Viable Leishmania parasites in the absence of an in vitro IFN-γ response in asymptomatic carriers.

Asymptomatic infection (the absence or inapparent signs and symptoms) has been observed in many endemic areas of leishmaniasis, however, little is known about the parasitological and immunological factors associated with this type of infection. This study aimed to identify the in vitro expression of IFN-γ in asymptomatic carriers of viable Leishmania parasites. Asymptomatic infection was identified using the Montenegro skin test in an at-risk population from Yucatan, Mexico. Parasite viability was evinced in the blood by 7SL RNA transcripts amplification. The expression of mRNA IFN-γ was analyzed in peripheral blood mononuclear cells stimulated with soluble Leishmania antigen, using RT-qPCR. Parasite viability was observed in 33.3 % (5/15) of asymptomatic subjects. No differences were found in the expression of IFN-γ between asymptomatic and healthy subjects, and no correlation was found between the presence of viable parasites and the expression of IFN-γ. This study demonstrates the persistence of Leishmania parasites in the absence of an in vitro IFN-γ response in asymptomatic carriers from Mexico.

Viable Leishmania parasites in the absence of an in vitro IFN-γ response in asymptomatic carriers

ABSTRACT Asymptomatic infection (the absence or inapparent signs and symptoms) has been observed in many endemic areas of leishmaniasis, however, little is known about the parasitological and immunological factors associated with this type of infection. This study aimed to identify the in vitro expression of IFN-γ in asymptomatic carriers of viable Leishmania parasites. Asymptomatic infection was identified using the Montenegro skin test in an at-risk population from Yucatan, Mexico. Parasite viability was evinced in the blood by 7SL RNA transcripts amplification. The expression of mRNA IFN-γ was analyzed in peripheral blood mononuclear cells stimulated with soluble Leishmania antigen, using RT-qPCR. Parasite viability was observed in 33.3 % (5/15) of asymptomatic subjects. No differences were found in the expression of IFN-γ between asymptomatic and healthy subjects, and no correlation was found between the presence of viable parasites and the expression of IFN-γ. This study demonstrates the persistence of Leishmania parasites in the absence of an in vitro IFN-γ response in asymptomatic carriers from Mexico.

Phlebotomine sand flies (Diptera: Psychodidae) from an emergent focus of localized cutaneous leishmaniasis in Yucatan, Southeast Mexico.

Localized cutaneous leishmaniasis (LCL) is endemic in the Yucatan Peninsula, with historical and contemporary records mainly in the states of Campeche and Quintana Roo. Recently, we reported autochthonous LCL cases and 27.6% of asymptomatic infection in the municipality of Tinum, Yucatan, where no studies of Phlebotominae (Diptera: Psychodidae) sand flies have been carried out. In this work, from November, 2019 to February, 2020, we conducted a field study in three areas of Tinum to document, for the first time, the species of Phlebotominae in areas with records of human leishmaniasis transmission. In order of abundance, the species identified were Pifanomyia serrana, Psathyromyia shannoni, Psathyromyia cratifer, Lutzomyia cruciata, Bichromomyia olmeca olmeca, and Dampfomyia deleoni. Most of the sand flies were captured in a Shannon trap where 77.8% of collected specimens were females. The distribution of sand fly species showed some degree of heterogeneity among sites, and the highest species richness was registered in a site located in Xcalakoop. We also discuss the medical importance of Lu. cruciata, Ps. shannoni, and Pi. serrana as potential vectors of causal agents of LCL in this area.

Preliminary epidemiological findings of Leishmania infection in the municipality of Tinum, Yucatan State, Mexico.

Localized cutaneous leishmaniasis (LCL) is endemic in Mexico, mainly in the states of Campeche and Quintana Roo, hyperendemic areas of Leishmania (Leishmania) mexicana transmission. In this report, epidemiological features of Leishmania infections in the municipality of Tinum, Yucatan State, Mexico are presented. Nine cases of LCL were diagnosed in 2015. Patients were men between 30 and 74 years of age, without a history of living or traveling to endemic areas (Quintana Roo or Campeche). Due to asymptomatic infection is the most common outcome after Leishmania inoculation, between November 2017 to June 2018, 47 men working in the forest were tested by Montenegro skin test (MST). Thirteen of them (27.6%) were identified MST positive, in absence of either lesion or typical scar, and evidence of exposure to vector. Findings in Tinum, Yucatan, supported the presence of specific environmental conditions that seem to favor Leishmania transmission in this region. Thus, active surveillance for the detection of new cases in the municipality of Tinum as well as the eco-epidemiological characterization to identify all the transmission components (parasite, vector, and reservoir species) are urgently needed.

Finding a model for the study of Leishmania (Leishmania) mexicana infection: The Yucatan Deer mouse (Peromyscus yucatanicus) as a suitable option.

For more than four decades, the murine model has been employed extensively to understand immunological mechanisms associated with Leishmania infection. Although the use of laboratory mice has been very informative, mainly for L. (L.) major infection, the extrapolation to other Leishmania species and more importantly to human disease has been limited. Particularly in the case of L. (L.) mexicana, most infected mouse strains are highly susceptible and never presented asymptomatic infection, which is the main outcome in human. Thus, we postulated the use of Peromyscus yucatanicus, a primary reservoir of L. (L.) mexicana in the Yucatan Peninsula of Mexico, as an experimental model to study Leishmania infection. This rodent species can produce both asymptomatic and clinical infections therefore they seem more appropriate for studying host-pathogen interactions. In this review, we recapitulate the immunological findings observed in the traditional murine model of L. (L.) mexicana highlighting the differences with humans' infection and demonstrate the pertinence of P. yucatanicus as the experimental model for studying L. (L.) mexicana infection.

Cytokine mRNA expression in Peromyscus yucatanicus (Rodentia: Cricetidae) infected by Leishmania (Leishmania) mexicana.

Peromyscus yucatanicus, the main reservoir of Leishmania (Leishmania) mexicana in the Yucatan peninsula of Mexico, reproduces clinical and histological pictures of LCL in human as well as subclinical infection. Thus, we used this rodent as a novel experimental model. In this work, we analyzed cytokine mRNA expression in P. yucatanicus infected with L. (L.) mexicana. Animals were inoculated with either 2.5×10(6) or 1×10(2) promastigotes and cytokine expressions were analyzed by real-time RT-PCR in skin at 4 and 12weeks post-infection (wpi). Independently of the parasite inoculum none of the infected rodents had clinical signs of LCL at 4wpi and all expressed high IFN-γ mRNA. All P. yucatanicus inoculated with 2.5×10(6) promastigotes developed signs of LCL at 12wpi while the mice inoculated with 1×10(2) remained subclinical. At that time, both IFN-γ and IL-10 were expressed in P. yucatanicus with clinical and subclinical infections. Expressions of TNF-α and IL-4 were significantly higher in clinical animals (2.5×10(6)) compared with subclinical ones (1×10(2)). High TGF-ß expression was observed in P. yucatanicus with clinical signs when compared with healthy animals. Results suggested that the clinical course of L. (L.) mexicana infection in P. yucatanicus was associated with a specific local pattern of cytokine production at 12wpi.

In situ cytokines (IL-4, IL-10, IL-12, IFN-γ) and chemokines (MCP-1, MIP-1α) gene expression in human Leishmania (Leishmania) Mexicana infection.

Crucial to the defense against Leishmania is the ability of the host to mount a cell-mediated immune response capable of controlling and/or eliminating the parasite. The composition of the cell populations recruited in the early phase of the infection seems to be essential for defining the infection outcomes. The signals that initiate and regulate the early immune response and local accumulation of cell subsets in the skin are poorly understood. We previously studied the in situ expression of cytokine genes in patients with localized cutaneous leishmaniasis (LCL) caused by Leishmania (Leishmania) mexicana. In the present study we examined in situ cytokine (IL-4, IL-10, IL-12, IFN-γ) and chemokine (MCP-1, MIP-1α) gene expression in L. (L.) mexicana active LCL lesions, and in the delayed type hypersensitivity (DTH) skin response to Leishmania antigen in subjects with healed lesion and subclinical infection. Data regarding cytokines were similar to previous studies in patients with active LCL. There were no significant differences in the profile of cytokine and chemokine gene expression in DTH from subjects with healed or subclinical infection. IL-12 gene expression detected in both groups was similar. High expression of MCP-1 was detected in all patients with active LCL. There was no difference in the level of MCP-1 expression between the healed lesion and the subclinical infection groups (p = 0.876). IL-12 and MCP-1 in the absence of IFN-γ might be playing a crucial role in infection outcomes at skin level.

Cloning and sequence analysis of Peromyscus yucatanicus (Rodentia) Th1 (IL-12p35, IFN-γ and TNF) and Th2 (IL-4, IL-10 and TGF-ß) cytokines.

The Yucatan deer mouse, Peromyscus yucatanicus (order Rodentia), is the principal reservoir of Leishmania (Leishmania) mexicana in the Yucatan peninsula of Mexico. Experimental infection results in clinical and histopathological features similar to those observed in humans with cutaneous leishmaniasis (CL) as well as peritoneal macrophage production of nitric oxide. These results support the possible use of P. yucatanicus as a novel experimental model to study CL caused by L. (L.) mexicana. However, immunological studies in these rodents have been limited by the lack of specific reagents. To address this issue, we cloned and analyzed cytokine sequences of P. yucatanicus as part of an effort to develop this species as a CL model. We cloned P. yucatanicus interleukin 4 (IL-4), IL-10, IL-12p35, gamma interferon, transforming growth factor beta and tumor necrosis factor partial cDNAs. Most of the P. yucatanicus sequences were highly conserved with orthologs of other mammalian species and the identity of all sequences were confirmed by the presence of conserved amino acids with possible biological functions in each putative polypeptide. The availability of these sequences is a first step which will allow us to carry out studies characterizing the immune response during pathogenic and nonpathogenic L. (L.) mexicana infections in P. yucatanicus.

Nitric oxide production by Peromyscus yucatanicus (Rodentia) infected with Leishmania (Leishmania) mexicana.

Peromyscus yucatanicus (Rodentia: Cricetidae) is a primary reservoir of Leishmania (Leishmania) mexicana (Kinetoplastida: Trypanosomatidae). Nitric oxide (NO) generally plays a crucial role in the containment and elimination of Leishmania. The aim of this study was to determine the amount of NO produced by P. yucatanicus infected with L. (L.) mexicana. Subclinical and clinical infections were established in P. yucatanicus through inoculation with 1 x 10 2 and 2.5 x 10 6 promastigotes, respectively. Peritoneal macrophages were cultured alone or co-cultured with lymphocytes with or without soluble Leishmania antigen. The level of NO production was determined using the Griess reaction. The amount of NO produced was significantly higher (p ≤ 0.0001) in co-cultured macrophages and lymphocytes than in macrophages cultured alone. No differences in NO production were found between P. yucatanicus with subclinical L. (L.) mexicana infections and animals with clinical infections. These results support the hypothesis that the immunological mechanisms of NO production in P. yucatanicus are similar to those described in mouse models of leishmaniasis and, despite NO production, P. yucatanicus is unable to clear the parasite infection.

Nitric oxide production by Peromyscus yucatanicus (Rodentia) infected with Leishmania (Leishmania) mexicana

Peromyscus yucatanicus (Rodentia: Cricetidae) is a primary reservoir of Leishmania (Leishmania) mexicana (Kinetoplastida: Trypanosomatidae). Nitric oxide (NO) generally plays a crucial role in the containment and elimination of Leishmania. The aim of this study was to determine the amount of NO produced by P. yucatanicus infected with L. (L.) mexicana. Subclinical and clinical infections were established in P. yucatanicus through inoculation with 1 x 10 2 and 2.5 x 10 6 promastigotes, respectively. Peritoneal macrophages were cultured alone or co-cultured with lymphocytes with or without soluble Leishmania antigen. The level of NO production was determined using the Griess reaction. The amount of NO produced was significantly higher (p ≤ 0.0001) in co-cultured macrophages and lymphocytes than in macrophages cultured alone. No differences in NO production were found between P. yucatanicus with subclinical L. (L.) mexicana infections and animals with clinical infections. These results support the hypothesis that the immunological mechanisms of NO production in P. yucatanicus are similar to those described in mouse models of leishmaniasis and, despite NO production, P. yucatanicus is unable to clear the parasite infection.

Preliminary study towards a novel experimental model to study localized cutaneous leishmaniasis caused bY Leishmania (Leishmania) mexicana / Estudio preliminar sobre un nuevo y original modelo experimental de leishmaniosis cutánea localizada causada por Leishmania (Leishmania) mexicana

There is not an experimental model of localized cutaneous leishmaniasis (LCL) caused by Leishmania (Leishmania) mexicana. The aim of the present study was to characterize the clinical and histological features of Peromyscus yucatanicus experimentally infected with L. (L.) mexicana. A total of 54 P. yucatanicus (groups of 18) were inoculated with 1x10(6) promastigotes of L. (L.) mexicana in the base of the tail. They were euthanized at three and six months post experimental infection. The control group was inoculated with RPMI-1640. The predominant clinical sign observed was a single ulcerated lesion in 27.77% (5/18) and in 11.11% (2/18) P. yucatanicus at three and six months respectively. The histological pattern described as chronic granulomatous inflammation with or without necrosis was found in 7/7 (100%) biopsies of euthanized P. yucatanicus at three (n = 5) and six (n = 2) months, respectively. These results resembled clinical and histological features caused by L. (L.) mexicana in humans, and support the possibility to employ P. yucatanicus as a novel experimental model to study LCL caused by this parasite.

No existe un modelo experimental de la leishmaniosis cutánea localizada (LCL) causada por Leishmania (Leishmania) mexicana. El objetivo del presente estudio fue el de caracterizar los cuadros clínico e histológico de Peromyscus yucatanicus infectados experimentalmente con L. (L.) mexicana. Un total de 54 P. yucatanicus (grupos de 18) fueron inoculados en la base de la cola con 1x10(6) promastigotes de L. (L.) mexicana. A los 3 y 6 meses posteriores a la infección experimental fueron sacrificados. El grupo control fue inoculado con RPMI-1640. El signo clínico predominante fue una lesión única ulcerada en 27.77% (5/18) y en 11.11% (2/18) P. yucatanicus a los 3 y 6 meses respectivamente. El patrón histológico descrito como inflamación crónica granulomatosa con o sin necrosis se observó en 7/7 (100%) biopsias de los P. yucatanicus a los 3 (n=5) y 6 (n=2) meses respectivamente. Los resultados son similares a los cuadros clínico e histológico de la infección por L. (L.) mexicana en humanos, y apoyan la posibilidad de utilizar P. yucatanicus como un nuevo y original modelo para el estudio de la LCL causada por L. (L.) mexicana.

Preliminary study towards a novel experimental model to study localized cutaneous leishmaniasis caused by Leishmania (Leishmania) mexicana.

There is not an experimental model of localized cutaneous leishmaniasis (LCL) caused by Leishmania (Leishmania) mexicana. The aim of the present study was to characterize the clinical and histological features of Peromyscus yucatanicus experimentally infected with L. (L.) mexicana. A total of 54 P. yucatanicus (groups of 18) were inoculated with 1x10(6) promastigotes of L. (L.) mexicana in the base of the tail. They were euthanized at three and six months post experimental infection. The control group was inoculated with RPMI-1640. The predominant clinical sign observed was a single ulcerated lesion in 27.77% (5/18) and in 11.11% (2/18) P. yucatanicus at three and six months respectively. The histological pattern described as chronic granulomatous inflammation with or without necrosis was found in 7/7 (100%) biopsies of euthanized P. yucatanicus at three (n = 5) and six (n = 2) months, respectively. These results resembled clinical and histological features caused by L. (L.) mexicana in humans, and support the possibility to employ P. yucatanicus as a novel experimental model to study LCL caused by this parasite.