Sequence homology of kinetoplast DNA in Leishmania studied by filter hybridisation of endonuclease digested fragments and in situ hybridisation of individual organisms

Abstract

Several methods are now available for DNA characterisation of Leishmania based on the detection of kDNA sequences in an unknown isolate, which are complimentary to kDNA sequences in a reference isolate. Kinetoplast DNA to be used as a probe has been purified from lO'" organisms of each internationally recognised isolate selected on epidemiological, biological, immunological and biochemical criteria. We have tested the application of three methods of DNA hybridisation sequence analysis to twenty-five isolates from Brazil. For Southern filter hybridisation, kDNA from lO' organisms was endonuclease digested, electrophoresed and Southern transferred on to nitrocellulose filters. In the « dot blot » method, 10^ organisms were dotted on to nitrocellulose filters, lysed and denatured. Hybridisation in both methods was with nick translated, radioactively labelled (P^^) kDNA from reference isolates. We have recently developed an « in situ » hybridisation method, in which less than 100 organisms were smeared on to microscope slides, fixed, and the kDNA denatured and hybridised with tritium labelled reference isolate kDNA. Under « blind » trial conditions, positive sequence homology with Leishmania guyanensis and L . braziliensis was recorded for six isolates in Brasília (LTB's 133, 194, 201, 259, 260, 276) and three with L . amazonensis (Cepa's 3, 3 R142, 4). LTB0055 hybridised with ali three probes and is thought to be a mixed infection. I n Belém under similar « blind » trial conditions, positive kDNA homology was recorded with L . guyanensis and L . braziliensis to M2903, M4147, M5533, M7535 and with L . amazonensis to PH8, M5487, M6302 and H18. Dot blot analysis detected sequences of the L . braziliensis complex in M50Õ3, M6033, M7649, M6426, LTB's 12, 300 and 250. The « in situ » results in both Belém and Brasília were identical to the filter results when promastigotes were used. Amastigotes of many isolates were so small that they were completely covered by silver grains whenever positive hybridisation occurred.