On the culture of malarial parasites and Piroplasma canis

ON

THE

CULTURE AND

OF

MALARIAL

PII~OPLASMA

13Y I~ROFESSOI~ t t A I ~ S Z I E M A N N

PARASITES

CANIS. (CHARLOTTENBURG).*

As you know, former experiments to cultivate malarial parasites

in vitro were fruitless, until in October, 1912, BAss and J o ~ s published in the Jo~trnal of Experimental Medicine their detailed methods of artificially cultivating those blood-parasites. BAss kihdly sent me the preparations for nay inspection, so that I was able to prove the correctness of his statements and to give some details which he had not communicated, f Unfortunately here in Berlin attenlpts to procure cases of malaria for the culture of parasites were at first fruitless. iV[Y OWN

CULTURE

OF

TEI~TIAN

PAI~ASIT]~8.

On January ]3th, 1918, [ was called to treat a case of ordinary tertian East-African infection, which led go a strong attack of fever at 2 o'clock a.m. In the blood taken on the 16th January, at i0 o'clock a.m., when the patient was h'ee from fever, I could, by using B~kss's method, ascertain a further development of the parasites, but unfortunately a growth of bacteria soon presented itself. Another specimen of blood taken on January 17th, at 6 p.m., proved that there were nearly fully developed parasites m the peripheral blood. I could then follow up the development of two-thirds of fully grown tertian parasites up to sporulation and the formation of young sehizonts again.:) Tertian gametes were s~ill to be seen in the evening of the 20th January, but in most of the culture tubes growth of bacteria had already begun, and these had caused the disappearance of the sehizonts. The reason why I did not succeed in cultivating more than two generations was probably that the malarial blood when transplanted cooled down, or that I had given tannate of quinine 1'0 gr. to the Presented

to ~he m e e t i n g

in the absence

of t h e A u t h o r .

H . ZIEMANN : " Oil BASS'S c u l t u r e of m a l a r i a l p a r a s i t e s Z e n t r a l b l . f. B a R t . B d . 67, H . 6. 1913.

,~z wt'ro a n d i t s r e s u l t s . "

+ I-I Z I E M A N N : " O n t h e a r t i f i c i a l s u b c u l t u r e {i~ vitro) of t e r t i a n s i t e s " D e u t s c h . Meal. W o e h e n s e h r i f t , 1913. No. 6 u. No. 8.

malarial para-

ON THE

CULTURE

OF MALARIAL

PAP~ASITES~

ETC.

elderly patient, who had a dmeased liver, before collecting t on J a n u a r y 16th. MY O W N

CULTURE

OF PLASMODIUM

FALCIPAI~UM.

On March 7tb a patient came to me who, a short time be been in the Cameroons, West Africa, for two years, and had had fe several times, but in general felt perfectly well. However, on his and after his arrival in Germany, he had fever for days and wee~ he either did not treat at all, or with quite small doses of qumii to the fear of blackwater fever. The patient said he had lost 8( during the last few weeks. General health moderate ; in the pc blood little and middle-sized signet ring-fornis; temperature at 87'2 ° C., at 6 p.m. chills, temperature 88'6 ° C., and at 8 p.m. " during the night, perspiration. A medium degree of polyehronia basophilia and anisozocytosis in the blood. N m n b e r of red blo 2,234,000 ; Hb, 60 per cent. On March 7th, at 12 p.m., blood w from the vein at the bend of the elbow by means of a Strauss' ne~ collected in a graduated and sterilized tube tilted to one side an¢ nated. Again, as i n the case of the tertian, the blood was mixe more or less large quantity of 50 per cent. solution of dextrose, t say, 0"1 e.c. of the 50 per cent. solution of dextrose was added t 8'10 and 12 c.c. of blood. To these mixtures were also added a cent. solution of sodium citrate in more or less large quantities. cation also took place, as the tubes were kept in dark rooms at a t ture of 37 ° C. or a temperature of 40 ° C. The mixtures thus 1 were previously centrifuged in centrifugal glasses at about 800 tions within five minutes, to drive the leneocytes from the bottoi top, and then to transfer the leucocyte-free malarial blood f middle or the bottom of the centrifugal tubes to the serum tubes had already been proved by previous control experiments with the that a distinct re-development of parasites is not possible wit addition of dextrose, only serum containing dextrose was emplo serum being non-inactivated and inactivated (one hour at ~13° C experiment was made with : 1. Horses' s e r u l n , 2. H u m a n ascitie serum, 3. H u m a n blood serum,

222 and

oN T~ it was

proved

CULWUaE

Or MALARIAL

that the development

vated dextrose-ascites sermu was added.

took

place best when

inacti-

The layer of sermn was most suitable at about five centimeters high, the height of the transferred layer of malarial blood being at least one-third centimeter. I kept this culture, not as BASS proposes at a temperature of 40 ° C., trot more advantageously at 37°C., so that one could bring it into the incubator just before the beginning of segmentation of the parasites, at a temperature of 40 ° C. The culture appears to be most successful when one puts leucocyte-free non-malarial dextrose-blood at the bottom of the serum tube, and then plants the malarial dextrose-blood upon it. To obtain subcultures T mixed one volmne of leucocyte-free dextrose malarial red blood corpuscles in sterilized pipett~es with about a ten-fold volmne of equally leucocyte-free normal dextrose red blood corpuscles and spread this mixture at the bottom of a serum tube. BAss has said that he very seldom succeeded in developing in one and the same culture lucre than two successive generations of parasites, but I found in my case, m the two tubes where there was dextrose malarial blood wflsh inactivated dextrose-aseltie fluid, even in a six days old culture, a constantly repeated increase of the parasites. Doubtless, also m the six days old plasmodium culture, a &stinet increase of the parasites took place. [t is possible that the addition of dextrose made it more easy for the young merozoites to settle down, the red blood ceils having become more viscous. [ have prewously stated that I, in many thousands of blood examinations in the Cameroons, succeeded only once in obtaining a form of sporulation of Plasmodiu'~n .falc@ctruvb in the peripheral blood. It was therefore very interesting to follow up, in the culture, all the different stages of the development. It w~s proved that the forms of sporulation of the Cameroon Plasvwdbu'm falciparum do not differ at all from the forms of sporulation found in other parts of the tropics and in italy. The n u m b e r of merozoltes were from 14 to 18, in most e0~ses 16. The development, from one form of sporulation to the other, lasted in the incubator at a ~emperagure of 37 ° C. about 40 to 48 hours. At a temperature of 40 ° C., the development apparently seemed to take a course only a few hours quicker. It is very interesting to note, that in the peripheral blood the infection of the red blood cells was very scanty, wherea~s in the culture a fern' to five-fold infection of the red

PAI~ASITES AND PIROP.LASMA

(JANIS.

2o~

blood cells was n o rarity, ft is important, that m tbe six days ol, culture containing a fair n m n b e r of crescents, males as well as females, n trace of a further development was to be seen, nor any indicatio~ of parthenogenesis. Sub-cultures could be obtained from the two-day old, and in one instance from the four-days old culture accordint to the method mentioned above. The patient had to take a cours of quinine, as the danger of blackwater fever of course existed the quantity of quinine was daily increased by 0'1 gr. (tannate, late the hydroehloride). Owing to the good food at the Sanatorium and th, treatment with small doses of quinine, the temperature" rose durin{ ~he next attack of fever on the 9th of March at 8 p.m. only to 38 ° C The n m n b e r of schizonts, too, diminished, whereas that of the cres cents augmented. On March 101~h tile blood was again taken for the purpose ol culture, i n the peripheral blood at this time, only crescents and very few schlzonts were observed, i n a seven days old culture no young schizonts were found, the development being poor, and the n u m b e r of dying-off sporulation forms larger than in the first experiment ; probably, because owing to the quinine cure and the stay at the Sanatorium, the virulence of the parasites had decreased, or the resistance of the patient increased. On MTareh 16th, at 12 a.m., the temperature being 87'~ ° C., blood was again taken for culture, at a time when there were very scarce signet rings, and rather a large n m n b e r of crescents in the peripheral blood. I n the last culture the development of the schizonts was still more sligh~ than before, nor was ~here any development of the crescents. The collecting of the peripheral blood had unfortunately taken place a little too late, when the majority of the parasites had ~lready got mt,o the inner organs for sporulation. On March 18th, at 6 p.m.. there was a temperature of ~31° C. J~'rom that time steady improvement of the patient's state of health took place, increased doses of quinine being given. On March 31st the patient was dismissed wi~h orders for further quinine treatment. The summary of the cases described above is as follows : - 1. I n a malarial blood--takei~ in a sterilized state, 5 e.c. of it being mixed with one-tenth of a 50 per cent. solution of dextrose--the malarial parasites shewed in the culture exactly the same morphological

~°~d_

ON T H E

CULTURE

OF N I A L A R I A L

and biological development as in the h u m a n organism. I n the culture, the development of the tertian parasites took place ordinarily at a temperature of 39"5° C. in about 34 hours, that of the Ph~smodiu~r~ /(dcipar~m ,~t a temperature of 37 ° C. in about 40 to 48 hours. Occasional differences occur in the development of parasites in cultures as well as in the peripheral blood, as some parasites develop sooner and others later. 5. Besides the normal, strongly-developed parasites, been observed in the cultures with vacuolised plasma pulverous solution of the ehromatin, fornis that were never pempheral blood of the patient. Between these dying-off forms there occur all kinds of transitions.

forms have bodies and seen in the and normal

8. Sometimes these degenerated forms remind one of the quinine forms in which, as is known, a pale colonring and rupture of the plasma body can be observed, whilst the chrolnatin at first preserves its

compactness. 4. Nit the dymg-off or degenerated forms are taken up by leucocytes. All kinds of leucocytes co-operate in the phagocytosis in the culture, unless they have been removed by an especially careful eentrifugalisation. 5. I could not observe in the culture a conjugation of yoking sch~zonts ~s described by I~VIANNAI~,:EP~@and later by Cl~Am. 6. Up to now, my experiments with tertian and falcipc~rum in cultures have shewn no trace of an indication of parthenogenesis or the formation of ookinets. 7. The young merozoites seem to stay only a short time extracorpuscular in the blood plasma. One can see them only whel sporulagion takes place, and near the sporulating bodies. 8. I have never seen, in many hundreds of preparations made from the cultures, that the parasites wandered from infected red blood cells to non-infected ones, as MTAI~YLAwso~-I~owLJ~Y maintains. 9. Since in my cultures of tertian parasites and of Plcesmodiu~ .fcdcipc~,r~m the same characteristic and morphological distinctions were i found as are adimtted for the different kinds of parasites--for instance,:i Sc~IOFP~E~'s dots in tertiana parasites, MAV~J~'s spots in Plasmodi~m i fcdc,ilJa~'t~,m--the belief of some authorities that the different n-ialari~l~:

PARASITES

AND

PII%OPLASMA

CANIS.

~5

parasites must be regarded only as varieties of one and ~he same kind may finally be refuted. 10.

As last year I had myself suffered from a slight recurrence of

PlasmodiuJ~ falciloa,~'um, I could not vaccinate myself with liwng cultures, bat I vaccinated myself with a culture of Plasmodi',~,faleiyarum which h~d died out. This vaccination led only to a slight rise of temperature, 37"8 ° C., but did not specially alter m y general state of health. 11. Until now I cannot share the belief of BAss that it is possible to cultivate malarial parasites ad infinit,zclTr without intervening sexual phases such as occur in Lhe bodies of anopheles. 12. A report wilt be made later about experimental influences of light, radium, electricity and the serological analysis as regards diagnosis. 14. I am prepared to recommend the cultivation of malarial parasites, whenever latent malaria is suspected, so that one might obtain a number of generations of parasites in the culture, and thus demonstrate either the parasites themselves or at least leucocytes containing pigment.

CULTURE

OF

PIROPLASMA

CANIS.

The information BASS has made about the culture o[ malarial parasites induced me to try, in December, 1912, the cultivation of Pb'ol)lasv~c~ c~¢~is. I soon found that the application of B a s s ' s method for the culture of Piropgas~c~ cauls by no means always leads to the desired effect. Some investigators have already believed that they have seen forms of development of t~irololasma ca~is in artifical cultures (I~LE[NE, NUTTAL). In the beginning of nay research I had no results, and only later, after many experiments, I learned that it was wrong to use dogs with a large number of parasites for the cultures ; strong, young dogs, having just got parasites in their peripheral blood, being best fitted for experiment. If there are only larger dogs (more than three to four months old) at disposal, I recommend that their spleen should be taken out and their blood collected three to four days after the operation (about 30 to 50 c.c. of blood according to the size of the dogs) from the ve~zajug~daris. The infection is then always successful.

ON T H E

CULTURE

OF M A L A R I A L

Similarly to what 1 found in cultivating tertian parasites and Plas~voclium falciparz~, my experiments again were extremely variable, for 1 mixed the Piro2plasn~a cauls blood with h u m a n ascitic fluid, human blood serum, dogs' blood serum, horses' blood serum, all of them partly reactivated, partly non-inactivated and partly mixed with sodium citrate in more or less large quantities, and kept the cultures at room temperature, at 37 ° C., and 40 ° C. The most favourable results were ~btained by nsmg the following technique: One lays ()pen the carotid of a young, strong dog, hawng yet very few parasites in its blood, and taking the blood by means of a sterilized glass canula, allows it to run into graduated cylinders with a small neck. i t is then defibrinated carefully and for a long time, because the production of air bubbles must be avoided. After this the coagulated fibrin is removed and to each 5 c.c. of blood is added I to 10 e.c. of 50 per cent. solutton of dextrose (formerly I added that quantity to 8 c.e. of blood), and to 10 c.c. of blood 0'3 c.c. of a 2 per cent. solutmn of sodimn citrate. T h e n one centrifuges just as in the culture of the malarial parasites, and takes wlth a pipette the leucocytefree piroplasma-blood out of the middle or the bottom of the centrifuged tubes and plan~s it at the bottom of the serum in the culture tubes, which are filled with at least 5 era. highly inactivated sodium citrate, dextrose dogs' blood serum, or, sodium citrate dextrose ascitie serum. I t is best not to make the layers of the red blood ceils too thin, and to proceed just as in the culture of the malarial parasites. It is possible also to get excellent cultures at a temperature of 40 ° C., and at room temperature. I n these a distinct augmentation of the parasites can be observed, but I saw the best development of parasites, especially of chromatins, at a temperature of 37 ° C., and when I added sodimn citrate. After 24 hours a considerable increase of the parasites can be observed in the same blood that in the dog itself shewed only a very slight infection. F o r m s of sporulation with more than four merozoites are very rarely to be seen in the peripheral blood, but in successful cultures forms of sehizogony with 16 and more merozoites are seen. I n this way it is possible to keep the culture alive up to the fifth, or even to the sixth day, but already after the second day some degenerated forms appear which become more frequent after the third day. Nevertheless I succeeded in mortally infecting a dog by an intravenous injectmn in the jugular vein taken

PARASITES AND PIROPLA~qMA CANIS.

from a three d&ys' old sub-culture, which in t u r n was taken fro days' old culture. I n one instance I also succeeded in getting sub. from a four days' old culture. Even a four days' old sub-eultur, by bacteria shewed itself virulent the other day by intravenous i If the growth of bacteria in a culture has lasted more than however, a quick dying-off of the parasites can be observed, just the cultures of malarial parasites. The contamination of the eu blastomycetes does not damage the parasites so quickly. As degeneration takes place, the piroplasmata lose thmr typical pea turn roundish, the plasma body becomes of a darker colour, colouring of the chromatin ceases. At the same time some part plasma body may come off m the form of small particles or little l later the degenerated paramtes are ingested by leucocytes. i shall report about serological, diagnostical, etc., expe elsewhere.

DISCUSSION.

Sir W~z. I]~zsH~aN : I should like to congratulate Dr. THomsoN on his interesting paper, and especially on the beautift he has shewn. The photographs do not by any means do ju the preparations, and I think that nobody looking at them cot~ any doubt that they represent a true multiplication of the p i% vitro. I was also particularly interested in his observations as tendency of the m a l i g n a n t tertian schizonts to run together i n , and his suggestion that this may account for the rarity with we encounter the segmenting forms in the peripheral blood form of malaria. Dr. J. G. THOMSON: Professor ZZI~lVIAN~T'Spaper is nmst int( to me, beeauso in a great many respects the results he has obtai~ almost identical with my own ; be has noticed that the parasites at a different rate m &fferent cases. I notice he says that leucocytes take up and ingest these p; very rapidly. I have found that it is scarcely necessary, at least development of one generation, to remove the leucocytes at all, t

228

DTSCUSSION.

not seem to ingest the parasites as long as they are in the corpuscles. W h e n the parasites degenerate and become free in the serum then the leucocytes take them up. As long as the plasmodia remain in the corpuscles they do not seem to be ingested, and you will see here under the microscope that they have gone right up to sporulation. These masses of sporulating forms occur even in cultures not free from leucocytes. W e found in a culture tube not free from leucocytes, that three generations of the malignant tertian parasite developed. We watched the leucocytes for six days in one culture tube, and although they took up some parasites they did not prevent the formation of new generations. I t is most interesting to hear that Professor ZIE~A~S has managed to cultivate loiroplasma c(u~is. I tried this on two separate occasions, but I was not able to get any growth. Dr. J. C~oPP~R: I should like to congratulate Dr. THOMSON very much on his experiments and the information he has obtained, and I should like to ask him what was the earliest date on record that ~he parasites were known to clump. I n 1908 1 reported a case at this Society, and published in the Lancet with coloured drawings, where clumping was found in the blood of a man who died in Palestine. M~tsses of 19 and 21 corpuscles were clumped together. There are many points raised in Professor ZIEMANN'S paper, and I notice that only once was he able to obtain sporulation in many thousands of examinations. I n Palestine, in 1,000 positive examinations, ] was successful in finding sporulation in 10 per cent. of the tropical form. I should like to have Dr. TttOMSON'S opinion on the cause of this difference of sporulation in the peripheral blood. I n Palestine it is easy to find, in other countries it is apparently difficult. Also [ should like his opinion on the difference in the growth of the parasites ; is it owing to the change of temperature ? It has always been interesting to me, and I think this may account for the variable hines the parasites take to develop, l should be glad of any information he may be able to suggest. Dr. J. (~. TKOMSO~: I n Liverpool we have several slides from the P a n a m a shewing sporulation of the malignant tertian parasite in the

DISCIISBION.

peripheral blood. I am quite unable to explain why this occurs, have noticed in the cultures of the malignant tertian parasite tt some eases there is no tendency to clump at all, in other words elui does not occur in all eases. W h e t h e r that has anything to do witk not I do not know, possibly it has. Ig is ,¢ery difficult of cour explain, and the only explanation 1 can give, is, that in some cases is no~ such a tendency to clump. Dr. J. CaopPEr~: I should also like to know whether Dr. THo~soi observed phagocytosis of the infected red cell, and ~f he can say wh he observed'any other forms of leucocyte 1)esides; the large mononuc acting in this way. Dr. J. (3. T~OMSOST: No, I have not observed in cultures polymorph leucocytes with ingested parasites, but mononuelears frequently seen with ingested parasites. Dr. G. C. L o w : I n reply to Dr. CrcO~PER's question, 1 say I that have never seen nor heard of such a large number of re'flit malarias as 10 per cent. shewing sporulating forms m the peript blood. I n m y own experience, which is ~ somewhat large one, I ] only seen it in three or four eases ; one of these being in a peripl~ blood film sent from I-Iong Xong. This specimen shewed large nmn of sporulating malignant parasites in the blood, and also the interesting condition of two complete rosettes ingested by a polymor[ nuclear leucocyte. Many of these latter cells also contained pigm, In another ease seen in the W e s t lndies a similar condition was for Why in a certain number of cases such an occurrence should take ptac by no means clear. I t is certainly uncommon. Dr. I). T ~ o ~ s o ~ : ] have seen sporulating forms of the malign tertian parasite in the peripheral blood frequently, but only in very he infections where there is a special tendency to coma. I n such eases often finds a tremendous increase of leucocytes. In these eases of v heavy infection, polymorphonuelear leucocytes, eontabling pigment, n be seen. The mononuelears, bowever, take up the pigment parasites far more frequently.

280

DISCUSSION.

I have ,%lso noticed, like Dr. C~oPr~R, that in malignant tertian mMaria one paroxysm may follow another in less than 48 hours; sometimes a longer interval elapses, but they are very irregular in point of time. I n the cultures of malignant tertian sometimes the parasites sporulated in 52 hours, sometimes in ~4 hours; so this irregularity might explain the irregularity in the fever paroxysms. I t seems to depend on the amount of the sugar and on favourable conditions or otherwise in the culture tube. W h e n the conditions are favourable ~hey sporulate quickly, in less than 48 hours, but where the conditions are less favoura.ble, tlne time required for ~rowth and spo|'ulation is prolonged. Professor W. J. SIMPSON: I am sure, gentlemen, that you will all join with me in thanking Dr. DAVID THOMSON and Dr. J. G. THOMSON for the papers they have given us this evening. Both papers, though on very different subjects, have been exceedingly in~eresting and~ instructive, and we must congratulate the IAverpool School of Tropical' Medicine in bringing these papers before the Society.