Application of qPCR method to hair and cerumen samples for the diagnosis of canine leishmaniosis in Araçatuba, Brazil.

Visceral leishmaniosis (VL) remains a serious public health problem in Brazil. Dogs are the main hosts of the parasite, developing canine leishmaniosis (CanL), hence the importance of an accurate diagnosis of the animals. Recently, the application of qPCR method to non-invasive samples obtained from dogs with CanL has shown high sensitivity. Thus, we analyzed by qPCR blood, hair (from healthy zones and cutaneous lesions) and cerumen of 16 dogs with confirmed leishmaniosis from Araçatuba, a Brazilian endemic area. Cerumen-qPCR showed the highest sensitivity (87.5%), followed by hair (lesions: 78.57%, healthy skin: 62.5%), and blood (68.75%). We also analyzed blood, hair and cerumen of 5 healthy dogs from a non-endemic area, obtaining 100% of specificity in all samples. The use of cerumen and hair for qPCR analysis provides high reliability, taking into account the sensitivity and total specificity of the method. The non-invasive sampling procedure without the need of specific conditions of storage and transport support the usefulness of hair and cerumen for the diagnosis of CanL.

First detection of Leishmania kDNA in canine cerumen samples by qPCR.

Nowadays, searching for alternative non-invasive methods for molecular diagnosis of canine visceral leishmaniosis is getting increasingly important. We previously described the presence of Leishmania kinetoplast DNA (kDNA) in canine hair; in this case we hypothesized whether foreign DNA might be present in cerumen of dogs with leishmaniosis, and be detected by Real time quantitative PCR (qPCR). A population of 38 dogs that lived in Leishmania endemic areas was divided in two groups: A (33 dogs with confirmed leishmaniosis by serological techniques) and B (5 healthy dogs). Blood, lymph node, bone marrow and cerumen samples from all animals were tested for the presence of parasite kDNA. Our method was 100% specific, and in dogs from group A, Leishmania infantum kDNA was detected and quantified in the 100% of lymph node samples, in 90.9% of cerumen samples, in 88.5% of the bone marrow samples and in 57.6% of the blood samples. The qPCR-cerumen is a new non-invasive method that shows a high potential for the diagnosis of zoonotic visceral leishmaniosis.

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.

First detection of Leishmania infantum kinetoplast DNA in hair of wild mammals: application of qPCR method to determine potential parasite reservoirs.

The data presented in this paper describe the application of a method for a reliable and non-invasive diagnosis of leishmaniosis in wild reservoirs, based on the detection of Leishmania infantum kinetoplast DNA (kDNA) in hair samples by Real Time PCR (qPCR). The study has been performed on 68 ear/leg hair samples from 5 different wild species (Vulpes vulpes, Canis lupus, Martes foina, Rattus norvegicus and Erinaceus europaeus) from several geographic areas of West and North Spain. The presence of Leishmania kDNA was detected in 14 of the 68 analyzed samples, being the highest quantity of DNA observed in foxes. This is the first report of the presence of Leishmania in a hedgehog. The kDNA remained stable under the exposure of hair to different environmental conditions (freezing or high temperature, ultraviolet rays or treatment with tanning salts). This detection method could constitute a suitable alternative for the search of the parasite in wild hosts, due to the numerous advantages that hair samples present for collection, transport and storage processes.

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.