Brun, R.

Jenni, L.

Schell, K-F

Schonenberger, M.

Tanner, M.

RERO DOC Digital Library

182 TRANSACTIONS OP THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 75, No. 1, 1981 Laboratory Demonstrations and Posters Scanning electron micrographs showing the early behaviour of macrophages and pro- mastigotes of Leishmania MUNA AL-TAQI AND A. HELMY MOHAMMED Department of Zoology, Faculty of Science, University of Kuwait, Kuwait Monolayered macrophage preparations from four common wild rodents in Kuwait (Gerbillus chees- mani, Jaculus jaculus, Meriones crassus and Mus musculus) were exposed in vitro for various periods of time to promastigotes of a Kuwaiti human isolate of Leishmania tropica. The behaviour of promastigotes and cells was then studied by SEM. A sequential picture of invasion was presented by selected SE micrographs taken from various preparations. Host cell differences, if any, are neglected in the present report. A proportion of the promastigotes soon become attached by the tips of their flagella to cells, mostly at the latter’s ectoplasmic active extensions. Stretched flagella at this stage suggest attempts by parasites to free themselves from a strong holdfast. A small tubular pseudopodium is produced at the point of attachment and grows gradually enveloping the flagellum to contain it completely within 4 to 10 min (or more). In most cases the tube is cylindri- cal and narrow, fitting rather closely around the flagellum; sometimes it is wider, possibly a response to an actively moving flagellum. The ingestion tube may now undergo lesser or greater retraction before it starts growing wider over the parasite’s body. The earliest complete ingestion of pro- mastigotes observed was within about 30 min of exposure in Mus musculus and J. jaculus. Previous records were: 2 to 5 min in a phase-contrast study of L. donovani and hamster macrophages (MILLER, H. C. & TWOHY, D. W., 1967; J. Protozool., 14, 781) and one hour in an SEM study of L. tropica and macrophages of laboratory mice (ZENIAN, A. et al., 1979; J. Cell. Sci., 39, 187). Newly ingested parasites may remain within a bulge outside the main body of the cell for some time. Filopodial projections or microspikes at the tip of the growing ingestive tube, as seen by ZENIAN et al., were not observed. Concerning whether the anterior or posterior end is ingested first it seems now that anterior end first is the rule (confirming ZENIAN et al., and some other authors). As to the penetration/engulfment controversy, it seems that the parasite plays an active role if at all, only very briefly, in initial attraction towards the macrophage; the rest of the ingestive process is actively taken by the cell. The greater part of ingestion may be described as an envelop&g or enclosing process (a special modi- fication of classical amoebic “circumfluence”) rather than the commonly used term “engulfment”: Finally, it may be added that other, but much less common, methods of ingestion seem to occur, and that the behaviour of promastigotes with peritoneal macrophages in vitro is not necessarily the same as their behaviour with macrophages at dermal lesions. Cultivation of bloodstream forms of pleo- morphic Trypanosoma brucei stocks R. BRUN, L. JENNI, M. TANNER, M. SCHONENBERGER AND K-F. SCHELL Swiss Tropical Institute, Socinstrasse 57, Base& Switzerland An in vitro system for the continuous cultivation of vertebrate-infective bloodstream forms of Try- panosoma brucei has been developed (BRUN -et al.. 1979; Acta troo.. 36. 387-390). This allows the propagation of &ombrphic, tsetse fly trans- missible stocks at 37°C. A feeder layer of rabbit embryo fibroblast-like cells was used in combina- tion with a modified Minimum Essential Medium (MEM) and lS”jh rabbit serum. MEM (Eagle) with Earle’s salts, sodium bicarbonate and L- glutamine was supplemented with HEPES (25 mM), glucose (2.0 g/l), Na-pyruvate (1 mM) and 17, MEM non-essential amino acids. The rabbit serum was freshly obtained from New Zealand Whites and could be stored for several months at - 20°C. Commercially available rabbit sera have so far given disappointing results. The rabbit embryo fibroblast-like cell line was isolated from 1%day-old New Zealand White embryos. The embryos were aseptically removed, washed with Earle’s BSS, finely chopped and incubated in 0.5”~; trypsin 1 : 250 (Difco) in Earle’s BSS (without Cal-/tMg++) at 37°C for 5 min. The remaining tissue pieces were disinte- grated by passing them several times through a Pasteur pipette. The cells were then removed from the trypsin by centrifugation, counted and seeded into T-25 tissue culture flasks. After two sub- cultures the cells were ready to be used as feeder layer. Cultures were initiated in 24-well tissue culture plates or T-25 flasks either with bloodstream forms from mice or metacyclic forms from infected Glossina m. morsitans. The metacyclic forms trans- formed to slender-like bloodstream forms and started to divide, leading to an established culture. Two trypanosome populations could be found in our culture system. The first one grew in between the feeder layer cells mainly as monomorphic slender-like forms and the second one in the super- natant where variable pleomorphism could be observed. Growth in culture was similar to that LABORATORY DEMONSTRATIONS AND POSTERS 183 in the mouse, with a GDT (generation doubling time) of 8 to 9 hours. So far, one T. rhodesiense and four T. brucei stocks have been tested (all pleomor- phic and tsetse fly transmissible) and all could be continuously grown as bloodstream forms in vitro. Different manual and automated methods in Chagas’s serology R. DIETRICH -- Hoffmann-La Roche & Co. AG, Bau 619, CH-4002 Basel, Switzerland Screening for Chagas’s antibodies in rural areas requires a simple and rapid method needing no suecial laboratorv eauiument. The rauid slide iocculation test iRS6Tj fulfils these conditions. The lyophilized antigen is very stable and ready to use after reconstitution of original volume by adding distilled water. After mixing one drop of reagent and one drop of serum on a plate and rock- ing for five minutes, the result is read visually. Compared with the indirect immunofluorescence test, an IF titre of 1 : 50 or higher reveals a clearly positive reaction (medium to strong flocculation). Sera with negative IF reaction show no flocculation. In blood donor centres a screening test for Chagas’s antibodies is very important to avoid infections by blood transfusion. The rapid haemagglutination test (RHT) adapted to GROUPAMATIC “ROCHE”, an automatic blood grouping machine, is an ideal tool to screen for Chagas’s antibodies. The reagent is prepared by coupling the reconstituted lyophilized soluble antigen to tanned human red cells of group 0, Rh negative. Results are automatically recorded. Specificity and sensitivity are comparable to the flocculation test. Sera revealing positive results with the floccula- tion or rapid haemagglutination tests are subse- quently analysed by ;ndirect immunofluorescence tests (IFTL Determination of titre and differentia- \ I tion of IgG and IgM type Chagas’s antibodies are a very helpful tool for precise diagnosis and facili- tate decisions on chemotherapy. The combination of the three methods RSFT, RHT and IFT permits a rapid, safe and reliable serological diagnosis of Chagas’s disease. These tests are sensitive, specific and well adapted to meet all requirements of routine serology in different fields of application. Dietary suppression of rodent malaria J. S. EDIRISINGHE, G. A. T. TARGETT AND E. B. FERN London School of Hygiene 0 Tropical Medicine, London IVClE 7HT The relationship between the level of dietary protein and the morbidity of l’lamrodium berghei infection in rats has not been clearly shown. This may be due to the use of diets of uncertain com- position such as those made to simulate human diets. We have examined the situation by using synthetic diets of precise composition to show the relationship of parasitaemia to the protein level and the protein to energy ratio of the diet. Our results show that the morbidity associated with I’. berghei infection in rats can be rklated to the protein content of the diet, but it is not related simply to the protein intake. The results are better explained by the relationship of the protein to energy consumption. Thus, while a low protein intake is effective in restraining the development of high parasitaemias, it will only do so if there is a low protein to energy ratio in the diet. Conse- quently an unrestricted intake of a low protein diet is more effective in controlling the degree of parasitaemia than a limited intake of a high protein diet. There are several possible mechanisms: (i) an enhanced immunological response, (ii) a decrease in reticulocytosis related to protein deprivation or (iii‘, some nutritional factors other than p-ABA. ’ $i are not yet in a position to comment on the possible importance of altered immunological responsiveness, Reticulocytosis appears not to be important as all rats showed normal or above normal levels of reticulocytes. The nutritional aspect would seem to be significant; recent studies that we have made with aminoacid mixtures indicate that there is competition for essential aminoacids between parasite and host. Correlation of clinical, parasitological and immunological data of patients with Schistosoma haematobium infections H. FELDMEIER, M. DIETRICH AND E. MANNWEILER Bernhard-Nocht-Institut fiir Sch$fs- u. Tropen- krankheiten, Bernhard-Nocht-Strasse 74, D-2000 Hamburg 4, German Federal Republic Clinical, parasitological and immunological find- ings in 35 patients with urogenital schistosomiasis, admitted to the Clinical Department between October 1978 and December 1979, were evaluated. The egg output was quantitatively assessed by the filtration trypan blue staining technique (FELD- MEIER et al., 1979; Tropenmed. Parasit., 30, 417). Nearly all patients originated from an area where uroeenital schistosomiasis is endemic. 28 patients werveinfested with Schistosoma haematobium; where- as seven patients had a mixed infection of S. haematobium and S. mansoni. 23 patients com- plained of a typical symptom, e.g., dysuria or abdominal pain or haematuria or a combination, whereas in 12 patients no symptoms were observed. Eosinophil counts varied considerably but only 13 patients had values above 500 per $. The demonstration of antibodies against the heter- ologous antigen (S. mansoni) assessed by indirect haemagglutination test (IHA) proved unreliable as a diagnostic method. In only 13 sera titres sug- gesting of Schistosoma infection were found. Endo- scopic-and histopathological findings characteristic of urogenital schistosomiasis were seen in only 15 cases out of 20, whereas negative findings were 

Cultivation of bloodstream forms of pleomorphic Trypanosoma brucei stocks

Abstract

Similar works

Full text

Available Versions

RERO DOC Digital Library