Dimorphism and Dissemination of Histoplasma capsulatum in the Upper Respiratory Tract after Intranasal Infection of Bats and Mice with Mycelial Propagules.

This article describes, for the first time, the role of the nasal mucosa (NM) as the initial site for the Histoplasma capsulatum mycelial-to-yeast transition. The results highlight that yeasts may arrive to the cervical lymph nodes (CLN) via phagocytes. Bats and mice were intranasally infected with H. capsulatum mycelial propagules and they were killed 10, 20, and 40 minutes and 1, 2, and 3 hours after infection. The NM and the CLN were monitored for fungal presence. Yeasts compatible with H. capsulatum were detected within the NM and the CLN dendritic cells (DCs) 2-3 hours postinfection, using immunohistochemistry. Histoplasma capsulatum was re-isolated by culturing at 28°C from the CLN of both mammalian hosts 2-3 hours postinfection. Reverse transcription-polymerase chain reaction assays were designed to identify fungal dimorphism, using mycelial-specific (MS8) and yeast-specific (YPS3) gene expression. This strategy supported fast fungal dimorphism in vivo, which began in the NM 1 hour postinfection (a time point when MS8 and YPS3 genes were expressed) and it was completed at 3 hours (a time point when only the YPS3 transcripts were detected) in both bats and mice. The presence of intracellular yeasts in the nasal-associated lymphoid tissue (NALT), in the NM nonassociated with the NALT, and within the interdigitating DCs of the CLN suggests early fungal dissemination via the lymph vessels.

First description of a clinical case of murine typhus in Campeche, Mexico.

Murine typhus is a flea-borne disease caused by Rickettsia typhi, which was first detected in Mexico in 1927. It was not until 1996 that the first systematized study involving this pathogen was conducted in two coastal states of Mexico. We now report the first confirmed case of murine typhus in the state of Campeche, which occurred in a male patient who exhibited fever, thrombocytopenia, hyperbilirubinemia, and a rash. Furthermore, the patient reported having had previous contact with Rickettsia reservoirs.

First description of a clinical case of murine typhus in Campeche, Mexico

Abstract Murine typhus is a flea-borne disease caused by Rickettsia typhi, which was first detected in Mexico in 1927. It was not until 1996 that the first systematized study involving this pathogen was conducted in two coastal states of Mexico. We now report the first confirmed case of murine typhus in the state of Campeche, which occurred in a male patient who exhibited fever, thrombocytopenia, hyperbilirubinemia, and a rash. Furthermore, the patient reported having had previous contact with Rickettsia reservoirs.

Ecology of phlebotomine sandflies and putative reservoir hosts of leishmaniasis in a border area in Northeastern Mexico: implications for the risk of transmission of Leishmania mexicana in Mexico and the USA.

Leishmaniases are a group of important diseases transmitted to humans through the bite of sandfly vectors. Several forms of leishmaniases are endemic in Mexico and especially in the Southeast region. In the Northeastern region, however, there have only been isolated reports of cases and scanty records of sandfly vectors. The main objective of this study was to analyze the diversity of sandflies and potential reservoir hosts of Leishmania spp. in the states of Nuevo León and Tamaulipas. Species richness and abundances of sandflies and rodents were recorded. A fraction of the caught sandflies was analyzed by PCR to detect Leishmania spp. Tissues from captured rodents were also screened for infection. Ecological Niche Models (ENMs) were computed for species of rodent and their association with crop-growing areas. We found 13 species of sandflies, several of which are first records for this region. Medically important species such as Lutzomyia anthophora, Lutzomyia diabolica, Lutzomyia cruciata, and Lutzomyia shannoni were documented. Leishmania spp. infection was not detected in sandflies. Nine species of rodents were recorded, and Leishmania (Leishmania) mexicana infection was found in four species of Peromyscus and Sigmodon. ENMs showed that potential distribution of rodent pest species overlaps with allocated crop areas. This shows that Leishmania (L.) mexicana infection is present in the Northeastern region of Mexico, and that previously unrecorded sandfly species occur in the same areas. These findings suggest a potential risk of transmission of Leishmania (L.) mexicana.

Can You Judge a Disease Host by the Company It Keeps? Predicting Disease Hosts and Their Relative Importance: A Case Study for Leishmaniasis.

Zoonoses are an important class of infectious diseases. An important element determining the impact of a zoonosis on domestic animal and human health is host range. Although for particular zoonoses some host species have been identified, until recently there have been no methods to predict those species most likely to be hosts or their relative importance. Complex inference networks infer potential biotic interactions between species using their degree of geographic co-occurrence, and have been posited as a potential tool for predicting disease hosts. Here we present the results of an interdisciplinary, empirical study to validate a model based on such networks for predicting hosts of Leishmania (L.) mexicana in Mexico. Using systematic sampling to validate the model predictions we identified 22 new species of host (34% of all species collected) with the probability to be a host strongly dependent on the probability of co-occurrence of vector and host. The results confirm that Leishmania (L.) mexicana is a generalist parasite but with a much wider host range than was previously thought. These results substantially change the geographic risk profile for Leishmaniasis and provide insights for the design of more efficient surveillance measures and a better understanding of potential dispersal scenarios.

First report of "Candidatus Rickettsia amblyommii" in west coast of Mexico.

We report the first case of "Candidatus Rickettsia amblyommii" detected in Amblyomma mixtum ticks on humans on the west coast of Mexico. This is the most western record of "Ca. R. amblyommii" in the Western Hemisphere, representing the first record for the western coast of the Americas. Even if the record is far from the previously known locations for the species it does not represent a new record regarding temperature, precipitation and topographic parameters. Since "Ca. R. amblyommii" antibodies have been detected in patients suspected of Rocky Mountain spotted fever, and the tick A. mixtum has been associated with humans, it is important to consider "Ca. R. amblyommii" as a potential risk for the human population that has not been considered at risk before.

Leptospirosis in Mexico: Epidemiology and Potential Distribution of Human Cases.

BACKGROUND: Leptospirosis is widespread in Mexico, yet the potential distribution and risk of the disease remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: We analysed morbidity and mortality according to age and gender based on three sources of data reported by the Ministry of Health and the National Institute of Geography and Statics of Mexico, for the decade 2000-2010. A total of 1,547 cases were reported in 27 states, the majority of which were registered during the rainy season, and the most affected age group was 25-44 years old. Although leptospirosis has been reported as an occupational disease of males, analysis of morbidity in Mexico showed no male preference. A total number of 198 deaths were registered in 21 states, mainly in urban settings. Mortality was higher in males (61.1%) as compared to females (38.9%), and the case fatality ratio was also increased in males. The overall case fatality ratio in Mexico was elevated (12.8%), as compared to other countries. We additionally determined the potential disease distribution by examining the spatial epidemiology combined with spatial modeling using ecological niche modeling techniques. We identified regions where leptospirosis could be present and created a potential distribution map using bioclimatic variables derived from temperature and precipitation. Our data show that the distribution of the cases was more related to temperature (75%) than to precipitation variables. Ecological niche modeling showed predictive areas that were widely distributed in central and southern Mexico, excluding areas characterized by extreme climates. CONCLUSIONS/SIGNIFICANCE: In conclusion, an epidemiological surveillance of leptospirosis is recommended in Mexico, since 55.7% of the country has environmental conditions fulfilling the criteria that favor the presence of the disease.

New wildlife hosts of Leptospira interrogans in Campeche, Mexico.

Leptospira interrogans has been identified to cause leptospirosis, a widespread zoonotic disease that has been identified in domestic and wild animals. This work analyzed kidneys from two species of wild rodents from the state of Campeche, Mexico. Analyses were made by PCR using specific primers for detection of Leptospira interrogans DNA. The rodent species that tested positive were Heteromys gaumeri and Ototylomys phyllotis, both of which are new hosts for the bacteria in Southeastern Mexico. These records provide new insights into the disease's transmission that should be studied carefully in order to identify other potential host species, including humans, which are at risk of becoming infected if they are in contact with infected wildlife.

Leishmania (L.) mexicana infected bats in Mexico: novel potential reservoirs.

Leishmania (Leishmania) mexicana causes cutaneous leishmaniasis, an endemic zoonosis affecting a growing number of patients in the southeastern states of Mexico. Some foci are found in shade-grown cocoa and coffee plantations, or near perennial forests that provide rich breeding grounds for the sand fly vectors, but also harbor a variety of bat species that live off the abundant fruits provided by these shade-giving trees. The close proximity between sand flies and bats makes their interaction feasible, yet bats infected with Leishmania (L.) mexicana have not been reported. Here we analyzed 420 bats from six states of Mexico that had reported patients with leishmaniasis. Tissues of bats, including skin, heart, liver and/or spleen were screened by PCR for Leishmania (L.) mexicana DNA. We found that 41 bats (9.77%), belonging to 13 species, showed positive PCR results in various tissues. The infected tissues showed no evidence of macroscopic lesions. Of the infected bats, 12 species were frugivorous, insectivorous or nectarivorous, and only one species was sanguivorous (Desmodus rotundus), and most of them belonged to the family Phyllostomidae. The eco-region where most of the infected bats were caught is the Gulf Coastal Plain of Chiapas and Tabasco. Through experimental infections of two Tadarida brasiliensis bats in captivity, we show that this species can harbor viable, infective Leishmania (L.) mexicana parasites that are capable of infecting BALB/c mice. We conclude that various species of bats belonging to the family Phyllostomidae are possible reservoir hosts for Leishmania (L.) mexicana, if it can be shown that such bats are infective for the sand fly vector. Further studies are needed to determine how these bats become infected, how long the parasite remains viable inside these potential hosts and whether they are infective to sand flies to fully evaluate their impact on disease epidemiology.

Genetic structure and divergence in populations of Lutzomyia cruciata, a phlebotomine sand fly (Diptera: Psychodidae) vector of Leishmania mexicana in southeastern Mexico.

The low dispersal capacity of sand flies could lead to population isolation due to geographic barriers, climate variation, or to population fragmentation associated with specific local habitats due to landscape modification. The phlebotomine sand fly Lutzomyia cruciata has a wide distribution throughout Mexico and is a vector of Leishmania mexicana in the southeast. The aim of this study was to evaluate the genetic diversity, structure, and divergence within and among populations of Lu. cruciata in the state of Chiapas, and to infer the intra-specific phylogeny using the 3' end of the mitochondrial cytochrome b gene. We analyzed 62 sequences from four Lu. cruciata populations and found 26 haplotypes, high genetic differentiation and restricted gene flow among populations (Fst=0.416, Nm=0.701, p<0.001). The highest diversity values were recorded in populations from Loma Bonita and Guadalupe Miramar. Three lineages (100% bootstrap and 7% overall divergence) were identified using a maximum likelihood phylogenetic analysis which showed high genetic divergence (17.2-22.7%). A minimum spanning haplotype network also supported separation into three lineages. Genetic structure and divergence within and among Lu. cruciata populations are hence affected by geographic heterogeneity and evolutionary background. Data obtained in the present study suggest that Lu. cruciata in the state of Chiapas consists of at least three lineages. Such findings may have implications for vector capacity and hence for vector control strategies.

Drug target validation of the trypanothione pathway enzymes through metabolic modelling.

A kinetic model of trypanothione [T(SH)(2)] metabolism in Trypanosoma cruzi was constructed based on enzyme kinetic parameters determined under near-physiological conditions (including glutathione synthetase), and the enzyme activities, metabolite concentrations and fluxes determined in the parasite under control and oxidizing conditions. The pathway structure is characterized by a T(SH)(2) synthetic module of low flux and low catalytic capacity, and another more catalytically efficient T(SH)(2) -dependent antioxidant/regenerating module. The model allowed quantification of the contribution of each enzyme to the control of T(SH)(2) synthesis and concentration (flux control and concentration control coefficients, respectively). The main control of flux was exerted by γ-glutamylcysteine synthetase (γECS) and trypanothione synthetase (TryS) (control coefficients of 0.58-0.7 and 0.49-0.58, respectively), followed by spermidine transport (0.24); negligible flux controls by trypantothione reductase (TryR) and the T(SH)(2)-dependent antioxidant machinery were determined. The concentration of reduced T(SH)(2) was controlled by TryR (0.98) and oxidative stress (-0.99); however, γECS and TryS also exerted control on the cellular level of T(SH(2)) when they were inhibited by more than 70%. The model predicted that in order to diminish the T(SH)(2) synthesis flux by 50%, it is necessary to inhibit γECS or TryS by 58 or 63%, respectively, or both by 50%, whereas more than 98% inhibition was required for TryR. Hence, simultaneous and moderate inhibition of γECS and TryS appears to be a promising multi-target therapeutic strategy. In contrast, use of highly potent and specific inhibitors for TryR and the antioxidant machinery is necessary to affect the antioxidant capabilities of the parasites.

Canine leishmaniasis in México: the detection of a new focus of canine leishmaniasis in the state of Guerrero correlates with an increase of human cases

Background. In Mexico, a steady increase of patients with visceral leishmaniasis has been reported, especially in the states of Chiapas and Guerrero, yet only limited information exists on canine leishmaniasis in areas of visceral leishmaniasis in Mexico. A veterinary report of dogs with nonhealing cutaneous lesions in Pungarabato, Guerrero led us to investigate the possible presence of Leishmania infection in an area where Lutzomyia longipalpis and Lutzomyia evansi, both vectors of Leishmania infantum, have been described. Methods. We analyzed skin lesions of 25 dogs by immunohistochemistry and PCR. Results. We found a 60% prevalence of Leishmania-infected dogs, the infection rate being higher in males than females. Thus, we established a new focus of canine leishmaniasis, and although to date no patients have been reported in this municipality, it is close to and shares the same ecological characteristics of dry tropical forests as regions where visceral leishmaniasis has been reported in Mexico. We also include updated information of localities of visceral leishmaniasis in Mexico as well as the distribution of possible sand fly vectors. Conclusions. Our data show the need to ascertain the magnitude of this new focus in view of the current data on human visceral leishmaniasis, a disease that is surging in Mexico.

Sand flies naturally infected by Leishmania (L.) mexicana in the peri-urban area of Chetumal city, Quintana Roo, México.

The surveillance of prevalent Leishmania sand fly vectors is an important issue for epidemiological studies in populated areas where leishmaniasis is endemic. In this study, we collected sand flies from a peri-urban area in the southeast of Mexico. Natural infection with Leishmania (L.) mexicana was studied by PCR using a Leishmania internal transcribed spacer of the ribosomal RNA gene for amplification. Infected Lutzomyia olmeca olmeca, Lu. shannoni and Lu. cruciata sand flies were collected mainly during the high transmission season (November to March), coinciding with the highest sand fly densities. Additionally, positive specimens of Lu. olmeca olmeca were also captured during July and August. The infected sand flies were from primary forest (subperennial forest) and secondary forest (18-25 years old and 10-15 years old respectively). Sand flies collected with Disney and Shannon traps were the ones found to be infected with L. (L.) mexicana. We conclude that the high-risk period in which L. (L.) mexicana is transmitted in the peri-urban area of Chetumal City is from July to March and that transmission is associated with both the subperennial forest and the secondary forest.

PCR for identification of species causing American cutaneous leishmaniasis.

Parasites of the complexes Leishmania (Leishmania) mexicana, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) chagasi coexist within the same endemic areas of the American Continent. They produce similar clinical manifestations, yet not all respond well to treatment with anti-leishmania drugs. Thus, high specificity and sensitivity are needed to improve diagnosis and treatment. We developed a highly specific and sensitive polymerase chain reaction based diagnostic method that permits the identification of parasites belonging to the genus Leishmania and the differentiation between parasites belonging to the L. (L.) mexicana and L. (V.) braziliensis complexes and the identification of species of the L. (L.) mexicana complex, such as L. (L.) mexicana, Leishmania (L.) amazonensis, and Leishmania (L.) venezuelensis. This PCR permits the specific identification of Leishmania species in tissues of patients with different clinical forms of leishmaniasis. Its high sensitivity and specificity allow a precise diagnosis in lesions of patients that harbor few parasites, where the microscopic evaluation is unreliable. Additionally, this PCR could be a valuable tool for the identification of Leishmania species in mammalian reservoirs and sand fly vectors present in the American Continent.

[Leishmania mexicana DNA activates murine macrophages and increases their TLR9 expression]. / El ADN de Leishmania mexicana activa al macrófago murino e induce aumento en la expresión de TLR9.

BACKGROUND: Macrophages are immune system cells that recognize pathogen associated molecular patterns (PAMPs) through receptors that can be located on the cell membrane or in intracellular compartments, such as the TLR (toll like receptors). Different TLRs bind to ligands shared among multiple pathogens. The binding of ligands to TLRs induces a signaling cascade that leads to cytokine and co-stimulatory molecule production due to the nuclear translocation of NF-kappaB. We demonstrated that Leishmania lipophosphoglycan (LPG) is a ligand for TLR2, leading to NK-cell activation. Schieicher et al. recently reported that genomic DNA from Leishmania infantum activates plasmacitoid dendritic cells through TLR9, leading to IFN type I production. OBJECTIVE: In the present study we explored wether Leishmania mexicana DNA contained non-methylated CpG motifs able to activate murine bone marrow derived macrophages, as previously described for bacterial DNA containing CpG motifs. RESULTS AND CONCLUSIONS: We observed that Leishmania mexicana DNA contains non-methylated CpG morifs able ofactivating murine bone marrow derived macrophages, leading to the production of proinflammatory cytokines such as TNFalpha and IL- 12(P40) as well as the over expression of mRNA for TLR9.

El ADN de Leishmania mexicana activa al macrófago murino e induce aumento en la expresión de TLR9 / Leishmania mexicana DNA activates murine macrophages and increases their TLR9 expression

Antecedentes: Los macrófagos son células de la respuesta inmune que reconocen patrones moleculares asociados a patógenos (PAMP) mediante receptores presentes en la superficie de la célula como en compartimentos intracelulares, como los TLR (toll like receptors). Distintos TLR reconocen ligandos que comparten múltiples patógenos. La unión de TLR con su ligando desencadena una cascada de señalización que termina en la producción de citocinas y moléculas coestimuladoras a través de la translocación de NF-κB al núcleo. Nuestro grupo demostró que el lipofosfoglucano de Leishmania es un ligando de TLR2 que activa células NK. Schieicher y cols.12 informo recientemente la activación de células dendríticas plasmacitoides con ADN genómico de Leishmania infantum a través de TLR9, con alta producción de IFN tipo I. Objetivo: En el presente trabajo exploramos si el ADN de Leishmania mexicana contiene motivos CpG no metilados capaces de activar al macrófago murino derivado de médula ósea, como ha sido descrito anteriormente para motivos CpG no metilados de ADN bacteriano. Resultados y conclusiones: Encontramos que el ADN de Leishmania mexicana posee motivos CpG no metilados que activan macrófagos murinos de la cepa BALB/c, llevando a la producción de citocinas proinflamatorias como TNFα e IL12P40 y a la sobreexpresión del mARN de TLR9.

BACKGROUND: Macrophages are immune system cells that recognize pathogen associated molecular patterns (PAMPs) through receptors that can be located on the cell membrane or in intracellular compartments, such as the TLR (toll like receptors). Different TLRs bind to ligands shared among multiple pathogens. The binding of ligands to TLRs induces a signaling cascade that leads to cytokine and co-stimulatory molecule production due to the nuclear translocation of NF-kappaB. We demonstrated that Leishmania lipophosphoglycan (LPG) is a ligand for TLR2, leading to NK-cell activation. Schieicher et al. recently reported that genomic DNA from Leishmania infantum activates plasmacitoid dendritic cells through TLR9, leading to IFN type I production. OBJECTIVE: In the present study we explored wether Leishmania mexicana DNA contained non-methylated CpG motifs able to activate murine bone marrow derived macrophages, as previously described for bacterial DNA containing CpG motifs. RESULTS AND CONCLUSIONS: We observed that Leishmania mexicana DNA contains non-methylated CpG morifs able ofactivating murine bone marrow derived macrophages, leading to the production of proinflammatory cytokines such as TNFalpha and IL- 12(P40) as well as the over expression of mRNA for TLR9.

Polymorphism analysis of the internal transcribed spacer and small subunit of ribosomal RNA genes of Leishmania mexicana.

Leishmania mexicana causes a wide spectrum of clinical diseases. In spite of the variety of clinical forms, no data exist regarding genetic polymorphism of L. mexicana. We analyzed the polymorphism of the internal transcribed spacer (ITS) and the small subunit rRNA genes of 3 reference strains and 24 Mexican isolates of L. mexicana, by means of polymerase chain reaction and subsequent digestion by restriction enzymes. All strains of L. mexicana had invariant patterns for both the ITS and the small subunit of rRNA genes. Leishmania amazonensis and Leishmania venezuelensis displayed polymorphism only in the ITS. The high degree of identity of this region was confirmed by sequencing DNA from three L. mexicana isolates. There was almost complete identity of the sequence for the ITS region of L. venezuelensis and that of strains of Leishmania major, suggesting that these species may be more closely related than previously thought.

Impacto de nuevas metodologías en el estudio de parásitos: III. Variación genética intraespecie de Leishmania mexicana mexicana y su repercusión sobre la fisiopatogenia de la enfermedad / Impact of new methodologies in the study of parasites: III

Las formas más frecuentes de leishmaniosis en México son la leishmaniosis cutánea localizada (LCL), un padecimiento relativamente benigno, y la leishmaniosis cutánea diseminada (LCD), de evolución generalmente mortal. La caracterización fenotípica de parásitos aislados de pacientes con LCL y LCD ha revelado, que el agente casual de ambos cuadros clínicos de Leishmania mexicana mexicana. Sin embargo la resistencia a medicamentos y la virulencia inesperada en algunos pacientes hacen sospechar una posible introducción de nuevas especies en México o bien mutaciones intraespecie. En este trabajo realizamos un análisis genotípico del kADN de leishmanias aisladas de pacientes con LCL y LCD, mediante endonucleasas de restricción (Hae II.I y Hpa II) y RAPD (amplificacion aleatoria de ADN polimórfico con oligonucleótidos no específicos). Encontramos polimorfismo en las digestiones sugestivas de la introducción de nuevas especies en México, lo cual se tendrá que confirmar con PCR especie-especificos. Mediante el RAPD detectamos una ligera diferencia entre un paciente con LCD y otros con LCL. Esta variación intraespecie pudiera ser una de las posibles causas de diseminación del parásito.