422

As the results in table 11 show, there was gross crenationof red blood-cells with all the other solutions tested but only10 g. per 100 ml. dextrose produced agglutination and thiswas only microscopic in degree.

Discussion

Red-blood-cell agglutination was the most striking andsignificant finding. It could happen easily in a drip set,if mixing of blood and mannitol was allowed, or in veinsinto which mannitol was infused rapidly. The danger ofsuch agglutinates lodging in branches of the pulmonaryartery is obvious and has been recorded as a complicationof cardioangiography with hypertonic agents (Read 1959).It follows therefore that hypertonic solutions of mannitolshould never be mixed with blood in a transfusion set andthat intravenous infusions should always be administeredat a carefully controlled slow rate.

Red-blood-cell crenation, by means of an associated fallin hxmatocrit, has been considered beneficial to the actionof mannitol (Lilien et al. 1963) but this has been refutedby Lucas and Read 1966. Although we found that manni-tol did not affect the mechanical and saline-osmotic-

fragility tests there seems little doubt that it permanentlydamages red blood-cells, for normal morphology couldnot be restored despite washing in saline and suspen-sion in serum.

Preliminary Communications

THE BIOLOGICAL PURPOSE OF THE

BLOOD-CYCLE OF THE MALARIA PARASITE

PLASMODIUM CYNOMOLGI

MALARIA parasites in the blood have a clearly definedcycle of development which is a multiple of 24 hours;and all parasites come to schizogony at approximatelythe same time every 24, 48, or 72 hours according to thespecies. Clinically, the synchronous division of the

parasites produces the well-known tertian or quartanparoxysms of fever which begin about midday. The

Fig. 1—The 48-hour variation in mosquito infectivity of the bloodof monkey 334, infected with P. cynomolgi.The figures for oocysts represent the mean for all mosquitoes in

each batch. The arrows below the base-line indicate the approximatetime of schizogony.

Our results were obtained with 10 g. per 100 mi.mannitol and the precautions suggested are even moreimportant if 25 g. per 100 ml. solutions are used.

SummaryMannitol can cause agglutination and irreversible crena.

tion of red blood-cells. The dangers of mixing mannîtû:and blood in a transfusion set are emphasised.We thank Dr. F. M. Parsons, consultant in clinical renal physiology

the General Infirmary at Leeds, for suggesting this work and for hconstructive criticism, and -Miss Marion Burnley for her technicalassistance.

Requests for reprints should be addressed to Dr. B. E. Rober;Department of Pathology, the General Infirmary, Great Geor;:Street, Leeds 1.

REFERENCES

Barry, K. G., Malloy, J. P. (1962) J. Am. Med. Ass. 179, 510.Boba, A., Gainor, J., Powers, S. R. Jr. (1962) Surgery, St. Louis, 52, 188Dacie, J. V., Lewis, S. M. (1963) Practical Hæmatology. London.Dawson, J. L. (1965) Br. med. J. i, 82.Lilien, O. M., Jones, S. G., Mueller, C. B. (1963) Surgery Gynec Obstet

117, 221.Lucas, C. E., Read, R. C. (1966) Surgery, St. Louis, 59, 408.Luck, R. J., Irvine, W. T. (1965) Lancet, ii, 409.Luke, R. G., Linton, A. L., Briggs, J. D., Kennedy, A. C. (1965) ibid.

980.

Powers, S. R., Jr., Boba, A., Hostnik, W., Stein, A. (1964) Surgery, St. Louis.55, 15.

Read, R. C. (1959) J. thorac. cardiovasc. Surg. 38, 685.

biological reason for this cycle has hitherto been obscure,The purpose of the present article is to show that the

cycle is designed to facilitate insect-vector transmissionby timing the formation of new gametocytes so that theyreached the short-lived peak of infectivity just at the timeof night when mosquitoes are most likely to suck blood,

METHODS

The malaria parasite used was the Langur M69 strain ofPlasmodium cynomolgi isolated by Prof. A. S. Dissanaike and

provided by Prof. P. C. C. Gamham. This produces an

infection of moderate intensity (maximum parasitaemia approxi-mately 10% of red blood-cells infected) in rhesus monkeys.The asexual cycle is 48 hours, and in our laboratory schizogon.vhappens at about 11 A.M. and 3 P.M., Greenwich Mean Time,The mosquitoes were a strain of Anopheles stephensi, establishedfrom eggs kindly provided by Mr. P. G. Shute and Mr, R,Killick-Kendrick. The experiments consisted of feeding a

new batch of mosquitoes on an infected monkey every 4 hoursMosquitoes which had fed were separated from the others:

they were dissected 6-7 days later, and the number of oocvscon the wall of the stomach were counted. The number e’

700

I I HOURS

Fig. 2-Mosquito infectivity of the blood of monkey 33b.

423

cocysts per mosquito, and the percentage of mosquitoes:nfected, were taken as measurements of the infectivity of thegametocytes at the time the mosquitoes fed. Thin blood---Ilms were also taken at 4-hour intervals, and the numbers ofsexual parasites and of gametocytes were counted by theasual methods.

RESULTS

The results of two experiments are shown in figs. 1

and 2 in table i. In the first experiment, monkey 334 wasinoculated intravenously on June 3, with 1-2 ml. lightlyinfected blood from monkey 332. Scanty parasites werefirst found in the blood on June 6. Feeding of mosquitoesbegan at 8 A.M. on June 8, the 5th day after inoculation.When these mosquitoes were dissected 7 days later it

was found that none of the mosquitoes which had fed at8 A.M., noon, or 4 P.M. had become infected. After that,the infectivity (as measured by the mean number ofobcysts for all mosquitoes) rose to a plateau from 8 P.M.to 4 A.M. (maximum at 4 A.M.), was low from 8 A.M. to4 P.M. (minimum at 4 P.M.), rose high again from 8 P.M.to 8 A.M. (with a peak at midnight), and fell to a low levelat noon (after which exhaustion of mosquito suppliesprevented further observations). During this period the

TABLE I-VARIATIONS IN THE INFECTIVITY OF P. cynomolgi WHENMOSQUITOES WERE FED 4-HOURLY ON MONKEYS 334 AND 336

:.’h monkey 334, 20 mosquitoes were dissected in each batch; with- 336, 10 mosquitoes were dissected in each batch except on day 4,

noon (20 mosquitoes), and on day 5, 8 A.M. and day 7, 4 A.M.’ .

Tne approximate time of schizogony.

gametocytes had only just begun to appear in the bloodand their number was irregular and low, bearing norelation to the cycle of infectivity just described. The

period of these feeds occupied the time from one

schizogony of the asexual parasites to the next.In the second experiment, monkey 336 was inoculated

intravenously on July 8 with 1 ml. blood containing 1

parasite per field. 3 days later, the blood at 8 A.M. con-tained 1 parasite in two fields. Feeding of mosquitoesbegan at 8 A.M. on July 12 the 4th day after inoculation.Again the infectivity was very low from 8 A.M. to noonon day 4. Then it rose rapidly, reaching a peak at 4 A.M.followed by a small depression (minimum at noon),after which it rose again to a higher peak at midnight andfell to a low figure at noon on the 3rd day; the next feedat 4 P.M. was omitted owing to lack of mosquitoes butthe next four feeds during the night showed a high andincreasing level of infectivity. (These high numbers ofoocysts [more than 500 per mosquito] were too large tobe counted accurately). The number of gametocytes inthe blood was about ten times higher than that in thefirst experiment; it remained approximately level until8 A.M. on the 3rd day and it was not parallel to the curveof infectivity; later it rose steeply as shown. The periodof feedings occupied the period from one schizogony ofthe asexual forms to the next, and part of the subsequentcycle. Crisis of the asexual forms had not started duringthe period of feeding.

DISCUSSION

Figs. 1 and 2 show that the infectivity of the bloodfollows a 48-hour cycle corresponding to the time of theasexual cycle. This cycle of infectivity is not due to a

cycle of the number of gametocytes in the blood, butmust be due to variation in their physiological state-i.e.,their suitability to develop in mosquitoes. Presumablythe gametocytes originate as small forms at schizogonyof . the asexual parasites. They spend approximately60 hours in growing and becoming mature (i.e., able todevelop in mosquitoes) and this state of maturityapparently persists (possibly with variations) for approxi-mately 30 hours, after which it diminishes. Thus the

gametocytes originating from one schizogony reach peakinfectivity for mosquitoes during the period 60-90 hourslater; and we suggest that the synchronisation of thecycle of the asexual forms (so that all individuals cometo schizogony at approximately the same hour of the day)is designed so that the subsequent period of high infec-tivity of the gametocytes will coincide with the hours atwhich the mosquitoes usually suck blood. In this way,the chance of transmission and of further multiplicationof the parasite would be greatest.

VARIATIONS OF INFECTIVITY DURING INFECTION

During this work, preliminary investigations were alsomade on day-to-day variations in the infectivity duringthe early course of the infection. For this purpose,batches of mosquitoes were fed on monkey 332 eachmorning at 8 A.M. starting from day 3 after the intravenousinjection of blood (day 0). The infectivity (as judged bythe mean number of oocysts), and the numbers ofasexual parasites and of gametocytes are shown in fig. 3

and table 11. (All these figures refer to 8 A.M., at whichhour infectivity is low relative to night time). Infectivitybegan to rise on day 6 and reached a peak on day 9,during which period the number of gametocytes alsorose to a maximum but more slowly at first. On day 10,infectivity had fallen again almost to zero, although the

424

Fig. 3-The day-by-day variation in mosquito infectivity of theblood of monkey 332.

number of gametocytes in the blood was still high (andthe asexual parasites soon began to fall in number,presumably as the result of a crisis produced by themonkey’s immune response). Probably this immune

response adversely affected the gametocytes besides theasexual forms; thus the gametocytes were not able todevelop in the mosquitoes although they were still visiblein the blood. On day 14, there is a striking contrastbetween the high number of gametocytes which haveappeared in the blood and their complete failure to

develop in mosquitoes. Subsequent observations on thecourse of this infection are incomplete owing to shortageof mosquitoes. Apparently, between days 14 and 18inclusive there were many gametocytes in the blood, andon day 19 and subsequent days these diminished rapidlyin number. The parasitaemia of asexual forms was

relatively low during this later period. Of the mosquitoesTABLE II-VARIATIONS IN THE INFECTIVITY OF PI cynomolgi WHEN

MOSQUITOES WERE FED DAILY ON MONKEY 332

Mosquito data are derived from 10 dissections per group except in thosemarked * when 20 dissections were made.

S = Day of principal schizogony, which took place after the day’s obser-vations had been made. There was also a minor brood with someschizogony on the alternate days.

which fed on days 18 and 19 at 8 A.M., only half developedoocysts; this contrasts with 100% infected on days ? and 8, when gametocytes were few; and contrasts wit.

no mosquitoes infected during the crisis period, whenvisible gametocytes were numerous. The ratio of oöcystsper mosquito to gametocytes in blood was especially loion day 18. The small number of mosquitoes infected ondays 18 and 19 was presumably due to inhibition of thegametocytes by immune bodies. The fact that a few of ithe gametocytes were still able to develop in mosquitoes.in spite of the immune bodies now accumulated in the imonkey’s plasma, may indicate that an antigenic variatiochad taken place in a few of the gametocytes, which migc:thus enable them to escape the inhibitory action of theimmune bodies elaborated against the initial type cmalaria parasite.

SUMMARY

During infections of Plasmodium cynomolgi in rhesusmonkeys, the infectivity of the gametocytes for mos-

quitoes (Anopheles stephensi) follows a 48-hour cycle,with a peak of infectivity about midnight, 84 hours afterthe schizogony at which they had been formed. The

cycle of asexual parasites seems to be arranged so thatthis phase of maximal infectivity of the sexual forms willhappen at the time when the insect vector normallysucks blood (i.e., night time). This seems to be the

biological purpose of the synchronisation of the develop-ment of individual asexual parasites which constitutesthe cycle.The infectivity of the gametocytes also follows a

day-to-day variation which is not proportional to thenumber of gametocytes in the blood. It is relatively highin the earliest days of parasitaemia and falls abruptlywhen the crisis of asexual forms begins, presumably dueto inhibition by developing antibodies. Later in the

infection the number of gametocytes in the blood may be

high, and infectivity may be present although relativelyless high than at the start; the presence of infectivity, in

spite of a high level -of antibodies, may indicate that

variation has occurred in the antigens of some (but notall) of the gametocytes.

F. HAWKINGD.M. Oxon., F.R.C.P.

M. J. WORMSB.SC. Lond.

K. GAMMAGE

P. A. GODDARDNational Institute for Medical Research,

Mill Hill, London N.W.7

QUINTUPLE VACCINEIN one of a number of small trials which we had made

recently with inactivated-measles-virus vaccines, vira

haemagglutinin obtained by Tween ’-ether treatment ofthe virus 1 was blended with a standard quadruple vaccine(poliomyelitis, diphtheria, pertussis, and tetanus) to makea quintuple vaccine. It has been recommended thatquadruple vaccine be given in a course of three doses Lv

intervals of 6 weeks and 6 months, the first dose beinggiven at 6 months of age,2 but because of doubts aboutthe potency of the measles component of the quintuplevaccine a four-dose schedule was used in this trial. Three1 ml. intramuscular doses were given at monthly inter-’1. Norrby, E., Lagercrantz, R., Gard, S., Carlström, G. Acta

Stockh. 1965, 54, 581.2. Dane, D. S., Dick, G. W. A., Haire, M., Briggs, E. M., Conn

J. Hyg., Camb. (in the press).