5sa. REPORT ON FURTHER EXPERIMENTS IN CONNEC TION WITH ANTI-TYPHOID INOCULATION.l

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1 5sa REPORT ON FURTHER EXPERMENTS N CONNEC TON WTH ANT-TYPHOD NOCULATON.l By BREVET LEUT.-COLONELW. B. LESHMAN MAJOR W. S. HARRSON MAJOR H. W. GRATTAN AND LEUTENANT R. G. ARCHBALD. Royal Army Medical Corps. STANDARDSATON OF THE VACCNE. THE method at present in use is a modification of Wright's method which was devised by one of us the details of which were published in this Journal. 2 The technique has been simplified in the following respects: (a) t has been found unnecessary to stain the bacteria as they are perfectly well seen unstained especially if one examines the films by artificial light; (b) the bent tube is no longer used; in its place the volume of blood is taken up into an ordinary straight capillary pipette and washed into a centrifuge tube by means of citrate of soda solution in saline; the pipette which has been used for measuring the blood is kept on one side and is subsequently used for measuring the culture which has to be counted. t has been fouid that any difficulties arising from the motility of the organisms can be overcome by the addition of a trace of formalin (of a strength of two or three drops in 10 cc. saline) to the fluid in which the mixture of blood cells and bacteria is suspended. The results that have been obtained have been fairly uniform and the error can be easily kept under ± 10 per cent. after a short experience of the method; it has however been found that when tried by those unaccustomed to the procedure the tendency is to under-estimate the strength of the culture. Lamb and Foster's Method. The authors of this method 3 aimed at measuring the strength of a vaccine by finding the smallest quantity of the vaccine which would remove the bactericidal power from a given quantity of normal goat serum for a given test dose of living bacteria. A series of experiments was undertaken to see if the principle could be The earlier experimental work of which this is a continuation appeared in articles by Lieutenant-Colonel Leishman and Major Harrison in the JOURNAL OF THE ROYAL ARMY MEDCAL CORPS May HarrisonJ OURNAL OF THE ROYAL ARMY MEDCAL CORPS March Scientific Memoirs Medical and Sanitary Officers Government of ndia New Series No

2 584 Further E.eperiments with Anti-Typhoid noculation relied on for the practical estimation of the strength of a vaccine. We were not in a position to use goat serum but substituted for it the fresh pooled sera of six normal rabbits the principle being unaffected- by this change. The process is a very laborious one much more so than the counting method and it was found that it could not be relied on for the detection of a variation of ± 25 per cent. in the strength of a vaccine. The following is an example of the results obtained. A forty-eight hours' culture of Bacillus typhosus was killed by heating to 53 C. for one hour and the following test vaccines were prepared from it :- Vaccine A = undiluted. Vaccine B = 3 parts vaccine A + 1 part sterile water. Vaccine C = 2 parts vaccine A + 2 parts water. Each of these was diluted to &c. up to 1-10; and the following mixtures made: 50 c.mm. fresh normal rabbit serum and 200 c.mm. of each of dilutions of the vaccine. The mixtures were incubated for one hour at 37 C. At the end of this time 50 c.mm. of a dilution of a living twenty-four hours' broth culture were added to each; the mixture was then incubated for twenty-four hours after which period the tubes were filled up with broth. The following were the results:- Dilution o 1-' Vaccine A Growth Sterile Vaccine B Sterile Vaccine C Growth. Sterile. No 'bservation. All the precautions prescribed by the authors of the method were scrupulously adopted in every experiment tried and as the results were unsatisfactory in every case we were compelled to abandon the method. Standardisation by means of a Photometer. n these experiments an attempt was made to arrive at a method for standardising a vaccine by measuring the interference to the passage of light caused by placing in front of an electric lamp a flat-sided vessel of definite thickness filled with vaccine. The instrument consisted of a long board along which one could slide a carrier holding a paper screen in the centre of which was a small grease spot made with a drop of castor oil. The ends of the

3 W. Leishman W. Harrison H. Grattan and R.Archibald 585 board were fitted with two Nernst lamps of equal power. When using the apparatus the point at which the two sides or the screen were equally illuminated (as shown by the disappearance of the image of the grease spot) was noted:--(a) with both lamps unobstructed Cb) with a flat-sided vessel full of broth in front of one lamp and (c) with the same yessel full of vaccine. t was hoped that one might be able thus to arrive at a means of measuring the obstruction of light caused by the bacterial bodies in the vaccine and from that to establish a means of standardising it. The apparatus was tested by comparing the influence of progressive dilution of a vaccine upon its opacity. t was found at first that the method. gave very encouraging results but after more prolonged observations the first promise was not fulfilled and the method was eventually abandoned because although one might obtain consistent results over quite a long period a very considerable error was shown every now and again due partly to variations in the light from the lamps during the time of the experiment and partly to personal factors fatigue eye-strain; &c. in the observer. Standardisation by Chemical Methods. An attempt was made to discover a means of standardising a vaccine by an' estimation of the chemical changes.which occur in broth as the result. of the growth of typhoid bacilli in it. A fortyeight hours' growth of the organism in broth was centrifuged till quite clear and a chemical analysis made of the supernatant fluid and of the original broth. t was found that there was a rise in specific gravity in free and albuminoid ammonia and in oxidisable matter. as a result of the growth of the bacteria in the broth. The total solids were increased but the residue after incineration was reduced and chlorine was likewise reduced. The changes however were not sufficiently constant to serve as a basis for standardisation. EXPERMENTS ON THE EFFECTS OF VAROUS EXPERMENTAL VACCNES. Comparison of Vaccines Killed by Heat and those Killed by. Lysol only. i As it had been found that 0'25 per cent. lysol would itself kill Bacillus typhosus in twenty-four hours the question arose as to whether it was necessary to heat the vaccine at all before adding the lysol. A forty-eight hours' growth of typhoid bacilli in broth

4 586 Further Experiments with Anti-Typhoid noculation was divided into two parts one part was killed by heating to 53 C. for one hour and 0'25 per cent. of 1yso1 was subsequently added; the other half was killed by the simple addition of 0'25 per cent. OEC JAN FEB MARCH DATE ' CHART.-Bactericidal action of the sera of a group of rabbits inoculated with vaccine killed by heat at 53 C. for one hourand subsequently mixed with 0'25 per cent. lysol compared with the bactericidal action of the sera of a group of rabbits inoculated with a correspondingdose of vaccine killed by the simple addition of 0'25 per cent. lysol. OEC JAN FES MAR DATE 17 [ ' ' S S t \ 4 4 4'0 'l. -'1' 3 8 i '.\ ' ' 3'2 <; i) '. \ 2'0.:\ ' Jj 1 8 '!! '-..'. 2 a r 11. 5\ 2 8 t 32 3'6 4'0 L.rso1ld.lkd Vacc=e_.._.. _ '.' Heatd Vaccuw ' '. CHART n.-phagocytic indices of a group of rabbits inoculated with vaccine killed by heat at 53 C. for.one hour and subsequently mixed with 0'25 per cent. lysol compared with the phagocytic indices of a group of rabbits inoculated with a corresponding dose of vaccine killed by the simple addition of 0'25 per cent. lysol. 1ys01 and was found to be sterile at the end of twenty-four hours. The vaccines were administered hypodermically to in each case three rabbits the dose given being 43 million bacteria and the subsequent observations were made on the pooled sera of the '

5 W Leishman W. Harrison H. Grattan and R. Archibald 587 three rabbits in each group the pooled sera of three normal rabbits serving as a control. n the first experiment there was a rise in' agglutinins bactericidal substances and in substances concerned with phagocytosis in the case of those rabbits which were inoculated with the vaccine killed by lysol only; but there was absolutely DEC JAN FEB MARCH OATE 1517r9(2r2312s27293J!2146'B \ 'OS 13'6 ' 32 2'8 :! ' 24..! l 2'0! ' l :f-'$2$_k -t- =t ' =i'- l_$' _L_.i--'''' =t' *$=.)-...''i:i'':-$$=$t=1$ Or: -2! i ; 1'6 -.l '! 20 l i 2-4 i!! 28 S 32 i ' ! i i! HeaLed f7itcdne.ly80lkilled fft.ccvte_.. _.. _ OHART.- Stimulin chart of the sera of rabbits inoculated with (a) vaccine killed by heat at 53 O. and subsequently mixed with ysol (0'25 per cent.); (b) vaccine killed by 0'25 per cent. ysol only. DEC JAN FEB MARCH OAT E !25'27 29:> OHART V.-Agglutininsproduced in rabbits by inoculation of (a) vaccine killed by beat at 53 O.and subsequently mixed with yso (0'25 per cent.); (b) vaccine killed by 0'25 per cent. ysolonly. no change in the case of those rabbits inoculated with the vaccine which had been killed by heat and subsequently mixed with lysol. This result was inconsistent with results previously obtained in 'rabbits by the use of a vaccine similarly prepared and it was suspected that it might be due to the fact that the lysol had been 40.!

6 5tl8 Furthm Emperirnoots with Anti-Typhoid noculation added before the heated vaccine had cooled down. As will be seen below this suspicion was confirmed and the accident has thus served to indicate a very necessary precaution in the practical pre-. paration of anti-typhoid vaccines. The experiment was repeated this time taking care to allow the heated vaccine to cool down before adding the lyso1. t will be seen from the accompanying charts (Nos. 1. to V.) that there is no great difference in the effect; of the two vaccines; so that it would appear so far as this single experiment goes that it is not necessary to heat the vaccine before adding the preservative and on the other hand that heating to 53 C. does not appreciably impair its efficacy. t is necessary however to have further evidence on this point before one can abandon the present method of preparing the vaccine which has been proved to give good results in actual practice. The Effect of Swallowing Dead Typhoid Bacilli Suspended in Fat. Emulsions of twenty-four-hurs' cultures of B. typhosus were made in sterile water and then desiccated over sulphuric acid. The resulting mass was found to be sterile after forty-eight hours and was then ground to a powder and mixed with lard to which a little stearine had been added in order to make a firmer mass. The fat was used in order to protect the bacterial bodies from the action of the gastric juice and it was hoped that they would subsequently be absorbed along with the fat particles from the small intestine. An experiment with a suspension of dead tubercle bacilli in fat had been previously made on a guinea-pig-the guinea-pig was killed three hours after swallowing the mixture and smears of its organs were made a few tubercle bacilli were found in the mesenteric glands and the spleen. The mixture of typhoid bacilli and fat was divided into pills each. containing the bacteria from one agar tube of culture. These pills were swallowed at short intervals by one of us (W. S. H.) who had already been the subject of other experiments of a similar nature and whose blood still showed evidence of the presence of substances antitropic to the typhoid bacillus. The pills were swallowed half an hour before breakfast in each case at intervals of about two days as shown in the charts (Nos. V. to V.); 'they caused no untoward symptoms with the exception of some slight nausea at times for an hour or so after swallowing them; this was attributed to the unpleasant flavour of the stearine. The results of the blood examinations

7 W Lei.hman W Harrison H. Grattan and R. Archibald 589 are shown in the accompanying charts; it will be seen that there is a general rise in agglutinins and in the substances concerned with phagocytosis and that the bactericidal power of the serum was depressed at one time but rose above normal on discontinuing the use of the pills. ngestion of Dead Typhoid Bacteria Suspended in Lard. n this experiment (Nos. X. to X.) the subject (R. G. A.) had not been previously inoculated and the dried bacteria were suspended in lard only the mixture being put into gelatine capsules each containing the bacteria from one agar slope. n the preparation of the fat suspension it was found that although one could not rely on the preliminary drying to kill the bacteria they died within twentyfour hours after admixture with the lard. The capsules were swallowed at first at intervals of about one month and towards the end of the experiment about every ten days. No agglutinins appeared at any time which is in contrast with the results of the former experiment and this seems to show that agglutinins are formed during such experiments only when the doses are repeated at short intervals. The bactericidal power of the serum was likewise not appreciably affected by the doses given. The power of the serum to encourage phagocytosis however was affected very distinctly by each dose; there was a rise in the phagocytic index thirteen days after taking the first capsule and this rise was repeated after each dose but at a shorter interval. There was a slight drop in phagocytosis on the fourth day after the third dose and this was followed by a rise above normal on the tenth day. On the twenty-sixth day after the third dose the phagocytic ratio fell below normal. This may be attributable to the effect of a severe cold from which the subject of the experiment was suffering at the time. n the experiments of J amiary 2nd and 14th the blood was taken six hours after swallowing a capsule and it will be seen that on both these occasions the phagocytic ratio fell below normal for the time being. The stimulin chart follows very closely the line taken by the chart of the phagocytic ratio a phenomenon which we have found to be very constant throughout our work; the figures for the construction of this stimulin chart are arrived at bya comparison of the phagocytosis in the following mixtures aftr exposure to 3T C. for fifteen minutes :-

8 590 Further Experimpnts with Anti-Typhoid noculation A. Washed blood cells.. Heated (60 ) normal serum Heated (60 )' normal serum (diluted to 1-5) Emulsion of B. typhosus B. Washed blood-cells.. Heated (60 U ) normal serum Heated (60 ) immune serum (diluted to 1-5) Emulsion of B. typhosus '.. 3 volumes volume volumes volume.. 1 n the estimation of the phagocytic ratio the control experiment has the same formula as (a) above and in the test experiment the whole of the normal serum is replaced by an equivalent volume of serum from the immunised subject. The Use of 'Washed Bacteria as a Vaccine. The object of this experiment was to see if the local reaction could be reduced by giving the bodies of the dead bacteria free from the fluid in which they had grown and in which they had been killed. A forty-eight-hours' broth culture of typhoid bacilli was killed by heating to 53 C. for one hour; it was then centrifuged and the resulting deposit was washed in normal saline being finally taken up in' such a quantity of 0'25 per cent. of lysol in norma- saline as would make the emulsion up to the same strength as the original culture viz bacteria per cubic centimetre. A dose of 1 cc. of the emulsion was given to one of us (W. S. H.) by hypodermic inoculation into the abdominal wall; the result was as follows: Pain on movement came on three hours after inoculation; it was not severe and the subject was able to take a long walk the same afternoon and to spend the evening gardening (not digging); rest was not disturbed at night and there was no difficulty in turning about in bed; a walk of 4 miles was taken the following day without great discomfort. The tenderness was increased by taking a glass of beer but it did not become severe. The whole of the local symptoms were gone at the end of thirtysix hours. General reaction consisted of slight fever and headache not at all severe; appetite and capacity for work were unimpaired. Taking them altogether the local and general reactions were much less severe than after a dose of standard vaccine taken by the same subject some two years previously. Several volunteers (officers on probatioll) were subsequently inoculated in the abqominal wall with the suspension of washed typhoid bacteria. Their reports were as follows ;-

9 W. Leishman W. Harrison H. Grattan and R. Archib(:Lld 591 (a) Continuous 'stitch' for eight hours: general symptoms very slight. (b) Tenderness at the site of inoculation and headache were the only symptoms. (c) Very slight local symptoms; felt very well. (d) Moderate local reaction felt feverish eight hours afterwards later well. (e) Pain bad same night; bad only on movement the following day no sleep the first night. (j) Mild local reaction; felt a little seedy nothing more. (g) Very mild local reaction; could get about; general symptoms very slight. (h) Moderately severe local reaction but could get about; general symptoms very slight. (i) Very severe local reaction same and a half hours and then disappeared. evening. quite well next day. evening; it lasted for one Slight faintness the same A number of other lieutenants on probation were inoculated at the same time with a corresponding dose of ordinary vaccine and a comparison of the effects in the two cases gave one the impression that the local symptoms were milder after the use of the 'washed bacterial vaccine than after the injection of the ordinary vacclne. All those who had received the washed bacterial vaccine on the first occasion received a dose of ordinary vaccine after an interval of ten days and their symptoms were taking them altogether more severe than on the first occasion and more severe also than in the case of those inoculated for the second time who had had the standard vaccine for their first dose. Whether this was because the first dose did not protect sufficiently or whether their reports were biassed by the comparison with their first experience it is difficult to say. Comparative estimations of the anti-tropic substances produced in the blood of those receiving washed bacterial vaccine and those who had been inoculated with standard vaccine showed no great differences except in the matter of agglutinins which were very much lower in the case of those who had been inoculated with the washed bacterial vaccine (1-80 as compared with 1-800). A very much simpler way of reducing the discomfort due to the local symptoms has been found to be to change the site of inoculation to the infra-clavicular region or to the outer side of the arm at the level of the insertion of the deltoid muscle. Either of those sites

10 592 Further Experiments with Anti-'Pyphoid noculation has been found to present such advantages over the abdominal site that we now recommend them for general use. t is ncessary to make certain that the needle enters the subcutaneous tissue for if the inoculation be made in the deeper layers of the derma the pain AUG SEP DATE i ' t)luton or SERUM 120 N 110 ;:' ' i S ':C GO '> \ l f-. 20 K' la j ' 10 f- 0 Treated Suhjeeb lvormaz S1..-Lhjet:P OHART V.-Showing the bactericidal action of the serum of a subject (W. S. H.) after swallowing a sllspension of dead typhoid bacilli in fat. AUG SEP DATE B la ' 1:S :; 2 2 ' ::'< :::: 2'1! 2 0 l-.-' g 1'8 1'7 1) l\. 1\ b' 1 4 \ 1'3 1 2 'i 1'1 e r1 1'1 ': 1 2 j11'3 14 _\ :--... \ - 1 CHART Vr.-Showing the phagocytic ratio of the serum of a subject (W. S. H.) after swallowing a suspension of dead typhoid bacilli in fat. at the time of the operation is considerable and the local reaction is apt to be much more severe a result which was noted by one of us who received accidentally a very small dose of vaccine into the derma instead of into the suhcutaneous tissue. t was noted at the

11 W. Leishmrw W. Harrison H. Grattan and R. Archibald 593 time of the operation that this was happening there being considerable smarting and the skin becoming puckered and bleached but the operation was continued in order to see the effect. Another of us received a full dose of standard vaccine intra-muscularly and in this case also the local reaction was abnormally severe. AUG DATE '2'3'415'6'7'8'9202' '30311' 2 3 ' SEP :;l ' i1 i' i l OHART V.-Showing the stimulin action of the serum of a subject (W. S. H.) after swallowing a suspension of dead typhod bacilli in fat. AUG SEP DATE ' ' DilUTON or SERUM! i i 550 ' 500 -& j '450 \ l' ' :;l' :is i'ii ' ii 11!:::' '- '- - '- '''' ' 'J ' 100 l- r-.. OHART V. - Showing the agglutinins in the serum of a subject (W. S. H.) after swallowing a suspension of dead typhoid bacilli in fat. 1\ i : i 'l'he Use of Vaccine Killed by Glycerine. As previously reported 1 it had been found that the addition of.20 per cent. of neutral glycerine to a culture of B. typllosus was 1 Harrison JOURNAL OF 'fhe ROYAL ARMY MEDCAL CORPS May :

12 594 Further Ewperiments with Anti-'l'yphoid noculation sufficient to kill it in about four days when the mixture was kept at blood heat. The culture when killed gradually loses the opaque appearance of a well-grown broth culture of typhoid bacteria and eventually becomes almost as transparent as the original broth. From microscopic examination of the resulting fluid it seems probable that this change in appearance is due to a solution of the greater portion of the organisms though a few roughly about a tenth of the original number remain unaltered in appearance. As this was the nearest approach to a complete solution of the bacteria in a culture that we had hitherto obtained and as the preparation of a vaccine by this method would be extremely simple it became of interest to find out whether the fluid so prepared possessed any value and if so how much value as a vaccine. Two vaccines were prepared A and B. Vaccine A was made by adding 20 per cent. of glycerine to a twenty-four-hours' broth culture of a strain of non-virulent B. typhosus ( R) which has been largely employed in the preparation of vaccine for the troops. The mixture was kept at 37 C. for a week when it was found to be sterile. The original culture contained bacteria per cubic centimetre. Vaccine B was prepared in the same way as vaccine A but was made from a culture of a virulent strain ( L ) of B. typhosus. The original culture contained bacteria per cc. Two groups of rabbits were inoculated one with vaccine A the other with vaccine B the doses being -1-5 cc. on December 31st 1906 '[\ cc. on January 16th 1907 and! cc. on February 2nd The estimations of anti-tropic substances were made from the pooled sera of each group of rabbits and the control was in each case the pooled sera of a similar group of normal rabbits. The test organism was of the non-virulent strain C R ) from which the vaccine A had been prepared. Agglutinins appeared to only a slight extent the highest point reached being 1-60 in the case of the vaccine B rabbits and this on one occasion only. (Charts Nos. X. to XV.) showing the bactericidal action of the sera of the experimental animals and the results of the phagocytosis experiments are. appended. t seems clear from these that a vaccine prepared in this way is effective and further that such a vaccine prepared from a virulent culture appears in some respects more effective than the one prepared from a non-virulent culture. n this connection it is to be noted that the test organism for the experiments was of the same strain as that from which the vaccine A (non-virulent strain) was prepared. Further experiments are

13 SEP OCT NOV DEC JAN DATE: Z B DLUTON L OF SERUM ::: l& '\. 'll D ' 1'100 ;\- 90 ' 1< ' ' ' 70 ' J f' :L 60 \ - - ' - \ - \ \ '!. '0. V \ - ' 40 \ r--bl \ \ 30 ' ' 0.KorrnaZ Sub.lea Treated Suo cb OHART X.-Showing the bactericidal action of the serum of a subject (R. G. A.) after swallowing suspension of typhoid bacilli in lard. 'l C'> ;;::-.... ;:.0 i;) i;).. ''' 6jramc on 1 June Downloaded from on 11 March 2019 by guest. Protected by

14 SEP OCT NOV DEC JAN DATE H2468WMH '3 3'2 3'1. 3'0 2'9 P. 2'8 2'7 2 5 N ' 2'41' l; -r 5 2'2 2.1 it 'r 11\ -r J 2'0 \ 2'3.. 1 t\ < 5 1'9 1\ \ 1'8 { { \ 1'6 _-'- L q 1'4-... \. j L 1'3 1 2 V- 1.. \l V V 0 :;::: i\ 2 H 1' '5 \ ::; 1 8 [ OHART X.-Showing the phagocytic ratio of the serum of a subject (R. G. A.) after swallowing dead typhoid bacteria suspended in lard. 6jramc on 1 June Downloaded from on 11 March 2019 by guest. Protected by

15 SEP OCT NOV OEe JAN DATE : la \ 1-9 \-8 \ ' -1-7! i fl r: \ \-6 r-- 7\ n: -s f f \ \-4 t V+--- \ -3 1\ \'\ 10 - \-2 \'3 1 4 l L 11 'V. L 1 e- J 5 \' r 1'6 1'7 \ 8 J 1'9 \i CHART XL-Showing the stimulin action ofthe serum of a subject (R G. A.) after swallowing dead typhoid bacteria suspended in lard. 6jramc on 1 June Downloaded from on 11 March 2019 by guest. Protected by \ --

16 598 FU'rther Er:cpe'rirnents with Anti-Typhoid noculation in progress to find out how far these results apply to man and whether this method of preparing a vaccine presents any practical advantages over that at present in use. THE MMEDATE EFFECT OF THE SECOND NOCULATON ON THE DEVELOPMENT OF PROTECTVE SUBSTANCES. Twelve officers who had received their first inoculation at the same'time were divided into two groups of six each; one group was re-inoculated at the usual time and in the other group the re-inoculation was postponed for a day or two and comparative observations were made on the pooled sera of the members of each group. t JAN. FEB MAR DATE LUTONH'\+-f t--+--Jf f J t 130 ' CHART X.-Bactericidal action of the sera of (1) two normal rabbits; (2) two rabbits vaccinated with B. typhosus (' R ) non virulent killed by glycerine (20 per cent.) for one week at 37 C. = Vaccine A; (3) two rabbits vaccinated with B. typhosus ( L) virulent killed by glycerine (20 per cent.) for one week at 37 C. = Vaccine B. was found that on the two days following re-inoculation the bactericidal power of the serum of those who had been reinoculated was a point lower (1-60 as compared with 1-70) than in the case of those who had up to that time only received one dose of vaccine but it did not fall to normal (1-40). Agglutinins were reduced from to on the day following re-inoculation but they had already recovered on the second day and were then higher than in the case of those men who had only received one dose of vaccine ( as compared with ). The phagocytic index was markedly lowered as a result of the second dose but it was unfortunately impossible owing to the exigencies

17 W Leishman W. Harrison H. Grattan and R. ArchibaZd 599 of the Service t() continue these observations beyond the second day after inoculation. JAN FES MAR DATE r: 8 i!! 9 1 t t!i <S 'S s1 5 ;;; c y. ;3i 4 3 g L ' -.' 0- r.-- j 2 d3 l S 4 ; 1hcci.neAtLVon-virulent) Ta.ccineB (Plr) OHART X.-Phagocytic ratio of the sera of two normal rabbits and (A) two rabbits vaccinated with B. typhosus ( R ) non-virulent killed by glycerine = Vaccine A; (B) two rabbits vaccinated with B. typhosus (H L ) virulent killed by glycerine = Vaccine B. JAf FES MAR DATE ' ' ' ' r-+. 1 _. -j-j 0 + H T 1- f )11 ; -+ + r+ t-.. t- t-t- t-t j-+-t-r-.l- S 5 \l ;;; 4 ' ' j-- -!. - l. d3 1.. i') :- -1- V ]'-- 1 ' 'j. 'H' ---i - l. 3 --! ' h...canea.hon-virulent}.. VaccUle B fpu-ulenb) OHART XV. -Effect of substituting 1 vol. of serum of vaccinated rabbits (diluted 1-5) for 1 vol. of similarly diluted normal serum in the following mixture: Washed cells = 3 vols.; normal serum = 2 vols.; normal serum (1-5) = 1 vol.; emulsion B.T.A. = 1 vol. Vaccine A = B.T.A. (R.) non-virulent killed by glycerine; Vaccine B = B.T.A. ( L.) virulent killed by glycerine. ACTON OF THE SERUM UPON PARATYPHOD BACLL AFTER TYPHOD NOOULATON. This appears to be a point of considerable practical importance m view of the fact that a certain number of cases of supposed

18 600 Further Ewperiments 'With Anti-Typhoid nogttlation typhoid fever occurring in inoculated men have been proved to be paratyphoid by the isolation of this germ from the blood-stream. For the purpose ot the experiment the blood of two men who had previously been inoculated against typhoid was tested as to its agglutinative bactericidal and phagocytic power against paratyphoid A and paratyphoid B respectively. (1) Paratyphoid A. Bactericidal Estimation.-The serum of an inoculated roan H. was found to kill this strain of paratyphoid in a dilution of 1-50 while the control serum derived from a non-inoculated man only killed in a dilution of On repeating this experiment however on another occasion no difference was found both the normal and the immune serum killing in a dilution of The technique was that employee for the estimation of the bactericidal substances throughout the former experiments. Agglutinins.-A slight group agglutination was found on the tw? occasions on which this was investigated the same figure being recorded in each instance viz. incomplete clumping of the paratyphoid bacilli in half an hour in a maximum dilution of The control normal serum showed no trace of agglutination in the same time in a dilution of Phagocytosis.-The phagocytic ratio of H. tested on two occasions was found to be 1'6 on the first inyestigation and 1'2 on the second. A similar experiment on the serum of a second inoculated man E whose ratio to the typhoid bacillus was at the moment 2 showed when tested against the paratyphoid A a ratio of 1'1. (2) Paratyphoid B.-Similar experiments were made with the same serum against this strain and with much the same results. n the case of the bactericidal substances the serum of H. on one occasion killed the paratyphoid bacilli in a dilution of 1-10 while the normal serum failed to kill in this degree of concentration. On a subsequent estirration both H's. serum and that of the control failed to kili in this dilution. Agglutination was incomplete in a dilution of 1-30 and the phagocytic ratio was found to be 1'1 against this strain on comparison with that of a non-inoculated control. t would appear then as far as may be concluded from these few experiments that inoculation with. anti-typhoid vaccine does not confer any marked immunity against infection by paratyphoid bacilli as judged by the development of protective substance's in the blood.

19 W. Leishman W. Har'rison H. Grattan and R. Archibald 601 ACTON OF THE SERUM AFTER TYPHOD NOCULATON UPON STRANS OF B. TYPHOSUS OTHER THAN THA'] EMPLOYED FOR MMUNSATON. ' The serum used was that of H. who had been inoculated with vaccine prepared from the strain R and who on a subsequent occasion had ingested pills and capsules containing dead typhoid bacilli of the same strain. The bactericidal action of H.'s serum was determined against the strain R with which he had been immunised against strain G of equally low virulence and against a third strain B of moderate virulence for guinea-pigs. The technique employed was to mix together one volume of the serum diluted in varying degrees with one volume of a twenty-four-hours' broth culture of each of these three strains of typhoid diluted with sterile broth to After incubation of the mixtures for one hour at 37 C. they were blown out into test tubes of 1\'[cConkey's mannite bile-salt medium which were then incubated for twenty-four hours. The results are shown in the following table and it will be seen that in each case there is practically the same difference in favour of the immune serum as compared with the bactericidal action of the serum of the normal individual used as a control. This result is in confirmation of similar experiments which had been made at an earlier date and points to the conclusion that a monovalent vaccine may be relied upon to induce the formation of protective substances which are effective not only against the a-virulent strain employed as a vaccine but also against the other and more virulent strains of B. typhosus. Dilution of sera no Normal ! } R mmune (H) strain. Normal } G mmune (H) strain. Normal l B mmune (H) ) strain. + = Growth. - = Sterile. 1 Experimental error.