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Background Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.Methodology/Principal FindingsThe T. rangeli haploid genome is ∼24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heat-shock proteins.Conclusions/SignificancePhylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets.

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.

Human sewage from Florianopolis (Santa Catarina, Brazil) was analyzed for severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) from October 2019 until March 2020. Twenty five ml of sewage samples were clarified and viruses concentrated using a glycine buffer method coupled with polyethylene glycol precipitation, and viral RNA extracted using a commercial kit. SARS-CoV-2 RNA was detected by RT-qPCR using oligonucleotides targeting N1, S and two RdRp regions. The results of all positive samples were further confirmed by a different RT-qPCR system in an independent laboratory. S and RdRp amplicons were sequenced to confirm identity with SARS-CoV-2. Genome sequencing was performed using two strategies; a sequence-independent single-primer amplification (SISPA) approach, and by direct metagenomics using Illumina's NGS. SARS-CoV-2 RNA was detected on 27th November 2019 (5.49 ± 0.02 log 10 SARS-CoV-2 genome copies (GC) L −1 ), detection being confirmed by an independent laboratory and genome sequencing analysis. The samples in the subsequent three events were positive by all RT-qPCR assays; these positive results were also confirmed by an independent laboratory. The average load was 5.83 ± 0.12 log 10 SARS-CoV-2 GC L −1 , ranging from 5.49 ± 0.02 log 10 GC L −1 (27th November 2019) to 6.68 ± 0.02 log 10 GC L −1 (4th March 2020). Our findings demonstrate that SARS-CoV-2 was likely circulating undetected in the community in Brazil since November 2019, earlier than the first reported case in the Americas (21st January 2020).

We analysed human sewage located in Florianopolis (Santa Catalina, Brazil) from late October until the Brazil lockdown on early March. We detected SARS-CoV-2 in two samples collected independently on 27th November 2019 (5.49 ± 0.02 log genome copies/L). Subsequent samplings were positive until 4th March 2020 (coinciding with the first COVID-19 case reported in Santa Catalina), with a SARS-CoV-2 RNA increase of one log (6.68 ± 0.02 log genome copies/L). Our results show that SARS-CoV-2 has been circulating in Brazil since late November 2019, much earlier than the first reported case in the Americas (21st January 2020, USA).

Two species of the genus Trypanosoma infective to humans have been extensively studied at a cell and molecular level, but study of the third, Trypanosoma rangeli, remains in relative infancy. T. rangeli is non-pathogenic, but is frequently mistaken for the related Chagas disease agent Trypanosoma cruzi with which it shares vectors, hosts, significant antigenicity and a sympatric distribution over a wide geographical area. In this study, we present the T. rangeli gene expression profile as determined by the generation of ESTs (Expressed Sequence Tags) and ORESTES (Open Reading Frame ESTs). A total of 4208 unique high quality sequences were analyzed, composed from epimastigote and trypomastigote forms of SC-58 and Choachí strains, representing the two major phylogenetic lineages of this species. Comparative analyses with T. cruzi and other parasitic kinetoplastid species allowed the assignment of putative biological functions to most of the sequences generated and the establishment of an annotated T. rangeli gene expression database. Even though T. rangeli is apathogenic to mammals, genes associated with virulence in other pathogenic kinetoplastids were found. Transposable elements and genes associated mitochondrial gene expression, specifically RNA editing components, are also described for the first time. Our studies confirm the close phylogenetic relationship between T. cruzi and T. rangeli and enable us to make an estimate for the size of the T. rangeli genome repertoire ( approximately 8500 genes).

BackgroundIt has been suggested that genes involved in the reproductive biology of insect disease vectors are potential targets for future alternative methods of control. Little is known about the molecular biology of reproduction in phlebotomine sand flies and there is no information available concerning genes that are expressed in male reproductive organs of Lutzomyia longipalpis, the main vector of American visceral leishmaniasis and a species complex.Methods/Principal FindingsWe generated 2678 high quality ESTs (“Expressed Sequence Tags”) of L. longipalpis male reproductive organs that were grouped in 1391 non-redundant sequences (1136 singlets and 255 clusters). BLAST analysis revealed that only 57% of these sequences share similarity with a L. longipalpis female EST database. Although no more than 36% of the non-redundant sequences showed similarity to protein sequences deposited in databases, more than half of them presented the best-match hits with mosquito genes. Gene ontology analysis identified subsets of genes involved in biological processes such as protein biosynthesis and DNA replication, which are probably associated with spermatogenesis. A number of non-redundant sequences were also identified as putative male reproductive gland proteins (mRGPs), also known as male accessory gland protein genes (Acps).ConclusionsThe transcriptome analysis of L. longipalpis male reproductive organs is one step further in the study of the molecular basis of the reproductive biology of this important species complex. It has allowed the identification of genes potentially involved in spermatogenesis as well as putative mRGPs sequences, which have been studied in many insect species because of their effects on female post-mating behavior and physiology and their potential role in sexual selection and speciation. These data open a number of new avenues for further research in the molecular and evolutionary reproductive biology of sand flies.

The objective of this study was to evaluate the effect of supplementation with 100ppm sodium monensin or 0.15% of a blend of functional oils (cashew nut oil + castor oil) on the intestinal microbiota of broilers challenged with three different Eimeria spp. The challenge was accomplished by inoculating broiler chicks with sporulated oocysts of Eimeria tenella, Eimeria acervulina, and Eimeria maxima via oral gavage. A total of 864, day-old male broiler chicks (Cobb) were randomly assigned to six treatments (eight pens/treatment; 18 broilers/ pen) in a 3 × 2 factorial arrangement, composed of three additives (control, monensin or blend), with or without Eimeria challenge. Intestinal contents was collected at 28 days of age for microbiota analysis by sequencing 16s rRNA in V3 and V4 regions using the Illumina MiSeq platform. Taxonomy was assigned through the SILVA database version 132, using the QIIME 2 software version 2019.1. No treatment effects (p > 0.05) were observed in the microbial richness at the family level estimated by Chao1 and the biodiversity assessed by Simpson's index, except for Shannon's index (p < 0.05). The intestinal microbiota was dominated by members of the order Clostridiales and Lactobacillales, followed by the families Ruminococcaceae, Bacteroidaceae, and Lactobacillaceae, regardless of treatment. When the controls were compared, in the challenged control group there was an increase in Erysipelotrichaceae, Lactobacillaceae, Bacteroidaceae, Streptococcaceae, and Peptostreptococcaceae, and a decrease in Ruminococcaceae. Similar results were found for a challenged group that received monensin, while the blend partially mitigated this variation. Therefore, the blend alleviated the impact of coccidiosis challenge on the microbiome of broilers compared to monensin.