576.8.07 : 576.851 . 555 616.36. 941-092 . 9 : 619. 31

THE IDENTIFICATION OF CLOSTRIDIUM G D E M - A T I E N S AND A N EXPERIMENTAL INVESTIGA- TION OF ITS ROLE IN THE PATHOGENESIS O F INFECTIOUS NECROTIC HEPATITIS (" BLACK DISEASE") OF SHEEP

S. JAMIESON VeteTinary Laboratom'es, Mill of Craibstone, Bucksbum, Aberdeen

(PLATES LXXXVII-LXXXIX)

TECHNICAL obstacles to the study of anaerobes, though not inherently serious, are troublesome enough to have curtailed or delayed the investigation of many important anaerobic infections. This is particularly true of C1. edematiens infection, because the organism is not only more exacting in its growth requirements than Clostridium welchii, but is also much more difficult to identify with precision. For this reason among others, infectious necrotic hepatitis (" black disease ") of sheep, although described in Australia by Gilruth as early as 1910, was not thought to be associated with Cl. edematiens until Albiston (1927) suggested this organism as the bacterial cause. It had previously been known, however, that infestation with the liver fluke, Fascioh hepatica, was in some way necessary for the production of the disease (Dodd, 191% and b, 1921). In Great Britain, edematiens infections in animals were rarely reported (Bosworth and Jordan, 1929 ; Roberts and McEwen, 1931 ; McEwen, 1931), although a condition known for many years in the north of Scotland as " watery braxy " of sheep was shown to be black disease (Jamieson et al., 1948).

The purpose of this paper is to record studies of the strains of Cl. edematiens isolated from the lesions of black disease, the latent distribution of the organism in different hosts and the experimental reproduction of the disease.

TECHNICAL METHODS

Strains. Seventeen strains of Cl. cedematiens isolated from the hepatic lesions of black disease were examined in detail. Twenty-eight strains obtained from the liver of apparently healthy sheep, cattle, rabbits and one crow were less fully investigated. Three type-A strains from human and bovine hosts, C.N. 637, C.N. 1780 and C.N. 929 from the Wellcome Laboratories' collection, were

J. PATH. BACl'.-VOL. LXI

390 S. JAMIESON

compared with the newly isolated strains. The most effective method of isolation was to inoculate Robertson's cooked-meat medium with material taken from the edge of a necrotic hepatic lesion, i.e. from the region of the leucocyte barrier between the necrotic lesion and normal liver tissue. Forty- eight-hour cultures in this medium were then inoculated into a series of glass tubes about 15 cm. long and 5 mm. in diameter containing Weinberg's " viandl- foie " (V.F.) agar, as described by Turner (1930).* Each series covered a range of tenfold dilutions t o and pure cultures were usually obtained from the highest dilution showing growth.

The irregularity of growth on solid media of Cl. cedemutiens recently isolated from animal hosts is well known, but toxin-antitoxin tests on solid media can be used at an early stage of identification. It was found that a method based on the recommenda- tions of Petrie and Steabben (1943) was highly successful. The method was modified so that the colonies grew immediately below the surface, and the antitoxin concentration was reduced from 75 to 4 I.U. per ml. of medium. In a positive test (fig. 1) the colony is surrounded by irregular zones of varying opacity.

Various media were examined for their ability to support good continuous growth of subcultures. Proteose-peptone 2.5 per cent. with 0.5 per cent. glucose in distilled water proved the most consistent and reliable. The medium was used in 100-ml. quantities, freshly autoclaved. Optimum growth was observed between p H 8 and 7.8; outside this range results were poor. During growth of the organisms the pH of the medium fell a t a rate roughly proportional to the speed of growth, e.g. from 8.0 to 5.4 in liver-saline medium within 4 days.

The method which proved most useful was that of Reed and Orr (1941). Tubes with 5 ml. of their semi-solid proteose-peptone medium containing 1 per cent. of fermentable substrate were inoculated from a vigorous 48-hour proteose-peptone culture and incubated at 37" C. for two days in a McIntosh and Fildes jar. All strains produced acid in glucose and some produced acid in maltose. Lactose, sucrose, mannitol, salicin and glycerol were not changed by any of the strains examined.

Bacterial suspensions in which 80 per cent. of the organisms carried spores were obtained by alternate pasteurisation and sub- culture. The procedure was t o heat cultures in Robertson's cooked-meat medium at 100' C. for 3 minutes and transfer 0.05 ml. to a fresh tube containing 10 ml. of the same medium which was then incubated at 37" C. for 4 days in a McIntosh and Fildes jar. From this, spore suspensions were prepared in bulk by using the 10 ml. of 4-day culture to inoculate 500 ml. of the same medium. This was incubated for 4 days at 37" C. in the anaerobic jar and allowed to stand for 14 days on the bench at room temperature. The spores were washed free from toxin by centrifuging thrice with glass-distilled water, and the deposit was suspended in saline to give an opacity four times that of Brown's tube no. 10 and heated for 30 minutes at 80" C. to kill vegetative forms. The total spores were counted by Breed's slide method and the number of viable spores was estimated roughly by inoculating 0-05 ml. of serial dilutions down to 2-1° into 10 ml. of cooked-meat medium. The suspensions were stored in the cold at * 5 O C. and remained viable for a t least 6 months.

Identification by toxin-antitoxin tests on solid media.

Media.

Fermentation reactions.

Spore suspensions.

Lecithinase differentiation The differentiation of GI. odematiens from other

anaerobes and types A and B from each other by their production of specific lecithinase was described by Oakley et al. (1947) and their methods were largely

* See appendix for details of preparation of the medium.

Preliminary identification.

CL. (EDEMATIENS IN BLACK DISEASE OF SHEEP 391

used in the present work. When the cultures under investigation had been brought to a reasonably pure state a preliminary identification test based on lecithinase production proved successful. The test consisted of the titration of supernatant fluids or filtrates of the culture against a single dilution of aidematiens anti-/3 and anti-y sera and of welchii anti-a serum. One ml. of the filtrate was mixed with 1-0 ml. of saline, and 0-5 ml. of lecitho-vitellin (L.V.) siispension was added. A control tube was set up with 2.0 ml. of saline and 0-5 ml. of L.V. suspension. Both tubes were placed in a water-bath at 37" C. for one hour. The presence of a lecithinase was indicated by the development of opalescence in the tube containing filtrate but not in the saline control.

To identify the lecithinase present, 1.0 ml. of the filtrate was then added to three tubes containing 1.0 ml. of antiserum diluted to contain 20 units respectively of cedematiens B and y antitoxin and of welchii a antitoxin, and after half-an-hour a t room temperature 0.5 ml. of L.V. suspension was added and the tubes were incubated at 37" C. in the water-bath for 1 hour. The possible results and their interpretations are given in table I.

TABLE I Possible results from method of preliminary testing for

speci$c lecithinases

I ' Tube I ' Tube 2 1 xcF:'t , (ademattens ' (ademtiens

anti-v serum) anti B serum) I '-

Probable lecithinase in flltrate

CGdematiens y (type A)

wekili a Possibility of C1. hamolyticum or

other bacterial species (Oakley et al., 1947 ; McGaughey and Chu, 1948)

B (type B)

+ = opacity in L.V. suspension - = no opacity in L.V. suspension

For the present work further tests were devised for confirmation of type lecithinase and to estimate the variation of lecithinase production in different media.

The range of antiserum dilutions in confirmation tests to differentiate cedematiens types A and B depends on the dilution used in the preliminary lecithinase identification test, which in the present examinations was 7.5 units of B antitoxin.

Titrations were made with 1.0 ml. of filtrate against 7-6, 5-0, 2.0, 1.0 and 0.1 B units of antitoxin. After these had stood on the bench for half- an-hour, 0.5 ml. of L.V. suspension was added as indicator to each tube and the tubes were placed in a water-bath at 37" C. for exactly one hour, after which they were examined for opacity in the usual way.

In qest 1, the f is t dilution of antiserum showing opalescence was usually in the range 1-0-0.1 j3 unit. The actual figure was used as a guide in preparing a further range of dilutions at closer intervals. This procedure was repeated twice with two sera : 0-5 ml. of L.V. suspension was added to each serum dilution and the rest of the procedure carried out as in test 1.

3. From the results of test 2 a close range of dilutions of filtrate was made and titrated against a constant antitoxin dilution of 0.1 /3 unlt. The filtrate dilutions ranged from 1-5 to 0.10 ml., an example of the usual spacing being 0.4, 0.36, 0-2, 0.15, and 0-10. These tests were replicated with two /3 antisera, R.X. 5325 and E.X. 117.

Type lecithinase GonJimnation tests.

Test 1.

Test 2.

Test

392 8. JAMIESON

L.V. suspension prepared on the w/v basis recommended by McGaughey and Chu (1948) proved most reliable. Filtrates for these examinations were obtained by inoculating 100-ml. amounts of 2.5 per cent. freshly autoclaved proteose-peptone containing 0-5 per cent. glucose with vigorous actively growing cultures in cooked-meat medium. The proteose-peptone cultures were then incubated for 4 days at 37" C. in the anaerobic jar. The cultures were centrifuged at 7000 r.p.m. for 15 minutes and the supernatant used as the " filtrate."

Lecithimse production tests

These tests were used to determine the growth conditions for maximum lecithinase production. The tests involved preparation of two series of two-fold dilutions down to 2-1° in Lambeth tubes measuring 8 x 0-75 em.

Series 1. An initial dilution of antitoxin was made to yield 20 B units per ml. One ml. of this dilution was added to tube 1 of a series of ten tubes containing 1 ml. of 0-85 per cent. saline. From tube 1 two-fold dilutions were extended along the series. To each tube 1 ml. of filtrate was added.

Series 2. This consisted of a series of ten tubes each containing 1 ml. of 0.85 per cent. saline. To tube 1, 1 ml. of filtrate was added and two-fold dilutions were continued to the end of the series.

0.5 ml. of L.V. suspension was added as indicator to each tube in both series and the tubes placed in the water-bath at 37" C. for one hour. Readings were made at t.his interval and again 12 hours later.

Cultivation of cercarim

Cercarize of Fmciola hepatica were cultivated in the laboratory according to the technique of Taylor and Mozley (1948) and were administered by mouth to guinea-pigs and rabbits in gelatine capsules containing wet bran.

EXPERIMENTAL OBSERVATIONS

Identi$cation of C1. cedematiens

In establishing the post-mortem diagnosis of black disease of sheep it was necessary to prove the identity and type of strain of C1. edematiens cultured from diseased livers. Cultures were made as already described ; thereafter procedure and results were as follows.

The toxin-antitoxin reactions of Cl. edematiens on the surface of solid medium have been recommended as a rapid and reliable means of identifying type-A strains of C1. edematiens (Petrie and Steabben, 1943 ; Nagler, 1945 ; Oakley et al., 1947). Petrie and Steabben tested only one type-A strain, however, and it seemed necessary to check their observation by examining not only further strains of type A but also strains of type B. With all my strains, 3 of type A and 17 of type B, the modified method proved a useful means of differentiating Cl. cedematiens from other maerobes, but it did not of course distinguish type A from type B. With strains of type B isolated from animal hosts, preliminary identification on the surface of solid media as suggested by Oakley et al. did not prove so reliable because of the uncertain growth obtained from recently isolated type-B strains.

Preliminary identijication.

CL. CEDEMATIENS I N BLACK DISEASE OP SHEEP 393

When the preliminary identification was made by examining for lecithinase activity by the methods recommended by Oakley et al., all my strains appeared to be type B, with inhibition of opalescence by 1.0 ml. of filtrate in the presence of 7.5 units of /3 antitoxin. Further evidence had to be obtained, however, since Oakley et al. demonstrated that qualitative examination of a filtrate could not be regarded as proof that this contained /3 or y toxin alone ; they maintained that it was necessary to show in type-confirmation tests that toxin in the filtrate was neutralised by several sera in proportion to their antitoxin content. They quoted the failure of /3 or y antitoxin to inhibit opalescence in filtrates from two strains of C1. hemolyticum before dilution as demonstrating the necessity for these tests.

Type-conJirmation tests. Such type-confirmation tests were accord- ingly applied to my strains with the results shown in table 11. These

TABLE I1

Results of eonfrmatory type-leeithime tests for C1. cedematiens -~

I i i Strain

I 1 Glengolly . I B.D.J./l .

B.D.J./I . €%.D.J./6 . B.D.J./9 . B.D.J./11 . ILD.J./12 . B.D.J./14 . E<.D.J./lS .

Volume of flltrate neutralised by 0.1

B antitoxin

0.4 0.15 0.4 0.4 0.2 0.7 0.8 0.4 0.2

Strain

B.D.J./19 B.D.J./ZO B;D.J./21 B.D.J./25 B.D.J./26 B.D.J./27 B.D.J./28 B.D.J./35

...

t'olume of flltrate neutralised by 0.1

B antitoxin

0.4 0.10 0.15 0.2 0.15 0.4 0.10 0.4 ...

results show the volume of toxin-containing filtrate completely neutralised by 0.1 unit of /3 antitoxin and they afford proof that the filtrates are neutralised by antitoxic sera in proportion to their /3 antitoxin content and therefore contain /3 toxin. This establishes that the strains are Cl. cedematiens type B.

OPTIMUM GROWTH CONDITIONS FOR PRODUCTION OF LECITHINASE

There are inherent difficulties in the expression of an end result when testing for variations in the lecithinase activity of filtrates. The two main difficulties are the variability of the crude L.V. suspension and the reduction of the final dilutions to absolute points. To over- come these difficulties methods were devised which had the advantage that the test filtrate was examined simultaneously in the presence and in the absence of antitoxin. As the strains were shown by the type-confirmation tests to belong to type B, it was sufficient to include a single tube with 150 units of cedematiens y antitoxin and 1 ml. of

A!. JAMlESON

z0.0 i + + + + +++t i t + + ++++ I I + + I I + ! I I I I ' f

m o j t t + t + - t + + + t i + t t + t t - ! 1 + t - i : + 1 I I I I .3 L _ -. - 2 5 + + t - + t + t - t i - t + + +is++- I I + + I I i I I I ! I

+ + t + ++i-i ++++ I + + + I : + I ! I f ! I I I I ' ' 6

.- I..- 2-5 5 I 9.01 ++++ ++++ At-++ I t + + I l l I I I t : I l l ! ; . $ 4

I -_ _.__ -- - ~ . _ _

___.-

5 3 I - t - + + - t . ++++ C + t + I + + + I I I I 1 1 + 1 I I I I I; 9

___~__

_ . ~ . _ . _ _ _ _ _ _ _ _ _ _ . .- 4 - - - -- -

F Z - 1 1 ++++ ++!I+ ++'+ l + i + I I ! I 1 1 - 1 I l l !

c . z ; I + t + + + I L ' + I i i ! I It I I I I : I I I I I I I

I __ ~ . _ _ _ _ _ _ I

__ g J I + - + + I + I I I I I I I I I I I I I I I I I I I I I I 1

C .- c! 4 ill

i C c

L z

a - c L c t

N I

CL. (EDEMATIENS I N BLACK DISEASE OF SHEEP 395

filtrate. Since this large amount of y antitoxin did not inhibit their lecithinase activity the strains could not belong to Cl. edematiens, type A.

Variations in lecithinase production in different media proved troublesome in the earlier examinations. Because of this, four strains were examined in 7 different media with the results detailed in table 111, from which it is obvious that the differences observed were due to growth conditions and not to strains. It appears significant that where growth was rapid, as in liver-saline, lecithinase production was barely detectable.

NATURAL INCIDENCE OF LATENT SPORES

From 1945 to 1948 a survey was made in the North of Scotland t o determine the presence or absence of latent spores of Cl. edematiem in the liver of apparently healthy sheep grazing pastures on farms where black disease had been diagnosed, and on farms where black disease had not been reported.

Since crows and lesser gulls are attracted to sheep dead from black disease the survey was extended to include the liver from these birds. The isolation of Cl. cedematiens from the beaks of crows by Edgar (1931) and from a swan dead of a septicEmic disease (Piening, 1932) supported the belief that latent Cl. ademtiens spores might be found in the tissues of birds.

Although the number of examinations may not be large enough to justify more than tentative conclusions, two significant points emerge from the results (table IV) :. first, that sheep grazing in black-disease

TABLE IV 'The incidence of latent spores of C1. cedematiens in the liver of apparently heulthy

animals in black-disease areas and in areas from which the disease i s absent

Black-discase areas , Host species ~ Livers with spores I SO. of livers 1

: rxamincd ~ S o . i Per cent.

I

Sheep . Cattle Starlings . Oulls . . Crows Rabbits .

38 8

24 8

10 35

11 6 0 0 1 3

28.9 75.0

0 0

10.0 8.6

Other areai

Livers nitli spores

SO. Pcr cent.

So. of livers examined

26 15 12 2

10 11

1

0 1 0 0 0 0

0 6.7

0 0 0 0

.areas have a much higher incidence of latent ademutiens spores in the liver than those grazing elsewhere ; and second, that there is a relatively high incidence of latent spores in the liver of cattle, suggesting that black disease probably affects bovines in this country.

396 8. JAMIESON

Experiments to show spore latency

Expt. 1. Six guinea-pigs, three rabbits and one sheep were given doses of washed spores of Cl. cedematiens by the routes shown in table V. Guinea-pig no. 3 became ill two days after receiving the spore suspension by mouth and died on the fourth day. Post-mortem examination revealed the typical appearances which follow the ingestion of whole cultures of Cl. cedematiens : a congested, haemorrhagic gastro-intestinal tract and gelatinous effusions into the serous cavities. The dose of

TABLE V

The distribution of latent C1. cedematiens spores in animal tissues after their experimental introduction (experiment 1 )

Animal

Guinea-pig no. 1 ,, 2 1 , 3 7, 4

, ,, 5 ,, 6

Rabdit no. 1 . ,, ,, 2 . ,, ,, 3 *

Sheep .

Route by which spores were given

Intravenous

OPal

Intraperitoneal

IntraGenous Intraperitoneal

Oral Intramuscular

Dose of spores

(millions)

25 10

200 100 10 5

25 15

100 10

Day of experiment on which

killed

50 50

50 50 50 50 50 50 60

4 (died)

Presence (+) or absence (-) of C1. mdematiens in

liver

+ + + + + + + +

- -

spleen

+ + + + + + + +

-

-

spores was certainly high, but there was no evidence that vegetative forms were present, and the same suspension was used intravenously in guinea-pig no. 2 without ill effect. All other animals remained well until killed. Spores were more often found in the spleen and liver than in the bone marrow or muscle.

Expt. 2. Two rabbits were each given two intravenous injections of 2,500,000 spores in saline with an interval of 8 days between the injections. Seven days after the last injection 10 cercariae of Fasciola hepatica were given by the mouth to one rabbit and 15 cercariae to the other. Twenty-one days later both rabbits appeared well and they were then killed with chloroform and examined. In both there was evidence of liver-fluke infestation but there were no necrotic lesions resembling those seen in black disease of sheep. Vegetative bacillary forms of C1. cedematiens were not found in smears or sections of the liver. From the liver and spleen of both rabbits C1. cedematiens was isolated in pure culture.

Expt. 3. One rabbit was given 5 million spores by one intravenous injection and another received 40 million spores in a single dose by mouth. These two rabbits and a control which had received no spores were given each 25 cercariae. Ten days later the rabbit died which had received the spores intravenously. Post-mortem examination

CL. (EDEMATIENS IN BLACK DISEASE OF SHEEP 397

showed that the liver was acutely congested and was the site of a single hsmorrhagic rupture of the capsule. No necrotic lesions were observed on the surface or in the substance of the organ. From the liver two immature liver flukes were recovered. C1. cedernutiens wa.s isolated in pure culture from the liver and spleen.

At the end of 36 days the other two rabbits of the experiment were still healthy. Five million spores were now given to each rabbit intravenously in a single injection. Ten days later the animals, which were still healthy, were killed by chloroform. Liver-fluke infestation was present in each but there was no evidence of necrotic lesions resembling black disease. C1. cedematiens was isolated from the liver in both animals.

A guinea-pig weighing 900 g. received toxin-free spores of Cl. cedernutiens to a total of 5 million by the intraperitoneal route. Spores were given on 19th, 22nd, and 29th January 1949, in doses of 1, 18 and 2+ millions. Twenty days later (18th February), 25 cercarize were given by mouth. Eighteen days later the animal died after an illness of less than 24 hours. Post-mortem examination revealed the general features typically observed in sheep dead of black disease, namely distension of the pericardium with clear fluid, zonal congestion of the pylorus and the presence of clear fluid in the peritoneal cavity. The liver showed numerous necrotic tracks and two small necrotic areas about 3 mm. in length situated in a zone of congestion. The necrotic areas were sharply demarcated by an intense bright red zone. Histological examination of the necrotic areas revealed necrosed hepatic cells surrounded by a leucocytic barrier (fig. 2) in which vegetative bacilli were evident (fig. 3). No bacilli were observed in the necrotic tracks made by the passage of flukes. Cl. cedematiens was isolated from the leucocytic barrier of these lesions.

Expt. 4 was repeated with a guinea-pig which received in one dose 5 million Cl. cedematiens spores by the intravenous route and, 30 days later, 25 cercaris by mouth. At the same time a control guinea-pig, not given spores, received 25 cercarize by mouth. The guinea-pig which received both spores and cercariae died 21 days after dosage with cercarize, with the typical lesions of black disease as described in expt. 4. Seven days later the control guinea-pig, which had not received spores, was killed by chloroform ; it showed evidence of fascioliasis only.

Expt. 4.

Expt. 5.

DISCUSSION

In the absence of a standard procedure and an accepted classifica- tion of Cl. cedernutiens by fermentation reactions, the value of these reactions is severely restricted. Turner (1930), using Weinberg’s V.F. broth rendered sugar-free by the growth of Cl. welchii and containing 10 per cent. of added fermentable substrate, concluded that strains isolated from cases of black disease were distinguishable

398 S. JAMIESON

from strains of human and equine origin by the failure of the black- disease strains to ferment glycerol. Scott et al. (1934) recommended this difference tw a basis for identification. Keppie (1944), on the other hand, using a medium containing 1 per cent. of horse serum and 0.5 per cent. of fermentable substrate, found three type-B strains which fermented glycerol. It is significant, however; that one of these strains was isolated from a horse and one from an ox, and tha t the origin of the third strain was unknown, whereas classical type-B strains “ Gigas I,” “ Tongala ’’ and “ Albiston,” all isolated from sheep, failed to ferment glycerol. Odrley et al., using a medium essentially similar to that of Keppie, found that 10 of 14 type-B strains fermented glycerol. No information is given about the host origin of these strains. The fermentation reactions of my strains conform more closely to those of Turner than to those reported by Keppie and by Oakley et al. The point of interest is that all Turner’s strains and all my 17 strains from sheep with black disease failed to ferment glycerol.

The rapid identification of C1. ademtiens is more difficult with type-B than with type-A strains because of the fastidious growth requirements of type-B strains. The modified method of Petrie and Steabben proved the most reliable, and, supplemented by preliminary lecithinase tests, should prove invaluable in the diagnosis of anaerobic diseases of sheep and cattle, where mixed infections are common. The typing of strains by the use of specific anti-lecithinase sera proved satisfactory with my black-disease strains. The variation in lecithinase production in different media needs further investigation. Keppie, using the turbidimetric method of van Heyningen (1941), showed that with Cl. cedematiem lecithinase production was not related to production of the main lethal toxin as i t is with C1. welchii. He showed that the addition of extract of horse muscle and glucose were both important for the demonstration of even slight lecithinase production by type-B dernutiens strains. My observations (table 111) show that the rate of growth of the strains, as judged by the lowering of pH in the culture, is not proportional to the production of lecithinase. Thus liver-saline medium, which invariably produced a low final pH and a rapid and luxuriant growth, was notable for low lecithinase production. Strains 12 and 35 generally yielded higher lecithinase levels than the other strains. The failure of lecithinase production in Brewer’s semi-solid medium with 0.5 per cent. agar was not surprising. In preliminary studies with this medium, I often observed spurious reactions in duplicate parallel tests, replicated with different batches of L.V. suspension. Oakley et al. recommended Brewer’s semi-solid medium for routine lecithinase testing, using high-speed centrifugation or filtration over Hyflosupercel (Johns-Mancille Ltd.) to obtain the filtrate. In my tests high-speed centrifugation gave obviously discrepant results, but these were corrected by growing the organism in Brewer’s broth without agar, or by filtration through kieselguhr.

FIG. 1.-Positive toxin- antitoxin reaction with 6’1. m?tder.naliens type B on solid medium. Note toxylin and eosin. x 20. irregular zones of vary- ing opacity xound the central pin-hea,d colony (natural size).

FIG. 2.--Necrotic lesion surrounded by a barrier of leucocytes in the liver of a guinea-pig produced by experimental i-p. inoculation of mdematiena spores followed by cercarirt: of Fasciola hepatica by mouth. Hzma-

FIG. 3.--IIigher-power view of fig. 2 to FIG. 4.-Lesion from natural black disease of sheep. Fluke track in liver ending a t the edge of an infective necrot,ic lesion. H. and E. x 60.

show vegetative and sporulating forms of GI. edematiens in the leucocyte barrier. Gram’s :stain. x 1200.

J. PATH. BACT.-VOL. LXL PLATE LXXXVII

CI,. ~f115’.~I.i1’lELVS IN DLACK DISEASE 08’ SHI’:KI’

FIG. 5.-Lesion from natural black disease of sheep. Remnants of fluke track within an area of hepatic necrosis which is surrounded by a dense zone of leucocytes (below). x 60.

PIG. 6.-Vegetative CZ. edematiens from the leucocyto FIG. 7.-Lesion from natural black barrier of fig. 5. Gram's stain. x 1200. disease of sheep. Immature fluke in

tissue adjacent to infective lesion. H. andE. x20.

J. PATH. BACT.-VOL. LXL PLATE LXXXVII

CI,. ~f115’.~I.i1’lELVS IN DLACK DISEASE 08’ SHI’:KI’

CL. (EDEMATIENS IN BLACK DISEASE OF SHEEP 399

The behaviour of strain 12 in 2-5 per cent. proteose-peptone plus 0.5 per cent. glucose medium suggests that other lecithinases might be present for which no anti-lecithinase existed in the sera; alter- natively the explanation might be that this particular lecithinase did not combine well with the antitoxin. The lecithinase tests which were devised, although not free from disadvantages, were more sensitive than turbidimetric estimations for C1. cedematiens and greatly reduced the danger of introducing other factors which might give spurious results.

Since the work of Dodd ( 1 9 1 8 ~ and b, 1921), when black disease was first shown to have a dual parasitic and bacterial origin, the pathogenesis of this disease has attracted considerable interest. Turner (1928, 1929, 1930) believed that the disease was brought about by the activation of latent spores of Cl. cedematiens by the immature wandering liver fluke. The evidence in support of this hypothesis was indeed weighty. Histological studies of the liver in black disease unfailingly demonstrated the various stages of fluke infestation as well as the infective necrotic lesion, which consisted of necrotic liver cells surrounded by a deep leucocytic barrier in which vegetative bacillary forms were easily demonstrated (figs. 4-7). That black disease was found only in known areas of fluke infestation further supported Turner’s view. The discovery of latent spores of Cl. Edematiens in the liver of apparently healthy sheep (Edgar, 1928; Turner, 1930) was additional evidence pointing to the probable method of infection.

In his experiments, Turner activated spores of Cl. cedematiens in the liver of guinea-pigs but his experimental method did not establish whether, in the natural infection, it was likely that the spores were taken in by the fluke or were already present. He fed 8,000,000 toxin-free spores to four guinea-pigs which, 85 days later, received 15 cercaris of Fascioh hepatica by mouth. Twenty-two days later, the animals were still alive. A control guinea-pig given only cercarie showed extensive liver damage by the young flukes. Turner concluded that the spore population in the four guinea-pigs was not adequate for production of the disease and accordingly his four animals were each given 12,500,000 spores in traperitoneally. Within three days all four guinea-pigs died. A control guinea-pig which received 25,000,000 toxin-free spores showed only a passing indisposition, but recovered. On histological examination Turner recorded the marked necrosis along the fluke tracks and the presence therein of CZ. cedematiens. He stated that it did not necessarily follow that the spores actually grew along the fluke tracks.

Turner’s experiments, however, do not prove that latent spores of Cl. cedernutiens in the liver are activated by wandering liver flukes. All that is certain is that when toxin-free spores were introduced a t the height of an acute fluke infestation of the liver, in an animal believed to be carrying latent spores, the guinea-pigs died, and tha t

J. PATH. BA0T.-VOL. LXX 2 0

400 S. JAMIESON

on post-mortem examination vegetative forms of C1. adematiens were found along the necrotic fluke tracks.

Certainly I have not seen a natural case of black disease in which vegetative bacilli were demonstrated in the necrotic tracks. It is significant that in the summary of a general paper on anaerobic infections in animals Scott et all. make the statement that black disease is due to the germination of latent spores of Cl. adematiens which have been carried to the liver by wandering liver flukes. The question has hitherto been unanswered as to whether latent spores of Cl. adematiens in the liver can be act,ivated by young wandering liver flukes or whether the spores must be introduced after or along with the liver-fluke infestation. Expts. 4 and 5 of this paper show con- clusively for the first time that the experimental disease in guinea-pigs can be produced by activation of latent spores by wandering flukes, and that there is no need to postulate that the adematiens spores are carried into the liver by the flukes. This makes the pathogenesis of the natural disease much more intelligible.

In confirmation of the earlier Australian work (Edgar, 1928; Turner, 1930), the present paper shows that 28.9 per cent. of healthy sheep from black-disease areas have latent spores of Cl. ademt iens in the liver and that the organism was not isolated from the liver of any of the healthy sheep from areas free of black disease. The positive findings in the liver of rabbits, cattle and one crow in black-disease areas (table IV) provide supporting evidence of the association between black disease and the spores of Cl. adematiens latent in the organs of animals.

Histological examination of the liver lesions from sheep with black disease confirms the Australian findings. The termination of a fluke track in a typical infective necrotic lesion (fig. 4) is evidence that wandering flukes activate already existing latent spores. The evidence indicates that when spores are activated a necrotic lesion is produced by the necrotising toxin liberated by Cl. adematiens. The size of the lesion and the intensity of the leucocytic response obviously depend upon the number of spores activated, the potency of the toxin, whether the conditions are optimal for bacterial growth, and the defensive response of the animal. The diffusion of toxin must rapidly reduce the lining of the track and the surrounding tissue to a necrotic mass. The animal dies within a few hours and histo- logically it can be shown that the fluke track terminates in the necrotic lesion.

The disease was reproduced in two guinea-pigs, into which spores were experimentally introduced and to which cercariae were fed after an interval of 21-30 days. The guinea-pigs died with the typical syndrome and the pathological changes associated with black disease in sheep. Histologically the lesions in the guinea-pig (figs. 2 and 3) were essentially similar to those of naturally occurring black disease (figs. 4-7). Minor differences in the guinea-pig lesions were the less

J. PATH. BSCT.--Vor,. LXT PLATE LXXXIX

CL. @DEMAl’IB:NS IN BLACK DISEASE O F SHEEP

FTC.. R.--CI. mdrwm/iens. Lenticular rolony grown in rnpillary tlthp of V.F. agar. x inn.

PI(;. 9.-Later stage than fig. 8. The “ rn grenade ” form. x 100.

FIG. lO.-Surf:tce colony of CI. wdem,atiens on V.F. agar plate. This :qqma.ra.nre is very mre. x 90.

CL. (EDEMATIENS I N BLACK DISEASE OF SHEEP 401

substantial leucocytic barrier and the greater disintegration of the necrotic cells. Within the leucocytic barrier of the experimental lesions large numbers of vegetative bacilli were seen. The minor differences in the experimental lesion could result from the wtivation of a larger number of spores than are present in the natural infection, with consequent death of the animals before the defence mechanism had reached the stage usually seen in the natural disease in sheep.

SUMMARY

1. Clostridium cedematiens type B is the bacterial cause of black disease of sheep in the North of Scotland.

2. Fermentation reactions cannot be relied upon completely for the identification of Cl. cedematiens ; but the 17 type-B strains which I isolated from black disease in the sheep failed to ferment glycerol -a finding which supports Turner’s results with strains from the same host.

3. Methods of identifying C1. Edematiens types A and B are described and an account is given of tests devised for lecithinase type confirmation. Type-B strains are more fickle in their growth require- ments than type-A strains. Consequently many of the tests on the surface of solid media are less reliable for type-B than for type-A strains. A modification of Petrie and Steabben’s method proved the most reliable.

4. The production of lecithinase depends upon the medium in which the strains are grown and the limited evidence available indicates that the more rapidly the organism grows the less lecithinase is produced.

5 . In black-disease areas 17.1 per cent. of apparently healthy livers from various host species harbour latent spores of Cl. cedematiens compared with 1.3 per cent. in healthy livers obtained in areas free from the disease.

6. The pathogenesis of black disease has been shown, by histo- logical evidence obtained from naturally occurring cases of the disease and from experimental evidence in guinea-pigs, to be the activation of latent spores of Cl. edematiens in necrotic foci in the liver caused by the wanderings of the immature liver fluke Fasciola hepatica. Attempts to reproduce the disease in rabbits were not successful.

I am indebted to Professor J. Cruickshank, Department of Bacteriology, University of Aberdeen, for his helpful guidance and advice ; to Miss Helen E. Ross and Mr Alexander Thomson of the Wellcome Research Laboratories, Beckenham, for the supply of antitoxic sera and for their invaluable advice on technical methods in lecithinase identification tests ; to the Agricultural Research Council for a grant which made this work possible ; and to Dr J. W. Howie of the Rowett Research Institute for assistance in preparing the manuscript.

402 S. JAMIESON

REFERENCES ALBISTON, H . E. . . . . . 1927. Austral. J . Exp. Biol. Med. Sci., iv,

BOSWORTH, T. J., AND JORDAN, L. 1929. DODD, S. . . . . . . . 1918a. N.S.W. Agric. Gaz., xxix, 657.

113. Vet. J. , Ixxxv, 393.

9 , . . . . . . . 1918b. J . Comp. Path and Therap., xxxi, 1. 9 , . . . . . . . 1921. Ibid., xxxiv, 1.

,, . . . . . . . 1931. Ibid., vii, 64. EDGAR. G. . . . . . . . 1928. Austral. Vet. J. , iv, 133.

GILRUTH, J. A. . . . . . . 1910. Vet. J. , lxvi, 254. VAN HEYNINGEN, W. E. . . . 1941. Biochem. J. , xxxv, 1246. JAMIESON, s., THOMPSON, J. J., 1948. Vet. Rec., lx, 11.

KEPPIE, J. . . . . . . . 1944. A study of the antigens of Cl. odematiens and Cl. gigas by in- vitro and in-vivo methods. Ph.D. thesis, Cambridge.

MCEWEN, A. D. . . . . . 1931. J . Comp.Path. and Therap., xliv, 149. MCGAUOHEY, C. A., AND CHU, 1948. J . Gen. Microbiol., ii, 334.

NAGLER, F. P. 0. . . . . . 1945. Austral. J . Exp. Biol. Med. Sci.,

OAKLEY, C . L., WARRACK, G. 1947. J . Gen. Microbiol., i, 91.

AND BROTHERSTON, J. G.

H. P.

xxiii, 59.

HARRIET AND CLARKE, PATRICIA H.

DOROTHY PETRIE, G. F., AND STEABBEN, 1943. Brit. Med. J. , i, 377.

PIENING, C. . . . . . . . 1932. Dtsch. tierartd. Wschr., xl, 466. REED, G. B., AND ORR, J. H. . 1941. War Medicine, i, 493. ROBERTS, R. s., AND MCEWEN, 1931. J . Comp. Path. and Therap., xliv,

SCOTT, J. P., TURNER, A. W., AND 1934. Proc. 12th Internat. Vet. Cong.,

TAYLOR, E. L., AND MOZLEY, A. . 1948. Nature, clxi, 894. TURNER, A. W. . . . . . . 1928. C.R. SOC. Biol., xcviii, 558.

A. D. 180.

VAWTER, L. R. New York, pp. 168.

,, . . . . . . 1929. Austral. Vet. J., v, 11. 9 , . . . . . . 1930. Austral. Counc. Sci. Indust. Res.,

bull. no. 46.

Appendix Preparation of Weinberg's V.F. broth and agar

To prepare Weinberg's V.F. broth and agar a stock solution is made up as follows. Fresh ox liver 5.6 lb., fresh ox muscle 5.5 lb. and fresh pig stomach 7 lb. are each minced Gnely and placed in a large earthenware vessel. Twenty- five litres of tap water and 250 ml. of pure concentrated HCL are now added and the mixture is kept at 48450°C. for 24 hours. The temperature is then raised to 80" C. to destroy the pepsin and the mixture allowed to stand for a further 24 hours, when it is decanted and filtered through moistened Chardin paper. The filtrate is filled into 500-ml. bottles, steamed at 100' C. for 15 minutes and stored. To prepare V.F. broth the stock solution is heated to 80-90" C., adjusted to pH 8 and heated at 120" C. for 15 minutes to precipitate earthy phosphates. It is then distributed into bottles of convenient size and agar is added in the proportion required, i.e. 0.5 per cent. for capillary-tube cultures and 2.5 per cent. for plate cultures.

If properly prepared the stock solution keeps for as long as nine months. Culture forms of C1. mdematiens in this medium are shown in figs. 8-10.