Detection of species-specific antibody response of humans and mice bitten by sand flies.

Sand fly saliva plays an important role in Leishmania transmission. We characterized the host antibody response to saliva from 3 sand fly species. Specific IgG was observed in sera from experimentally bitten mice as well as in sera from individuals living in the endemic area of Leishmania tropica in Sanliurfa, Turkey. Sera of Sanliurfa inhabitants showed high IgG levels against saliva of Phlebotomus sergenti and P. papatasi, the 2 most abundant sand fly species in this area, but did not react with saliva of the New World sand fly, Lutzomyia longipalpis. Patients with active Le. tropica lesions possessed significantly higher anti-P. sergenti IgG levels than the healthy individuals from the same place while anti-P. papatasi IgG levels were equal in both groups. Major protein bands in P. papatasi and P. sergenti saliva reacted with both, human and mice sera; in P. papatasi, however, mouse IgG recognized preferentially the 42 kDa protein band while the human IgG reacted strongly with the 30 kDa band. Our data suggest that the antibody response to sand fly saliva could be used for monitoring the exposure of humans and other hosts to sand flies and might be used as a marker of risks for Leishmania transmission in endemic areas.

Oxidative stress and non-enzymatic antioxidative status in dogs with visceral Leishmaniasis.

Leishmaniasis is a potentially fatal chronic protozoan disease in human, canine and rodent species. The infection by Leishmania is endemic in the Mediterranean Sea region, Africa, Asia and South America. Canine visceral leishmaniasis (CanVL) is a systemic disease caused by Leishmania infantum and Leishmania chagasi from the Leishmania donovani complex group. The blood glutathione (GSH), plasma malondialdehyde (MDA), ascorbic acid (AA), beta-carotene, retinol and ceruloplasmin levels of dogs with CanVL were investigated to establish the status of the antioxidant defense mechanism in the infected animals. Dogs diagnosed as CanVL with amastigotes in lymph node smear examination and/or antibody titers > or = 128 were used as subjects, while those with no serological response against leishmaniasis were used as healthy controls. The glutathione and retinol amounts were decreased although not significantly (p > 0.05), but the MDA levels were significantly higher in dogs with VL, suggesting increased lipid peroxidation.

Development of a fast agglutination screening test (FAST) for the detection of anti-Leishmania antibodies in dogs.

A fast agglutination screening test (FAST) for the detection of anti-Leishmania antibodies in serum samples from dogs with visceral leishmaniosis was developed. The test is based on the direct agglutination test (DAT), but combines a higher parasite concentration with a smaller test volume. In contrast to the DAT, the FAST makes use of only one serum dilution and the results can be read within 3 h as opposed to 18-20 h for the DAT. The FAST was evaluated using serum samples of confirmed cases of the disease and healthy controls collected in the most important endemic regions of canine visceral leishmaniosis, import cases of canine leishmaniosis in a non-endemic country, from non-endemic healthy controls and from dogs with other diseases. The performance of the FAST was compared with standard DAT. In the present study, the FAST had a sensitivity of 93.6% and a specificity of 89.0%. The DAT had a sensitivity of 88.6% and a specificity of 96.7%. Furthermore, using a large panel of serum samples of previously examined DAT positive or negative dogs it was shown that degree of agreement between the two tests was high (95.7%; kappa value = 0.91). The FAST offers the advantages of the DAT based on freeze-dried antigen with respect to stability of the antigen, sensitivity and specificity. Moreover, the FAST allows the rapid screening of a large number of samples, which makes the test very useful for epidemiological screening of large populations of dogs.

Leishmaniasis in Turkey.

Leishmaniases are widespread in most countries in the Mediterranean basin, including Turkey. Two forms are observed in Turkey; Leishmania infantum is responsible from visceral leishmaniasis (VL), and L. tropica causes cutaneous leishmaniasis (CL). Phlebotomus sergenti, P. papatasi, P. major and P. syriacus are considered to be the probable vectors, and dogs are the main reservoir of L. infantum, while P. sergenti is the main suspected vector of L. tropica.VL is sporadically seen mainly in the Aegean, Mediterranean, and Central Anatolia Regions, but CL is endemic, especially in the Southeastern and Mediterranean Regions. Major touristic sites are free of both infections, and no infection is reported in any tourist. Mean number of annual VL and CL cases reported to Ministry of Health are 40 and 1,204, respectively, in the last four years. These data suggest that both VL and CL represent a public health problem in Turkey, but a decline is observed in the number of cases with both infections in recent years.

Multi-site DNA polymorphism analyses of Leishmania isolates define their genotypes predicting clinical epidemiology of leishmaniasis in a specific region.

Leishmania isolates from 57 cases of human cutaneous (CL), human visceral (VL), and canine visceral (CVL) leishmaniasis in Turkey were grouped by multi-site DNA polymorphism analyses into five genotypes. The initial grouping was based on DNA heterogeneity of the faster-evolving mitochondrion (kinetoplast) minicircles and the intergenic regions of two nuclear repetitive genes. Taxonomic affiliation and phylogenetic relationships of the five genotypes were inferred by comparing them with reference species for sequence heterogeneity in a approximately 1.4 kb conserved single-copy gene, encoding N-acetylglucosamine-1-phosphate transferase (NAGT). Alignment of the available sequences revealed no gap, but up to 7% scattered base substitutions, suggesting that this functionally important gene is a suitable marker. Three genotypes are completely identical to the NAGTs of the reference species, identifying them as L. infantum, L. tropica. and L. major, respectively. The remaining two are recognized as L. major NAGT variants with one and four base substitutions, respectively. As expected, Maximum Likelihood analysis of the NAGT sequences separates them into three clades, corresponding to the three species. The majority of the isolates obtained are L. infantum and L. tropica, which have been known to cause infantile VL and anthroponotic CL in western and southeastern Turkey, respectively. Unexpected is the finding of Leishmania major variants and their dispersal, possibly as previously unrecognized clinico-epidemiologic entities of CL and VL.

A survey on canine leishmaniasis in western Turkey by parasite, DNA and antibody detection assays.

Human visceral leishmaniasis (VL) caused by Leishmania infantum is found throughout the Mediterranean Region, including Turkey, where dogs are considered to be the main reservoir host for this parasite. In the district of Manisa, western Turkey, 37 human VL cases were reported from June 1993-August 1997. Twenty-four villages in this district were chosen for a survey of disease prevalence in dogs. The dogs, 490 in total, were examined using either the indirect immunofluoresence assay (IFAT) or direct agglutination test (DAT). Anti-Leishmania antibodies were found by at least one test in 5.3% (26/490) of the dogs. Infections were confirmed by parasitological examination of or polymerase chain reaction (PCR) on lymph node aspirates in 65% (13/20) and 76.4% (13/17) of the seropositive dogs tested, respectively. The confirmation rate was 85% by combining the results of PCR and microscopy. Our results demonstrate that canine VL is wide spread in western Turkey where human VL is endemic, and that serodiagnosis is a valuable tool for monitoring the infection.

Serodiagnosis and epidemiology of visceral leishmaniasis in Turkey.

Infantile Mediterranean visceral leishmaniasis (IVL) and anthroponotic cutaneous leishmaniasis (ACL) have long been known to exist in the western and southeastern Turkey, respectively. To further study these and other related diseases, a recombinant antigen (rK39) specific to VL was used in an ELISA for serodiagnosis of selected patients and for screening dog reservoir populations in several endemic sites. Among 24 confirmed VL cases from western Turkey, the rK39 ELISA proved to be more sensitive than a combination of cultivation and microscopy of bone marrow aspirates. The specificity of rK39 for leishmaniasis was demonstrated by its lack of cross-reactivity with sera from other human diseases in the same sites. Interestingly, six of the 83 parasitologically proven ACL cases from southeast Turkey were also rK39 positive. The end point titers of the positive VL and CL cases vary from 10(-2) to 10(-5) and from 10(-2) to 10(-3), respectively. The rK39 ELISA was also used to screen 494 apparently healthy dogs from Urfa in southeast Turkey, Manisa/Alasehir near the Aegean Sea, and Karabuk near the Black Sea. Eighteen rK39-positive cases (3.6%), all from the latter two areas, were found to have varying endpoint titers (10(-2)-10(-4)). The high titers predicted increased severity and frequency of the clinical symptoms (i.e., lymphadenopathy, depilation, skin lesion, weight loss and/or death), which were manifested subsequently in 16 of these 18 cases. In addition, more positive canine cases were diagnosed by the rK39 ELISA preclinically than the procedures to detect parasites postsymptomatically in the lymph node aspirates. The use of the rK39 ELISA as a sensitive tool makes it possible to demonstrate coendemicity of canine and human VL, as expected in the case of IVL. The results also point to the possible presence of additional VL types in western Turkey and cutanovisceral type in the southeast part of this country.

Dog-DAT: a direct agglutination test using stabilized, freeze-dried antigen for the serodiagnosis of canine visceral leishmaniasis.

We have evaluated the use of an improved direct agglutination test (DAT) based on stable, freeze-dried antigen for the detection of anti-Leishmania antibodies in canine serum samples. With a cut-off value of 1:640, the sensitivity of the DAT was shown to be 100% and the specificity of the test was 98.8%.

Epidemiology, diagnosis and control of leishmaniasis in the Mediterranean region.

The leishmaniases are a widespread and medically important group of parasitic diseases, some of which pose a serious health threat in communities throughout the Mediterranean basin. In 1993, a joint, collaborative study of the Mediterranean leishmaniases was initiated by scientists from Israel, Turkey, Portugal and the Netherlands. The aim of this project was the development of a multi-component approach to the successful control of all forms of leishmaniasis, with special emphasis on the more severe, visceral leishmaniasis (VL). The need for highly sensitive and accurate new tools to facilitate diagnosis and epidemiological surveys of endemic areas and for studies on the immunology of VL in laboratory models (dogs and mice) was soon recognized. It is anticipated that the development of these tools and the associated technology will provide a better understanding of the disease and improve its control.