Al was 4 times stronger than A4 toxin on testing it against Al serum, and again 4 times stronger when tested against A4 antiserum in line 2.

The toxins were diluted so that 0.1 ml. contained an arbitrarily selected test dose in the region of 50 to 100 LDao/ml. The antitoxins were appropriately diluted according to their previously estimated potencies, so that they would neutralise this dose of homologous toxin in a dose of O-1 ml., and three further doubling dilutions were made from this. Each toxin was mxed in equal volumes with each of the four dilutions of each serum, held at 38°C. for 30 minutes, and 0.2 ml. of each mixture was injected intramuscularly into each of two 18 to 22 g. mice. To provide statistical data for the evaluation of the results, the dose response relationship was ascertained by observing mortality in mice injected with toxin and graded doses of corresponding antiserum falling in 20 per cent. steps from 1 / 1 to l/2. Two titrations were made, using groups of 20 mice per level. Total mortality per cent. per level was 0, 3 1, 81, 100, 100, thus indicating a low probability of error in a test in which the serum levels differed by x 2. It will be seen in Table 2 that in each cross protection test, both toxins and sera had the same relative values, whether measured by homologous or heterologous toxin or serum, e.g. in the first test in the table, Al toxin was 4 times stronger than A4 toxin, whether measured by neutralisation by its own or by A4 antiserum, while Al serum was 100 times stronger than A4 serum, whether measured against Al toxin or A4 toxin. Toxicity of alpha Toxin Producers after Neutralisation of alpha Toxin In-vitro tests had shown the absence of any trace of beta anti-haemolysin in the Al serum and only a trace of gamma anti-haemolysin in Bl serum. Therefore

R.

S. ROBERTS

et

cd.

13

these sera, mixed with heterologous toxins, would act as anti-alpha sera. To ascertain whether the remaining haemolysins, or any other agent not neutral&d by the serum of the heterologous type, had any toxicity, the toxins as listed in Table 1 were mixed, type A toxins with Bl antiserum and type B toxins with Al antiserum, in the proportion of 2 parts of toxin to 1 part of serum, and 0.3 ml. was injected intramuscularly in each of 2 mice. The Al and A3 toxins were not neutralised by this amount of Bl serum (Table 2) and for these the amount of toxin in the mixture was reduced to one-fifth. With this adjustment, all mixtures were innocuous. In this experiment up to 2,000 LD,, of alpha toxin were injected into each mouse and it can be concluded, therefore, that if anything lethal other than the alpha toxin was present it accounted for less than 0.05 per cent. of the toxicity. Pathological

Efects

of beta Haemolysin

Haemoglobinuria following intravenous injection of culture filtrate and necrosis following intramuscular injection of Cl. haemolyticum culture have been reported repeatedly since the early investigation of Vawter and Records (1925), who used doses of 35 to 40 ml. of culture to demonstrate necrosis in cattle. Jasmin (1947) found that mortality following intramuscular injection of cultures was correlated with the haemolysin titres of filtrates. No observations on necrotic effects were made in this work. Cultures of Cl. haemolyticum were grown with adjustment of pH to give titres of 10,000 haemolysing doses/ml. or more against horse r.b.c.‘s. It was found that the minimum lethal dose of beta haemolysin for mice by intravenous injection was approximately 1,000 haemolysing dosesand that mice died in approximately 4 hours with severe haemoglobinuria, tumefied tissuesand a spectacular reddening of the limbs, face and ears. This dose accords with the tabulated data of Lozano and Smith (1967). These symptoms were different from the mild haemoglobinuria and respiratory and nervous symptoms which follow intravenous injection of the toxin of Cl. welchii type A in mice. Filtrates of all Cl. haemolyticum strains were innocuous on intramuscular injection in mice in dosesup to 0.4 ml. when given intramuscularly. ,Ctability of Haemolysin and Lecithinase In cultures grown with pH adjustment, the titre of beta haemolysin was high and stable. It appeared possible that the alleged instability of Cl. haemolyticum haemolysin was associated with acid cultures. Four Cl. haemolyticum strains were therefore grown, in duplicate, in meat broth and in meat broth with O-5 per cent. of glucose, and haemolysin and lecithinase tests were carried out after 1, 3, 6 and 9 days. In the glucose medium the mean titre of haemolysin fell to 4 per cent. and the lecithinase to 0.4 per cent. In the control tubes without sugar haemolysin fell to 75 per cent. and lecithinase to 20 per cent. Type A haemolysin and lecithinase were stable also in glucose-free cultures, but deterioration could not be measured in the glucose cultures because non-specific haemolysin and lecithin loss occurred as the cultures aged, presumably due to greater acidity (Table 1). B

14

STUDIES ON Clostridium oedematiens

Susceptibility

of Horse

and Sheep Erythrocytes

to gamma and beta Haemolysins

Four Cl. oedematiens type A and four Cl. haemolyticum strains were grown for 2 days in meat broth. Haemolysin titres were assayed using sheep erythrocytes in one assay and horse erythrocytes in another. The gamma haemolysin in the type A strains was greater in each strain when measured with horse cells and the difference between the mean values was x 9. The beta haemolysin in each Cl. haemolyticum strain was greater when measured with sheep cells and the difference between the means was x 4. 3

TABLE NEUTRALISATION

OF

HAEMOLYSIN TYPE

Haemolysin Type and strain JVo.

In culture

units

320 160

20i

640 160 320

ml.

12:: 160 E 320 8 0 0 0

160 1::

BY

Neutraliscd

units

__-

per I ml. serum

A

D

640 320

320

z

320 640

320

:i

320 160 320

E 40

38200 80 640

z

it 80 160 80

;:

all

Liz 160 320 160 1280

160

CULTURES

Lecithinare

20

1 2 3 4

7

Q-DAY

320 160

B B B B

E D

IN

320 320 E

1: 11 12 13 14 15 16 17

ANTISERA

In culturt? per ml.

43

i B B B B B B B

SPECIFIC

I ml. serum D

A

~~ B 7 3 8

LECITHINASE

Neutralisedpr A

per

2:

AND

0” 0

all

., >. ,, 3, ,>

all

E

ii 40

ii 40 1280 160

:“o

1280 320 1280 1280 1280 320

80

all

20 40 40 40

I. *, I> 3,

2: 80

40

-..

?’ . .

Table 3 shows titres of haemolysin and lecithinase, read left to right along the range of x 2 dilutions, if any by O-05 ml. antisera, read right to left, ‘/20,1/40 . . . . r/2560, and the titre of neutralization from the indicating tube, also on the scale 20, 40 . . . . 2560 units, because by definition there were 20 units ml. in the indicating tube. This gave the sim le rule that if an antiserum was lower in titre than the antigen, 1 tube would remain haemolyse cf for each ~2 of deficiency e.g. type A antihaemolysin in the table. “0” indicates that the titre of antigen was unaffected by the serum and “all” indicates that the antiserum neutralised the haemolysin at dilutions of’/20 and greater. The high titres of lecithinax in the type D cultures were fortuitous.

Haemolysin and Lecithinase Antisera were available against strains Al and Bl, Dl and A3. The type of strains Al, Bl and A3 was well known and it had been confirmed in biochemical

R. s. ROBERTS

et al.

15

and cultural studies. Some CL haemolyticum antiserum from Dr. A. M. Jasmin was also available. Titrations of haemolysin in filtrates of all strains in the presence of these antisera gave clear differentiation into two groups corresponding with cultural and biochemical characters as shown in Table 1. The absence of cross neutralisation was complete, except for an occasional slight neutralisation of type A haemolysin by heterologous antisera. Table 3 shows the results of an experiment in which the super-n&ant fluids from S-day cultures in meat broth of all strains, grown under comparable conditions, were titrated, alone and in the presence of 10 and 50 units of Al and Dl anti-haemolysin respectively. All assays were duplicated using lecithin substrate; in these there was a consistent non-specific neutralisation of approximately 40 units/ml. of lecithinase activity (Table 3), and a similar neutralisation, or interference, was observed in the sera of 4,‘4 normal sheep. While it would be possible to discount this neutralisation it appeared preferable, in the absence of a more precise understanding, to avoid the reaction and to rely only upon the observation of neutralisation of haemolysin. Identification

and Routine

Typing

In accordance with the above results the following procedure was used for the identification of cultures. (1) Purity was ensured by plating on blood agar and incubating aerobically and anaerobically for 3 days. (2) Sensitivity to air was determined. (3) Clear supernatant fluid from a well grown 3-day culture in meat broth was obtained. (4) Haemolysin in this was titrated alone and in the presence of 0.2 ml. of Cl. oedematiens type A antiserum and of type B or type D antiserum, containing 160 or more anti-haemolysin units per ml. If the titre of haemolysin was inadequate, the test was repeated, using sheep r.b.c.‘s; these titrations differentiated type A from types B and D. (5) Two mice were each injected intramuscularly with 0.1 ml. of the supematant fluid, and two mice with 0.2 ml. of a mixture of equal parts of supematant fluid and a type A antiserum containing 50 international units/ml. of gas gangrene (Oedematiens) antiserum. Survival of only the two mice which received antiserum indicated either type A or type B according to which haemolysin had been identified in the in vitro tests. DISCUSSION

Cross protection against the lethal effects of filtrates of types A and B was reported by Albiston (1927), Keppie (1944), and Oakley, Warrack and Clarke (1947), and it is clear from their results that the lethal agent was the alpha toxin. The cross protection experiments described here confirm this. The gamma and beta haemolysin were, in fact, unlikely to be concerned because the quantity in the test dose was less than one thousandth of a lethal dose for mice by intravenous There injection, assessed from the lethality of beta toxin in Cl. haemolyticum. appear to be no grounds, therefore, for supposing that beta haemolysin could assist the pathogenic action of Cl. oedematiens type B, and the likely explanation of its role in nature is that in type B infection death occurs before it has been produced in harmful quantities, and that in bacillary haemoglobinuria the lesion progresses until it is large enough to produce a lethal dose in a few hours. The main reason for carrying out these tests was the unexpected immuno-

16

STUDIES

ON Clostridium oedematiens

logical results obtained with strain B16 which had been acquired as a CL haemolyticum strain for the preparation of vaccine in Turkey. It had to be ascertained whether this was a Cl. haemolyticum producing some lethal antigen in common with type B cultures. It was shown conclusively that it and another strain, B17, classified as a Cl. haemolyticum and recently acquired, were, in fact, type B strains, which must be presumed to have been isolated from black disease in bovines. Strain B16 had been in Turkey for several years and its original number is not known. It cannot, therefore, be determined whether B16 and B17 were different strains. It can be seen from the literature that this confusion has existed from the earliest days, because the classical syndrome of overwhelming haemolysis was sometimes absent in cases presented as bacillary haemoglobinuria (Vawter and Records, 1925) and cultures were not always typical (Sordelli and Ferrari, 1937). It became an objective of this work to discover satisfactory procedures to avoid this confusion. The likely reason was that type B strains, many of which produce abundant haemolysin, are mistaken for type D because the haemolysin was neutral&d in vitro by Cl haemolyticum antiserum; moreover, the identification appeared to be confirmed by pathogenicity on intramuscular injection of culture in guinea pigs. Cultures were identified as Cl. oedematiens on the basis of colony formation on blood agar, absence of growth on a corresponding plate incubated in air and other easily observed cultural characters. Though they were only used occasionally in this work there appeared to be advantages in deep agar cultures, which, if the agar was autoclaved to compensate for its low heat conductivity, gave characteristic colonies after overnight incubation. Type A could be distinguished from types B and D by three simple observations. (1) Type A organisms were small, type B were larger and had pleomorphic batons, and type D were large; identification was not helped by fluorescein-antibody staining and may even have bee? hindered, because the staining revealed cell division nodes in batons. (2) Sensitivity to air in deep cultures in nutrient agar autoclaved for a prolonged period under controlled conditions, could be measured, with an error of perhaps only 5 per cent. ; the test depended on the incomplete reduction of the medium so that even the less sensitive type A strains left 10 mm. of clear agar above the limit of growth. Other workers who may wish to confirm these results might need to determine by experiment the autoclaving conditions giving a clear zone of convenient length of about 10 mm. with type A cultures. The consistency of the results suggest that this may be a measurement of a fundamental type character, to which fermentation may be secondary. (3) Many type B and type D strains grew weakly even in meat broth and qualitative observation of fermentation was difficult. In the method used, the pH did not on any occasion fall by 0.2 in two experiments in culture medium alone or in culture medium with lactose, sucrose or salicin; a positive reading was recorded only when in each of two experiments, the pH fell by 0.2. If smaller falls had been recorded as positive, qualitative assessment of fermentation would seldom have differentiated type A from types B and D. Serological confirmation of the type depended on a procedure devised to identify gamma and beta haemolysins without incurring a risk of confusion with other antigens of confirmed or unconfirmed existence acting upon lecithin or erythrocytes. The gamma and beta haemolysins were stable, and the oxygen labile delta haemoly-

R.

S. ROBERTS

et cd.

17

sin of type A was absent from the 3-day cultures in sugar-free medium used as a source of antigen. The use of lecithin as a substrate was avoided because of non-specific neutral&&ion, and the opalescing factors, epsilon in type A and theta in type ID, would not be encountered. The zeta haemolysin of type B was not detected because all type B haemolysins were neutralised by type D antisera. Types B and D were distinguished by intramuscular injection of filtrate in mice. The information gained from this firmly established the type. Type A, a toxigenic culture produced specific gamma haemolysin; type B, a toxigenic culture produced specific beta haemolysin; type D, a non-toxigenic culture produced specific beta haemolysin. Nevertheless, it was felt that it might be desirable to identify the toxin, and this was done by demonstrating its neutralisation by gas gangrene (CL oedematicns) International Standard antitoxin. The possibility has also to be considered of cultures with unusual characters as, for example, type A strains producing no toxin, strains producing toxin and no haemolysin and type D strains producing beta haemolysin in high titre, lethal to mice by intramuscular injection. These situations are detectable by the procedure described. SUMMARY

Twenty-eight strains of CL oedematiens, 4 type A, 17 type B and 7 type D, were studied by cultural, biochemical, immunological and serological procedures. A culture classified as Cl. oedematiens by cultural characteristics, could be identified as either type A or type B or D, by its morphology, sensitivity to air and the fall in pH in cultures containing glucose, maltose or glycerin. Types B and D were differentiated with difficulty on morphology grounds. The differentiation of type A from types B and D could be confirmed by cross neutralisation of haemolysin in mature cultures in sugar-free media. There was no evidence that any antigens other than gamma and beta participated in these reactions. To distinguish type B from type D, the presence or absence of alpha toxin was established by intramuscular injection of supernatant fluid from cultures in mice. Antisera were prepared against 6 strains, namely, two from human gas gangrene, one from gas gagrene in sheep, one from black diseasein Turkey, one from black disease in Australia, and one type B strain, previously regarded as Cl. haemolyticum, from bacillary haemoglobinuria. There was no evidence of preferential protection by homologous sera. The high lethality of alpha toxin, compared with that of beta haemolysin, su,ggeststhat the latter takes no part in Cl. oedcmatiens type B infection. It is pointed out that bacillary haemoglobinuria is a massive haemolysis due to rapid production of abundance of beta. haemolysin in a massive liver infarct, and attention is drawn to the importance of distinguishing it from black disease. ACKNOWLEDGMENTS

The authors are indebted for strains to Dr. B. IyigGren, Etlik Research Laboratories, Ankara; Dr. A. M. Jasmin, Department of Agriculture Diagnos& Laboratories, Kissimee, Florida; Dr. K. D. Claus, National Animal Disease Laboratory, Ames. Iowa; Dr. E. A, Lozano, College of Agriculture, Montana State University, Bozeman, Montana; and Dr. Max Sterne, Wellcome Laboratories. Reckenham, Kent; the)

18

STUDIES

ON Clostridium

oedematiens

further thank Dr. Jasmin for Cl. haemolyticum antiserum. They thank Mr. N. Giiney and Mrs. F. Yazgan for technical

assistance.

REFERENCES

Albiston, H. (1927). Amt. J. exp. Biol. and med. Sci., 4, 113. Batty, I., and Walker, P. D. (1964). J. Path. Bact., 88, 327. Erdogan, I., Worrall, E. E., Roberts, R. S., and Giiven, S. (1968). J. zet. Control and Res. Inst. Pendik, 1, 29. Iyigben, B., Durkan, S., and Kiliqoglu, G. (1966). Etlik Bakt. Enst. Dergisi, 3, 3. Jasmin, A. M. (1947). Amer. J. vet. Res., S, 289. Keppie, J. (1944). Ph.D. Thesis; Cambridge. Lozano, E. A., and Smith, L.D.S. (1967). Amer. J. z:et. Rcs., 28, 1567. McEwen, A. D. (1931). J. camp. Path., 41, 149. Oakley, C. L., and Warrack,, Harriet G. (1959). J. Path. Bact., 78, 543. Oakl;~~lCiL~lWarrack, Harriet G., and Clarke, Patricia H. (1947). J. gcn. MicraSordelli,“A.: and Ferrari, J. (1937). Folia biologica, 74, 320. Van Kampen, K. R., and Kennedy, P. C. (1968). Amer. J. vet. Res., 29, 2173. Vawter, R. L., and Records, E. (1925). .I. Amer. vet. med. Ass., 68, 494. Walburn, L. E., and Reymann, G. C. (1938). J. Path. Bact., 44, 379. Wilson, G. S., and Miles, A. A. (1964). P rinciples of Bacteriology, Topley and Wilson, 1, p. 1073, Arnold; London. Zeissler, J., and Rassfeld, L. (1929). Arch. Tierheilk, 59, 419. [Received

for

publication,

April

lOth,

19691