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The RNA-binding PUF proteins are post-transcriptional regulators found throughout the eukaryotic domain. In Trypanosoma cruzi, ten Puf genes termed Puf1 to Puf10 have been identified. Considering that the control of gene expression in this parasite is mainly at the post-transcriptional level, we characterized the PUF3 protein by knocking out and overexpressing the gene in T. cruzi epimastigotes and studied different genetic and biological features. The RNA-seq analyses in both genotypes showed significant changes in the number of regulated transcripts compared with wild-type parasites. Thus, the number of differentially expressed genes in the knockout (ΔTcPuf3) and the overexpressor (pTEXTcPuf3) were 238 and 187, respectively. In the knockout, a more significant proportion of genes was negatively regulated (166 out of 238). In contrast, in the overexpressor, positively regulated genes were predominant (149 out of 170). Additionally, when we predicted the subcellular location of the differentially expressed genes, the results revealed an important representation of nuclear genes encoding mitochondrial proteins. Therefore, we determined whether overexpression or knockout of TcPuf3 could lead to changes in both mitochondrial structure and cellular respiration. When mitochondria from ΔTcPuf3 and pTEXTcPuf3 parasites were analyzed by transmission electron microscopy (TEM), it was observed that the overexpressor had an abnormal mitochondrial morphology, evidenced by swelling. The results associated with cellular respiration showed that both the ΔTcPuf3 and pTEXTcPuf3 had a lower efficiency in routine respiration and the electron transport system capacity. Likewise, the mitochondria from overexpressing parasites showed a slight hyperpolarization. Additionally, several biological features, depending on the function of the mitochondria, were altered, such as growth, cell division, metacyclogenesis, ROS production, and response to benznidazole. In conclusion, our results suggest that although PUF3 is not an essential protein in T. cruzi, it influences mitochondrial transcripts, affecting mitochondrial morphology and function.
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Parasites of the genus Leishmania pose a global health threat with limited treatment options. New drugs are urgently needed, and genomic screens have the potential to accelerate target discovery, mode of action, and resistance mechanisms against these new drugs. We describe here our effort in developing a genome-wide CRISPR-Cas9 screen in Leishmania, an organism lacking a functional nonhomologous end joining system that must rely on microhomology-mediated end joining, single-strand annealing, or homologous recombination for repairing Cas9-induced double-stranded DNA breaks. A new vector for cloning and expressing single guide RNAs (sgRNAs) was designed and proven to be effective in a small pilot project while enriching specific sgRNAs during drug selection. We then developed a whole-genome library of 49,754 sgRNAs, targeting all the genes of Leishmania infantum. This library was transfected in L. infantum expressing Cas9, and these cells were selected for resistance to two antileishmanials, miltefosine and amphotericin B. The sgRNAs the most enriched in the miltefosine screen targeted the miltefosine transporter gene, but sgRNAs targeting genes coding for a RING-variant protein and a transmembrane protein were also enriched. The sgRNAs the most enriched by amphotericin B targeted the sterol 24 C methyltransferase genes and a hypothetical gene. Through gene disruption experiments, we proved that loss of function of these genes was associated with resistance. This study describes the feasibility of carrying out whole-genome CRISPR-Cas9 screens in Leishmania provided that a strong selective pressure is applied. Such a screen can be used for accelerating the development of urgently needed antileishmanial drugs.IMPORTANCELeishmaniasis, a global health threat, lacks adequate treatment options and drug resistance exacerbates the challenge. This study introduces a CRISPR-Cas9 screening approach in Leishmania infantum, unraveling mechanisms of drug resistance at a genome-wide scale. Our screen was applied against two main antileishmanial drugs, and guides were enriched upon drug selection. These guides targeted known and new targets, hence validating the use of this screen against Leishmania. This strategy provides a powerful tool to expedite drug discovery as well as potential therapeutic targets against this neglected tropical disease.
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Antiprotozoários , Sistemas CRISPR-Cas , Resistência a Medicamentos , Ensaios de Triagem em Larga Escala , Leishmania infantum , Leishmania infantum/genética , Leishmania infantum/efeitos dos fármacos , Resistência a Medicamentos/genética , Antiprotozoários/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Fosforilcolina/farmacologia , Fosforilcolina/análogos & derivados , Anfotericina B/farmacologia , RNA Guia de Sistemas CRISPR-Cas/genética , Genoma de ProtozoárioRESUMO
Cellulose nanofibrils from the banana rachis are a good alternative as packaging materials, food packaging, stabilizing agents, and functional food ingredients. To address the potential effects of ingested banana rachis cellulose nanofibrils (BR-CNFs), their toxicity in vitro and in vivo was evaluated using Caco-2 intestinal cells and mice, respectively. The results showed that BR-CNFs did not cause cytotoxic effects at the concentrations evaluated on Caco-2 cells. In addition to cytotoxicity tests, genotoxicity assays using comet assay indicated that Caco-2 cells showed no DNA damage at the concentrations of CNFs tested. Finally, acute in vivo cytotoxicity assays indicated that mice showed no sign of pathogenesis or lesions in the liver, kidney, or small intestine when treated with a single dose of BR-CNFs. Moreover, when the mice were treated daily for a month with BR-CNFs no hyperplasia or hypertrophy was observed in any of the organs evaluated. Additionally, biochemical parameters such as blood chemistry, creatinine, liver enzymes, and renal function showed that the BR-CNFs do not cause organ damage. Overall, this study shows that BR-CNFs are neither cytotoxic nor genotoxic. In conclusion, these studies are essential to guarantee the safety of this high value-added product in the food industry.
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Ehrlichia canis and Babesia vogeli are vector-borne pathogens that infect blood cells and produce the diseases Canine Monocytic Ehrlichiosis (CME) and Babesiosis in dogs. Considering the lack of studies on these pathogens in Colombia, this study aims to determine the molecular prevalence and genetic characterization of E. canis and Babesia spp., in dogs from the Metropolitan Area of Bucaramanga (MAB), Santander, a region with one of the greatest pet densities in Colombia. One hundred eighty-five dogs were surveyed and analyzed through molecular, clinical, and hematological approaches. The molecular detection of E. canis and Babesia spp., was performed by conventional PCR targeting the dsb and 18S rRNA genes, respectively. To identify genogroups, E. canis positive samples underwent a hemi-nested PCR of the trp36 gene, and the PCR products were subsequently sequenced. Molecular analyses showed a prevalence of 13% (24/185; CI 95%, 8.1 - 18.0%) and 1.09% (2/185; CI 95,% -0.43 - 2.6%) for E. canis and B. vogeli respectively, as well as the presence of the genogroups US (USA), BR (Brazil), and CR (Costa Rica), in 62.5, 16.6, and 16.6% of E. canis positive samples, respectively. Values of hematocrit, hemoglobin, platelets, erythrocytes, white blood cell (WBC) count, lymphocytes, and eosinophils showed significant differences between animals infected with the different genogroups of E. canis (p< 0.05). In contrast, hematocrit values, hemoglobin, platelets, red blood cells, and creatine kinase MB isoenzyme (CK-MB) were lower in B. vogeli positive animals. Statistical analysis indicated that E. canis infection was associated with specific socioeconomic sectors as well as with some household features (p< 0.05). In conclusion, our results present evidence of the circulation of multiple genogroups of E. canis in the MAB, which is associated with different geographical origins and clinical traits. Epidemiological analyses suggest a need to increase molecular surveillance and prevention campaigns especially in lower socioeconomic sectors.
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Babesia , Babesiose , Doenças do Cão , Ehrlichiose , Animais , Cães , Babesia/genética , Ehrlichia canis/genética , Colômbia/epidemiologia , Babesiose/diagnóstico , Ehrlichiose/epidemiologia , Ehrlichiose/veterinária , Genótipo , Doenças do Cão/diagnósticoRESUMO
Background: Rhodnius prolixus is considered the most relevant Trypanosoma cruzi vector in Colombia and Venezuela due it is responsible for domestic transmission in both countries. However, a wild population of this species is distributed in the eastern plains of the Orinoco region and Amazonia jungle, where its epidemiological importance has not been sufficiently elucidated. This study aimed to assess epidemiological parameters of T. cruzi transmission in the Department of Vichada, Colombia. Methods: We determined the characteristics of T. cruzi transmission using entomological studies in domestic and sylvatic ecotopes. We analyzed the T. cruzi infection in triatomine insects, identified blood meal sources, and conducted a serological determination of T. cruzi infection in scholar-aged children, domestic dogs, and wild hosts. Results: Fifty-four triatomine bugs, 40 T. maculata and 14 R. prolixus were collected in peridomestic and sylvatic ecotopes. Infected R. prolixus was observed in La Primavera, Santa Rosalia, and Cumaribo municipalities. All the T. maculata bugs were not infected. Serological analysis indicated that two of 3,425 children were T. cruzi positive. The seroprevalence in domestic dogs was 10,5% (49/465). Moreover, 22 synanthropic mammals were sampled, being Didelphis marsupialis the most common. TcI genotype was detected in seropositive dogs, R. prolixus, and D. marsupialis. Conclusion: The present work describes extra domestic R. prolixus and D. marsupialis in a sylvatic T. cruzi transmission cycle with transmission to humans and domestic dogs in Colombia's Vichada Department.
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Doença de Chagas , Rhodnius , Trypanosoma cruzi , Criança , Humanos , Cães , Animais , Idoso , Estudos Soroepidemiológicos , Colômbia/epidemiologia , Insetos Vetores , Doença de Chagas/epidemiologia , Doença de Chagas/veterinária , Trypanosoma cruzi/genética , MamíferosRESUMO
INTRODUCTION: In Colombia, organochloride, organophosphate, carbamate, and pyrethroid insecticides are broadly used to control Aedes aegypti populations. However, Colombian mosquito populations have shown variability in their susceptibility profiles to these insecticides, with some expressing high resistance levels. MATERIALS AND METHODS: In this study, we analyzed the susceptibility status of ten Colombian field populations of Ae. aegypti to two pyrethroids; permethrin (type-I pyrethroid) and lambda-cyhalothrin (type-II pyrethroid). In addition, we evaluated if mosquitoes pressured with increasing lambda-cyhalothrin concentrations during some filial generations exhibited altered allelic frequency of these kdr mutations and the activity levels of some metabolic enzymes. RESULTS: Mosquitoes from all field populations showed resistance to lambda-cyhalothrin and permethrin. We found that resistance profiles could only be partially explained by kdr mutations and altered enzymatic activities such as esterases and mixed-function oxidases, indicating that other yet unknown mechanisms could be involved. The molecular and biochemical analyses of the most pyrethroid-resistant mosquito population (Acacías) indicated that kdr mutations and altered metabolic enzyme activity are involved in the resistance phenotype expression. CONCLUSIONS: In this context, we propose genetic surveillance of the mosquito populations to monitor the emergence of resistance as an excellent initiative to improve mosquito-borne disease control measures.
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Aedes/efeitos dos fármacos , Aedes/genética , Resistência a Inseticidas , Inseticidas/farmacologia , Animais , Colômbia , Proteínas de Insetos/genética , Mutação , Nitrilas/farmacologia , Permetrina/farmacologia , Piretrinas/farmacologiaRESUMO
In South America, Colombia is the third-largest livestock producer with approximately 28.8 million cattle, of which Colombian Creole cattle represent around 1% of the livestock population. Animal Trypanosomiasis (AT) is one of the most critical problems in the livestock industry, reducing its production by about 30 %. Considering the paucity of information to understand the epidemiological features of AT in Colombian Creole cattle, the present study reports the molecular prevalence and clinical traits associated with the infection of Trypanosoma spp. in three Colombian Creole breeds. From 2019 to 2020, cross-sectional surveillance in farms of central and west of Colombia was designed to evaluate the mentioned characteristics in Casanareño, Chino Santandereano, and Sanmartinero Creole breeds. Molecular analysis showed an AT prevalence of 60.2 % (95 % CI = 54.2 % - 66.2 %). The Chino Santandereano population presented the highest value (Trypanosoma spp., 75.2 %, T. theileri 59.6 % and T. evansi 15.6 %), followed by Casanareño (Trypanosoma spp., 65.3 %, T. theileri 38.6 %, T. evansi 24.0 %, and T. vivax 5.3 %) and Sanmartinero (Trypanosoma spp., 33.3 %, T. theileri 24.0 % and T. evansi 9.3 %). Features such as breeds, age, and feeding system were significantly associated with AT prevalence (P < 0.05). Additionally, a low level of serum total proteins was observed during T. evansi infection in Sanmartinero (P < 0.05). To our knowledge, this is the first cross-sectional survey that evaluates using molecular methods the infection of Trypanosoma spp. in Colombian Creole breeds, showing significant variations in the prevalence and clinical signs associated with the infection. These results suggest different degrees of trypanotolerance in these breeds, as well as a possible effect of environmental variables on the prevalence and clinical characteristics associated with the infection. The epidemiological and economic implications of these findings are discussed here.
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Doenças dos Bovinos , Trypanosoma , Tripanossomíase , Animais , Cruzamento , Bovinos/parasitologia , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/parasitologia , Colômbia/epidemiologia , Estudos Transversais , Gado , Trypanosoma/genética , Trypanosoma/isolamento & purificação , Tripanossomíase/epidemiologia , Tripanossomíase/veterináriaRESUMO
Several species of trypanosomes can infect bats (Chiroptera), but current information about bat trypanosomes in Colombia is scarce. The objectives of this study were to estimate the infection rate and to characterize the trypanosome species infecting bats from three rural regions near the municipality of Cumaribo in Vichada, Colombia. Blood samples were collected from 39 bats. DNA was extracted from the blood samples and analyzed using nuclear genetic markers (SSU rDNA, ITS rDNA, and cathepsin genes) to discriminate among trypanosome species. Trypanosomes were detected in 66.7 % (26/39) of blood samples using PCR; 61.5 % (24/39) of infections were identified as Trypanosoma theileri and 5.1 % (2/39) as T. wauwau-like parasites. The phylogeographic analysis revealed that our T. theileri sequences were associated with the TthIIB genotype from cattle in Brazil and Venezuela. The T. wauwau-like parasites represent a new genotype of the species and were found in Molossus molossus and Platyrrhinus helleri bats. These data represent the first evidence of this trypanosome in both Colombia, and in these species of bats. Bat infections with T. theileri suggest an important role of these hosts in maintaining this genotype, probably acquired by ingesting insect vectors. The T. wauwau-like genotype in new mammalian host species supports the 'bat seeding' hypothesis of the T. cruzi clade. The epidemiological and evolutionary implications of these findings are discussed.
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Quirópteros , Monitoramento Epidemiológico/veterinária , Interações Hospedeiro-Parasita , Trypanosoma/isolamento & purificação , Tripanossomíase/veterinária , Animais , Quirópteros/sangue , Colômbia/epidemiologia , DNA de Protozoário/análise , DNA Ribossômico/análise , Genótipo , Prevalência , Trypanosoma/genética , Tripanossomíase/sangue , Tripanossomíase/epidemiologia , Tripanossomíase/parasitologiaRESUMO
Insecticide resistance in Aedes aegypti populations is a problem that hinders vector control and dengue prevention programs. In this study, we determined the susceptibility of Ae. aegypti populations from six Colombian regions to the pyrethroid lambda-cyhalothrin and evaluated the presence of the V1016I mutation in the sodium channel gene, which has been broadly involved in the resistance to this insecticide. The diversity of the gut microbiota of these mosquito populations was also analyzed. Only mosquitoes from Bello were susceptible to lambda-cyhalothrin and presented a lower allelic frequency of the V1016I mutation. Remarkably, there was not an important change in allelic frequencies among populations with different resistance ratios, indicating that other factors or mechanisms contributed to the resistant phenotype. Treatment of mosquitoes with antibiotics led us to hypothesize that the intestinal microbiota could contribute to the resistance to lambda-cyhalothrin. Beta diversity analysis showed significant differences in the species of bacteria present between susceptible and resistant populations. We identified 14 OTUs of bacteria that were unique in resistant mosquitoes. We propose that kdr mutations are important in the development of resistance to lambda-cyhalothrin at low insecticide concentrations but insect symbionts could play an essential role in the metabolization of pyrethroid insecticides at higher concentrations, contributing to the resistant phenotype in Ae. aegypti.
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Surra is a zoonotic disease caused by Trypanosoma evansi, affecting the health and production of the livestock significantly. There are several methods to diagnose this disease, which have different principles, sensitivity, and specificity. Among them, the serological techniques using T. evansi as antigen are powerful tools for its epidemiological surveillance. However, they are poorly used due to inefficient in vitro propagation of T. evansi, which requires the use of laboratory animals for antigen production. In the present study, whole cell lysate of T. brucei brucei propagated in vitro was used as an antigen for the detection of anti-T. evansi immunoglobulin G in cattle through an indirect-ELISA. Based on a total of 45 samples from non-infected and 45 samples from T. evansi infected cattle, the sensitivity and specificity were estimated as 100% and 97.7%, respectively. After the validation, serological and molecular surveys were carried out in 710 cattle samples from two endemic Colombian regions (Antioquia and Arauca departments) for T. evansi where molecular prevalences of Ë7.0% were detected through the year and sporadic outbreaks of T. vivax infections have been associated to low prevalence of this species (<1%). A total of 424 (59.7%) samples were positive by indirect-ELISA T. b. brucei, while PCR test for T. evansi and T. vivax, showed 49 (6.9%) and no positive samples, respectively. Interestingly, categories of animals aged>1â¯year, Bos taurus breed, and those raised under intensive farming system exhibited a higher seroprevalence to T. evansi (Pâ¯<â¯0.05). The results displayed a new alternative for antibody detection anti-T. evansi in livestock, using parasites propagated in vitro as antigen, which presents the advantage of higher standardization potential, and avoid the use of live animal for antigen production. A larger availability of this ELISA will generate useful information for a better understanding of the epidemiologic aspects, as well as for the management and control of these diseases in Colombia. However, the ability of the test to detect and/or cross react with T. vivax infections remains to be investigated.
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Antígenos de Protozoários/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/veterinária , Reação em Cadeia da Polimerase/veterinária , Trypanosoma brucei brucei/isolamento & purificação , Animais , Bovinos , Colômbia , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G , Gado , Reação em Cadeia da Polimerase/métodosRESUMO
Dengue is the second most prevalent vector-borne disease after malaria in Colombia. It is caused by dengue virus, an arbovirus that exhibits high epidemic power, which is evidenced by its occurrence in more than 80% of the country, largely because of the extensive dispersion of the mosquito vector Aedes aegypti. The existence of two lineages of Ae. aegypti has been proposed based on genetic differences at the mitochondrial level, and they have been reported to circulate in similar proportions in the municipality of Bello (Colombia). It has been suggested that the differentiation of these lineages could influence features such as vector competence (VC) and life table. With the aim of testing this hypothesis, female mosquitoes from both lineages collected from Bello were orally challenged with dengue virus serotype 2 (strain D2-HAN) to measure infection, dissemination, survival and fecundity. Analysis of VC showed an increase in viral titer over time; however, no significant differences were observed between the lineages. The survival rate was not different between the infected lineages, but comparing lineages, it was lower in infected mosquitoes, which may affect the intensity of transmission. Finally, we conclude that the genetic differentiation of Ae. aegypti into lineages did not confer differences in epidemiological status when the mosquitoes were infected with this D2 serotype strain.
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Aedes/virologia , Vírus da Dengue/fisiologia , Animais , Colômbia , Efeito Citopatogênico Viral , Vírus da Dengue/genética , Interações Hospedeiro-Patógeno , Mitocôndrias/genéticaRESUMO
BACKGROUND: Babesia bigemina and B. bovis are two economically important hemoparasites affecting both cattle and buffaloes involved in dairy and beef production. In Colombia, although some parasitological and serological studies suggest an endemicity of these pathogens in areas under 1000 m, little is known about its molecular prevalence in different host. The objective of this study was to estimate the prevalence and molecular traits of these parasites in cattle and buffaloes from two Colombian regions. METHODS: Between 2014 and 2016, a three-point longitudinal survey was designed in farms from Caribbean and Orinoquia regions to evaluate the molecular prevalence of B. bigemina and B. bovis using a nested PCR (n-PCR) targeting hypothetical protein (hyp) and rhoptry-associated protein (RAP-1) genes, respectively. A total of 1432 cattle, 152 buffalo and 1439 Rhipicephalus microplus samples were analyzed. Moreover, phylogenetic relationship of isolates was analyzed using the 18S rRNA gene. RESULTS: A molecular prevalence of 31.6% (24.2% for B. bigemina and 14.4% for B. bovis), 23.6% (6.5% for B. bigemina and 17.7% for B. bovis) and 4.3% (3.5% for B. bigemina and 1.0% for B. bovis) was observed in cattle, buffaloes and Rhipicephalus microplus, respectively. Higher values of infection were observed during the wet season and late wet season; nevertheless, other variables such as age, production type, sex, breed and babesiosis control were also significantly associated with infection. Prevalence analysis showed that B. bovis infection was higher in cattle that coexist with buffaloes, when compared to those which did not. For each species, phylogenetic analyses revealed a high genetic diversity of isolates without clusters related to the isolation source. CONCLUSIONS: To our knowledge, this is the first longitudinal survey that evaluates through molecular methods, the infection of B. bigemina and B. bovis in two important livestock regions from Colombia. This study reveals that the prevalence of infection by Babesia spp., in cattle and buffaloes are modulated by seasonal variations, host factors and vector traits. Our results provide new insights on the epidemiological aspects of infection of Babesia spp., in cattle and buffaloes, which must be taken into consideration when babesiosis control programs are implemented in the study area.
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Babesia/isolamento & purificação , Babesiose/epidemiologia , Búfalos/parasitologia , Doenças dos Bovinos/epidemiologia , Animais , Babesia/genética , Babesia bovis/genética , Babesia bovis/isolamento & purificação , Babesiose/parasitologia , Bovinos , Doenças dos Bovinos/parasitologia , Colômbia/epidemiologia , Feminino , Variação Genética , Estudos Longitudinais , Masculino , Fenótipo , Filogenia , Reação em Cadeia da Polimerase/veterinária , PrevalênciaRESUMO
Anaplasma marginale is the most prevalent vector-borne pathogen in the livestock industry in Colombia, causing economic losses of approximately USD 4.2 million per year. The present study reports the seasonal transmission patterns, genetic diversity and phylogeographic traits of A. marginale strains in cattle and buffaloes from Colombian livestock areas. A three-point longitudinal survey was designed to evaluate the above characteristics of farms in the Caribbean and Orinoquía regions. The A. marginale prevalence was evaluated in 1432 cattle blood samples, 152 buffalo blood samples and the hemolymph of 439 ticks using semi-nested PCR (sn-PCR) targeting the msp5 gene. The molecular prevalence in cattle and buffaloes was 54.8% and 13.1%, respectively, with higher values during the wet and late wet seasons. Factors such as age and production system were significantly associated with the infection. Rhipicephalus microplus was the only carrier of A. marginale DNA, with an infection rate of 17.2%. On the other hand, the tandem repeat and microsatellite analyses of the msp1α gene showed high genetic diversity and new tandem repeats that suggested strain adaptation to different transmission modes. Phylogeographic analysis using the msp4 gene showed a relationship between Colombian isolates and Mexican, Brazilian, Venezuelan, European and Asian isolates, as well as two worldwide haplogroups that were associated with the geographical origin of each isolate. In conclusion, this study shows that A. marginale occurs under enzootic stability in both hosts, with a high prevalence of infection during wet months and in animals dedicated to beef production. The genetic variability analyses suggest that a high strain diversity is associated with multiple selective pressures in the study area, while phylogeographic traits suggest a high genetic similarity between Mexican and South American strains.
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Anaplasma marginale/crescimento & desenvolvimento , Anaplasmose/transmissão , Búfalos/microbiologia , Doenças dos Bovinos/microbiologia , Variação Genética , Anaplasma marginale/genética , Anaplasma marginale/isolamento & purificação , Anaplasmose/sangue , Anaplasmose/epidemiologia , Anaplasmose/microbiologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Bovinos , Doenças dos Bovinos/epidemiologia , Meio Ambiente , Proteínas de Membrana/genética , Repetições de Microssatélites/genética , Fenótipo , Filogenia , Filogeografia , Reação em Cadeia da Polimerase , Prevalência , Rhipicephalus/microbiologia , Sequências de Repetição em Tandem/genéticaRESUMO
Resistance to pyrethroids in mosquitoes is mainly caused by target site insensitivity known as knockdown resistance (kdr). In this work, we examined the point mutations present in portions of domains I, II, III, and IV of the sodium channel gene in Aedes aegypti mosquitoes from three Colombian municipalities. A partial region coding for the sodium channel gene from resistant mosquitoes was sequenced, and a simple allele-specific PCR-based assay (AS-PCR) was used to analyze mutations at the population level. The previously reported mutations, V1016I and F1534C, were found with frequencies ranging from 0.04 to 0.41, and 0.56 to 0.71, respectively, in the three cities. Moreover, a novel mutation, at 419 codon (V419L), was found in Ae. aegypti populations from Bello, Riohacha and Villavicencio cities with allelic frequencies of 0.06, 0.36, and 0.46, respectively. Interestingly, the insecticide susceptibility assays showed that mosquitoes from Bello were susceptible to λ-cyhalothrin pyrethroid whilst those from Riohacha and Villavicencio were resistant. A positive association between V419L and V1016I mutations with λ-cyhalothrin resistance was established in Riohacha and Villavicencio. The frequency of the F1534C was high in the three populations, suggesting that this mutation could be conferring resistance to insecticides other than λ-cyhalothrin, particularly type I pyrethroids. Further studies are required to confirm this hypothesis.
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Animal Trypanosomiasis (AT) is one of the most important problems in the Colombian livestock industry reducing its production around 30%. Caribbean and Orinoquia regions play a significant role in the development of this industry, having about 6.9 million cattle and 113,000 buffaloes. Considering the paucity in studies to understand the epidemiological features and control of AT in Colombia, the present study reports the seasonal transmission patterns and phylogeographic traits of the causal agents of AT in cattle and buffaloes from these regions. Between 2014 and 2016, a three-point longitudinal survey was designed to evaluate the mentioned characteristics. Molecular analysis in cattle showed an AT prevalence of 39.2% (T. theileri 38.6%, T. evansi 6.7% and T. vivax 0.2%), with higher values during wet and late wet seasons, while in buffaloes the prevalence was 28.2% (T. theileri 28.2% and T. evansi 1.3%), with higher values during the dry season. Additionally, variables such as tabanid abundance, vector control, breeding system, age and anemia signs were significantly associated with AT prevalence (P<0.05). Only T. theileri infection was higher in cattle with anemia signs than those with normal packed cell volume. Finally, phylogeographic analysis revealed that Colombian T. theileri isolates were associated to specific host genotypes IA and IIB, described worldwide; T. vivax isolates were related to the genotype from West Africa; while T. evansi isolates are related to the South American genotypes and to new genotypes. This is the first longitudinal survey that evaluates through molecular methods, the infection of Trypanosoma spp. in two important livestock regions from Colombia, showing that the clinical effects and prevalence of these trypanosomes in cattle and buffaloes are modulated by seasonal variations, host factors, and parasite traits. The results suggest that these factors have to be taken into account to successfully control AT in these regions.
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Búfalos , Doenças dos Bovinos/parasitologia , Doenças dos Bovinos/transmissão , Filogenia , Trypanosoma/classificação , Tripanossomíase/veterinária , Animais , Bovinos , Doenças dos Bovinos/epidemiologia , Colômbia/epidemiologia , DNA de Protozoário/genética , Especificidade de Hospedeiro , Estudos Longitudinais , Estações do Ano , Trypanosoma/genética , Tripanossomíase/epidemiologia , Tripanossomíase/parasitologia , Tripanossomíase/transmissãoRESUMO
Nifurtimox (Nfx) and benznidazole (Bz) are the current drugs used for the treatment of Chagas disease. The mechanisms of action and resistance to these drugs in this parasite are poorly known. Prostaglandin F2α synthase or old yellow enzyme (OYE), an NAD(P)H flavin oxidoreductase, has been involved in the activation pathway of other trypanocidal drugs such as Nfx; however, its role in the mechanism of action of Bz is uncertain. In this paper, we performed some experiments of functional genomics in the parasite Trypanosoma cruzi with the aim to test the role of this gene in the resistance to Bz. For this, we overexpressed this gene in sensitive parasites and evaluated the resistance level to the drug and other chemical compounds such as hydrogen peroxide, methyl methanesulfonate and gamma radiation. Interestingly, parasites overexpressing OYE showed alteration of enzymes associated with oxidative stress protection such as superoxide dismutase A and trypanothione reductase. Furthermore, transfected parasites were more sensitive to drugs, genetic damage and oxidative stress. Additionally, transfected parasites were less infective than wild-type parasites and they showed higher alteration in mitochondrial membrane potential and cell cycle after treatment with Bz. These results supply essential information to help further the understanding of the mechanism of action of Bz in T. cruzi.
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The improvement of Chagas disease treatment is focused not only on the development of new drugs but also in understanding mechanisms of action and resistance to drugs conventionally used. Thus, some strategies aim to detect specific changes in proteins between sensitive and resistant parasites and to evaluate the role played in these processes by functional genomics. In this work, we used a natural Trypanosoma cruzi population resistant to benznidazole, which has clones with different susceptibilities to this drug without alterations in the NTR I gene. Using 2DE-gel electrophoresis, the aldo-keto reductase and the alcohol dehydrogenase proteins were found up regulated in the natural resistant clone and therefore their possible role in the resistance to benznidazole and glyoxal was investigated. Both genes were overexpressed in a drug sensitive T. cruzi clone and the biological changes in response to these compounds were evaluated. The results showed that the overexpression of these proteins enhances resistance to benznidazole and glyoxal in T. cruzi. Moreover, a decrease in mitochondrial and cell membrane damage was observed, accompanied by a drop in the intracellular concentration of reactive oxygen species after treatment. Our results suggest that these proteins are involved in the mechanism of action of benznidazole.
Assuntos
Nitroimidazóis/metabolismo , Receptores de Neurotensina/genética , Trypanosoma cruzi/metabolismo , Álcool Desidrogenase/metabolismo , Aldo-Ceto Redutases/metabolismo , Animais , Doença de Chagas/parasitologia , Resistência a Medicamentos/genética , Eletroforese em Gel Bidimensional , Imunidade Inata/genética , Proteínas de Protozoários/metabolismo , Receptores de Neurotensina/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Surra disease is a zoonosis caused by Trypanosoma (Trypanozoon) evansi, a salivary trypanosome, originally from Africa, which affects a wide range of mammalian worldwide. Dogs are highly susceptible to T. evansi infection and they often exhibit strong clinical signs than can lead to death, even within weeks in untreated acute cases. The present survey is the first report through clinical, parasitological and molecular approaches, of two fatal cases of T. evansi in Colombian dogs. After analysing two presumptive cases of infection with Trypanosoma spp., in dogs by parasitological methods, we confirmed by molecular techniques the presence of T. evansi, finding clinical signs such as anaemia, thrombocytopenia and hepatosplenomegaly, with fatal outcomes within a week even after the treatment. A phylogenetic and phylogeographic analysis of both isolates from T. evansi, suggest a complex evolutionary relationship with species of Trypanozoon subgenus. Moreover, the haplotype H2 was observed for the first time in Colombia, in common areas where human cases of T. evansi infection has been reported. These findings imply a relevant problem for animal health in the country, and highlight the importance of this infection in domestic animals and the possibility of human cases.
Assuntos
Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Trypanosoma/genética , Tripanossomíase/veterinária , Animais , Colômbia/epidemiologia , Diminazena/uso terapêutico , Doenças do Cão/tratamento farmacológico , Doenças do Cão/transmissão , Cães , Evolução Fatal , Geografia , Haplótipos , Masculino , Filogenia , Reação em Cadeia da Polimerase/veterinária , Tripanossomicidas/uso terapêutico , Trypanosoma/isolamento & purificação , Tripanossomíase/diagnóstico , Tripanossomíase/tratamento farmacológico , Tripanossomíase/transmissãoRESUMO
Currently, the only available treatments for Trypanosoma cruzi are benznidazole (Bz) and nifurtimox (Nfx). The mechanisms of action and resistance to these drugs in this parasite are not complete known. In order to identify differentially expressed transcripts between sensitive and resistant parasites, a massive pyrosequencing of the T. cruzi transcriptome was carried out. Additionally, the 2D gel electrophoresis profile of sensitive and resistant parasites was analyzed and the data were supported with functional genomics. The results showed 133 differentially expressed genes in resistant parasites. The transcriptome analysis revealed the regulation of different genes with several functions and metabolic pathways, which could suggest that resistance in T. cruzi is a multigenic process. Additionally, using transcriptomics, one gene, adenine phosphoribosyltransferase (APRT), was found to be down-regulated in the resistant parasites and its expression profile was confirmed by 2D electrophoresis analysis. The role of this gene in the resistance to Bz was confirmed overexpressing it in sensitive and resistant parasites. Interestingly, both parasites became more sensitive to Bz and H2 O2 . This is the first RNA-seq study to identify regulated genes in T. cruzi associated with Bz resistance and to show the role of APRT in T. cruzi resistance. Although T. cruzi regulation is mainly post-transcriptional, the transcriptome analysis, supported by 2D gel analysis and functional genomic, provides an overall idea of the expression profiles of genes under resistance conditions. These results contribute essential information to further the understanding of the mechanisms of action and resistance to Bz in T. cruzi. J. Cell. Biochem. 118: 1936-1945, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Adenina Fosforribosiltransferase/metabolismo , Nitroimidazóis/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/patogenicidade , Adenina Fosforribosiltransferase/genética , DNA de Protozoário/genética , Resistência a Medicamentos/genética , Resistência a Medicamentos/fisiologia , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Genômica , Filogenia , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismoRESUMO
In Colombia, vector-borne diseases are one of the most important problems in the livestock industry. The present study reports parasitological and molecular surveys of vector-borne pathogens in cattle from two high-value livestock areas in Colombia. A total of 464 samples (226 from Antioquia and 238 from Arauca) were analyzed. While the blood smear analysis identified 98 (21.1%), 14 (3.0%) and 30 (6.5%) positive samples for Anaplasma spp., Babesia spp. and Trypanosoma spp., respectively, the molecular methods indicated that 275 (59.3%), 146 (31.5%), 64 (13.8%), 236 (50.9%) and 43 (9.3%) of the samples were positive for Anaplasma marginale, Babesia bigemina, Babesia bovis, Trypanosoma theileri and T. evansi, respectively. Mixed infections were detected in 250 (53.9%) samples. Interestingly, animals aged ≤1 year had higher probabilities of being infected with A. marginale and Babesia spp., and lower probabilities of being infected with Trypanosoma spp., while the animals raised under intensive system breeding had higher probabilities of being infected with all pathogens. Additionally, T. theileri infection was found in higher prevalence in anemic animals than animals with normal packed cell volume (PCV). This is the first molecular report that evaluated the infection with three genders of vector-borne pathogens in cattle in Colombia and provides useful information for a better understanding of the epidemiologic aspects, as well as for the management and control, of these diseases.