V. Díaz-Sáez

Risk maps for the presence and absence of Phlebotomus perniciosus in an endemic area of leishmaniasis in southern Spain: implications for the control of the disease

The aim of this study was to construct risk maps for the presence of the dominant Leishmania infantum vector, P. perniciosus, and check its usefulness (a) to predict the risk of canine leishmaniasis and (b) to define effective leishmaniasis control measures. We obtained data for the presence/absence of P. perniciosus at 167 sampling sites in southern Spain, from which we also took a series of ecological and climate-related data. The probability of P. perniciosus presence was estimated as a function of these environmental variables and generated spatial risk maps. Altitude, land use and drainage hole features (with or without PVC piping) were retained as the only predictors for the distribution of this vector species. Drainage hole features in retaining walls, with or without PVC piping, produce significant variations in the probability of P. perniciosus presence, varying from 2·3 to 91·8% if PVC piping is absent and from 0·4 to 66·5% if all holes have PVC piping. It was concluded that the use of PVC piping in drainage holes could help to reduce leishmaniasis transmission.

Epidemiological implications of the use of various methods for the diagnosis of canine leishmaniasis in dogs with different characteristics and in differing prevalence scenarios

Our aim was to establish the influence on the prevalence of canine leishmaniasis (CanL) of the following: (1) the use of different diagnostic techniques; (2) different positivity thresholds; (3) selection of animals either at random from a population or focused on symptomatic individuals, (4) the function which the dog performs; and (5) scenarios with differing epidemiological characteristic. Three groups of dogs were analysed (416 sampled at random from an endemic area, 71 with symptomatology compatible with CanL and 15 from a non-endemic area) using three serological techniques (indirect fluorescence antibody test (IFAT), Kalazar DetectTM and Q Letitest ELISA) and a PCR-ELISA. The diagnostic technique had a considerable influence on the CanL prevalence value obtained. Uncertain antibody titres were more representative in dogs sampled at random and with the IFAT technique. Although employing different capture antigens, correlation of results was higher between the two commercial techniques in the group of dogs with symptomatology compatible with CanL. The sensitivity and specificity values of the different diagnostic techniques were affected by the epidemiological characteristic of the area under study, the presence of clinical signs and the function which the dog performs. This must be taken into account when comparing endemicity in different geographical areas, such as in studies carried out for the construction of risk maps. Using more than one technique, and adopting the criterion of considering an animal to be positive only when it has been diagnosed as such by more than one technique, considerably raises the prevalence values but maintains the differences between areas with different characteristics.

High rates of Leishmania infantum and Trypanosoma nabiasi infection in wild rabbits (Oryctolagus cuniculus) in sympatric and syntrophic conditions in an endemic canine leishmaniasis area: epidemiological consequences.

Leishmania infantum infection has been reported in various host species, both domestic and wild, in some cases with high prevalence rates. However, until the recent discovery of infected hares, no studies had provided clear evidence of any significant reservoir other than domestic dogs. Our focus was on another lagomorph, Oryctolagus cuniculus or wild rabbit. This species is native to the Iberian Peninsula and its presence and abundance gave rise to the name of Spain. In an endemic area for canine leishmaniasis in the southeast of Spain, 150 rabbits were captured over a period of three years. Samples of blood, bone marrow, liver, spleen, heart and skin were taken and analysed through parasitological, serological and molecular techniques in order to detect Leishmania and Trypanosoma. 20.7% of the rabbits were infected with L. infantum and 82.4% with Trypanosoma nabiasi, and 14.8% of mixed infections were detected. Both parasites were found in all the animal organs analysed, a factor which, along with the presence of serological cross-reactions, must be taken into account in epidemiological studies on leishmaniasis. O. cuniculus is an abundant and gregarious species, with a long enough average lifespan to ensure L. infantum transmission. The presence of the parasite in the skin and blood of these rabbits with no acute manifestation of disease ensures its contact with the vector, which finds in their warrens a suitable biotope to inhabit. The rabbit therefore seems to meet the most of conditions for being considered a reservoir host of L. infantum.

(−)-α-Bisabolol, a Promising Oral Compound for the Treatment of Visceral Leishmaniasis

The aim of the present study was to assess the in vitro and in vivo activity of (-)-α-bisabolol (1) against the etiological agents of visceral leishmaniasis. Bone-marrow-derived macrophages were infected with Leishmania infantum or L. donovani promastigotes and incubated with (-)-α-bisabolol at different concentrations. Pentamidine isethionate and meglumine antimoniate were used as reference drugs. Inhibitory concentration 50% (IC50) and cytotoxic concentration 50% (CC50) were calculated. Balb/c mice were infected intraperitoneally with stationary-phase promastigotes. They were treated with (-)-α-bisabolol at different doses orally, meglumine antimoniate at 104 mg Sb(V)/kg, or a combination of both. (-)-α-Bisabolol proved to be innocuous to mammal cells and active against L. infantum and L. donovani intracellular amastigotes (IC50 55 and 39 μM, respectively). Compound 1 also proved to be active in an in vivo model of visceral leishmaniasis due to L. infantum, as it reduced parasite load in the spleen and liver by 71.60% and 89.22%, respectively, at 200 mg/kg without showing toxicity. (-)-α-Bisabolol (1) is a nontoxic compound that was proven to be active against visceral leishmaniasis in an in vivo murine model orally. It was more effective than meglumine antimoniate at reducing spleen parasite load and as effective as this antimonial drug in the liver.

Predicting the risk of an endemic focus of Leishmania tropica becoming established in south-western Europe through the presence of its main vector, Phlebotomus sergenti Parrot, 1917

The aim of the study was the construction of risk maps for exposure to Phlebotomus sergenti, the main vector of Leishmania tropica, with a view to identifying hot spots for the potential establishment of this parasite in the southwest of Europe. Data were collected on the presence/absence of this vector and the ecological and climatic characteristics of 662 sampling sites located in the southeast, centre and northeast of the Iberian Peninsula (south-western Europe). The environmental factors associated with the distribution of P. sergenti were determined. The best predictors for the presence of this dipteran were ‘altitude’, ‘land use’, ‘land surface temperature’, ‘aspect’, ‘adjacent land cover’, ‘absence of vegetation in wall’ and the ‘absence of PVC pipes in the drainage holes of retaining walls’. Risk maps for exposure to the vector were drawn up based on these variables. The validation of the predictive risk model confirmed its usefulness in the detection of areas with a high risk of P. sergenti being present. These locations represent potential hot spots for an autochthonous focus of L. tropica becoming established. The risk maps produced for P. sergenti presence revealed several areas in the centre and south of the Iberian Peninsula to be the most prone to this process, which would make it possible for the disease to enter south-western Europe.

Topical Treatment of Leishmania tropica Infection Using (−)-α-Bisabolol Ointment in a Hamster Model: Effectiveness and Safety Assessment

There is currently no reliable treatment for the management of cutaneous leishmaniasis, and intralesional antimonial injections remain the main treatment. The present work aims at evaluating the antileishmanial effectiveness and safety of (-)-α-bisabolol (1) in a novel topical formulation on a cutaneous leishmaniasis model involving Leishmania tropica-infected Syrian hamsters. The topical treatment with 1 reduced lesion thickness to 56% at 2.5%, showing a higher efficacy than the reference control, meglumine antimoniate. Other regimens (ointment at 1% and 5% and oral treatment at 200 mg/kg) reduced the footpad thickness as well. The skin parasite load decreased after the experiment in all treatment groups, particularly in those animals treated with the 2.5% formulation (83.2%). Treatment with (-)-α-bisabolol at different concentrations or through an oral route did not lead to the appearance of toxicity or side effects in healthy hamsters or infected animals. Therefore, topical (-)-α-bisabolol was more effective than meglumine antimoniate in this cutaneous leishmaniasis model without showing toxicity effects on the hamsters. These results are of great interest and might be used for the development of alternatives for the treatment of cutaneous leishmaniasis, either in monotherapy or in combination with other drugs whose skin permeability could be enhanced by this sesquiterpene.

Hair parasite load as a new biomarker for monitoring treatment response in canine leishmaniasis

Canine leishmaniasis treatment focuses on the reduction of parasite load, the clinical improvement of the animal, and the avoidance of relapses, in a scenario where the definitive parasite clearance is not achievable. Therefore, monitoring is crucial during the treatment of this disease. Quantitative PCR has been shown as an ideal tool for the treatment monitoring when quantifying parasite load in target organs such as lymph node or bone marrow, tissues that are too invasive for regular evaluation. This study aims to prove the potential of hair parasite load in the treatment monitoring of canine leishmaniasis. Six dogs were treated with meglumine antimoniate and monitored up to four months after the end of the treatment. Parasite loads in bone marrow, blood, lymph node and hair were quantified by real-time quantitative PCR. Total IgG, IgG1, and IgG2 antibody titres were analysed by immunofluorescent assay and a clinical assessment was carried out. Treatment consisted of two 28-day courses of meglumine antimoniate (100mg/kg/day) separated by an one-month interval. Analyses were performed before (day 0), during (day 60) and after treatment (day 120), and at the end of a follow-up period (day 210, four months after the end of treatment). Hair parasite load turned out to be strongly correlated with bone marrow, lymph node and blood parasite loads and with the clinical score and the IgG1 antibody titre. The evolution of this biomarker reflects the evolution of the parasitological, immunological and clinical state of the dog, highlighting its potential as a non-invasive marker for the treatment monitoring in canine leishmaniasis.

SPERMATOGENESIS IN THE IXODID TICK HAEMAPHYSALIS (HERPETOBIA) SULCATA (ACARINA: IXODIDAE)

Haemaphysalis sulcata (Acarina, Ixodidae) is a common ectoparasite of wild artiodactyls (mouflon and wild goat) in Sierra Nevada (Granada, southern Spain). A study of the spermatogonial meiosis of 15 H. sulcata males was carried out. The diploid complement is 2n = 21 and its sex determination is XX:XO. The behavior of the chromosomes in the different stages of meiosis was also investigated, and the possible presence of a secondary nucleolar organizer region in H. sulcata is discussed.

Differential ecological traits of two Phlebotomus sergenti mitochondrial lineages in southwestern Europe and their epidemiological implications

The introduction of leishmaniasis in a new area requires a well‐established population of the sandfly vector species of the parasite. No autochthonous cases of anthroponotic cutaneous leishmaniasis have been detected in southwestern Europe, and Leishmania infantum is the only causative agent of leishmaniasis in this area. Phlebotomus sergenti, the main vector of Leishmania tropica, is commonly found in the Iberian Peninsula at sufficient densities to be able to act as a vector. It is characterised by high genetic diversity and classified in four mitochondrial lineages. Our aim was to analyse the composition and distribution of P. sergenti mitochondrial lineages in southwestern Europe given the possibility of phenotypic differences of biomedical importance between them.

Comparison of PCR-based methods for the diagnosis of cutaneous leishmaniasis in two different epidemiological scenarios: Spain and Morocco

Cutaneous leishmaniasis (CL) is a disfiguring and stigmatising disease occurring in more than 70 countries across the world including Spain and Morocco. The use of sensitive tests that can differentiate Leishmania species is advised.