Comparative chromatin analysis of Trypanosoma congolense

The chromatin of Trypanosoma congolense was analyzed by electron microscopy. The chromatin is organized as nucleosome filaments but does not form a 30 nm fiber. There are five groups of histones, including a histone H1-like protein, which has a molecular weight within the range of the core histones, and is extremely hydrophilic. Weak histone-histone interaction, a typical feature of trypanosoma chromatin, was found. These results are similar to those for T. cruzi and T. b. brucei, but differ significantly from those for higher eukaryotes. The results confirm the model of trypanosome chromatin, and support the theory of their early separation from the other eukaryotes during the evolution. T. congolensis is an excellent model for chromatin research on trypanosomes, because it is easy to cultivate and its chromatin has, a relatively high stability, compared to that of other trypanosomes.

Trypanosoma congolense procyclins: unmasking cryptic major surface glycoproteins in procyclic forms

In the tsetse fly, the protozoan parasite Trypanosoma congolense is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These include a protease-resistant surface molecule (PRS), which is expressed by procyclic forms early in infection, and a glutamic acid- and alanine-rich protein (GARP), which appears at later stages. Since neither of these surface antigens is expressed at intermediate stages, we investigated whether a GPI-anchored protein of 50 to 58 kDa, previously detected in procyclic culture forms, might constitute the coat of these parasites. We therefore partially purified the protein from T. congolense Kilifi procyclic forms, obtained an N-terminal amino acid sequence, and identified its gene. Detailed analyses showed that the mature protein consists almost exclusively of 13 heptapeptide repeats (EPGENGT). The protein is densely N glycosylated, with up to 13 high-mannose oligosaccharides ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) linked to the peptide repeats. The lipid moiety of the glycosylphosphatidylinositol is composed of sn-1-stearoyl-2-lyso-glycerol-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol. Heavily glycosylated proteins with similar repeats were subsequently identified in T. congolense Savannah procyclic forms. Collectively, this group of proteins was named T. congolense procyclins to reflect their relationship to the EP and GPEET procyclins of T. brucei. Using an antiserum raised against the EPGENGT repeat, we show that T. congolense procyclins are expressed continuously in the fly midgut and thus form the surface coat of cells that are negative for both PRS and GARP.

Desenvolvimento de métodos moleculares para a deteção de Trypanosoma spp em glossinas (Diptera: Glossinidae) da República da Guiné Bissau

As moscas do género Glossina são vetores de patologias provocadas por várias espécies de parasita do género Trypanosoma, como a Tripanossomose Humana Africana e as Tripanossomoses Animais. A correta identificação destes tripanossomas é um ponto fulcral quando se procura determinar o risco de doença que determinadas populações de glossinas podem provocar. No presente trabalho foram utilizadas e adaptadas diversas metodologias de diagnóstico molecular, nomeadamente o PCR em tempo real e o PCR-RFLP, que permitiram, através da utilização de primers genéricos, a determinação da carga parasitária e a correta identificação dos tripanossomas presentes no vetor. Os primers denominados Tryp18SF e Tryp18SR foram desenvolvidos especificamente para serem utilizados na identificação das espécies de tripanossoma utilizando PCR em tempo real com metodologia SYBR® Green I e PCR-RFLP. Os primers denominados Tryp18S2F, Tryp18S2R e sonda Tryp18S2P foram desenvolvidos para a quantificação dos parasitas presentes nas amostras utilizando PCR em tempo real com metodologia Taqman®. Foi estudada uma amostra de 762 glossinas provenientes do território da República da Guiné-Bissau, zona atualmente considerada como estando livre de Tripanossomose Humana Africana, mas onde não são realizadas atividades de recenseamento de casos desde o final da ocupação portuguesa. Das 762 glossinas utilizadas, 241 estavam infetadas com tripanossomas, o que representa uma percentagem de infeção geral de 31,6 %. Destas glossinas, 28.63 % encontravam-se infetadas com Trypanosoma grayi, 14.11 % com Trypanosoma congolense, 7.05 % com Trypanosoma vivax e 0.83 % com Trypanosoma brucei brucei, tendo as infeções mistas representado 1.66 %. Não foi possível a identificação conclusiva ao nível da espécie em 48.13 % das amostras positivas, tendo estas ficado consideradas como Trypanosoma spp. Não foram identificados vetores infetados com tripanossomas responsáveis pelas patologias humanas. A utilização de primers genéricos para a identificação permitiu obviar o número de reações necessárias para identificar corretamente o parasita responsável pela infeção, e este trabalho é de resto a primeira descrição da utilização de primers genéricos que permitam a identificação de Trypanosoma grayi. Esta é também a primeira descrição da utilização de PCR em tempo real com metodologia Taqman® para quantificação de tripanossomas, e a sua utilização com primers genéricos aumenta a aplicabilidade em estudos epidemiológicos de grande escala. Palavras-chave: PCR em tempo real; PCR-RFLP; República da Guiné-Bissau; Glossina; Trypanosoma; Tripanossomose.

Laboratory and Field Evaluation of Biodegradable Polyesters for Sustained Release of Isometamidium and Ethidium

An overview is presented of the results obtained with biodegradable sustained release devices (SRDs) containing a mixture of polymers and either isometamidium (ISMM) or ethidium. Under controlled laboratory conditions (monthly challenge with tsetse flies infected with Trypanosoma congolense) the protection period in SRD treated cattle could be extended by a factor 2.8 (for ethidium) up to 4.2 (for ISMM) as compared to animals treated intramuscularly with the same drugs. Using a competitive drug ELISA ISMM concentrations were detected up to 330 days after the implantation of the SRDs, whereas after i.m. injection the drug was no longer present three to four months post treatment. Two field trials carried out in Mali under heavy tsetse challenge showed that the cumulative infection rate was significantly lower in the ISMM-SRD implanted cattle than in those which received ISMM intramuscularly. Using ethidium SRD, however, contradictory results were obtained in field trials in Zambia and in Mali. The potential advantages and inconvenients of the use of SRDs are discussed and suggestions are made in order to further improve the currently available devices.

Immune Response of Cattle Infected with African Trypanosomes

Trypanosomosis is the most economically important disease constraint to livestock productivity in sub-Saharan Africa and has significant negative impact in other parts of the world. Livestock are an integral component of farming systems and thus contribute significantly to food and economic security in developing countries. Current methods of control for trypanosomosis are inadequate to prevent the enormous socioeconomic losses resulting from this disease. A vaccine has been viewed as the most desirable control option. However, the complexity of the parasite's antigenic repertoire made development of a vaccine based on the variable surface glycoprotein coat unlikely. As a result, research is now focused on identifying invariant trypanosome components as potential targets for interrupting infection or infection-mediated disease. Immunosuppression appears to be a nearly universal feature of infection with African trypanosomes and thus may represent an essential element of the host-parasite relationship, possibly by reducing the host's ability to mount a protective immune response. Antibody, T cell and macrophage/monocyte responses of infected cattle are depressed in both trypanosusceptible and trypanotolerant breeds of cattle. This review describes the specific T cell and monocyte/macrophage functions that are altered in trypanosome-infected cattle and compares these disorders with those that have been described in the murine model of trypanosomosis. The identification of parasite factors that induce immunosuppression and the mechanisms that mediate depressed immune responses might suggest novel disease intervention strategies.

Improved Methods for the Diagnosis of African Trypanosomosis

The diagnosis of trypanosomosis in animals with low parasitaemia is hampered by low diagnostic sensitivity of traditional detection methods. An immunodiagnostic method based on a direct sandwich enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies, has been examined in a number of African laboratories for its suitability for monitoring tsetse control and eradication programmes. Generally, the direct sandwich ELISAs for the detection of trypanosomal antigens in serum samples have proved to be unsatisfactory with respect to diagnostic sensitivity when compared with traditional parasitological methods such as the dark ground/phase contrast buffy-coat technique. Consequently, antigen-detection systems exploiting various other direct, indirect and sandwich ELISA systems and sets of reagents are being developed to improve diagnosis. In addition, an existing indirect ELISA for the detection of antibodies has been improved and is being evaluated in the field in order to detect cattle that are or have been recently infected with trypanosomes. Developments and advantages of other diagnostic techniques, such as dip-stick assay and tests based on the polymerase chain reaction are also considered.

Phylogenetic analysis of Trypanosoma vivax supports the separation of South American/West African from East African isolates and a new T-vivax-like genotype infecting a nyala antelope from Mozambique

In this study, we addressed the phylogenetic and taxonomic relationships of Trypanosoma vivax and related trypanosomes nested in the subgenus Duttonella through combined morphological and phylogeographical analyses. We previously demonstrated that the clade T. vivax harbours a homogeneous clade comprising West African/South American isolates and the heterogeneous East African isolates. Herein we characterized a trypanosome isolated from a nyala antelope (Tragelaphus angasi) wild-caught in Mozambique (East Africa) and diagnosed as T. vivax-like based on biological, morphological and molecular data. Phylogenetic relationships, phylogeographical patterns and estimates of genetic divergence were based on SSU and ITS rDNA sequences of T. vivax from Brazil and Venezuela (South America), Nigeria (West Africa), and from T. vivax-like trypanosomes from Mozambique, Kenya and Tanzania (East Africa). Despite being well-supported within the T. vivax clade, the nyala trypanosome was highly divergent from all other T. vivax and T. vivax-like trypanosomes, even those from East Africa. Considering its host origin, morphological features, behaviour in experimentally infected goats, phylogenetic placement, and genetic divergence this isolate represents a new genotype of trypanosome closely phylogenetically related to T. vivax. This study corroborated the high complexity and the existence of distinct genotypes yet undescribed within the subgenus Duttonella.

Highly debilitating natural Trypanosoma vivax infections in Brazilian calves: epidemiology, pathology, and probable transplacental transmission

Clinical, epidemiological, and pathological aspects of trypanosomiasis caused by Trypanosoma vivax in calves were reported for the first time in northeast Brazil. Clinical and epidemiological data, packed cell volumes (PCV), and parasitemia were assessed in 150 calves in May 2009 (rainy season-survey 1) and in 153 calves in November 2009 (dry season-survey 2) in three farms (A, B, and C). Prevalence of T. vivax in calves examined in the survey 1 was 63.3%, 65.0%, and 80.0% in farms A, B, and C, respectively. Morbidity varied from 63.3% to 80%, mortality from 15% to 30% and lethality from 23% to 37.5%. In survey 1, for all farms, high parasitemia (from 30.3 to 26.2x10(6) parasites/mL), fever (from 39.8 to 40.3 degrees C), low PCV (from 15.7% to 18.1%), and body score (from 2.5 to 3.5) were detected. Calves showed depression, weight loss, pale mucous membranes, enlarged lymph nodes, edema of the dewlap, cough, coryza, and diarrhea. The animals from farms A and B were treated with diminazene aceturate. Six months after, in survey 2, non-treated calves from farm C showed values for prevalence (81.82), morbidity (81.82), mortality (12.73), and lethality (15.55) similar to those in survey 1 (P>0.05). Also in survey 2, four calves aging merely 1-3 days old presented high parasitemia levels (from 32x10(6) to 74x10(6) parasites/mL), suggesting transplacental transmission. In conclusion, trypanosomiasis by T. vivax constitutes high prevalent disease for calves raised in Brazilian semiarid and may have transplacental transmission.

Prevalence and associations of Trypanosoma spp. and Sodalis glossinidius with intrinsic factors of tsetse flies

Trypanosomiasis has been identified as a neglected tropical disease in both humans and animals in many regions of sub-Saharan Africa. Whilst assessments of the biology of trypanosomes, vectors, vertebrate hosts and the environment have provided useful information about life cycles, transmission, and pathogenesis of the parasites that could be used for treatment and control, less information is available about the effects of interactions among multiple intrinsic factors on trypanosome presence in tsetse flies from different sites. It is known that multiple species of tsetse flies can transmit trypanosomes but differences in their vector competence has normally been studied in relation to individual factors in isolation, such as: intrinsic factors of the flies (e.g. age, sex); habitat characteristics; presence of endosymbionts (e.g. Wigglesworthia glossinidia, Sodalis glossinidius); feeding pattern; host communities that the flies feed on; and which species of trypanosomes are transmitted. The purpose of this study was to take a more integrated approach to investigate trypanosome prevalence in tsetse flies. In chapter 2, techniques were optimised for using the Polymerase Chain Reaction (PCR) to identify species of trypanosomes (Trypanosoma vivax, T. congolense, T. brucei, T. simiae, and T. godfreyi) present in four species of tsetse flies (Glossina austeni, G. brevipalpis, G. longipennis and G. pallidipes) from two regions of eastern Kenya (the Shimba Hills and Nguruman). Based on universal primers targeting the internal transcribed spacer 1 region (ITS-1), T. vivax was the predominant pathogenic species detected in flies, both singly and in combination with other species of trypanosomes. Using Generalised Linear Models (GLMs) and likelihood ratio tests to choose the best-fitting models, presence of T. vivax was significantly associated with an interaction between subpopulation (a combination between collection sites and species of Glossina) and sex of the flies (X2 = 7.52, df = 21, P-value = 0.0061); prevalence in females overall was higher than in males but this was not consistent across subpopulations. Similarly, T. congolense was significantly associated only with subpopulation (X2 = 18.77, df = 1, P-value = 0.0046); prevalence was higher overall in the Shimba Hills than in Nguruman but this pattern varied by species of tsetse fly. When associations were analysed in individual species of tsetse flies, there were no consistent associations between trypanosome prevalence and any single factor (site, sex, age) and different combinations of interactions were found to be significant for each. The results thus demonstrated complex interactions between vectors and trypanosome prevalence related to both the distribution and intrinsic factors of tsetse flies. The potential influence of the presence of S. glossinidius on trypanosome presence in tsetse flies was studied in chapter 3. A high number of Sodalis positive flies was found in the Shimba Hills, while there were only two positive flies from Nguruman. Presence or absence of Sodalis was significantly associated with subpopulation while trypanosome presence showed a significant association with age (X2 = 4.65, df = 14, P-value = 0.0310) and an interaction between subpopulation and sex (X2 = 18.94, df = 10, P-value = 0.0043). However, the specific associations that were significant varied across species of trypanosomes, with T. congolense and T. brucei but not T. vivax showing significant interactions involving Sodalis. Although it has previously been concluded that presence of Sodalis increases susceptibility to trypanosomes, the results presented here suggest a more complicated relationship, which may be biased by differences in the distribution and intrinsic factors of tsetse flies, as well as which trypanosome species are considered. In chapter 4 trypanosome status was studied in relation to blood meal sources, feeding status and feeding patterns of G. pallidipes (which was the predominant fly species collected for this study) as determined by sequencing the mitochondrial cytochrome B gene using DNA extracted from abdomen samples. African buffalo and African elephants were the main sources of blood meals but antelopes, warthogs, humans, giraffes and hyenas were also identified. Feeding on multiple hosts was common in flies sampled from the Shimba Hills but most flies from Nguruman had fed on single host species. Based on Multiple Correspondence Analysis (MCA), host-feeding patterns showed a correlation with site of sample collection and Sodalis status, while trypanosome status was correlated with sex and age of the flies, suggesting that recent host-feeding patterns from blood meal analysis cannot predict trypanosome status. In conclusion, the complexity of interactions found suggests that strategies of tsetse fly control should be specific to particular epidemic areas. Future studies should include laboratory experiments that use local colonies of tsetse flies, local strains of trypanosomes and local S. glossinidius under controlled environmental conditions to tease out the factors that affect vector competence and the relative influence of external environmental factors on the dynamics of these interactions.

The Cratylia Mollis Seed Lectin Induces Membrane Permeability Transition In Isolated Rat Liver Mitochondria And A Cyclosporine A-insensitive Permeability Transition In Trypanosoma Cruzi Mitochondria.

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca(2+) and mitochondrial dysfunction due to matrix Ca(2+) overload. In order to investigate the mechanism of Ca(2+) -induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (∆Ψm ) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 μM Ca(2+) was significantly decreased by 50 μg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 μM Ca(2+) this lectin, at 50 μg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ∆Ψm disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca(2+) dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA-insensitive MPT in T. cruzi mitochondria.

Trypanosoma Cruzi Strains From Triatomine Collected In Bahia And Rio Grande Do Sul, Brazil.

Collection of triatomines in domestic, peridomestic and sylvatic environments in states of Bahia and Rio Grande do Sul, Northeastern and Southern Brazil respectively, and isolation of Trypanosoma cruzi strains. First, the captured triatomines were identified using insect identification keys, then their intestinal content was examined by abdominal compression, and the samples containing trypanosomatid forms were inoculated in LIT medium and Swiss mice. Six triatomine species were collected in cities in Bahia, namely Panstrongylus geniculatus (01), Triatoma melanocephala (11), T. lenti (94), T. pseudomaculata (02), T. sherlocki (26) and T. sordida (460), and two in cities in Rio Grande do Sul, namely T. circummaculata (11) and T. rubrovaria (115). Out of the specimens examined, T. cruzi was isolated from 28 triatomine divided into four different species: T. melanocephala (one), T. lenti (one), T. rubrovaria (16) and T. sordida (10). Their index of natural infection by T. cruzi was 6.4%. The isolation of T. cruzi strains from triatomines found in domestic and peridomestic areas shows the potential risk of transmission of Chagas disease in the studied cities. The maintenance of those T. cruzi strains in laboratory is intended to promote studies that facilitate the understanding of the parasite-vector-host relationship.

Design, Synthesis And Biological Evaluation Of Hybrid Bioisoster Derivatives Of N-acylhydrazone And Furoxan Groups With Potential And Selective Anti-trypanosoma Cruzi Activity.

Hybrid bioisoster derivatives from N-acylhydrazones and furoxan groups were designed with the objective of obtaining at least a dual mechanism of action: cruzain inhibition and nitric oxide (NO) releasing activity. Fifteen designed compounds were synthesized varying the substitution in N-acylhydrazone and in furoxan group as well. They had its anti-Trypanosoma cruzi activity in amastigotes forms, NO releasing potential and inhibitory cruzain activity evaluated. The two most active compounds (6, 14) both in the parasite amastigotes and in the enzyme contain the nitro group in para position of the aromatic ring. The permeability screening in Caco-2 cell and cytotoxicity assay in human cells were performed for those most active compounds and both showed to be less cytotoxic than the reference drug, benznidazole. Compound 6 was the most promising, since besides activity it showed good permeability and selectivity index, higher than the reference drug. Thereby the compound 6 was considered as a possible candidate for additional studies.

Primeiro registro de Trypanosoma vivax em bovinos no Estado do Rio Grande do Sul, Brasil

Este estudo teve o objetivo de relatar a ocorrência de Trypanosoma vivax no Sul do Brasil. O protozoário foi diagnosticado em esfregaço sanguíneo de um bovino e a identificação baseada na morfologia das formas tripomastigotas e confirmada pela técnica de reação em cadeia de polimerases (PCR). O animal infectado apresentou sintomatologia compatível com a forma nervosa da infecção por T. vivax. Outros bovinos que compartilhavam o mesmo ambiente apresentaram resultados negativos para T. vivax por PCR. Este é o primeiro registro de T. vivax no Estado do Rio Grande do Sul e na região Sul do Brasil.