Natural canine infection by Leishmania infantum and Leishmania amazonensis and their implications for disease control

Sanches, Letícia da Cruz; Martini, Cristina; Nakamura, Alex Akira; Santiago, Maria Emília Bodini; Lima, Beatriz Dolabela de; Lima, Valéria Marçal Felix de

doi:10.1590/s1984-29612016071

Cited by 24 publications

(20 citation statements)

References 27 publications

(25 reference statements)

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“…Notably, Sanches et al [28] showed a high percentage of L. ( L. ) amazonesis infection in naturally infected dogs in an endemic area, underlining the necessity to discriminate these two species not only in human but also in veterinary medicine. Since HRM analysis resulted inconclusive for discrimination of L. ( L. ) infantum and L. ( L. ) amazonensis in about 53% of samples, a new SYBR-green qPCR assay (qPCR-ama) was designed to amplify a minicircle subclass preponderant in L. ( L. ) amazonensis, rather than targeting a hypothetical species-specific sequence.…”

Section: Discussionmentioning

confidence: 99%

The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis

et al. 2017

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BackgroundLeishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area.MethodsDNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed.ResultsThe qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the Cq values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of Cq values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples.ConclusionsA new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2181-x) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis

et al. 2017

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“…Leishmania species compatibility was evaluated according to a previously described melting curve analysis [19], and subgenus confirmation was performed using a conventional PCR to amplify fragments of the ITS1 region, followed by restriction fragment length polymorphism (PCR-RFLP) analysis by HaeIII digestion as previously described [22,23]. All reactions were performed by a researcher blinded to the other exams and to the patients' clinical conditions.…”

Section: Index Test (Qualitative and Quantitative Qpcr)mentioning

confidence: 99%

Accuracy of qPCR for quantifying Leishmania kDNA in different skin layers of patients with American tegumentary leishmaniasis

Sevilha-Santos

Júnior

Medeiros-Silva

et al. 2019

Clinical Microbiology and Infection

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“…Determination of the Leishmania species was performed by PCR-RFLP (Restriction Fragment Length Polymorphism) [20], comparing the restriction profiles of the sample with a PCR restriction profile obtained from L . infantum (IOC / L0575-MHOM / BR / 2002 / LPC-RPV), L .…”

Section: Methodsmentioning

confidence: 99%

Relationship of peripheral blood mononuclear cells miRNA expression and parasitic load in canine visceral leishmaniasis

et al. 2018

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Visceral leishmaniasis (VL) in humans is a chronic and often fatal disease if left untreated. Dogs appear to be the main reservoir host for L. infantum infection, however, in many regions other canids such as jackals, foxes, wolves and other mammals, such as hares or black rats, have been implicated as wild reservoirs. Most dogs cannot form an effective immune response against this infection, and this could be modulated by small non-coding RNAs, called microRNAs, responsible for post-transcriptional control of gene expression. Here, we evaluated the expression of miRNAs in peripheral blood mononuclear cells (PBMC) of symptomatic dogs naturally infected with Leishmania (L.) infantum (n = 10) and compared to those of healthy dogs (n = 5). Microarray analysis revealed that miR-21, miR-424, miR-194 and miR-451 had a 3-fold increase in expression, miR-192, miR-503, and miR-371 had a 2-fold increase in expression, whereas a 2-fold reduction in expression was observed for miR-150 and miR-574. Real-time PCR validated the differential expression of miR-21, miR-150, miR-451, miR-192, miR-194, and miR-371. Parasite load of PBMC was measured by real-time PCR and correlated to the differentially expressed miRNAs, showing a strong positive correlation with expression of miR-194, a regular positive correlation with miR-371 expression, and a moderate negative correlation with miR-150 expression in PBMC. These findings suggest that Leishmania infection interferes with miRNAs expression in PBMC, and their correlation with parasite load may help in the identification of therapeutic targets in Canine Visceral Leishmaniasis (CVL).

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Natural canine infection by Leishmania infantum and Leishmania amazonensis and their implications for disease control

Cited by 24 publications

References 27 publications

The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis

The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis

Accuracy of qPCR for quantifying Leishmania kDNA in different skin layers of patients with American tegumentary leishmaniasis

Relationship of peripheral blood mononuclear cells miRNA expression and parasitic load in canine visceral leishmaniasis

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