451

THE PATHOGENICITY OF THE MENINGOCOCCUs..

By LIEUTENANT-COLONEL M. H. GORDON, C.M.G., C.B.E. Late Royal Army Medical Corps.

(Ooncluded from p. 38.5.)

FURTHER OBSERVATIONS ON THE REDUCTASE AND ENDOTOXIN OF THE MENINGOCOCCUS.

'A.-COMPARISON OF THE REDUCTASE OF THE MENIN(JOCOCCUS WI'l'H THAT OF STAPHYLOCOCCUS EPIDERMIDIS AND OF BACILLUS 'COLI

RESPECTIVELY. ,

(a) Effect oj\Exposure to a Temperature of 70° C.

A slope culture on trypagar was ma~e of Staphylococcus epide1'midisand another of Bacillus coli, and after 'eighteen hours at 37° C., the growth on each wa,s suspended in 2'5eubic centimetres of broth and on~ cubic centi­metre of the \suspension then heated to 70° C. for one ,hour. On testing the reducing power of the suspensions before and after they had been heated, it was found that while,before heating both bacteria up to at least I-t"O of 'a slope reduced' methylene blue, after the heating no-reduction' was produced even b'y t of a slope. The reductase of both these bacteria, therefore, is no more r,esistant to a temperature of 70· C. than is that of

, the meningococcus. '-, Cb) Effect of Desiccation.

, , ,-The growths on three one-day slopes, of the meningococcus (T. 1. Smith)

S. epideniddis and J3. coli respectively' were each suspended in 2'5 cubic centimetres of broth, a~d 0'5 cubic' centimetre of this suspension was then placed in a watch glass and dried in vacuo at 37° C. overnight. Next mbriling the dry deposit in each watch glass was taken up in one cubic centimetre of distilled water, a culture made, and the reducing power tested in the usual way. The result was as follows :,-

Fractions of slope '. - I Reducing power Vitality

,',

1/10 . , 1/20. I 1/40" 1/10' . -------------------------

". - .

Meningococcus " - -, -." - '. -. Stapylococcus " + + +

I +

Bacillus coli + - + , + -,

+ ..

It appears that, whereas after desiccation the meningococcus had lost both its vitality and reducing power, the two hardier bacteria had retained both qualities.

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452 The Pathogenicity of the ,Meningococcus

B.-SERVICEABILITY OF THE REDUCTASE FOR DETECTING 'rHE PRESENCE

OF LIVING MENINGOCOCCI IN BODY EXUDATES.

Experiment .I.-In order to determine if th!') reductase test can be applied to detect the presence of livil1g,bacteria in an exudate an experiment was 'made ih- the . first place to see i£an exudate free of bacteria reduces methylene biue. .

A guinea-pig w'eighing 350 grammes was injected intraperitoneally with ten cubic centiJ;l1etres of ten per cent Witte peptone dissolved in saline, and kIlled py ether next morning. The peritoneal cavity was found tobe filled with a large exudate rich in polymOl:phonuclear cells, but free of bacteria. By diluti~g 0;5 cubiC centimetre of the exudate through successive 0'5 cubic centimetre amounts of broth a series of dilutions was obtained of 1 : 2, 1 : 4, 1 : 8, 1: 10, etc., and methylene blue was added to each of these and the tubes kept fortw'o hours at 37° C. Although the leucocytes formed a thick white precipitate in the first tubes no definite reduction of the methylene .blue was observed except for a trace in case of 0'5 cubic cent~­metre of the undiluted exudate. On now adding a rich suspension of living

. meningococci to the exudate and repeating the test, reduction of the methylene blue was effected up to the sev.enth successive dilutIOn (1: 128). It appeared, therefore, that whereas the exudate per se failed to reduce when

. diluted, in broth 1 : 2 and more, this exudate did not prevent the m~ningo­cocci from exercising their reducing power.

Experiment n.-The' growth on twoone":day-old plates of a virulent Type II meningococcus (Lindenbaum) was suspended in ten cubic centi­metres of '~qual parts..broth arrdsaline and injectedintraperitoneally into a guinea-pig of 390 'grammes weight. The dose thus given represented an

'eight-fold fatal dose. After five, hours the guinea-pig wa~ killed by ether and its peritoneal fluid and blood collected and e~amined fo~ reduction of. methylene blue in the usual way. Cultures were also made from measured amounts of the peritoneal exudate and the blood of the guinea~pig. The results were as follows :-

Number of living meniJ;1gococci. The blood of the guinea-pig contained over one hundred but less than one tho.usand living meningococci per cubic.centimetre, and the peritoneal exudate over one thousand but less than ten thous~ha: living cocci per cubic centimetre, as judged by cultures made from quantitative dilutions of these materials.

rhe reducing power of the suspension of cocci injected, of the perito­neal exudate before and after being centrifuged, and of the blood before and after dotting is shown in the 'table on next page .

. ' It would appear that (1) The suspension was eight times as active as the exudate; but its

dilution by the exudate must be ta-ken into account, also the elapse of five hours 'since the injection .

. ,(2) The exudate after being centrifuged had lost seven-eighths of its red~cing power byremoval of a la!ge I?~rtion of itsparticulate matter .

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-M. H. Gordon 453

,I No. i Material

Dilution ip broth

, I 1/2 I 1/4 ~------I---

1 Suspension injected I + - + 2 Peritoneal exudation + + 3 Peritoneal exudation + +

after centrifugation I 4 I Blood before clotting I + 5 Serum'.. .. I + +

I I

+ = reduction o'f methylene blue. ~ I

(3) The blood of th!3 same guinea-pig had some slight reducing action on methylene blue better seen when the red corpuscles were removed­possibly because they to some extent neutralized the reducing agent.

Conclnsion.-;-The reductase of the meningococcus can, be -applied to ,detect the presence of this micro-organism when'present in a body exudate in living condition and in large numbers.

C.-THE ENDOTOXIN OF THE MENINGOCOCCUS. ,

The experimental study of the pathogenic ac'tion of the meningococcus described in a preceding section led to the conclusion that two different 'factors are here involved, namely, (1) a labile element, especially prominent in the most virulent specimens of the meningococcus, and intimately bound up with an' ability on the part of the coccus to multiply in the tissues of its host; and (2) a more stable toxic element, intracellular in origin, and still in' evidence when the vitality of the coccus has been destroyed. The object of the present section is to describe the result of further study of this endotoxin of the meningococcus. .

,This investigation was determined by demands of a very practical nature. During earlier stages of the outbreak an undesirabl,efluctuation -in the therapeutic potency of the anti meningococcus serum then available was the cause o{much concern. This concern was increased when it was found, on comparing in the laboratory the serum that had proved most pot'ent clini,cally with numerOl'j.S samples of antimeni·ngococctis serum of inferior therapeutic value, that none of the _ three' indices of potency chiefly advocated up to tha,t time served to distinguish bet'ween them .

. Thus the superiority of the successful serum lay neither in its opso~in content, nor in its agglutinin content, nor i-\l its yield of complement­fixing antibodies. When, the'refore, towards the end of 1917 it was found that of all the spe~ime~s of antimeningococcus ~erum under investigation, the clinically potent serum alone possessed the ability to neutralize the toxic action of the dried meningococcus in the peritoneal cavity of the, mouse; and also, that this toxin-neutralizing capacity/of the serum' extended tb both of the commonest types of the meningococcus, it seemed­that the long-sought-for clue to potency ha.d been obtained, and further

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/1

The Pathogenicity of the Meningococcus " /

study,of the endotoxin of. the meningococcus was indicated. This in- ' ,vestigat~on, however, was prevented for a considerable time by the more pressing, need first of testing numerous samples of antimeningococcus serum so. as to sele<;t the hest for. immediate clinical use; and secondly by the necessity of obtaining some information a~ to. the conditions under which' meningococcus antiendotoxin is to be found in the circulating blood of an aIlimal in course of immu~ization: . Accordingly it was not until the

, middle of 1918 that an investigation could be begun for the purpose of obtain­ing further information concerning the endotoxin of the meningococcus.

In the meantime, the method used for, determining the antiendotoxic capacity of various samples of s~rum-though the best that could be eh1borated'-at the time-was far from perfect;' The serum that had proved most efficient clinically w~s f~und to neutralize one M.L.D. of the dried coccus of each of the' two commonest types £If the meningococcus 'with 0'5 cubic ,centimetre of the serum. In testing sam'ples of serum to see if they came up to this standard, it was found that if a dose of powdered dry coccus was adm:inistered that,exceeded the M.L.D. for the mouse, very few samples·' indeed of antimeniIl:gococcus serum' then available were capable 01 neutrali'zing-' it; and if a subminimal lethal dose was used, the controls lost inconclusiveness. It was clear that this dilemma would only, be disposed of satisfactorily when in addition to improved knowledge of the, conditions, under which antiendotoxin is generated-information

,indispensable for the -preparation of more potent serum in quantities" sufficient to meet demands-a method could De ar~ived at of obtaining the endotoxin in a form th'at would permit of more ·accurate measurement of its toxic effect. It seemed possible ,also that a s.atisfactory solution of the latter problem might even go some way towards resolving the former as well. '

For these reasons, the present study of the endotoxin of the meningo­coccus has been influenced largely by a desire to obtain if possible a nlethod ofgettirig ,the endotoxin of this micro-organism into solution. In the first place, however, 'an investigation was made in 9rder to ascertai,n whether the '.endotoxin contained by the meningococcus 'can be increased, by, raising the virulen~e of the coccus by animal passage. "

(a) Exper.iments to dete;mi?w (1) if the Vi1'1tielice of the Meningococcus can be increased by Animal Passage; and (2) if in the event ofsucli

, Increase of ViJ"ulence, the Endotoxin contained by the Coccus is also increased. '

Four, specimens of the meningococcus-two of Type I and two of , Type II-were each injected into mice, and on recovery from the ,heart's blood of the first mouse passed through a succession of further mice in the same way: After several passages the virulence of each coccus for the mouse was againmeal'?ured., In only one of these cocci could any increase

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'M. H.' Gordon 455

be detected; namely, in the case of Type I, strain Howes, that before mouse passage killed when alive in a dose of 5,000 millions, not.in a dose of 2,500 millions ; but on recovery from the fourth mouse' in succession

. killed in a dose of 500 millions. The virulence of this particular coccus, therefore, had4ncreased tenfold. ,

In order to determine if this increase of virulence was accompanied by an increase in the endotoxin content of the coccus two procedures were tried. In the first place a portion 6f the identical suspension of the Howes coccus (that on recovery from the fourth mouse proved fatal in a dose of 500 millions when living) had been dried, and the toxici~y of .this was

. compared with that of the same coccus before mouse passage wben dried in the same way. No difference, however, could be detected in the toxicity of these two specimens of dried coccus. Secondly, the growth on trypagar plates of the' coccus before and after mouse passage was collected, washed in saline, dried, and the toxicity of the watery extracts of the dried cocci, and also the dried cocci' themselves, compared.' Again .the t'oxicity was equal. This experience indicates therefore: (1) that since three of the fOUl; cocci failed to show increase of virulence, it is exceptional to get an increase'in the virulence of the meningococcus by passing it through mice; (2) that when such increase of virulence does take place, it is nptneces­sarilyaccompanled by an incryase in the portion of the endotoxin of the coccus that survives drying,

, ,

(b) Comparison .of the Value of Various Solvents for Extracting . the Toxin from the Dried Meningococcus,

, A comparison was made of the value of distilled water, saline, absolute alcoholvacetone, and ether for this purpose by grinding up 0'1 gramme of the dried coccus (Type I, strain Howes) in an agate mortar and slowly' adding five cubic centimetres of the solve.nt. The mixture of each solvent with the dried coccus was then tramlferredto-aeentrifuge tube, stood over night in the Ice chest and centrifuged. The results were as follows :--(i) 'N aked-eyeappearance of the extracts,

(a) Water extract. The fluid is more opaque and the deposit is less abundant than in any of the others.

(b) Saline extract. The fluid is slightly opalescent and the deposit is over twice as large as in the case of water.

(c) All of the remaining extracts are clear and the cocci completely deposited. The acetone extract was slightly yellow, apparently from some substance extracted from the cocci which deposited in yellow drops .when the acetone was evaporated off. . (ii) Microscopic appearance of the deposits;

Films were made of each deposit, fixed, stained, and examined. The results were as follows -

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The Pathogenicity of the Meningococcus

Solvent : Deposit Water Sludge. No formed cocci seen. Saline Sludge. Acetone Mostly sludge. A few cocci. Alcohol Many cocci seen. Ether Cocci not disintegrated at all.

(iii) 'Amount of the ~ried coccus tal{en up by each solvent .. , The ,deposits were collected from the centrifuge tubes, dried, and

re-weighed. Tbe results were as follows :-llHlligraIf!mes of dried coccus

~ , Solvent: Water Saline Acetone Alcohol Ether

Before extraction. . 100 ioo 100 100 'lOO , After 14 34 , 57 55 55

A good deal of the dried coccus was unavoidably lost ·in pouring the m aterials from the agate mort.ar into the centrifuge tubes, and in tmns-

, ferring the deposits in the latter to, watch glasses for drying. In spite of this the main result is clear, the water having obviously taken up mor~ than the other solve:r'J.ts. ' (iv) Toxicity of the residues.

No material difference could be found in the toxicity of the dried COCCI

recovered after extraction by.the 'various solvents. Jv) Toxicity' of/the extracts.

Both the water and saline extracts proved toxic to mice' intraperi. '. toneally, the former being the more ?octive. The toxicity of the other

ext'ractswas not tested on this occasion; but a test of similar extracts was made later by evaporating off the solvent, dissolving up the dry deposits in water, and injectipg them intraperitoneally into"mice. These mice re'mained unaffected . . . Co.nclusion.-Distilled water. takes up more of the dried coccus arid, of

its endotoxin than any of the other solvents. In consequence distilled water was adopted in the first place for, the routine purpose of extracting the' toxin from the dried meningoeoccus when testin~ samples of serUIl) f~r antiendotoxin.

(c) The Value of Improved Mechanical Disintegrationjor Extracting Endotoxin fr~mthe Dried Meningococcus.

Comparison was now made of the toxicity of extracts prepared with distilled water~ and (a) grinding up the coccus in the usual way·; (b) adding an equal amount of we'll-washed and sterilized sand before grinding.

, Both extracts were lightly centrifuged to throw down the larger particles and the sand before testing them for toxicity. As it was found that the addition of sa1;ld inoreased the amount of toxin in the extract, this proce-dure was adopted for routine purposes.' ,

At this stage, therefore, the method used for testipg sera was to weigh 'out 0'1 gramme of the dried coccus, to add an equal amount of sand, and after thoroughly grin?ing up the mixture to add slowly five cubic centi·

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M. H. Gm'don 457

,metres of distilled water while continuing the grinding. The mixture was then centrifuged lightly in order to throw down the sand. The super­ilatantfiuid which was opalescent in colour was fatal to the mouse intra­peritoneally in a dose of 0'1 to 0'2 cubic centimetre. For preserving it; a few drops of ether were added.

(d) The Val~te of Sodium Hydroxide for getting the Endotoxin of the JJ1eningococcus'into Solution.

Numerops experirnents were now made in ~:Jrder to deterniine whether the well-known solvent properties of sodium hydroxide co~ld be made use of to obtain the endotoxin of the meningococcus in solution. In this

· relation importance attaches to the following points:- , ' (1) The toxicity of 'NaOH per se when administeredintraperitoneally

to the mouse. ' . (2) The degre'e to which it dissolves the meningococcus, ttnd (3) Tbe ex£e~tto which it alters the endotoxin. '

(1) Toxicity of NaOH for the mouse. The M.L.D. of NaOH intraperitoneally for a inouse of 15-20 grammes

was found to be 0'5 cubic centimetre of a N/10 solution, or 2 milligrammes of NaOH. ' (2) The value of NaOH for dissolving the meningococcu~ and' obtaining

. its endotoxin in solution~ , Experiments with the Dried Coccus.-N/1NaOFJ; was tried in the first

place. In a preliminary experiment it was found 'that by grinding up 0'1 gramme of the dried cocc,us in ]'25 cubic centimetre, NIl NaOH, by now adding 1'25 cubic centimetre of water; and then placing the fluid in a water bath at 37° C. for one hour, and finally reducing the~ alkalinity to N 150, a solution of the' bacterial protein and~ endotoxin was obtained with the endotoxin unaltered, as proved by the ability of antiendotoxic

· serum, but not of other serum to neutraJize it. Nevertheless, in further expe~iments' this 'method proved too uncertain for routine use owing to the teddency of the strong N aOH to destroy the toxin.

An attempt was next made to get. the dry coccus into solution by grinding the powder up directly in falling amounts of 'N aOH, e.g., N /'2,

· N/4, NjlO, Nj20 j etc., subsequently reducing the alkalinity to N/50 where necesSfl.ry by' dilution or neutralization, or by both. As only imperfect solution of' the ,dry coccus took place, however, the method was abandoned.

Tbe effect was now tried of adding falling' amounts of NaOH to a s~spensioll of the dried coccus already made by grinding it. up in water. As before, the stronger solutions of NaOH were found to destroy, the endotoxin, but the weaker solutions (N /20, N /40, etc.), were found to liberate toxin from the particles of dried cocci without destroying it, and to considerablY- improve.the .surenessni thBJ>:illingpower of the suspension .

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458 The Pathogenicity of the Meningococcus

For this r~ason, and also because it saves so much time and "labour, the addition o~ dilute NaOH to the suspension of the dry coccus was hence~ f9rth adopted for routine use. .. In order to avoid lo.,s of toxicity on, keeping, a smaller amount of the

dried coccus is used, and the samples of 'serum tested shortly after pre­paring the endotoxin. .Procedure is as follows: 0'05 gramme of 'the drie,d coccus is ground up in an agate mortar with 1'25 cubic centimetre of distilled water, and after grinding for a few minutes 1'25 cubic centimetre o( N/20 NaOH is 'added. '1'he suspension undergoes rapid clearing when the N aOH is added. This· fluid is then transferred to watch glasses already set out fbreach serum. The dose of dissolved endotoxin, used is generally 01 cubic centimetre or 0'15 cubic centimetre, according to the toxicity of the dried coccus in use; 0'5 c,ubic centrimetre of serum is then added to each watch glass, normal serum being added to the first pair, and each of the sanlples of serum for test to the others. "The mixtures of serum and toxinfLre placed in the incubator for thirty minutes, and then the contents of ,each watch glass are injected intraperitoneally into a mouse. The experiment is carried out in duplicate, so as to reduce error due to. the varying susceptibility of individual mice. TIle II\ice are kept under observation for two to three days and the result recorded. Since tliis meth,od was elabmated, it has been III routine use' for ,the purpose -of· testing samples of serum for antiendotoxin. . '

Experiments 'with. the Raw Coccus:-Kra,us and Doerr observed .that the addition. of N/10 NaOH to the rayV coccus renders the killing power of its endotoxin more sure.' This solution, however, is unnecessarily strong, anti is certainly too, strong when mice are used, sintJe 0'5 cubic centimetre of the N /10 solution per se is fatal to them .

. SoIpe experiments made with a suspension containing approximately 200,006 ~aw meningococci per cubic centimetre, showed that when present. iua concentration of N /40, the NaOH produc(:ld partial solution of the cocci and increased the sureness of the killing power of the suspension.' This toxicity also wa~ retained by the fluid of the suspension when, the cocci were centrifuged out of it. It would appear, therefore, that in case of need endotoxiI\ can be obtained from the raw COCc\;S by thifl procedure fOf" the purpose of testing the antiendotoxic value of sampit's of serum. It was observed that the addition of dilute alkali, e.g., N/80 or N/160, to . Huspensionfl of the raw meningococcus rendered'them very viscid, a change due probably to the liberation of mucin from the coccus.

Cc) The Value of Autolysis for the purpose C?F obtaining a Solution , of Menirtgococcus Endotoxin.

Some experiments -were made in which 8uspensioIlfl of the raw cocc'us in water or sallne were incubated with toluol or ether as preservative in order to ascertain the, comparative value of autolysis 'for the purpose of obtajning a s()1ution of .tpe endotoxin. Althol:1gh active endotoxin was

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M. H. Gm'don 459

found free in the fluid in some cases after twenty-four hours at either 37° C. or laboratory temperature, it was apt to disappear later; and as on repeating the experiments this' method was fonnd to' be less sure as a source of active, endotoxin than the dried coccus, it was not adopted.

(f) The Effect of Heat on the Endotoxin of the Meningococcus.

Expm'iment I.-The effect of heat was tried in the first place on the toxicity of a suspension of Type I meningococcus, strain Smitl), prepared by suspending the growth of each of two trypagar plates of eighteen hours' incubation at 37°C. in five cubic centimetres of distilled water, and then pooling them. After microscopical examination the suspension was divided into four parts, one of which served as control, while the other three were heated for half an hour to 55° C., 100° C., and 120° C. respec­tively. ,The two later temperatures were obtained by heating the tubes containing the suspension in a water bath of boiling water, Ij,nd in the autoclave respectively. At the same time also the effect of heat was determined in the same way on the Smith coccus after it had been dried, the suspension used for this purpose being made ,up 'by grinding up tw,o lots of 0'1 gramme of'the dried coccus in 5 cubic centimetres of distilled water, and then pooling them. Both watery suspensions of the Smith coccus, raw and dried respectively, failed to kill am~use in a dose below 0:1 cubi6, centimetre. The effect of heat ~n :their toxicity was found to be as follows:-

____ Treatment _____ ~~_I_Raw coccus_' ___ D~::~_ Unheated •. .. ." ..•. 0'1, I' + 'R

, 0'2 + +

Heated to 5511 O. for 30 minutes ..

Heated to 1000 O. for 30 minutes ..

Heated to 1200 O. for 30 minutes ..

, + = mouse died .

0'3 I + + 0'1 I + +, 0'2 + + 0'3 + +

0'1 0'2 0'3

+ + + + R ' '+

R = mouse ill; recovered.

R + + + + +

. The mice that succumbed to Jhe raw unheated coccus grew meningo­cocci from their hearts' blood; the blood of the other mic~ that died 'Was sterile.

The Smith coccus, therefore, whether raw or dried, withstood heating to temperatures up to 1200 C. for. thirty minutes without loss of toxicity.

Experiment 2.':"'-The experiment was next repeated on Type II, strain Pngh, in the same way. The results were as follows: ' .

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460 Phe Pathog~nicity of the' Meningococcus

11reatmenn. . _ - . . I· Dose' Raw coccus. _ Dried ~occus ---'----_._-----------'--~---' - -------, ,

Unheated 0'1 + + 0'2 + + 0'3 + +

Heated to 55° C, ,for 30 minutes 0'1 + + 0'2 + i-0'3 +

Heated to 1000 C. for 30 minutes " 0'1 R + 0'2 + + 0'3 '+ +

Heated to 120" C. for 30 minutes .. iH + 0 0,2 + 0 0'3 +- 0

+ = mouse died. R '= mouse ill; recovered. o = mouse unaffected.

Whereas the Pugh coccus, when raw, withstood heating to 1200 C. with­out loss of toxicity, the suspen,sion' of the same coccus when dried lost its toxicity within half an hour at this temperature.

Experiment a.-The experiment was repeated on two further specimens of Types I,and II respectively, strains' Howes and Morgan. Su~pensions of the ia:.w coccus were u~ed, exposure to 550 C. was omitted" and the effect of exposure to 1200 C. tor two hours. was' tried -instead. The results were as fol,lows :

Treatment Dose Howes Morgan

Unheated 0'1 R +-0·2 R R 0'3, + +

Heated to 1000 C. fo~ 30 minutes " 0'1 . 'R + 0·2 + R 0'3 + 'R

" Heated to 1200 C. for 30 minutes .. 0'1, R 0 0'2 It 0 0'3 + 0

Heated to 1200 C. fdr 2 hours

"I 0.'1 It 0 0·2 0 0 0'3 0 0

+ = :r;nouse died. R = mouse m; recovered. o =, mouse unaffected.

It woul<l appear that the Howes COCCU$ retained Home of its toxicjty after exposure to 1200. C. for thirty minutes, but lost' it after two hours at this temperature. The toxicity ,of the Morgan coccus .was destroyed within half an hour at 1200 C. '

Experiment 4.~The' effect was now determined of heating the raw s.uspensions used in experiments I' and 2 'to i200 C. for two hours. Sufficien~ of tile suspenSIOn of each that -had been heat{\d to 55° C. for ,

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,thirty minutes remained for this purpose. It was ,tound that whereas both these suspensions were toxic to mice before being autoclaved j neither

, of them showed 'any evidence of toxicity aftflr being exposed for two hours to 1200 C" .'

,Gonclusion.-These experiments on mice led to the following conclu­sions. The endotoxin of the meningococcus withstands heating to 100° C. for thirty minutes, and in some cases to 1'.20° C. for this time. At 1200 C., however, it is destroyed within two hours. As might, perhaps, have been' expected, the loss of toxicity produced by heat ,is more evident in cocci of low than in those of high toxicity.

Gon!i?:znatory Experiment on Guinea-pigs.~At a later date, when, investigating the question of the' secretion of soluble toxin by the meningo­coccus in the peritoneal cavity of the guinea-pig the opportunity was taken, of repeating the above experiment on guinea-pigs. Four ~arge guinea-pigs' were selected of between 350 and 450 'gramIl?-es weight~ A recentlY' isolated meningococcus (Type II Lindenbaum) of good virulence was used and

. eight plates of it of eighteen hours' growth suspended in altogether sixteen cubic c'entimetres of equal parts of saline and broth. This suspen­sion was divided· into foul' equal portions, the first of which was used as control, the remainder heated for half an hour to 55° C., 100° C., and 1200 C. respectively; , Ej1ch portion was then injected into the peritoneal cavity of a differe.nt guinea-pig. Further' details of this experiment (l,nd fhe results are seen in the following table : -

No. Treatment of coccus' Weight

Vitality ,of guinea­

pig

Peritoneal fluid Result

Lesion Film I Culture -------' -------------~ -'--------,--,-

1 ,Raw .. I + 380 Dead in 18 hours H[Einorrhagic Swarm.ing with + pe.ritonitis COCC1,

2 Heated for 30 minutes ,450 Hffimorrhagic Swarming' with '-

to 55'1 0: peritonitis 'cocci ' 3 Heated for SO minutes 390

" 'Serous peri. Swarm.i?g wit~ . ~

to 1000.0. tonitis COCCl

4 Heated for, 30 minutes 350 ,', Serous- peri'l Swarming with to 1200 0., tonitis : (Shadows) "

, .

The dose given to each of these guinea-pigs was about eight times the M.L.D. of the living coccus for a guinea-pig, of t4is size ; the dose therefore was a large one. The result, however,goes,to confirm the previous obser~ 'va~ion on mice to the e'ffect that the endotoxin of the meumgococcus may withstand heating for 'thirty minutes' to 1200 C. It IS noteworthy that although the meningococcus in this experiment produced hmmorrhagic peritonitis raw, and when heated to 55° C., it failed to do so after being heated to 100° C.

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462 The Pathogenicity of the Mem;ngococcus

,(g) Trial of Vaughan's Method for the p7lrpose of obtaining a Solution of the Endotoxi~ of the Meningoco-ccus. '

The principle of this method which has been applied ,by V. C: Vaughan and S.M. "Wheeler' to. B. coli and other bacteria is, to boil up the dr,ied bodies of tne micro-qrganism with absolute alcohol containing'two per cept of NaOH. By 'repeated' boilings of an, hour's duration under a reflux condenser the bacterial protein' is split ~p and the intracellular poison is taken up by the alkaline alcohol, frolll which it can be recovered by neutral­izing the alkali with HCI, and_ after removing the salt thus formed, evaporating off the alcohol. T~e deposit left is soluble in water and very toxic. '

As this method does not appear to hav'e been applied previously t6 the meningococcus, an attempt was made to extract the endotoxin from that micro-organism by means of it. The method as described by Wheeler (Journal of Arnerican Medical Association, April 22,1908) was followed. A gramme of the dried meningococcus (Type I, strain Smi~h) was powdered well,extr'acted witb e'tber in a: soxhlet, 'and again dried. The weight of dried coccus recovered from the soxhlet was 0'8 gramme. This powder was next boiled three times over £0.1' • one hour in absolute alc6~01 containing 1: 50 of NaOH. The alcohol was then neutralized/with HCl, filtered;and evaporated to dryness. The yellow scaly deposit thus obtaIned was dissolved

, up in water and injected intra peritoneally into mice, but no toxic effect was observed. The residue of the bacteria-I bodies was now tested in the same way, but again with no result. The prolonged boiling in alkaline alcohol apparently had' destroyed, the endotoxin of the meningococcus. The antigenic value of .tlie-extracted bodies was examined by Major A. S.·G Bell"who found its specific agglutinogenic properties to beintact. . The method was tried once more, but again without succes~. In this c,ase,however, while the deposit from the alcoh()lic expractwas without.

,,'toxic action, a little endotoxin was still left in the coccus bodies. . " - - ~, >'. ?" " . , ", )

, (h) A Method of obta'ining 'the Endotoxin in 'Solution. . n, .

.-AI,though Vaughah's particular method of combi~ing heat with alkali , was not successful, nevertheless,jn view of the ascertained resistance of the

endotoxin of the meningococcus -to he[l,t and-to dilute alk~li separately, it still seemed possible to obtain a solution of it by this,means. Accordingly, before the Central Cerebrospinal Fever ,Laboratory, was, demobilize.dthe following experiments werecarri~dout. '. .,.,., , Experirnent I.-The effeCt was first tried «fboiling the dried.coccus in ,

N/40 }'faOIr' (i.e., NaOH 1: 1;0(0) di,ssolvedin water. " Op.e gramme of the ~' , dried meningococcus (Type I, Smith) was put into a flask wit4fifty cubic centimetres of N/40 NaOH and,'boiled for one hour in the steamer:- rhe reaction which was now N/110 N aOH was increased to N/40 and the boiling repeated for one hour. After the second boiling the alkalinity, which was.

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M. H. Gordon 463

~gain NjllO, was increased to Nj40 and t,he boiling repeated forone:hou:r: The mixture was now neutralized with, HOt The coccus bodies had' largely gone into solution, a clear brown-coloured fluid re'suIting. This fluid, mixed well with water, entering apparently into complete solution with it. On testing the toxicity, however, it was found that 0'5 cubic centimetre of the fluid was required in order to kill a ,mous~ by ~he intra­peritoneal route. As the M.L.D. of the suspensioI;l before beirigheated had been found to be 0'1 cubic centimetre, it was ,evident that the pr'olonged boiling even in N /40 N aOH had destroyed a large proportion of the endotoxin,

Experiment, 2.~The experiment was now repeated, but the dried coccus' was only boiled for half an hour in the Nj40 NaOH, and then neutralized. Although solu~ion was less perfect than in Experiment 1, and the resulting fluid far more opaque, the bacterial protein, neverth~less, was found to keep up well in it on standing. As the M.L.D. for the' mouse intraperitoneally was found to lie between 0'1 and 0'2 cubic centimetre, but ,little of the endotoxin had been lost '; and the toxicity was 'found to be practically the same after the solution.had stood for 'two weeks at laboratory temperatur:e. Since serum containing antiendotoxin for Type I, and neutralizing a . suspension of this same Smith coccus in cold, water, was also found tb

, neutralize the toxic action of the present solution, the specificity.of th~ endotoxin was unimp~ired. While it is not" cl"aimea, therefore, that an' 'entirely satisfa~torymethod ,of obtaining meningococcus endotoxin, in, " 'Solution has yet been obtained, the present procedure would appear to constitute a material ad vance in that direction ..

. SummaTy.-Incre~se in"the virulence of the meningococcus resulting frqm ani-mal passage is not neDessarily accompanied by a rise in its endo­toxin content. As a means of obtaining m~ningococcus endotoxin, auto­lysis is not sure enough to be s'atisfactory. Ifor extrac'ting endotoxin from

. the meningococ~us, distilled water' proved superior to the other sblvents ' tested. The additien Qf/di,Jute N aOH increases the-solvent power of water, and does not destroy the endot'9xin: ' Meningococcus endotoxin w~thstands , ~eatingfor' halt an hour to 1000 C.; and sometimes for half an hour to 1200 C., " but is destroyed within two hours, at, 120° O. By heating" the coccus for not too long a tim~ to 100° O. in dilut~ alkali its ,endotoxin ~ecoilles distri-' buted through th!3 fluid and, this method, or some de\;elopment qf it, promises ' to be of considerable value in further studies of m'Elriingocpccus end~toxin. '

. "'- ~ ,

,ANTIBO,QIES TO THE- MENINGOCOCCUS.,

The specific antibodies present inantimeningococcus serum include' agglutinin; ,precipitin, 'complement-fixing substances, .opsonin, lysin: and

, antiendotoxin. ' .' It has already been related how agglutinin was found to have'a direct

practical ; application for the purpose' of defining and identifying the meningocqccus of· o'ne or another type; ithus provmg of ,the greatest use.

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464, . The Pathogenicity of the Meningococc~~s ,. , '

not only in increasing-the accuracy of the procedure provisionally adopted for limiting the spread of cerebrospinal fever, but also in enabl(nga watch' to he kept on, the general prevalence of the meningococcus in a cO,mmunity when cerebrospinal fever is present, and when it is abs.ent; and during the rise and fall of an outbreak. .

Th~ practical~pplication of bacteriology for the purpose of 'dealing ;yith cerebrospinal fever,' however, is not limited to prevention. Before the recent outbreak began the)ntrathecal administration of potent antimeningococcus

, serllm had been conclusively'shown by Flexner and others to be capable of materially reducing the mortality from this disease. ~ut as yet DO sure method of standardizing the serum had been itrrived at; and largely as a

- re,sult of this defect most of the antimeningococcus serum used clinically during earlier stages of the ~ecent outbreak proved ineffective. i

A study, therefore, was undertake~of ~he ~ntibodies present in anti- '~ meningococcus serurn,' and when a consignment of this serum came to

.: hand, that wasJound clinically 'to possess a:' high degree of therapeutic effic~cy, the antibody content of this batch of serum was compared at length with that of specimens of less successful serum with a view, to defining the cause of its therapeutic superiority. For.a, considerable ;time this attempt to discover the antibody chiefly concerned in the therapeutic potency, of antimeningococcus serum was in vain. I.Is>w the desired )nformation eventually came to light, the practical' 'application!TIade ·of .this knowledge, and with what degree of success, will be seep. later. The result ,of .the studies that· have been made of individual antibodies' to the, meningococcus will now"be described.,

,AGGLUTINIK

A .-c;A ntigens.

The chief preparation employed was a saline'suspension of the men- '. 'ingococcusprepared'in the following manner. The growth from trypagar ' plates' of eighteen hours' in,cubatipn at 37 0 C.·is'susp'endedin saline ,and heated,: to' .65 0 C. )6r thirty IIlinutes in oraer to inactivate the autolytic enzyme of the do'ccus. '. The density of the suspension ,is then' determined, by transferring 0'1' cubic cen'timet~e of It to a clean test tube and' adding

',water (fl'om a fi,ve cubic centimetre pipette graduated in tenths of a cubic centimetre) -until 'the suspension is only just. perceptibly trirb"id, when compared .with a cop.trol of water alone. This end-point ~ay 'be takEm to represent a Qensity of 100 million cocci per cubic centimetre, and the

,amount ()f water nE).ededto dilute the suspension to this pQint' is :!,eadily deter,rnined ill' a Jew minutes. '1'he calculation,is a perfectly simple, one,

. e.g., 0'1 cubic centimetre: of the suspension requir~s ten cubiccentimetre~ -".of water fo :reach'the end-point; it contains ~herefOJ;e 10 X 100,=1,000

millioll: cocci, and-a, cubic centimetre' of the suspension therefore contains 10,000 minion cocci per cubic centimetre. After a little practice the end-

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. :

M. H. Gordon 465 \ . <-

pDint is £:lasily' ~ecognized and the calculation is simplified by realizing that' , every c1;lbic centimetre of water required by 0"1 cubic centimetre 6£ the

suspension in order to reach the end-po{nt i~dicates the:"presence of. 1,000 .. million cocci per cubic centimetre of it. The' density of the suspension h~ving been determined, it is diluted with saline until it contains 2,000, million cocci per cubic .centimetre, 0'5 per cent of phenol is then added and the suspension transferred to a glass-stoppered bottle.' Suspensions made in this way ~eep for several months and can be used either as antigens, or for testing the agglutinating power ·of sera. ','

Other antigens tested at one time or another have been the sensitized raw coccus, tne dried coccus', the raw coccus after being autoclaved for,half-an'- , hour at 120° C., and the dried coccus after most of the endotoxin' contained by it had been destroyed by boiling it,up for three hours in absolute alcohol

. containingl!50 of NaOH. . ,

, \ B.-Mode 01 Dosage.

Rabbits weighing from 1,000 to 1,500 grammes were used, as older' rabbits react less specifica,lly. The injections are made intravenously and· ' the rabbits weighed daily.. A, suitable first dose is 500 million of the raw' coccus. Hine's method consists in giving on the first day two doses of 250 . million at an hour's interval, and on the fifth day a dose of 1,000 million: By this procedure the animal's serum .may give a clear-cut titre of 1j400·to 1/800 by the tenth day. Another I11ethodis to' gIve the" rabbit a' dose every

) , . _... ,

forty-eight hours, beginning with 500 million imd repeati~gthepreceaing " dose before raising it. ' ' , ', " " ., .. " '

C.-Mode of conducting the Agglutination Test .

Small glass tubes t inch bioadby three inches' long, plugged with . wool, are used ; they are held in' square. wOQden racks designed by Major Hin!3 which ,fit into the; 55° C. incubator. Each rack holds nine rows of twelve tubes, and' a, strip of celluloid is nailed between each ro'w and at' either end. The principle al ways followed/has been first to. dilute the serum in saline, each dilution bulking to 0'5 cubic centimetre. When the dilutions have been made, 0'5 cubic centimetreo£ the!laline: suspension is run into e!bCh tube. An example is as follows :-' .

It is proposed to test a serum at 1/50, 100, 200, and AOO. Four, tubes. are put into the rack, the first is left _ empty, and 0'5 cubic ce~tillletre of saline is run into'each of the three other'S; 0'1 cubic centimetre of, the serum is' now diluted with' 2'4 cubic centimetres ()f saline, and 0'5 cubic centimetre transferred to each of the first two tubes; after shaking, 0'5 cubic centimetre from the 'second tube is transferred to the third" and from that 0'5 cubic centimetre to' the fourth~0'5 cubic centimetre from', ' the latter being then thrown,away. A dilution ist.hus obtained of 1/25,50, 100, and 200 of the serum; , 0'5 cubic centimetre of the suspension is then, added all round, the rack placed at 55° C., and the results read off ~fter

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, , -466 . The Pdtlibgenicity oj the Meningococcus

/ ., . . twenty-four hours. . A control with normal serum in the same way should always be carried out. ,

D.-;Absorption Tests .

. The .object of these is' to' determine whether a gIven coccus removes the specific ,agglutinin from a specimen of agglutinating serum III the same' manner as the ho~ologous coccus, or not. > T he titre of the serum for' the homG'logous coccus must be kp6wn. 'A low dilution of ~be serum is' then made and divided into thre~ parts, one of which. is saturated by adding to it an ,equal quantity o£ the suspension of the homologous

'coccus, another is saturated inthes~me way. with the testc'occus, the third receives an equal amount of saline only.' After standing in the incubator, at 37° O. over night, the tubes are Gentrifuged,. and the agglutinating titre of the unsaturated serum and of the two satur~t,ed ser~ respectively determined for each of the two cocci. The homologous

o coccus is found to,have removed' its own agglutinin. If th~ test coccus is of the same type, it removes this -agglutinin also, but if of another type, the agglutiniri is untouched. 'It is advisable to use freshly m~de suspensions

, . for absorption .tests,and Major Tulloch prefers to use a suspension .of 4:009 million cocci per Ci:!llic centimetre for, saturatingth~ serum rather than. a suspension of 2,000 milliori per ~uliic ce~tin:{etr~. " .

E;~Agglutinogen'icValtles"of various. Pr:eparatiqns oj the ,same ( . . M ening~coccus..· ,

. ,·It has 'alre~dy:"bee~' stated th~t theantigeri with whi~h th~ agglutinating '. serum .v,as prepared -(that was used. for identifying the meningococcus

during the outbreak) .was a'. saline suspension of'tJ:le raw coccus heated at 65°0. ·and then.phenolat~d.. . " . " " Recently, wberi comparing the value of various prepa:r;ations .of the same coccus' for exciting the production' of antiendotoxin, the opportunity was taken of, ooserving also their_ agglutinogenic capacity. Th~ coccus used was Type I, strain Howes; and the antigens compared were (1) a suspension' of the raw coccus ; (2) the same s~spension 'after being ,sensitlzed by homologous serum subsequently removed; and (3) a standard suspension- of, O'lgram~e of the dried coccus. in five cubic centimetres of distilled w.ater. . .

The experiment was performed in duplicate, three rabbits recelvIpg a stationary dose throughout, while in .three other rabbits the dose was slowly raised. :Further particulars are as follows :---=-

, Five- doses in all were ,given of each antigen, the interval between' the first two being seventy-two hours, and between the remaining doses

,forty-eight hours. Tpe . first dOf;ie in cas~ o£.the raw and sensitized co'ccus respectively', was 500 millism, and in the two rabbits ih which the dose was raised the last dose was 700 million. In case of the dried cocc

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M. H. Gm'don 467

the, first' dbse was 0'Q5 cubic centimetre of the standard 0 suspension representing one milligramme of the dried coccus, and in case, 'of the rabbit in'which the dose was, raised the last .dose was 0'07 cubic centimetre "-6f the suspension or 1'2 miUigramme of the dried coccus. Oh the eleventh day of the 'experiment, and twenty-four hours after the fifth \

,dose, all six ,rabbits were bled and their serum tested against, the homologous Type I coccus and also against a representative coccus of each of tHe other three types. All of the six sera proved nega~ive ·in a dilution of 1/50 against these cocci of heterologous types, The resnlts

, against the homologous coccus were as follows :-. - " ". . . \

I ,Antigen Dosage· 1 ---

Dilution of Serum

Raw coc~~-. --.-.,--,-, Stationa~~ 1:~ Ij:~ ,1:0 ~:o l/~I~ 1/~'~~11!4: .o~ , , Raised, , , , + + . + . + + + I ..

Sensitized coccus . . . . Stationary . . +. \ '. '. _ .. . . . . . .

Dried coccus.. ..~ .. ~t~:i~~~~y :: t ,1

1

t t (t) + It + I::: I Raised.. .. + + . + + +.' + ..

+= weU.markedagglutination.

Inferencesdra~n fr'om these results were as fbllO'ws:~' . (1) Since none of the six sera contained: aggl¥ltinin.£or heterologous

~. ,types, all of the antigens we~~ specific. .". . ! .,

.. (2) As regards the' bes~ antigen, clearly the raw coccus was superior ' tQ the, sensitized Coccus for exciti~g the production of agglutinin, though this superiority was reduced when the dose of the sensitized coccus was

raised. The two rabbits receiving the dried coccus both gave a full response; but in th~ircase the dose given was relatively much larger .

. (3) As regards the effect of raising the dose, it is noteworthy that in case of.both raw:and sensitized coccus, the rabbit gave a better production of agglutinin with the raised dose. In case of ,the dried coccus, on the other hand, the' stationary dose . consisting of one milligramme ,of the dried coccus was sufficient·toevoke a maximum response .. '

F.~The Efject of Autoclaving the Meningococcus O1Lits Antigenic Specificity. , . .

. Shortly pefore the Ce~traJ Cerebrospinal Fever Laboratory was closed' Major Tulloch very kindly carried out the following experiment, Saline suspensions of representatives of each of the four ,types of the meningo­coccus respectively were autoc1aved at 1200 C: for half an hour, and 'a rabbit was' then prepared against each. In -all cases agglutinin' was formed; but while . this agglutinin was active again~t the· ordinary suspensi~n of tl1e ravy coccus of the h'omo'togous type. (heated to 65°, C" fo! thirty minut~s and ,then phenolated),and w3tS strictly specific, it failed

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468 The Pathogenicity of the Meningococcus

to affect the autoclaved suspension which had stimulated. its production. 'l'he specific'agglutinogenic substance of the meningococcus therefore ~ould appear to withstand not 'only desiccatio~, but also exposure in the autoclave for half an hour, at 1200 O. The. fact that the specific agglutinogen of the raw COCCllfl withstands heating for thirty minutes to 120°,0. was confirmed later by Major Bell who found,however, that this exposure Had the effect of reducing it, and that exposure at 1:300 O .

. foritwo' hours abolished it altogether. ,It inay be recalled that in an observation previously recorded when applying Vaughan's method of extracting endqtoxinto the meningococcus, it was found that the dried bodies of me:ningococci after being boiled, three times over for, an hour each time in. absolutealcohol'~'containing 1/50 NaOH,although their ~ndotoxin had been to a ,large extent destroyed, still retained their specific

. agglutinogenibproperty. . . . . .

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