SOME OBSERVATIONS ON BALANTIDIUU / COLI AND ENTAMCEBA HISTOLYTICA " OF MACAQUES

By R. KNOWLES

LIEUTENANT-COLONEL, I.M.S.

Professor of Protozoology

and

Assistant Surgeon B. M. DAS GUPTA, l.m.p.

Assistant Professor of Protozoology, Calcutta School of Tropical Medicine

Barret and Yarbrough (1921) claim to have successfully cultivated Balantidium coli of man in a medium consisting of inactivated human serum and 0.5 per cent salt solution, in the proportion of one part of the serum to sixteen parts of salt solution. But when faeces from the monkey Silenus

(Macacus) rhesus, which are invariably crammed with blastocystis, are inoculated into the above medium and incubated at 37?C.?the

optimum temperature for growth of most of the intestinal protozoa of warm-blooded hosts?the fungus develops with very great rapidity, with consequent destruction of any Balantidium coli which may be present. Jameson (1927) has shown that Balantidium

coli from the domestic pig can be readily culti- vated in inspissated horse serum and egg white to which a little solid rice starch has been added ?a medium recommended by Dobell and Laid- law (1926) for the cultivation of the entozoic

amcebse, and especially of Entamoeba histolytica. Macaques of species Silenus rhesus obtained

from dealers in the Calcutta markets are very

heavily parasitized with a ciliate protozoon which corresponds morphologically with Balan- tidium coli of man, and some months ago we commenced attempts to cultivate this organism and to transfer it to human volunteers. Our earlier experiments at culture were not too suc- cessful, as the cultures were frequently over- grown with blastocystis, in spite of solid rice starch being added to the medium before inocu- lation.

It was noted that the starch, before it could exert its restraining influence on the develop- ment of blastocystis, was split up by the starch- fermenting bacteria which are very often present in monkey's faeces. In order to keep down the very troublesome

growth of blastocystis, a few drops of acriflavine solution were added to the medium, as suggested by Dobell and Laidlaw (1926). In the majority of instances this gave excellent results, but sometimes even this measure was of no avail, as some of the starch-splitting bacteria were

able to withstand a much stronger solution of acriflavine than were the balantidium. We have isolated from monkey's faeces an organism which appears to correspond to ' bacillus 1' of Dobell and Laidlaw (1926), and which these authors claim to be highly detrimental to blastocystis,

ISOSPORA IN INDIAN CATS

fj

1 to 4 ?Isospora 'type BShowing stages of development outside the body of the host.

5 Jsospora 'type A'. As seen in freshly-passed 1 J=' fteces.

-n- a T mv nower view of a preparation after concen- fration by th? 'D. C. P.' method of Clayton Lane.

BALANT1D1UM COLI FROM A MACAQUE

9 Figs. 7 and 8.?Undergoing binary fission. The ciliates are loaded with ingested rice starch from the culture medium Figs. 9 and 10.?Showing encystation and excystation respectively

Plate VII

ISOSPORA IN INDIAN CATS

17,Vs 1 to 4 ?Isospora 'type BShowing stages of

development outside the body of the host. Vicr 5?Isospora 'type A'. As seen in freshly-passed

h' feces.

tti; fi tm power view of a preparation after concen-

fration by the 'D. C. F/ method of Clayton Lane.

balantidium coli from a macaque

Figs. 7 and 8.?Undergoing binary fission. The ciJiates are loaded with ingested rice starch from the culture medium.

Figs. 9 and 10?Showing encystation and excystation respectively

T t TV TNDTAN CATS : KNOWT.ES & DAS GUPTA 391

July, 1934] ISOSrORA INFECTION IN INI)1 aim i/Aip 0*1

^llt 6VCI1 Medium wifh+i^-01!8 i.noculation of the culture

nionkev blicf 1IS .aciUus fails to keep down the

splitting bnni?C^1S w^en the resistant starch- faeces.

' Cna are present in the monkey's , Reaction of //

serum is vprv 11 v me^lum-?The inspissated

the hydroo-pn ? a"ne- In twelve samples tested 8-8 to 9.2 rv\?n c?ttcentration varied from pH about neufril i! Par^ the medium is

with the spnirvi 1 w.^en it is left in contact

line, with n nTr ?Pe it becomes definitely alka-

linity is nof 0 about 8.0. This initial alka-

ciliate. Snnr.anr?^ec^on to cultivation of the ^uid part of th

*s inoculated the

acid, owing f medium invariably becomes

bacteria in +k? r Presence of acid-producing

bacteria i<? n^ 80ces- If the growth of these a little acrifln -now checked by the addition of ttiav be r^ir,aJlne, a rich culture of balantidium At the?? -Very 1uickly-

f?r a week unnin^. subcultures were made daily tidium W)<! ^ ̂ kich time the strain of balan- cultures wprWe established. After this sub- this wav 1 finm^die. only every third day. In coli of the m Yr *n? culture of Balantidium and utilizer] ?n^ey was maintained for 44 days, In cuS^r ,exPerimental work.

by binarv t multiplication of the ciliate axis of the ura?sverse fission in the transverse the body of tl ^as ver^ frequently observed, shape. This ciliate assuming an hour-glass Photomicrofrr P^0cess illustrated in plate VII, tl?n occurred +jS 7 and 8* Occasionally encysta- tinuing to r 1

ci^ate within the cyst con- as illustnfp^V- Ve sI?wly within the cyst wall, Some of th

ln photomicrograph 9.

granules in tf -encysted ciliates showed starch

ty been in l61fr cytopIasm; these had apparent- trophozoifp 5? ** ^ the ciliate in its motile

Prior to eno ^+aS-6' ^ut bad not been extruded

with anv r a^9n* Encystation did not occur investigqfp

U ai'ity, and we were not able to Jameson flQ97\^?.rs wl"ch induced it to occur, tidium of whilst working with the balan- in vitro PlgS never encountered encystation p

cess is shnn?n-Was observed once only; the pro- ?from frpc]11 111 P^ate VII, photomicrograph 10 VaPour ] Jnateria 1 fixed with osmic acid

Although th keystation was occurring. a very tlii 1? c^s^s ?f Balantidium coli possess

culturp K f"5? Wa^ the cysts do not survive Though' ? ?e.nerate within 24 hours.

?f the pU jfCOnj u^ation was a prominent feature seen bv T Balantidium coli of pigs, as

?ccurrenpo aineson> it was of rather infrequent tidium Af11 ?Ul cult"res of the monkev balan- Rhowing a vp?,n0itinie we had one culture tube eilcystinff of ^ar?G number of cysts, but this failiirp +n Lr?ln Was unfortunately lost through In order ?me,y transplant.

exPerimental ain cysts in large numbers for encvHfqt;n Purposes, the method of inducting 1

,0n of Entamoeba histolytica devised by |

Dobell (1928) was followed. Monkey's stools containing the ciliate were inoculated into rice- free medium, with the result that blastocysts commenced to overgrow the ciliates. At this moment transplantation into medium containing rice starch greatly improved the growth of balan- tidium, but no great degree of success was

achieved in inducing encystation. Although a

very flourishing culture strain of the monkey balantidium was maintained for over six weeks, the ciliate only encysted in vitro on five occa- sions.

Attempts to inject human beings with the Balantidium of monkeys

Dobell (1931) has proved that the entamoeba of macaques, resembling Entamoeba histolytica of man, is identical with the latter parasite. In a later paper, Dobell (1933) has shown that the amoeba of genus Endolimax of macaques is

morphologically and culturally identical with

Endolimax nana of man; that man can be para- sitized with the Endolimax nana of macaques, and macaques with the Endolimax nana of man; the two are therefore identical.

It thus appears that possibly the other intes- tinal protozoa of macaques may be transmis- sible to man. Walker (1913) has shown that

monkeys when fed or injected per rectum with Balantidium coli of man become infected. It thus seems possible that the balantidium of

macaques and man are interchangeable, and we decided to investigate this possibility. The stools of a human volunteer were care-

fully examined daily for a period of five weeks. At no time was any balantidium found, and the only protozoon present was Entamoeba coli. This volunteer was now fed on cultures of Balantidium coli from Silenus rhesus showing numerous cysts?the culture medium containing rice starch, and being administered in gelatine capsules. This attempt ended in failure; the volunteer has now been under observation for two months since the feed, but has not showed balantidium at any time.

It was next decided to feed a second volunteer on cysts discharged in the monkey's faeces. This volunteer's stools were examined microscopically and culturally daily for twelve days before the experiment was commenced; at no time were

any protozoa encountered,?the only parasites present being ova of Ascaris lumbricoides and

Blastocysts hominis. About two grammes of a freshly-passed stool were collected in a Petri dish from a Silenus rhesus. On thorough examination they showed numerous cysts of the monkey balantidium and more scanty cysts of Entamoeba histolytica of the monkey. The stool was emulsified in normal saline, filtered through glass wool, centrifuged, and the centri- fuged deposit placed in the cool incubator at

22?C. for 24 hours. On very careful examina- tion of this deposit after it had been cooled, numerous cysts of Balantidium coli were found,

392 THE INDIAN MEDICAL GAZETTE [July, 1934

and a fair number of cysts of both Entamoeba coli and Entamoeba histolytica (of the monkey). The centrifuged deposit was now swallowed by the volunteer. The volunteer's stools were now examined

daily. On the eighth day after the -infective feed he had loose stools with some abdominal discomfort. Examination of the stools showed a fairly large number of both motile trophozoite forms and cysts of Entamoeba histolytica, with some mucus but no blood or cellular exudate. The diarrhoea persisted for one day, but then cleared up without any treatment. This volun- teer has now been under observation daily for two months after the infective feed. No balan- tidium has ever been seen, but he continues to

pass Entamoeba histolytica, chiefly in the

encysted stage, in varying numbers. The strain of Entamoeba histolytica is a small one, with

cysts down to 6 to 7 microns in diameter. On one occasion he has also shown cysts of Enta- moeba coli?apparently also of monkey origin. Although both experiments failed in their

original intention, the transmission of infection with Entamoeba histolytica from a macaque to man is of interest as affording further proof that this parasite is identical in both the

macaque and man. The second volunteer is still under daily observation, and, if possible, it is intended to keep him off all treatment and study the course of this monkej'- infection in a human host.

Summary

1. The medium advocated by Barret and

Yarbrough (1921) proved quite unsuitable in

our hands for the cultivation of Balantidium coli of macaques.

2. The ' HSre -f- S' medium devised by Dobell and Laidlaw (1920) for cultivation of the

entozoic amcebse of macaques and man proved an excellent one in our hands for the cultiva- tion of Balantidium coli of macaques, provided that a small quantity of acriflavine was added to

keep down the growth of starch-splitting bacteria and blastocystis.

3. Division, encystation, excystation and con- jugation of the ciliate were observed at the height of the growth.

4. Two attempts to infect human volunteers with cvsts of Balantidium coli of Silenus rhesus, one made with cysts in culture, and the other with cysts in freshly-passed faeces, both failed.

5. In the second instance, however, the Silenus rhesus was also infected with Entamoeba histolytica, and this infection was transmitted from the monkey to man and has now persisted in the human volunteer for a period of two months.

References

Barret, H. P., and Yarbrough, N. (1921). A Method for the Cultivation of Balantidium coli. Amer. Joum. Trop. Med., Vol. I, p. 161.

Dobell, C. (1928). Researches on the Intestinal Protozoa of Man and Monkeys. Parasitology, Vol. XX, p. 357.

Dobell, C. (1931). Researches on the Intestinal Protozoa of Man and Monkeys. Vol. IV. An Experi- mental Study of the Histolytica?like Species of Entamoeba living naturally in Macaques. Parasitology, Vol. XXIII, p. 1.

Dobell, C. (1933). Researches on the Intestinal Protozoa of Man and Monkeys. Vol. V. The Endolimax of Macaques. Parasitology, Vol. XXV, p. 436.

Dobell, C.. and Laidlaw. P. P. (1926). On the Culti- vation of Entamoeba histolytica and Some Other Enfozoic Amoeba. Parasitology, Vol. XVIII, p. 283.

Jameson, A. P. (1927). The Behaviour of Balantidium coli Malm, in Cultures. Parasitology, Vol. XIX, p. 411. Walker, E. L. (1913). Experimental Balantidiasis.

Philippine Joum. Sci., Vol. VIII, p. 333.