Detection and identification of Leishmania species within naturally infected sand flies in the Andean areas of Ecuador by a polymerase chain reaction

The surveillance of prevalent Leishmania and sand fly species in endemic areas is important for prediction of the risk and expansion of leishmaniasis. In this study, we developed a polymerase chain reaction (PCR)-based method for detection of Leishmania minicircle DNA within individual sand flies. Using this method, we detected minicircle DNA in 6 (3.3%) of 183 sand flies, while 5 (3.5%) of 143 were positive for Leishmania promastigotes in the same areas by microscopic examination. The species were identified as Leishmania (Leishmania) mexicana by nucleotide sequencing of the cytochrome b gene. Additionally, all the Leishmania-positive sand flies were identified as Lutzomyia ayacuchensis by the restriction enzyme digestion of the PCR-amplified 18S ribosomal RNA gene fragments. Since this combined method is relatively easy and can process a large number of samples, it will be a powerful tool for the rapid identification of prevalent sand fly and Leishmania species as well as monitoring the infection rate in sand fly populations in endemic areas.Fil: Kato, Hirotomo. Yamaguchi University; JapónFil: Uezato, Hiroshi. University of the Ryukyus; JapónFil: Katakura, Ken. Hokkaido University; JapónFil: Calvopina, Manuel. Kochi University. Kochi Medical School; JapónFil: Marco, Jorge Diego. Kochi University. Kochi Medical School; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Barroso, Paola Andrea. Kochi University. Kochi Medical School; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Gomez, Eduardo. Universidad Católica de Guayaquil; EcuadorFil: Mimori, Tatsuyuki. Kumamoto University; JapónFil: Korenaga, Masataka. Kochi University. Kochi Medical School; JapónFil: Iwata, Hiroyuki. Yamaguchi University; JapónFil: Nonaka, Shigeo. University ok the Ryukyus; JapónFil: Hashiguchi, Yoshihisa. Kochi University. Kochi Medical School; Japó

A mouse model offers novel insights into the myopathy and tendinopathy often associated with pseudoachondroplasia and multiple epiphyseal dysplasia

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are relatively common skeletal dysplasias belonging to the same bone dysplasia family. PSACH is characterized by generalized epi-metaphyseal dysplasia, short-limbed dwarfism, joint laxity and early onset osteoarthritis. MED is a milder disease with radiographic features often restricted to the epiphyses of the long bones. PSACH and some forms of MED result from mutations in cartilage oligomeric matrix protein (COMP), a pentameric glycoprotein found in cartilage, tendon, ligament and muscle. PSACH-MED patients often have a mild myopathy characterized by mildly increased plasma creatine kinase levels, a variation in myofibre size and/or small atrophic fibres. In some instances, patients are referred to neuromuscular clinics prior to the diagnosis of an underlying skeletal dysplasia; however, the myopathy associated with PSACH-MED has not previously been studied. In this study, we present a detailed study of skeletal muscle, tendon and ligament from a mouse model of mild PSACH harbouring a COMP mutation. Mutant mice exhibited a progressive muscle weakness associated with an increased number of muscle fibres with central nuclei at the perimysium and at the myotendinous junction. Furthermore, the distribution of collagen fibril diameters in the mutant tendons and ligaments was altered towards thicker collagen fibrils, and the tendons became more lax in cyclic strain tests. We hypothesize that the myopathy in PSACH-MED originates from an underlying tendon and ligament pathology that is a direct result of structural abnormalities to the collagen fibril architecture. This is the first comprehensive characterization of the musculoskeletal phenotype of PSACH-MED and is directly relevant to the clinical management of these patients

Mild myopathy is associated with COMP but not MATN3 mutations in mouse models of genetic skeletal diseases.

<div><p>Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are skeletal disorders resulting from mutations in COMP, matrilin-3 or collagen IX and are characterised by short-limbed dwarfism and premature osteoarthritis. Interestingly, recent reports suggest patients can also manifest with muscle weakness. Here we present a detailed analysis of two mouse models of the PSACH/MED disease spectrum; ΔD469 T3-COMP (PSACH) and V194D matrilin-3 (MED). In grip test experiments T3-COMP mice were weaker than wild-type littermates, whereas V194D mice behaved as controls, confirming that short-limbed dwarfism alone does not contribute to PSACH/MED-related muscle weakness. Muscles from T3-COMP mice showed an increase in centronuclear fibers at the myotendinous junction. T3-COMP tendons became more lax in cyclic testing and showed thicker collagen fibers when compared with wild-type tissue; matrilin-3 mutant tissues were indistinguishable from controls. This comprehensive study of the myopathy associated with PSACH/MED mutations enables a better understanding of the disease progression, confirms that it is genotype specific and that the limb weakness originates from muscle and tendon pathology rather than short-limbed dwarfism itself. Since some patients are primarily diagnosed with neuromuscular symptoms, this study will facilitate better awareness of the differential diagnoses that might be associated with the PSACH/MED spectrum and subsequent care of PSACH/MED patients.</p> </div

Analysis of salivary gland transcripts of the sand fly Lutzomyia ayacuchensis, a vector of Andean-type cutaneous leishmaniasis

The saliva of blood sucking insects contains potent pharmacologically active components that assist them in counteracting the host hemostatic and inflammatory systems during blood feeding. In addition, sand fly salivary proteins affect host immunity and have the potential to be a vaccine against Leishmania infection. In the present study, the salivary gland transcripts of Lutzomyia (Lu.) ayacuchensis, a vector of cutaneous leishmaniasis in Ecuadorian and Peruvian Andes, were analyzed by sequencing randomly selected clones of the salivary gland cDNA library of this sand fly. This resulted in the identification of the most abundant transcripts coding for secreted proteins. These proteins were homologous to the salivary molecules present in other sand flies including the RGD-containing peptide, PpSP15/SL1 family protein, yellow-related protein, putative apyrase, antigen 5-related protein, D7 family protein, and 27 kDa salivary protein. Of note, homologues of maxadilan, an active vasodilator abundantly present in saliva of Lu. longipalpis, were not identified. This analysis is the first description of salivary proteins from a sand fly of the subgenus Helcocyrtomyia and from vector of cutaneous leishmaniasis in the New World. The present analysis will provide further insights into the evolution of salivary components in blood sucking arthropods

Genetic diversity of the mitochondrial cytochrome b gene in Lutzomyia spp., with special reference to Lutzomyia peruensis, a main vector of Leishmania (Viannia) peruviana in the Peruvian Andes

The genetic divergence caused by genetic drift and/or selection is suggested to affect the vectorial capacity and insecticide susceptibility of sand flies, as well as other arthropods. In the present study, cytochrome b (cyt b) gene sequences were determined in 13 species circulating in Peru to establish a basis for analysis of the genetic structure, and the intraspecific genetic diversity was assessed in the Lutzomyia (Lu.) peruensis, a main vector species of Leishmania (Viannia) peruviana in Peruvian Andes. Analysis of intraspecific genetic diversity in the cyt b gene sequences from 36 Lu. peruensis identified 3 highly polymorphic sites in the middle region of the gene. Haplotype and gene network analyses were performed on the cyt b gene sequences of 130 Lu. peruensis in 9 Andean areas from 3 Departments (Ancash, Lima and La Libertad). The results showed that the populations of La Libertad were highly polymorphic and that their haplotypes were distinct from those of Ancash and Lima, where dominant haplotypes were observed, suggesting that a population bottleneck may have occurred in Ancash and Lima, but not in La Libertad. The present study indicated that the middle region of the cyt b gene is useful for the analysis of genetic structure in sand fly populations

Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection

Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1 h from a crude sand fly template without DNA purification. Amplicon detection could be accomplished by the newly developed colorimetric malachite green (MG)—mediated naked eye visualization. Pre-addition of MG to the LAMP reaction solution did not inhibit amplification efficiency. The field applicability of the colorimetric MG-based LAMP assay was demonstrated with 397 field-caught samples from the endemic areas of Ecuador and eight positive sand flies were detected. The robustness, superior sensitivity, and ability to produce better visual discriminatory reaction products than existing LAMP fluorescence and turbidity assays indicated the field potential usefulness of this new method for surveillance and epidemiological studies of leishmaniasis in developing countries

Population genetics of Leishmania (Leishmania) major DNA isolated from cutaneous leishmaniasis patients in Pakistan based on multilocus microsatellite typing

Background: Cutaneous leishmaniasis (CL) is a major and fast increasing public health problem, both among the local Pakistani populations and the Afghan refugees in camps. Leishmania (Leishmania) major is one of the etiological agents responsible for CL in Pakistan. Genetic variability and population structure have been investigated for 66 DNA samples of L. (L.) major isolated from skin biopsy of CL patients. Methods: Multilocus microsatellite typing (MLMT), employing 10 independent genetic markers specific to L. (L.) major, was used to investigate the genetic polymorphisms and population structures of Pakistani L. (L.) major DNA isolated from CL human cases. Their microsatellite profiles were compared to those of 130 previously typed strains of L. (L.) major from various geographical localities. Results: All the markers were polymorphic and fifty-one MLMT profiles were recognized among the 66 L. (L.) major DNA samples. The data displayed significant microsatellite polymorphisms with rare allelic heterozygosities. A Bayesian model-based approach and phylogenetic analysis inferred two L. (L.) major populations in Pakistan. Thirty-four samples belonged to one population and the remaining 32 L. (L.) major samples grouped together into another population. The two Pakistani L. (L.) major populations formed separate clusters, which differ genetically from the populations of L. (L.) major from Central Asia, Iran, Middle East and Africa. Conclusions: The considerable genetic variability of L. (L.) major might be related to the existence of different species of sand fly and/or rodent reservoir host in Sindh province, Pakistan. A comprehensive study of the epidemiology of CL including the situation or spreading of reservoirs and sand fly vectors in these foci is, therefore, warranted

Quantification of the muscle fibers with central nuclei at 3 weeks of age.

<p>Quantification based on images obtained using the modified Gomori trichrome stain on transverse sections of mouse legs at 3 weeks of age. Inset shows an example of stained tissue skeletal muscle is stained red, collagenous tissue blue, nuclei black - yellow arrow indicates a muscle fibre with central nucleus. Mutant COMP mice (<b>A</b>) have more muscle fibers with central nuclei around the myotendinous and perimysial junction than their wild type littermates. Matrilin 3 mutant mice (<b>B</b>) show similar distribution of fibers with central nuclei as their wild type littermates. Error bars; SEM (standard error of the mean; n≥3). Key: * P<0.05, ** P<0.01, *** P<0.001.</p

Grip strength measurement at 3 and 9 weeks of age.

<p>COMP mutant mice (<b>A</b>) become weaker with age compared to their littermates whereas the grip strength of matrilin 3 mutant mice (<b>B</b>) remains similar to the wild type littermates at all ages. Error bars; SEM (standard error of the mean; n≥10). Key: * P<0.05, ** P<0.01, *** P<0.001.</p

Genotyping of sand fly species in Peruvian Andes where leishmaniasis is endemic

Genotyping of sand fly species circulating in Peru was established on the basis of PCR-restriction fragment length polymorphisms (RFLPs) of the 18S ribosomal RNA (rRNA) gene. The sequences of 18S rRNA gene fragments from 12 Lutzomyia and 1 Warileya species were determined and their RFLP-patterns were analyzed. Consequently, RFLP analysis with the restriction enzyme AfaI and then HapII or KpnI, followed by XspI successfully differentiated them. Intraspecific genetic diversity affecting RFLP-patterns was not detected in the specimens collected from 24 areas of 8 departments. The genotyping was applied to the surveillance of sand flies collected from Andean areas where leishmaniasis is endemic, and its usability was verified. The present method promises to be a powerful tool for the classification and surveillance of sand flies circulating in Peru