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African trypanosomes proliferate as bloodstream forms (BSFs) and procyclic forms in the mammal and tsetse fly midgut, respectively. This allows them to colonise the host environment upon infection and ensure life cycle progression. Yet, understanding of the mechanisms that regulate and drive the cell replication cycle of these forms is limited. Using single-cell transcriptomics on unsynchronised cell populations, we have obtained high resolution cell cycle regulated (CCR) transcriptomes of both procyclic and slender BSF Trypanosoma brucei without prior cell sorting or synchronisation. Additionally, we describe an efficient freeze-thawing protocol that allows single-cell transcriptomic analysis of cryopreserved T. brucei . Computational reconstruction of the cell cycle using periodic pseudotime inference allowed the dynamic expression patterns of cycling genes to be profiled for both life cycle forms. Comparative analyses identify a core cycling transcriptome highly conserved between forms, as well as several genes where transcript levels dynamics are form specific. Comparing transcript expression patterns with protein abundance revealed that the majority of genes with periodic cycling transcript and protein levels exhibit a relative delay between peak transcript and protein expression. This work reveals novel detail of the CCR transcriptomes of both forms, which are available for further interrogation via an interactive webtool., Competing Interests: EB, CM, GO, JH, TO, KM No competing interests declared, (© 2023, Briggs et al.)

Trypanothione is the primary thiol redox carrier in Trypanosomatids whose biosynthesis and utilization pathways contain unique enzymes that include suitable drug targets against the human parasites in this family. Overexpression of the rate-limiting enzyme, γ-glutamylcysteine synthetase (GSH1), can increase the intracellular concentration of trypanothione. Melarsoprol directly inhibits trypanothione and has predicted the effects on downstream redox biology, including ROS management and dNTP synthesis that require further investigation. Thus, we hypothesized that melarsoprol treatment would inhibit DNA synthesis, which was tested using BrdU incorporation assays and cell cycle analyses. In addition, we analysed the effects of eflornithine, which interfaces with the trypanothione pathway, fexinidazole, because of the predicted effects on DNA synthesis, and pentamidine as an experimental control. We found that melarsoprol treatment resulted in a cell cycle stall and a complete inhibition of DNA synthesis within 24 h, which were alleviated by GSH1 overexpression. In contrast, the other drugs analysed had more subtle effects on DNA synthesis that were not significantly altered by GSH1 expression. Together these findings implicate DNA synthesis as a therapeutic target that warrants further investigation in the development of antitrypanosomal drugs.

Protozoan parasites are responsible for severe disease and suffering in humans worldwide. Apart from disease transmission via insect vectors and contaminated soil, food, or water, transmission may occur congenitally or by way of blood transfusion and organ transplantation. Several recent outbreaks associated with fresh produce and potable water emphasize the need for vigilance and monitoring of protozoan parasites that cause severe disease in humans globally. Apart from the tropical parasite Plasmodium spp., other protozoa causing debilitating and fatal diseases such as Trypanosoma spp. and Naegleria fowleri need to be studied in more detail. Climate change and socioeconomic issues such as migration continue to be major drivers for the spread of these neglected tropical diseases beyond endemic zones. Due to the complex life cycles of protozoa involving multiple hosts, vectors, and stringent growth conditions, studying these parasites has been challenging. While in vivo models may provide insights into host-parasite interaction, the ethical aspects of laboratory animal use and the challenge of ready availability of parasite life stages underline the need for in vitro models as valid alternatives for culturing and maintaining protozoan parasites. To our knowledge, this review is the first of its kind to highlight available in vitro models for protozoa causing highly infectious diseases. In recent years, several research efforts using new technologies such as 3D organoid and spheroid systems for protozoan parasites have been introduced that provide valuable tools to advance complex culturing models and offer new opportunities toward the advancement of parasite in vitro studies. In vitro models aid scientists and healthcare providers in gaining insights into parasite infection biology, ultimately enabling the use of novel strategies for preventing and treating these diseases., Competing Interests: The authors have declared that no competing interests exist.

Trypanosoma cruzi, and the T. brucei group of parasites cause neglected diseases that affect millions of people around the world. These unicellular microorganisms have complex life cycles involving an insect vector and a mammalian host. Both groups of pathogens possess an inositol 1,4,5-trisphosphate (IP 3 )/diacylglycerol (DAG) signaling pathway, and an IP 3 receptor, but with lineage-specific adaptations that make them different from their mammalian counterparts. The phospholipase C (PLC), which hydrolyzes phosphatidyl inositol 4,5-bisphosphate (PIP 2 ) to IP 3 is N-terminally myristoylated and palmitoylated. Acidocalcisomes, which are lysosome-related organelles rich in polyphosphate, are the main intracellular Ca 2+ stores. The inositol 1,4,5-trisphosphate receptor (IP 3 R) localizes to acidocalcisomes instead of the endoplasmic reticulum. The trypanosome IP 3 R is stimulated by luminal phosphate and pyrophosphate, which are hydrolysis products of polyphosphate (polyP), and inhibited by tripolyphosphate (polyP 3 ), which is the most abundant polyP in acidocalcisomes. Ca 2+ signaling is important for host cell invasion and differentiation and to maintain cellular bioenergetics., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Differentiation is a central aspect of the parasite life cycle and encompasses adaptation to both host and environment. If we accept that evolution cannot anticipate an organism's needs as it enters a new environment, how do parasite differentiation pathways arise? The transition between vertebrate and insect stage African trypanosomes is probably one of the better studied and involves a cell-cycle arrested or 'stumpy' form that activates metabolic pathways advantageous to the parasite in the insect host. However, a range of stimuli and stress conditions can trigger similar changes, leading to formation of stumpy-like cellular states. We propose that the origin and optimisation of this differentiation program represents repurposing of a generic stress response to gain considerable gain-of-fitness associated with parasite transmission., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Nanoencapsulation is the promising approach to enhance the therapeutic potential of a drug. In the present investigation, piperine-loaded nanocapsules (NCs) was prepared and evaluated for antitrypanosomal activity against the parasite Trypanosoma evansi, a causative agent of trypanosomiasis. Piperine, a bioactive compound was selected as an alternative for drugs that have been used for the treatment of the disease from decades to overcome the toxic effects or drug resistance effect. Moreover, piperine has reported to possess therapeutic potential against other Trypanosoma spp. and has also been reported to cause reactive oxygen species (ROS) mediated effect in cancer cells that was the other reason for the selection. To date, piperine and its nanoformulations have not been evaluated for their growth inhibitory effect against T. evansi. Piperine-loaded NCs exhibited more significant antitrypanosomal effect at approximately three-times less IC 50 value 5.04 μM as compared to piperine (IC 50 -14.45 μM). Moreover, increased production of reactive oxygen species observed in the case of piperine-loaded NCs as that of pure piperine in the axenic culture of T. evansi. Furthermore, different concentrations of piperine-loaded NCs showed less cytotoxicity on horse peripheral blood mononuclear cells as liken to pure piperine. In conclusion, our results demonstrated that piperine-loaded NCs induced more generation of ROS that contributed inhibitory effect on the growth of Trypanosoma evansi as compared to pure drug., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Trypanosoma evansi, an extracellular haemoflagellate, has a wide range of hosts receptive and susceptible to infection, in which it revealed highly inconsistent clinical effects. Drugs used for the treatment of trypanosomosis have been utilized for more than five decades and have several problems like local and systemic toxicity. In the present investigation, imatinib and sorafenib were selected as drugs as they are reported to have the potential to cause reactive oxygen species (ROS)-mediated effect in cancer cells. Both have also been reported to have potential against T. brucei, T. cruzi and Leishmania donovani. To date, imatinib and sorafenib have not evaluated for their growth inhibitory effect against T. evansi. Imatinib and sorafenib showed significant (p < 0.001) inhibition on parasite growth and multiplication with IC 50 (50% inhibitory concentration) values 6.12 μM and 0.33 μM respectively against T. evansi. Both the drug molecules demonstrated for the generation of ROS in T. evansi and were found up to 65% increased level of ROS as compared with negative control in the axenic culture system. Furthermore, different concentrations of imatinib and sorafenib were found non-toxic on horse peripheral blood mononuclear cells and Vero cell lines. Also, in conclusion, our results demonstrated that imatinib- and sorafenib-induced generation of ROS contributed inhibitory effect on the growth of Trypanosoma evansi in an axenic culture system.

Trypanosoma species (Trypanosomatida, Kinetoplastea) are almost exclusively heteroxenous flagellated parasites, which have been extensively studied as the causative agents of severe trypanosomiasis in humans and domestic animals. However, the biology of avian trypanosomes remains insufficiently known, particularly in wildlife, despite information that some species might be pathogenic and affect the fitness of intensively infected individuals. Avian trypanosomes are cosmopolitans. Due to regular bird seasonal migrations, this host-parasite system might provide new insight for better understanding mechanisms of transcontinental dispersal of pathogens, their ecological plasticity, specificity and speciation. Trypanosoma everetti parasitizes numerous bird species globally, but data on its biology are scarce and its vectors remain unknown. This study aimed to test experimentally whether widespread Culicoides (Diptera: Ceratopogonidae) biting midges are susceptible to infection with this parasite. Two common house martins Delichon urbicum and two sedge warblers Acrocephalus schoenobaenus naturally infected with T. everetti were caught in the wild after arrival from African wintering grounds. Laboratory reared Culicoides nubeculosus and wild-caught Culicoides impunctatus biting midges were exposed by allowing them to take infected blood meals. The experimentally infected and control insects were maintained in the laboratory and dissected at intervals to follow the development of the parasite. Infections were determined using microscopic examination and PCR-based testing. Four closely related haplotypes of T. everetti were found, and each was present in different individual parasite-donor birds. These parasites readily developed and produced metacyclic trypomastigotes in C. nubeculosus and C. impunctatus biting midges. Molecular characterisation of T. everetti was developed. According to Bayesian phylogenetic analysis using a DNA fragment encoding 18S rRNA, the five species of small avian trypanosomes were closely related. Wild caught Culicoides biting midges were also collected and screened for the presence of natural infections. In all, 6.8% of wild-caught biting midges belonging to five Culicoides species were PCR-positive for kinetoplastids, including Trypanosoma species. Culicoides biting midges are readily susceptible and likely naturally transmit avian trypanosomes and thus, should be targeted in epidemiology research of avian trypanosomiasis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Malaria, babesiosis, trypanosomosis, and leishmaniasis are some of the most life-threatening parasites, but the range of drugs to treat them is limited. An effective, safe, and low-cost drug with a large activity spectrum is urgently needed. For this purpose, an aryl amino alcohol derivative called Alsinol was resynthesized, screened in silico, and tested against Plasmodium, Babesia, Trypanosoma, and Leishmania. In silico Alsinol follows the Lipinski and Ghose rules. In vitro it had schizontocidal activity against Plasmodium falciparum and was able to inhibit gametocytogenesis; it was particularly active against late gametocytes. In malaria-infected mice, it showed a dose-dependent activity similar to chloroquine. It demonstrated a similar level of activity to reference compounds against Babesia divergens, and against promastigotes, and amastigotes stages of Leishmania in vitro. It inhibited the in vitro growth of two African animal strains of Trypanosoma but was ineffective in vivo in our experimental conditions. It showed moderate toxicity in J774A1 and Vero cell models. The study demonstrated that Alsinol has a large spectrum of activity and is potentially affordable to produce. Nevertheless, challenges remain in the process of scaling up synthesis, creating a suitable clinical formulation, and determining the safety margin in preclinical models.

Trypanosomes are ubiquitous blood parasites of fishes and at least 16 species were originally described infecting African freshwater fishes. This number was later reduced to six and in the late 1990s it was proposed that most records of freshwater fish trypanosomes across Africa are Trypanosoma mukasai Hoare, 1932. Recently, results from a molecular analysis of fish trypanosomes from the Okavango Delta, Botswana, reported the presence of at least two genotypic groups and concluded that the identification of T. mukasai remains problematic. The aims of the present study were thus to elucidate the life cycle of a freshwater fish trypanosome from southern Africa and to do a morphological and molecular characterization of this parasite from both the fish host and leech vector. To locate trypanosome stages, leeches were removed from fishes captured in the Phongolo River, South Africa, and fish blood films and leech squashes were Giemsa-stained and screened. To determine whether trypanosome stages in fishes and leeches were of the same genotype, DNA was extracted and fragments of the 18S rDNA gene were amplified and sequenced. Trypanosomes were detected in the fish families Cichlidae, Clariidae, Mochokidae and Schilbeidae. Sequence data showed that the trypanosome from one of the leeches, identified as Batracobdelloides tricarinata (Blanchard, 1897), was highly similar to those obtained from the plain squeaker, Synodontis zambezensis, with 0.7% difference recorded between them. From morphological and molecular data presented here, it is clear that the trypanosomes from Phongolo are closely related to those of the Okavango and should be considered as a single diverse species with genetic differentiation between 0.4-2.9%, under the 3-5% differences expected to be seen between true distinct species within the rRNA. Developmental stages of the trypanosome found in the leech B. tricarinata supports its status as the vector and the molecular evidence shows the relationship between the trypanosome in the fish and leech, but also illustrates the exceptional genetic and morphological diversity of a single species of trypanosome between host species. The work presented here provides us with clear information to take further steps in resolving the taxonomy and systematics of African freshwater fish trypanosomes., (Copyright © 2020 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

African trypanosomes are mainly transmitted by tsetse flies. In recent years there has been good progress in understanding how the parasites prepare for transmission, detect their changed environment through the perception of different environmental cues, and respond by changing their developmental gene expression. In this review, we discuss the different signals and signaling mechanisms used by the parasites to carry out the early events necessary for their establishment in the fly. We also compare Trypanosoma brucei and Trypanosoma congolense, parasites that share a common pathway in the early stages of fly colonization but apparently use different mechanisms to achieve this., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Trypanosoma evansi appears to have a significant tropism for brain tissue in its chronic and acute phases. The most common symptoms of this brain infection are motor incoordination, meningoencephalitis, demyelination, and anemia. There have only been few studies of the effects of T. evansi infection on neuronal differentiation and brain plasticity. Here, we investigated the impact of the congenital T. evansi infection on brain development in mice. We collected telencephalon-derived neural progenitor cells (NPCs) from T. evansi uninfected and infected mice, and cultivated them into neurospheres. We found that T. evansi significantly decreased the number of cells during development of neurospheres. Analysis of neurosphere differentiation revealed that T. evansi infection significantly increased neural migration. We also observed that T. evansi promoted expression of glial fibrillary acidic protein (GFAP) in infected cells. These data suggest that congenital T. evansi infection may affect embryonic brain development., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: A considerable amount of evidence has favored ecological host-fitting, rather than coevolution, as the main mechanism responsible for trypanosome divergence. Nevertheless, beyond the study of human pathogenic trypanosomes, the genetic basis of host specificity among trypanosomes isolated from forest-inhabiting hosts remains largely unknown., Methods: To test possible scenarios on ecological host-fitting and coevolution, we combined a host capture recapture strategy with parasite genetic data and studied the genetic variation, population dynamics and phylogenetic relationships of Trypanosoma terrestris, a recently described trypanosome species isolated from lowland tapirs in the Brazilian Pantanal and Atlantic Forest biomes., Results: We made inferences of T. terrestris population structure at three possible sources of genetic variation: geography, tapir hosts and 'putative' vectors. We found evidence of a bottleneck affecting the contemporary patterns of parasite genetic structure, resulting in little genetic diversity and no evidence of genetic structure among hosts or biomes. Despite this, a strongly divergent haplotype was recorded at a microgeographical scale in the landscape of Nhecolândia in the Pantanal. However, although tapirs are promoting the dispersion of the parasites through the landscape, neither geographical barriers nor tapir hosts were involved in the isolation of this haplotype. Taken together, these findings suggest that either host-switching promoted by putative vectors or declining tapir population densities are influencing the current parasite population dynamics and genetic structure. Similarly, phylogenetic analyses revealed that T. terrestris is strongly linked to the evolutionary history of its perissodactyl hosts, suggesting a coevolving scenario between Perissodactyla and their trypanosomes. Additionally, T. terrestris and T. grayi are closely related, further indicating that host-switching is a common feature promoting trypanosome evolution., Conclusions: This study provides two lines of evidence, both micro- and macroevolutionary, suggesting that both host-switching by ecological fitting and coevolution are two important and non-mutually-exclusive processes driving the evolution of trypanosomes. In line with other parasite systems, our results support that even in the face of host specialization and coevolution, host-switching may be common and is an important determinant of parasite diversification.

From September 2012 to May 2018, blood samples from 364 raptors (mostly adults) were collected and screened for trypanosomes and haemosporidians by microscopic examination and nested polymerase chain reactions (PCR). Trypanosoma spp. were identified in 15 birds from eight different species. Light microscopy revealed 14 cases of infection with Trypanosoma cf. corvi, including one each in black-shouldered kite (Elanus caeruleus, n = 49), Brahminy kite (Haliastur indus, n = 50), and spotted owlet (SO, Athene brama, n = 27); two mountain hawk-eagles (Spizaetus nipalensis, n = 3); and three each in Asian barred owlets (ABO, Glaucidium cuculoides, n = 27), barn owls (BO, Tyto alba, n = 65) and collared scops owls (CSO, Otus lettia, n = 41). In addition, one case of infection with T. avium was identified in an oriental scops owl (OSO, Otus sunia, n = 2). All infected raptors showed very low parasitemia levels. The PCR detected more three positives in one CSO, one Japanese sparrowhawk (Accipiter gularis), and one OSO. The sensitivity and specificity of the PCR method were 93.3% and 99.1%, respectively. The overall infection rate was very low (4.9%). The highest infection rate was recorded in cold-dry season (9.9%). Coinfection of Plasmodium with trypanosomes was found in all three ABOs. Coinfection with Haemoproteus spp. was found in one BO, three CSOs, and one SO. Coinfection with Haemoproteus spp. and Leucocytozoon danilewskyi was found in the OSO. Microfilarias were detected in one ABO and one CSO. The ultrastructure of trypomastigotes of T. cf. corvi in an ABO revealed fine structures. All small subunit ribosomal RNA (SSU rRNA) sequences belong to two clades: T. avium and T. corvi-culicavium complex/group. SSU rRNA gene amplification was not successful in one BO. The raptors with trypanosome infections showed normal hematological values and healthy appearance. Furthermore, this is the first report of T. avium in a nocturnal raptor from Thailand.

Trypanosomatid parasites are infectious agents for diseases such as African sleeping sickness, Chagas disease, and leishmaniasis that threaten millions of people, mostly in the emerging world. Trypanosomes compartmentalize glycolytic enzymes to an organelle called the glycosome, a specialized peroxisome. Functionally intact glycosomes are essential for trypanosomatid viability, making glycosomal proteins as potential drug targets against trypanosomatid diseases. Peroxins (Pex), of which Pex3 is the master regulator, control glycosome biogenesis. Although Pex3 has been found throughout the eukaryota, its identity has remained stubbornly elusive in trypanosomes. We used bioinformatics predictive of protein secondary structure to identify trypanosomal Pex3. Microscopic and biochemical analyses showed trypanosomal Pex3 to be glycosomal. Interaction of Pex3 with the peroxisomal membrane protein receptor Pex19 observed for other eukaryotes is replicated by trypanosomal Pex3 and Pex19. Depletion of Pex3 leads to mislocalization of glycosomal proteins to the cytosol, reduced glycosome numbers, and trypanosomatid death. Our findings are consistent with Pex3 being an essential gene in trypanosomes., (© 2019 Banerjee et al.)

In trypanosomes, RNA polymerase II transcription is polycistronic and individual mRNAs are excised by trans-splicing and polyadenylation. The lack of individual gene transcription control is compensated by control of mRNA processing, translation and degradation. Although the basic mechanisms of mRNA decay and translation are evolutionarily conserved, there are also unique aspects, such as the existence of six cap-binding translation initiation factor homologues, a novel decapping enzyme and an mRNA stabilizing complex that is recruited by RNA-binding proteins. High-throughput analyses have identified nearly a hundred regulatory mRNA-binding proteins, making trypanosomes valuable as a model system to investigate post-transcriptional regulation.

Trypanosoma caninum is a parasite isolated from domestic dogs, of which several biological aspects remain unknown, including evolutive forms found in vertebrate hosts. The objective of this study was to evaluate co-cultures of T. caninum with different cell lines as feeder layers to monitor the differentiation process and investigate infective potential. The study was performed using DH-82, MDCK, and Lulo cell lines. T. caninum from axenic culture was added to the cultured adherent cells. At intervals over 30 days, aliquots of the supernatant were collected for quantification and assessment of differentiation. Infectivity assays were performed on the aforementioned cell lines seeded on glass coverslips and evaluated after 6, 24, and 72 h. In the supernatant of the feeder layer, T. caninum presented similar growth profiles, with epimastigote and trypomastigote forms in binary and multiple divisions. During co-culture with DH-82 and MDCK cells, a higher level of differentiation to trypomastigotes was observed. This study shows that the differentiation process of this parasite can vary according to culture conditions and that DH-82 and MDCK lineages could be applied to the study of trypomastigote forms. All forms of T. caninum described until now (aflagellar epimastigotes, typical epimastigotes, or trypomastigotes) were unable to infect the cell line Finally, this study provides additional data about morphobiological aspects. Although the biological cycle of T. caninum has not been established, the present data suggest the importance of feeder layers in promoting the growth and differentiation of this new parasite., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Trypanosoma cruzi invades and replicates inside mammalian cells, which can lead to chronic Chagas disease in humans. Trypanosoma copemani infects Australian marsupials and recent investigations indicate it may be able to invade mammalian cells in vitro, similar to T. cruzi. Here, T. cruzi 10R26 strain (TcIIa) and two strains of T. copemani [genotype 1 (G1) and genotype 2 (G2)] were incubated with marsupial cells in vitro. Live-cell time-lapse and fluorescent microscopy, combined with high-resolution microscopy (transmission and scanning electron microscopy) were used to investigate surface interactions between parasites and mammalian cells., Results: The number of parasites invading cells was significantly higher in T. cruzi compared to either genotype of T. copemani, between which there was no significant difference. While capable of cellular invasion, T. copemani did not multiply in host cells in vitro as there was no increase in intracellular amastigotes over time and no release of new trypomastigotes from host cells, as observed in T. cruzi. Exposure of host cells to G2 trypomastigotes resulted in increased host cell membrane permeability within 24 h of infection, and host cell death/blebbing was also observed. G2 parasites also became embedded in the host cell membrane., Conclusions: Trypanosoma copemani is unlikely to have an obligate intracellular life-cycle like T. cruzi. However, T. copemani adversely affects cell health in vitro and should be investigated in vivo in infected host tissues to better understand this host-parasite relationship. Future research should focus on increasing understanding of the T. copemani life history and the genetic, physiological and ecological differences between different genotypes.

Trypanosomatids such as Leishmania and Trypanosoma are digenetic, single-celled, parasitic flagellates that undergo complex life cycles involving morphological and metabolic changes to fit them for survival in different environments within their mammalian and insect hosts. According to current consensus, asymmetric division enables trypanosomatids to achieve the major morphological rearrangements associated with transition between developmental stages. Contrary to this view, here we show that the African trypanosome Trypanosoma congolense, an important livestock pathogen, undergoes extensive cell remodelling, involving shortening of the cell body and flagellum, during its transition from free-swimming proventricular forms to attached epimastigotes in vitro. Shortening of the flagellum was associated with accumulation of PFR1, a major constituent of the paraflagellar rod, in the mid-region of the flagellum where it was attached to the substrate. However, the PFR1 depot was not essential for attachment, as it accumulated several hours after initial attachment of proventricular trypanosomes. Detergent and CaCl2 treatment failed to dislodge attached parasites, demonstrating the robust nature of flagellar attachment to the substrate; the PFR1 depot was also unaffected by these treatments. Division of the remodelled proventricular trypanosome was asymmetric, producing a small daughter cell. Each mother cell went on to produce at least one more daughter cell, while the daughter trypanosomes also proliferated, eventually resulting in a dense culture of epimastigotes. Here, by observing the synchronous development of the homogeneous population of trypanosomes in the tsetse proventriculus, we have been able to examine the transition from proventricular forms to attached epimastigotes in detail in T. congolense. This transition is difficult to observe in vivo as it happens inside the mouthparts of the tsetse fly. In T. brucei, this transition is achieved by asymmetric division of long trypomastigotes in the proventriculus, yielding short epimastigotes, which go on to colonise the salivary glands. Thus, despite their close evolutionary relationship and shared developmental route within the vector, T. brucei and T. congolense have evolved different ways of accomplishing the same developmental transition from proventricular form to attached epimastigote., Competing Interests: The authors have declared that no competing interests exist.

Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU- and EdU-incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and "click" chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re-estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring., (© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.)

A serie of isostructural complexes with general formula [M(ftpO) 2 (H 2 O) 4 ] have been obtained from reaction between the first time characterized triazolopyrimidine derivative 5-phenyl-1,2,4-triazolo[1,5-a]pyrimidi-7(4H)-one (HftpO) (1) and first row transition nitrates (M=Cu (2), Co (3), Ni (4) and Zn (5)). A copper complex with formula [Cu(HftpO) 2 (NO 3 ) 2 (H 2 O) 2 ]·H 2 O (6) was also isolated. HftpO and their metal complexes have been characterized by spectroscopic and thermal analysis and their crystal structures have been solved by X-ray diffraction methods. The isostructural compounds are mononuclear complexes where the triazolopyrimidine ligand acts as monodentate ligand through N3 nitrogen position. The crystal structure of these novel bis-5-phenyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one-tetraaquo metal complexes offers an excellent opportunity at these complexes to acts as potential building blocks. Also, the antiparasitic activity of HftpO ligand against different leishmania and trypanosome strains has been studied., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Dourine is caused by Trypanosoma equiperdum via coitus with an infected horse. Although dourine is distributed in Equidae worldwide and is listed as an internationally important animal disease by the World Organization for Animal Health (OIE), no effective treatment strategies have been established. In addition, there are no reports on drug discovery, because no drug screening system exists for this parasite. A new T. equiperdum strain was recently isolated from the genital organ of a stallion that showed typical symptoms of dourine. In the present study, we adapted T. equiperdum IVM-t1 from soft agarose media to HMI-9 liquid media to develop a drug screening assay for T. equiperdum. An intracellular ATP-based luciferase assay using CellTiter-Glo reagent and an intracellular dehydrogenase activity-based colorimetric assay using WTS-8 tetrazolium salt (CCK-8 reagent) were used in order to examine the trypanocidal effects of each compound. In addition, the IC 50 values of 4 reference trypanocidal compounds (pentamidine, diminazene, suramin and melarsomine) were evaluated and compared using established assays. The IC 50 values of these reference compounds corresponded well to previous studies involving other strains of T. equiperdum. The luciferase assay would be suitable for the mass screening of chemical libraries against T. equiperdum because it allows for the simple and rapid-evaluation of the trypanocidal activities of test compounds, while a simple, inexpensive colorimetric assay will be applicable in developing countries for the evaluation of the drug sensitivity of epidemic trypanosome strains., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Kinetochores are multiprotein complexes that couple eukaryotic chromosomes to the mitotic spindle to ensure proper segregation. The model for kinetochore assembly is conserved between humans and yeast, and homologues of several components are widely distributed in eukaryotes, but key components are absent in some lineages. The recent discovery in a lineage of protozoa called kinetoplastids of unconventional kinetochores with no apparent homology to model organisms suggests that more than one system for eukaryotic chromosome segregation may exist. In this study, we report a new family of proteins distantly related to outer kinetochore proteins Ndc80 and Nuf2. The family member in kinetoplastids, KKT-interacting protein 1 (KKIP1), associates with the kinetochore, and its depletion causes severe defects in karyokinesis, loss of individual chromosomes, and gross defects in spindle assembly or stability. Immunopurification of KKIP1 from stabilized kinetochores identifies six further components, which form part of a trypanosome outer kinetochore complex. These findings suggest that kinetochores in organisms such as kinetoplastids are built from a divergent, but not ancestrally distinct, set of components and that Ndc80/Nuf2-like proteins are universal in eukaryotic division., (© 2017 D'Archivio and Wickstead.)

Neglected tropical diseases (NTD) are treated with toxic therapy of limited efficacy. Previously, we studied the antimicrobial effect of Dinoponera quadriceps venom (DqV) against bacteria. To continue the study, we report in this short communication the antimicrobial effect of DqV against Leishmania amazonensis and Trypanosoma cruzi. DqV inhibits the promastigote forms of L. amazonensis and all T. cruzi developmental forms, with low toxicity in host cells. DqV causes cell death in T. cruzi through necrotic and apoptotic mechanisms observed by staining the cells with annexin V-FITC (AX) and propidium iodide (PI), loss of mitochondrial membrane potential by flow cytometry analyses and confocal microscopy and morphological alterations, such as loss of membrane integrity and cell shrinkage by scanning electron microscopy (SEM). In conclusion, we suggest there is an antimicrobial effect also on parasites., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.)

This study was conducted to investigate the occurrence of oxidative stress in the heart tissue of rats infected with Trypanosoma evansi. Rats were divided into 2 groups (A and B) with 12 animals each, and further subdivided into 4 subgroups (A1 and A2, 6 animals/each; and B1 and B2, 6 animals/each). Animals in the groups B1 and B2 were subcutaneously inoculated with T. evansi. Thiobarbituric acid reactive substances (TBARS), superoxide dismutase activity (SOD), glutathione S-transferase activity (GST), reduced glutathione activity (GSH), and non-protein thiols (NPSH) in the heart tissue were evaluated. At day 5 and 15 post-infection (PI), an increase in the TBARS levels and a decrease in the SOD activity (P<0.05) were observed. GSH and GST activities were decreased in infected animals at day 15 PI (P<0.05). Considering the proper functioning of the heart, it is possible that the changes in the activity of these enzymes involved in the oxidative stress may be related, at least in part, in the pathophysiology of rats infected with T. evansi.

Trypanosomatids are the causative agents of African sleeping sickness, Chagas' disease, and the different forms of leishmaniasis. This family of protozoan parasite possesses a trypanothione-based redox metabolism that provides the reducing equivalents for various vital processes such as the biosynthesis of DNA precursors and the detoxification of hydroperoxides. Almost all enzymes of the redox pathway proved to be essential and therefore fulfil one crucial prerequisite for a putative drug target. Trypanothione synthetase and trypanothione reductase are present in all trypanosomatids but absent from the mammalian host which, in addition to the essentiality, renders them highly specific. The chemotherapy research on both enzymes is further supported by the availability of high-throughput screening techniques and crystal structures. In this review we focus on the recent advances and limitations in the development of lead compounds targeting trypanothione synthetase and trypanothione reductase. We present an overview of the available inhibitors and discuss future perspectives including other components of the parasite-specific redox pathway., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Trypanosoma copemani has been found infecting several threatened/endangered marsupial species within Australia and is thought to be a key player in the rapid decline of the woylie (Bettongia penicillata). To better understand the biology and life cycle of this parasite, the growth requirements, and kinetics of infection of two newly described genotypes, T. copemani G1 and G2, were investigated and compared with the T. cruzi strain-10R26 in vitro. Both G1 and G2 were able to infect all four cell lines tested. The number of infected cells where at least one intracellular amastigote of T. copemani G1 and G2 was seen was below 7% and 15% respectively in most cell lines. However, in VERO cells the rate of infection for T. copemani G2 was 70%-approximately seven and two times higher than for G1 and T. cruzi respectively. Despite the higher infection rate, the number of intracellular forms of T. copemani G2 was lower compared with T. cruzi, and intracellular replicating forms were not observed. The capability of T. copemani G2 to infect cells may have important consequences for pathogenicity and suggests it might employ similar strategies to complete its life cycle in the vertebrate host to those seen in T. cruzi., (Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Trypanosomatidae are a dangerous family of Euglenobionta parasites that threaten the health and economy of millions of people around the world. More precisely describing the population biology and reproductive mode of such pests is not only a matter of pure science, but can also be useful for understanding parasite adaptation, as well as how parasitism, specialization (parasite specificity), and complex life cycles evolve over time. Studying this parasite's reproductive strategies and population structure can also contribute key information to the understanding of the epidemiology of associated diseases; it can also provide clues for elaborating control programs and predicting the probability of success for control campaigns (such as vaccines and drug therapies), along with emergence or re-emergence risks. Population genetics tools, if appropriately used, can provide precise and useful information in these investigations. In this paper, we revisit recent data collected during population genetics surveys of different Trypanosoma species in sub-Saharan Africa. Reproductive modes and population structure depend not only on the taxon but also on the geographical location and data quality (absence or presence of DNA amplification failures). We conclude on issues regarding future directions of research, in particular vis-à-vis genotyping and sampling strategies, which are still relevant yet, too often, neglected issues.

Trypanosomes are blood-borne parasites that can cause severe disease in both humans and animals, yet little is known of the pathogenicity and life-cycles of trypanosomes in native Australian mammals. Trypanosoma copemani is known to be infective to a variety of Australian marsupials and has recently been shown to be potentially zoonotic as it is resistant to normal human serum. In the present study, in vivo and in vitro examination of blood and cultures from Australian marsupials was conducted using light microscopy, immunofluorescence, scanning electron microscopy and fluorescence in situ hybridization. Promastigote, sphaeromastigote and amastigote life-cycle stages were detected in vivo and in vitro. Novel trypanosome-like stages were also detected both in vivo and in vitro representing an oval stage, an extremely thin stage, an adherent stage and a tiny round stage. The tiny round and adherent stages appeared to adhere to erythrocytes causing potential haematological damage with clinical effects similar to haemolytic anaemia. The present study shows for the first time that trypomastigotes are not the only life-cycle stages circulating within the blood stream of trypanosome infected Australian native marsupials and provides insights into possible pathogenic mechanisms of this potentially zoonotic trypanosome species.

Trypanosoma copemani is known to be infective to a variety of Australian marsupials. Characterisation of this parasite revealed the presence of stercorarian-like life-cycle stages in culture, which are similar to T. rangeli and T. cruzi. The blood incubation infectivity test (BIIT) was adapted and used to determine if T. copemani, like T. cruzi and T. rangeli, has the potential to grow in the presence of human serum. To eliminate any effects of anticoagulants on the complement system and on human high density lipoprotein (HDL), only fresh whole human blood was used. Trypanosoma copemani was observed by microscopy in all human blood cultures from day 5 to day 19 post inoculation (PI). The mechanism for normal human serum (NHS) resistance in T. copemani is not known. The results of this study show that at least one native Australian trypanosome species may have the potential to be infective for humans., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Fish trypanosomes are widely distributed in commercially important fish, with high prevalence in some Brazilian species. This study provides the first record of the isolation and in vitro maintenance of trypanosomes from Brazilian fish. We produced 49 trypanosome isolates from naturally infected catfish (Hypostomus affinis and Hypostomus luetkeni), using 9 different culture media (out of 31 tested). Trypanosomes were maintained in culture for at least 15 mo and were successfully cryopreserved. Culture forms-epimastigotes and short trypomastigotes-were capable of dividing in vitro. Our study is an important step in the investigation of ultrastructure, taxonomy, and phylogeny of trypanosomes from commercially important Brazilian fish.

The data on the fauna of trypanosomes of Anura of the Leningrad Province are given. The initial development stages of Trypanosoma rotatorium in peripheral blood and internal organs of the frog are described for the first time.

Representatives of the genus Trypanosoma have been traditionally found in epimastigote, espheromastigote and trypomastigote flagellated forms in axenic cultures. Trypanosoma caninum is a trypanosomatid that has recently been reported infecting dogs in endemic areas of canine leishmaniasis in Brazil. It presents specific biological characteristics and it is found exclusively on healthy skin. Here, we describe the evolutive forms of this parasite showing not only the forms commonly found in culture, but also epimastigote forms with no free flagellum. The study was conducted using scanning and transmission electron microscopy and, we demonstrate that typical flagellated epimastigotes originate from forms without flagellum, although the latter may remain without differentiation in the culture. Two hypotheses are considered and discussed in this paper: (i) the aflagellated epimastigotes are a typical developmental forms of T. caninum and (ii) the emergence of these aflagellated forms could be resultant from a disturbed process during cell division caused by interfering specific proteins, which leads to inability to form and regulate the flagellum length. In any case, considering that T. caninum is a parasite that is still little studied, the information brought by our study adds data which may be useful to clarify aspects on the cell cycle of this intriguing parasite that has been found in different regions of Brazil., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Trypanosoma caninum constitutes the most recent trypanosomatid species infecting dogs in Brazil. Due to the limited data available about this parasite, this study aims to disclose clinical and laboratory findings from 14 dogs naturally infected. The dogs were diagnosed during a cross-sectional survey in Cuiabá (Mato Grosso, Brazil) and followed up at an interval of 3, 6, and 12 mo in order to evaluate the clinical evolution and to investigate the parasite, the DNA, or both in different biological samples (intact skin, cutaneous scar, blood, bone marrow, and lymph node aspirate) by parasitological (culture and smear exam) and molecular (DNA-based tests) methods. Specific anti-T. caninum and anti-Leishmania antibody production was also evaluated. Ten of 14 dogs infected by T. caninum showed a good general state at the time of diagnosis, and this status did not vary during the follow-up. Anti-T. caninum and anti-Leishmania IgG antibodies were detected by IFAT in 10 and 2 animals, respectively. Concomitant infection by Leishmania chagasi was confirmed in 2 dogs, indicating an overlap of endemic areas in Cuiabá. Trypanosoma caninum (parasite or DNA) was found only in the intact skin in all animals examined. Our results suggest that T. caninum infection can be manifested as an asymptomatic case with low humoral immune response.

We examined the immunotoxic effects of acute and subchronic exposures of goldfish to aged, fresh, and ozonated oil sands process-affected water (OSPW) using a flow-through exposure apparatus. We measured the expression of proinflammatory cytokine genes, the antimicrobial responses of primary macrophages isolated from OSPW-exposed fish, and the ability of the goldfish to control infection with a protozoan parasite, Trypanosoma carassii. After acute (1 week) exposure to aged OSPW, we observed upregulation in the expression of interferon gamma (IFN-γ), tumor necrosis factor alpha-2 (TNF-α2) in the kidney and spleen but not in gills of the fish. After subchronic (12 weeks) exposure to aged OSPW, we observed significant increases in mRNA levels of proinflammatory genes in the gill (IFN-γ, interleukin-1 beta 1 [IL1-β1], TNF-α2), kidney (IL1-β1, TNF-α2), and spleen (IL1-β1). An upregulation of immune gene expression in the gill and kidney (IFN-γ, IL1-β1, TNF-α2) and spleen (IL1-β1, TNF-α2) was observed after acute exposure of fish to diluted fresh OSPW. Following subchronic exposure to diluted fresh OSPW, we observed high mRNA levels of IL1-β1 in all tissues examined. However, there were significant decreases in the mRNA levels of IFN-γ and TNF-α2 in the kidney and spleen and gill and spleen (IL-12p35 and IL-12p40) of exposed fish. There were no changes in the expression of anti-inflammatory cytokine IL-10 after both acute and subchronic exposures to diluted fresh OSPW. In fish exposed to ozonated fresh OSPW, immune gene expression was similar to nonexposed control fish in all organs examined, with exception of IL1-β1. The ability of primary kidney macrophages to generate reactive oxygen and nitrogen intermediates was significantly reduced in fish exposed to fresh OSPW. The enhanced proinflammatory response after acute exposure to diluted fresh OSPW was confirmed by the parasite challenge experiments, where OSPW-exposed fish controlled the infection better than nonexposed fish.

We develop a Bayesian nonparametric mixture modeling framework for quantal bioassay settings. The approach is built upon modeling dose-dependent response distributions. We adopt a structured nonparametric prior mixture model, which induces a monotonicity restriction for the dose-response curve. Particular emphasis is placed on the key risk assessment goal of calibration for the dose level that corresponds to a specified response. The proposed methodology yields flexible inference for the dose-response relationship as well as for other inferential objectives, as illustrated with two data sets from the literature., (© 2013, The International Biometric Society.)

Endemic animal diseases such as tsetse-transmitted trypanosomosis are a constant drain on the financial resources of African livestock keepers and on the productivity of their livestock. Knowing where the potential benefits of removing animal trypanosomosis are distributed geographically would provide crucial evidence for prioritising and targeting cost-effective interventions as well as a powerful tool for advocacy. To this end, a study was conducted on six tsetse-infested countries in Eastern Africa: Ethiopia, Kenya, Somalia, South Sudan, Sudan and Uganda. First, a map of cattle production systems was generated, with particular attention to the presence of draught and dairy animals. Second, herd models for each production system were developed for two scenarios: with or without trypanosomosis. The herd models were based on publications and reports on cattle productivity (fertility, mortality, yields, sales), from which the income from, and growth of cattle populations were estimated over a twenty-year period. Third, a step-wise spatial expansion model was used to estimate how cattle populations might migrate to new areas when maximum stocking rates are exceeded. Last, differences in income between the two scenarios were mapped, thus providing a measure of the maximum benefits that could be obtained from intervening against tsetse and trypanosomosis. For this information to be readily mappable, benefits were calculated per bovine and converted to US$ per square kilometre. Results indicate that the potential benefits from dealing with trypanosomosis in Eastern Africa are both very high and geographically highly variable. The estimated total maximum benefit to livestock keepers for the whole of the study area amounts to nearly US$ 2.5 billion, discounted at 10% over twenty years--an average of approximately US$ 3300 per square kilometre of tsetse-infested area--but with great regional variation from less than US$ 500 per square kilometre to well over US$ 10,000. The greatest potential benefits accrue to Ethiopia, because of its very high livestock densities and the importance of animal traction, but also to parts of Kenya and Uganda. In general, the highest benefit levels occur on the fringes of the tsetse infestations. The implications of the models' assumptions and generalisations are discussed., (Copyright © 2013 Food and Agriculture Organization of the United Nations. Published by Elsevier B.V. All rights reserved.)

Background: Trypanosomes are ubiquitous blood parasites of marine and freshwater fishes, typically transmitted by aquatic leeches. Phylogenetic studies have been dominated by examples derived from freshwater fishes, with few marine representatives. Furthermore, life cycle studies on marine fish trypanosomes have focused on those of the northern hemisphere. In this investigation, we have examined the life cycle and molecular taxonomy of a marine fish trypanosome from South Africa., Methods: To locate trypanosome stages, leeches were removed from fishes captured on the west and south coasts of South Africa, and fish blood films and leech squashes were Giemsa-stained and screened; leeches were also examined histologically. To determine whether trypanosome stages in fishes and leeches were of the same genotype, DNA was extracted from Giemsa-stained fish blood films and leech squashes, and from fish whole blood. Fragments of the 18S rRNA gene were amplified by PCR using trypanosome-specific primers and sequenced. Resulting sequence data were compared with each other and with published trypanosome 18S rDNA sequences, and used for phylogenetic analysis., Results: Trypanosomes were detected in blood films from fishes of the families Clinidae, Blenniidae and Gobiidae. The flagellates ranged in size and staining properties within the films and across fish hosts. In squashes and histological sections of adult and juvenile leeches, identified as Zeylanicobdella arugamensis, trypanosome developmental stages were predominantly slender epimastigotes. Sequence data showed that trypanosomes derived from fishes were identical, irrespective of whether they were small or large forms; sequences derived largely from leech epimastigotes were also identical to those obtained from fish trypanosomes. Fish and leech trypanosome sequences fell into a marine fish aquatic clade, and aligned most closely with two trypanosome sequences from marine fishes off Norway., Conclusions: Combined morphological and molecular methods indicate that the trypanosomes examined here represent a single pleomorphic species, rather than the three species described originally. This species is identified as Trypanosoma nudigobii Fantham, 1919 with the leech Z. arugamensis as its vector, and T. capigobii Fantham, 1919 and T. blenniclini Fantham, 1930 are regarded as junior synonyms of the species. Phylogenetic analysis establishes its affinity with marine fish trypanosomes off Norway.

Atoxyl, the first medicinal drug against human African trypanosomiasis (HAT), also known as sleeping sickness, was applied more than 100 years ago. Ever since, the search for more effective, more specific and less toxic drugs continued, leading to a set of compounds currently in use against this devastating disease. Unfortunately, none of these medicines fulfill modern pharmaceutical requirements and may be considered as therapeutic ultima ratio due to the many, often severe side effects. Starting with a historic overview on drug development against HAT, we present a selection of trypanosome specific pathways and enzymes considered as highly potent druggable targets. In addition, we describe cellular mechanisms the parasite uses for differentiation and cell density regulation and present our considerations how interference with these steps, elementary for life cycle progression and infection, may lead to new aspects of drug development. Finally we refer to our recent work about CNS infection that offers novel insights in how trypanosomes hide in an immune privileged area to establish a chronic state of the disease, thereby considering new ways for drug application. Depressingly, HAT specific drug development has failed over the last 30 years to produce better suited medicine. However, unraveling of parasite-specific pathways and cellular behavior together with the ability to produce high resolution structures of essential parasite proteins by X-ray crystallography, leads us to the optimistic view that development of an ultimate drug to eradicate sleeping sickness from the globe might just be around the corner.

Tsetse flies are the most important vectors of African trypanosomiasis but, surprisingly, are highly refractory to trypanosome parasite infection. In populations of wild caught flies, it is rare to find mature salivarian and mouthpart parasite infection rates exceeding 1 and 15%, respectively. This inherent refractoriness persists throughout the lifespan of the fly, although extreme starvation and suboptimal environmental conditions can cause a reversion to the susceptible phenotype. The teneral phenomenon is a phenotype unique to newly emerged, previously unfed tsetse, and is evidenced by a profound susceptibility to trypanosome infection. This susceptibility persists for only a few days post-emergence and decreases with fly age and bloodmeal acquisition. Researchers investigating trypanosome-tsetse interactions routinely exploit this phenomenon by using young, unfed (teneral) flies to naturally boost trypanosome establishment and maturation rates. A suite of factors may contribute, at least in part, to this unusual parasite permissive phenotype. These include the physical maturity of midgut barriers, the activation of immunoresponsive tissues and their effector molecules, and the role of the microflora within the midgut of the newly emerged fly. However, at present, the molecular mechanisms that underpin the teneral phenomenon still remain unknown. This review will provide a historical overview of the teneral phenomenon and will examine immune-related factors that influence, and may help us better understand, this unusual phenotype.

African trypanosomes are sustained in the bloodstream of their mammalian hosts by their extreme capacity for antigenic variation. However, for life cycle progression, trypanosomes also must generate transmission stages called stumpy forms that are pre-adapted to survive when taken up during the bloodmeal of the disease vector, tsetse flies. These stumpy forms are rather different to the proliferative slender forms that maintain the bloodstream parasitaemia. Firstly, they are non proliferative and morphologically distinct, secondly, they show particular sensitivity to environmental cues that signal entry to the tsetse fly and, thirdly, they are relatively robust such that they survive the changes in temperature, pH and proteolytic environment encountered within the tsetse midgut. These characteristics require regulated changes in gene expression to pre-adapt the parasite and the use of environmental sensing mechanisms, both of which allow the rapid initiation of differentiation to tsetse midgut procyclic forms upon transmission. Interestingly, the generation of stumpy forms is also regulated and periodic in the mammalian blood, this being governed by a density-sensing mechanism whereby a parasite-derived signal drives cell cycle arrest and cellular development both to optimize transmission and to prevent uncontrolled parasite multiplication overwhelming the host. In this review we detail recent developments in our understanding of the molecular mechanisms that underpin the production of stumpy forms in the mammalian bloodstream and their signal perception pathways both in the mammalian bloodstream and upon entry into the tsetse fly. These discoveries are discussed in the context of conserved eukaryotic signaling and differentiation mechanisms. Further, their potential to act as targets for therapeutic strategies that disrupt parasite development either in the mammalian bloodstream or upon their transmission to tsetse flies is also discussed.

In Venezuela, horses are indispensable for extensive cattle raising, and extensive cattle raising prevails in all regions. This determines the numerical relationship between horses and cattle (r = 0.93) to be relatively constant nationwide. At regional level, the average extension of cattle ranches varies greatly. However, in relation to the area covered by pastures, the numbers of horses (r = 0.95) and cattle (r = 0.93) are relatively uniform nationwide. Water buffalo occupy small fractions of the territory; therefore, their numbers are related to the area of pastures less strongly (r = 0.56). There is no information on the numerical relationship between the numbers of horses and water buffalo. In the Llanos region of the country, equine trypanosomiasis is responsible for a high mortality in horses, causing considerable financial losses to cattle ranches. So far, such losses have not been assessed. For this region, in 2008, it can be calculated that: (1) with no treatment, losses owing to horse mortality caused by this hemoparasitosis would have amounted to US$7,486,000; (2) the diagnosis and treatment of affected horses would have required an investment of US$805,000; and (3) in terms of horses saved, this investment would have resulted in benefit of US$6,232,000. Therefore, for every monetary unit invested, there would be a benefit 7.75 times greater, this ratio being applicable to any year and all regions of the country. It follows that the profitability of investing in the diagnosis and treatment of equine trypanosomiasis is guaranteed.

African trypanosomes are unicellular flagellated parasites causing trypanosomiases in Africa, a group of severe diseases also known as sleeping sickness in human and nagana in cattle. These parasites are almost exclusively transmitted by the bite of the tsetse fly. In this review, we describe and compare the three developmental programs of the main trypanosome species impacting human and animal health, with focus on the most recent observations. From here, some reflections are made on research issues concerning trypanosome developmental biology in the tsetse fly that are to be addressed in the future.

Human African trypanosomiasis (HAT) is caused by trypanosomes transmitted to humans by the tsetse fly, in which they accomplish their development into their infective metacyclic form. The crucial step in parasite survival occurs when it invades the fly midgut. Insect digestive enzymes and immune defenses may be involved in the modulation of the fly's vector competence, together with bacteria that could be present in the fly's midgut. In fact, in addition to the three bacterial symbionts that have previously been characterized, tsetse flies may harbor additional bacterial inhabitants. This review focuses on the diversity of the bacterial flora in Glossina, with regards to the fly species and their geographical distribution. The rationale was (i) that these newly identified bacteria, associated with tsetse flies, may contribute to vector competence as was shown in other insects and (ii) that differences may exist according to fly species and geographic area. A more complete knowledge of the bacterial microbiota of the tsetse fly and the role these bacteria play in tsetse biology may lead to novel ways of investigation in view of developing alternative anti-vector strategies for fighting human--and possibly animal--trypanosomiasis.

The aim of this study was to evaluate the changes in hematological and biochemical parameters of blood during acute Trypanosoma evansi infection in Wistar rats. The end points studied were hematologic parameters, red blood cell fragility, iron content, and glutathione and lipid peroxidation levels. Forty-eight animals were infected with trypomastigotes and distributed into five groups according to the level of parasitemia. Twelve non-inoculated animals were used as control. Parasitemia increased progressively, reaching highest scores at 15 days post-inoculation. At this point, several deleterious effects were observed such as an increase in iron content, in osmotic fragility, and in lipid peroxidation index, while glutathione decreased drastically. These changes were highly correlated to parasitemia (p < 0.0001) and among each other (p ≤ 0.001). Hematological indices (Hb, packed cell volume (PCV), red blood cells (RBC), and mean corpuscular hemoglobin concentration) were also correlated to parasitemia (p ≤ 0.0003) but failed to correlate to the other variables. Along with increase in iron, RBC fragility produced a decrease in RBC, PCV, and Hb, but not in mean corpuscular volume. Decrease in glutathione was negatively correlated to the end products of lipid peroxidation, clearly indicating the establishment of a pro-oxidant condition. The results show that the infection causes hematological impairments, increases iron and osmotic fragility, along with marked oxidative stress in red blood cells of rats inoculated with T. evansi.

The aim of this study was to evaluate biochemical parameters of iron metabolism in rats experimentally infected with Trypanosoma evansi. To this end, 20 rats (Wistar) were intraperitoneally inoculated with blood containing trypomastigotes 10(6) (Group T) and 12 animals were used as negative control (Group C) and received saline (0.2 mL) through same route. Blood samples were collected by cardiac puncture on day 5 (C5, T5) and 30 (C30, T30) post-inoculation (pi) to perform complete blood count and determination of serum iron, transferrin, ferritin, total and latent iron fixation capacity, transferrin saturation and prohepcidin concentration. Also, bone marrow samples were collected, to perform Pearls staining reaction. Levels of iron, total and latent iron binding capacity and prohepcidin concentration were lower (P<0.05) in infected rats (T5 and T30 groups) compared to controls. On the other hand, levels of transferrin and ferritin were higher when compared to controls (P<0.05). The transferrin saturation increased on day 5 pi, but decreased on day 30 pi. The Pearls reaction showed a higher accumulation of iron in the bone marrow of infected animals in day 5 pi (P<0.01). Infection with T. evansi in rats caused anemia and changes in iron metabolism associated to the peaks of parasitemia. These results suggest that changes in iron metabolism may be related to the host immune response to infection and anemic status of infected animals., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The purpose of this study was to verify the in vitro development of Trypanosoma sp. isolated from Leptodactylus ocellatus frogs under a new protocol using a biphasic medium composed of Novy, McNeal, and Nicolle (NNN) blood agar medium as a solid phase and liver infusion, brain heart infusion, and tryptose (LIBHIT) medium as a liquid phase. Blood forms, collected by cardiac puncture or after the maceration of different organs, were inoculated in culture tubes containing the biphasic medium composed by NNN and LIBHIT. Trypanosomes were observed 4 days postinoculation; most bloodstream trypomastigotes had differentiated into epimastigotes and amastigotes by this time. Trypomastigotes were again observed in older cultures (7 days). Parasites were successfully subcultured for 8 mo in this medium and successfully cryopreserved. The present study provides a new protocol medium for the isolation and culture of anuran trypanosomes.

Temporins are a family of short antimicrobial peptides (8-17 residues) that mostly show potent activity against Gram-positive bacteria. Herein, we demonstrate that temporin-SHd, a 17-residue peptide with a net charge of +2 (FLPAALAGIGGILGKLF(amide)), expressed a broad spectrum of antimicrobial activity. This peptide displayed potent antibacterial activities against Gram-negative and Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus strains, as well as antiparasitic activity against promastigote and the intracellular stage (amastigote) of Leishmania infantum, at concentration not toxic for the macrophages. Temporin-SHd that is structured in a non-amphipathic α-helix in anionic membrane-mimetic environments, strongly and selectively perturbs anionic bilayer membranes by interacting with the polar head groups and acyl region of the phospholipids, with formation of regions of two coexisting phases: one phase rich in peptide and the other lipid-rich. The disruption of lipid packing within the bilayer may lead to the formation of transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. To our knowledge, Temporin-SHd represents the first known 17-residue long temporin expressing such broad spectrum of antimicrobial activity including members of the trypanosomatidae family. Additionally, since only a few shorter members (13 residues) of the temporin family are known to display antileishmanial activity (temporins-TA, -TB and -SHa), SHd is an interesting tool to analyze the antiparasitic mechanism of action of temporins., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

This paper reviews the transmission modes of Trypanosoma evansi. Its worldwide distribution is attributed to mechanical transmission. While the role of tabanids is clear, we raise questions on the relative role of Haematobia sp. and the possible role of Stomoxys sp. in delayed transmission. A review of the available trypanocidal drugs and their efficacy in various host species is useful for understanding how they interact in disease epidemiology, which is complex. Although there are similarities with other mechanically transmitted trypanosomes, T. evansi has a more complex epidemiology due to the diversity of its hosts and vectors. The impact of clinical and subclinical disease is difficult to establish. A model was developed for buffaloes in the Philippines, which could be transferred to other places and livestock systems. Since Trypanosoma evansi was reported in humans, further research is required to investigate its zoonotic potential. Surra remains a potentially emerging disease that is a threat to Australia, Spain, and France. A number of questions about the disease have yet to be resolved. This brief review of the basic knowledge of T. evansi suggests that there is renewed interest in the parasite, which is spreading and has a major economic impact.