Demonstrations

(The previous number of these Transactions, Vol. 50, No. 6, was published on 30th November, 1956).

LABORATORY MEETING of t h e ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE h e l d at t h e Liverpool School of Tropical M e d i c i n e , on Thursday, 15th N o v e m b e r , 1956, a t 7.30 p . m .

The President, PROFESSOR R. M . GORDON, O.B.E., M.D., D.SC., F.R.C.P., in the Chair

DEMONSTRATIONS LIVERPOOL SCHOOL OF TROPICAL MEDICINE DEPARTMENT OF TROPICAL ~V~EDICINE

Dr. D. C. Torpy and Professor B. G. Maegraith : Entamoeba histolytica lesions in guinea-pig caecum. 1. Extension of lesion from surface. 2. Technique (cine film). (1) T h e demonstration illustrated the various stages of penetration of the caecal wall by Entamoeba histolytica. T h e r e is an initial superficial colonization of the entamoebae, followed by penetration and destruction of the epithelium and deep colonization, superficial to the muscularis mucosae. A t a later stage the muscularis mucosae is penetrated and the entamoebae spread into the submucous tissues. Serial section of distant lymphoid follicles has revealed infection with the entamoebae, suggesting that there may be a spread along lymphatic channels. Evidence of enzymatic activity was demonstrated by histological techniques. (2) A 16-ram. colour cinematograph film was shown, illustrating intracaecal inoculation of E. histolytiea, and showing some examples of active E. histolytica in scrapings from the lesions so produced.

Dr. C. S. and Mrs. L. A. Leithead : Comfort and efficiency in a hot dry climate. Physiological and medical studies. (In collaboration with Kuwait Oil Company,) Research into comfort, health and efficiency in a hot dry climate was carried out in the summer months of 1956 in Kuwait, in collaboration with the Kuwait Oil Company. T h e general plan of this research was outlined, along with actual or photographic presentation of some of the methods used. A large-scale map indicated the area of the survey, which included an inland desert region and a coastal port. Two graphs, each showing daily wet and dry bulb, and maximum and minimum shade readings, illustrated the difference in ambient temperature between the desert and the coast. T h e principal aspects of the research were : (1) T h e physiological processes of acclimatization to heat, with particular reference to circulatory and biochemical adjustments, and the over-all time factor involved. (2) Tropical fatigue and deterioration over the summer, in permanent residents. (3) Heat illness, as regards aetiology, clinical and biochemical data, and classification of cases. T h e findings of this research have yet to be published.

LABORATORY MEETING

Dr. T. D e e g a n and Dr. Kanjika Devakul : Fate of radioactive haemoglobin injected intravenously into Macaca mulatta. T h e fate of extracellular haemoglobin has been of interest in connection with the production and metabolism of methaemalbumin. DUESBERO(1934) failed to obtain proof of the presence of methaemalbumin after injection of 5 g. of haemoglobin into the plasma of normal individuals, but FAIRLEY (1938) working with higher concentrations of haemoglobin, 15-20 g., demonstrated its presence 6 to 12 hours after injection. DRABKIN (1935), working with dogs, showed that the renal threshold for haemoglobin was approximately 130 rag. per 100 ml. of plasma, and demonstrated that the rate of removal of the pigment from the plasma could not be explained by " urinary spillage." In the work reported, the fate of Fe59-1abelled haemoglobin injected at a concentration slightly below Drabkin's value for the renal threshold, viz. at 110 mg. per 100 ml., was followed in the plasma and in the red cells of M. mulatta monkeys. T h e results showed the rapid removal of the introduced pigment from the blood stream - - after 8 hours only a trace of the pigment remained in the p l a s m a - and the reappearance of the isotope in the red cells of the monkeys, in increasing amounts, 10-20 days later. It appears that the introduced pigment is quickly metabolized and its iron fraction enters the metabolic pool where it is re-utilized in the production of fresh erythrocytes. R E F E R E N C E S : DRABKIN, D. L., WIDERMAN, A. H. and LANDOW, H. (1935). ft. biol. Chem., 109, 1935. DUESBERa, RICHARD (1934). Arch. exp. Path. Pharmak., 174, 305. FAIRLEY, N. H. (1938). Proc. R. Soc. Med., 32, 2, 1278. Dr. T. Deegan, Dr. H. M. Gilles and Dr. I. A. McGregor : Serum and plasma protein patterns of Gambian Africans. M c G a E a o a and DEEGAN (1954) recorded the prevalence of high erythrocyte sedimentation rates in Gambian African males and females, all of whom when examined were engaged in their normal daily duties. It was suggested that a reason for this increased sedimentation rate compared with the accepted values for normal Europeans was to be found in a different blood protein pattern. T h e results of two investigations of this postulation were shown. In the initial studies, carried out at Keneba and Jali, in the West Kiang district, where the investigation was restricted to normal male Africans as it was easier to obtain men than women, sera were analysed for total and differential protein by chemical methods and by paper electrophoresis ; the latter technique was also applied to plasmas. Preliminary results showed an increase in ~f-globulin content of both sera and plasmas in the case of African tissues compared with the corresponding factor in European tissues. A second investigation was undertaken at Fajara, near Bathurst, involving both men and women, in which the total and differential serum proteins and the y-globulin content were determined solely by chemical methods. F o r the purpose of the studies the normal African was considered as an individual living and conducting his normal daily existence in his natural environment, who was apyrexial a n d whose blood did not contain malaria parasites in high density. T h e results of the investigations revealed that, compared with the same factors from a control group of Europeans, members of the staff of the Medical Research Council Laboratories, the general serum protein characteristics of the African sera, viz. total protein level and albumin-globulin ratio, were within " normal " limits. However, the distribution of the globulin fractions was different ; in particular the y-globulin contributed a larger proportion in the African sera. Further, a larger fibrinogen content was detected in the plasmas of the African volunteers. R E F E R E N C E : McGm~aoa, I. A. and DE'~aAN, T . (1954). Ann. trop. Med. Parasit., 48, 220.

DEPARTMENT OF CLINICAL MEDICINE

Dr. D. R. Seaton : Treatment of tapeworm infection with Dichlorophen. Dichlorophen (Anthiphen, May and Baker) is 2 : 2'-dihydroxy-5 : 5' dichlorodiphenylmethane. It was introduced in 1946 for the treatment of taeniasis in dogs and cats. Unlike other taenifuges it has a lethal action upon the worm, which after being killed is more or less digested. T h e recommended dose is 0.5 gramme per 16-1b. body-weight, given all at once without any preliminary starvation or subsequent purgation. T h e patient can take the treatment without any interruption of

LABORATORY MEETING his ordinary activities. There have been no untoward symptoms attributable to the drug, though a number of patients have had slight colic and looseness of the bowels during the 12 hours after taking the dose. It is not possible to find the scolex or anterior segments of the worm in the stools after treatment, so the result cannot be known except by waiting for 3 months, when if segments have not reappeared a cure can be assumed. Of 14 cases of infection with Taenia saginata observed for 3 or more months after treatment nine have been cured and five have not, which means that, in my hands, the drug is less efficient than mepacrine but better than filix mas. DEPARTMENT OF ENTOMOLOGY AND PARASITOLOGY

P r o f e s s o r R. M . G o r d o n : The deposition by the tsetse fly of the infective forms of Trypanosoma rhodesiense, the subsequent migration to the general circulation and their d e v e l o p m e n t to the blood forms. P a r t . I.

( P r o f e s s o r G o r d o n , D r . W . C r e w e a n d D r . K . C. W i l l e t t ) : The m e t h o d of feeding adopted by the tsetse fly and its relation to the transm i s s i o n of trypanosomiasis.

A colour film was shown which illustrated the process of feeding adopted by the tsetse fly, as observed in living mammalian tissue ; the apparatus used in making the film and the various techniques employed to observe the exact site of deposition of the saliva in the tissues were also demonstrated. In addition, sections cut through the bitten area were exhibited ; these showed the nature of the lesions produced by the probing fascicle and the probable distribution of the saliva. As a result of these combined observations, the authors conclude that many of the metacyclic trypanosomes deposited with the saliva of an infective fly will be widely distributed in the host's tissues, but that it is probable that the majority will be deposited in the neighbourhood of previously torn capillaries. P a r t II.

( D r . K . C. W i l l e t t a n d P r o f e s s o r G o r d o n ) :

The

migration

of the

metacyclic

trypanosomes

from the site of their

inoculation. Sections cut through tissues which had been excised from the site of the infective bite were exhibited. Trypanosomes were never found in the bitten tissues so long as these were removed from the mammalian host after the fly had finished its bloodmeal. On the other hand, if the animal was killed and the tissues excised while the fly was still probing, but prior to its having taken up any blood, numerous trypanosomes could be demonstrated in the spaces between the connective tissue fibres and in the muscle layers. I n the latter circumstances, they were sometimes found at sites beyond the direct reach of the fascicle of the feeding insect.

Part III.

(Professor Gordon and Dr. Willett) : The d e v e l o p m e n t of the m e t a e y c l i c forms of T. rhodesiense to the blood forms. Stained smears made from cultures of the metacyclic forms of T. rhodesiense were exhibited.

These showed that at the end of 4 hours a proportion of the metacyclic trypanosomes had assumed forms morphologically indistinguishable from the slender forms with free flagella found in the blood after the establishment of the disease. At the end of the 24 hours a high proportion of the free flagella forms had undergone division resulting in a steady increase in the numbers in culture. If these culture forms were inoculated into a susceptible animal, they reproduced the disease.

P r o f e s s o r R. M . G o r d o n : P a r t i a l blocking of the proventriculus of Xenopsylla cheopis by Pasteurella pestis. Doctor M. Baltazard, Director of the Institut Pasteur de l'Iran, recently sent us several hundred fleas collected from wild rodents trapped during a plague survey in India. Serial sections were cut through some 120 of the fleas, but in only one was blocking of the proventriculus clearly visible. The original drawings by members of the Indian Plag~le Commission were shown for comparison. These drawings had been copied into many text-books but the work did not appear to have been commonly repeated.

4

LABORATORY MEETING

Dr. W. E. K e r s h a w : T h e population d y n a m i c s of Onchocerca volvulus in the v e c t o r , Simulium damnosum. Whilst it is not possible at present to build up a comprehensive mathematical model of the infection with Onchocerca volvulus, quantitative studies should not be deferred. T h e construction of mathematical models is of great value in experimentation and research, and a comparison of such models with nature (even though the models may be incomplete) reveals which assumptions should be rejected and which fnally be accepted in our concepts of epidemiology. T h e fate of populations of microfilariae after ingestion by flies feeding on infections of different intensity, can be followed by constructing frequency distribution curves of the numbers surviving in different stages of development. About one-tenth of the microfilariae ingested by those flies which survive long enough for infective forms to develop - - survive to the infective form ; and if about half the flies survive long enough to produce these forms, then about one-twentieth of the microfilariae originally ingested complete their development. (These findings will be published in full elsewhere.)

Dr. M. J. G. Furnell a n d Dr. W. E. K e r s h a w : An o u t b r e a k of t r i c h i n o s i s in m a n in L i m e r i c k , in 1956. I n September, 1956, an outbreak of human trichinosis occurred in Limerick, in which three cases were diagnosed, one of which proved fatal. It seemed likely that the infection had been acquired from a small butcher's shop. Except for an outbreak in 1944 amongst German prisoners of war, this infection had not previously been described in Ireland. In an investigation of the epidemiological background, rats from the City D u m p and from bacon factories, and cats and dogs, were examined as an index of circulating infection in the past. T e n of the 136 rats caught on the City Dump were infected. It was suspected that a cycle in wild animals might occur, and foxes, badgers and mice were examined, but none was found to be infected. T h e fact that the rats on the City Dump were infected means only that infected material had been deposited there. Investigations are proceeding to determine how the pigs were infected.

Dr. W. E. K e r s h a w and Mr. P. W i l l i a m s : T h e effect of filariasis on e x e r c i s e t o l e r a n c e in the cotton-rat. We have no accurate information concerning the effect of filariasis on the cotton-rat. T h e physical fitness, as assessed by exercise tolerance, has been compared with the weight of the cotton-rat, and the course of the infection. Fitness is inversely related to weight in an uninfected animal, and from preliminary experiments the course of the infection does not seem to influence this basic relation. Further tests of performance are being devised.

Dr. G. Crisp (introduced by Professor R. M. Gordon) : T h e distribution of Simulium damnosum in the Gold Coast. I n the Northern Territories, S. damnosum is present along stretches of all the main rivers. It also occurs in the tributaries and in streamlets which flow for only a few weeks in the year. Outside the Northern Territories and Togoland, the insect has a limited distribution. It has been found in a small area round Kintampo in Ashanti ; in the Gold Coast Colony it is confined to a stretch of the Volta river north east of Accra. T h e distribution of S. damnosum was examined in relation to climate and vegetation, physical features and geology. D r . M . M . J. L a v o i p i e r r e

:

S o m e e c t o p a r a s i t e s of Chrysops silacea the v e c t o r of Loa loa. During the course of studies carried out at the Liverpool School of Tropical Medicine on Chrysops silacea, a vector of Loa loa, several hundred female flies were examined, and four of these were found (1)

LABORATORY MEETING to harbour ectoparasites. I n the case of three flies, the parasites were the larval stage of a mite belonging to the family Erythraeidae and in the case of the fourth fly they were the planidial larvae of a hymenopterous insect. There appears to be no previous record in the literature of the occurrence of erythraeid mites on flies of the genus Chrysops although they have previously been collected from other tabanids. One of the mites was removed from its host and on subsequent examination proved to be an undescribed species of the genus Achorolophus. It had to be removed from the fly by dissecting away one of the abdominal sternal plates to which it was firmly fixed by its chelicerae ; it was observed that at the point of attachment of the mite to the fly, a stylostome, remarkably similar to those produced by trombiculid mites in mammalian tissues, was present. Planidial larvae have previously been reported as occurring on Simulium but never on Chrysops or other tabanid flies. T h e specimens demonstrated were found accidentally during the examination of the labella of a female C. silacea. Two planidia were found attached to the inner surface of the right labellum of the fly ; at the point of attachment of each of the larvae the labellum was seen to be damaged.

(2)

A n e w species of Chorioptes collected in the British Cameroons.

A new species of Chorioptes, the male and the female of which were demonstrated, was found in ear scrapings taken from a red-flanked duiker, Cephalophus rufilatus Gray, 1846, which was shot at the Bombe Rest House, near K u m b a in the British Cameroons on the 30th May, 1956. T h e new species is qtiite distinct from all the described species of the genus Chorioptes, particularly as regards the male sex. The male of the new species is readily distinguishable from the males of other species of Chorioptes, in having very much stouter first and second pairs of legs and in being provided with longer abdominal lobes, the setae of which are long and slender ; the female resembles those of other Chorioptes spp., and differs from them only in minor characters.

(3)

A note on the escape of the infective forms of L. loa from the head of C. silacea.

Although the emergence of worms from the feeding C. silacea has been observed, the precise site of emergence has never been seen. A study of the mouthparts of the fly has revealed several possible routes of escape. One such route is via the labella, a second possible route is by way of the individual elements of the biting fascicle of the mouthparts and the membranes which bind them to the head capsule, or thirdly, the worms may escape from the labio-hypopharyngeal membrane which connects the root of the labium with the underside of the pharynx. Sections of infected flies were demonstrated showing that although some of the worms may escape from the head of the feeding fly via the labella and the biting fascicle, most of them escape from the head by rupturing the labio-hypopharyngeal membrane. I n addition, sections of several infected and non-infected flies were demonstrated showing that the kinking of the labium as it is withdrawn into the head, and the displacement forwards towards the head of the fluid of the abdominal haemocoele and the gaseous contents of the abdominal air sacs, during feeding, probably increases the intracranial pressure of the fly. Tl~e writer believes that the increase in the intracranial pressure coupled with the active movements of the worms in the sub-cibarial haemocoelic space may bring about rupture of the delicate labio-hypopharyngeal membrane which is stretched as the labium is withdrawn into the head of the fly.

Dr. W. A. F. W e b b e r : An improved technique for the filtration of microfilariae from venous blood, and a m e t h o d of recovering the mierofilariae alive. Dr. G l a d y s R u t h e r f o r d ( D e m o n s t r a t e d b y P r o f e s s o r T. H. D a v e y ) : Methods of Health Education and Sanitation in rural India. Audio-visual methods of health education were demonstrated. These included the flarmelgraph, cartoon film strips and cards showing " stick " figures. Sanitary appliances included a bore-hole cutter, an enclosed well and a modified aqua-privy. These appliances can all be made by village artisans. The bore-hole cutter is made of sheet metal and rivetted to form a truncated cone, 6½ inches diameter at the bottom and 5½ inches at the top. T w o strips of heavy iron are rivetted to its sides and bolted to a handle. I n use it is driven into the ground, lifted out and the impacted soil removed. T h e enclosed well contains two buckets on a spindle. When the handle operating the spindle is turned, one bucket sinks and the other rises until it is seen, through a peep-hole, to lift and pass a

6

LABORATORY MEETING

trigger. T h e movement of the handle is then reversed and the trigger catches the bottom of the bucket and tips it to empty the water into a trough which discharges through the parapet of the well-top into the individual's water container. The parapet surrounding the well is covered over so that contamination cannot be introduced from the surface. T h e modified aqua-privy consists of a concrete latrine stance and basin discharging through an angled pottery pipe into the top of a large pottery container. Another pottery pipe, with its end some distance below the influent, discharges the contents from the container into an adjacent bore-hole. This appliance can be produced and installed at a cost of 12 rupees. It causes no nuisance and under ordinary family conditions has a life of about 2 years. It meets the requirements of a rural Indian family and has proved popular.

LONDON

SCHOOL

OF HYGIENE

AND

TROPICAL

MEDICINE

DEPARTMENT OF ENTOMOLOGY

Dr. D. S. B e r t r a m : Illustrations of places where day biting by ceratopogonids occurs in the Gambia. Photographs showed a tree-platform where Culicoides nigeriae bit human bait before sunset, besides a palm and another tree at the roadside where, respectively, Dasyhelea sp. and Forcipomyia sp. also bit man at ground level in the afternoon before 4 p.m. DEPARTMENT OF PARASITOLOGY

P r o f e s s o r P. C. C. G a r n h a m a n d Dr. R. L a i n s o n : Anopheles aztecus as a highly efficient vector of malaria parasites. Anopheles aztecus, indigenous to the Mexican highlands, is now established as a colony at the London School of Hygiene and Tropical Medicine. This species has been Shown by Mexican workers to be a suitable vector of Plasmodium berghei, while Burgess in the U.S.A. has found that it will transmit well human malaria. We tested A. aztecus for its power to transmit (a) Plasmodium cynomolgi and (b) Plasmodium knowlesi. When mosquitoes of this species were fed on monkeys infected with P. cynomolgi, practically 100 per cent. became infected with very heavy gut infections. Anopheles stephensi, fed at the same time, showed a much lower rate of infection. A' aztecus fed on monkeys infected with P. knowlesi became heavily infected with this malaria parasite with enormous numbers of oocysts in the mid gut and numerous sporozoites in the salivary glands.

Dr. R. L a i n s o n a n d P r o f e s s o r P. C. C. G a r n h a m : Stages of Plistophora culicis encountered during dissections of Anopheles stephensi. Plistophora culicis is a microsporidian infecting different species of mosquitoes, causing enormous accumulations of parasites in the body cavity of the insects. T h e intensity of the infections appears to interfere with the development of malaria parasites in the mid gut. T h e organism superficially might be mistaken for oocysts of malaria parasites by someone not well acquainted with such forms. P. culicis apparently occurs in Anopheles gambiae in West Africa, and this is the presumed source of the infection in our colony of Anopheles stephensi.

P r o f e s s o r P. C. C. G a r n h a m , Dr. R. L a i n s o n a n d Dr. A. E. G u n d e r s : Plasmodium rdchenowi : o c c u r r e n c e of developing crescents and schizonts after splenectomy of the host. A chimpanzee sent to us from Liberia by Dr. Avery-Jones in 1954 was found to have on arrival infections of Plasmodium schwetzi and Plasmodium reichenowi. T h e animal was treated with primaquine and quinine in 1954 when the parasites disappeared. Two years later, a splenectomy was performed on the animal and P. reichenowi returned to the circulation, presenting aS schizonts and later as developing gametocytes.

LABORATORY MEETING

7

Professor P. C. C. G a r n h a m , Dr. R. L a i n s o n a n d Mr. W. Cooper : Pre-erythroeytie stages of Plasmodium hnowlesi. A sfrain of Plasmodium hnowlesi recently isolated by D r . Hawking from a Macaca irus sent from Malaya was established in our laboratory and passed through Anopheles stephensi three times to further monkeys. On the last occasion, A. stephensi and Anopheles aztecus were allowed to feed on the monkey while gametocytes were present in the blood. Both species of mosquitoes became infected and on the 12th day guts containing ripe oocysts were lightly ground in Locke's serum solution and the sporozoites were inoculated intravenously into a rhesus monkey. Five days later, a liver biopsy was performed and, after a very lengthy search, a pre-erythrocytic schizont was found. The following day (the 6th day) rings appeared in the blood and the monkey was kiUed. Sections of liver of the 6th day showed loci consisting of phagocytes around merozoites of the bursting schizonts. A full description of these stages will be published later.

Mr. B. D a s g u p t a : Cytoehemistry of the basophilie material in the ooeysts of Plasmodium gaUinaceum. T h e oocysts of Plasmodium gaUinaceum stained with certain standard eytochemical techniques have been demonstrated. With toluidene blue the basophilic material in the oocysts stained orthochromatically and also metachromatically. The basophilic material stained red with pyronin in the Unna-Pappenheim stain. One hour incubation with ribonuclease at 37 ° C removed the orthochromatic stain, while the metachromasia persisted in some oocysts. Similarly, the affinity for pyronin also persisted in the metachromatic areas. I n control sections incubated in distilled water, no reduction i n staining properties was noticed. Perchloric acid extraction completely eliminated the orthochromatic stain, the metaehromasia, and also the affinity for pyronin. T h e usual sites of metachromasia were found to be devoid of Feulgen-positive reaction. Using the alcian blue method, granules staining blue-green were noticed in the cytoplasm of some oocysts. Negative result was obtained with periodic acid/Schiff reaction. It is concluded that the orthochromatic part of the basophilic material in the oocysts of P. gallinaceum represented ribonucleic acid. It is difficult to come to a conclusion about the significance of metachromasia in these oocysts. The evidence available at present suggests that the metachromatically stained part of the basophilic material may represent ribonucleic acid or mucopolysaccharide or both. MISCELLANEOUS

Dr. P. E. Clinton M a n s o n - B a h r : Coloured slides of (1) Dyssebacia pellagra and gynaecomastia in the same patient, (2) depigmented lesions of post kala-azar dermal leishmanoid in Kenya. (1) Signs of malnutrition and excess of oestrin in the same male patient. (2) T h e depigmented lesions of post kala-azar dermal leishmanoid found chiefly on the shoulders, upper part of the back and limbs.

Dr. C. P o l g e and Dr. M. A. Soltys : Preservations of trypanosomes in the frozen state. This communication deals with a method of preserving trypanosomes which may render many practical advantages in storing trypanosomes in the frozen state, as compared with more conventional methods of maintenance in animals. I n addition, studies on drag resistant strains and on antigenic structure of trypanosomes can be carried out without previous difficulties. Method. T h e organisms are collected form anaesthetized mice by bleeding direct from the carotid artery at the peak of infection when there is a high concentration of trypanosomes in the blood. Each mouse is bled into 8 ml. of Alsever's solution containing 10 per cent. horse serum. Since citrate is present in the Alsever's solution, the blood does not d o t and the tubes containing the diluted trypanosomes are centrifuged at 500 to 1,000 r.p.m, for 2 to 3 minutes in order to speed the sedimentation of the red cells which have been agglutinated by the horse serum. A clear suspension containing the trypanosomes is then transferred in the proportion of 1 : 1 to Alsever/serum containing l0 per cent. glycerol. After dilution, 0.5 ml. quantities are put into glass test tubes 8 cm. x 1 cm., which are then sealed in a special gas burner and the preparations are ready for freezing.

LABORATORY MEETING

The sealed tubes are then placed in a vacuum jar filled with absolute achohol at room temperature. The alcohol is then cooled slowly by adding small pieces of solid CO2. T h e rate of cooling is controlled to about 2 degrees per minute from room temperature to - 30 ° C, and to about 5 degrees per minute from - 30 ° C to 79 ° C. After reaching - 79 ° C, additional pieces of solid CO2 are put into the jar which is stored in a insulated chest with blocks of solid CO2. T h e results of the experiment on the preservation of trypanosomes reported elsewhere showed that all examined strains of trypanosomes were still motile, pathogenic and maintained all their biological characteristics after 10 months of storage.

Dr. D. J. Lewis : Phletobomus lesleyae, an interesting African sandfly. Phlebotomus (Phlebotomus) lesleyae Lewis and Kirk is only known to occur in the Republic of the Sudan where it was very seldom seen till 1953. In that year many cases of kala-azar were reported in the Sennar area, and P. lesleyae was found to be abundant there although it had not been seen in previous collections from Sennar. Nothing is known of its habits but it merits some further study in relation to kala-azar. P. lesleyae shows some resemblance to P. argentipes Annandale and Brunetfi, the Indian vector of the disease ; it is known from several infected areas in the Sudan ; and it appears to occur at Sennar in the absence of P. orientalis Parrot which is probably a vector in the Sudan. Males of P. lesleyae and P. argentipes were exhibited to show points of similarity between them.

Dr. Elizabeth U. Canning: Plistophora culicis W e i s e r (Protozoa, Microsporidia) : its d e v e l o p m e n t in Anopheles gambiae. Plistophora culids, a parasite of the Malphigian tubules, was originally described from Culex pipiens in Czechslovakia (WEISER, 1946). At the London School of Hygiene and Tropical Medicine it was found infecting colonies of mosquitoes in which it was primarily parasitic in the Malphigian tubules but capable of infecting other tissues including the fat body. The mosquitoes affected were

Anopheles gambiae and A. stephensi. T h e infection begins With the emergence of the sporoplasm from the spore probably under the influence of digestive juices of the larval mosquito. The uninucleate sporoplasm develops by nuclear division into a schizont with not more than 16 nuclei, usually less. T h e merozoites are uninucleate, spherical bodies, measuring 3-3.5~ diameter with an area of clear cytoplasm round the nucleus. These may develop into further schizonts or into sporonts. T h e sporonts are of two types : (1) Large sporonts, often oval, measuring up to 25 x 26.5~ and having as many as 100 closely packed nuclei. These give rise to sporoblasts mad finally to microspores measuring 3.7 x 2.2~ (fresh). (2) Small sporonts, usually spherical, measuring from 10-15~t diameter and containing from 8-30 nuclei. These give rise to sporoblasts and finally to macrospores measuring 5 x 3.5~t (fresh). T h e sporoblast is uninucleate with the nucleus elongate and occupying a position at one pole of the spore. During the development of the spore a large vacuole appears at the pole away from the nucleus and a metachromatic granule appears in the vacuole. T h e granule increases in size while the nucleus undergoes division. Later a second vacuole appears in the spore causing the cytoplasm to move to the centre of the spore where the nuclei undergo autogamy. Meanwhile some of the cytoplasm has been used up in the development of the polar filament, which at maturity lies coiled within the vacuoles. In the adult mosquito the ceils of the Malphigian tubules are distended and brittle owing to the complete replacement of the cytoplasm and nuclei by parasitic spores. Spores are presumed to be liberated by passing into the gut and out with the faeces or simply by death and disintegration of the host. On ingestion by a new host, the spore hatches by shooting out the polar filament dragging with it the sporoplasm which is attached to the tip. Extrusion of the polar filament can be effected by mechanical pressure under a coverslip. T h e demonstration slides of the parasite in _4. gambiae showed : (1) Mature sehizont and group of merozoites (Giemsa stained). (2) Microsporous sporont and macrosporous sporont and numerous cysts of microspores at various stages of development (Giemsa stained). (3) Spores with filaments extruded (Giemsa stained). (4) Section of whole adult A. gambiae showing Malphigian tubule heavily infected (Giemsa/colophonium). R E F E R E N C E : WEISER, J. (1946). Vestn. csl. zool. Spol., 10, 1.

LABORATORY MEETING

Dr. W. L. N i c h o l a s a n d Dr. H y n e s : The life-cycle of Polymorphus minutus ( A c a n t h o c e p h a l a ) , a parasite of the duck and other birds. Polymorphus minutus (Goeze, 1782) ( = P. boschadis Schrank , 1788) is an intestinal parasite of various water birds including the domestic duck, which may occasionally be killed by the parasite. Its life-cycle, including Gammarus pulex (L.), the freshwater shrimp as an intermediate host, has been known in outline since the work of GRE~FF (1864). Recently, one of us (HYNES, 1955), has found that all three of the native species of Gammarus may act as intermediate hosts, but our preliminary experiments suggest that strains of the parasite occur which differ in their infectiveness for the three species of shrimp. We have also found that shrimps, of the appropriate species, are readily infected by feeding them with gravid female worms taken from the domestic duck, but we have failed to infect them with eggs freed from the female worm. We suspect that in nature, shrimps normally become infected by feeding on the female worms which are defaecated while distended with eggs. Infections among shrimps are widespread in this country (they can readily be detected because of the bright pink colour of the parasite), although records of infections in the duck are rare. Many of these shrimp infections are clearly dependent on wild birds, but it is surprising to find, as we have done, ducks feeding on waters where infected shrimps occur in large numbers without any apparent symptoms of infection in the ducks, while in other cases ducks may suffer from heavy infections. T h e demonstration showed each of the stages in the life-cycle of the parasite. T h e embryonated eggs (Acanthors) ; the larvae removed from infected shrimps (Aeanthellae), both the early stage with an everted proboscis and the later stage in which both anterior and posterior ends are invaginated ; and adult males and females removed from the duck. Living infected shrimps and a list of the sites from which we have collected such shrimps were also shown. R E F E R E N C E S : GREEFF, R. (1864). Arch. Naturg, 1, 98. H ~ E s , H. B. N. (1955). J. anim. Ecol., 24, 352.

Dr. R. S. Bray : (1) Developing schizonts and gametoeytes of Plasmodium reichenowi in the blood

of a chimpanzee. T h e infection demonstrated was found in a chimpanzee naturally infected with P. reichenowi after splenectomy. I n heavy infections with this parasite aU erythrocytic stages can be seen in the peripheral system whereas in lighter infections only rings and mature gametocytes can be seen.

(2) Pre-erythrocytic schizonts of Plasmodium vivax found in the liver of a chimpanzee inoculated with 142 g l a n d s of P. vivax-infeeted Anopheles gambiae 8 days previously. Dr. E. J. L. Soulsby : Antigenic analysis of Asearis tissues by the double diffusion precipitin test. Preliminary investigations into the antigenic structure of Ascaris lumbricoides were demonstrated. T h e technique consisted of placing antiserum containing 0.4 per cent. agar in the bottom of serological tubes. Layering over this 0.5 ml. of 0.4 per cent. agar in saline and superimposing on this 0.5 ml. of antigen (homologous to the antiserum) in the fluid phase. T h e antigens were saline extracts of various tissues of A. lumbricoides. Reactions were read after 7 and 14 days' incubation at room temperature. Zones or bands of precipitate form for each portion of the antigen at fixed sites in the interposed column of agar in saline according to the zone of optimal proportions of the reacting antigenantibody system. Reactions after incubation for 14 days were given in whole worm, intestine, cuticle and polysaccharide antigens. Whole worm antigen showed nine bands with homologous antibody. Intestine antigen showed 14 bands with homologous antibody. When such antibody was absorbed with sheep erythrocytes, eight bands disappeared. Such absorption removes Forssman antibody and consequently Forssman antigen is present in extracts of the intestine of Ascaris. Previous work with other serological tests has detected this Forssman antigen in intestinal extracts. Cuticle antigen showed nine bands. A polysaccharide antigen showed four bands with its homologous antiserum but with normal

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LABORATORY MEETING

serum from several species two distinct zones are seen. These are also present in serum taken from the umbilical cord of cattle and pigs. T h e reaction occurs very rapidly (within a few hours) and is probably associated with low molecular components present in both antiserum and antigen. T h e reaction in normal serum with the polysaccharide cannot yet be removed by aborption but can be removed by diluting the antigen. Saline (protein) extracts of other tissues of 21scaris give negative reactions with normal serum. T h e density of the bands of precipitate may be a general guide to the nature of the antigen, dense rings tending to occur with protein antigens and fine rings with polysaccharide antigens.

Dr. L. J. Bruee-Chwatt and Mr. M. W. Service : An aberrant form of A. gambiae Giles from Southern Nigeria (Preliminary communication). It was thought that eggs of 2t. gambiae gambiae could nearly always be distinguished from eggs of A. gambiae melas by the differences in their morphology. T h e demonstration indicated that, in Nigeria, eggs of 21. gambiae gambiae collected some 100 miles inland may have a typical morphology of 21. gambiae melas. T h e results of salinity tests carried out on the corresponding larvae indicate that they are not resistant to salt water containing over 10 g. NaC1 per litre. It was also found that these eggs measure 0.515 =t: 0.016 ram. in length and thus are by 0.32 num. and 0.043 ram. longer than the eggs of 21. gambiae melas and A. gambiae gambiae respectively. T h e pecten of larvae produced from these eggs is typical of 21. gambiae gambiae.

Dr. B. O. L. Duke and Dr. F. H a w k i n g : Mite ( ? Pneumonyssus) in lung of monkey. Recently pieces of the lung of a drill were taken at postmortem examination by Dr. B. O. L. Duke, of the Helminthiasis Research Unit at Kumba, British Cameroons, West Africa. They were sent to Dr. F. Hawking of the National Institute for Medical Research, for histological examination of the microfilariae which they contained. During this examination, the lung was found also to contain certain small creatures which form the subject of this demonstration. T h e y lie in dilated bronchi not far from the pleural surface of the lung, and they have been identified by Prof. D. S. Bertram as probably mites of the Pneumonyssus type. T h e mites appear to feed on the columnar epithelium lining the dilated bronchi. They excite considerable cellular reaction in the walls of the bronchi, above and below the layer of muscle fibres ; many of the cells are eosinophils. R E F E R E N C E S : VITZTHUM,H. G. (1932) Z. Parasitenk, 4, 48. WtmM, H. (1926). Zbl. Bakt. I. Orig., 98, 514. Dr. J. MeArthur : Further contributions to microscope design. I n previous demonstrations a new form of microscope has been shown, designed particularly for tropical medicine and research, which is only the size of a miniature camera. One of the disadvantages of this microscope has been that it is rather low for comfort when used on a bench, and two solutions to this difficulty were shown. T h e first is a simple " table-top " photographic tripod, which raises the microscope to a convenient height and tilts it to any convenient angle, and which is of value also for drawing with a camera lucida. T h e " head " for this tripod also allows the microscope to be used on any photographic tripod for field work. T h e second design consists of a standard 35 ram. camera - - actually a " Wrayflex " was shown - - w h i c h forms a rigid base for the microscope, raising it to a convenient height. This camera does not interfere with the operation of the microscope, but is instantly ready to take photographs, singly or in rapid succession, in monochrome or colour, of the object as seen in the eyepiece.