Desarrollo de nuevos métodos no invasivos aplicados al diagnóstico mediante PCR a tiempo real de la leishmaniosis

En la actualidad el diagnóstico de la leishmaniosis está basado en una combinación de varios métodos: detección del parásito por microscopía, tests inmunológicos y técnicas moleculares como la PCR a tiempo real. El uso de esta última proporciona resultados reproducibles y muy sensibles. Las muestras utilizadas habitualmente para este tipo de análisis se obtienen de manera invasiva, causando estrés al animal y rechazo por parte de los propietarios. Por ello es importante el uso de métodos no invasivos como una alternativa para el diagnóstico de la leishmaniosis. En este trabajo se plantearon distintos objetivos enfocados a la evaluación del pelo, cerumen y queratinocitos epidérmicos de una población de perros con Lcan adquirida de forma natural, al estudio del pelo de diversas especies de animales silvestres y al análisis del pelo de ratones infectados experimentalmente con la especie de tropismo cutáneo Leishmania major, todo ello utilizando el método molecular qPCR. Los resultados obtenidos muestran por primera vez la detección y cuantificación del ADN del kinetoplasto (ADNk) de L. infantum en el pelo, cerumen y queratinocitos epidérmicos de perros con Lcan y en el pelo de diversas especies de mamíferos silvestres, junto la cronología de la incorporación del ADNk de L. major al pelo en un modelo ratón durante el curso de una infección experimental. En conclusión, se ha realizado una nueva aportación al diagnóstico molecular de la leishmaniosis mediante el desarrollo de métodos fiables y precisos empleando muestras no invasivas, aplicables en estudios epidemiológicos e infecciones experimentales.Clinical diagnosis of canine leishmaniosis is based on different methods: parasite detection through microscope, immunological tests and molecular techniques as Real Time PCR. The use of this method provides the ability to perform sensitive and reproducible analysis. The samples commonly used for this kind of analysis are obtained by invasive methods that cause animal distress and owner reluctance. Therefore, it is important to test non-invasive samples as an alternative for the diagnosis of leishmaniosis. Thus, the objectives of the work were, using the molecular method qPCR: to evaluate hair, cerumen and epidermal keratinocytes from a population of dogs with CanL naturally infected, to study hair from different wild animals and to analyze hair from mice experimentally infected with Leishmania major (cutaneous leishmaniosis model). The results obtained in this study show the first detection of L. infantum kinetoplast DNA (kDNA) in hair, cerumen and epidermal keratinocytes of dogs with CanL and in hair from different wild animals. It has been also determined the chronology of the appearance of L. major kDNA in the hair of a mouse model during a follow up period of infection. To conclude, the present work has made a new contribution to the molecular diagnosis of leishmaniosis by developing reliable and accurate methods using non-invasive samples, which can be applied in epidemiological studies and experimental infections

Detection of Leishmania infantum kinetoplast DNA by Real Time PCR in hair of wild rabbits

The study of potential wild mammal reservoirs is necessary for the surveillance of leishmaniosis, as Leishmania protozoans have been isolated from a wide range of wild and domestic animal species, including Leporidae. Recently, it has been demonstrated that both hares and wild rabbits can act as sylvatic reservoirs of Leishmania. In Spain, most of the research involving wild rabbits has been developed in the central area of Madrid and in the southeastern Mediterranean coast. We studied the presence of Leishmania infantum in 116 wild rabbits (Oryctolagus cuniculus) captured in Santovenia de Pisuerga, Valladolid, Spain. Hair samples were analyzed by real time PCR. L. infantum kinetoplast DNA (kDNA) was detected and quantified in 4 out of 116 analyzed animals. The estimated number of parasites obtained were quite variable, ranging from 2.60 to 276.60. Hair samples can be collected by non-invasive methods, being a proper sample for Leishmania detection in wild Leporidae, which have an important role as reservoirs of Leishmania. Our findings enhance the need for more extensive studies in different geographical areas.S

Detection of Leishmania infantum kinetoplast minicircle DNA by Real Time PCR in hair of dogs with leishmaniosis

It is known that hair can accumulate environmental toxics and excrete foreign chemical or biological substances. In this context, we hypothesized that foreign DNA could be found in the hair of an infected organism, and thus, be detected by Real Time PCR in the hair of Leishmania infantum naturally infected dogs. A population of 28 dogs living in Leishmania endemic areas was divided into two groups: A (13 Leishmania infected dogs) and B (15 healthy dogs). Blood, lymph node and ear hair samples from all of them were tested for the presence of parasite kinetoplast DNA (kDNA). For the same purpose, hair of several body areas and hair sections of two infected dogs were also analyzed. Epidermal keratinocytes from an infected animal were also analyzed for reactivity against Leishmania antigens by ELISA and for the presence of kDNA. Regarding to dogs from group A, parasite kDNA was detected in the 100% of lymph node samples. The sensitivity of Real Time PCR in ear hair was similar to that obtained in blood (9 positive out of 13 versus 8 positive out of 13, respectively). Moreover, the presence of L. infantum kDNA was also detected in the hair of all the analyzed body zones, in all hair sections and in epidermal keratinocytes. In infected dogs, parasite kDNA could be detected and quantified from just one single hair, whereas it was not detected in any of the samples of the healthy dogs. This work describes a new method for a reliable and non-invasive diagnosis of canine leishmaniosis using hair samples of infected animals. The data presented also provide some insights for the understanding of the physiology of keratinocytes and the role of hair as a specialized tissue in the kidnapping and removal of foreign DNA. © 2012 Elsevier B.V.Research agreement SGTRI 112/06 between LETI Laboratories S.L.U. and University of Extremadura (Spain)Peer Reviewe

Experimental model for reproduction of canine visceral leishmaniosis by Leishmania infantum

In this report an experimental model of Leishmania infantum (L. infantum) infection in dogs is described. The data presented are derived from an overall and comparative analysis of the clinical outcomes of three groups of dogs intravenously infected with 500,000 promastigotes on different dates (2003, 2006 and 2008). The parasites used for challenge were isolated from a dog having a patent form of leishmaniosis, classified as MCAN/ES/1996/BCN150 zymodeme MON-1. Late-log-phase promastigote forms derived from cultured amastigotes obtained from the spleen of the heavily infected hamsters were used for infection. Only one single infective dose was administered to each dog. After challenge, the animals were monitored for 12 months. To analyze the disease outcome, several biopathological, immunological and parasitological end-points were considered. The analysis of the infected dogs indicated that the development of the clinical disease was very similar in the three experimental challenges, as shown by the immune response, the parasite load and the clinical and histopathological lesions detected at necropsy. A high similarity was also observed between the disease development after the experimental challenge and the one reported to occur in endemic natural infection areas, as various degrees of susceptibility to the disease and even resistance were observed in the experimentally infected animals. We believe that this challenge model faithfully reproduces and mimics the course of a natural infection and that it could be used as a suitable tool for analyzing the efficacy of anti-Leishmania drugs and vaccines. © 2012 Elsevier B.V.Unit of Immunology and Vaccines for Global Health; Laboratorios LETI S.L.U. (Madrid, Spain)Peer Reviewe

Detection and chronology of parasitic kinetoplast DNA presence in hair of experimental Leishmania major infected BALB/c mice by Real Time PCR

Hair can accumulate foreign chemical or biological substances. Recently, it has been reported that parasite DNA can also be detected in the hair of Leishmania infantum infected dogs. The aim of this work has been to find out whether parasite DNA incorporates in the hair of Leishmania major experimentally infected animals. For this purpose, a group of 4 BALB/c mice, intradermally inoculated in both ears with 1000 L. major V1 strain promastigote forms, was monitored for parameters associated to the infection during 35 days. Weekly, ear swelling was measured, and hair samples from ears and leg were collected. Blood samples were obtained before challenge and at day 35 post infection, when parasite load was measured in ear, lymph node and spleen by limit dilution. Ear swelling and other parameters observed in the infected mice were consistent with those described for this model. The presence of parasite kinetoplast DNA (kDNA) was detected by Real Time PCR in all ear and leg hair samples at the final timepoint. These data suggests that hair is a specialized tissue in the sequestration and removal of foreign DNA. Detection of DNA in hair could be, therefore, a useful tool to chronologically record the infection process during experimental mice assays. © 2013 Elsevier B.V.Ministerio de Ciencia e Innovación (FIS PI11/00095); Instituto de Salud Carlos III within the Network of Tropical Diseases Research (RICETRD06/0021/0008); University of Extremadura, Spain (SGTRI112/06); Fundación Ramón ArecesPeer Reviewe

Strength and medium-term impact of HisAK70 immunization in dogs: Vaccine safety and biomarkers of effectiveness for ex vivo Leishmania infantum infection

HisAK70 candidates have successfully been tested in cutaneous (CL) and visceral leishmaniosis (VL) mouse models. Here, we analyse different biomarkers in dog trials after a heterologous immunization strategy with a HisAK70 candidate (plasmid DNA plus adoptive transfer of peripheral blood-derived dendritic cells (DCs) pulsed with the same pathoantigen and CpG ODN as an adjuvant) to explore the antileishmanial activity in an ex vivo canine co-culture system in the presence of Leishmania infantum parasites. In the canine model, the heterologous HisAK70 vaccine could decrease the infection index in the DC-T cell co-culture system by up to 54% after 30 days and reach almost 67% after 100 days post-immunization, respectively, compared to those obtained in the control group of dogs. The observed security and potential to fight ex vivo L. infantum infection highlight a HisAK70 heterologous immunization strategy as a promising alternative to evaluate its effectiveness against canine VL.Ministerio de Economía y Competitividad (España)Comunidad de MadridDepto. de Sanidad AnimalFac. de VeterinariaTRUEpu