Superparamagnetic iron oxide nanoparticles as a tool to track mouse neural stem cells in vivo.

Cell transplantation offers a promising approach in many neurological disorders. Neural stem (NS) cells are potential candidates for cell therapy. The ability to track the grafted cells in the host tissue will refine this therapy. Superparamagnetic iron oxide nanoparticles (SPION) have been suggested as a feasible method, but there is no consensus about its safety. Here we investigated the feasibility of label NS cells with SPION and track by MRI after transplantation into mouse striatum with SPION cells and its therapeutic effects by grafting the cells into mouse striatum. We demonstrated that SPION-labeled NS cells display normal patterns of cellular processes including proliferation, migration, differentiation and neurosphere formation. Transmission electron microscopy reveals SPION in the cytoplasm of the cells, which was confirmed by microanalysis. Neurons and astrocytes generated from SPION-labeled NS cells were able to carry nanoparticles after 7 days under differentiation. SPION-labeled NS cells transplanted into striatum of mice were detected by magnetic resonance imaging (MRI) and microscopy 51 days later. In agreement with others reports, we demonstrated that NS cells are able to incorporate SPION in vitro without altering the stemness, and can survive and be tracked by MRI after they have been grafted into mice striatum.

Development and fate of the residual body of Toxoplasma gondii.

As the tachyzoite form of Toxoplasma gondii divides inside the parasitophorous vacuole, the daughter cells remain attached to each other at the posterior end through the so-called residual body (RB). Here, we studied this process using field emission scanning electron microscopy of dry scraped infected cells, transmission electron microscopy of random ultrathin sections, X-ray microanalysis, and 3-D modelling of tomographic volumes and slice and view series obtained by FIB SEM at 7, 24, and 48 h post infection. Combining these methods of observation, we traced a timeline of events for the formation, development, and fate of the RB. The RB is formed as the first endodyogenic division is complete. Before that, finger-like invaginations at the posterior end of the tachyzoite secrete tubules from the intravacuolar network. The RB is roughly spherical and measures 1 µm in diameter at random. Its size does not vary considerably as the division cycles that form the rosette proceed. The contents of the RB are similar to the cytoplasm of the parasites. It contains ER membranous profiles and vacuolar structures identified as acidocalcisomes. This was confirmed by microanalysis. Mitochondrial profiles seen inside the RB are actually branches of mother cell mitochondrion not yet split between the two daughter cells. Acidocalcisomes of a mother cell are distributed between the two daughter cells, but as the rosette of parasites grow, acidocalcisomes seem to concentrate inside the RB where they are usually larger and tend to fuse to each other, filling most of the space in the RB. Here we hypothesize that, upon egress, the acidocalcisomes would ultimately fuse with the RB membrane liberating its contents inside the parasitophorous vacuole (PV) and, by doing so; the RB would disintegrate, releasing its contents in the PV.

New advances in scanning microscopy and its application to study parasitic protozoa.

Scanning electron microscopy has been used to observe and study parasitic protozoa for at least 40 years. However, field emission electron sources, as well as improvements in lenses and detectors, brought the resolution power of scanning electron microscopes (SEM) to a new level. Parallel to the refinement of instruments, protocols for preservation of the ultrastructure, immunolabeling, exposure of cytoskeleton and inner structures of parasites and host cells were developed. This review is focused on protozoan parasites of medical and veterinary relevance, e.g., Toxoplasma gondii, Tritrichomonas foetus, Giardia intestinalis, and Trypanosoma cruzi, compilating the main achievements in describing the fine ultrastructure of their surface, cytoskeleton and interaction with host cells. Two new resources, namely, Helium Ion Microscopy (HIM) and Slice and View, using either Focused Ion Beam (FIB) abrasion or Microtome Serial Sectioning (MSS) within the microscope chamber, combined to backscattered electron imaging of fixed (chemically or by quick freezing followed by freeze substitution and resin embedded samples is bringing an exponential amount of valuable information. In HIM there is no need of conductive coating and the depth of field is much higher than in any field emission SEM. As for FIB- and MSS-SEM, high resolution 3-D models of areas and volumes larger than any other technique allows can be obtained. The main results achieved with all these technological tools and some protocols for sample preparation are included in this review. In addition, we included some results obtained with environmental/low vacuum scanning microscopy and cryo-scanning electron microscopy, both promising, but not yet largely employed SEM modalities.

Identification of new palmitoylated proteins in Toxoplasma gondii.

Protein palmitoylation has been shown to be an important post-translational modification in eukaryotic cells. This modification alters the localization and/or the function of the targeted protein. In recent years, protein palmitoylation has risen in importance in apicomplexan parasites as well. In Toxoplasma gondii, some proteins have been reported to be modified by palmitate. With the development of new techniques that allow the isolation of palmitoylated proteins, this significant post-translational modification has begun to be studied in more detail in T. gondii. Here we describe the palmitoylome of the tachyzoite stage of T. gondii using a combination of the acyl-biotin exchange chemistry method and mass spectrometry analysis. We identified 401 proteins found in multiple cellular compartments, with a wide range of functions that vary from metabolic processes, gliding and host-cell invasion to even regulation of transcription and translation. Besides, we found that more rhoptry proteins than the ones already described for Toxoplasma are palmitoylated, suggesting an important role for this modification in the invasion mechanism of the host-cell. This study documents that protein palmitoylation is a common modification in T. gondii that could have an impact on different cellular processes.

Developmental and Ultrastructural Characterization and Phylogenetic Analysis of Trypanosoma herthameyeri n. sp. of Brazilian Leptodactilydae Frogs.

We described the phylogenetic affiliation, development in cultures and ultrastructural features of a trypanosome of Leptodacylus chaquensis from the Pantanal biome of Brazil. In the inferred phylogeny, this trypanosome nested into the Anura clade of the basal Aquatic clade of Trypanosoma, but was separate from all known species within this clade. This finding enabled us to describe it as Trypanosoma herthameyeri n. sp., which also infects other Leptodacylus species from the Pantanal and Caatinga biomes. Trypanosoma herthameyeri multiplies as small rounded forms clumped together and evolving into multiple-fission forms and rosettes of epimastigotes released as long forms with long flagella; scarce trypomastigotes and glove-like forms are common in stationary-phase cultures. For the first time, a trypanosome from an amphibian was observed by field emission scanning electron microscopy, revealing a cytostome opening, well-developed flagellar lamella, and many grooves in pumpkin-like forms. Transmission electron microscopy showed highly developed Golgi complexes, relaxed catenation of KDNA, and a rich set of spongiome tubules in a regular parallel arrangement to the flagellar pocket as confirmed by electron tomography. Considering the basal position in the phylogenetic tree, developmental and ultrastructural data of T. herthameyeri are valuable for evolutionary studies of trypanosome architecture and cell biology.

New views of the Toxoplasma gondii parasitophorous vacuole as revealed by Helium Ion Microscopy (HIM).

The Helium Ion Microscope (HIM) is a new technology that uses a highly focused helium ion beam to scan and interact with the sample, which is not coated. The images have resolution and depth of field superior to field emission scanning electron microscopes. In this paper, we used HIM to study LLC-MK2 cells infected with Toxoplasma gondii. These samples were chemically fixed and, after critical point drying, were scraped with adhesive tape to expose the inner structure of the cell and parasitophorous vacuoles. We confirmed some of the previous findings made by field emission-scanning electron microscopy and showed that the surface of the parasite is rich in structures suggestive of secretion, that the nanotubules of the intravacuolar network (IVN) are not always straight, and that bifurcations are less frequent than previously thought. Fusion of the tubules with the parasite membrane or the parasitophorous vacuole membrane (PVM) was also infrequent. Tiny adhesive links were observed for the first time connecting the IVN tubules. The PVM showed openings of various sizes that even allowed the observation of endoplasmic reticulum membranes in the cytoplasm of the host cell. These findings are discussed in relation to current knowledge on the cell biology of T. gondii.

Three dimensional reconstruction by electron microscopy in the life sciences: An introduction for cell and tissue biologists.

Early applications of transmission electron microscopy (TEM) in the life sciences have contributed tremendously to our current understanding at the subcellular level. Initially limited to two-dimensional representations of three-dimensional (3D) objects, this approach has revolutionized the fields of cellular and structural biology-being instrumental for determining the fine morpho-functional characterization of most cellular structures. Electron microscopy has progressively evolved towards the development of tools that allow for the 3D characterization of different structures. This was done with the aid of a wide variety of techniques, which have become increasingly diverse and highly sophisticated. We start this review by examining the principles of 3D reconstruction of cells and tissues using classical approaches in TEM, and follow with a discussion of the modern approaches utilizing TEM as well as on new scanning electron microscopy-based techniques. 3D reconstruction techniques from serial sections and (cryo) electron-tomography are examined, and the recent applications of focused ion beam-scanning microscopes and serial-block-face techniques for the 3D reconstruction of large volumes are discussed. Alternative low-cost techniques and more accessible approaches using basic transmission or field emission scanning electron microscopes are also examined.

The dispersant Corexit 9500 and (dispersed) oil are lethal to coral endosymbionts.

Endosymbionts (Symbiodiniaceae) play a vital role in the health of corals. Seawater pollution can harm these endosymbionts and dispersants used during oil spill cleanup can be extremely toxic to these organisms. Here, we examined the impact of oil and a specific dispersant, Corexit-9500, on two representative endosymbionts - Symbiodinium and Cladocopium - from the Southwestern endemic coral Mussismilia braziliensis. The survival and photosynthetic potential of the endosymbionts decreased dramatically after exposure to the dispersant and oil by ~25 % after 2 h and ~50 % after 7 days. Low concentrations of dispersant (0.005 ml/l) and dispersed oil (Polycyclic Aromatic Hydrocarbons, 1132 µg/l; Total Petroleum Hydrocarbons, 595 µg/l) proved highly toxic to both Symbiodinium and Cladocopium. These levels triggered a reduction in growth rate, cell size, and cell wall thickness. After a few hours of exposure, cellular organelles were damaged or destroyed. These acute toxic effects underline the fragile nature of coral endosymbionts.

Biological characterization and next-generation genome sequencing of the unclassified Cotia virus SPAn232 (Poxviridae).

Cotia virus (COTV) SPAn232 was isolated in 1961 from sentinel mice at Cotia field station, São Paulo, Brazil. Attempts to classify COTV within a recognized genus of the Poxviridae have generated contradictory findings. Studies by different researchers suggested some similarity to myxoma virus and swinepox virus, whereas another investigation characterized COTV SPAn232 as a vaccinia virus strain. Because of the lack of consensus, we have conducted an independent biological and molecular characterization of COTV. Virus growth curves reached maximum yields at approximately 24 to 48 h and were accompanied by virus DNA replication and a characteristic early/late pattern of viral protein synthesis. Interestingly, COTV did not induce detectable cytopathic effects in BSC-40 cells until 4 days postinfection and generated viral plaques only after 8 days. We determined the complete genomic sequence of COTV by using a combination of the next-generation DNA sequencing technologies 454 and Illumina. A unique contiguous sequence of 185,139 bp containing 185 genes, including the 90 genes conserved in all chordopoxviruses, was obtained. COTV has an interesting panel of open reading frames (ORFs) related to the evasion of host defense, including two novel genes encoding C-C chemokine-like proteins, each present in duplicate copies. Phylogenetic analysis revealed the highest amino acid identity scores with Cervidpoxvirus, Capripoxvirus, Suipoxvirus, Leporipoxvirus, and Yatapoxvirus. However, COTV grouped as an independent branch within this clade, which clearly excluded its classification as an Orthopoxvirus. Therefore, our data suggest that COTV could represent a new poxvirus genus.

A simple and efficient method to observe internal structures of helminths by scanning electron microscopy.

Morphological studies of helminths by scanning electron microscopy are generally limited to the external topography of the organisms. In this work, we present a simple technique using ethanol as a cryoprotectant without postfixation in osmium tetroxide that allows for observation of the inner organization of helminths and preserves cellular structures. We tested the technique in three helminths: Echinostoma paraensei, Cruzia tentaculata, and Hassalstrongylus epsilon. The results show that this technique could potentially be applied to study the morphology, ultrastructure, and taxonomy of helminths.

Collapse of scallop Nodipecten nodosus production in the tropical Southeast Brazil as a possible consequence of global warming and water pollution.

Mollusc rearing is a relevant global socioeconomic activity. However, this activity has faced severe problems in the last years in southeast Brazil. The mariculture scallop production dropped from 51,2 tons in 2016 to 10,2 tons in 2022 in the Baia da Ilha Grande (BIG; Rio de Janeiro). However, the possible causes of this collapse are unknown. This study aimed to analyze decadal trends of water quality in Nodipecten nodosus spat and adult production in BIG. We also performed physical-chemical and biological water quality analyses of three scallop farms and two nearby locations at BIG in 2022 to evaluate possible environmental stressors and risks. Scallop spat production dropped drastically in the last five years (2018-2022: mean ± stdev: 0.47 ± 0.45 million). Spat production was higher in colder waters and during peaks of Chlorophyll a in the last 13 years. Reduction of Chlorophyll a coincided with decreasing spat production in the last five years. Warmer periods (>27 °C) of the year may hamper scallop development. Counts of potentially pathogenic bacteria (Vibrios) and Escherichia coli were significantly higher in warmer periods which may further reduce scallop productivity. Shotgun metagenomics of seawater samples from the five studied corroborated these culture-based counts. Vibrios and fecal indicator bacteria metagenomic sequences were abundant across the entire study area throughout 2022. The results of this study suggest the collapse of scallop mariculture is the result of a synergistic negative effect of global warming and poor seawater quality.

Dynamics and 3D organization of secretory organelles of Toxoplasma gondii.

Micronemes, rhoptries and dense granules are secretory organelles of Toxoplasma gondii crucial for host cell invasion and formation of the parasitophorous vacuole (PV). We examined whether their relative volumes change during the intracellular cycle. Stereological analysis of random ultrathin sections taken at 5min of interaction, 7 and 24h post-infection demonstrated that the relative volume of each type of organelle decreases just after the respective peak of secretion. Micronemes are radially arranged below the polar ring, while rhoptries converge to but only a few reach the inside of the conoid. In contrast to the apical and polarized organelles, dense granules were found scattered throughout the cytoplasm, with no preferential location in the parasite cell body. Extensive observation of random sections indicated that each organelle probably secretes in a different region. Micronemes secrete just below the posterior ring and probably require that the conoid is extruded. The rhoptries passing through the conoid secrete at a porosome-like point at the most apical region. Dense granules secrete laterally, probably at fenestrations in the inner membrane complex. Immunocytochemistry showed that there are no subpopulations of rhoptries or dense granules, as a single organelle can contain more than one kind of its specific proteins. The vacuolar-like profiles observed at the apical portion of parasites just after invasion were confirmed to be empty rhoptries, as they were positively labeled for rhoptry proteins. These findings contribute for a better understanding of the essential behavior of secretory organelles.

Remarkable kinetoplast, cytostome-cytopharynx complex, and storage-related structures as dissected by three-dimensional reconstruction of Trypanosoma sp. 858 isolated from a toad (Amphibia: Anura).

In Brazil, the Trypanosoma sp. 858 was isolated from a toad (Anura: Bufonidae: Rhinella ictericus) and successfully maintained in cultures. We previously demonstrated that this trypanosome is different but tightly clustered phylogenetically with other trypanosomes from anurans. In this study, we addressed the ultrastructural features of cultured epimastigotes of this new trypanosome. Our results showed very long and thin free motile forms exhibiting a long flagellum and remarkable large and loose K-DNA network. In addition, the anterior portion contained many acidocalcisomes and a well-developed spongiome tubules-contractile vacuole system. One of the main morphological features of this anuran trypanosome was the presence of a complex cytostome-cytopharynx with a specialized membrane coating at the entrance, which is often hidden by the flagellum. Other conspicuous features are the presence of lipid-like droplets, lamellar membrane limited inclusions, and one very large reservosome, all at the posterior portion of the cell body. This new trypanosome may constitute an excellent model for organelles studies related to endocytosis and lipid storage, as demonstrated herein using scanning and transmission electron microscopy and three-dimensional models obtained by either electron microscopy tomography or dual-beam slice and view series.

Endoplasmic Stress Affects the Coinfection of Leishmania Amazonensis and the Phlebovirus (Bunyaviridae) Icoaraci.

Viral coinfections can modulate the severity of parasitic diseases, such as human cutaneous leishmaniasis. Leishmania parasites infect thousands of people worldwide and cause from single cutaneous self-healing lesions to massive mucosal destructive lesions. The transmission to vertebrates requires the bite of Phlebotomine sandflies, which can also transmit Phlebovirus. We have demonstrated that Leishmania infection requires and triggers the Endoplasmic stress (ER stress) response in infected macrophages. In the present paper, we tested the hypothesis that ER stress is increased and required for the aggravation of Leishmania infection due to coinfection with Phlebovirus. We demonstrated that Phlebovirus Icoaraci induces the ER stress program in macrophages mediated by the branches IRE/XBP1 and PERK/ATF4. The coinfection with L. amazonensis potentiates and sustains the ER stress, and the inhibition of IRE1α or PERK results in poor viral replication and decreased parasite load in macrophages. Importantly, we observed an increase in viral replication during the coinfection with Leishmania. Our results demonstrated the role of ER stress branches IRE1/XBP1 and PERK/ATF4 in the synergic effect on the Leishmania increased load during Phlebovirus coinfection and suggests that Leishmania infection can also increase the replication of Phlebovirus in macrophages.

Morphological and biochemical repercussions of Toxoplasma gondii infection in a 3D human brain neurospheres model.

BACKGROUND: Toxoplasmosis is caused by the parasite Toxoplasma gondii that can infect the central nervous system (CNS), promoting neuroinflammation, neuronal loss, neurotransmitter imbalance and behavioral alterations. T. gondii infection is also related to neuropsychiatric disorders such as schizophrenia. The pathogenicity and inflammatory response in rodents are different to the case of humans, compromising the correlation between the behavioral alterations and physiological modifications observed in the disease. In the present work we used BrainSpheres, a 3D CNS model derived from human pluripotent stem cells (iPSC), to investigate the morphological and biochemical repercussions of T. gondii infection in human neural cells. METHODS: We evaluated T. gondii ME49 strain proliferation and cyst formation in both 2D cultured human neural cells and BrainSpheres. Aspects of cell morphology, ultrastructure, viability, gene expression of neural phenotype markers, as well as secretion of inflammatory mediators were evaluated for 2 and 4 weeks post infection in BrainSpheres. RESULTS: T. gondii can infect BrainSpheres, proliferating and inducing cysts formation, neural cell death, alteration in neural gene expression and triggering the release of several inflammatory mediators. CONCLUSIONS: BrainSpheres reproduce many aspects of T. gondii infection in human CNS, constituting a useful model to study the neurotoxicity and neuroinflammation mediated by the parasite. In addition, these data could be important for future studies aiming at better understanding possible correlations between psychiatric disorders and human CNS infection with T. gondii.

Cidofovir inhibits genome encapsidation and affects morphogenesis during the replication of vaccinia virus.

Cidofovir (CDV) is one of the most effective antiorthopoxvirus drugs, and it is widely accepted that viral DNA replication is the main target of its activity. In the present study, we report a detailed analysis of CDV effects on the replicative cycles of distinct vaccinia virus (VACV) strains: Cantagalo virus, VACV-IOC, and VACV-WR. We show that despite the approximately 90% inhibition of production of virus progeny, virus DNA accumulation was reduced only 30%, and late gene expression and genome resolution were unaltered. The level of proteolytic cleavage of the major core proteins was diminished in CDV-treated cells. Electron microscopic analysis of virus-infected cells in the presence of CDV revealed reductions as great as 3.5-fold in the number of mature forms of virus particles, along with a 3.2-fold increase in the number of spherical immature particles. A detailed analysis of purified virions recovered from CDV-treated cells demonstrated the accumulation of unprocessed p4a and p4b and nearly 67% inhibition of DNA encapsidation. However, these effects of CDV on virus morphogenesis resulted from a primary effect on virus DNA synthesis, which led to later defects in genome encapsidation and virus assembly. Analysis of virus DNA by atomic force microscopy revealed that viral cytoplasmic DNA synthesized in the presence of CDV had an altered structure, forming aggregates with increased strand overlapping not observed in the absence of the drug. These aberrant DNA aggregations were not encapsidated into virus particles.

A contiguous compartment functions as endoplasmic reticulum and endosome/lysosome in Giardia lamblia.

The dynamic evolution of organelle compartmentalization in eukaryotes and how strictly compartmentalization is maintained are matters of ongoing debate. While the endoplasmic reticulum (ER) is classically envisioned as the site of protein cotranslational translocation, it has recently been proposed to have pluripotent functions. Using transfected reporter constructs, organelle-specific markers, and functional enzyme assays, we now show that in an early-diverging protozoan, Giardia lamblia, endocytosis and subsequent degradation of exogenous proteins occur in the ER or in an adjacent and communicating compartment. The Giardia endomembrane system is simple compared to those of typical eukaryotes. It lacks peroxisomes, a classical Golgi apparatus, and canonical lysosomes. Giardia orthologues of mammalian lysosomal proteases function within an ER-like tubulovesicular compartment, which itself can dynamically communicate with clathrin-containing vacuoles at the periphery of the cell to receive endocytosed proteins. These primitive characteristics support Giardia's proposed early branching and could serve as a model to study the compartmentalization of endocytic and lysosomal functions into organelles distinct from the ER. This system also may have functional similarity to the retrograde transport of toxins and major histocompatibility complex class I function in the ER of mammals.

Toxoplasma gondii Mechanisms of Entry Into Host Cells.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan parasite. Toxoplasma can invade and multiply inside any nucleated cell of a wide range of homeothermic hosts. The canonical process of internalization involves several steps: an initial recognition of the host cell surface and a sequential secretion of proteins from micronemes followed by rhoptries that assemble a macromolecular complex constituting a specialized and transient moving junction. The parasite is then internalized via an endocytic process with the establishment of a parasitophorous vacuole (PV), that does not fuse with lysosomes, where the parasites survive and multiply. This process of host cell invasion is usually referred to active penetration. Using different cell types and inhibitors of distinct endocytic pathways, we show that treatment of host cells with compounds that interfere with clathrin-mediated endocytosis (hypertonic sucrose medium, chlorpromazine hydrochloride, and pitstop 2 inhibited the internalization of tachyzoites). In addition, treatments that interfere with macropinocytosis, such as incubation with amiloride or IPA-3, increased parasite attachment to the host cell surface but significantly blocked parasite internalization. Immunofluorescence microscopy showed that markers of macropinocytosis, such as the Rab5 effector rabankyrin 5 and Pak1, are associated with parasite-containing cytoplasmic vacuoles. These results indicate that entrance of T. gondii into mammalian cells can take place both by the well-characterized interaction of parasite and host cell endocytic machinery and other processes, such as the clathrin-mediated endocytosis, and macropinocytosis.

The life-cycle of Toxoplasma gondii reviewed using animations.

Toxoplasma gondii is a protozoan parasite that is the causative agent of toxoplasmosis, an infection with high prevalence worldwide. Most of the infected individuals are either asymptomatic or have mild symptoms, but T. gondii can cause severe neurologic damage and even death of the fetus when acquired during pregnancy. It is also a serious condition in immunodeficient patients. The life-cycle of T. gondii is complex, with more than one infective form and several transmission pathways. In two animated videos, we describe the main aspects of this cycle, raising questions about poorly or unknown issues of T. gondii biology. Original plates, based on electron microscope observations, are also available for teachers, students and researchers. The main goal of this review is to provide a source of learning on the fundamental aspects of T. gondii biology to students and teachers contributing for better knowledge and control on this important parasite, and unique cell model. In addition, drawings and videos point to still unclear aspects of T. gondii lytic cycle that may stimulate further studies.

Phylogenetic analyses based on small subunit rRNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase genes and ultrastructural characterization of two snake Trypanosomes: Trypanosoma serpentis n. sp. from Pseudoboa nigra and Trypanosoma cascavelli from Crotalus durissus terrificus.

We sequenced the small subunit (SSU) rRNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes of two trypanosomes isolated from the Brazilian snakes Pseudoboa nigra and Crotalus durissus terrificus. Trypanosomes were cultured and their morphometrical and ultrastructural features were characterized by light microscopy and scanning and transmission electron microscopy. Phylogenetic trees inferred using independent or combined SSU rRNA and gGAPDH data sets always clustered the snake trypanosomes together in a clade closest to lizard trypanosomes, forming a strongly supported monophyletic assemblage (i.e. lizard-snake clade). The positioning in the phylogenetic trees and the barcoding based on the variable V7-V8 region of the SSU rRNA, which showed high sequence divergences, allowed us to classify the isolates from distinct snake species as separate species. The isolate from P. nigra is described as a new species, Trypanosoma serpentis n. sp., whereas the isolate from C. d. terrificus is redescribed here as Trypanosoma cascavelli.