G. J. J. M. Van Eys

SEQUENCE ANALYSIS OF SMALL SUBUNIT RIBOSOMAL RNA GENES AND ITS USE FOR DETECTION AND IDENTIFICATION OF LEISHMANIA PARASITES

The sequence of the most variable part of the small subunit ribosomal RNA (SSU rRNA) gene, comprising 800 bases, was analysed for 9 Leishmania taxa and compared with those of Trypanosoma brucei, Trypanosoma cruzi and Crithidia fasciculata. Considerable differences were observed between the sequence of the Leishmania taxa on the one hand and those of Crithidia and Trypanosoma on the other. Amongst the Leishmania taxa only a few point mutations were found, all located within 2 sequence blocks in the central part of the SSU rRNA gene, which are unique for Kinetoplastida. These unique sequences were used for the development of kinetoplastid-specific probes and a Leishmania-specific PCR assay of high sensitivity (less than 10 parasites could be detected). Based on the observed point-mutations an identification of the Leishmania parasites, according to complex, could be achieved by direct sequencing, restriction fragment analysis or single-stranded conformation polymorphism of the PCR-generated fragments.

Detection of seven species of pathogenic leptospires by PCR using two sets of primers

Two sets of primers derived from genomic DNA libraries of Leptospira serovars icterohaemorrhagiae (strain RGA) and bim (strain 1051) enabled the amplification by PCR of target DNA fragments from leptospiral reference strains belonging to all presently described pathogenic Leptospira species. The icterohaemorrhagiae-derived primers (G1/G2) enabled amplification of DNA from L. interrogans, L. borgpetersenii, L. weilii, L. noguchii, L. santarosai and L. meyeri, whereas the bim-derived primers (B64-I/B64-II) enabled the amplification of L. kirschneri. Southern blot and DNA sequence analysis revealed inter-species DNA polymorphism within the region spanned by primers G1 and G2 between L. interrogans and various other Leptospira species. Using a mixture of primer sets G1/G2 and B64-I/B64-II, leptospires of serovars icterohaemorrhagiae, copenhageni, hardjo, pomona, grippotyphosa and bim were detected in serum samples collected from patients during the first 10 days after the onset of illness.

Comparison ofGiardia isolates from different laboratories by isoenzyme analysis and recombinant DNA probes

A total of 13 newGiardia isolates were established in axenic culture. All of the new isolates were obtained by excystation ofGiardia cysts from the feces of patients in Dutch hospitals. These isolates were subjected to isoenzyme and DNA analysis together with isolates from Poland, Belgium, and various other parts of the world. Isoenzyme analysis revealed that nearly all of the newly established isolates exhibited unique zymodemes. Isolates obtained from individuals from Belgium and Poland, on the other hand, displayed single zymodemes. Genomic DNA libraries were constructed from isolates belonging to the latter two zymodemes; specific and common recombinant DNA clones were selected from these libraries. Differential screening revealed that the two isolates had only 80% of the clones in common. Restriction-fragment-length polymorphism analysis using three different probes together with two synthetic probes that are complementary toGiardia structural protein genes led to the separation of all isolates into two major groups; within these groups, a further division could be made by application of other techniques or probes. The results of DNA analysis and zymodeme classification were in general agreement; in the present report they are compared with the data in the literature and discussed.

DNA hybridization with hardjobovis-specific recombinant probes as a method for type discrimination of Leptospira interrogans serovar hardjo.

Restriction endonuclease analysis of DNA of Leptospira interrogans, serovar hardjo, showed two distinct types within this serovar. These two types, hardjoprajitno and hardjobovis, cannot be differentiated by monoclonal antibodies. Application of 32P- or biotin-labelled total DNA probes in dot-blot or in situ hybridization assays showed a high sensitivity of the assays but also considerable cross-hybridization. Therefore, a genomic library of hardjobovis was constructed and a number of hardjobovis-specific recombinant clones were isolated. Finally, four clones were selected on the basis of a strong hybridization signal and a high specificity for hardjobovis as compared to hardjoprajitno. In a dot-blot assay as well as in in situ hybridization experiments all four clones gave strong signals, and no cross-hybridization with hardjoprajitno was observed in either type of assay. Our results indicate that specific recombinant DNA probes might provide tools for routine diagnosis and classification in cases of hardjo infections.

Detection ofLeishmania parasites by DNA in situ hybridization with non-radioactive probes

In situ hybridization techniques develop rapidly into diagnostic tools of considerable value for detection of viruses and bacteria. Here we report the application of this technique for the detection ofLeishmania parasites. Biotin-labelled total promastigote DNA was hybridized to culturedLeishmania parasites and to blood and impression smears of infected mice. In promastigotes kinetoplasts were strongly stained, nuclei somewhat more diffuse. In amastigotes both nuclear and kinetoplast DNA hybridized strongly. Amastigotes were easily detected in tissue of infected mice by their stable configuration of kinetoplast and nuclei. Cross-hybridization was observed betweenLeishmania donovani andL. tropica, but not between these two andL. braziliensis orTrypanosoma cruzi. A minor aspecific staining of host cell nuclei in the smears did not interfere with the detectability of the parasites.

A NEW SPECIES OF LEISHMANIA ISOLATED FROM THE GREAT GERBIL RHOMBOMYS OPIMUS

Leishmania turanica n.sp., found infecting the desert rodent Rhombomys opimus in the southern territories of the USSR and the Mongolian Peoples Republic, is described. This parasite exists sympatrically with L. major and L. gerbilli in R. opimus and is the predominant species. A total of 284 isolates of L. turanica from R. opimus, 3 from naturally infected Phlebotomus andrejevi and 1 from P. papatasi were characterized and found to be clearly distinguishable on isoenzyme and nuclear DNA characteristics from all other Old World taxa of Leishmania.

Detection and identification of Leishmania parasites by in situ hybridization with total and recombinant DNA probes

In situ hybridization on cultured promastigotes and sandfly smears were performed with nonradioactively labeled total DNA and recombinant DNA probes containing minicircle kinetoplast DNA (kDNA) or nuclear DNA inserts. Total DNA probes lack specificity whereas recombinant nuclear DNA probes work only if they contain repetitive sequences. Minicircle kDNAs of five Leishmania isolates, representative of five Leishmania taxa found in Kenya, were sequenced. Comparison of the sequences showed a 150-bp region with around 80% homology, whereas the rest of the minicircles had about 50% homology. Nevertheless, application of these probes in in situ hybridization assays as tested on Leishmania promastigotes in the vector gave good specificity and hybridization signal. Two types of labeling were tested: incorporation of biotin-labeled dUTP or directly horseradish peroxidase (HRP)-labeled nucleotides. Both techniques provided good sensitivity and signal-to-noise ratio on cultured promastigotes. Hybridization with HRP-labeled kDNA probes gave a superior signal-to-noise ratio if tested on sandfly preparations. This method provided a reliable and fast identification and facilitated the detection of promastigotes in sandflies. The technique presented here may be helpful in rapid identification of Leishmania promastigotes, and thus make epidemiological studies easier and less time consuming.

Application of Total and Recombinant DNA Probes in the Diagnosis of Leishmania Infections

Detection and classification of Leishmania parasites is still laborious and time-consuming. Detection of Leishmania infections depends largely on microscopic screening of smears and tissue sections, whereas classification relies on geographical area, pathology of the infection and more recently immunological and biochemical properties of the parasite. At this moment isoenzyme analysis is the most satisfactory method for the determination of Leishmania parasites. The main disadvantage of this method is that one has to culture the parasites.