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.

New State Record for Culex rejector and Cx. restrictor in Yucatan, Mexico.

Several known mosquito species occur in the Mexican state of Yucatan, including competent and suspected vectors responsible for transmitting zoonotic arboviruses. Between February and July 2022, mosquito collections were carried out in several forested areas in Yucatan. As part of the ongoing monitoring, we present the first reports of Culex (Microculex) rejector and Cx. (Anoedioporpa) restrictor. Another 14 species were identified during the monitoring: Aedes albopictus, Ae. bimaculatus, Ae. tormentor, Ae. cozumelensis, Anopheles albimanus, Cx. coronator s.l., Cx. erraticus, Cx. lactator, Cx. salinarius, Coquillettidia venezuelensis, Limatus durhamii, Psorophora ciliata, Toxorhynchites theobaldi, and Wyeomyia mitchellii. Currently, the mosquito fauna in Yucatan consists of 65 species. The subgenera Microculex and Anoedioporpa had not been documented in Yucatan State prior to the current investigation.

Acute oral toxicity in BALB/c mice of Tridax procumbens and Allium sativum extracts and (3S)-16,17-didehydrofalcarinol.

ETHNOPHARMACOLOGICAL RELEVANCE: Approximately 80% of people in developing countries depend on medicinal plants for their health care. Tridax procumbens (T. procumbens) and Allium sativum (A. sativum) have beneficial effects against parasitic and bacterial diseases. On the other side, the biological activity of the oxylipin (3S)-16,17-didehydrofalcarinol isolated from T. procumbens against the parasite Leishmania mexicana has been verified. AIM OF THE STUDY: To evaluate the acute oral toxicity of the methanolic extract of T. procumbens, the aqueous extract of A. sativum, their mixture, and pure oxylipin (3S)-16,17-didehydrofalcarinol in BALB/c mice. MATERIALS AND METHODS: Doses of 2000 and 5000 mg/kg of the methanolic extract of T. procumbens, the aqueous extract of A. sativum, and their mixture (1:1), and doses of 300 and 500 mg/kg of pure oxylipin were administered orally to female mice of the strain BALB/c, which were observed for 72 h in search of signs of toxicity. After 14 days, the animals were euthanized, blood was extracted for the measurement of transaminases, and the livers were recovered and stained with hematoxylin/eosin for histopathological analysis. RESULTS: No clinical signs of toxicity were observed in any of the animals dosed with T. procumbens and A. sativum extracts, while the majority of the animals dosed with pure oxylipin showed signs of toxicity and died. There was no difference in the weight index in most of the animals, except for the animals treated with T. procumbens at doses of 2000 mg/kg who presented an increase in the weight index, nor was there a correlation between the dose of A. sativum and the mixture and food consumption; however, a direct proportional correlation was observed between T. procumbens dose and food consumption. In none of the animals dosed with T. procumbens, A. sativum, and the mixture there was a difference in the levels of transaminases. In the histopathology study, slight lesions were observed in the hepatocytes of the mice treated with T. procumbens, A. sativum, and their mixture at doses of 2000 and 5000 mg/kg. On the other side, moderate injuries were observed in animals treated with pure oxylipin and it was considered as toxic due to almost all the animals died. CONCLUSION: The extracts of T. procumbens and A. sativum evaluated and applied orally did not cause signs of acute toxicity or severe liver damage, suggesting to evaluate their chronic toxicity including other biochemical parameters in the future. However, pure oxylipin caused signs of acute toxicity and death so it is recommended to work with lower doses.

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.

The role of monocytes/macrophages in Leishmania infection: A glance at the human response.

Leishmania are obligate intracellular parasites of mononuclear phagocytes transmitted by Phlebotomine sandflies. Monocytes are one of the main cell types recruited to the site of the bite having an important role in the defense against Leishmania parasites in the first hours of infection. In the tissue, macrophages play a pivotal role as both the primary replication sites and the major effector cells responsible for parasite elimination. Many authors have reviewed the monocyte/macrophage-Leishmania interactions from results derived in mice, however, given the important differences between mice an humans we considered vital to discuss the role of these cells in human leishmaniasis. In this review, we recapitulated the most important studies carried out to understand the different roles of human monocyte/macrophages in Leishmania infection and how they can participate in both control and the immunopathogenesis of the disease.

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.

Asymptomatic infection with American cutaneous leishmaniasis: epidemiological and immunological studies.

American cutaneous leishmaniasis (ACL) is a major public health problem caused by vector-borne protozoan intracellular parasites from the genus Leishmania, subgenera Viannia and Leishmania. Asymptomatic infection is the most common outcome after Leishmania inoculation. There is incomplete knowledge of the biological processes explaining the absence of signs or symptoms in most cases while other cases present a variety of clinical findings. Most studies of asymptomatic infection have been conducted in areas of endemic visceral leishmaniasis. In contrast, asymptomatic ACL infection has been neglected. This review is focused on the following: (1) epidemiological studies supporting the existence of asymptomatic ACL infection and (2) immunological studies conducted to understand the mechanisms responsible for controlling the parasite and avoiding tissue damage.

Asymptomatic infection with American cutaneous leishmaniasis: epidemiological and immunological studies

American cutaneous leishmaniasis (ACL) is a major public health problem caused by vector-borne protozoan intracellular parasites from the genus Leishmania, subgenera Viannia and Leishmania. Asymptomatic infection is the most common outcome after Leishmania inoculation. There is incomplete knowledge of the biological processes explaining the absence of signs or symptoms in most cases while other cases present a variety of clinical findings. Most studies of asymptomatic infection have been conducted in areas of endemic visceral leishmaniasis. In contrast, asymptomatic ACL infection has been neglected. This review is focused on the following: (1) epidemiological studies supporting the existence of asymptomatic ACL infection and (2) immunological studies conducted to understand the mechanisms responsible for controlling the parasite and avoiding tissue damage.

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.

Animal models for the study of leishmaniasis immunology.

Leishmaniasis remains a major public health problem worldwide and is classified as Category I by the TDR/WHO, mainly due to the absence of control. Many experimental models like rodents, dogs and monkeys have been developed, each with specific features, in order to characterize the immune response to Leishmania species, but none reproduces the pathology observed in human disease. Conflicting data may arise in part because different parasite strains or species are being examined, different tissue targets (mice footpad, ear, or base of tail) are being infected, and different numbers ("low" 1 × 10(2) and "high" 1 × 10(6)) of metacyclic promastigotes have been inoculated. Recently, new approaches have been proposed to provide more meaningful data regarding the host response and pathogenesis that parallels human disease. The use of sand fly saliva and low numbers of parasites in experimental infections has led to mimic natural transmission and find new molecules and immune mechanisms which should be considered when designing vaccines and control strategies. Moreover, the use of wild rodents as experimental models has been proposed as a good alternative for studying the host-pathogen relationships and for testing candidate vaccines. To date, using natural reservoirs to study Leishmania infection has been challenging because immunologic reagents for use in wild rodents are lacking. This review discusses the principal immunological findings against Leishmania infection in different animal models highlighting the importance of using experimental conditions similar to natural transmission and reservoir species as experimental models to study the immunopathology of the disease.

Animal models frr the study of leishmaniasis immunolggy / Modelos animales para el estudio de la inmunologia de la leishmaniosis

Leishmaniasis remains a major public health problem worldwide and is classified as Category I by the TDR/WHO, mainly due to the absence of control. Many experimental models like rodents, dogs and monkeys have been developed, each with specific features, in order to characterize the immune response to Leishmania species, but none reproduces the pathology observed in human disease. Conflicting data may arise in part because different parasite strains or species are being examined, different tissue targets (mice footpad, ear, or base of tail) are being infected, and different numbers (“low” 1×102 and “high” 1×106) of metacyclic promastigotes have been inoculated. Recently, new approaches have been proposed to provide more meaningful data regarding the host response and pathogenesis that parallels human disease. The use of sand fly saliva and low numbers of parasites in experimental infections has led to mimic natural transmission and find new molecules and immune mechanisms which should be considered when designing vaccines and control strategies. Moreover, the use of wild rodents as experimental models has been proposed as a good alternative for studying the host-pathogen relationships and for testing candidate vaccines. To date, using natural reservoirs to study Leishmania infection has been challenging because immunologic reagents for use in wild rodents are lacking. This review discusses the principal immunological findings against Leishmania infection in different animal models highlighting the importance of using experimental conditions similar to natural transmission and reservoir species as experimental models to study the immunopathology of the disease.

Las leishmaniosis siguen siendo un importante problema de salud pública a nivel mundial y se clasifican como categoría I por el programa TDR/WHO, debido principalmente a la ausencia de control. Muchos modelos experimentales tales como roedores, perros y monos han sido desarrollados, cada uno con características específicas, para caracterizar la respuesta inmune a las diferentes especies de Leishmania, sin embargo ninguno reproduce la patología observada en la enfermedad humana. La diversidad en los resultados obtenidos podría deberse en parte a que diferentes cepas de parásitos o especies están siendo examinadas, diferentes tejidos (cojinete plantar, oreja o base de la cola) han sido infectados y diferente número (“bajo” 1×102 y “alto” 1×106) de promastigotes metacíclicos han sido inoculados. Recientemente, nuevos enfoques han sido propuestos con el fin de obtener datos más significativos en cuanto a la respuesta inmune del huésped y a la patogénesis, de tal forma que reproduzcan lo que ocurre en la enfermedad humana. El uso de la saliva del insecto y de un número de parásitos menor en las infecciones experimentales ha permitido reproducir la transmisión natural, identificar nuevas moléculas, así como mecanismos inmunes que deberían ser considerados en el diseño de vacunas y estrategias de control. Adicionalmente, se ha propuesto como una buena alternativa el uso de roedores silvestres como modelos experimentales tanto para el estudio de las relaciones huésped-patógeno como para probar nuevas vacunas. A la fecha, el uso de reservorios naturales para estudiar la infección por Leishmania ha sido un reto, debido a la carencia de reactivos inmunológicos para uso en roedores silvestres. Esta revisión describe los principales hallazgos inmunológicos ante la infección por Leishmania, en los diferentes modelos animales, destacando la importancia del uso de condiciones experimentales similares a la transmisión natural y de reservorios como modelos experimentales para el estudio de la inmunopatología de la enfermedad.

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.