We report Trypanosoma cruzi infection in wild and domestic mammals from three orally acquired Chagas disease outbreak areas in Brazil. Cachoeiro do Arari (Pará) displayed a panzootic scenery (positive mammals in all ecologic strata), and human cases were probably the consequence of their exposure within the sylvatic T. cruzi transmission cycle. In Navegantes (Santa Catarina), Didelphis spp. was the main reservoir host, given that 93% were infected. In Redenção (Ceará), Monodelphis domestica and Thrichomys laurentius were also important for parasite maintenance. TCI was present in the three studied areas. Additionally, Z3 was detected in an armadillo from Pará and TCII in a triatomine from Navegantes. Domestic animals showed a high seroprevalence and should be considered sentinels in surveillance programs. The importance of a reduction in wild mammalian fauna diversity and selection of suitable T. cruzi reservoir hosts are discussed as risk factors for the re-emergence of Chagas disease.

Abbreviation: PCR, polymerase chain reaction; RT-qPCR, reverse transcriptase quantitative polymerase chain reaction; YFV, yellow fever virus; NS5, protein of the viral polyprotein, it is the largest and the most highly conserved among the flaviviral proteins; 17DD, strain used to yellow fever vaccine; YF, yellow fever; RNA, ribonucleic acid; C, capsid protein; prM/M, membrane protein; E, envelope protein; NS, nonstructural protein; ELISA, enzyme-linked immunosorbent assay; DNA, deoxyribonucleic acid; DL, detection limit; QL, quantification limit; ANVISA, Brazilian Health Surveillance Agency; CV, coefficient of variation; RNAse P, human constitutive gene; Ct, cycle threshold; EXO IPC, exogenous internal positive control; PFU, plaque former unit; FDA, food and drug administration agency; JEV, japanese encephalitis virus; DENV, dengue virus; MuV, mumps virus; MV, measles virus; MOI, multiplicity of infection; WNV, West Nile VirusThe development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.

This is a long-term follow-up of infection by Trypanosoma cruzi (TC) and Trypanosoma evansi (TE) in the free-ranging coatis (Procyonidae: Nasua nasua) from Pantanal region (Mato Grosso do Sul, Brazil). We evaluated TC and TE infection by immunofluorescence assay, hemoculture (HC), and microhematocrit centrifuge techniques (MHCT). We also examined coatis health by quantifying hematological parameters including packed cell volume (PCV), white blood cell (WBC) count, and differential leukocyte count. TC isolates thought HC were typed by miniexon gene. Mixed infections by both parasites and the two main lineages of TC (76% TCI, 3% TCII, and 14% TCI/TCII) were observed. Trypanosoma rangeli was also isolated (7%). Overall, seroprevalence of TC and TE infection were 53.5% and 42.0%, respectively. Positive HC (indicating high TC parasitemia) occurred in 34% of seropositive coatis for TC, and positive MHCT (high TE parasitemia) were observed in 36.4% of seropositive coatis for TE. We detected higher prevalence of positive HC in females (72%) than males (43%), and also during the dry season, indicating a seasonal potential of this host species on TC transmission. These features did not occur for TE infection. However, prevalence of TE based on serology and MHCT was higher among adults than subadults. Coatis with positive HC or MHCT displayed a slight decrease in their WBC. In contrast to the animals with positive HC, coatis with positive MHCT displayed a decrease on their PCV. Moreover, concurrent high TC and TE parasitemia caused a larger decrease of PCV values. This study corroborates the importance of coatis in the maintenance of TC and TE transmission cycles in the southern Pantanal and shows a seasonal character of TC transmissibility to its vector by the coati population from the study area.

BackgroundAsymptomatic individuals are one of the major challenges for malaria elimination programs in endemic areas. In the absence of clinical symptoms and with a lower parasite density they constitute silent reservoirs considered important for maintaining transmission of human malaria. Studies from Brazil have shown that infected individuals may carry these parasites for long periods.ResultsPatients were selected from three periurban endemic areas of the city of Manaus, in the western Brazilian Amazon. Symptomatic and asymptomatic patients with positive thick blood smear and quantitative real-time PCR (qPCR) positive for Plasmodium vivax were invited to participate in the study. A standardised pvs25 gene amplification by qPCR was used for P. vivax gametocytes detection. Anopheles aquasalis were fed using membrane feeding assays (MFA) containing blood from malaria patients. Parasitemia of 42 symptomatic and 25 asymptomatic individuals was determined by microscopic examination of blood smears and qPCR. Parasitemia density and gametocyte density were assessed as determinants of infection rates and oocysts densities. A strong correlation between gametocyte densities (microscopy and molecular techniques) and mosquito infectivity (P < 0.001) and oocysts median numbers (P < 0.05) was found in both groups. The ability to infect mosquitoes was higher in the symptomatic group (41%), but infectivity in the asymptomatic group was also seen (1.42%).ConclusionsAlthough their infectivity to mosquitoes is relatively low, given the high prevalence of P. vivax asymptomatic carriers they are likely to play and important role in malaria transmission in the city of Manaus. The role of asymptomatic infections therefore needs to be considered in future malaria elimination programs in Brazil.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2749-0) contains supplementary material, which is available to authorized users.

Whole mitogenome sequences (mtDNA) have been exploited for insect ecology studies, using them as molecular markers to reconstruct phylogenies, or to infer phylogeographic relationships and gene flow. Recent Anopheles phylogenomic studies have provided information regarding the time of deep lineage divergences within the genus. Here we report the complete 15,393 bp mtDNA sequences of Anopheles aquasalis , a Neotropical human malaria vector. When comparing its structure and base composition with other relevant and available anopheline mitogenomes, high similarity and conserved genomic features were observed. Furthermore, 22 mtDNA sequences comprising anopheline and Dipteran sibling species were analyzed to reconstruct phylogenies and estimate dates of divergence between taxa. Phylogenetic analysis using complete mtDNA sequences suggests that A . aquasalis diverged from the Anopheles albitarsis complex ~28 million years ago (MYA), and ~38 MYA from Anopheles darlingi . Bayesian analysis suggests that the most recent ancestor of Nyssorhynchus and Anopheles + Cellia was extant ~83 MYA, corroborating current estimates of ~79–100 MYA. Additional sampling and publication of African, Asian, and North American anopheline mitogenomes would improve the resolution of the Anopheles phylogeny and clarify early continental dispersal routes.

Abstract. We analyzed the development of Leishmania (Leishmania) infantum chagasi in its natural sandfly vector Lutzomyia longipalpis. In addition, we compared sandfly infections initiated with axenic amastigotes or promastigotes. Our data showed no important difference between Lu. longipalpis infection rates resulting from either type of infections. Furthermore, development of infection was equivalent in both cases. All promastigote forms were found inside the sandfly and, after blood digestion, most of the population consisted of procyclics and nectomonads. A low percentage of metacyclic forms was coincident with a high number of nectomonads during late stages of infection, but which form gives rise to metacyclic forms in L. infantum chagasi is unknown. These results also show that the promastigote infection model, at least for this situation, is suitable for obtaining of infected sandflies because it is easier and less laborious.

Dengue virus (DENV) and Zika virus (ZIKV) belong to the same viral family, the Flaviviridae. They cause recurring threats to the public health systems of tropical countries such as Brazil. The primary Brazilian vector of both viruses is the mosquito Aedes aegypti. After the mosquito ingests a blood meal from an infected person, the viruses infect and replicate in the midgut, disseminate to secondary tissues and reach the salivary gland (SG), where they are ready to be transmitted to a vertebrate host. It is thought that the intrinsic discrepancies among mosquitoes could affect their ability to deal with viral infections. This study confirms that the DENV and ZIKV infection patterns of nine Ae. aegypti field populations found in geographically separate health districts of an endemic Brazilian city vary. We analyzed the infection rate, disseminated infection, vector competence, and viral load through quantitative PCR. Mosquitoes were challenged using the membrane-feeding assay technique and were tested seven and fourteen days post-infection (early and late infection phases, respectively). The infection responses varied among the Ae. aegypti populations for both flaviviruses in the two infection phases. There was no similarity between DENV and ZIKV vector competencies or viral loads. According to the results of our study, the risk of viral transmission overtime after infection either increases or remains unaltered in ZIKV infected vectors. However, the risk may increase, decrease, or remain unaltered in DENV-infected vectors depending on the mosquito population. For both flaviviruses, the viral load persisted in the body even until the late infection phase. In contrast to DENV, the ZIKV accumulated in the SG over time in all the mosquito populations. These findings are novel and may help direct the development of control strategies to fight dengue and Zika outbreaks in endemic regions, and provide a warning about the importance of understanding mosquito responses to arboviral infections.

BackgroundMalaria is transmitted when an infected mosquito delivers Plasmodium sporozoites into a vertebrate host. There are many species of Plasmodium and, in general, the infection is host-specific. For example, Plasmodium gallinaceum is an avian parasite, while Plasmodium berghei infects mice. These two parasites have been extensively used as experimental models of malaria transmission. Plasmodium falciparum and Plasmodium vivax are the most important agents of human malaria, a life-threatening disease of global importance. To complete their life cycle, Plasmodium parasites must traverse the mosquito midgut and form an oocyst that will divide continuously. Mature oocysts release thousands of sporozoites into the mosquito haemolymph that must reach the salivary gland to infect a new vertebrate host. The current understanding of the biology of oocyst formation and sporozoite release is mostly based on experimental infections with P.berghei, and the conclusions are generalized to other Plasmodium species that infect humans without further morphological analyses.ResultsHere, it is described the microanatomy of sporozoite escape from oocysts of four Plasmodium species: the two laboratory models, P. gallinaceum and P. berghei, and the two main species that cause malaria in humans, P.vivax and P. falciparum. It was found that sporozoites have species-specific mechanisms of escape from the oocyst. The two model species of Plasmodium had a common mechanism, in which the oocyst wall breaks down before sporozoites emerge. In contrast, P. vivax and P. falciparum sporozoites show a dynamic escape mechanism from the oocyst via polarized propulsion.ConclusionsThis study demonstrated that Plasmodium species do not share a common mechanism of sporozoite escape, as previously thought, but show complex and species-specific mechanisms. In addition, the knowledge of this phenomenon in human Plasmodium can facilitate transmission-blocking studies and not those ones only based on the murine and avian models.