Molecular tools for the classification and identification of members of the Leishmania donovani 'complex'
Visceral leishmaniasis is a potentially lethal disease. It is clinically presented by
viscerotropic dissemination to internal organs. The agents causing the disease are all
grouped within the L. donovani "complex" (Lainson and Shaw, 1987). Many issues in
the taxonomy within this complex are still controversial, such as the agents causing
visceral leishmaniasis in Sudan and the validity and the identity of L. "archibaldi".
The agents causing VL in Sudan are at present defined on the basis of an isoenzyme
classification as Leishmania donovani, L. infantum and L. "archibaldi". L. "archibaldi"
only differs from L. donovan; in the mobility of one enzyme (GOT). The presence of
all three species in Sudan has been contested by many authors who suggested grouping
the zymodemes belonging to these two taxa in a single group L. donovan; sensu lato
(Ashford et al. 1992).
In this study we have obtained the sequence of the chitinase gene of 37 stocks of these
parasites from the Sudan and elsewhere to construct a phylogenetic tree. A panel of
microsatellite markers suitable for classifying these species was also developed. Two
strategies were used to develop the panel of microsatellites.
Firstly, the Leishmania major genome sequence was used to identify microsatellite
markers. Twenty-seven independent microsatellite loci were identified by BLAST
search of the L. major genome. 13 out of 27 primers designed against the L. major
sequences also amplified a single product of approximately the expected size from L.
donovani. These 13 microsatellites were tested for variation in a panel of the L.
donovan; "complex". Only two out of 13'loci that were polymorphic in L. major were
also polymorphic in L. donovani. Almost all microsatellite peR products were
significantly smaller in all the test strains than they were in L. major. Therefore, the
use of the L. major genome sequence to identify microsatellite loci is unlikely to be an
efficient method of identifying microsatellite loci in other Leishmania strains.
Secondly, forty microsatellites were identified in L. donovani by an enrichment method
(http://WWW.liv.ac.uk/-kempsj/genomics.htmI.).Primers for twenty of these
amplified a single sharp band from L. donovani DNA and were found to be
polymorphic between L. donovani stocks. Phylogenetic trees were constructed using
Both chitinase and microsatellite phylogenies showed that stocks of all three species
isolated from Sudan form a single monophyletic group within the Leishmania
donovani, / L. infantum clade. We conclude that L. "archibaldi" is not a valid species
and the definitions of L. donovani and L. infantum may have to be revised in the light
of this data.