Diphtheria bacilli in the horse

GENERAL ARTICLES.

DIPHTHERIA BACILLI IN THE HORSE. By Captain F. C. MINETT, M.B.E., B.Sc., R.A.V.C., Royal Army Veterinary School, Aldershot. IT is intended in this article to bring forward evidence to prove that certain micro-organisms cultivated from horses are in reality identical with those causing true diphtheria infection in man. During the last year of the war and for some months after the armistice very numerous specimens of pus were received for examination at the Army Veterinary School, these being derived mostly from cases suspect of ulcerative lymphangitis. I n a certain number of cases, apart from such common contaminants as staphylococci and streptococci, the Preisz-Nocard bacillus was isolated and the suspicion proved to be correct. At the same time the opportunity was taken of obtaining in pure culture any organism encountered which was evidently a member of the large group of bacteria usually described as the diphtheroid group. Quite a number of diphtheroid strains have in this way been collected, but in this article it is intended to confine attention to those which subsequent investigation showed to be identical with the true Klebs-Lbffier bacillus. It is hoped to publish later the results of work with other diphtheroids, including the bacillus of Preisz-N ocard. Before going into the details of the evidence, it is as well to state that the horse is susceptible to the diphtheria bacillus, and, as is well known to those engaged in the preparation of anti-diphtheritic serum, is especially sensitive to the toxin produced by it. Further, on one occasion the diphtheria bacillus itself has been isolated from the nasal discharge of a horse, viz., by Cobbett. 1 As that appears to be the only previous occasion on which the diphtheria bacillus has been isolated from the horse with any degree of certainty, it is as well to give the essential details of the case. A little girl fell ill with diphtheria and her medical attendant cast about to find if possible the source from which infection was derived. He discovered that a pony belonging to the girl's father was suffering from a purulent and slightly hcemorrhagic nasal discharge, and accompanying this symptom there was some enlargement of "the glands beneath the tongue» and also some laryngeal obstruction. The medical man made a culture from the nasal discharge, from which Cobbett succeeded in isolating an organism similar in appearance to Bacillus diphtherice and capable of producing acid in glucose broth but incapable of liquefying gelatine. He found further that the organism was pathogenic for the guinea-pig and gave rise to the general symptoms of diphtheria in that animal. The organism produced an extracellular toxin ot which ·05 cc. was approximately the minimum lethal dose in one case, and 5 cc. (or about IOO M.L.D.) of this same toxic filtrate were neutralised by 5·5 units of human anti-diphtheritic serum. He concluded that the organism isolated was the human diphtheria bacillus, and that horses were subject to 1

"Lancet," 15th August 1900.

268

GENERAL ARTICLES.

diphtheria infection in the same way as man and ought therefore to be looked upon as a possible source of infection to the human subject. Another fact adduced in support of this opinion was that some apparently normal horses (ten out of thirteen examined by Cobbett) showed the presence of diphtheria anti-toxin in their blood. In Table 1. are given details of the strains studied-twelve in all. In most cases the strains from equines were derived from suspect cases of ulcerative lymphangitis, i.e., cases in which swelling of the lower parts of a limb or limbs was accompanied by abscess formation followed by ulceration. In none of these cases was the organism is.olated in pure culture, but always accompanied by other organisms. In the case of one strain, viz., F, the organism appeared to be present in pure culture in the pus, and it is necessary to give a fuller description of this case. The animal in question had been working in the transport of a cavalry regiment at A - - for about two years and had always been in good condition. On 3rd May 1919 he was sent to the Army Veterinary School and considered by the Veterinary Officer to be suffering from a form of acne. The lesions on the skin, which numbered about a dozen, were mostly confined to the region of the withers, and consisted of rather superficial suppurating points covered by scabs, each about the size of a threepenny piece. A particle of pus from a lesion was sown out direct on solid horse serum, which after incubation developed an apparently pure growth of a Gram + bacillus, which was at first considered to be that of Preisi-Nocard. This organism was found to be capable of producing omental abscesses in a guinea-pig on intraperitoneal injection, and from these abscesses the organism was again recovered in pure culture on solid serum. The animal was kept under observation for twentyfive days, when all the lesions were found to have healed. During this period pus from several other abscesses was cultivated on serum and always with the same result, viz., the development of an organism which was considered at the time to be the Preisz-Nocard bacillus. The following methods were adopted for identifying the strains and for comparing them with control diphtheria cultures of human origin : (a) Examination of the morphological characters, including staining. (b) Examination of the cultural characters, including ha:molysis tests, etc. (c) Sugar fermentation tests. (d) Serological tests. (e) Animal inoculation with bacteria themselves. Toxin producing power and neutralisation by antitoxin.

(a) MorpllO!ogy.-All the twelve strains studied conformed to the type species, Bacillus diphtheria: (see fig. J). They were all non- motile and non - sporulating and showed no capsules. They were all Gram-positive. They varied considerably in morphology even in the same preparation. Many were evenly rod - shaped and generally rather slender, others were markedly clubbed at

GENERAL ARTICLES.

TABLE

Strain.

Date Isolated.

Origin.

I.

Animal.

Remarks.

-----1-----------------------------D

Pus from suspect ca' e of ulcerative lymphangitis at W--.

7.5.19

Horse

F

Pus from skin lesions on horse sent for examination Army 'Veterinary School.

13.5.19

Horse

G

Pus from suspect case of ulcerative lymphangitis at l\LH--.

Prior to 24.6.18

Horse

II

Pus from suspect case of ulcerative lymphangitis at C--.

6.2.19

Horse

I

Pus from suspect case of ulcerative lymphangitis at 1V--.

29.5.19

Horse

L

Pus from suspect case of ulcerative lymphangitis at A--.

2.6.19

Horse

0

Pus from suspect case of ulcerative lymphangitis at L--.

G.9.19

Mule

P

Pus from contused fetlock at Co--.

10.9.19

Horse

D14

Pus from suspect case of ulcerative Iymphangitis at 0--.

23.10.19

Horse ( All three animals were cavalry chargers which had returned from Germany and had been quarantined in 0 - Remount Depot before being admitted to hospital. The two from which D 14 and D 15 strains were do. I isolated showed no response to treatment and were destroyed. The animal from which D ]6 was isolated showed some swelling of one leg with do. lesions which quite healed up, and in consequence the horse was discharged as cured on 21.11.19. l Horse This animal was an officer's charger which had suffered from a nasal discharge for which trephining had been practised prior to February 1920. Though the discharge lessened and the animal remained in excellent condition, the trephine opening refused to heal, and was still in existence on 17.10.20 when BaciJIus diphtherire and streptococci were isolated from it. Man Stock culture received from Lister Institute.

D 15

Do.

do.

23.10.19

D16.

Do.

do.

28.10.19

D34

Lister Elstree

Swab from trephine opening in horse's cheek at 1V--.

17.10.20

23.8.19

Recently repatriated from France, and admitted to hospital on 7.4.19 suffering from contusion of near hind leg.

On 2.1.19 horse was suffering from contused fetlock owing to a bale injury. On 6.1.19 a small abscess developed in the same region and discharged pus. On 20.1.19 the swelling of the fetlock extended to the stille, and a line of small ulcers appeared at the front of the hock. No nasal dis· charge. Mallein gave a negative result. Pus taken 011 swab On 6.2.19. Animal later destroyed. Admitted to hospital at 1V-- on 23.5.19, having been recently repatriated from France.

Admitted to hospital from L-- Remount Depot on 25.8.19 with abscess near hind leg, which was very swollen from the coronet to the hock and also extremely painful. (Not described as simulating lymphangitis.)

I ,I I I

l\lan

strain received from Dr 1\1 'Conkey.

GENERAL ARTICLES.

one extremity. The tendency to club formation was the most constant variation, but uniformly thickened or fusiform individuals were commonly met with (see figs. 2 and 3). Sometimes the organisms were wedge - shaped, and often so in pairs with the broad ends of the wedge in apposition, an appearance rather common to Hoffmann's throat bacillus. A certain amount of variation occurred from strain to strain, some showing very obvious tendency to clubbing, others very slight. On the different culture media again the morphology varied considerably, eg., on agar media the clubs were often very thick and short, on old blood agar media many individuals had become Gram-negative. An interesting change in morphology occurred in strain F (from

FIG. 1.

Diphtheria bacilli, Lister Institute strain, fourth day on solid serum, Gram stain.

x 1000.

horse's skin). In the primary cultures in all media used the pre·· domillating form was a shortish rod very like the Preisz-N ocard bacillus and without any clubs. In later cultures clubs became notable features and the forms appeared to be larger. When stained by a simple stain, e.g., Loffler's methylene blue, coloration was not as a rule of uniform intensity, the organisms being beaded or showing barred or striped appearances. The clubs as a rule stained uniformly and intensely. Metachromatism was never a very pronounced feature, though occasionally observed. Of special staining procedures the well known method of Neisser has been most practised, and according to some is of the highest value for differentiating the Klebs-Loffler bacillus from other diphtheroid bacilli which may be present in the human throat. It appears to be certain that the method has been over-rated by

GENERAL ARTICLES.

many,! but most observers are agreed that positive Neisser staining is a point in favour of any doubtful organism being the true diphtheria bacillus. Of the strains studied all were N eisser-positive to a greater or less extent; the granules were as a rule terminal; often a third granule was placed centrally (see fig. 4). N eisser staining was carried out on twenty-four-hour solid ox serum cultivations according to the following technique : Methylene b l u e . 9 6 per cent. alcohol { Distilled water.. Glacial acetic acid Stain B {Bismarck brown Boiling distilled water

Stain A

gramme cc. 950 cc. 50 cc. 2 grammes I litre I

20

FIG. 2. Diphtheria bacilli, Strain H (horse), third day on serum agar. Gram stain.

x 1000.

Preparations were stained in A for half-a-minute, washed and stained in B in the cold for one minute, washed and dried. (b) Cultural.-At 37° C. under cerobic conditions growth in suitable media became visible in twenty-four hours. I n plain beef broth growth occurred as a faint uniform turbidity. with a whitish finely powdered deposit at the bottom or on the side of the tube and rising to a uniform cloudiness on being shaken. Only very slight film formation was the rule. Sometimes with continued incubation the cloudiness completely cleared up, but generally clearing was only partial. Growths were more meagre in broth to which 5 per cent. glycerine, 2 per cent. maltose, or 2 per 1

In my hands many Priesz·Nocard bacilli have been found to be Neisser-positive.

27 2

GENERAL ARTICLES.

cent. glucose had been added. On the other hand, development was considerably enhanced by the addifion of liquid serum up to IO per cent. On plain agar slant growth was rather poor, taking the form of a grey, dry, non-spreading film. Growth was still further diminished by addition of glycerine, maltose, and glucose, and often failed entirely on glucose agar. As with broth, addition of 10 per cent. liquid serum to agar leads to a more abundant and whiter growth. Blood also forms a very good adjuvant, added as defibrinated horse blood up to IO per cent. Growths on blood agar made in this way become slightly brown or ochre-tinted with continued incubation. All strains grew on both ox and horse solid serum but more

FIG. 3.

Diphtheria bacilli, Strain D (horse), fourth day on solid serum. Gram stain.

x 1000

abundantly and yellower on the former, growth on horse serum being greyish-white; growths dryish and not spreading. On Dorset egg medium growth is without special characters. On naturally acid potatoes, no growth. On the other hand if the portions of potato be soaked in I per cent. sodium carbonate solution overnight before being tubed one may notice an extremely slight white growth, but generally not. In gelatine stab if the temperature is maintained at about 20° C. a moderately abundant growth takes place, confined to the needle track, and sometimes with slight growth on the surface. No liquefaction. All strains show definite zones of hcemolysis when grown on agar plates to which about 5 per cent. defibrinated horse blood has been added immediately before setting. The production of hcemolytic substances can also be shown, but not so satisfactorily, by incubating together at 37° C. a mixture of I per cent. washed horse corpuscles and the fairly clear fluid obtaining by centrifuging

GENERAL ARTICLES.

a week old broth culture. On microscopic examination of the red cells after such treatment one can note distortion and buckling of the cells, while no such change can be observed in a control tube containing unsown broth. All strains usually produced acid in litmus milk, the amount being just sufficient to slightly redden the litmus. At other times no appreciable acid production could be observed. An alkaline reaction was never obtain ed in milk. When grown in beef broth as ordinarily prepared . to which a sufficiency of litmus or neutral red has been added as an indicator after sterilisation, one notes first a change to acid reaction, followed after about a month by the appearance of an alkaline reaction. For some reason which was not clear the change occurred with greater

FIG. 4. Diphtheria bacilli, Strain G (horse), third day on serum agar, Neisser stain.

x 1000.

constancy in the case of neutral red broth than with litmus broth, i.e., the reaction in the latter medium sometimes remained acid even when the neutral red tubes had assumed a definite yellow ( alkaline) tint. (c) Sugar Fermentation Tests.- The medium used was sugarfre e broth (reaction + 10 phenol phthalein ) containing 10 per cent. sterile heated ox serum. The different test subst ances were added to the serum broth up t o I per cent. strength, either before the medium was tubed or after sterilisation, fro m stron g (10 per cent. ) solutions in distilled water. The difference in the procedure did not appear to affect the results. To some tubes a few drops of sterile litmus solution were added, others were reserved for titration against ,1'lf N aOH., using ~ per cent. phenolphthalein in 50 per cent. alcohol as indicator. T

GENERAL ARTICLES.

Duplicate sets of those for titration were always put up. Proper control tubes both sown and unsown were always introduced for the purpose of making colour and titration comparisons. The test substances were obtained from Messrs Baird & Tatlock and were guaranteed to be suitable. Those used were glucose, galactose, maltose, dextrin, lactose, saccharose, glycerine, inulin, salicin, and mannite. The litmus tubes were examined as a rule on the second, fourth, seventh, eleventh, fifteenth, and twenty-first days. Titrations were mostly carried out on the fifth and fifteenth days. In the case of lactose the second titration was made on the fourteenth day and in the case of saccharose and glycerine on ninth day. All cultures were heated to 60° C. for half-an-hour, and from each tube there were made two titrations of 5 cc. of culture, each diluted in 4S cc. distilled water (neutral to phenolphthalein). Titrations were made at the boiling point and the first appearance of a permanent pink tinge taken as the neutral point. Table II. illustrates the result of such a titration experiment. Concordant results were obtained with litmus as the indicator, and in addition no fermentation was noted with inulin, mannite, and salicin. No gas production occurred with any strain. From Table 11. it will be seen that all cultures produce acid in the presence of glucose, galactose, maltose, dextrin, and glycerine, but fail to do so with lactose and saccharose. It is to be noticed particularly that no fermentation of saccharose occurs.

Immunological. (a) Complement Fixation.-From a review of previous serological work with the diphtheria bacillus one gained the impression that it was a matter of difficulty to obtain satisfactory emulsions of this organism owing to the characters of its growth in artificial media. As a result it was thought that complement fixation would be the method most likely to give results, since this test permits the use of various extracted antigens. Specific immune sera were obtained by injecting rabbits intravenously (vide infra, p. 277). . The following were the principal methods tried with the object of finding the most suitable antigen : I. Simple antigen. The forty-eight-hour growth on solid serum or plain agar was very carefully emulsified in a small quantity of normal saline, pipetted off, well shaken, heated to 60° C. half hour in a water-bath, again shaken and placed on ice for two to four days. When required for use saline was added to give a slightly hazy emulsion. II. Eberson's 1 antigen. The three-day growth from serum agar contained in a Roux bottle was washed off in 10 cc. saline and the hacilli precipitated in clumps by adding absolute alcohol. The clumps were deposited by centrifuging, removed to a watch glass (previously weighed), and dried in vacuo. When required for use a weighed amount was removed and emulsified in saline to give an antigen of a definite strength. 1

"Journal Inf. Dis." July 19]8.

_.

~

day.

~

day,

~

day,

1'5

1'7

1'7

1'2

1'25

0

P

D 14

D 15

D 16

1'3

145

Lister

Un.own control

1'(;

1'1

1'1

1'3

1'33

1'55

1'5

1'6

1'25

1'1

1

UJ

1'2

x

1'75

2'6

3

3'15

3'1

4'15

3'2

3'35

3'95

2'25

4'05

3'6

3'05

1

I

i

!

I

I I

I

1'8

3'2

2'8

4'15

3'9

4li

3'n

4'2

4'8

2'2

3'9

4'05

3'S

~

day,

~

day,

I

~

day,

~

day,

I

~

day,

I

day,

m

I

day,

~

Lactose.

-------1 -Dextrir,.

1'7

1'65

1'5

x

1'55

2'9

3'25

2'85

2,95

3'35

2'8

345

2'9

2'45

1'75

2'4

2'2

2'5

3'7

3'5

3'4

3'3

3'8

2'8

3

2'S

2'1

1'6

2'85

2'65

x

2'95

3'4

4'4

2'25

2'6

2'55

3'3

4'35

3'3

3'S

I

1'75

3'45

3'3

1'15

2'6

2'9

x

2'8

I "

I

3'05

3'7

2'1

3'1

3'25

2'3

4'05

2S

3'85

4'2

I

5'2

4'15

3'S5

4

3'2

4'1

3'6

4

1'55

2'9

2'0

3'4

4'3

4'45

::I

2'75

4'3

3'4

3'85

3'S

3'75

1'7

1'05

1'1

2'3

2

2'1

2'05

2'25

2'5;j

2,1

2'7

1'05

4

1'6

'n5

'95

1'25

1'15

1'5

1'35

'()5

1'1

'9

1'4

1'75

1'1

-;3-~;- ~~5-1-~- -2~;-- -;'5~-1~;----;;;-

~

day,

,-

.Maltose.

- - - - - - . --_.-

Galactose.

II

\

I

:

9th day,

1'5

1'45

1'21\

1'4

1'4

1'55

15

1'6

1'5

1'25

1'6

1'55

1'4

l'S

1'7

1'3"

1'65

1'45

1'5

1'4

2'1

I'!)

1'3

1'45

175

1'8

1'55

l'S5

9th day,

1'25

2'95

2'2

x

1'9

2'45

2'2

2'45

2'7

2'85

2'6

3'15

l'S5

1'35

2'95

1'6

2'4

2'35

2'8

2'8

3'05

3'35

2'8

2'7

3'1

2'85

3'1

2'55

2'85

---

5th day,

- - - --

5th day,

Glycerine. --------

Saccha'J'ose. --------

Notes,-The figures represent the amount in cc, of 2"0- NaOH, required to neutralise 5 cc, of culture fluid diluted with 45 cc, neutral distilled water, Each figure is the average of two estimations, The results with inulin, mannite, and salicin are not included, these test substances being of less importance, D 34 was a recent addition to the series, and no titration experiments were conducted, the strain being passed only through the litmus tubes, ( - ;;;;No fermentation, x=fermentation,) A repeat test was made in the case of any tube which failed to grow or which was obviously contaminated,

1

Elstree ,

-

1'6

L

D 34

1'55

I

1'15

1'85

'

1'65

,

H,

,

G,

F,

~

day,

-;.;-

i

Glucosc.

~-, - ,- - ,- ~6~ ~;;- - 3 -

Strain.

Ox Serum Broth. I

TABLE

Z

:;0

'"

->


'!'

l'J

r-'

(")

j

;,.

t"

:;0

'" ;,.

C'l l'J

GENERAL ARTICLES.

III. Ten Broeck's 1 antigen. One five-day. Roux bottle agar growth was detached in 10 cc. of distilled water by means of a bent pipette into small centrifuge tubes, heated to 60° C. for one hour, thoroughly shaken, and placed on ice for seven days with shaking each day. The antigen was made isotonic when required for use by adding ICC. 8 per cent NaCI. to every 9 cc. of emulsion and centrifuged at high speed. The supernatant fluid was used as antigen without further dilution. Of these three, the simple antigen made from serum cultures appeared to give the most satisfactory results, so far as actual fixation was concerned. It was soon found, however, that by the ordinary technique employed the results were not sufficiently specific to be of value, a good deal of cross-fixation occurring \\ jth allied antigens, including the Preisz-Nocard bacillus. The work on complement fixation was not pressed, partly owing to this non-specificity but more particularly to the fact that it was found that the more easily conducted agglutination test would yield far better results. Owing to these facts, details of the complement fixation experiments are purposely omitted. (b) AggZutination.-As stated above, it is hardly possible by the usual method to prepare satisfactory emulsions with the diphtheria bacillus and allied bacteria. The rea50n for this is that the growths on solid media tend to be dryish and coherent, and in liquid media, e.g., broth, are often of a granular type. The following simple method was found, however, in most cases to give quite satisfactory results. Three or four-day growths on horse-serum agar were scraped off into a few cc. of normal saline by means of a pipette slightly bent near the drawn-out extremity, care being taken not to tear the agar. The somewhat granular emulsion was pipetted off into a tube and centrifuged at high speed to throw down as abundant a deposit as possible. This was then washed once or twice in fresh saline. The final deposit was mixed with as small a quantity of saline as possible, heated 60° C. half an hour, and pi petted on to a watch glass, which was placed in a desiccator containing calcium chloride. \\'hen the deposit was quite dry the watchglass was stored on ice. The emulsion for the test was made up from this dry deposit by patient grinding in an agate mortar, and, when finally powdered, saline was very gradually added, drop by drop at first and later more quickly, until one obtained an emulsion roughly twice the ultimate density required, which was rather thicker than a faint haze. 1 cc. 5 per cent. carbolic acid in saline was then added to every 9 cc. of emulsion as a preservative. Emulsions made in this way were in most cases quite satisfactory and could be stored on ice and used for several weeks if necessary. Sedimentation was sometimes considerable if the tubes were left standing for a few days, but a thorough shake before use was all that was necessary to give a homogeneous emulsion. Emulsions were made twice the ultimate density required owing to the technique followed in the tests (vide t"nfra). It is to be remarked however, that some strains were not so 1

"Journal Exp. Med." December 1918.

GENERAL ARTICLES.

readily emulsified as others. This was always found to be the case with the Elstree strain, and consequently this could not be used for tests. The same thing occasionally happened with some of the equine strains, particularly D 16, but as a rule a second or third trial was followed by success. The specific agglutinating serum was prepared from healthy rabbits weighing 5 or 6 lbs. Weekly injections of dead bacilli (60° C. half hour) were given intravenously, commencing usually with one quarter of the growth from an average-sized horse-serum agar slant and gradually working up to the growth from a whole Roux bottle culture. The {ollowing example is given of one such experiment : 29 th January 1920 and 5th February 1920. Rabbit 12 received intravenously half a growth three-day serum agar slant, 60° C. half hour, Strain H. 14th February 1920 and 21st February 1920. Rabbit 12 received intravenously one growth three-day serum agar slant, 60° C. half hour, Strain H. 27th February 1920. Rabbit 12 received intravenously two growths three-day serum agar slant, 60° C. half hour, Strain H. 4th March 1920. Rabbit 12 received intravenously half growth three-day serum agar (Roux bottle), 60° C. half hour, Strain H. 11th March 1920. Serum showed no agglutinin when tested. 24th March 1920, 31st March 1920, 8th April 1920, and 15th April 1920. On each date Rabbit 12 received intravenously surface growth (three-day) from one Roux bottle serum agar, 6d C. half hour, Strain H. 22nd April 1920. Serum showed agglutination up to I in 80 when tested. 4th May 1920 and 15th May 1920. Two further injections on these dates, on each occasion dead growth from one Roux bottle serum agar. 22nd May 1920. Serum showed stronger power of agglutination than on 22nd April 1920, but titre still only 1 in 80. Similar agglutinating sera were prepared from Strains Land D 15. The titre of such sera was never very'high, rarely over 1 in 320, but, on the other hand, the results were marked by a large measure of specificity. Technique of Test.- The test was carried out by the macroscopic method, using tubes 70 by 7 mm. The emulsion was delivered from a Pa~teur pipette graduated to '25 cc. and a similar quantity of serum dilution (I in 5, I in 10, I in 20, and so on up to 1 in 640) added, giving final dilutions of 1 in 10, 1 in 20, up to I in 1280. Normal rabbit serum was used as control in 1 in 10 and 1 in 20 final dilutions. The results appeared to be more specific at 55° C. (water-bath) than at 37° C. (water-bath), and consequently in all the tests the former temperature was chosen. Agglutination was generally strongly marked after one hour, and the results were read after four to five hours incubation. Total liquid in each tube '5 cc. Tables II l. and IV. give the results of actual tests in which DIS and H sera were employed. Incubation at 55° C. in a water-bath and results noted at the fourth hour.

GENERAL ARTICLES.

A, B, E, and R were strains of Bacillus Preisz-Nocard; J and Q were (horse) wound diphtheroid bacilli of two different types; D I, D 2, and D 26 were diphtheroid strains of two or three types isolated from the conjunctival sac of normal horses, and Hoffman was a TABLE

III.

IV.

TABLE

Carbolised D 15 Serum.

Carbolised H Serum.

Strain.

: -----------1----------

-------~--

N,O

1'80 \1'160 \1'320 1'640 1'128 N,O

R

1'2)

xxx

-- -

- -

1'80

1'160 1'820 1'20

xxx

xxx

-x--x-~--

xx

x

.J Q D 12 D 26 Hoffmann

D1 D2

x

D

'" xxxx

xxx x

F

xx

x

G

xxxx

H

xx

~

xxx

'x"

xxxx

xxxx

xx

xx

x

L

xxxx

xxxx

xxxx

0

xxxx

xx

xx

p

xx

x

D14

xxx

xxxx

xxxx

D 15

xxxx

xxxx

xx

?x

xx

x

"

xxxx

xxx

xx

xxx'!!:

xxx

xx

x

xxxx

xxx

~

xxx x

xxx

xx

"x

xxxx

xxx

xx

x

xxxx

xxxx

xxx

x

xxx

xx

xxx

xx

"x

xxxx

xxx

xxx

x

D 16

xxxx

xxxx

xx

x

Lister

xxxx

xxx

x

xx

--

N,O

x?

strain of Hoffman's (human) throat bacillus obtained from the Lister Institute. These were all included as additional controls to the specificity of the reaction.

1'40

GENERAL ARTICLES.

The following signs are used to indicate the degree of agglutination : xxxx = Complete agglutination and sedimentation, liquid water-clear. xxx = Agglutination practically complete and sedimentation nearly so, liquid nearly water-clear. xx = Partial agglutination. x = Distinct agglutination. x = Agglutination just appreciable on very close inspection without the use of a lens. - =No agglutination . ... = Not done. Table I II. shows quite clearly the marked specificity or the test serum prepared from Strain DIS. The strains under consideration all agglutinate without exception, while control emulsions of diphtheroid bacilli from various sources uniformly fail to do so. In the case of the serum prepared by the use of Strain H (Table IV.) the results are not quite so good. It will be seen that Strains Band R showed considerable agglutination. This may have been due partly to the tendency to spontaneous sedimentation which these particular emulsions exhibited, or more probably to the serum not being quite specific. i.e., containing minor agglutinins for these strains. The same tendency to non-specific agglutination was met with on several occasions during the course of these researches, and the fact that such agglutination was due to the existence of minor agglutinins was proved by means of absorption tests. Table V. gives the result of such an absorption test carried out with the same serum H and using the same emulsions as in Table IV. Four centrifuge tubes were set up. Tube I. contained H serum diluted 1 in 10 with carbolised saline. Tube II. contained H serum dil uted 1 in 10 with carbolised saline in which had been emulsified the growths from two Roux-bottle serum agars sown with Strain H. The growths from the Roux bottles were detached in carbolised saline, deposited by centrifuging, washed, heated to 60° C. half-hour, dried on a watch-glass, and well ground up before being added to the serum. Tube III. contained H serum diluted I in 10 with carbolised saline in which had been emulsified an amount of Strain R (PreiszNocard) approximately equal to the growth used for Tube II. Total volume of liquid in each tube was 10 cc. The tubes were placed in a water-bath at 37° C. for thirty-six hours. A preliminary test showed that absorption was then complete in the case of Tubes II. and III. Tube IV. contained a. similar dilution of carbolised normal rabbit serum. Absorption by H has thus removed all the agglutinin from the serum, while R has removed the minor agglutinin for Band R, but has affected the agglutinin for the Strains G, H, L, 0, P, DIS and D 16 only slightly in comparison.

280

GENERAL ARTICLES.

V.

TABLE

Strain.

Unabsorbed H Serurn in Final Dilutions of

H Serurn Aosorbed H Final Dilutions of

1 1 0 1 "_'\______ __ 1_'2{_} _ _ 1_'4_ 1__'8_°__ 1___'2_°__ 1___'4_°__

B : 2

G

o

'~I':'i 1~ _

xx

xx

xx

i

X:X

xx

x

I

::~

:

H Serum AbsorbedR in Final Dilutions of

in

x

I

180

x

x

m

I

I '::'

':'

I ,:: I xxx

x

x

x

x

xx

x

xxx

xx

xx

p

xxx

x

x

D 15

xxx

xxx

xx

x

xxx

xx

x

D 16

xxxx

xxx

xxx

x

xxx

XXY

x

x

Lister * . "Emulsion largely sedimented spontaneously. water-bath.

Incnbation for six hours at 55' C. in

In order to demonstrate finally the antigenic similarity between a strain of human diphtheria bacillus (Lister) 'and certain of the strains isolated from horses, direct absorption tests were carried out and the results are presented in Table VI. TABLE

Stmin.

Unabsorbed Carbolised H Serurn.*

HSer,J/ffI, Absorbed with H.

* As used for test shown in Table V.

t

VI. H Serurn Absorbed with Lister.

HSe"urn A bsorbed with D19.

Emulsions of P and D 16 accidentally mixed.

GE:-;ERAL ARTICLES.

A bsorption was carried out in the manner before described (except that the mixtures were incubated for twenty-five hours only), usi~g Strains H, Lister, and D I9. The last was a diphtheroid bacillus which had been originally isolated from pus from a human wound, and which for certain reasons must be regarded as most probably not identical with the Bacillus diphtheri~. Incubation for four hours at 55° C. Results were read on this occasion after the tubes had been standing at room temperature overnight. It will be noted that absorption with D I9 has failed to remove a very appreciable amount of agglutinin for any strain. L was evidently a very easily agglutinable organism, and consequently the serum proved to be incompletely absorbed for it. It will also be seen that Lister is practically as efficient for removing the major agglutinin from the test serum as the strain used for the preparation of the serum, viz. H. It cannot be, therefore, that the agglutinin for Lister is of the nature of a minor agglutinin. Animal Inoculation. Table VI I. gives a resume of inoculation experiments with the equine and human strains. From a survey of this table it becomes apparent (I) that some strains (F, G, I, 0) are quite harmless on inoculation; (2) that short of this result in the case of Strains P, D I4. D I 5, the animals became emaciated and died without exhibiting any very noticeable lesions, or that any ill effects were quite transient; (3) that the two strains (L and Elstree) exhibited only very slight virulence but, as will be seen later, produced a good deal of toxin when growing in vitro; (4) that with the remaining strains death of the experimental animals when it occurred was due not to widespread multiplication of the bacteria but to tox;emia as the result of their localised growth (see for example Guinea-pig 180, Strain H).

Toxin Production and Neutralisation of Same by Antitoxin. Of the means employed for determining the true nature of the bacilli under consideration, the question of toxin production (if any) and neutralisation of the same by serum 1 prepared from known human diphtheria toxin is the most interesting and important. It was found very early in the work that some of the equine strains were capable of excreting an active filterable toxin into the medium in which growth was taking place. The next step was to find out whether this toxin was identical with human diphtheria toxin, allied to it in nature, or something entirely different. Vv'hichever of these alternatives had proved to be correct the result would have been equally interesting, in the first place on account of the probably very rare occurrence of Bacillus diphtheri;e in the horse, and in the second place because, apart from the Preisz - N ocard bacillus, no other diphtheroid has been shown to produce an active exotoxin. 1 The expression" human autidiphtheritic serum" is used in this article for such serum to distinguish from that prepared by the use of "equine toxin."

Do.

Do.

Do.

Do.

Guinea-pig 57 .

Mouse

Guinea-pig 7

Rabbit 4.

8.12.19

F

13.10.19

H

Horse 53.

2 cc. 4-day serum culture 1 cc. 4-day serum culture 1 ce. 4-day serum culture Washed bacilli from 7· day Dean's broth culture

Material and Dose. / Post-rnort(rn Findin g./ R(m((rks.

Diell in emaciated condition on 15th day

Died 17.10.19

except

Local abscess at point of inoculation, smears from which showed Gram + bacilli; adrenal gland. slightly reddened and lungs congested

No lesions

and showed :.:-symp-I----=-----I----=---toms or loss of weight Do. do.

Result.

Anim~ survived,

VIt.

3-day serum agar Animal survived, and shoWell no sympemulsion (10th genertoms or los8 of weight ation) 1 cc. serum broth (9th Do. do. generation) 1 cc. emulsion of 4-day Survived serum culture 1 cc. emulsion of 4-day Animal survived, and showed no sympserum culture toms or loss of weight 2 cc. emulsion of 4-day Do. do. serum culture No lesions 1 cc. emulsion of 4-day Died 3.11.19 serum culture 1 cc. emulskn of 4-day Animal survived, and showed no sympserum culture toms or loss of weight 1 cc. emulsion of 4-day Died 25.10.19 No lesions serum culture 1 cc. emulsion of 4-day Animal died on 20th day, probably due No lesions emaciation serum agar cuI ture to cold Slight swelling of the injected limb up 2~ cc. emulsion of 4·day to the carpal region, bnt insufficient serum agar to cause lameness. Swelling disappeared in a few days

. I l~ ce.

Subcut. Fetlock (O.F.)

Do.

Do.

Mouse 8 .

H

Guinea-pig 25 .

2.9.19

13.10.20

H

Do.

Do.

Mouse 7 .

Guinea-pig 8

2.9.19

2.9.19

G

2.9.19

G

H

2.9.19

G

F

Intrap.

Guiuea-pig 37 .

23.10.19

Subcut.

F

Do.

Mouse 4 .

Guinea-pig 111 (350 gram.)

2.9.19

16.6.20

D

Do.

Guinea-pig 4

2.9.19

D

D

Intrap.

----------

Animal.

Rabbit 3.

Date.

2.9.19

I

D

Strain.

Route of 1noculation.

TABLE

:r,

'"

~

r

r;

..;

:>;J

;l>

~

;..

t;:

Z

"

...

w

00

Date.

I

Animal.

I

In~:~~~t~t.1. I

Mate1'ial and Dose.

I

R~sult. 1

Pvst-mortem Finding. 1

Remarks.

L

I

13.10.19

Guinea - pig 26 (450 gram.)

Do.

Do.

]\[ouse 50

8.12.19

I

Do.

2.9.19

Guinea-pig 58 .

Do.

8.12.19

Guinea-pig

mucous

membrane of vulva

on

Scarified area

Guinea - pig 91 Intrap. (350 gram.) ]\[ouse 9 . Do.

2.10.20

H

Gninea-pig

2.9.19

26.8.20

H

1

1

1

1.

1

A

loopful of 2 - day serum agar culture, rubbed into scarified area cc. 4-day serum culture cc. 4-day serum culture cc. serum broth culture cc. serum broth culture cc. emulsion from 4 - day serum agar culture

area Loopful from 3 - day serum agar cuI ture

I Died on 28th day

Animal survived, and showed no symptoms Survived

Animal ~urvived, lost no weight and showed no symptoms DiEd 27.9.19

Died on 2nd day with fibriuous membrane and considerable swelling of the part

Rather sligh~ fibrinons deposit fur 2 days and slight swelling of the part, which then disappeared. Animal survived

of

Condition rather emaciated, but no lesions

No lesions

Typical lesions toxaemia

mucous

mem-

the brane was made by touching with hot glass and scraping with scalpel

Erosion of

----,----- --------,-------- ------------ ------------------- ----------- --------H 18.10.20 Guinea-pig 179 Subcut. 1 cc. 48 - hour broth Died on 2nd day after showing symp- Typical lesions of culture toms of acute intoxication (310 gram.) toxaemia H 18.1O.~0 Do. 1 cc. emulsion of washed Died 6 or 9 hours later than Guinea-pig Guinea-pig 180 Do. bacilli, from culture (420 gr'1m.) I7l), after showing symptoms of acute as for Guinea-pig 179 intoxication H 24.8.20 Horse Scarified area ~ ce. 4-day serum agar Slight swelling for a day or two, which The area on the on back culture rubbed into then disappeared. back was first scarified area shaved and then slightly scarified with poin t of knife H 24.8.20 Guinea-pig Do. cc. 4 - day culture Death on 3rd day of acute intoxication. Typical lesions of toxaemia rubbed into scarified

Strain. I

T ABLE V I I.-continued.

~

'"

00 CN

~

fxl

(;

>-l

>

> t""'

~

t>l

Z

t>l

Cl

!

I

.

Ammal.

I

Route of Inoculation.

Material and Dose.

Result.

VI I.-continued.

I

Horse 59.

Guinea - pig 52 (510 gram.)

24.10.19

24.10.19

19.11.19

D14

D14

D 15

I

Do.

Mouse 43

24.10.19

D14

IntraI'.

Subcut. At fetlock (N. :1<'. )

no

IntraI"

Guinea-pig 46 .

13.10.19

p

Do.

Emaciation but lesions No lesions

Subcut. Near fetlock (O.F.)

Horse 49.

13.10.19

I\Iouse 27

emulsion from I No swelling or lameness 4 - day serum agar culture 1 cc. emulsion from I Died on 17th day I-day serum agar 1 cc. emulsion from Died 11th day i-day serum agar 2 cc. emulsion from Some swelling up to the carpal region and very slight lameness, which persisted 1 - day serum agar culture (3rd genera- , for a few days tion) 1 cc. 5-day serum broth Survive,l, and appeared to be healthy. culture (5th genera- , tion) .

A questionable lesion in neighbourhood of the testis

Died 14th day

1 cc. 4-day serum agar culture

p

2~ cc.

Some emaciation but no lesions

Died 20th day

1 cc. 4-day serum agar culture

13.10.19

p

Intrap.

2 cc. emulsion from serum culture

Subcut. Above fetlock (N-F.)

Horse 53.

269.19

Guinea - pig 27 (590 gram.)

No swelling or lameness

1 cc. serum culture

Do.

lIlouse UI

26.9.19

Animal remained healthy and lost no weight Survived

Intrap.

Scarifications healed, ami animal showed no untoward symptoms

A questionable lesion in the neighbourhood of the testis

Guinea - pig 19

Scarified area on back

Guinea-pig 150 (350 gram)

No swelling or lameness

Died on 14th day

26.9.19

Loopfulof 2-day serum culture, rubbed into scarified area 1 cc. serum culture

S ubcu t. .J us t above fetlock

cc. emulsion from 4 - day serum agar culture 2! cc. emulsion from 4 - day serum agar culture

Horse 54.

(N.F.)

IntraI'.

o o o

7.9.20

L

I

!

13.10.1ll !l\Iouse 26

1

Remarks. Post-mortem Finding. _ _ _ _ _ _ __

--------------1----------1-------------1------

13.10.19

I

Date.

L

L

__ -

Stl·ain.

TABLE IV

00

t%J

'!'

~

t'"

(')

"j

>

t'"

>

:;
t%J

Z

C'l

.j>.

Date.

~11.19

19.11.19

19.11.19

1!l.11.1!)

HI.n.1!)

Strain.

D]5

D 15

D 16

D 16

D 16

I

Subcut. Just above fetlock (N.F.)

Do.

Subcut. At fetlock (N,F.)

lIIouse 46

Horse 62.

I Material and Dose.

I

5 cc. 5-day serum broth culture (4th genera· tion)

1 cc. 5-day serum broth culture (4th generation)

1 cc. 5-day serum broth culture (4th generation)

5 cc. 5-day serum broth culture (5th generation)

cnlture (5th genera· tion)

Intra;---.-I~~-day serum broth

Intrap.

--

b~~~f~t!on.

Guinea·pig 53 (520 gram.)

Horse 52.

lIIouse 45

Animal.

I Post·mortem Finding. Remarks.

Considerable oedematous swelling of the limb, at first reaching only to the knee but gradually extending to the elbow. Animal dead lame. A well· marked gummy eruption appeared below the knee on the 4th clay, with some oozing at the point of inoculation. Temperature rose to 103'6° F. on the 1st day and to ]04'8° F. on 4th; then it gradu· ally dropped to subnormal on 11th day, when death supervened.

Deacl 3rd day

0

I

An abscess in omen· tum, from which bacilli resembling D 16 were grown. No bacilli in blood. Also characteristic lesions of toxremia Lungs greyish and sol i d if i e d i n patches. Bacilli resembling D 16 cultivated from the spleeu but not from the blood Beyond very pronouncecl oedema of of the limb there were no well· marked lesions

which contained Gram + bacilli resembling those injected -

I

-

-

-

d:;------------I~:_:mental~scess, ---~--

Next day there was a marked swelling of the limb up to the knee, and the animal was dead lame. S welling and lameness disappeared in 4 days, the animal showing no other symptoms except rise of temperature to 102'2 F. on 2nd day Dead 2nd day

Died 23rd

Result.

T ABLE V I I.-continued.

'"

()O Ol

~

t>l

t""

n

:>0 >-l

:.-

t""

:.-

:>0

t>l

Z

Q t>l

2.9.19

2.9.19

2.9.19

Lister I

Lister I

Lister

Scarified area on back

Guinea-pig 149 (350 gram.)

Guinea·pig 156 (385 gram.)

Guinea-pig 190 (370 gram.)

7.9.20

Elstree I 15.9.20

D 34 I 30.10.20

Do.

Scarified area on skin of back SUbCllt.

Do.

Elstree I

Do.

Guiuea . pig (old)

Scarified area on vulva

Subcut.

Do.

Do.

Do.

Do.

I Intra]!.

II

I

3 loopsful of washed deposit from 7 - clay Martin's broth cultUre 1 cc. 2·day ox serulll broth

Loopful 4 - day serum agar culture rubbed in

Loopful 3· day serum cultUl'e rubbed in Loopful 3· clay serum culture rubbed in

Loopful 2 - day serum agar culture rubbed in

1 cc. 4-day serum broth culture Thrice washed bacilli frolll 2-day broth

1 cc. 4·day serum broth culture

1 cc. emulsion frolll 4·day serum culture

1 cc. emulsion from 4·day serum culture

Several loopsful from 8-day ox serum cui· ture 2 cc. emulsion from 4-day .crum culture do.

Died same night

Survived, and lost no weight. abscess fornoed locally

A small

Scarified area healed, and animal presented no symptoms

Survi ved, after showing slight fibrinous deposit and swelling of part Do. do.

Died at 48th hour

Died 6th day

Died on 9th day

Animal survived, and lost no weight

Survived

Do.

Animal survived, and lost no weight

healed up without any disturbance locally or generally

Sc~rifications

No markedlesiolls

False membrane on scarified area and lesions of toxremia

Lesions of toxremia

No lesions

RCSUlt. _ _ _ _ _ _ 1 Post· mortem Finding.

J_________

ll':::~1~ I M.',,'.'."" n..

Guinea-pig

2.10.20

Elstree

26.8.20

Guinea (large)

Lister I 13.10.20

I

Guinea·pig 185 (430 gram.)

Lister I 23.10.20

Elstree

}Iouse 48

Lister I 8.12.19

pig

Guinea-pig 53 .

Mouse

Guinea·pig 13 .

Rabbit 6.

Horse

__Anima_l·__

Lister I 8.12.19

I

23.3.20

Dat~I

D 16 I

Strain.j

T ABLE V I I.-continued. Remarks.

Z

tol ~

ri""t'"

'"

:>-

t'"

':>-"

l'l

Cl l'l

'"

0>

00

GENERAL ARTICLES.

The methods adopted for demonstrating the true nature of the toxins produced by the equine strains were as follows;(a) By comparing the lesions produced in young guinea-pigs with those produced by known diphtheria toxins. (b) By seeing whether these toxins were completely neutralised in vitro by human antidiphtheritic serum. The word" completely" in the preceding sentence is important owing to the evidence brought forward by Dassonville 1 to show that the toxin of the Preisz-N ocard bacillus is rather similar to diphtheria toxin and is partially neutralised by human antidiphtheritic serum. (c) By seeing whether human antidiphtheritic serum was potent in producing a passive immunity against the equine strains. (d) By preparing an antitoxic serum against one of the equinetoxins and seeing whether such a serum was capable of neutralising human diphtheria toxin. In the first place it must be said that only some of the equine strains were capable of producing a toxin. Strains F, G, I, 0, P,. D r 4, and D r 5 were atoxic. Strain D seemed to be rather feebly toxic. The remainder, viz., H, L, D r6, and D 34 were definitely toxic, as were also the two human strains, Lister and Elstree. Preparatz'on of tlte Toxin.- Two different broths were employed; (a) Dean's broth. This is prepared exactly as ordinary nutrient broth, except that the beef used should be left to stand at room temperature for a week. Actually one used frozen beef which was kept two days before being minced up. After the broth has been made slightly alkaline to litmus one adds 7 cc. N. NaOH. per litre. (b) Martin's broth. Prepared by the usual technique from pigs" stomachs, except that beef was used in place of veal, which was unobtainable. Adjusted to + r 5 (Eyre scale). The medium was distributed in roo cc. amounts in Roux bottles, which were laid flat during incubation to give a thin layer of liquid with a broad surface exposed to the air. Steam sterilisation for half an hour on each of three successive days was employed. The seed material was always taken from a two or three-day borse serum or serum-agar culture, but no flask was used unless growth was abundantly present. At the end of seven to nine days the culture was tested for purity by microscopic examination and filtered through a Doulton candle which gave rapid filtration and which had proved itself many times to be reliable. r cc. of the filtrate was always transferred to a tube of serum broth and incubated as a sterility test. The toxic fluid was distributed into ampoules and stored on ice without the addition of any preservative.

(a) Effect of tlte Toxins on Guinea-pigs. Throughout this part of the work young .guinea-pigs from 250 to· 350 grammes were employed in nearly all cases. All the toxins, including those of Lister and Elstree, produced identical effects on small guinea-pigs if given subcutaneously in the abdominal region in at least the minimum lethal dose. Death occurred in from about sixteen hours to four or five days, depending upon the dose. The following lesions were present with great constancy; More or less. 1

"Journal of Comparative Pathology and Therapeutics," Vol. XXI., p. 181, Abstracts.

288

GENERAL ARTICLES.

abundant hcemorrhagic, gelatinous <:edema in the subcutaneous tissues at the seat of inoculation and extending in many cases to the upper part of the chest. Moderate enlargement of the spleen. Darkening of the adrenal glands due to hcemorrhage into the cortex or into the substance of the glands. Greyish patches of solidification in the lungs and extremely abundant effusion of clear watery fluid into the pleural cavities.

(b) Toxin Neutralisation Experiments in vitro. The principle of all these experiments was the same. One found in the first place the minimum lethal dose, i.e., the smallest amount of toxic fluid which was capable on subcutaneous inoculation of producing death in guinea-pigs (250 to 350 grammes weight) in four or five days. When this had been determined one mixed in a tube this dose of toxin with the smallest amount of anti diphtheritic serum which in similar circumstances would just completely neutralise one minimum lethal dose (M.L.D.) of known diphtheria toxin. This quantity of serum was found to be approximately what was to be expected, viz., the amount equivalent to -d-o unit. The mixture was immediately injected subcutaneously into a guinea-pig. The result of all these tests was the same, viz., that human antidiphtheritic serum was quite as efficient for neutralising in vitro the toxins of the strains isolated from horses as for neutralising the toxins of the human diphtheria bacillus. In the earlier experiments on this part of the subject it was not found possible to employ what are probably the more usual test standards, viz., IOO times the M.L.D. of toxin + I unit of antitoxin, owing to the fact that the M.L.D. rarely fell below '2 to '4 cc. This was with Dean's broth, which was used most extensively. Later, however, with Martin's broth, more potent toxins were prepared, the M.L.D. being as small as '01 cc. With such potent toxins, therefore, one was able to employ IOO M.L.D.'s as the test quantity. The following protocol of experiments is given in support of these statements:Elstree Toxin.-M.L.D. found to be '9 cc. 7th September 1920. Guinea-pig 144 (270 grammes) received subcutaneously '9 cc. Elstree toxin. This was followed by an <:edematous swelling. On the third day the animal was very sick, and on the fourth morning was dead. Post-mortem examination showed the usual lesions of toxcemia. 7th September 1920. Guinea-pig 141 (250 grammes) received subcutaneously a mixture of Th unit antidiphtheritic serum and '9 cc. Elstree toxin. A slight swelling developed locally but the animal remained lively for thirty days. L Toxin.-M.L.D. found to be '4 to '5 cc. 7th September·192o. Guinea-pig 148 (325 grammes) received subcutaneously '4 cc. L toxin. Animal died on sixth morning after being very sick for two days. Post-mortem showed the usual lesions of diphtheritic intoxication, with local <:edema and deeply congested lungs but no pleural effusion. 11th September 1920. Guinea-pig 153 (3IO grammes) received subcutaneously a mixture of Th unit antidiphtheritic serum and

GENERAL ARTICLES.

'5 cc. L toxin (slightly largt'r' amount owing to the guinea - pig 148 having survived a trifle too long). A moderate swelling developed locally and later sloughed off, but the animal remained lively for thirty days. 7th September 1920. Guinea-pig 146 (280 grammes) received subcutaneously a mixture of 1 unit antidiphtheritic serum and '4 cc. L toxin. No trace of swelling developed locally, but the animal was noted to be completely paralysed in the hind quarters on the twentieth day and was killed next day. No lesions seen post-mortem except slight patchy congestion of the lungs. (Note.- This was the only result of the kind noted, and is rather curious considering the large amount of serum used in the experimellt; although, of course, it is well known that diphtheria toxin may be responsible for such accidents if given in a dose less than the M.L.D.) H Torin.-M.L.D. found to be '2 cc. 14th October 1920. Guinea-pig 173 (250 grammes) received subcutaneouslY'2 cc. H toxin (stored since IOth July 1920). Animal gradually became very sick and maintained an arched-back attitude owing to the abdominal cedema. Died on the fifth night. Postmortem examination showed the abdominal muscles to be very dry and necrotic-looking, and the lungs were found to be intensely congested, but there was no actual pleural effusion at the time of death. 18th October 1920. Guinea-pig In (250 grammes) received subcutaneously a mixture of T~O unit antidiphtheritic serum and '3 cc. H toxin (as used for guinea-pig 173). Except for a very slight cedema, the animal remained quite healthy for thirty days. D I6 Torin.-M.L.V. found to be about '75 cc. 9th November 1920. Guinea-pig 203 (250 grammes) recei\'ed subcutaneouslY'75 cc. D 16 toxin (stored since 3rd November 1920). The animal was sick on the first day and dead on the third day, showing a moderately marked cedematous lesion locally and pronounced lung lesions (congestion). 12th November 1920. Guinea-pig 207 (4IO grammes) received subcutaneously a mixture of r&o unit antidiphtheritic serum and '75 cc. D 16 toxin (as used for guinea-pig 203). The animal remained lively and showed no trace of local swelling. D 34 Torin.-M.L.D. found to be about '3 cc. 11th November 1920. Guinea-pig 206 received subcutaneously '3 cc. D 34 toxin (Dean's broth, seven day). Dead third day, with pleural effusion and grey patches in lungs. 24th November 1920. Guinea-pig 229 received subcutaneously a mixture of Th unit human antidiphtheritic serum and '3 cc. D 34 toxin (as used for guinea-pig 206). Animal survived. D Torin.-M.L.D. not determined, but I cc. eleven-day Martin's broth culture produced death in forty-eight hours thus : 17th November 1920. Guinea-pig 2 I 5 (3 IO grammes) received subcutaneously 1 cc. as stated. Death occurred forty-eight hours later, and post-mortem examination showed slight local cedema and pleural effusion. 19th November 1920. Guinea-pig 220 (260 grammes) received subcutaneously a mixture of ~ unit human antidiphtheritic serum and u

GENERAL ARTICLES.

I cc. thirteen-day Martin's broth culture. and showed no symptoms.

Animal remained lively

(c) Experiments showing the Protective Power oj Human Antidiphtheritic Serum against Fatal Infection by the Equine Strains. Roux in his early work on diphtheria found that with many strains of the bacillus it was possible to produce a typical false membrane and fatal intoxication if a small amount of culture was rubbed into a small scarified area on the vulval mucous membrane of female guinea-pigs. His method of scarification was by the use of a hot glass rod. In the following experiments very young (one or two-day) serum agar cultures were used and slight erosion of the mucous membrane was produced in the same way, the burnt layers of epithelial cells being finally scraped away lightly with the point of a knife. In this way it was found that one of the human strains (Elstree) and two of the toxic equine strains (L and D) were incapable of producing a fatal intoxication, a slight membrane forming which ultimately sloughed off and the animal evincing no untoward symptoms. Strains Lister, H, D 16, and D 34, however,. nearly constantly produced a fatal intoxication in two or three days with a local formation of false membrane. A subcutaneous injection three to six hours before rubbing in the culture of t unit human antidiphtheritic serum uniformly succeeded in protecting the guinea-pig from this fatal result. All that happened in such cases was some swelling and slight membrane formation of the vulva. Details of these experiments were as follows:Lister Strain.-13th October 1920. Large female guinea-pig received on scarified area on vulva a loopful of a two-day serum agar culture. The animal was sick next day and showed a yellowishwhite false membrane on the eroded area. Death occurred in less than forty-eight hours. A fairly pronounced membrane was present at the seat of inoculation, and post-mortem examination showed an enlarged spleen, slightly darkened adrenals, greyish solidification of the lungs, and abundant pleural effusion. 23rd October 1920. Guinea-pig 184 (350 grammes) was inoculated in the same manner, but had received subcutaneously three and a half hours earlier t unit antidiphtheritic serum. The animal survived after showing slight membrane formation on the scarified area. H Strain.-9th October 1920. Large female guinea-pig received on scarified area on vulva a loopful of a twenty-four hours serum agar culture. The animal was dead at the forty-eighth hour with a well-marked false membrane, h;:emorrhage into the cortex of the adrenals, and congested lungs. A six hours previous injection of -is unit antidiphtheritic serum failed to protect a similar guinea-pig inoculated by scarification at the same time, no doubt owing to the dose of serum being insufficient. 2nd November 1920. Guinea-pig 193 (250 grammes) received subcutaneously ~- unit antidiphtheritic serum and six hours later was scarified in the usual way with a loopful of twenty-four hours serum agar culture. Considerable swelling and slight membrane

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formation occurred in the vulva, but the animal remained quite lively. D I6 Strain.-I9th November, 1920. Guinea-pig 221 (250 grammes) received on scarified area on vulva a loopful of one-day serum agar culture. Local swelling and slight membrane formation occurred, with death about the thirtieth hour. Post-mortem examination showed pleural effusion. 23rd November 1920. Guinea-pig 226 (250 grammes) inoculated exactly as guinea-pig 22 I, but three and a half hours earlier a subcutaneous injection of ~ unit of human antidiphtheritic serum had been given. Slight membrane formation occurred on the vulva, but the animal remained lively. D 34 Strain.-I9th November 1920. Guinea-pig 222 (250 grammes) treated exactly as guinea-pig 221, except that D 34 strain was used. Death occurred at about the thirtieth hour, but the animal showed pleural effusion and slight membrane formation on the scarified area. 23rd November 1920. Guinea-pig 227 inoculated exactly as guinea-pig 222, except that three and a half hours previously a subcutaneous injection of ~ unit human antidiphtheritic serum had been given. Slight membrane formation occurred on the scarified area, but the animal remained lively.

(d) Preparation of an Antitoxic Serum against the Toxin of an Equine Strain and Neutralisation of tlte Toxin of Human Bacillus Diphtherice by the same. For the production of an antitoxic serum a healthy, full-grown goat was employed and was injected at intervals with gradually increasing doses of L toxin. Injections commenced on 21st July 1920, the animal receiving subcutaneously in the neck region '4 cc. toxin to which had been added just before inoculation'l cc. Gram's iodine. The result was the development of a painful swelling the size of the palm of the hand at the point of inoculation and a rise in temperature to 105 F. The swelling disappeared and the temperature dropped to normal in three days. The animal lost 3 lbs. in weight during the first week. On 26th July 1920 the inoculation of 21st July 1920 was repeated with much the same result except that no loss of weight was recorded and the highest temperature reached was 104'2° F. On 30th July 1920, 5th August 1920, and loth August 1920, the injections of 21st July 1920 were repeated, the reaction, local and temperature, being a little less each time. On 16th August 1920 a mixture of I cc. of L toxin and a few drops of Gram's iodine were injected subcutaneously with a resulting very slight reaction. The injections were continued at intervals of four to six days up to 4th November 1920, and the doses being gradually increased in quantity and given without the addition of Gram's iodine. From 13th October 1920 about half of the toxin to be injected was given intravenously. On 4th November 1920 the animal received 20 cc. intravenously and 25 cc. subcutaneously of L toxin, the minimum lethal dose of which for the guinea-pig was '01 cc. A rise of temperature to 103 F. and a small swelling at the point of inocula0

0

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tion were recorded after some of these injections, but there was no reaction following the injection on 4th November 1920, and the animal had slightly gained in weight at the end of the immunisation. The goat was bled on 11th November 1920, and its serum was separated and stored in sterile ampoules on ice. No preservative added. The serum was standardised in the usual manner, and it was found that '5 cc. of a 1 in 25 dilution was sufficient to neutralise about 50 M.L.D. of L toxin, as the following experiment shows : 22nd November I920. Guinea-pig 224 (250 grammes) received subcutaneously a mixture of '5 cc. of goat serum diluted 1 in 25 with saline and '5 cc. L toxin, of which '01 was the M.L.D. There resulted a slight local cedema, but the animal survived. The following experiment shows that the goat serum was equally efficacious for neutral ising human diphtheria toxin:24th November 1920. Guinea-pig 228 (250 grammes) received subcutaneously a mixture of'5 cc. of goat serum diluted 1 in 25 with saline and 2 cc. Elstree toxin, of which '04 was the M.L.D. The animal survived. DISCUSSION.

After perusing the experiments described in this paper no one can have any doubt but that the five toxic equine strains (D, H, L, D 16, and D 34) are identical with the bacillus of human diphtheria. The section of the work which indicates the complete neutralisation of the toxin by human antidiphtheritic serum proves this to be the case. In the case of the atoxic strains, there might be some reasonable doubt as to their nature. Here one has to rely upon evidence of less weight, viz., appearances in cultures, biological properties, and immunological data. Nevertheless it is at once recognised that in all respects except the actual production of toxin the atoxic strains behaved in a fashion exactly analogous to those which were toxic. In this connection it is probably most important to lay stress on the non-fermentation of saccharose and the results of the agglutination tests. Finally, it has to be borne in mind that similar atoxic bacilli are comparatively commonly met with in the-human throat, and that such are generally considered to be atoxic diphtheria bacilli.1 As to the significance of these findings, it is to be made clear in the first place that this paper is not intended to support the idea, which is possibly still accepted in some quarters, that horses commonly suffer from a disease identical with human diphtheria. As has been mentioned, Cobbett, as the result of his finding in one case, came to the conclusion that the horse is to be considered a possible source of diphtheritic infection in man. Against this view it can only be said that the last twenty years have brought no experience in support of it. When one considers that the diphtheria bacillus has been isolated from the horse in one previous instance only, and that at the same time the disease is of comparatively common occurrence in human beings, it is surely much more reasonable to suppose that the reverse 1 Such harmless bacilli have also been isolated from human wounds. tors, "Jl. Canadian Med. Assoc.," Vol. VIII., p. 9.

Adami and collahora

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293

view is true, viz., that man is an actual source of danger to the horse in this respect. Here it is to be pointed out again that the diphtheria bacillus is known to be pathogenic for the horse, and one can readily understand that a wound on any part of a horse's body might easily become infected with diphtheria bacilli from a human attendant who happened to be in some stage of a diphtheritic attack or an actual "carrier." Such transferred bacilli might multiply quite well in the wound secretions of such an animal. Reasoning in this way it may be held that the equines which provided the material for this work were in a manner accidentally harbouring diphtheria bacilli which had been passed on to them in the first place by a human being. I have endeavoured to gather facts which might shed light on the problem, but without much success. This is not surprising, however, when one bears in mind the movements to which men and horses in the Army are necessarily subjected. In the case of the animal from which Strain DI6 was isolated some very slender evidence on this point was forthcoming. This animal was under treatment in a veterinary hospital situated 3 miles from 0--, which was the nearest town, and men were of course passing constantly to and fro between the hospital and 0--. While there were no cases of human diphtheria reported in the hospital, there was at the time an epidemic of this disease at 0--, and consequently it is possible that some of the men may have acted as" carriers." The only case in my series which resembled clinically the one reported by Cobbett was the animal from which Strain D 34 was obtained. It cannot be said how long this animal had been harbouring Bacillus diphtherice, but it is certain that the trephine wound had been in existence for at least ten months before the strain was isolated. Enquiry showed that during this time no cases of human diphtheria had occurred which could possibly have originated from this animal. SUMMARY.

Bacilli proved to be identical with human diphtheria bacilli have been isolated from eleven horses and one mule, all of which animals, with the exception of two, presented on the limbs lesions that were described as simulating those of ulcerative lymphangitis. Of the two exceptions, one animal showed cutaneous lesions somewhat resembling those of acne, and the other was a case of suppuration of a nasal sinus in which treatment by trephining had been tried without success. Of the twelve strains isolated, five proved to be toxic and the remainder exhibited no evidence of toxicity. My acknowledgements are due to Lieut.-Colonel H. WatkinsPitchford, C.M.G., Commandant, Royal Army Veterinary School; to Bt.-Major C. A. Murray, RA.V.C., for kindly making certain enquiries; to Captain G. E. Oxspring, R.A.V.C., for assistance at busy times; to Drs M'Conkey and Brooks of the Lister Institute for cultures of Bacillus diphtherice and a supply of anti diphtheritic serum; and to Dr Duncan Reid, late of the Lister Institute, for valuable assistance.