Morphology does not allow differentiating the species of the Phlebotomus perniciosus complex: Molecular characterization and investigation of their natural infection by Leishmania infantum in Morocco

Morphological and DNA-based complemented approaches were applied for characterization of sympatric populations of Phlebotomus longicuspis and Phlebotomus perniciosus in Morocco. Both sand fly species are generally recorded in sympatry in North Africa but on few occasions have been molecularly characterized. The diagnostic confusion of these species has led to errors in their geographical distribution and probably, in the assignment of their role in the transmission of L. infantum. Sand flies were caught inside households in El Borouj, central Morocco, in 2014–2015. For female sand flies, detection of L. infantum natural infection and blood meal identification were carried out. According to morphological identification, Phlebotomus longicuspis s.l. (34.7%) was the second most abundant Phlebotomus species after P. sergenti, followed by atypical Phlebotomus perniciosus (7.1%); 11.6% of the male specimens of P. longicuspis s.l. were identified as P. longicuspis LCx according to the number of coxite setae. The density of Larroussius species was very high (31 Larroussius/light trap/night) in the peripheral neighbourhood of Oulad Bouchair (p = 0.001) where the first case of cutaneous leishmaniasis due to Leishmania infantum was detected in 2017. Phylogenetic trees based on three independent genes highlighted three well-supported clusters within P. perniciosus complex that could be interpreted as corresponding to P. perniciosus, P. longicuspis s.s. and an undescribed species, all coexisting in sympatry. Some females with typical morphology of P. longicuspis were genetically homologous to P. perniciosus. The taxa cannot be differentiated by morphological methods but characterized by a distinctive genetic lineage for which the synapomorphic characters are described. Leishmania infantum was detected in females of all clusters with a low parasite load. Population genetics will help to assess the threat of the geographical spreadUniversity of Granada (Centro de Iniciativas de Cooperación al Desarrollo, CICODE, 2013)Universidad de Granada/CBU

Role of wild rabbits as reservoirs of leishmaniasis in a non-epidemic Mediterranean hot spot in Spain

We would like to thank the hunters of the municipality of Montefrio for their support in the study. This study was funded by the Project, Ministry of Economy and Competitiveness, Instituto de Salud Carlos III, Madrid and Feder Funds for Regional Development from the European Union, "One way to make Europe". Funding for open access charge: Universidad de Granada/CBVA.There is limited information regarding the role of wild mammals in the transmission dynamics of Leishmania infantum. A potential human leishmaniasis hot spot was detected in southern Spain that could not be explained solely by canine leishmaniasis prevalence. The aim of this work was to analyse the involvement of wild rabbits as the main factor affecting this Mediterranean hot spot. A survey of wild rabbits, dogs and sand flies was conducted in the human cases environment. A nearby region without clinical leishmaniasis cases was used as reference control. 51 wild rabbits shot by hunters were analysed by molecular techniques. 1100 sand flies were captured and morphologically identified. Blood collected from patients’ relatives/ neighbours (n = 9) and dogs (n = 66) was used for molecular analysis and serology. In Mediterranean leishmaniasis hot spots such as Montefrío municipality (average incidence of 16.8 human cases per 100,000 inhabitants/year), wild rabbits (n = 40) support high L. infantum infection rates (100%) and heavy parasite burdens (average value: 503 parasites/mg) in apparently normal ear skin directly accessible to sand flies, enabling the existence of heavily parasitized Phlebotomus perniciosus females (12.5% prevalence). The prevalence of infection and median parasite load were very low among rabbits captured in Hu´escar (n = 11), a human clinical leishmaniasis-free area for the last 18 years. P. perniciosus was the most abundant Phlebotomus species in all the domestic/peridomestic microhabitats sampled, both indoors and outdoors. Accordingly, leishmaniasis is clustering in space and time at this local scale represented by Montefrío due to the proximity of two competent host reservoirs (dogs and heavily parasitized wild rabbits) associated with overlapping sylvatic and domestic transmission cycles through the main vector, P. perniciosus. We highlight the usefulness of determining the prevalence of infection and parasite burden in wild rabbits as a control leishmaniasis measure with the advantage that the use of the ear offers.Ministry of Economy and Competitiveness, Instituto de Salud Carlos III, MadridFeder Funds for Regional Development from the European Union, "One way to make Europe"Universidad de Granada/CBV

Leishmaniasis vectors in the environment of treated leishmaniasis cases in Spain

Transmission of leishmaniasis in endemic areas is characterized by microfocality related to the presence of the vector. Most entomological studies in southwestern Europe have focused on sylvatic areas and town outskirts, very few have sampled town or urban centres, and no survey has investigated inside households. The aim of this study was to determine the sand fly species diversity and vector density in the surroundings of human leishmaniasis cases compared with environments in which there was no association. Sand flies were captured in 26 households associated with recently treated leishmaniasis patients, 15 neighbouring houses without associated cases, and in others environments. Overall 7495 sand flies belonging to six species were captured. The highest sand fly density was found in farmhouses where there is a great availability of blood sources and breeding sites. In the environment of human leishmaniasis cases, Sergentomyia minuta was the most prevalent species followed by Phlebotomus perniciosus. Nevertheless, lower Leishmania infantum infection rates and lower intensity of infection were detected in S. minuta sand flies than in P. perniciosus. The density of P. perniciosus in households with recently treated leishmaniasis patients varies between 0 and 108 sand flies per light trap/night, with the maximum values corresponding to farmhouses. This species appears to be adapted to both indoors and outdoors domestic biotopes, including urban households

Wild rabbits are Leishmania infantum reservoirs in southeastern Spain

We contribute to the understanding of the transmission dynamics of Leishmania infantum suggesting the involvement of rabbits as wild reservoirs. Results: The prevalence of infection was 86.0% (270/314 wild rabbits) ranging from 18.2% to 100% in natural geographical regions. The estimated average parasite load was 324.8 [CI 95% 95.3–554.3] parasites per mg of ear lobe ranging from 0 to 91,597 parasites/mg per tissue section. Conclusions: A positive correlation was found between skin parasite load in wild rabbits and human incidence with evidence of the presence of the same L. infantum genotypes in rabbits and humans, providing new epidemiological and biological basis for the consideration of wild rabbits as a relevant L. infantum wild reservoir. Molecular parasite surveillance reflects the great genotypic variability of the parasite population in wild rabbits. Most of these genotypes have also been found to infect humans, dogs and sandflies in the region. Our findings also highlight that direct genotyping of the parasite in host tissues should be used for molecular surveillance of the parasite instead of cultured isolates.Ministerio de Ciencia e Innovación, Spain (MICIU/AEI/10.1339/50110011033)FEDER, UE through the project, Grant/ Award Number: PID2022-142230NB. 100Universidad de Granada/CBU

Effectiveness of an O-Alkyl Hydroxamate in Dogs with Naturally Acquired Canine Leishmaniosis: An Exploratory Clinical Trial

Canine leishmaniosis is a challenge in veterinary medicine and no drug to date has achieved parasite clearance in dogs. Histone deacetylase inhibitors are a drug class widely used in cancer chemotherapy. We have successfully used O-alkyl hydroxamates (vorinostat derivatives) in the treatment of a laboratory model of visceral leishmaniasis without showing toxicity. In order to test the effectiveness of a particular compound, MTC-305, a parallel-group, randomized, singlecentre, exploratory study was designed in naturally infected dogs. In this clinical trial, 18 dogs were allocated into 3 groups and were treated with either meglumine antimoniate (104 mg SbV/kg), MTC-305 (3.75 mg/kg) or a combination of both using a lower MTC-305 dose (1.5 mg/kg) through a subcutaneous route for 2 treatment courses of 30 days, separated by a 30-day rest period. After treatment, a follow-up time of 4 months was established. Parasite burden in bone marrow, lymph node and peripheral blood were quantified through qPCR. Antibody titres were determined through an immunofluorescence antibody test, and cytokine expression values were calculated through RTqPCR. Treatment safety was evaluated through the assessment of haematological and biochemical parameters in blood, weight, and gastrointestinal alterations. Assessment was carried out before, between and after treatment series. Treatment with MTC-305 was effective at reducing parasite burdens and improving the animals’ clinical picture. Dogs treated with this compound did not present significant toxicity signs. These results were superior to those obtained using the reference drug, meglumine antimoniate, in monotherapy. These results would support a broader clinical trial, optimised dosage, and an expanded follow-up stage to confirm the efficacy of this drug.University of GranadaAndalusian GovernmentEuropean Commission B-CTS-270UGR18 P20_0013

Decreased antimony uptake and overexpression of genes of thiol metabolism are associated with drug resistance in a canine isolate of Leishmania infantum

Visceral leishmaniasis (VL) caused by the protozoan parasite Leishmania infantum, is one of the most important zoonotic diseases affecting dogs and humans in the Mediterranean area. The presence of infected dogs as the main reservoir host of L. infantum is regarded as the most significant risk for potential human infection. We have studied the susceptibility profile to antimony and other anti-leishmania drugs (amphotericin B, miltefosine, paromomycin) in Leishmania infantum isolates extracted from a dog before and after two therapeutic interventions with meglumine antimoniate (subcutaneous Glucantime®, 100 mg/kg/day for 28 days). After the therapeutic intervention, these parasites were significantly less susceptible to antimony than pretreatment isolate, presenting a resistance index of 6-fold to SbIII for promastigotes and >3-fold to SbIII and 3-fold to SbV for intracellular amastigotes. The susceptibility profile of this resistant L. infantum line is related to a decreased antimony uptake due to lower aquaglyceroporin-1 expression levels. Additionally, other mechanisms including an increase in thiols and overexpression of enzymes involved in thiol metabolism, such as ornithine decarboxylase, trypanothione reductase, mitochondrial tryparedoxin and mitochondrial tryparedoxin peroxidase, could contribute to the resistance as antimony detoxification mechanisms. A major contribution of this study in a canine L. infantum isolate is to find an antimony-resistant mechanism similar to that previously described in other human clinical isolates.This work was supported by the Spanish Grants Proyecto de Excelencia, Junta de Andalucía Ref. CTS-7282 (to F.G.), SAF2012-34267 (to F.G.), FEDER funds from the EU to S.C. and F.G., and by the University of Granada (CEI-Biotic project 2013/1/4 and Pilot Prototypes and Experiences PR/12/011)Peer reviewe

Nuevas perspectivas en el tratamiento de la Leishmaniosis: Evaluación de derivados hidroxámicos y (-)-α-bisabolol

The aim of this work was to put forward new treatment perspectives for leishmaniasis. In order to accomplish it, the objectives of the study were to screen new synthetic HDAC inhibitors and the natural compound (-)-α-bisabolol, to characterize HDAC enzymes in Leishmania, to get an insight into the mechanism of action of (-)-α-bisabolol, to evaluate in vivo the most active structures; and finally, to assess the potential effectiveness and safety of these molecules in a pilot clinical trial involving dogs with canine leishmaniasis (CanL) naturally acquired.Tesis Univ. Granada. Departamento de ParasitologíaEste trabajo de tesis doctoral ha sido subvencionado por: El grupo de investigación de la Junta de Andalucía BIO-176 El proyecto del Ministerio “Sobre el posible establecimiento de Leishmania tropica en España y la expansión altitudinal de los vectores de Leishmania infantum en el sudeste de España” del Ministerio de Ciencia e Innovación (CGL2010-22368-C02.02), de 3 años de duración (2011-2013). El proyecto “Valoración de un nuevo inhibidor de la histona desacetilasa con actividad leishmanicida in vitro en el tratamiento de la leishmaniosis experimental en modelos animales”, del subprograma GREIB Translational Projects de la Universidad de Granada, de un año de duración (2011). El proyecto “Síntesis y evaluación de nuevos inhibidores de las histona desacetilasas” del CEI-Biotic Granada, de un año de duración (2012). El proyecto “Evaluación de la eficacia y seguridad de un nuevo fármaco antiparasitario” del CEI-Biotic Granada, de un año de duración (2013). El Programa de Ayudas a la Transferencia de Investigación, de la Oficina de Transferencia de Resultados de Investigación de la Universidad de Granada, a través de la concesión de ayudas para la Realización de Prototipos y Experiencias Piloto (2013)

Phlebotomus langeroni Nitzulescu (Diptera, Psychodidae) a new vector for Leishmania infantum in Europe

Burrows of the wild rabbit, Oryctolagus cuniculus, a lagomorph that has been recently suggested as a Leishmania infantum reservoir, constitute an unspoilt biotope in phlebotomine studies in Europe. We hypothesize that Phlebotomus langeroni, a proven vector of L. infantum in North Africa, is associated with rabbits and may have been overlooked in Europe. Sandfly captures were carried out with CDC light traps in an L. infantum endemic area of southern Spain with a high density of lagomorphs and a large numbers of burrows. The stable, permanent, and highly abundant presence of P. langeroni was assessed. After morphological identification, this sandfly species was characterized by comparing it with P. perniciosus and other P. langeroni populations from North Africa through molecular techniques. P. langeroni had not been found in southern Spain to date, despite being a highly investigated area, except for this particular biotope. Its activity period turned out to begin in mid- July, ending in late October, accounting for a maximum activity during this month. This study shows that P. langeroni is associated with the existence of rabbit burrows and has been overlooked in Europe. L. infantum DNA was found in almost half of the female specimens (47.6%) captured inside a biotope where wild rabbits are infected as wel

Implementación y evaluación de un curso práctico piloto para la captura y recolección de parásitos en grados que contemplen la parasitología como estrategia para innovar en el proceso enseñanza-aprendizaje de esta disciplina

Afrontamos el reto de innovar en la enseñanza-aprendizaje de la Parasitología diseñando una metodología adaptada al nuevo enfoque del EESS, buscando: la participación activa del estudiante, la adquisición y evaluación de competencias y la retroalimentación. Las enseñanzas prácticas de la Parasitología son deficitarias en recogida de muestras y sin embargo estas funciones las realizan nuestros egresados. El modelo de enseñanza/aprendizaje que proponemos supone la realización de tareas fuera de las aulas, proporcionando a los alumnos experiencias de aprendizaje enriquecedoras. Acercamos a los alumnos al entorno de los parásitos y buscamos su competentencia en la toma de muestras en diferentes contexto