Targeting Trypanosoma evansi with disulphide-rich peptides derived from a phage display library.

A phage-display library was generated using a Bus thalamus scorpion toxin (BTK-2) as a peptide scaffold. BTK-2 belongs to the disulfide-rich family of proteins with pronounced structural stability due to the presence of three disulfide bridges that connects antiparallel beta-sheets and one alpha helix. Using BTK-2 as a phage display scaffold, we introduced mutations in five residues located in the alpha-helix and two residues located in the smaller loop, keeping intact the disulfide bridges to create a peptide phage-displayed library with disulfide-rich family properties. The library was subjected to in vivo and in vitro phage display selections against Trypanosoma evansi, the etiological agent of "Surra", a disease that affects a wide range of mammals. The development of T. evansi specific biomarkers is essential to improve diagnostic methods and epidemiological studies leading to a more accurate clinical decision for the treatment of this disease of economic impact for commercial livestock production. In this study, we identified two disulfide-rich peptides targeting T. evansi parasites. Further specificity studies are necessary to investigate the potential of selected peptides as new biomarkers to aid diagnostic and treatment procedures of T. evansi infections.

Hematobiochemical and Immunological Responses of Rats Treated with Multi-strain Probiotics and Infected with Trypanosoma brucei.

The effects of treatment with probiotics on the immunological and hematobiochemical changes in Trypanosoma brucei infection were investigated. Probiotic strains used are Bifidobacterium BB-12, Lactobacillus acidophilus LA-5, Lactobacillus delbrueckii LBY-27, Lactobacillus paracasei LC-01, and Streptococcus thermophilus STY-31. Thirty rats randomly assigned to five groups were used in the experiment. Groups A to C received 1 × 109 CFU, 5 × 109 CFU, and 10 × 109 CFU of the multi-strain probiotics daily and respectively from day 0 post-supplementation (PS) to termination. Group D and E were the infected and uninfected controls respectively. On day seven PS, groups A to D were challenged intraperitoneally with approximately 1 × 106 trypanosomes. Parasitemia, nitric oxide level, hematobiochemical parameters, and antibody titer to heterologous antigen stimulation were monitored post-infection. By days 7 and 16 PS, probiotics-treated groups had significantly lower (p < 0.05) mean creatinine concentration than the controls; however, on day 7 PS, there were no significant variations in the leukocyte counts (LC), total erythrocyte counts (TEC), and the packed cell volume (PCV) in all experimental groups. Following infection, by day 16 PS, the pre-patent period, parasitemia levels, and antibody titer were similar in all infected groups. Furthermore, the probiotics-treated groups and the infected control had significantly lower PCV, TEC, and LC values when compared to the uninfected control, and probiotics treated groups (A and C) had only marginally lower nitric oxide levels than the infected control. Treatment with the probiotic strains gave a creatinine-lowering effect, was innocuous to the hematopoietic system, but was not sufficiently immunostimulatory in trypanosomosis.

Clinical and therapeutic aspects of an outbreak of canine trypanosomiasis / Aspectos clínicos e terapêuticos de um surto de tripanossomíase canina

Abstract Trypanosomiasis caused by Trypanosoma evansi can seriously affect both domestic and wild animals. This article reports on an outbreak of canine trypanosomiasis on a farm in the Pantanal region of Brazil. The farm had 38 dogs, 20 of which died before receiving veterinary care. The remaining 18 dogs were underwent anamnesisn, clinical examination, hematological and biochemical evaluations. Blood smears and PCR analysis were performed for the diagnosis. The treatment protocols used according to the clinical recovery or parasitological cure of the dogs, using diminazene diaceturate, isometamidium chloride or quinapyramine sulfate. Post-treatment parasitological evaluation was performed by the microhematocrit technique. 7/18 dogs were PCR positive for T. evansi (confirmed by sequencing). There was clinical findings, which were consistent with both the acute and chronic stages of the disease in dogs. The infected dogs all exhibited at least one clinical sign of the disease. The hematological findings were compatible with trypanosomiasis, highlighting the hypochromic microcytic anemia as the main outcome. No treatment protocol was fully effective and the prolonged use of diminazene diaceturate caused the death of an animal. The trypanosomiasis can cause high rates of morbidity and mortality in dogs and difficulty in establishment an effective and safe therapeutic protocol.

Resumo A tripanossomíase causada por Trypanosoma evansi pode acometer gravemente os animais domésticos e selvagens. Este artigo relata um surto de tripanossomíase canina em uma fazenda na região do Pantanal, Brasil. Na fazenda havia 38 cães, 20 dos quais morreram antes de receber cuidados veterinários. Os 18 cães restantes foram submetidos a anamnese, exame clínico, avaliação hematológica e bioquímica. Esfregaços de sangue e análise da PCR foram realizados para o diagnóstico. Os protocolos de tratamento foram utilizados de acordo com a recuperação clínica ou cura parasitológica dos cães, utilizando diaceturato de diminazeno, cloreto de isometamídio ou sulfato de quinapiramina. A avaliação parasitológica pós-tratamento foi realizada pela técnica de microhematócrito. 7/18 cães foram PCR positivos para T. evansi (confirmado por sequenciamento). Os achados clínicos encontrados, foram consistentes com os estágios agudo e crônico da doença em cães. Todos os cães infectados exibiram pelo menos um sinal clínico da doença. Os achados hematológicos foram compatíveis com a tripanossomíase, destacando a anemia microcítica hipocrômica como principal consequência. Nenhum protocolo de tratamento foi totalmente eficaz e o uso prolongado de diaceturato de diminazeno causou a morte de um animal. A tripanossomíase pode causar altas taxas de morbidade e mortalidade em cães e dificultar o estabelecimento de um protocolo terapêutico eficaz e seguro.

Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.

Trypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vaccines are available. Disease eradication requires sensitive diagnostic tools and efficient treatment strategies. Immunodiagnostics based on antigen detection are preferable to antibody detection because the latter cannot differentiate between active infection and cure. Classical monoclonal antibodies are inaccessible to cryptic epitopes (based on their size-150 kDa), costly to produce and require cold chain maintenance, a condition that is difficult to achieve in trypanosomiasis endemic regions, which are mostly rural. Nanobodies are recombinant, heat-stable, small-sized (15 kDa), antigen-specific, single-domain, variable fragments derived from heavy chain-only antibodies in camelids. Because of numerous advantages over classical antibodies, we investigated the use of nanobodies for the targeting of trypanosome-specific antigens and diagnostic potential. An alpaca was immunized using lysates of Trypanosoma evansi. Using phage display and bio-panning techniques, a cross-reactive nanobody (Nb392) targeting all trypanosome species and isolates tested was selected. Imunoblotting, immunofluorescence microscopy, immunoprecipitation and mass spectrometry assays were combined to identify the target recognized. Nb392 targets paraflagellar rod protein (PFR1) of T. evansi, T. brucei, T. congolense and T. vivax. Two different RNAi mutants with defective PFR assembly (PFR2RNAi and KIF9BRNAi) were used to confirm its specificity. In conclusion, using a complex protein mixture for alpaca immunization, we generated a highly specific nanobody (Nb392) that targets a conserved trypanosome protein, i.e., PFR1 in the flagella of trypanosomes. Nb392 is an excellent marker for the PFR and can be useful in the diagnosis of trypanosomiasis. In addition, as demonstrated, Nb392 can be a useful research or PFR protein isolation tool.

Acidocalcisomes of Trypanosoma brucei have an inositol 1,4,5-trisphosphate receptor that is required for growth and infectivity.

Acidocalcisomes are acidic calcium stores rich in polyphosphate and found in a diverse range of organisms. The mechanism of Ca(2+) release from these organelles was unknown. Here we present evidence that Trypanosoma brucei acidocalcisomes possess an inositol 1,4,5-trisphosphate receptor (TbIP(3)R) for Ca(2+) release. Localization studies in cell lines expressing TbIP(3)R in its endogenous locus fused to an epitope tag revealed its partial colocalization with the vacuolar proton pyrophosphatase, a marker of acidocalcisomes. IP(3) was able to stimulate Ca(2+) release from a chicken B-lymphocyte cell line in which the genes for all three vertebrate IP(3)Rs have been stably ablated (DT40-3KO) and that were stably expressing TbIP(3)R, providing evidence of its function. IP(3) was also able to release Ca(2+) from permeabilized trypanosomes or isolated acidocalcisomes and photolytic release of IP(3) in intact trypanosomes loaded with Fluo-4 elicited a transient Ca(2+) increase in their cytosol. Ablation of TbIP(3)R by RNA interference caused a significant reduction of IP(3)-mediated Ca(2+) release in trypanosomes and resulted in defects in growth in culture and infectivity in mice. Taken together, the data provide evidence of the presence of a functional IP(3)R as a Ca(2+) release channel in acidocalcisomes of trypanosomes and suggest that a Ca(2+) signaling pathway that involves acidocalcisomes is required for growth and establishment of infection.

Canine Trypanosoma evansi infection introduced into Germany.

A 9-year-old male Jack Russell Terrier with a history of travel to Thailand was presented with chronic lethargy, weight loss, unilateral anterior uveitis, pancytopenia, hyperglobulinemia, and proteinuria. Numerous trypomastigotes were found on a blood smear, and using molecular methods the parasite was identified as Trypanosoma evansi. After initial response to treatment, the dog experienced a relapse with central neurologic signs 88 days after initial presentation and died. Antibodies to T evansi were detected in both serum and cerebrospinal fluid (CSF) using a card agglutination test (CATT/T evansi), and PCR analysis of CSF for T evansi was positive. Findings at necropsy included marked non-purulent meningoencephalitis. Chronic infection with T evansi in a dog that returned to Germany following international travel highlights the risk associated with introduction of foreign animal diseases to Europe and the possibility of these infections becoming endemic. Detection of chronic infection and curative therapy of trypanosomiasis are challenging, and infection is usually fatal in the dog.

Productos naturales con actividad leishmanicida y tripanocida / Natural products with leishmanicidal and trypanocidal activity

Las enfermedades parasitarias constituyen un importante problema de salud, y muchas de ellas están emergiendo en países donde se consideraban erradicadas. La leishmaniasis, la enfermedad del sueño y la enfermedad de Chagas, causadas por los parásitos Leishmania spp, Trypanosoma brucei y Trypanosoma cruzi, respectivamente, se encuentran entre las enfermedades parasitarias más prevalentes. La principal alternativa para tratarlas es la quimioterapia. Sin embargo, los tratamientos actuales se encuentran lejos de ser satisfactorios. La toxicidad de los fármacos, la vía de administración, la duración de los tratamientos y la aparición de resistencias hacen necesario el desarrollo de nuevas moléculas activas, más seguras y eficaces. Estudios recientes ponen de manifiesto la actividad leishmanicida y tripanocida in vivo de una amplia variedad de compuestos fenólicos, alcaloides y terpenos. En este artículo se revisan los productos naturales activos frente a leishmaniasis, enfermedad del sueño y enfermedad de Chagas (AU)

Diseases caused by protozoan parasites are still an important human health problem, since many of them are becoming “emerging” infectious sickness in geographical areas where they were considered eradicated. Leishmaniasis, African sleeping sickness and Chagas disease, caused by the parasites Leishmania spp, Trypanosoma brucei and Trypanosoma cruzi, respectively, are among the most important parasitic diseases. The main alternative to control such parasitosis is chemotherapy. Nevertheless, the current drug treatments are far from being satisfactory. Toxic side effects, route of administration, long-term treatments and the apparition of resistance, highlight the urgent need of developing new active molecules, more safe and effective. Recent studies report the leishmanicidal and trypanocidal activities of a wide variety of phenolic compounds, alkaloids and terpenes that have shown activity in vivo. This review outlines the current understanding of natural products against leishmaniasis, African sleeping sickness and Chagas disease (AU)

Susceptibility of Trypanosoma evansi to human blood and plasma in infected mice.

Around 1900 Laveran and Mesnil discovered that African trypanosomes do not survive in the blood of some primates and humans. The nature of the trypanolytic factor present in these sera has been the focus of a long-standing debate between different groups. The aim of this study was to investigate the susceptibility of T. evansi isolates to therapy using human blood and plasma in experimentally infected mice. Forty-eight 2-month-old female mice (Mus musculus) were divided into six groups of eight animals per group (A, B, C, D, E and F). Plasma was obtained after blood collection in order to perform therapy. Animals from group A (positive control) were inoculated with T. evansi and treated with 0.2mL of saline solution. Animals from groups B and C were infected with the flagellate and received a curative treatment with 0.2mL of human blood (group B) and 0.2mL of human plasma (group C), 24h after infection. Animals from groups D and E received a prophylactic treatment with 0.2mL of human blood and 0.2mL of human plasma, respectively, 24h prior to the infection. Animals from group F (negative control) were not infected and received 0.2mL of saline solution. The four treatments (B, C, D and E) increased animals longevity when compared to group A. Prepatency period was longer in groups D (15 days) and E (37.7 days) under prophylactic immunotherapy. Moreover, no parasites were found in most of the animals 60 days post-inoculation (PI). Besides the longer longevity, treatments were capable of curing 50% of mice of group B, 37.5% of group C, 37.5% of group D and 25% of the animals from group E.

Therapeutic DNA vaccine against Trypanosoma cruzi infection in dogs.

Chagas' disease is an important health problem in most Latin American countries, and a concern in dog populations, which act as a reservoir. We showed in previous studies that a therapeutic DNA vaccine could partially control the pathology after Trypanosoma cruzi infection in mice, and this vaccine may represent an alternative treatment for Chagas' disease. Here we evaluated the therapeutic efficacy of this vaccine in experimentally infected dogs for up to 2 months after infection. Our results suggest that DNA vaccine treatment may affect the immune response and delay Chagas' disease progression in T. cruzi-infected dogs, and confirm the potential of this novel treatment.

[Strokes caused by infection in the tropics]. / Ictus de causa infecciosa en el trópico.

INTRODUCTION: Almost three out of every four people in the world who suffer a fatal stroke live in developing countries. A number of different tropical diseases may appear in Europe in the coming years as a consequence of the demographic change that is being brought about by migratory flows. We review the main infectious causes of strokes in the tropics. DEVELOPMENT: There are estimated to be 500 million cases of malaria every year. Cerebral malaria can cause cerebral oedema, diffuse or focal compromise of the subcortical white matter and cortical, cerebellar and pontine infarctions. Chagas disease is an independent risk factor for stroke in South America. At least 20 million people have the chronic form of Chagas disease. The main prognostic factors for Chagas-related stroke are the presence of apical aneurysms, arrhythmia and heart failure. Vascular complications of neurocysticercosis include transient ischemic attacks, ischemic strokes due to angiitis and intracranial haemorrhages. The frequency of cerebral infarction associated with neurocysticercosis varies between 2% and 12%. Gnathostomiasis is a cause of subarachnoid haemorrhage in south-east Asia. Other less common causes of stroke are viral haemorrhagic fevers due to arenavirus and flavivirus. CONCLUSIONS: Several diseases that are endemic in the tropics can be responsible for up to 10% of the cases of strokes in adults.

Congenital trypanosomiasis.

The last successfully treated case of congenital trypanosomiasis in Zambia was in October 1978, with detailed analysis of immunoglobulins, illustrating the waning of blood and serum levels of IgA, IgG, and IgM during treatment, up to 99 days after treatment. Twenty-five years later, we report on a case of congenital trypanosomiasis. The disease is now rare and can be missed or dismissed as retroviral disease, particularly in adults. The main unusual symptoms were the prolonged intermittent convulsions in an otherwise well infant. Management of the disease is now more interdisciplinary, resources for laboratory support are fewer, lumbar puncture is more relevant, and antitrypanosomal drugs are more difficult to obtain. The mother died within one week of hospitalization and the infant initially responded to three doses of suramin and 3 weeks of melsopropol. Convulsions ceased during the second round of melsopropol. Unfortunately, the infant died of nosocomial infection.

Paediatric Sleeping Sickness in Kenya : A Case Report

Sleeping sickness is often considered a disease of adults rather than children due to their reduced exposure to the vector. Presumptive diagnosis of sleeping sickness was however difficult since the clinical signs observed were non-specific. This makes clinical diagnosis difficult. Often the disease in children masquerades as a pulmonary infection that is undetectable on x-ray or auscultation. A male child aged two years and eight months was diagnosed with the disease in western Kenya. The patient presented with severe respiratory distress; hepatosplenomegay and neurological symptoms. The disease transmission was associated with the socio-cultural habit of placing children under bushes whilst farming. The implications of delayed diagnosis on response to treatment are discussed

Trypanosoma evansi and T. equiperdum: distribution, biology, treatment and phylogenetic relationship (a review).

Trypanosoma evansi and T. equiperdum were compared regarding their ultrastructure, their mammalian hosts, way of transmission, pathogenicity, diagnosis and treatment, and biochemical and molecular characteristics. Electron microscopic investigation revealed no ultrastructural differences between the two species except that there were more coated vesicles in the flagellar pocket of T. equiperdum. Biological, biochemical and molecular studies were reviewed and exhibited many similarities between T. evansi and T. equiperdum. The most prominent differences between the two species are the presence of maxicircles in T. equiperdum, which are missing in T. evansi, and the route of transmission. While T. evansi is transmitted by biting flies, T. equiperdum is transmitted from one equine host to another during copulation when mucous membranes come into contact. Otherwise the two species are remarkably similar. The phylogenetic relationship between the two species and T. b. brucei is being discussed, and the hypothesis is proposed that T. evansi arose from a clone of T. equiperdum which lost its maxicircles.

In vitro magnesium- and calcium-induced sialidase activity in caprine intestinal mucosa cells.

Sialidase activity identical to 0.0196 mg per ml of liberated sialic acids (2.11 Units) was detected in small intestinal contents of goats. Mg++ and Ca++ induced in vitro detection in mucosa cells of both small and large intestines. Magnesium alone induced sialidase activities identical to 0.0128 mg per ml of liberated sialic acids (1.38 Units) and 0.0166 mg per ml (1.79 Units) in small and large intestinal mucosal cells, respectively. Mg++ and Ca++ together induced higher sialidase activities identical to 0.0191 mg per ml (2.06 Units) and 0.0194 mg per ml (2.09 Units) in small and large intestinal mucosal cells, respectively. The enzyme activity was slightly higher in large than in small intestinal mucosal cells. The results are discussed in relation to control of African animal trypanosomiasis.

Further characterization of the curative antibodies in Trypanosoma musculi infection.

The ability of immune plasma (IP) taken from different donor strains of mice to cure Trypanosoma musculi infection in various recipient mouse strains, when given during the plateau phase of infection, was examined. C57BL/6, B10.A/SgSn, B10.D2/oSn, B10.D2/nSn, DBA/2, and BALB/c strains could be cured of parasitemia (giving 0.4 to 0.8 ml of IP per mouse), whereas A/J and C3H/HeN strains could not (giving up to 1.2 ml of IP per mouse). Noncure appeared to be associated with the high-plateau parasitemias (approximately 10(8] that developed in the latter strains since IP administered early in infection, when the parasite burden was similar to the plateau parasitemias (approximately 10(6] of strains that could be cured, was at least partially effective in A/J and C3H/HeN mice. The IP of any strain tested (C57BL/6, B10.D2/oSn, B10.D2/nSn, DBA/2, A/J, or C3H/HeN) could bring about elimination of trypanosomes in strains able to be cured. The potency of IP from different strains varied, being greater in the strains that developed higher-plateau parasitemias. Potency of IP appears to correlate positively with the titers of trypanosome-specific antibody of the immunoglobulin G2a isotype (the curative antibody). The role of the late-acting complement components was examined. In C5-deficient mice the course of infection was normal, although the elimination phase was delayed by a few days. Cure of parasitemia by IP administered during the plateau phase was equally effective in the presence or absence of C5 in either the donor or the recipient. When tested in vitro, however, IP only exhibited antitrypanosomal activity when added to infected blood taken from C5-sufficient strains of mice. We conclude that in vitro, under the conditions used in the assay, antibody-mediated destruction of the trypanosomes is brought about by complement-mediated lysis. This process, although it probably occurs to some extent, is unlikely to be the major mechanism of trypanosome elimination in vivo.