Francisco Morillas-Márquez

Sandflies of the Phlebotomus perniciosus complex: mitochondrial introgression and a new sibling species of P. longicuspis in the Moroccan Rif

Abstract.  The bloodsucking adult females of Phlebotomus perniciosus Newstead and P. longicuspis Nitzulescu (Diptera: Psychodidae) are important vectors of the protozoan Leishmania infantum Nicolle (Kinetoplastida: Trypanosomatidae) in western Mediterranean countries. The species status of the two phlebotomine sandflies was assessed, along with the epidemiological implications. Individual sandflies from three Moroccan Rif populations were characterized morphologically, isoenzymatically (by the isoelectrofocusing of alleles at the polymorphic enzyme loci of HK, GPI and PGM), and by comparative DNA sequence analysis of a fragment of mitochondrial Cytochrome b (mtDNA). By reference to the character profiles of specimens from other locations, including southern Spain and the type‐locality countries, the Moroccan flies were placed in three lineages: first, the lineage of P. perniciosus, which contained two mtDNA sublineages, one (pnt) widely distributed and associated with the morphology of the male types from Malta, and the other (pna) associated with a P. longicuspis‐like male morphology; second, the lineage of P. longicuspis sensu stricto, including typical forms from Tunisia; and third, a new sibling species of P. longicuspis. The mtDNA sublineage (pnt) of typical P. perniciosus was also found in some P. longicuspis from Morocco, indicating interspecific hybridization. The typical race of P. perniciosus occurs in Italy as well as in Malta, Tunisia and Morocco. It is replaced in southern Spain by the Iberian race (with the pni mtDNA sublineage). The discovery of interspecific gene introgression and a new sibling species mean that previous records of the two morphospecies do not necessarily reflect their true vectorial roles or geographical and ecological distributions.

ITS 2 sequences heterogeneity in Phlebotomus sergenti and Phlebotomus similis (Diptera, Psychodidae): possible consequences in their ability to transmit Leishmania tropica.

An intraspecific study on Phlebotomus sergenti, the main and only proven vector of Leishmania tropica among the members of the subgenus Paraphlebotomus was performed. The internal transcribed spacer 2 (ITS2) sequences of 12 populations from 10 countries (Cyprus, Egypt, Italy, Lebanon, Morocco, Pakistan, Portugal, Spain, Syria, and Turkey) were compared. Samples also included three species closely related to P. sergenti: Phlebotomus similis (three populations from Greece and Malta), Phlebotomus jacusieli and Phlebotomus kazeruni. Our results confirm the validity of the taxa morphologically characterised, and imply the revision of their distribution areas, which are explained through biogeographical events. At the Miocene time, a migration route, north of the Paratethys sea would have been followed by P. similis to colonise the north of the Caucasus, Crimea, Balkans including Greece and its islands, and western Turkey. Phlebotomus sergenti would have followed an Asiatic dispersion as well as a western migration route south of the Tethys sea to colonise North Africa and western Europe. This hypothesis seems to be well supported by high degree of variation observed in the present study, which is not related to colonisation or to intra-populational variation. Two groups can be individualised, one oriental and one western in connection with ecology, host preferences and distribution of L. tropica. We hypothesise that they could be correlated with differences in vectorial capacities.

Toscana Virus in Spain

At least 2 virus lineages are circulating in the Mediterranean basin.

Canine Leishmaniasis in Southeastern Spain

To examine prevalence changes and risk factors for canine leishmaniasis, we conducted a cross-sectional seroprevalence study and a survey during April–June 2006. Seroprevalence had increased at the meso-Mediterranean bioclimatic level over 22 years. Risk was highest for dogs that were older, large, lived outside, and lived at the meso-Mediterranean level.

Diagnosis of infections with Leishmania infantum using PCR-ELISA

On the basis of partial amplification of a cloned fragment of kDNA of Leishmania infantum which is specific for this species, we developed a PCR-ELISA technique which avoids the problems associated with classical diagnostic techniques. This technique was tested on 33 L. infantum strains from 19 different zymodemes, which were recognized equally. It was also used on human and canine clinical samples. PCR-ELISA has a higher sensitivity than the other techniques used (IFAT, parasite cultures, optical microscopy of stained samples) and permits detection of a minimum of 0.1 promastigotes or 1 fg of genomic DNA. PCR-ELISA can be used to diagnose human cutaneous leishmaniasis using material obtained by scraping the lesion margin, and human visceral leishmaniasis in HIV(+) individuals and canine leishmaniasis with peripheral blood samples. The presence of L. infantum in dogs with low antibody titres with IFAT technique (20 and 40) was demonstrated indicating that seroprevalence data from epidemiological studies underestimate the true rates of infection.

Predicting the distribution of canine leishmaniasis in western Europe based on environmental variables.

The domestic dog is the reservoir host of Leishmania infantum, the causative agent of zoonotic visceral leishmaniasis endemic in Mediterranean Europe. Targeted control requires predictive risk maps of canine leishmaniasis (CanL), which are now explored. We databased 2187 published and unpublished surveys of CanL in southern Europe. A total of 947 western surveys met inclusion criteria for analysis, including serological identification of infection (504, 369 dogs tested 1971-2006). Seroprevalence was 23 2% overall (median 10%). Logistic regression models within a GIS framework identified the main environmental predictors of CanL seroprevalence in Portugal, Spain, France and Italy, or in France alone. A 10-fold cross-validation approach determined model capacity to predict point-values of seroprevalence and the correct seroprevalence class (<5%, 5-20%, >20%). Both the four-country and France-only models performed reasonably well for predicting correctly the <5% and >20% seroprevalence classes (AUC >0 70). However, the France-only model performed much better for France than the four-country model. The four-country model adequately predicted regions of CanL emergence in northern Italy (<5% seroprevalence). Both models poorly predicted intermediate point seroprevalences (5-20%) within regional foci, because surveys were biased towards known rural foci and Mediterranean bioclimates. Our recommendations for standardizing surveys would permit higher-resolution risk mapping.

Distribution of sandfly species in relation to canine leishmaniasis from the Ebro Valley to Valencia, northeastern Spain

In Spain, only two of the 12 recorded species of sandflies, Phlebotomus (Larroussius) ariasi Tonnoir and P. (L.) perniciosus Newstead, are proven vectors of Leishmania infantum Nicolle, the causative agent of endemic leishmaniasis. Studies of the distributions of phlebotomine sandflies are important for evaluating the possible effects of climate warming on any northward or altitudinal range shifts of leishmaniasis or the other diseases they transmit. We describe a recent sandfly survey in Spain, starting in the northern Ebro Valley and continuing southeast into the Levante region of the Mediterranean coast. Sandflies (P. ariasi only) were found for the first time in the northern province of Alava, in the upper Ebro Valley, where cases of canine leishmaniasis have been described during the last decade. Throughout the provinces sampled, P. ariasi predominated over P. perniciosus in cooler bioclimatic zones, and this statistically significant pattern was more marked than that with higher altitudes.

Isoenzymatic polymorphism of Leishmania infantum in southern Spain.

Over a period of more than 10 years we have isolated and classified 161 Leishmania strains from cases of human visceral, cutaneous and mucosal leishmaniasis in immunocompetent subjects, from cases of visceral leishmaniasis in immunocompromised individuals with HIV, from dogs with leishmaniasis (visceral and cutaneous), from Rattus rattus and from sandflies. The strains were all L. infantum, the only species endemic in Spain, and corresponded to 20 different zymodemes. We describe the life cycle of these zymodemes for which, in most cases, we only partially know the hosts involved. We also discuss possible reasons for the greater polymorphism of L. infantum in southern Spain.

Genetic variability within the species Leishmania infantum by RAPD. A lack of correlation with zymodeme structure

The infraspecific variability of the species Leishmania infantum is studied by using genetic markers generated by random amplified polymorphic DNA (RAPD). We have applied this technique, using 18 primers of arbitrary sequence, to 33 strains of the parasite belonging to 18 zymodemes isolated in different clinical forms and hosts. Other strains belonging to the species L. donovani, L. major, L. tropica and L. mexicana were used as a reference. The RAPD technique produced very different genetic profiles between L. infantum and L. major, L. tropica and L. mexicana with all primers used, whereas 11 of the 18 primers distinguished L. infantum strains from the species L. donovani. All primers except 1 (TAF 300), generated polymorphism in the L. infantum strains. The dendrograms constructed with the isoenzyme data and with RAPD are congruent in relation to the separation of the different species but show little agreement within the L. infantum species, reflecting the genetic heterogeneity of the strains belonging to one zymodeme. A geographical structuralisation is observed with two diverging groups that evolve independently whereas there is no relation between the genotype of the parasite and the host or between the former and the clinical form of the disease.

Use of Noninvasive Markers To Detect Leishmania Infection in Asymptomatic Human Immunodeficiency Virus-Infected Patients

ABSTRACT Visceral leishmaniasis (VL) caused by Leishmania infantum is a common disease in human immunodeficiency virus (HIV)-infected people in the Mediterranean basin. However, most such cases are asymptomatic, and little information about the prevalence of these infections in HIV-infected individuals is available. The aim of this study was to assess the prevalence of subclinical infection and the relationship between several Leishmania infection markers by noninvasive methods in asymptomatic HIV-infected patients from Southern Spain. Ninety-two HIV-infected patients, who were consecutively attended at the participant hospitals in 2004, were invited to participate in this study. These patients were asymptomatic and without any history of cutaneous or visceral leishmaniasis. Leishmania kinetoplast DNA (kDNA) was amplified from peripheral blood samples from 28 (30.4%) of these HIV-infected subjects. Sera from three (3.5%) patients tested positive for Leishmania by an enzyme-linked immunoassay. Two patients (2.4%) showed a specific 16-kDa band by Western blotting. In contrast, none of the patients showed a positive agglutination of urine. The leishmanin skin test was positive for four (4.3%) patients. None of the patients with a PCR-positive result showed a positive reaction by enzyme-linked immunoassay or by specific bands in Western blotting or had a positive leishmanin skin test. In conclusion, L. infantum kDNA was detected in a large proportion of asymptomatic HIV-infected patients, although a demonstrable cellular or humoral immune response to this parasite was not shown. Conversely, Leishmania antigen in urine was not detected in these patients.