Phlebotomine sand fly (Diptera: Psychodidae) fauna, blood meal source, and detection of Leishmania (Kinetoplastida: Trypanosomatidae) DNA in the Gurupi Biological Reserve, Eastern Amazon, Brazil.

This study was conducted in the Gurupi Biological Reserve (REBIO-Gurupi), the largest area of Amazon rainforest in Maranhão State, Brazil. The objectives were to survey the sand fly (Diptera: Psychodidae) fauna of REBIO-Gurupi, identify blood meal sources, and investigate the presence of Leishmania (Ross, 1903) (Kinetoplastida: Trypanosomatidae) DNA. Individuals were collected using Centers for Disease Control (CDC) light traps and black and white Shannon traps in May and Jun 2022 and Jan 2023. DNA was extracted from female sand flies and subjected to amplification and sequencing of cytochrome b molecular marker (CYTB) for identification of blood meal sources and the first internal transcribed spacer (ITS-1) of ribosomal DNA for Leishmania detection. A total of 514 sand flies individuals were sampled, of which 93 were identified at the genus or series level (9 taxa) and 421 were identified at the species level (24 taxa). Psychodopygus davisi (Root, 1934) (41.1%), Nyssomyia antunesi (Coutinho, 1939) (10.3%), and Psychodopygus (Mangabeira, 1941) Chagasi Series Barretto, 1962 (9.7%) were the most frequently collected. Human (Homo sapiens, Primates, Hominidae) and tapir (Tapirus terrestris, Perissodactyla, Tapiridae) DNA was detected in 10 female sand flies. Leishmania (Leishmania) infantum Cunha and Chagas, 1937 DNA was detected in 2 specimens of Ps. davisi. Given the presence of vectors of Leishmania in REBIO-Gurupi, it is imperative to conduct more comprehensive studies on the interactions among sand flies, Leishmania, and pathogen reservoirs in the area.

Infection by Trypanosoma cruzi (Chagas, 1909) in a colony of Triatoma sherlocki Papa et al. 2002 (Reduviidae: Triatominae) present in a subsistence pig farm in Northeast Brazil.

Triatomines of the species Triatoma sherlocki are considered sylvatic; however, household invasion appears imminent, potentially carrying Trypanosoma cruzi, the causative agent of Chagas disease. The aim of this study was to report the first occurrence of a colony of T. sherlocki infected by T. cruzi in a subsistence pig farm. Triatomines collected underwent polymerase chain reaction (PCR) technique for T. cruzi detection and determination of blood meal source. The 19 triatomines collected in the pig farm were of the species T. sherlocki, comprising 26.3% nymphs (5/19), 52.6% males (10/19) and 21.1% females (4/19). PCR showed that 15.8% (3/19) of triatomines were infected by T. cruzi. The only detected blood meal source in triatomines (n = 11) was the domestic mammal Sus scrofa, commonly known as domestic pig, indicating that T. sherlocki is an opportunist, feeding on available vertebrates in the environment, including domestic animals such as pigs. These results highlight the possibility of domiciliation of the species T. sherlocki and its potential role in bridging the transmission of T. cruzi between sylvatic and domestic environments.

Blood meal sources of mosquitoes (Diptera: Culicidae) in domestic and open green environments from two urbanisations of temperate Argentina.

Understanding the blood-feeding patterns of mosquitoes is essential for evaluating their potential as disease vectors, especially in urban areas where mosquitoes coexist with humans, domestic animals and wildlife. This study aimed to bridge a substantial gap in regional knowledge by identifying the blood meal sources of field-collected mosquitoes in domestic and open green environments from two urbanisations of temperate Argentina, the Área Metropolitana de Buenos Aires (AMBA) and Tandil, using molecular techniques. Female mosquitoes were collected from November 2019 to March 2020 and April-May 2021. A bipartite network analysis was performed for each environment and urbanisation. A total of 103 blood meals from Aedes (2 species) and Culex (7 species) were identified. Among these, five mammal and 18 bird species were recognised as hosts. Aedes mosquitoes exclusively fed on mammals, while Culex mosquitoes exhibited a broader host range including both birds and mammals. In AMBA, the open green environments were composed by more mosquito species than the domestic environments, while both presented similar numbers of vertebrate species. In contrast, in open green environments from Tandil only blood-fed Aedes albifasciatus were collected. For open green environments of AMBA and domestic environments of Tandil, results suggested some degree of host selection. For the three main vectors of diseases in the region, Aedes aegypti, Ae. albifasciatus and Culex pipiens molestus, we present the first molecular evidence of human blood meals in South America. Epidemiological significance of the present findings is discussed.

Blood feeding patterns of the Culex pipiens complex in equestrian land uses and their implications for arboviral encephalitis risk in temperate Argentina.

Blood feeding patterns of mosquitoes are a key component in the dynamics of arboviral encephalitides transmission. In temperate Argentina, the members of the Culex pipiens complex include Cx. pipiens molestus, Cx. quinquefasciatus and their hybrids. To characterize their blood feeding patterns, adult resting mosquitoes were collected monthly during the warm season in urban and rural equestrian fields. The availability of birds and domestic mammals per site was characterized. The blood source and the complex member were successfully identified for 89 specimens using PCR. Blood of 19 vertebrate species was isolated including four mammals (most common feeds from dog, Canus lupus 19% of the blood meals; and horse, Equus caballus 18%) and 15 birds (picazuro pigeon, Patagioenas picazuro 11%; eared dove, Zenaida auriculata 10%; chicken, Gallus gallus 9%). The Forage Ratio (FR), calculated as the proportion of feeds taken from a given host species with respect to that host availability in the environment, suggested preference for dog by all members of the complex (FR ≥4.5). On the contrary, FR values suggested avoidance for horse by Cx. quinquefasciatus and the hybrid (FR ≤0.8), and a use proportional to its abundance by Cx. pipiens molestus (FR = 1.1-1.2 in urban and rural sites, respectively). FR values suggesting preference were obtained for avian species of the orders Passeriformes (7 species in total) and Columbiformes (5) by all members of the complex (FR ≥ 3.3), whereas values for monk parakeet (Myiopsitta monachus, Psiitaciformes) suggested avoidance by Cx. quinquefasciatus in urban sites (FR = 0.4) and by Cx. pipiens molestus in rural sites (FR = 0.3) but not in urban sites (FR = 1.4). A mammal-bird index (MBI, from -1 all avian to +1 all mammalian blood meals) was calculated for each member of the complex and urbanization category. Values were negative for Cx. quinquefasciatus (MBIurban  = -0.60, MBIrural  = -0.33) and positive for Cx. pipiens molestus (MBIurban  = 0.20, MBIrural  = 0.60), indicating a higher proportion of feeds taken on birds and mammals, respectively, regardless of the urbanization category. In temperate Argentina, the members of the Cx. pipiens complex fed both on horses and on birds, thus representing a real risk of transmission of arboviral encephalitides from avian enzootic cycles to horse epizootics.

The antennal transcriptome of Triatoma infestans reveals substantial expression changes triggered by a blood meal.

BACKGROUND: Triatoma infestans is the main vector of Chagas disease in the Americas, currently transmitting it in Argentina, Paraguay, and Bolivia. Many T. infestans populations present insecticide resistance, reducing the efficiency of control campaigns. Alternative vector control methods are needed, and molecular targets mediating fundamental physiological processes can be a promising option to manipulate kissing bug behavior. Therefore, it is necessary to characterize the main sensory targets, as well as to determine whether they are modulated by physiological factors. In order to identify gene candidates potentially mediating host cue detection, the antennal transcripts of T. infestans fifth instar larvae were sequenced and assembled. Besides, we evaluated whether a blood meal had an effect on transcriptional profiles, as responsiveness to host-emitted sensory cues depends on bug starvation. RESULTS: The sensory-related gene families of T. infestans were annotated (127 odorant receptors, 38 ionotropic receptors, 11 gustatory receptors, 41 odorant binding proteins, and 25 chemosensory proteins, among others) and compared to those of several other hemipterans, including four triatomine species. Several triatomine-specific lineages representing sensory adaptations developed through the evolution of these blood-feeding heteropterans were identified. As well, we report here various conserved sensory gene orthogroups shared by heteropterans. The absence of the thermosensor pyrexia, of pickpocket receptor subfamilies IV and VII, together with clearly expanded takeout repertoires, are revealed features of the molecular bases of heteropteran antennal physiology. Finally, out of 2,122 genes whose antennal expression was significantly altered by the ingestion of a blood meal, a set of 41 T. infestans sensory-related genes (9 up-regulated; 32 down-regulated) was detected. CONCLUSIONS: We propose that the set of genes presenting nutritionally-triggered modulation on their expression represent candidates to mediate triatomine host-seeking behavior. Besides, the triatomine-specific gene lineages found represent molecular adaptations to their risky natural history that involves stealing blood from an enormously diverse set of vertebrates. Heteropteran gene orthogroups identified may represent unknown features of the sensory specificities of this largest group of hemipteroids. Our work is the first molecular characterization of the peripheral modulation of sensory processes in a non-dipteran vector of human disease.

Rhodnius (Stål, 1859) (Hemiptera, Triatominae) genus in Bolivian Amazonia: a risk for human populations?

BACKGROUND: Chagas disease, one of the most important neglected tropical diseases in the countries of Latin America, is considered to be a particularly important public health concern in the Amazon region due to increases in the number of outbreaks of acute Chagas disease and increased local transmission in the last 20 years. However, relative to other countries, in Bolivia there is little information available on its transmission in the Amazon region. The aim of this study was to investigate the infestation of palm trees, the main habitat of Triatominae in the region, in several localities, to evaluate the danger they represent to inhabitants. METHODS: Triatominae were collected using live bait traps left overnight in six localities in Pando and Beni Departments, Bolivia. DNA extraction and sequencing were used to establish the Triatominae species (Cytb, 16S and 28S-D2 gene fragments), and the blood meal sources (Cytb fragment). Trypanosoma sp. infection was analyzed by sequencing gene fragments (GPX, GPI, HMCOAR, LAP, PDH and COII) or by mini-exon multiplex PCR. RESULTS: A total of 325 Rhodnius were captured (97.3% of nymphs) from the 1200 traps placed in 238 palm trees and 32 burrows/ground holes. Sequence analyses on DNA extracted from 114 insects and phylogeny analysis identified two triatomine species: Rhodnius stali (17%) and Rhodnius montenegrensis (equated to Rhodnius robustus II, 83%). These were found in palm trees of the genera Attalea (69%), Astrocaryum (13%), Copernicia (12%), Euterpe (2%) and Acrocomia (1%). The infection rate was around 30% (165 analyzed insects), with 90% of analyzed insects infected by Trypanosoma cruzi (only the TcI discrete typing unit was detected), 3% infected by Trypanosoma rangeli (first time found in Bolivian Triatominae) and 7% infected by mixed T. cruzi (TcI)-T. rangeli. Rhodnius specimens fed on Didelphidae, rodents, gecko and humans. CONCLUSIONS: The results of this study highlight the epidemiological importance of Rhodnius in the Bolivian Amazon region. The huge geographical distribution of Rhodnius and their proximity to the human dwellings, high infection rate and frequent meals on the human population highlight a risk of transmission of Chagas disease in the region.

Anopheles blood meal sources and entomological indicators related to Plasmodium transmission in malaria endemic areas of Colombia.

Malaria is an important public health problem, caused by Plasmodium parasites which are transmitted by female Anopheles mosquitoes that bite humans to obtain blood. The aim of this work was to identify the blood feeding sources of Anopheles female mosquitoes and calculate their entomological indices in relation to Plasmodium transmission. Mosquitoes were collected in malaria endemic localities of the Bajo Cauca and Pacific regions of Colombia using human landing catch and barrier screens, from 18:00 to 24:00 hr, in 2018-2021. Animal censuses within a radius of ∼250 m were carried out at each sampling site. A total of 2018 Anopheles specimens were collected and the most abundant species were Anopheles (Nys.) darlingi and Anopheles (Nys.) nuneztovari. The highest human biting rate was 77.5 bites per person per night (b/p/n) for An. nuneztovari in Córdoba-Pacific and 17.5 b/p/n for An. darlingi in Villa Grande-Bajo Cauca. Both species were active mainly in indoor unwalled rooms of the houses. Only An. nuneztovari from Córdoba-Pacific was infected with Plasmodium, with an entomological inoculation rate of 91.25 infective bites per year. Detection of blood feeding sources demonstrate that humans were the most common host, however, An. nuneztovari showed a preference for feeding on dogs and An. darlingi on pigs, dogs and Galliformes, rather than humans. These results contribute to entomological surveillance information and provide valuable data that can be used to tailor effective control interventions to minimize human-vector contact in these malaria endemic regions.

Blood feeding habits of mosquitoes: hardly a bite in South America.

Mosquito blood feeding plays a key role in epidemiology. Despite its importance and large number of studies worldwide, less attention has been paid in South America. We summarized some general concepts and methodological issues related to the study of mosquito blood feeding habits, and compiled and analyzed all published information regarding the subject in the continent until 2020. Available literature comprised 152 scientific studies, that pursued different approaches: human landing catches (102 studies), baited trap (19), and blood meal analyses of collected specimens (38). Among the latter, 23 used serological and 15 molecular techniques. Species most frequently studied were those incriminated in malaria transmission, whereas relevant vectors such as Aedes aegypti, Ae. albopictus, and Haemagogus janthinomys were surprisingly neglected. Brazil was the leading country both in number of works and species studied. For over 70% of the species and three out of 13 South American countries there is no single information on mosquito blood feeding habits. Data from baited traps included 143 mosquito species, 83.9% of which were attracted to humans, either exclusively (10.5%) or in combination with other vertebrates (73.4%). Host blood identification of field collected specimens provided data on 102 mosquito species, and 60.8% of these fed on humans (55.9% combined with other vertebrates). Only 17 of the 73 species assessed by both methods yielded similar feeding patterns. Finally, supplementary tables are provided in a comprehensive summary of all information available and information gaps are highlighted for future research in the continent.

Identification of blood meal sources in species of genus Rhodnius in four different environments in the Brazilian amazon.

Chagas disease is a zoonotic disease caused by the hemoflagellate Trypanosoma cruzi and transmitted primarily by triatomine vectors. Triatomines are hematophagous insects that feed on a variety of vertebrate hosts. The Chagas disease transmission cycle is closely related to the interactions between vectors, parasites, and vertebrate hosts. Knowledge of triatomine food sources is critical to understanding Chagas disease transmission dynamics. The aim of this study was to identify blood meal sources used by triatomines from different environments in the Brazilian Amazon. A total of 25 captures were conducted in four environments. Triatomine specimens were captured on palm trees and were identified by morphological and morphometric characters. Blood meal sources identification was conducted using a traditional PCR followed by Sanger sequencing of mtDNA cytb gene. Sequencing was successful in 167 specimens and a total of 21 blood meal sources were identified: two reptilians, six birds, and 13 mammals. Among these 21 species, three (Tamandua tetradactyla, Didelphis marsupialis and Rattus rattus) are considered reservoir of T. cruzi. Knowledge of the relationship between triatomines and possible reservoirs can help to elucidate the enzootic cycle of T. cruzi in the Amazon region and guide control strategies for Chagas disease transmission in that region.

Diversity of Trypanosoma cruzi parasites infecting Triatoma dimidiata in Central Veracruz, Mexico, and their One Health ecological interactions.

Triatoma dimidiata is the main vector of Trypanosoma cruzi parasites in Veracruz, Mexico, and its association with human housing appears variable. Also, in spite of a high seroprevalence of T. cruzi infection in humans, parasite transmission remains poorly understood. Therefore, we aimed to identify T. dimidiata blood feeding sources and its parasite and microbial diversity to reconstruct T. cruzi parasite transmission ecology in central Veracruz, Mexico, within a One Health/Ecohealth framework. We used a metabarcoding and deep sequencing approach of specific markers for the simultaneous identification of T. dimidiata haplogroup (ITS-2), vertebrate blood meals (12 s gene), T. cruzi parasites (mini-exon gene), and gut microbiota (bacterial 16 s). Twelve species of domestic/synanthropic animals and humans were identified as blood sources, with multiple feeding on 4.2 ± 0.4 hosts per bug. The feeding/parasite transmission network was strongly centered on humans, emphasizing a significant risk of infection. We also unambiguously confirmed the presence of TcI, TcII, TcV and TcVI DTUs in T. dimidiata, and sequences from Veracruz tended to cluster apart from parasites from other regions, suggesting some level of local differentiation. Analysis of T. dimidiata microbiota suggested that several bacterial families may be associated with the presence/absence of T. cruzi, and some of these associations may also be parasite DTU-specific. Such integrative approaches within the EcoHealth/One Health framework provide key insights on T. cruzi transmission and potential novel strategies for disease control.

Relationship between vertical stratification and feeding habits of mosquito (Diptera: Culicidae) assemblages collected in conservation units in the green belt of the city of São Paulo, Brazil.

São Paulo is one of the largest cities in the world and has several characteristics that favor a diversity of urban and wild mosquitoes. Little is known about how variations in mosquito diversity and feeding preferences for different hosts in different vegetation strata can influence the risk of pathogen transmission to humans. We investigated vertical stratification of mosquitoes and its relationship with vertebrate hosts in environments with different degrees of conservation in two conservation units in the city of São Paulo. Adult mosquitoes were collected using CDC traps, aspiration and Shannon traps. After morphological identification, host blood in engorged females was analyzed by PCR with a vertebrate-specific primer set based on mitochondrial cytochrome b DNA of vertebrates commonly found in the two conservation units. Although a higher abundance of the species Anopheles cruzii and Culex nigripalpus was found in the canopy, blood not only from birds but also from humans and rodents was identified in these mosquitoes. In one of the units, Wyeomyia confusa and Limatus durhamii were found occupying mainly niches at ground level while Culex vaxus was frequently found in the canopy. Haemagogus leucocelaenus, the main vector of yellow fever, was found in low abundance at all collection points, particularly in the canopy. Species richness and composition tended to vary little between canopy and ground level in the same environment, but the abundance between canopy and ground level varied more depending on the species analyzed, the most abundant and frequent species exhibiting a predilection for the canopy. Even those mosquito species observed more frequently in the canopy did not show an association with hosts found in this stratum as most of the blood identified in these species was from humans, suggesting opportunist feeding behavior, i.e., feeding on the most readily available host in the environment. The two most common species in the study, An. cruzii and Cx. nigripalpus, may be able to act as bridge vectors for pathogens to circulate between the forest canopy and ground level.

Molecular ecology of Triatoma dimidiata in southern Belize reveals risk for human infection and the local differentiation of Trypanosoma cruzi parasites.

OBJECTIVE: In Belize, the main vector for Trypanosoma cruzi, the agent of Chagas disease, is Triatoma dimidiata, but transmission cycles and the risk for human infection are unclear. Therefore, the aim of this study was to identify T. dimidiata blood feeding sources and its parasite and microbial diversity, in order to reconstruct T. cruzi parasite transmission ecology in southern Belize. METHODS: A metabarcoding approach based on deep sequencing of markers was used for bug taxonomy, blood meal sources, T. cruzi genotypes, and microbiota composition. Bugs were collected in 13 villages of Toledo district. RESULTS: Bugs fed on at least 13 species, from domestic hosts such as humans, dogs, cows, and pigs, to synanthropic species such as mice, rats, and opossums, and sylvatic species such as deer, peccary, and kinkajou, in agreement with an opportunistic feeding behavior. Nonetheless, most feeding focused on a few species, including humans. Infection with T. cruzi was detected in 24 of 39 bugs (62%), and the analysis of 242 T. cruzi mini-exon sequences (average 10 ± 5 haplotypes per bug) indicated the presence of TcI and TcIV parasite discrete typing units (DTUs). However, for both DTUs, sequences from Belize mostly clustered apart from sequences from North and South America, suggesting the local differentiation of parasites. T. dimidiata also harbored a diverse bacterial microbiota, with ontogenic changes suggesting microbiota maturation during nymphal development. CONCLUSIONS: Together, these results indicate a significant risk for T. cruzi infection in humans. They also highlight the need to better characterize the diversity of T. cruzi strains in the region and its impact on disease epidemiology.

Triatomine Feeding Profiles and Trypanosoma cruzi Infection, Implications in Domestic and Sylvatic Transmission Cycles in Ecuador.

Understanding the blood meal patterns of insects that are vectors of diseases is fundamental in unveiling transmission dynamics and developing strategies to impede or decrease human-vector contact. Chagas disease has a complex transmission cycle that implies interactions between vectors, parasites and vertebrate hosts. In Ecuador, limited data on human infection are available; however, the presence of active transmission in endemic areas has been demonstrated. The aim of this study was to determine the diversity of hosts that serve as sources of blood for triatomines in domestic, peridomestic and sylvatic transmission cycles, in two endemic areas of Ecuador (central coastal and southern highland regions). Using conserved primers and DNA extracted from 507 intestinal content samples from five species of triatomines (60 Panstrongylus chinai, 17 Panstrongylus howardi, 1 Panstrongylus rufotuberculatus, 427 Rhodnius ecuadoriensis and 2 Triatoma carrioni) collected from 2006 to 2013, we amplified fragments of the cytb mitochondrial gene. After sequencing, blood meal sources were identified in 416 individuals (146 from central coastal and 270 from southern highland regions), achieving ≥ 95% identity with GenBank sequences (NCBI-BLAST tool). The results showed that humans are the main source of food for triatomines, indicating that human-vector contact is more frequent than previously thought. Although other groups of mammals, such as rodents, are also an available source of blood, birds (particularly chickens) might have a predominant role in the maintenance of triatomines in these areas. However, the diversity of sources of blood found might indicate a preference driven by triatomine species. Moreover, the presence of more than one source of blood in triatomines collected in the same place indicated that dispersal of vectors occurs regardless the availability of food. Dispersal capacity of triatomines needs to be evaluated to propose an effective strategy that limits human-vector contact and, in consequence, to decrease the risk of T. cruzi transmission.

Standardization of blood meal production process at a rendering plant using design of experiments / Padronização do processo de produção de farinha de sangue em uma usina de processamento por meio do planejamento de experimentos

This research focuses on the standardization of the blood meal production process at a Colombian rendering plant through a design of experiments. Initially, 108 samples of blood meal were taken where only 23% achieved the moisture target (7.5% to 8.5%). Therefore, an analysis of the measurement system was performed using a repeatability and reproducibility (R&R) study. Results showed that 39.96% of the observed variability was caused by the measurement system that was out of control. So, it was necessary to improve the method of sampling reducing the participation of the measurement system in the variability of the process to only 3.79%. Later, several experiments were accomplished with a 2k factorial design. Each experiment consisted of a response variable (blood meal moisture), two controllable factors (drying chamber temperature and percentage of rotation of the metering screw), and an uncontrollable factor (initial blood meal moisture). Finally, experiments were carried out and validated observing that, with a drying chamber temperature of 160 °C and a percentage of screw rotation of 29%, more than 97% of the blood meal was according to the moisture target. In conclusion, is confirmed that the design of experiments is a tool that allows a clear path towards optimization and standardization of processes.(AU)

Esta pesquisa se concentra na padronização do processo de produção de farinha de sangue de uma usina de beneficiamento colombiana por meio de um planejamento de experimentos. Em primeiro lugar, foi realizado um estudo de 108 amostras de farinha de sangue onde apenas 23% atingiram a meta de umidade (7.5% a 8.5%). Portanto, uma análise do sistema de medição foi realizada usando um estudo de repetibilidade e reprodutibilidade (R&R). Os resultados mostraram que 39.96% da variabilidade observada foi causada pelo sistema. Assim, foi necessário aprimorar o método de amostragem, reduzindo a participação do sistema na variabilidade para apenas 3.79%. Posteriormente, vários experimentos foram realizados com um planejamento fatorial 2k. Cada experimento consistiu em uma variável de resposta (umidade da farinha de sangue), dois fatores controláveis (temperatura da câmara de secagem e porcentagem de rotação da rosca dosadora) e um fator incontrolável (umidade inicial da farinha de sangue). Finalmente, experimentos foram realizados e validados observando que, com uma temperatura da câmara de secagem de 160 ° C e um percentual de rotação da rosca de 29%, mais de 97% da farinha de sangue estava de acordo com a umidade desejada. Em conclusão, confirma-se que o planejamento de experimentos é uma ferramenta que permite um caminho claro para a otimização e padronização de processos.(AU)

No evidence for sylvatic cycles of chikungunya, dengue and Zika viruses in African green monkeys (Chlorocebus aethiops sabaeus) on St. Kitts, West Indies.

BACKGROUND: Dengue, chikungunya and Zika viruses (DENV, CHIKV and ZIKV) are transmitted in sylvatic transmission cycles between non-human primates and forest (sylvan) mosquitoes in Africa and Asia. It remains unclear if sylvatic cycles exist or could establish themselves elsewhere and contribute to the epidemiology of these diseases. The Caribbean island of St. Kitts has a large African green monkey (AGM) (Chlorocebus aethiops sabaeus) population and is therefore ideally suited to investigate sylvatic cycles. METHODS: We tested 858 AGM sera by ELISA and PRNT for virus-specific antibodies and collected and identified 9704 potential arbovirus vector mosquitoes. Mosquitoes were homogenized in 513 pools for testing by viral isolation in cell culture and by multiplex RT-qPCR after RNA extraction to detect the presence of DENV, CHIKV and ZIKVs. DNA was extracted from 122 visibly blood-fed individual mosquitoes and a polymorphic region of the hydroxymethylbilane synthase gene (HMBS) was amplified by PCR to determine if mosquitoes had fed on AGMs or humans. RESULTS: All of the AGMs were negative for DENV, CHIKV or ZIKV antibodies. However, one AGM did have evidence of an undifferentiated Flavivirus infection. Similarly, DENV, CHIKV and ZIKV were not detected in any of the mosquito pools by PCR or culture. AGMs were not the source of any of the mosquito blood meals. CONCLUSION: Sylvatic cycles involving AGMs and DENV, CHIKV and ZIKV do not currently exist on St. Kitts.

Blood Meal Sources of Anopheles spp. in Malaria Endemic Areas of Honduras.

Malaria remains a life-threatening disease in many tropical countries. Honduras has successfully reduced malaria transmission as different control methods have been applied, focusing mainly on indoor mosquitoes. The selective pressure exerted by the use of insecticides inside the households could modify the feeding behavior of the mosquitoes, forcing them to search for available animal hosts outside the houses. These animal hosts in the peridomicile could consequently become an important factor in maintaining vector populations in endemic areas. Herein, we investigated the blood meal sources and Plasmodium spp. infection on anophelines collected outdoors in endemic areas of Honduras. Individual PCR reactions with species-specific primers were used to detect five feeding sources on 181 visibly engorged mosquitoes. In addition, a subset of these mosquitoes was chosen for pathogen analysis by a nested PCR approach. Most mosquitoes fed on multiple hosts (2 to 4), and 24.9% of mosquitoes had fed on a single host, animal or human. Chicken and bovine were the most frequent blood meal sources (29.5% and 27.5%, respectively). The average human blood index (HBI) was 22.1%. None of the mosquitoes were found to be infected with Plasmodium spp. Our results show the opportunistic and zoophilic behavior of Anopheles mosquitoes in Honduras.

Eco-epidemiological study reveals the importance of Triatoma dimidiata in the Trypanosoma cruzi transmission, in a municipality certified without transmission by Rhodnius prolixus in Colombia.

Boyaca department is an endemic area for Chagas disease in Colombia, where 24 of its municipalities have been certified by the PAHO with interruption of Trypanosoma cruzi transmission by R. prolixus. However, the presence of secondary vectors, represent a risk of parasite transmission for citizens and a challenge for the health care institutions. The aim of this work was to investigate eco-epidemiological features of Chagas disease in the municipality of Socota (Boyaca), in order to improve control and surveillance strategies. To understand the transmission dynamics of T. cruzi in this area, we designed a comprehensive, multi-faceted study including: (i) entomological survey in five villages (La Vega, Comaita, Chusvita, Guaquira and Pueblo Nuevo), (ii) blood meal source determination, (iii) T. cruzi infection rate in collected triatomines, (iv) identification of circulating T. cruzi genotypes, (v) serological determination of T. cruzi infection in domestic dogs; and (vi) evaluation of infection in synanthropic mammals. A total of 90 T. dimidiata were collected, of which 73.3% (66/90) and 24.4% (22/90) were collected inside dwellings and peridomestic areas, respectively, while the rest (2/90) in Chusvita Elementary School. T. cruzi infection was evidenced in 40% (36/90) of triatomine bugs using PCR analyses, in which only DTU I was found, and TcI Dom was the most distributed. Blood-meal analysis showed that T. dimidiata only fed of humans. Seroprevalence in domestic dogs was 4.6% (3/66), while that two Didelphis marsupialis captured showed no infection. In conclusion, the high dispersion and colonization of T. dimidiata shown in this municipality, along the high rate of T. cruzi (TcI) infection and its anthropophilic behavior constitute a risk situation for Chagas disease transmission in this municipality certified without R. prolixus. The epidemiological implications of these findings are herein discussed.

Fast multiplex real-time PCR assay for simultaneous detection of dog and human blood and Leishmania parasites in sand flies.

BACKGROUND: The blood-feeding behaviour of female sand flies may increase their likelihood of acquiring and transmitting Leishmania parasites. Studies on the host usage by these insects may thus improve our understanding of the Leishmania transmission risk in leishmaniasis-endemic areas. Here, we developed a fast multiplex real-time PCR assay for simultaneous detection of dog, human and Leishmania DNA in sand flies. METHODS: Primers and TaqMan probes targeting the mitochondrial cytochrome c oxidase subunit 1 and cytochrome b genes of dog and human, respectively, were combined in a multiplex assay, which also includes primers and a TaqMan probe targeting the Leishmania minicircle kinetoplast DNA. RESULTS: The multiplex assay was 100% specific, with analytical sensitivities of 103 fg/reaction for dog and human and 1 fg for Leishmania. By testing field-collected engorged female sand flies (95 Migonemyia migonei and two Nyssomyia intermedia), 50 M. migonei were positive for one or two targets (positivity rates: 45.4% for human, 4.1% for dog and 12.4% for Leishmania DNA). CONCLUSIONS: This multiplex real-time PCR assay represents a novel fast assay for detecting dog, human and Leishmania DNA in female sand flies and therefore a tool for assessing the risk of Leishmania transmission to these hosts in areas of active transmission.

Técnicas moleculares integradas a la vigilancia entomológica de vectores de la enfermedad de Chagas: Estudio del vector secundario Triatoma sordida en la Región Oriental del Paraguay / Molecular techniques integrated to the entomological surveillance of Chagas disease vectors: Study of the secondary vector Triatoma sordida in the Eastern Region of Paraguay

Las técnicas moleculares para la detección de infección natural y fuente de alimentación en vectores secundarios de la enfermedad de Chagas cuando son aplicadas a ejemplares capturados en áreas endémicas, históricamente ocupadas por Triatoma infestans, proporcionan a las investigaciones epidemiológicas respuestas más exactas con relación a la transmisibilidad de la enfermedad. El presente estudio tiene como objetivo emplear biomarcadores moleculares para evaluar el impacto de la infestación intra y peridomicilar de Triatoma sordida en viviendas bajo vigilancia entomológica de departamentos de la Región Oriental del Paraguay en el período 2007 al 2015. Un total de 559 ejemplares de T. sordida capturados en 253, 91 y 52 viviendas de los departamentos Paraguarí, San Pedro y Cordillera, respectivamente fueron analizados. La infestación detectada fue del 24% al 48% así como una elevada colonización intradomiciliar del 5% al 36% en los tres departamentos. La detección molecular de infección natural osciló entre el 14% y 44%; y en 111 ejemplares se determinó la fuente de alimentación. El marcador molecular citocromo b permitió demostrar por vez primera un elevado porcentaje de triatominos con sangre humana como fuente de alimentación, principalmente en Cordillera con un 82% (28/34 T. sordida capturados). Estos hallazgos dejan en evidencia el avance del T. sordida en la ocupación del nicho ecológico de T. cruzi y la capacidad de esta especie secundaria como vector en la transmisión de T. cruzi en comunidades de la Región Oriental

When molecular techniques for the detection of natural infection and blood meal source in secondary vectors of Chagas disease are applied to specimens captured in endemic areas, historically occupied by Triatoma infestans, provide more accurate answers to questions about transmissibility of the illness and further contribute to the epidemiological studies. The aim of this study was to evaluate the impact of intra and peridomiciliary infestation of Triatoma sordida in households from the departments of the Eastern Region of Paraguay, under entomological surveillance during the period 2007 to 2015, by using the molecular biomarkers technology. A total of 559 specimens of T. sordida captured in 253, 91 and 52 households from Paraguarí, San Pedro and Cordillera departments, respectively, were analyzed. The infestation detected was from 24% to 48% as well as a high intradomicialiary colonization from 5% to 36% in the three departments. The molecular detection of natural infections ranged from 14% to 44% and in 111 specimens the meal source was identified. The molecular marker cytochrome b allowed to demonstrate, for the first time, high frequency of triatomines with human blood as a food source, mainly in Cordillera as it was determined in 82% (28/34) of the T. sordida captured. These findings demonstrate a progress of T. sordida into the ecological niche of T. cruzi and the abillity of this secondary species as a vector of the transmission of T. cruzi in communities from the Eastern Region of Paraguay

An overview of the sandfly fauna (Diptera: Psychodidae) followed by the detection of Leishmania DNA and blood meal identification in the state of Acre, Amazonian Brazil

BACKGROUND In Acre state, Brazil, the dissemination of cutaneous leishmaniasis has increased in recent years, with limited knowledge of the potential Leishmania spp. vectors involved. OBJECTIVES Here, data concerning the sandfly fauna of Brasiléia municipality, Leishmania DNA-detection rates and the identification of blood meal sources of insects captured in 2013-2015 are presented. METHODS Parasite detection in female sandflies was performed individually by multiplex polymerase chain reaction (PCR) (Leishmania kDNA/sandfly cacophony-gene), with the identification of Leishmania spp. by hsp70-PCR and sequencing. The identification of blood gut-content from fed females was performed by cyt b-PCR and sequencing. FINDINGS A total of 4,473 sandflies were captured. A subgroup of 864 non-blood-fed females evaluated for the presence of Leishmania DNA showed 2.9% positivity for Leishmania (Viannia) braziliensis and L. (V.) guyanensis. The identification of blood meal sources was performed in 96 blood-fed females, allowing the identification of 13 vertebrate species. In nine/96 fed females, DNA from L. (V.) shawi, L. (V.) guyanensis, L. (V.) braziliensis and Endotrypanum sp. was detected. MAIN CONCLUSIONS In Brumptomyia sp. and Evandromyia termitophila, the first report of Leishmania DNA-detection is provided in Acre; Nyssomyia shawi is implicated as potential vector of L. (V.) braziliensis and L. (V.) guyanensis for the first time in Brazil.