Demonstrations

ROYAL

SOCIETY

OF TROPICAL LABORATORY

MEDICINE

AND

HYGIENE

MEETING

Liverpool School of Tropical Thursday, 21 November,

Medicine 1974

The President : PROFESSOR A. W. WOODRUFF, M.D., P.R.c.P., in the Chair. DEMONSTRATIONS Trypanocidal

activity of antitumour antibiotics and other metabolic inhibitors J. WILLIAMSON AND T. J. SCOTT-FINNIGAN National Institute for Medical Research, Mill Hill, London NW7 1AA

New drugs are urgently required for African human and animal trypanosomiasis, especially the latter. In the absence of large-scale commercial drug screening, laboratory investigation can be usefully directed to examination of aspects of trypanosome metabolism which may be vulnerable to chemotherapeutic attack. Recent intensive work on cancer chemotherapy has specified the locus of action of a number of antibiotics with increasing precision. Many are especially active on stages of macromolecular synthesis, so that rapidlydividing cells such as proliferative turnours, tend to be selectively inhibited. For this reason, pathogenic trypanosomes, malaria parasites and schistosomes are likely to be susceptible to agents of this type, and particularly to inhibitors of nucleic acid synthesis, as all 3 parasites appear to be unable to make their own purine bases. A number of representative inhibitors of this kind has been tested for effects on the motility and infectivity of suspensions (lOB/ml.) of Tqpanosotna congolense kept for 4 hours at 37” in 50/50 inactivated calf serum/Krebs-Ringer-glucose medium. O-2 ml. suspensions were distributed in transparent plastic Microtest trays and test compounds were serially diluted with an automatic microlitre pipette (KOLK-VEGTER, et al., 1973). Motility was assessed in situ with an inverted microscope, and infectivity was checked by inoculation of the well contents after incubation, into mice which were subsequently examined daily for development of parasitaemia. Trypanocidal titres are expressed as log,, M- l; titres less than 3 were not considered significant. Following a recent classification of nucleic acid synthesis inhibitors (GALE et al., 1972), the compounds tested and found active may be grouped as follows : Inhibition

locus

Inhibitor

Nucleotide utilization

Arabinosyl adenine Adenosine N-oxide

Nucleotide incorporation into polynucleotides

Tubercidin Cordycepin Nucleocidin Puromycin Puromycin aminonucleoside 8-Azaguanine

Template DNA inactivation

“RNA

synthesis”

Ribosome function

Acriflavine Ethidium Daunorubicin Actinomycin D Miracil D Distamycin A Chromomycin Mitomycin C (8-Hydroxyquinoline) Puromycin Emetine

Trypanocidal Motility 3-z 2 10 ; <3

titre Infectivity 34 4 8 8 3: 3-4 3 5-6

<

6 6 6 3-4 4

Inactive compounds included several which act primarily on purine and pyrimidine synthesis, interconversion and utilization (6-mercaptopurine, 6-azauridine, trimethoprim, Psicofuranine, Decoyinine, 5fluorouracil, Hadacidin, arabinosyl cytosine and iododeoxyuridine). In contrast, and as a likely reflection of the parasite’s dependence on exogenous adenine, 718 adenine analogues are actively inhibitory.

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2

In these circumstances, and as the cell content of RNA is much higher than that of DNA, trvnanosomal RNA synthesis is likely to be particularly sensitive to inhibition. Our te& show that 12/16 compotmds known to inhibit RNA svnthesis in other tvnes of cell. mainlv bv inactivation of the DNA temnlate mimer of RNA polymerase, have marked trypanocidal activity. Exceptibnally high in vitro activity (&res k-10) is shown by the antibiotics Daunorubicin, Actinomycin D, Chromomycin and Mitomycin C, but activity in vivo was limited by toxicity or rapid metabolic degradation; the high activity of 8hydroxyquinoline may also be related in part to RNA synthesis inhibition (FRASER and CREANOR, 1974). The uniquely high trypanocidal titre of Daunorubicin places it, with Nucleocidin, among the most highly active trypanocidal substances so far described. Activity at a lower level is shown by Primaquine and Tyrothricin (template DNA inactivation), and HBB and Methisazone (active respectively on RNA virus and viral RNA synthesis). Other compounds with low but significant activity were Camptothecin (affecting both DNA and RNA synthesis), chlortetracycline and Erythromycin (affecting ribosome function); the “structural” inhibitors Amantadine (affecting viral penetration), colchicine and vinblastine (affecting microtubules), were active, but Cytochalasin B was not. Inactive compounds included cyclophosphamide, methyl nitro-nitroso-guanidine, Rifampicin, Streptovaricin, nalidixic acid, hydroxyurea and cyclohexamide. Most of the trypanocidally active compounds have antitumour and antiviral activity; the common factor would seem to be that they are inhibitors of RNA synthesis. In the case of pathogenic trypanosomes (and of malaria parasites and schistosomes) their deficiency in adenine biosynthetic capacity should make attack with this kind of inhibitor a desirable form of chemotherapeutic research. REFERENCES FRASER,R. S. S. & CREANOR, J. (1974). Eur. J. Biochem., 46, 67. GALE, E. F., CUNDLIFFE, E., REYNOLDS, P. E., RICHMOND, M. H. & WARING, M. J. (1972), The molecular basis of antibiotic action. London: John Wiley & Sons. KOLK-VEGTER, A. J., KOLK, A. H. J., KREDIET, R. T. & STAUGAART-KLOOSTERZIEL,W. (1973). 3. immun. Methods, 3, 375.

Immunoglobulins M. J. CLARKSON, of Veterinary Parasitology,

in mice infected with trypanosomes M. CHOUDHRY AND T. J. CULLINGHAM Liverpool School of Tropical Medicine, Pembroke Place, Liverpool Department L3 5QA. LAC/A mice were infected with Trypunosomu brucei (TREU 667) and killed at intervals. Serum IgM concentration increased to over 5 times normal by Day 8 and reached over 8 times normal by Day 35. The spleen weight increased rapidly from 3 mg./g. body weight to over 20 mg./g. on Day 8 and almost 40 mg/.g. on Day 28. Athymic nude mice infected with the same strain showed an increase in IgM to twice normal by Day 20 but nude/ + heterozygotes showed no increase even up to Day 28. The mean time to death (14.2 + 3.4 days) was significantly less in 17 nude mice than in LAC/A (33.7 •t~ 8.3) or nude/ + (28.5 & 6.5). Injection of homogenised trypanosomes, which did not result in infection, into LAC/A mice produced a 2-fold rise in IgM, as did injection of Escherichiu coli lipopolysaccharide, a known thymus independent antigen.

Serum protein changes in cattle infected with Trypanosoma vivax M. J. CLARKSON, W. J. PENHALE”, G. EDWARDS AND I’. J. FARRELL Department of Veterinary Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA. *MRC Clinical Endocrinology Unit, Forrest Road, Edinburgh EH1 2QW. Graphs were presented showing the effect of infection with a West African Strain (64/23) of Trypanosomu vivux on the concentrations of IgM, IgG and a 7Sy, protein in British calves. Heterophile antibody levels were also measured to chicken, human, rabbit and sheep red blood cells. Calf 75, which died of an acute infection on Day 24, showed an increase in IgM from 2 mg./ml. to 5 mg./ml., no change in IgG and a fall in 7Sy, from 33% to 3% on Day 19. Heterophile antibody to chicken cells rose, parallel with the increase in IgM, from a titre of 4 to 64 but no antibody to the other cells was detected. Calf 316, which died of a chronic infection on Day 122, showed a progressive rise in IgM to over 30 mg./ml. on Day 45. IgM then fluctuated between 10 and 25 mg./ml. IgG commenced to rise 65 days after infection and was 45 mg./ml. on Day 110, over twice normal. 7Sy, fell from 55% to 2% on Day 20, coinciding with a sustained peak of parasitaemia and remained between 2 and 15% until death. Low titres of heterophile antibody before infection rose to between 32 and 128, depending on the species of red cell, with the highest titre to rabbit cells. Calf 456 was treated on several occasions with a new drug but relapse occurred each time. IgM rose

LABORATORY MEETING

3

initially to 20 mg./ml. but then fell to almost normal levels after treatment even though parasites were still present. IgG did not change and the fluctuations in 7Sy, were related to the peaks of parasitaemia, rising immediatelv after treatment and falling as soon as relapse occurred. Only low titres (maximum 16) of heterophile antibody occurred. Calf 99 was successfully treated on Day 20 with diminazene aceturate. IgM, which had risen to 8 mg./ml., fell after treatment to normal values and remained low till Day 90, when the experiment was terminated. No change in IgG occurred. The possible mechanism of the increased IgM was discussed especially in relationship to increased titres of natural antibodies. A comparison

of soluble

antigens from Trypanosoma vivax variants D. MATTHEWS Department of Veterinary Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA. Variable antigens from 2 antigenically distinct variants of a rodent adapted (Desowitz) strain of Trypanosoma vivax were identified, purified and characterized. Soluble antigen was fractionated on CM cellulose columns, using a linear sodium chloride gradient in 5 mM phosphate-buffer, pH 6.0. Variant specific antigen was eluted ai a single small peak for both variants, although the variant 1 specific peak also contained small amounts of common antigens. The variable antigens appeared to be glycoproteins which dissociated readily on SDS polyacrylamide gel electrophoresis. The protein component of these antigens showed slight differences in molecular weight (both 40-50 x 103) and isoelectric point (pH 6.5-6.8 for Vl, pH 74-8.5 for V2). The multiple bands seen on isoelectric focusing compared with a much more homogeneous appearance on SDS gel electrophoresis. The sedimentation coefficient of the Vl variable antigen was approximately 4.7 S. The timing

of antigenic variation in Trypanosomo v&ax T. W. JONES Department of Veterinary Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA. Trypanosome populations were isolated at daily intervals and stored in liquid nitrogen from each of 12 calves infected with a West African strain of Trvbanosoma vivax (fW23). Sera were also collected daily. Antigenically distinct populations were identified”by means of immune lysis, testing trypanosomes direct from liquid nitrogen storage against sera from the homologous animal. New antigen types appeared at 2-5 day intervals after the first appearance of trypanosomes in the blood and bore little relationship to any peaks of parasitaemia. Behaviour

of

evotomys under experimental conditions M. HOMMEL Department of Parasitology, Liverpool School of Tropical Medicine. For the first time, Trypanosoma (Herpetosoma) evotomys kudickei, KRAhWITZ, 1961, the natural trypanosome of the bank vole, Clethrionomys glareolus, was cultivated in vitro. Voles were caught by Dr. R. W. Ashford at Thornton Manor Estate, Cheshire (with kind permission of Lord Leverhulme) and heart blood was inoculated into Falcon Flasks (Biocult) containing a monolayer of hamster kidney cells (B.H.K.) in a medium of R.P.M.I. 1640, H.E.P.E.S. and 10% foetal calf serum. The culture took place at 26°C. in a single flask, in which half the medium was changed at intervals of 2 or 3 days. The behaviour of T. evotomys, observed daily under an inverted microscope, is similar to that of other trypanosomes of the same subgenus, cultivated in the same system (HOWL and MILTGEN, 1974). After an initial period of 8-15 days when they stay in the overlay as a trypomastigote, the trypanosomes attached to the cell layer, change into the epimastigote form and start dividing. Daughter epimastigotes form two types of rosette : “free rosettes,” floating in the overlay and “plaques” or rosettes attached to the bottom of the culture flask, in spaces between the cells. Whereas “free rosettes” can be observed in most trypanosomatid cultures, the “plaques” seem to be a special feature of Herpetosoma. These formations contain various morphological forms: rare long slender epimastigotes, many small pyriform epimastigotes, but mainly small round forms which seem to have lost their flagella. After 6 weeks, the overlay contains only trypomastigotes and small “metatrypomastigotes.” It is not clear whether the forms produced in this in vitro system more closely resemble those in the vertebrate or in the vector hosts, however, it seems to us that the “plaques” which maintain the infection in the flasks by their solid attachment., may be a good model of what happens in the flea vector (MOLYNEUX, 1969). The overlay forms are infective, and can be subinoculated into culture (i.e., 4N); when inoculated intraperitoneally into a laboratory mouse they give rise to a low parasitaemia lasting more than 3 weeks. Trypanosoma

(Herpetosoma)

REFERENCES

HOWL, M., & MILTGEN, F. (1974). Protistologica, MOLYNEUX, D. H. (1969). Parasitology, 59, 843.

10, 17.

4

LABORATORY MEETING

The cause of death in acute murine trypanosomiasis W. J. HERBERT, M. G. MUCKLOW AND B. LENNOX University Department of Bacteriology and Immunology, Western Infirmary, Glasgow. The actual moment of death of mice infected with highly virulent strains of Trypanosoma brucei sub spp. is rarely observed. This is because the animals behave normally even with parasitaemias exceeding lOa organisms per ml. and death occurs suddenly. After a few minutes of unease, there is a generalized convulsion. The animal may die during this convulsion, or within 10 to 15 minutes of it. As these symptoms are reminiscent of hypoglycaemia in man, we measured the blood-glucose level during the convulsions. Using “Dextrostix”, readings of less than 25 me. ner 100 ml. were reneatedlv obtained. though normal mice recorded 175 mg. per 100 ml-Furthermore, we &&id that mice treated withparenteral glucose saline when in the convulsive stage, returned to normal behaviour within a minute or two of administration. Subsequently they survived for up to 17 hours after the first convulsion had been observed. However, further administration of glucose did not prevent death, and the animals died with normal blood glucose levels. Violent convulsions of the initial type, were not seen in these mice. After SCHERN (1925) showed that the in vitro activity of apparently moribund trypanosomes comd be revived by the application of glucose, many workers investigated the role of this sugar in trypanosomiasis. They showed that a lowering of the blood-glucose level, associated with disturbances of the enzyme and carbohydrate storage mechanisms of the liver, occurred during infections in guinea-pigs and rats. However these effects did not seem to play much part in pathogenesis. Agonal glucose levels in highly parasitaemic mice do not seem to have been reported previously. We consider that our observations show that the primary cause of death in acute murine trypanosomiasis, characterized by generalized convulsions, is hypoglycaemia. However, another, unidentified, pathogenic process is also active, and this becomes the lethal factor if the hypoglycaemic state is corrected. SCHERN, K. (1925). iiber Trypanosomen.

REFERENCE Zentralblatt fiir Bakteriologie,

96, 356.

The effect

of a new anti-malarial compound on Plasmodium fdciparum in Aotus J. M. BAFORT* AND A. VOLLER *University of Antwerp, Belgium. Department Clinical Tropical Medicine, London School of Hygiene and Tropical Medicine, and Nufleld Institute of Comparatrve Medicine, The Zoological Society of London. Aotus trivirgatus monkeys were infected with the virulent Palo-Alto (East African) strain of Plasmodium falciparum. Samples of blood were taken for in vitro cultivation just prior to treatment when the parasitaemia was between 5 and 40%. Two methods of cultivation were used to determine the in vitro activitv of B1304. in flasks (COHEN and BUTCHER, 1970) and in haemagglutination plates (BIDWELL and VoLLRR-unpublished). After 18-20 hours blood smears were made from the cultures containing B1304 and these were compared with blood films made from the control cultures. Cultures containing 20, 50 and 100 r*g/ml. of B1304 showed more or less complete inhibition of parasite development, with morphological dege’n&ation. In contrast the control cultures and those containing O-1,0.5, 1.0 and 5.0 pg/ml. of B1304 contained parasites which appeared morphologically normal, some of which had matured. We are grateful to Dr. Mitchell and Dr. Bidwell for setting up the cultures. REFERENCE COHEN, S. & BUTCHER, G. A. (1970). Immunology, 19, 369. Cerebral Malaria in a virulent rodent plasmodial infection YOELI*, HARRY MOST*, BRUCE HARGREAVES*, AND LUIS FAJARDOt *New York University School of Medicine. tstanford University School of Medicine. A mutated strain of Plasmodium berghei yoelii 17 x causes fulminating and fatal infections in CFl and A/J mice accompanied by very high parasitaemias. Intraperitoneal inoculation of 1 million parasitized red cells produces death in the inoculated mice within 5 to 7 days. Higher parasite inocula kill the animals within 3 to 4 days. The characteristics of this virulent mutated P.B.Y. 17 x strain are 1) The preference for invasion of mature erythrocytes, 2) the very high soaring parasitaemia, 3) the tendency to develop within and block brain capillaries and vessels. In 8 out of 10 infected animals, severe brain involvement can be observed. Fine capillaries permitting the passage of a single cell and larger vessels are found completely or partially obstructed by sequestrated infected red cells. Between 20% and 40% of all brain capillaries may be involved. Many of the infected mice show an absence of light reflexes, and drag their hind legs in the last stages of their infection (last 2 days). MEIR

LABORATORY MEETING

5

In Giemsa stained brain crush preparations and in histopathological sections, one clearly observes the extent of the cerebral involvement. The sequestration of infected erythrocytes, the very high number nf intravascular schizonts (34% as compared to 4-5% in the peripheral blood) and the damage to the capillary and vessel endothelium. Adherence of infected cells to the vessel wall can also be seen. In general the picture is very similar to that encountered in fatal cerebral malaria infections of Plasmodium fulciparum in man. Studies are now under way to elucidate the phenomenon of cerebral malaria in the mutated P. b. yoelii 17 x strain and its underlying causes. It is hoped through future collaborative investigation to endeavour to use this virulent strain as a model for studies of the pathophysiology, treatment and prevention of cerebral malaria in man. A number of giemsa stained brain crush preparations and brain histological sections from mice which succumbed to their P.B. Y. 17 x infections were demonstrated. A number of colour photomicrographs showing the occlusion of capillaries and brain vessels, and the pseudo-aneurism formations in certain cerebral vessels were exhibited. This work was supported by the U.S. Army Medical Research and Development Command under research contract DAMD 17 74 C 4110 and by a research grant from the World Health Organization. REFERENCE YOELI, M. & HARGRBAVES, B. (1974). Science, 184, 572.

Studies on the structure L. H. BANNISTER,

and invasive behaviour of merozoites of Plasmodium knowlesi G. A. BUTCHER, E. D. DENNIS AND G. H. MITCHELL Guy’s Hospital Medical School, London, S.E.l. Schizonts of Plasmodium knowlesi were separated by differential centrifugation from whole blood of infected rhesus monkeys (Macaca mulatta) and were incubated at 37°C. in Medium 199 in a specially constructed filter chamber (description to be published). Merozoites released from rupturing schizonts passed through a micro-filter and were collected in incubation medium. Some were then fixed immediately in 3% glutaraldehyde (phosphate buffered at pH 7.3) and others were mixed with fresh erythrocytes and fixed in glutaraldehyde at intervals to demonstrate stages of invasion. Electron microscopy showed that the filtration method preserved the normal features of merozoite structure, and that invasion of red cells was largely complete within one minute of mixing. It was confirmed that entry occurs by the invagination of the erythrocyte membrane rather than by puncture (LADDA, et al., 1969) and that at the noint of invasion the cell coat of the merozoite adheres to the red cell. but is left outside as the parasite invades. Paired organelles are retained until after invasion has occurred, but these, with other dense bodies and micronemes, disappear when the rounded merozoite extends to form the early trophozoite; the dense bodies pass to the periphery of the parasite and release their contents into the space between the parasite and erythrocyte membranes, apparently causing the red cell membrane to expand rapidly. It is suggested that the paired organelles and other dense membrane-bound cytoplasmic structures are concerned with causing the initial expansion of the red cell membrane in the early stages of entry, and that once inside they cause the further increase in area of internalised red cell membrane which allows the parasite to extend into a ring stage trophozoite. REFERENCE LADDA, R., AIKAWA,

M. & SPRINZ, H. (1969). J. Parasit., 55, 633.

Application

of an enzyme-linked immune sorbent assay (E.L.I.S.A.) to malaria. *A. VOLLER, G. HULDT, C. THORS AND E. ENGVALL *Department of Clinical Tripical Medicine, London School of Hygiene and Tropical Medicine and *N@& Institute of Comparative Medicine, The Zoological Society of London. State Bacteriological Laboratory, Stockholm. The ELISA test originally developed by ENGVALL and PERL~~ANN (1971, 1972) was adapted for the measurement of malarial antibodies. Plastic tubes were coated with soluble P. knowlesi antigen (prepared from infected erythrocytes) and the test sera from Tanzanians and a control group of Europeans were incubated in them. The tubes were then washed and antihuman globulin labelled with alkaline phosphatase was added. After further washing the enzyme substrate was added and its change of colour (measured at 409 nm) was used to indicate the amount of enzyme in each tube. That amount was proportional to the concentration of malarial antibody in the test serum. The ELISA appears to be a very sensitive means of detecting malarial antibody. All the Tanzanian sera, except one, gave much higher values than the European control group. There was no difference in mean values between the parasitologically positive and parasitologically negative Tanzanians. REFERENCES ENGVAL~

E. & PBRLMANN,

-

P. (1971). Immunochemistty,, (1972). J. Zmmun., 109, 129.

8,871.

6

LABORATORY

MEETING

Counter

immunoelectrophoresis studies on human malaria parasites D. BIDWELL AND A. VOLLER Nufield Insitute of Comparative Medicine, The Zoological Society of London and Department of Clinical Tropical Medicine, London School of Hygiene and Tropical Medicine. The development of precipitating antibodies to malaria in owl monkeys (Aotus trivirgatus) infected with P. falciparum or P. brasilianum has been studied by counter-immunoelectrophoresis. Sera from animals infected with P. falciparum reacted only with homologous antigen but sera from I’. malariae infections reacted with antigens prepared from both I’. falciparum and I’. malariae. Preliminary tests with sera from human malaria infections have given similar results. Gels are prepared from agarose using barbitone buffer pH 8.2. Two lines of wells are cut-l mm. in diameter and 4 mm. apart. Antibody samples are applied to the wells nearest the anode and antigen to the well nearest the cathode and electrophoresis is carried out at 100 volts for 45 minutes. Results are obtained quickly and only small amounts of serum and antigen are needed since no peripheral diffusion occurs. The method is particularly useful when weak antigens are encountered. Studies on the metabolism of chloroquine in rhesus monkeys and human subjects. K. A. FLETCHER, J. D. BATY, D. A. PRICE EVANS AND H. M. GILLES Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA and University of Liverpool. (A) Preliminary studies of chloroquine metabolism and excretion were carried out in rhesus monkeys. Following administration of the drug (10 mg./kg. body wt., i.m.) to animals (4-5 kg. body wt.), analysis by gas chromatography/mass spectrometry was performed on samples of various tissues to determine the metabolic profile. Chloroquine was detected in all the tissues studied. Desethyl chloroquine, the principal metabolite, was found in the liver in approximately the same concentration as chloroquine. The carboxylic acid metabolite of chloroquine was identified by mass spectrometry but was present only in trace amounts in the urine. Similar profiles were found in urine, liver and kidney tissue of both normal and Plasmodium knowlesi-infected monkeys. These studies confirm in part previously published work (MCCHESNEY et al., 1966). However, the his-desethyl, the 4’-hydroxy, the 4’-aldehyde derivatives of chloroquine and 4-amino-7-chloro-quinoline were not detected. (B) Since the monkey studies showed that desethyl chloroquine was the main metabolite of chloroquine, excretion patterns of these 2 compounds in a group of 7 volunteer white British subjects were examined. Urine excretion of these compounds was followed for 3 days following a single dose of 600 mg. chloroquine base. Chloroquine was estimated fluorometrically using the method of MCCHESNEY et al. (1966) with some modifications. There was a mean total 3-day excretion of 23.3 mg., the mean percentage of the total dose excreted being 3.9%. The excretion of desethyl chloroquine was up to 5% of that of chloroquine by weight. Some indigenous volunteers in Thailand were also studied. Full urine collection was not possible and chloroquine excretion was related to that of creatinine. Despite the obvious limitations of investigating drug excretion in random urine specimens, there were indications, when chloroquine excretion was related to urine creatinine values, that generally the Thai subjects excreted chloroquine at a faster rate than their British counterparts. The chloroquine/desethyl chloroquine ratio was of a similar order in the 2 groups. (C) Studies of chloroquine and desethyl chloroquine excretion in human subjects were next carried out under more controlled conditions of fluid intake, timing of urine collection, and diet, during a 7-hour period following administration of 600 mg. chloroquine base. Under these controlled conditions the excretion of chloroquine in terms of mg. per kg. body weight was 0.26 and 0.21 for 2 British subjects, and 0.53 and 0.75 wt. chloroquine . for 2 Thai subjects. The ratio wt creatinine m each hourly urine specimen was higher for the Thais than for the British subjects, but the proportion of desethyl chloroquine to chloroquine in the urine of all 4 subjects was very similar. Confirmation of these results of an increased excretion of chloroquine in Thai compared with British subjects is in progress. Similar studies in other ethnic groups will also be performed. REFERENCE

MCCHESNEY, E. W., CONWAY, W. D., BANKS, W. F. JR., ROGER, J. E. & SHEKOSKY,J. M. (1966). J. Pharmac. exp. Ther., 151, 482. A rodent babesia from Pakistan M. YOELI* AND C. L. WISSEMAN JR.* *New York University School of Medicine, and j-University of Maryland School of Medicine. In the course of a scientific mission and investigations by members of the University of Maryland School of Medicine in West Pakistan during the summer of 1963 on the prevalence of diverse pathogens in wild rodents, a babesia was isolated from 2 Rattus (Rattus) sp. in the Lahore District on the plains and from one Rattus rattoides from gupis in Gilgit Agency, W. Pakistan.

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7

The parasite was isolated by routine inoculation of spleen and liver tissues intraperitoneally into white mice. Intraerythrocytic parasites appeared in the peripheral blood 3 days after inoculation. Parasitaemia rose rapidly, reaching 60%-85% within 8-12 days and terminating in the death of the animals. Severe anaemia was one of the main characteristics of the infection. At post mortem, the spleen was found enlarged, haemorrhages were observed in internal organs, the bladder showed haemoglobinuria. It must be pointed out that additional pathogenic or symbiotic agents were also isolated at the same time in the same mice, and all 3 isolated lines were contaminated with other organisms such as Borrelia, Spirillum, possibly Nosema and/or Streptobacillus moniliformis. An abundance of ticks was observed in the region where the wild rodents were trapped. Following 32 passages in white mice, the babesia strains were stored for a period of 9-t years, frozen as a tissue homogenate at -70”. Following removal from the deep freeze in March 1974 and reinoculation of the thawed material into white mice, the babesia was successfully reisolated and maintained both by blood passages and by liver homogenate inoculation. The babesia has maintained its biological characteristics, pathogenicity and virulence in CFl and A/J mice. Endeavours have been made to clean the blood inoculum from the spirochete contamination by penicillin treatment of the inoculated animals. A study has been carried out on the morphology and pathogenesis of the babesia which has been temnorarilv designated as Babesia n. soec. A detailed descrintion of the narasite will be submitted for publication. The demonstration includes blood smears from experimentally infected mice showing the pleomorphic parasite in the red cells. Single ring forms and multiple parasitic infections in red cells as well as the typical cross formation can be seen. Histological sections of liver, lung, kidney and brain from mice which succumbed to their babesia infection are presented. The liver pathology is most conspicuous with areas of parenchymatous degeneration clearly seen. Inhibition

of

of Naegleria gruberi by a swimming pool additive D. C. WARHURST” AND M. SINGER* *Department of Medical Parasitology, Liverpool School of Tropical Medicine, and torganics Division, Imperial Chemical Industries Ltd. Baquacil S.B.** contains 20% of a polymeric biguanide which is the active ingredient. The formulation has been recommended as an inhibitor of bacterial, fungal and algal growth in swimming pools, when used at a concentration of 50 ppm. In view of recent reports of primary amoebic encephalitis caused by Naegleria spp., where infection has been acquired by swimming (CARTER, 1972) we have tested Baquacil against the non-pathogenic N. griiberi (Cambridge Collection of Type Cultures). 2000 trophozoites were added to 2 ml. of 0.1% NaCl containing nutrient-agar-grown Esch. coli (OD 650 = 0*3), with or without the additive. After 7 days at 21”C., when the amoebic population in control tubes had reached its peak, 5 samples were examined from each tube for the presence of trophozoites, cysts or flagellates. Centrifuged sediment from each tube was cultivated to detect viable organisms. Under these conditions, in 2 experiments using duplicate tubes, no trophozoites or flagellates were found at 7 pg./ml. active ingredient, but viable cysts were detected in one experiment, No viable organisms were detected at 23 pg./ml. in either experiment. In a further experiment, 2 sterile all glass fish tanks were each filled with 4.7 litres of boiled tap water. To each tank, 25,000 trophozoites, 2 ml. of an Esch. coli suspension (OD 650 = 1.1) and an O-5 ml. block of nutrient agar were added. 50 ppm Baquacil was added to one tank (10 pg./ml. active ingredient). The tanks were kept in subdued daylight, lightly covered, for 2 weeks. In samples taken from the control tank N. griiberi could be easily detected, whilst none could be found in the experimental tank. When centrifuged sediment from 500 ml. of mixed content of this tank was inoculated onto culture plates, no growth took place. The results we have obtained, though imperfect, indicate that Baquacil S.B. should be active at the recommended concentration in controlling N. griiberi in swimming pools. growth

REFERENCE CARTER, R. F. (1972). Trans. R. Sot. trop. Med. Hyg., 66, 193.

We are grateful to Professor W. Peters for his encouragement. assistance. *#Imperial Chemical Industries Limited.

Mr. S. C. Thomas gave invaluable technical

Localization of mepacrine in Plasmodium berghei D. C. WARHURST” AND S. C. THOMAS-J. *Research Group on the Chemotherapy of Protozoa1 Diseases and Drug-Resistance, t Department of Medical Parasitology, Liverpool School of Tropical Medicine. BOCK and OESTERLIN (193819) observed the concentration of mepacrine by blood forms of Plasmodium inui and P. knowlesi, using fluorescence microscopy. They reported numerous, usually peripheral, bright points of light, in the general fluorescence of the cytoplasm, but were unable to photograph the phenomenon.

8

LABORATORY MEETING

We have attempted to repeat these observations using P. berghei in vitro. It was our expectation that fluorescence due to the drug might be localized in the pigment-containing digestive vacuoles. Parasitized mouse erythrocytes were incubated in vitro in 199 medium (WARHURST et al., 1974) without serum. After incubation with or without mepacrine (lo-’ or 3 x lo-*M) for varying lengths of time at 37”C., the cells were spun down and thin films were prepared, to be examined directly, when dry, under oil immersion. Incident UV and transmitted phase-contrast illumination were used, and photographs were taken in both modes. Fluorescence was strikingly localized in the parasite but at no stage in the time-course (up to 80 minutes) was fluorescence detectable within pigment-containing digestive vacuoles. In trophozoites the parasiteirbc interface fluoresced and general cytoplasmic fluorescence developed very rapidly. The parasite nucleus was not detectable as a fluorescing entity. The most surprising observations were made on merozoites and developing merozoites. From less than 5 minutes after exposure to the drug the area in or around the outer end of each merozoite showed a fluorescent spot which occasionally appeared paired. This clearly represents the phenomenon seen by Bock and Oesterlin. Our observations have been repeated upon P. fulciparum from the Aotus monkey, with similar results. These findings suggest that material in merozoites, possibly associated with the paired organelles, has a high affinity for mepacrine. Care must however be exercized in the interpretation of these results because absence of fluorescence does not necessarily mean absence of drug. Moreover, concentration of a drug in an area does not necessarily mean that this is the site of action. We are grateful to Professor W. Peters for his encouragement. REFERENCES BOCK, E. & OESTERLIN, M. (1938/g). ZentraZblutt fiir B&t. (Orig.), 143, 306. WARHURST, D. C., HOMEWOOD, C. A. & BAGGALEY, V. C. (1974). Ann. trop. Med. Parasit., 68,41.

Effect

of intraerythrocytic parasites on the permeability of the red-cell membrane K. D. NEAME”, C. A. HOMEWOODt AND H. MOMEN-/ *Department of Physiology, University of Liverpool, and tDepartment of Parasitology, Liverpool School of Tropical Medicine. Properties of the red cell membrane, such as fragility and the characteristics of various transport systems, may be changed after infection with either Plasmodium or Babesia. In addition, L-glucose, a substance which hardly enters normal cells, readily enters infected cells. With normal cells, the %-L-glucose space of a packed cell pellet after incubation at 37°C for 30 minutes was 0.22 ( f 0.02 S.D., n = 6), similar to the inulin space (O-18 f 0.05 S.D., n = 6); the latter is assumed to represent merely the extra-cellular volume. With cells infected with P. berghei, the L-glucose space was 0.62 ( % 0.05 S.D., n = 6), and with cells infected with B. rodhaini it was 0.52 (i 0.07 S.D., n = 8). Although these values are somewhat similar, the uptake by cells infected with B. rodhuini was slower than that by cells infected with P. berghei. This increase in the permeability of infected cells may be an advantage to the parasite by allowing the entry of otherwise non-penetrating nutrients. Nuclear

activity

in macrogametocytes

of

Hepatocystis nigrovittatus

sp. from

an Asian

tree squirrel,

Callosciurus

ELIZABETH U. CANNING, R. KILLICK-KENDRICK AND P. C. C. GARNHAM When blood containing gametocytes of Hepatocystis is removed to a slide or taken up by the intermediate hosts, the uninucleate microgametocytes undergo exflagellation to give rise to 8 uninucleate microgametes. Under similar conditions the macrogametocytes normally apparently remain unchanged. In a SDeCieS of Heaatocvstis in the blood of tree squirrels, Callosciurus nigrovittatus, from Kota Belud in Sabah (KI~LICK-KEND~ICK & al., 1973) there was a stril&g difference between pale-staining and dark-staining macrogametocytes. Both were clearly distinguished from microgametocytes which had the usual pink-staining cytoplasm and large nuclei. The macrogametocytes could have belonged to 2 different species but the presence of both types and intermediates in all 3 squirrels examined and the lack of any features to distinguish the microzametocvtes fdvoured the conclusion that they represented developmental stages of a single species. The palelstaining macrogametocytes which were considered immature showed signs of nuclear division. They often had 2 nuclei, though specimens with from 1 to 4 nuclei could be found and occasionally a smaller area of nuclear material was present near a nucleus. Almost every nucleus showed one or more dark-staining dots on the nuclear membrane. These were possibly equivalent to the kinetic centres ( = centriolar plaques), which have been shown bv electron microscopy of various Haemosporina to be amorphous electron-dense structures in the pores of the nuclear envelope at the points of origin of the intra-nuclear spindle microtubules (VIVIER and VICKERMAN. 1974). The dark-staining gametocytes always had a single, peripherally-situated, . compact nucleus without kinetic centres. They were-considered to be mature macrogametes. REFERENCES KILLICK-KENDRICK, R., GARNHAM, P. C. C. & RAJAPAKSA, N. (1973). Trans. R. Sot. trop. Med. Hyg., 67, 2. VIVIER, E. & VICKERMAN, K. (1974). Resume des discussions des tables rondes du 4e Congrds international

de Protozoologie, Clermont-Ferrand,

1973, pp. 16 1.

LABORATORY MEETING

9

Biochemical

and morphological differentiation within the genus Leishmania M. L. CHANCE AND I’. J. GARDENER Department of Parasitology, Liverpool School of Tropical Medicine. Members of the genus Leishmania are generally considered to be morphologically identical. However, preliminary observations and a search of the literature indicated that size differences exist between amastigotes of Leishmania. An accurate survey was therefore undertaken at the electron microscope level. Lesions were prepared under standard conditions and sections examined with an electron microscope carefully calibrated with a diffraction grating replica. Measurements were made only on sections containing a nucleus and cut approximately transversely to the sub-pellicular microtubules. 4 significantly different groups were found in terms of cross section diameter. These consisted of, (1) 2 isolates of L. donovani, 1 from Ethiopia and 1 from an Indian seaman; (2) A single isolate of L. 6. braziliensis; (3) A group containing L. mexicana amazonensis from Brazil, L. mexicana from Panama and L. tropica major isolated in the U.S.S.R., and (4) L. enriettii. The first 2 groups are characteristically small parasites while the Leishmania of the second 2 groups are much larger, L. enriettii being the largest parasite studied. These groups correspond to major subdivisions of the genus as determined by DNA buoyant density measurements (CHANCE et al., 1974), and malate dehydrogenase enzyme electrophoretic variants (GARDNER et al., 1974). REFERENCES CHANCE, M. L., PETERS,W. & SHCHORY, L. (1974). Arm. trop. Med. Parasit., 68,307. GARDENER, P. J., CHANCE, M. L. &PETERS, W. (1974). Ibid., 68, 317. Unidentified

granule

characterizing an isolate of Leishmania braziliensis braziliensis I’. J. GARDENER Department of Parasitology, Liverpool School of Tropical Medicine. The amastigote stages of an isolate of Leishmania braziliensis braziliensis (H9, LV63), isolated in Brazil from a human cutaneous lesion by Drs. R. Lainson and J. J. Shaw, are characterized by a cytoplasmic granule visible with the light microscope. Preliminary investigations have established the following points : (1) The granule occurs in almost all of the amastigotes. It is not present, or is extremely rare, in other species of Leishmania and in other isolates of L. braziliensis braziliensis. It occurs in infections in different hamsters, and persists during syringe passage. It is not, however, pronounced in culture promastigotes. (2) It does not absorb Giemsa stain, and in stained smears appears as a refractile body varying in colour between yellow, amber and black according to the illumination. It is clearly visible in unfixed and unstained smears. (3) Comparative electron microscope studies on this and other isolates have shown no inclusions peculiar to isolate LV63 which could be interpreted as representing the granule. (4) It is not digested by RNA-ase. It rotates plane-polarized light, and is refractile under dark-field illumination. Microscopical comparison indicates similarities between this granule from L. braziliensis braziliensis (LV63) and pigment from malarial parasites and from Trypanosoma cyclops (WEINMAN, 1972). In particular, under plane-polarized light it appears very similar to granules of malaria pigment. REFERENCE WEINMAN, 0. (1972). Trans. R. Sot. trop. Med. Hyg., 66, 628. brasiliensis in immunosuppressed hamsters and nude mice M. HOMMEL, W. PETERS AND M. L. CHANCE Department of Parasitology, Liverpool School of Tropical Medicine. ~~~ “slow strains” Leishmania braziliensis braziliensis (VIANNA, 191 l), LAINSON and SHAW, 1972, isolated from man in Brazil (strains LV63 and LV64) were inoculated into hamsters, previously treated with cyclophosphamide (300 mg./kg. intraperitoneally 24 hours before infection). When the parasites are inoculated subcutaneously, the granuloma develops much more rapidly than in normal animals. The lesions reach a maximum as early as 6 to 8 weeks after infection, as compared with 3 to 4 months in normal animals. These lesions are rich in parasites, but are still locllized at the point of inoculation 6 months after infection. When the parasites are inoculated intraperitoneally, a visceral involvement occurs before parasites can be demonstrated in the skin, i.e., 3 months after inoculation (nose and mouth lesions appearing first). During the visceral stage liver, spleen, peritoneum and bone marrow are fairly heavily infected, but the infection in the epididymis and the tunica vaginalis of the testis is particularly heavy. The subcutaneous infection at the root of the tail of 3 nude mice (Nu/Nu) with LV64 produced a generalized, extremely heavy infection after 2 months, involving the monocytes at the reticuloendothelial system Leishmania

brasiliensis

10

LABORATORY MEETING

throughout the animal without producing any apparent reaction in the host organs and without obvious lesions, other than a small plaque at the point of inoculation. A large number of parasites could be demonstrated in the peripheral blood, both within neutrophils, monocytes, and in vacuoles of cells that had lost their nuclei, and sometimes completely free. An ultrastructural study of these bloodstream amastigotes, showed that some of the parasites which were not contained in a cellular vacuole, were surrounded by a “surfacecoat” very much like bloodstream trypanosomes. Parasites from the skin of the immunosuppressed hamsters could be grown in culture on NNN agar with Basal minimum Eagle H.E.P.E.S., 10% foetal calf serum and O-3 mg./ml. cyclic AMP. The buoyant density of kinetoplast DNA of this strain had conserved the L. braziliensis bruziliensis characteristics (CHANCE et al., 1974), even after adaptation in vitro, proving the unchanged nature of the strain. This strain (LV63) has now been maintained on routine rabbit 4N medium for 6 months. The ability of a mucocutaneous strain of Leishmaniu to give rise to a systemic infection sheds new light on the belief that temperature-sensitivity of the parasite is the major factor in determining its dermatotropism (ZELEDON et al., 1969). REFERENCES CHANCE, M. L., PETERS, W. & SHCHORY, L. (1974). Ann. trop. Med. Pat&t., 68, 307. LAINSON, R. & SHAW, J. J. (1972). Br. med. Bull., 28, 44. ZELEDON, R., BLANCO, E. & DE MONGE, E. (1969). Actu Tropica, 26, 136. Axoneme

structure in Leishmania amastigotes J. ALEXANDER Department of Zoology, University of Glasgow. Studies on the fine structure of Leishmania enrietta and Leishmania mexicana have revealed several unusual arrangements of microtubules in the flagellar axoneme. The anterior end of the flagellum in these 2 species was found to contain disarranged axonemal doublets (d) in the absence of the 2 central singlets (s). The 2 central microtubules were present for only a short distance beyond the transitional zone and thereafter 1 or 2 outer doublets tended to collapse into the centre of the flagellum to give either an 8d + 2s structure or, more usually, an arrangement consisting of 7 outer doublets surrounding 2 central doublets (7d + 2d). The collapse of the axoneme structure and the loss of the 2 central microtubules preceded constriction of the flagellum from 250-140 nm as it emerged through the flagellar canal. This constriction of the flagellum and apparent sealing-off of the flagellar pocket does not occur in Leishmaniu promastigotes and may play some part in the survival of amastigotes in macrophages. Leishmaniu amastigotes do not use their flagellum as do promastigotes for propulsion, and persistence of the usual 9d + 2s microtubular arrangement may not be necessary. Further studies with L. tropica major amastigotes demonstrated that, unlike L. mexicana and L. enriettij amastigotes, the 2 central microtubules persist along much of the length of the flagellum; only at the extreme anterior end are they lost and an 8d + 2s structure found. AS yet, a 7d + 2d structure has not been demonstrated in L. tropicu and this may possibly be used as a taxonomic marker between Old and New World species. An enzyme linked immune sorbent assay (E.L.I.S.A.) to schistosomiasis +B. LAGERQVIST, #G. HULDT, T. PHILLIPS, C. C. DRAPER AND A. VOLLER *State Bacteriological Laboratory, Stockholm. Department of Clinical Tropical Medicine and Ross Institute, London School of Hygiene and Tropical Medicine. ELISA tests (ENGVALL and PERLMANN, 1971, 1972) were set up for schistosomiasis antibody using plastic tubes coated with purified schistosoma egg antigen (PHILLIPS and DRAPER, 1974) and with crude adult worm antigen. Sera from Europeans with S. mansoni infections were tested against both antigens, and the results were compared with those obtained on sera from Europeans never exposed to schistosomiasis. The infected group gave very strong reactions with the egg antigen and less strong reactions with the adult worm antigen. The control sera gave low level reactions with both antigens. REFERENCES ENGVAL; E. & PERLMANN, P. (1971). Immunochemistry, 8, 871. -(1972). J. Zmmun., 109, 129. PHILLIPS, T. & DRAPER, C. C. (1974). Unpublished. Observations on malarial and schistosomal pigment G. A. MOORE AND C. A. HOMEWOOD 1. Both schistosomes and malaria parasites produce pigment as a result of the digestion of haemoglobin and it has previously been impossible to disl inguish between these two types of pigment. The pictures demonstrated that they can in fact be distinguished, in the parasites, in the liver, and in preparations of purified pigment (MOORE et al., 1974).

11

LABORATORY METING

A micrograph of the crystal lattice of malarial pigment demonstrate the basis of structural differences between malarial and schistosome pigments (MOORE and BOOTHROYD, 1974). 2. The pigment was purified by washing with sodium dodecyl sulphate solution, followed by incubation with pancreatin solution (HOMEWOOD et al., 1975). 10% of the purified malarial pigment. All the iron detected by chemical 3. Haemin was approximately and x-ray microanalysis could be accounted for as the iron of haemin. Preliminary results indicate that malarial pigment contains more nitrogen than would be expected from the measured haemin content. REFERENCES HOMEWOOD, C. A., MOORE, G. A., WARHURST, D. & ATKINSON, E. (1975). Ann. @op. Med. Par&t., press). MOORE, G. A. & BOOTHROYD, B. (1974). Ibid., 68, 489. -, HOMEWOOD, C. A. & GILLES, H. M. (1974). Ibid., (in press).

(in

The effect of dietary fat on the host-parasite relations in cotton rat filariasis. Host-parasite relations in the progeny of cotton rats fed protein deficient diets and infected with filariasis-a preliminary report. levels in the early stages of infection with filariasis in cotton rats with and 3. Immunoglobulin without dietary protein deficiency. 4. Action of aliphatic alcohols on aquatic stages of mosquitoes. W. E. KERSHAW, D. M. STOREY, I’. D. WELLS, J. B. ALEXANDER, I’. VAN DER ZEIL, F. A. K. AL-BALDAWI, S. A. MOHAMMED AND B. SINNIAH Department of Biology, University of Salford 1. Cotton rats were maintained from 3 weeks of age on diets containing 10% arachis oil (80 : 20 unsaturated: saturated fatty acids), 10% lard (50: 50) or 10 % glycerol (0 : 0). The diets were otherwise identical and adequate in all respects. At 7 weeks of age half the animals were exposed quantitatively to infection with Litomosoides carinii by the bite of the natural vector Liponyssus bacoti. They were killed and autopsied 55 days later The growth rate of animals was related to their dietary content of unsaturated fatty acids. Infection had no effect on growth rate. Total lipid levels were higher in animals fed the glycerol diet than in the other two groups. There was no difference between groups for cholesterol, phospholipids and lipoproteins. Infection had no effect on the level of any of these lipids. The development of L. car&ii was influenced by the dietary fatty acid content of their hosts diet. Markedly fewer worms developed from known exposure in cotton rats fed the 10% glycerol diet than in animals fed the other two diets. There were slightly fewer worms in cotton rats fed the 10% lard diet than in those fed the 10% arachis oil diet. The growth of female parasites was most retarded in animals fed a diet containing 10% glycerol, and slightly retarded in those fed the 10% lard diet. There was no significant difference between the length of male worms from any of the dietary groups. 2. An experimental plan was adopted: 1. 2.

Offspring’s

Parent’s Diet

diet

Colony diet

8% Protein diet

Colony diet

10% Protein diet

Colony diet

12% Protein diet

Colony diet

15% Protein diet 8% Protein diet

8% Protein diet 10% Protein

10% Protein diet

12% Protein diet

12% Protein diet

15% Protein diet

15% Protein diet

0

2 Litters Born

1 Weaned

4

6

$‘I

8

10

Infected Time in weeks.

12

14

a Killed and Autonsied

16

12

LABORATORY MEETING

Growth in animals was related to the protein content of their diet. Also, animals born of protein deficient parents were smaller than those of parents fed a normal diet, but their subsequent rate of growth was increased. The development of Litomosoides carinii in cotton rats was influenced by the dietary protein deficiency of their parents as well as their own deficiency. That is to say, the rate of development of the infection was retarded in protein deficient animals but more so if their parents had also been protein deficient. 3. Cotton rats were maintained from 5 weeks of age on diets containing 15% or 5% protein by weight. At 7 weeks, half of them were infected with Litomosoides carinii. Groups of them were killed and autopsied at levels were measured by single radial immuno10, 25, 55 or 70 days after infection. Serum immunoglobulin diifusibn with rabbit anti-cotton rat IgG. There was no detectable difference in the total serum immunoglobulin levels in infected and uninfected animals fed the same diet at 10,20, 55 or 70 days after infection. In previous work, we have shown that serum IgG levels 5 days after infection were higher in infected than in u&fected cotton rats. In our latest work we also found differences between the total body immunoglobulin levels of infected animals fed the 15% protein diet and those fed the 5% protein diet at 10, 55 and 70 days after infection. The cotton rat is the natural host for L. carinii and the humoral response to primary infection is minimal. This is not the case in re-infection where we have shown that the microfilarial count falls within the first week and never reaches the level found before re-infection. We do not yet know whether such effects are mainly humoral or cellular or both. We have shown recently that cotton rats do produce precipitating antibodies against L. carinii. The number of precipitin lines is greatest at 1,2 or 3 months after infection. Work is in progress to measure against the antibody levels in the infection with immunoglobulin class specific antisera and also by passive cutaneous anaphylaxis. 4. 10 different aliphatic alcohols, with the number of carbon atoms ranging from 2 to 18, were tested against the aquatic stages of Aedes aegypti as ovicides, larvicides and pupicides. These alcohols were effective against all stages of Aedes aegypti but required different dosages for each alcohol to achieve the same percentage mortality. Toxicity of the alcohols increased with increase in length of the carbon chain until it reached a maximum after which an increase in chain length resulted in decreased toxicity. Maximum toxicity was reached at a carbon atom chain length of 11. For 24 hours exposure, n-decanol, I-undecanol and n-dodecanol proved to be very effective, giving 50% mortality at a dosage of 0.6 gallons per acre. Toxicity was increased with increased temperature. These effects were due to the direct action of the alcohols on the mosquitoes and not via an effect on the surface film of the water in which the mosquitoes lived.

A device for inoculating mosquitoes with larval filariae H. TOWNSON (introduced by DR. W. W. MACDONALD) A number of devices have been described in the literature which might be used for inoculation of mosquitoes. A simple system for inoculating microfilariae into mosquitoes was described by NELSON (1962). The technique illustrated is a refinement of that method including an improved method of holding and restraining the mosquitoes during the process of inoculation. The mosquitoes for inoculation are held by the thorax on a small perspex plate. A small hole in the plate connects to tubing which is attached to a water operated pump. A non essential but useful addition is the inclusion of a meter to aid control of the force of suction. The mounting of the restraining device in a conventional microscope mechanical stage facilitates the manipulation of the mosquito under a dissecting microscope. Mosquitoes are inoculated in the membraneous area below the parategite with a fine needle of 60-80 pm. tip diameter. Suitable needles can be drawn from Pasteur pipettes although for routine work over an extended period it is convenient to use capillary tubing which has been pulled to the appropriate diameter on an electrode-puller of the type used by neurophysiologists. The needle is connected by tubing to a 1 ml. Luer fitting syringe. By pressure of thumb and forefinger on the tubing and operation of the piston of the syringe, the uptake and expulsion of small quantities of inoculum is controlled. For Aedes aegypti and Ae. malayensis filariae can be inoculated suspended in the saline developed by HAYES (1953) to which sodium penicillin G (50 pg/ml.) streptomycin sulphate (50 pg/ml.) and nystatin (100 pg/ml.) have been added. Survival rates of better than 85% over 10 days can be obtained with these 2 species. Survival has been less satisfactory with Culex pipiens fatigans and Anopheles gambiae. The technique is being used to study the nature of susceptibility of mosquitoes to infection with filariae. It can be shown that in susceptible strains of Ae. aegypti both males and females are able to support development of inoculated Brugia pahungi to the infective forms, the females in the absence of the blood meal which normally accompanies infection. Females of refractory strains are as insusceptible to inoculated filariae as they are to filariae acquired naturally in an infecting blood meal. Filariae removed from refractory females and inoculated into susceptible females will develop normally provided that this is done within 6 hours of the ingestion of the microfilariae. It has been found that even when filariae are removed from susceptible mosquitoes their ability to infect successfully other susceptible mosquitoes falls off from the 7th-hour after ingestion. Presumably the infective capacity of the developing filariae is quickly lost after their entry into the flight muscles.

LABORATORY MEETING

13

By replacing the hollow inoculation needle in the apparatus with a solid needle of electrolytically sharpened tungsten, the apparatus can be used for the insertion of infective larvae into mosquitoes of varying susceptibility. Infective larvae which were transferred to the abdomen of mosquitoes of a refractory stock have survived for 34 days and successfully migrated to the proboscis of those refractory mosquitoes. One curious observation which is currently the subject of further investigation has been the development of filariae in a proportion of inoculated males of stocks in which the females are refractory. This phenomenon has been observed in both Ae. aegypti and Ae. malayensis on inoculation with B. pahangi. REFERENCES HAYES, 0. H. (1953). J. econ. Em., 46, 624. NELSON, G. S. (1962). J. Hekninth., 36, 281.

A microfilaria from rats (Rottus spp) in Egypt M. S. ARAFA, A. M. SALIT AND T. HILAL (introduced by DR. R. MULLER) Faculties of Medicine and Agriculture, Assiut University, Egypt Unsheathed microtilariae have been found in the blood of 2 of 18 black rats (Rattus norvegicus), 2 of 71 R. rattus frugivorus, and 1 of 69 R. r. alexandrinus, all of which were caught in a suburban area of Assiut, Middle Egypt. The microfilariae measure 160-195 pm. in length and cannot be differentiated from those of Dzpetalanema witeae, a natural parasite of the jird (Meriones) in Iran and the neighbouring parts of the Soviet Union. It may also be identical to a microfilaria described from the blood of the black rat in Azerbaidzhan by ABUSALIMOV (1964). A natural filarial parasite of the rat may be very useful as a laboratory model and attempts are being made to recover adults and to determine the life cycle. REFERENCE fbUsALIMOV (1964). Invest. Akad. Nauk, Axerbaidxhan, 3, 55.

Hybridization of Brugia pahangi and Brugio patd D. A. DENHAM, P. B. McGREEVY AND R. R. SUSWILLO Department of Medical Helminthology, London Sc$.bi;f 7HH$ene and Tropical Medicine, Keppel Street, Londan Brugia pahangi and Brugia patei develop to maturity and produce microfilariae in the peritoneal cavity of jirds (Meriones unguiculatus) (ASH and RILEY, 1970; ASH, 1973). The developing stages are easy to collect from the peritoneal cavity and can be re-inoculated into further jirds where they will continue to live and grow normally (BUTTS and RARALAIS, 1974). It is, therefore, simple to establish single sex infections in the peritoneal cavity. The only other problem to be overcome before attempting a hybridization study was to ensure that the single sex female infections were composed of worms which had not mated. SCHACIIER (1962) described the development of B. pahangi from the 3rd to adult stages. Most of the criteria needed to separate the sexes required such careful examination that there was a grave risk that the worm would be injured. However, there was a size difference between the sexes which became more pronounced as the worms grew older. This difference was detectable 35 days after infection for B. patei and 24 days after infection for B. pahangi. To ensure that the separation of the sexes was accurate the procedure adopted was to perform a separation at 35 or 24 days for the 2 species and inoculate the worms into further jirds. These were killed 3 days later when the size difference was greater and the males had developed a fully curled tail. These worms were then inoculated into further jirds. These jirds were killed 75 days after the inoculation of infective larvae into the first jirds. When the jirds with implanted single sex worms were killed no microfilariae were present in the peritoneal cavity. The adult worms were recovered and further jirds were inoculated with adult worms as follows: Group 1 3 female B. patei 3 male B. pahangi Group 2 3 female B. patei 3 msle B. patei Group 3 3 female B. pahangi 3 male B. patei Group 4 3 female B. pahangi 3 male B. pahangi Group 5 3 female B. pahangi Group 6 3 female B. patei Microfilariae were found within 33 days in the peritoneal cavity of 4 of 5 jirds in Group 1, all 3 jirds in Group 2, all 3 jirds in Group 3 and in the single jird in Group 4. Microfilariae were not found in any animals

14

LABORATORY MEETING

of Groups 5 and 6. Several females of each species were dissected at the time when the fmal transplantations were performed and none of these contained sperm or microfilariae. It seems clear from these results that hybrid microfilariae were obtained by cross mating B. patei and B. pahangi. These species originate from widely separate geographical areas and it will be of interest to see the results of the crosses of B. pahangi and B. malayi (sub-periodic) which we are currently undertaking as these species co-exist in the same environment in Malaya. REFERENCES ASH, L. R. (1973). J. Parasit., 59, 692. & RILEY, J. M. (1970). Ibid., 56, 962. BUTTS, J. A. & RABALAIS, F. C. (1974). Ibid., 60, 436.

Some

preliminary

results tracing fluorescent drugs in the scanning electron microscope G. A. MOORE (introduced by PROFESSOR H. M. GILLES) Liverpool School of Tropical Medicine Since some antimalarial drugs and their metabolites may be detected and assayed by their fluorescence in short wavelength electromagnetic irradiation (particularly ultraviolet) it seemed reasonable to attempt in some drugs the detection of fluorescence resulting from irradiation by an electron beam. Micrographs taken in a scanning electron microscope show the secondary electron image and adjacent to it the fluorescent image of (1) Chloroquine (2) Hycanthone (3) Hycanthone treated schistosome (4) Untreated schistosome. These results could be extended to qualitative detection in sections, and could possibly become quantitative.

Possibility

of detecting

drugs (hycanthone) by x-ray microanalysis G. A. MOORE Liverpool School of Tropical Medicine Of the possible methods available for studying the movement of hycanthone in Schistosoma mansoni the physical technique of x-ray microanalysis was chosen. Using the primary electron beam of an electron microscope to excite x-radiation characteristic of the sulphur atom contained in each molecule of hycanthone, an existing stock of specimens prepared in a conventional manner for electron microscopy was analysed at medium resolution (0.5 pm. max.). Electron micrographs indicating the analysis sites within the muscle layers, gut cells, cuticle and surrounding Araldite were displayed together with the associated sulphur levels. Of the areas analysed, only the cuticle showed an “above background” sulphur content in comparisons of treated and untreated worms. Although this is possibly linked to ultrastructural damage observed in the cuticle of treated worms, it does not follow that this is the prime cause of their death. What is clearly demonstrated is the value of this technique in detecting accumulations of any drug molecules having an atomic number greater than fluorine (Z = 9).

Viper venoms, haemorrhage and thrombosis R. D. G. THEAKSTON, M. J. MORRISON AND H. A. REID Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA. The main systemic effects of viper venoms in man are haemorrhagic. Echis carinatus venom (ECV) is one of the most haemorrhagic venoms, whereas Vipera berus venom (VBV) appears to have slight haemorrhagic effects. Allied to haemorrhagic effects are changes in coagulation, fibrinolysis, platelets, complement, kinins, and so on; acute renal f&tre is a major problem. The venom research project is investigating the pathogenesis of poisoning by these two contrasting viper venoms , especially aspects of haemorrhage and thrombosis, so that the medical care of human victims may be improved. Preliminary studies LDSOs in mice injected intravenously were 0.125 mg. ECV/kg. and 0.025 mg. VBV/kg. in rats injected intramuscululy 4.7 mg. ECV/kg. and 3.6 mg. VBV/kg. VBV is thus more toxic than ECV. Electron microscope studies in mice killed 2-5 minutes after half to two LD50s ECV showed large fibrin deposits in the space of DissC and in sinusoid11 capillaries in the liver, between capillary endothelial cells and basement membrane of proximal tubule cells, and in the glomerulu capillaries of the kidney. The blood vessel endothelium was damaged; distention of endoplasmic reticulum and mitochondrial changes were observed both in hepatocytes and in proximal renal tubule cells. Fibrin was occasionally seen in the brain,

LABORATORY MEETING

15

deposited beneath the capillary endothelium. Gaps in the damaged endothelium were often plugged by platelets. In mice killed 2-5 minutes after half to two LDSOs VBV, fibrin deposits were similar though smaller. However, tissue damage, especially in the liver, was more extreme. In the brain, capillary endothelial cells showed “blebbing” into the capillary lumen, a feature also described by OWNBY et al., (1974) in capillaries of muscle from mice injected with rattlesnake venom. REFERENCE OWNBY, C. L., KAINER, R. A. & Tu, A. T. (1974). Am.J. Parh., 76, 401. We thank the Medical

Research Council for financial support.

Lung

mast cells in rats following a superchallenge of Nippostrongylus brasilicnsis PHYLLIS D. WELLS Department of Biology, University of Salford Rats were given an initial inlection of 5,000 larvae of N. brasiliensis followed by infections of 2,000 larvae at 2 and 3 weeks. At 5 weeks they were given a superchallenge of 5,000 larvae. The mast cell numbers were counted in the pleural region, until week 50 of this infection. Viable larvae were present in the lungs until day 15 of the superchallenge; thereafter all larvae found were encapsulated. Egg-laying adult worms were present in the intestine at week 50; the eggs laid produced adult worms. Mast cell numbers, which had been elevated during the previous infections, remained so during the superchallenge and the cells showed degranulation. There is no evidence to suggest that the presence of elevated numbers of degranulating mast cells causes a sudden expulsion of larvae from the lungs or of adult worms from the intestine.

Host

in Syngamus trachea infection in birds D. E. PACKER Department of Veterinary Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA. An investigation into the effect of repeated challenge of chicks with infective eggs of Syngamus trachea was carried out. 2 groups, A and B, were challenged daily for 21 days and 5 birds from each group were killed and examined every 7 days for 5 weeks. Group A birds were given 10 h 3 eggs per day and B were given 200 f 40 eggs per day. Both groups exhibited self-cure and a loss of worms. Regression analysis of log,, mean worm numbers against time gives a rate of loss which is greater in B than A. Egg counts show greater egg production in A than B. When the experiment was repeated with the inclusion of group C receiving 500 f 50 eggs per day and birds were killed over 7 weeks, results followed the same pattern in A and B but group C had a lower rate of loss than B and a higher egg production. Thus it appears that rate of loss of worms increases with size of challenge and egg production decreases, but a state is reached when the size of challenge is such that the mechanism causing worm expulsion cannot effectively deal with increased worm burden and the rate of loss decreases and egg production increases.

Host

parasite

parasite

relationships

relationships in M. J. CLARKSON Parasitology, Liverpool

obsignata infection in the fowl AND A. ESFANDIARI Department of Veterinary School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA. JARRETT, JARRBTT and URQUHART (1968) described the kinetics of Nippostrongylus brasiliensis infection in rats by regression analysis of the logarithm of worm numbers against time after infection. The analysis could be split into 4 phases which they called Loss Phase 1, Plateau Phase, Loss Phase 2 and Threshold Phase. 90 week-old fowls were infected with 5,000 infective eggs of Capillaria obsignata and were killed at different times up to 115 days after infection. Regression analysis showed that 4 phases could be described and they were named as follows: Phase l-which indicates the proportion of the infecting dose which became established over the first day. Phase 2-a slow loss over the first 21 days, at which time the worms became mature. Phase 3-a more rapid loss from Day 21 to 56. Phase 4-a very slow loss from Day 56 to 115, the population largely consisting of male worms. Results from 500 birds gave quantitative measurement of the rate of loss during these phases in primary Copilloria

16

LABORATORY MEETING

and secondary infections, indicating that the loss in Phase 1 was slightly greater in resistant birds, and in Phase 2 was 5 times greater in resistant birds so that very few worms of the secondary infection attained maturity. The larvae of the secondary infection had no effect on the rate of loss of the original population during Phase 2, indicating that “selfcure” of an existing infection was not influenced by the reinfection. This analytical technique helps to indicate the points at which a host immune response may be acting. REFERENCE JARRETT, E. E. E., JARRETT, W. F. H. & URQUHART, G. M. (1968). Parasitology,

58, 625.

A quantitative technique for the estimation of helminth eggs in urine and faeces R. J. PITCHFORD AND P. S. VISSER (presented by DR. M. G. TAYLOR) Bilharzia Field Unit, S.A. Medical Research Council, Nelspruit, South Africa. A rapid, simple, cheap and accurate method for the recovery of helminth eggs from humans and animals was demonstrated. The apparatus consists basically of 2 concentric cylindrical nylon-gauze filters, one hanging inside the other: the inner filter has a larger mesh aperture than the outer. For faecal egg counts, between 5 and 10 g. of weighed formalinized faeces are placed in the inner filter (mesh 95 pm.) and broken up with a strong jet of water. Eggs and small faecal debris pass through the inner filter into the outer filter (mesh 50 pm.) and are collected in a large plastic container by opening the tap at the bottom of the outer filter. The sample is washed 4 or 5 times in the inner filter and all the washings collected. The contents of the large container, plus all the washings, are poured into a Waring blender fitted with a tot measure. The blender is filled with water to one of the calibrated markings, and turned on. The first aliquot is run to waste by opening the tot measure but the second aliquot is collected in a small, thin-walled plastic container. The contents are stained by adding 5 ml. acid fuchsin to 50 ml. fluid and heating to 70°C. After decolourising with Na OH the solution is poured through a No. 90 Whatman filter paper in a bacteriological filter, acidified, decolourised, reacidified and washed with water to obtain an even distribution of eggs over the filter paper. The eggs stain brilliant red or pink while faecal debris remains unstained. For urine the inner nylon filter and the blender are dispensed with but the rest of the procedure is the same. The whole of the measured formalinized urine sample is filtered through the outer filter to remove debris and reduce the volume. Eggs of Schistosoma mansoni, S. rohdaini, S. haematobium. S. mattheei, S. leiperi and S. margrebowiei all take up the stain but other species of schistosomes have not yet been tested. Ascaris and hookworm eggs stain but eggs of Fasciola and paramphistomes do not. REFERENCES PITCHFORD, R. J. ET AL. (1973). 3Z. S. Afr. vet. Ass., 44, 405. VISSER, P. S. & PITCHFORD, R. J. (1972). S. Afr. med. J., 46, 1344.

The effects

of

(Protozoa, Microsporida) on the larval stages of Fasciola hepatica G. C. HIGBY AND ELIZABETH U. CANNING Nosema eurytremae, a microsporidian recovered from various trematodes in Malaysia, was tested for its ability to depress cercarial production of Fasciola hepatica in Lymnaea truncatula with a view to its possible use in biological control. In 3 separate experiments, doses of 4 x 103, lo4 and 2 x lo5 N. eurytremae spores per snail were added to rearing dishes containing 50 Lymnaea truncatula previously infected with miracidia of Fasciola hepatica. No spores were added to control dishes. The flukes were allowed to develop until cercariae were produced. In 2 of the experiments, the snails were separated into individual vials and were induced to shed their cercariae by cooling to 4°C. and re-warming. There was no significant difference between the numbers of cercariae shed by the test and control snails, though, when dissected at the conclusion of the 2 experiments, they showed 55% and 80% infection of rediae respectively. In the third experiment, the snails were dissected after 6 weeks, and the numbers of cercariae were counted. Only 17% of the rediae were infected, and again there was no significant difference in counts between the two groups. Spores were demonstrated in the tissues of the snails, including the liver, and rediae were assumed to become infected either through the cuticle or when the spores were ingested. Concentration of the sites of infection around the guts of the rediae suggested that the hyper-infections were more often acquired by ingestion. Redial tissues were often opaque with close-packed spores but spores rarely extended to cercarial embryos and produced only light infections in them. Nosemo

eurytremae

17

LABORATORY MEETING of a parasitic nematode Isotopic SCannlng in pursuit AND D. C. JENKINS+. P. J. PRESTON”, M. A. MACLEOD *, D. J. BAKER” *Royal Naval Hospital, Haslar. tParasitology Department, May & Baker Ltd., Dagenham.

Following the demonstration of 14C labelling of free living nematodes by BALL and BARTLETT (1969) we have investigated the application of other radio-nuclides to various nematodes (PRESTON and MACLEOD, 1973). This demonstration showed the results of labelling adult and larval Nippostrongylus brasiziensis in vitro and also in vivo in albino Sprague-Dawley rats. In vitro studies of labelled adult parasites revealed a biological half-life of 14 hours for 67o,, while similar studies of larvae obtained on culture according to the method described by KEELING (1960), showed a biological half-life of 69.4 hours, suggesting either a much slower metabolic turnover or a lower ability to excrete the radio-nuclide. Auto-radiographic studies are in progress to elucidate this aspect. In vivo labelling of adult parasites was demonstrated with scintigrams, using a Nuclear Chicago Pho/ Gamma camera linked with a Varian 62OL/lOO computer to show the behaviour of the worms in the live host. Similarly, the migration of inoculated labelled larvae was shown, the scintigrams demonstrating early migration of 50% of the larvae to the lungs at 22 hours and arrival of 4th stage larvae in the gut at 72 hours after infection. The scintigram appearances were confirmed by morphological identification and by well counting after killing the hosts. We feel this nuclear tracing technique has considerable potential for the study of parasites in a living host. REFERENCES BALL, P. A. J. & BARTLETT, A. (1969). Trans. R. Sot. trop. Med. Hyg., 63. KEELING, J. E. D. (1960). Ann. trop. Med. Parasit., 54, 182. PRESTON, P. J. & MACLEOD, M. A. (1973). Third Symposium Naval Medicine. Exhibit. We wish to thank Dr. J. A. McFadzean, advice.

Low dosage

aerial

applications

Chemotherapeutic

Research Manager,

of insect growth regulators against Aedes dense mangrove canopy M. E. C. GIGLIOLI Mosquito Research and Control Unit, incorporating the Natural Resources Laboratory,

May & Baker Ltd. for

Taeniorhynchus

through

Grand Cayman, B.W.Z.

Although used successfully as high volume sprays (5 gls./ac.) against pasture mosquitoes, little is known about the efficiency of Insect Growth Regulators (IGR), applied in low volume airsprays through dense canopy, against flood water mosquitoes like Aedes taeniorhynchus. The present tests were conducted at dusk and dawn with a Cessna Agwagon fitted with 4 Micronair AV 3000 atomizers callibrated to yield: 0.6 fluid ounces/acre active ingredient of Altosid SRlO (Zoecon Ltd.) in a total aqueous emission of 15 oz./acre, and 0.025 lb./acre active ingredient of HT 6040 (Hayward Thompson Ltd.) in a 15 oz./acre aqueous emission. Droplet size and density across the 100 yard wide swath, showed an uneven distribution with heavy transport downwind to 60-75 yards (2,500 drop~/ft.~) with NMD,, ranging from 17-10 p with distance from the flight path. This distribution was closely reflected by the Altosid mortalities collected every 25 yards across the swath; they ranged up to 95% at + 75 yards and fell to their lowest, 10% at 100 yards downwind. HT 6040 gave higher and more even mortalities across the swath ranging from 80-100’$0. Collections were isolated 62 hours after treatment with Altosid and observed until all had died or emerged. Results showed mortalities ranging from 95-98% in the isolates, while similar isolates taken after 32 hours of exposure to HT 6040 showed lower mortalities ranging from 64-85%. On the other hand serial collections at lengthening periods after treatment highlighted the transient nature of Altosid SR 10 where mortality dropped from 78% at + 64 hours to 16% at + 72 hours. In contrast HT 6040 still yielded 71% mortality at + 84 hours. It is hoped this Altosid deficiency has been overcome in the new SR 10F slow release formulation. Altosid is a larval moulting hormone mimic, only affecting the IVth instar 10-30 hours before pupation, and assessable only through pupal mortality which in Aedes taeniorhynchus is characterized by dead pupae showing larvoid, adultoid or melanic characteristics, and more rarely the production of a heteromorph. HT 6040 is not a true IGR, but affects the deposition of the endocuticle and thus can be lethal during any moult or at adult emergency. Dead larvae were characterized by segmental herniation and impeded moulting; pupae by larvoid or adultoid appearance, and impeded emergency of the adult was sufficiently common to be observable in situ in the swamps.

LABORATORY MEETING

18

An improved method for sectioning the mosquito thorax E. B. BECKETT (introduced by DR. W. W. MACDONALD) Department of Histology and Cell Biology (Medical), Liverpool University, and Department of Medical Entomology, Liverpool School of Tropical Medicine. The method which, until recently, I have used for preparing sections of the mosquito thorax involves fixation with trichloracetic acid in ethanol, followed by ethanol dehydration. Double embedding is by Peterfi’s technique, modified only in that the celloidin concentration is increased and paraffin wax is replaced by Ester 1960 wax (BECKETT, 1974). This technique allows one to cut serial 4~ sections satisfactorily. However, the hardness of the cuticle sometimes causes scoring of the sections and there is some evidence of tissue shrinkage. The improved method eliminates ethanol from the fixation and dehydration stages and benzene from the embedding procedure. This results in better structural preservation and also reduced tissue hardness, so that serial sections of consistently good quality are more readily obtained. 1) Fix either in Newcomer’s fluid (NEWCOMER, 1953) or in the fixative of de Giusti and Ezman (DE GIUSTI and EZMAN, 1955). The latter gives slightly better preservation, but makes the tissue rather resistant to eosin staining. 2) Wash out fixative and dehydrate in isopropyl alcohol. 3) Clear in methyl benzoate, and infiltrate with 3% and then 5% celloidin in methyl benzoate. 4) Drain specimens briefly, and harden celloidin in isopropyl alcohol. 5) Embed in Ester 1960 wax via 50/50 isopropyl alcohol/wax. Since neither isopropyl alcohol nor methyl benzoate cause hardening, stages 2, 3 and 4 can be prolonged if desired. REFERENCES BECKETT, E. B., (1974). J. Ent., 48, 135. DE GIUSTI, D. L. & EZMAN, L. (1955). Trans. Am. Mic. Sot., 74, 197. NEWCOMER, E. H. (1953). Science, 118, 161.

Sub-lethal

activity

of insecticides

against larvae of the sheep blowfly, Lucilia sericata Meig. W. N. BEESLEY Department of Veterinary Parasitology, Liverpool School of Tropical Medicine Insecticides may affect newly-hatched blowfly larvae in the wool of dipped sheep by (a) a rapid kill, (b) a delayed kill, or (c) sublethal phenomena (especially in the pupae or adults as deformities, semi-eclosion or sterility). Semi-eclosion and sterility have been demonstrated following the exposure of larvae to dieldrin (BEESLEY, 1960), thiourea and Bakthane (unpublished). Sublethal effects have now been shown to occur with the organo-phosphorus insecticides pyrimithate (Diothyl) and diaznon (Nucidol). This suggests that for some time after the accepted 8-12 weeks’ protective period afforded by the modern sheep blowfly OP dips, sublethal effects may be possible following exposure of newly-hatched larvae to very small residual amounts of insecticide. This could happen towards the end of the blowfly season and perhaps at the beginning of the next season. REFERENCE BEESLEY, W. N. (1960). Vet. Rec., 72, 638.

Blood

parasites

detected in Trinidad Livestock M. W. SMITH Formerly of The Department of Veterinary Parasitology, Liverpool School of Tropical Medicine* During an assignment concerned with the control of parasitic disease in cattle, the examination of blood samples from domestic livestock in Trinidad revealed the presence of Babesia bzgemina, Anaplasma marginale, Trypanosoma theileri, and a species of Eperythrozoon in cattle. Also found were Babesia caballi in horses and Babesia canis and Dirojilaria immitis in dogs. T. theileri, Eperythrozoon, B. cabal& B. canis and D. immitis have not so far been reported from Trinidad. Acridine orange was used as the standard stain for routine screening of samples for blood parasites other than trypanosomes. Acknowledgements are due to Prof. Holman Williams, Faculty of Agriculture, University of The West Indies, for his help in isolating T. theileri. *Present address: C/o Department of Veterinary Pathology, P.O. Box 147, The University of Liverpool, Liverpool L69 3BX.

19

LABORATORY MEETING

Diurnal

activity

of Gryon sp., hymenopteran parasite of triatomine bug eggs D. S. BERTRAM Department of Entomology, London School of Hygiene and Tropical Medicine. The exhibit, an artificial wall containing triatomid bug eggs in holes and crevices, illustrated with photographs and graphs, that the adult hymenopteran parasite, Gryon sp., of triatomid bug eggs is particularly active by day, and that this activity promotes migration of the adult wasps to new areas of wall in search of further bug eggs to parasitize.

An improved method for the membrane feeding of mosquitoes. in the blood food on the survival and fecundity (a) The effects of heparins and crystamycins of Aedes aegypti (SS) mosquities. (b) The effects of heparins and crystamycins in the blood food on the development of Brugia pahangi microfilariae in Ac. aegypti (SS). J. 0. WADE (introduced by DR. W. W. MACDONALD) Conventional membrane feeding-units often incorporate design faults that, owing to the phenomenon 1. of erythrocyte sedimentation, can introduce unwanted bias into quantitative experimental work. Microfilariae of Brugia pahangi suspended in the blood-food redistribute themselves during the course of sedimentation and are eventually almost excluded from the volume of packed erythrocytes. A water-heated glass membrane feeding-unit with a cylindrical blood chamber of 8 mm. radius, -6 mrn~ height and approximate volume of 1-O ml. was constructed which allowed the insertion of a 2-bladed elastic naddle. A Meccanon flexible drive connected the paddle to a Smith@ industrial synchronous motor- (240 volt, 50~ 2 watt) contained in a moisture-proof -cabinet. A stirring speed of 60 revolutions/minute prevented erythrocyte sedimentation, and discouraged microfilarial redistribution. The uptake of both erythrocytes and microfilariae by Aedes aegypti (SS) was demonstrated to remain constant during 3 successive feeding intervals over a go-minute period.

1. 2.

2(a) The number of eggs laid by A. aegypti (SS) mosquitoes after a single blood meal was altered by the addition of both heparinn and crystamycinn, an antibiotic mixture of penicillin (Sod.) and sodium streptomycin to the blood-food. Egg-laying was promoted over that in the control by the addition of 300 units of heparinR or 600 units of crystamycinn to the blood-food. An increased quantity of either agent was deleterious to egg oroduction, esoeciallv in the case of the antibiotic mixture. Blood-food having a high henarinR content (UD ;o 2,500 &t&l.) did not adversely affect mosquito survival whereas crystamycinn was considered toxi’c to mosquitoes when its concentration in the blood-food was high (above 2,500 units/ml.). 2(b) The migration and development of B. pahangi microfilariae in the mosquito A. aegypti (SS) was not adversely affected by blood-food heparinn concentrations of up to 2500 units/ml. The deleterious effects of crystamycinn on microfilarial migration and/or development were proportional to the concentration of the agent in the blood-food.

1. 2.

Susceptibility Susceptibility with

Litomosoides

of Delhi white of un-irradiated

rats to quantitative infections with and irradiated golden hamsters

Litomosoides

to quantitative

caridi

infections

carinii.

M. A. SIDDIQUI AND W. E. KERSHAW Department of Biology, University of Salford. 1. Delhi strain of white rats, as a group, were “poor” hosts as compared to cotton rats (the natural hosts) to quantitative infections with Litotnosoides carinii. Batches of potentially infective mites, Liponyssus bacoti, were used for transmission. The dose of infective larvae transmitted by batches of mites to each animal was estimated by using 5 methods of calculation. The relation was found between the calculated dose of exposure ( = transmission intensity) and the number of adult worms recovered from each of the animals at autopsy. The number of adult worms recovered at autopsy from the majority of Delhi white rats was considerably less than the calculated dose of exposure. There, however, were considerable variations in the susceptibility of the individual rats: a point of genetic interest. Young golden hamsters (tm-irradiated and irradiated) were highly susceptible to quantitative infections with Litomosoides carinii. Their susceptibility was similar to cotton rats, the natural hosts. Batches of potentially infective mites, Liponyssus bacoti, were used for transmission. In one group the hamsters were first exposed to varying doses of whole body x-irradiation and then exposed to infective mites at different intervals of time after radiation. In the other group animals were exposed to infection without the use of irradiation. The dose of infective larvae transmitted by batches of mites to each animal was estimated by using 5 2.

20

LABORATORY MEETING

methods of calculation. The relation between the calculated dose of exposure (= transmission intensity) and the number of adult worms recovered at autopsy from both un-irradiated and irradiated golden hamsters was similar. Within the limits and conditions of experiment neither the whole body x-irradiation nor the variation in the interval of time between radiation and exposure to infection appeared to show any detectable effect on the susceptibility of golden hamsters to infections with L. carinii.

Lung events in mice infected with rodent Plasmodia M. SUZUKI (Introduced by bOFESSOR W. PETERS) Department of Parasitology, School of Medicine, Tokai University, Japan The initial ultrastructural alteration of the lungs in mice infected with Plasmodium yoelii nigeriensis (KILLICK-KENDRICK, 1974) or with N strain of P. berghei berghei has been consecutively studied for 5 days since inoculation. On the 2nd day of inoculation, focal aggregation of platelets was detected in the capillary lumina. On the 4th day, a considerable number of hypertrophied polymorphonuclear leucocytes (PMN) occupied the blood spaces. Distended cytoplasmic extensions from endothelial cell were occasionally noted in the same specimens. In the air spaces, many alveolar macrophages were generally detected. On the 5th day of inoculation, multi-layered cytoplasmic sheets from hypertrophied endothelial cells together with PMN occupied the capillary space, causing obliteration of the blood vessel, which resulted in the diminished red blood cells in the lumina. The morphodynamics of the lungs from the infected mice are similar to that of kidney events from the same animal (SUZUKI, 1975), which implies the same aetiology of both disorders associated with malaria. REFERENCES

R. (1974). Parasitology, 69, 225. SUZUKI, M. (1975). Bull. Wld Hlth Org., in press.

KILLICK-KENDRICK,

(? Rayella spj of the Himalayan Flying Squirrel Parasites 1. Section of spleen showing merocyst 2. Section of lung showing merocyst B. DASGUPTA AND N. L. PAL (Demonstrated by Prof. P. C. C. GARNHAM) Ruyellu and Hepatocystis are parasites of flying squirrels and other arboreal mammals, which belong to the family Haemoproteidae as the blood stages are confined to gametocytes. Schizogony typically occurs in the form of merocysts in the liver, but in this demonstration, asexual forms were found in the spleen and the lung.

Malaria