D.A. Evans

Trypanosoma brucei: miniature anion-exchange centrifugation technique for detection of low parasitaemias: adaptation for field use

The miniature anion-exchange/centrifugation (AEC) method, originally developed for the detection of submicroscopic trypanosomaemias in laboratory rodents, has been adapted for the diagnosis of trypanosomiasis in man in the field using blood samples obtained by finger-prick. It has been tested in a survey in The Gambia. The method is shown to be highly sensitive and to fulfil the first essential criteria for exploitation in the field, namely, that it can be operated in the open air under tropical conditions, and that an adequate number of subjects can be examined in a normal working day at an acceptable cost. The method also offers two advantages over the other highly sensitive method applicable to small blood samples, the microhaematocrit buffy-coat microscopy (MBCM) method, namely, that it minimized the requirements for highly critical microscopy and provides, in the same operation, samples of diluted plasma which can be used for serological study.

Evidence of genetic recombination in Leishmania.

In the genus Leishmania there has been no convincing demonstration of genetic exchange, and it has been proposed that reproduction is clonal. However, preliminary characterization of two strains of Leishmania isolated from wild animals in a zoonotic focus of cutaneous leishmaniasis in the Eastern Province of Saudi Arabia, has suggested that they may represent hybrids of Leishmania major and Leishmania arabica. Evidence presented here strongly supports this hypothesis. Isoenzyme analysis and molecular karyotyping of cloned organisms indicated that the putative hybrids are distinct from other species of Leishmania, and possess characteristics of both L. major and L. arabica. Experiments using highly specific probes demonstrated that kinetoplast minicircle DNA from the putative hybrid contained L. major-specific, but not L. arabica-specific sequences. DNA fingerprinting data obtained using 6 genomic DNA probes were consistent in all cases with a L. major/L. arabica recombinant genotype, and implied both diploidy and allelic segregation. These observations suggest that sexual reproduction may generate genetic diversity within natural Leishmania populations.

A comparison of the direct agglutination test and enzyme-linked immunosorbent assay in the sero-diagnosis of leishmaniasis in the Sudan

The sensitivity and specificity of the direct agglutination test (DAT) and the enzyme-linked immunosorbent assay (ELISA) were compared for the sero-diagnosis of visceral (VL) and cutaneous (CL) leishmaniasis in Sudanese patients. All the sera from parasitologically confirmed cases of VL were positive in both ELISA and DAT. Some minor discrepancies were apparent between the two tests in patients with clinical signs of VL, but in whom VL was not confirmed parasitologically. In parasitologically confirmed CL both tests performed equally badly, with the DAT detecting 67% of cases and ELISA 60%. For the sero-diagnosis of VL, ELISA and DAT performed equally well, but on grounds of simplicity and low cost the DAT was preferred.

Leishmania infecting man and wild animals in Saudi Arabia 1. General survey

Using up to 13 enzymes, biochemical characterization of 75 isolates of Leishmania made from man, wild animals and sandflies from a wide variety of localities in the Kingdom of Saudi Arabia has revealed the presence of L. major (two similar zymodemes), L. tropica (two zymodemes) and a parasite of the L. donovani-L. infantum complex. Zymodeme LON-4 of L. major has been found in 52 of 53 isolates so far characterized from man, from one specimen of Phlebotomus papatasi, from 15 Psammomys obesus, from one Meriones libycus and from one dog. One isolate from man has been identified as a new variant of L. major. This variant, zymodeme LON-65, varies from zymodeme LON-4 in a single enzyme. While this is the only example of zymodeme LON-65 identified so far, zymodeme LON-4 has also been obtained from Kuwait and Iraq. These are the first reports of L. major in Meriones libycus from Saudi Arabia and the first proven isolate from the dog in any country. L. tropica was identified from only two foci, whereas L. major appears to be widely distributed in the Kingdom. Two infants with kala-azar were found to be infected with a parasite apparently identical to zymodeme LON-42 of L. donovani (sensu lato) which also occurs in the highlands of Ethiopia.

Identification of 'Old World' Leishmania by DNA recombinant probes.

Leishmania are usually identified by iso-enzyme analysis. This method works well, but there is a need for an additional, more simple, method of identification. Here we present data that show that in a Southern blot analysis, recombinant DNA probes in combination with certain restriction enzymes can differentiate between taxa of Leishmania. Probes based on clones selected from a L. infantum cDNA library gave characteristic patterns on Southern blots for reference strains of the different types of Leishmania found in Europe, Africa and Asia. Within the different taxa little or no variation was observed. Although the L. infantum derived probes showed a somewhat stronger hybridization for strains of the L. donovani complex, the signal obtained with most probes was satisfactory for L. major, L. aethiopica and L. tropica. Within the L. donovani complex none of the selected probes differentiated between isolates belonging to L. infantum, L. chagasi or L. donovani. Probes containing kinetoplast DNA showed considerable variation in hybridization within a taxon.

Characterization of Leishmania spp. from Kuwait by isoenzyme electrophoresis

Abstract Leishmania stocks isolated from skin lesions of 26 patients in Kuwait were compared among themselves and with Leishmania stocks collected from other parts of the Old and New Worlds on the basis of their isoenzyme patterns for seven enzymes by means of thin layer starch gel electrophoresis. The enzymes examined were: alanine aminotransferase (ALAT) E.C.2.6.1.2; aspartate aminotransferase (ASAT) E.C.2.6.1.1; glucose-phosphate isomerase (GPI) E.C.5.3.1.9; glucose-6-phosphate dehydrogenase (G6PD) E.C.1.1.1.49; malate dehydrogenase (MDH) E.C.1.1.1.37; malic enzyme (ME) E.C.1.1.1.40 and phosphoglucomutase (PGM) E.C.2.7.5.1. The isoenzyme patterns obtained fell clearly into 11 groups for the stocks of Leishmania tested. The Kuwaiti stocks separated into six groups. The patterns obtained with 15 Kuwaiti stocks were identical with those obtained with Leishmania tropica major (identified on clinical and geographical characteristics); seven stocks were identical with L. tropica minor similarly identified; three stocks had isoenzyme patterns different from each other and from all other leishmanias examined in this study; and one stock gave isoenzyme patterns identical with those of an L. donovani isolated in the Sudan. The isoenzyme patterns were the same in three stocks classified as L. tropica minor, L. aethiopica and a Leishmania isolated in Baghdad which caused a visceral disease in rats.

MIXED LEISHMANIAL INFECTIONS IN RHOMBOMYS OPIMUS: A KEY TO THE PERSISTENCE OF LEISHMANIA MAJOR FROM ONE TRANSMISSION SEASON TO THE NEXT

An important feature of the foci of zoonotic cutaneous leishmaniasis (ZCL) in Turkmenistan and Uzbekistan is a 6-10-month break in transmission when Leishmania parasites persist in great gerbils (Rhombomys opimus)--the main host for three species (L. major, L. turanica and L. gerbilli). Almost all (95%) of the laboratory-maintained R. opimus experimentally infected with L. major cured their infections within 6 months, a situation which, if mirrored in field conditions, cannot provide reliable persistence of the infection to the next transmission season. However, infections with L. turanica alone persisted for a mean of 15 months, and mixed infections of L. major and L. turanica persisted even longer (mean = 25 months), parasites of both species remaining detectable in the skin for at least 18 months. Isoenzyme identification of 664 isolates obtained from wild-caught R. opimus, and of 58 cloned strains developed from them, showed that L. turanica, which is non-pathogenic for humans, tends to predominate in the gerbils from all types of natural ZCL foci, including those which are hyper-endemic; in June, L. turanica may be present in 80%-100% of the R. opimus in the foci. In contrast, infections with L. major alone occur far less commonly, and are especially hard to find at the beginning of the transmission season. However, 5%-25% of great gerbils in these foci are each infected with a mixture of L. major and L. turanica. In hyper- and meso-endemic foci, the proportion of L. major within mixed infections of Leishmania increases significantly towards the end of transmission season (August-September). It would appear, therefore, that mixed L. major/L. turanica infections in R. opimus promote the persistence of L. major between transmission seasons.

Isoenzyme variation within the genusCryptosporidium

Soluble extracts of the oocysts ofCryptosporidium parvum had demonstrable, but low, activities of malate dehydrogenase (MDH, EC. 1.1.1.37), carboxylesterase (ES, EC 3.1.1.1) and lactate dehydrogenase (LDH, EC. 1.1.1.27) following thin-layer starch-gel electrophoresis. Much higher activities of glucose phosphate isomerase (GPI, EC. 5.3.1.9) and phosphoglucomutase (PGM, EC. 2.7.5.1) were found, and zymograms of these two enzymes were used to characterise isolates ofC. parvum from human, bovine, ovine and cervine sources,C. muris from the brown rat andC. baileyi from young turkeys. PGM and GPI zymograms clearly distinguished betweenC. parvum, C. muris andC. baileyi. The five isolates ofC. parvum showed the same electrophoretic mobility for GPI, whereas the PGM mobilitiy of the single human isolate ofC. parvum examined was clearly different from that of the other isolates. This is the first report of the use of isoenzymes to distinguish between species and isolates ofCryptosporidium.

Recent observations on the behaviour of certain trypanosomes within their insect hosts.

Publisher Summary The parasites responsible for the diseases, such as sleeping sickness and related trypanosomiases belong to the Trypanosoma brucei group. The methods used for studying the developmental cycles of trypanosomes within the tsetse fly are summarized in the chapter. The penetration of the fully developed peritrophic membrane can occur in the midgut region, and this penetration is outward into the ectoperitrophic space. It is related to the concentration of parasites within the lumen—that is, it occurs in those areas of the midgut that contain the highest concentration of trypanosomes–– and its frequency tails off on either side of these areas of maximum parasite density. The mechanics of penetration require a minimum of two trypanosome enzymes: a protease to allow passage across a chitin-containing layer of the peritrophic membrane and an obligatory enzyme, such as lipase, to attack the inner lipid-containing layer of the peritrophic membrane. A lipase is also necessary for the trypanosomes to cross the cell membranes of the tsetse gut cells. There is also the possibility that the parasites may travel up (or down) within the haemocoelomic membrane; outside the gut epithelial cells, to some suitable point of exit; or possibly remain within the membrane near the external membrane of the salivary gland.

Zoonotic cutaneous leishmaniasis in Saudi Arabia: lesions healing naturally in man followed by a second infection with the same zymodeme of Leishmania major

A patient with a previous history of an infection with Leishmania b. braziliensis contracted zoonotic cutaneous leishmaniasis (ZCL) in the Al-Hassa oasis, Eastern Province, Saudi Arabia. Five lesions healed spontaneously over a period of 40 weeks without treatment. A year after acquiring ZCL he became infected again in the same focus. Isolates of parasites at both episodes were identified as L. major, zymodeme LON-4. Compared with the first infection of ZCL, parasites were fewer in the lesions on the second occasion, the lesions were smaller and healing was quicker (10 weeks). This work and a previous report of patients with active lesions and leishmanial scars suggest that second infections of L. major are not uncommon in the oasis where no autochthonous infections of other species of Leishmania have yet been recorded in man and only one species of Phlebotomus (P. papatasi) is known.