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.

Survey of trypanosoma (kinetoplastida: trypanosomatidae) infection in Monte Negro Municipality, State of Rondônia, western amazon, with first record of T. evansi in the state

Abstract INTRODUCTION: Trypanosomes can infect humans and animals. This is the first record of the occurrence of Trypanosoma evansi in Rondônia. METHODS: Blood samples were collected from 7 dogs and 22 humans. Furthermore, triatomines and tabanids were collected. RESULTS: It was observed that 42.8% of the dogs tested positive for T. evansi and 14.3% presented mixed infection; 15% of the triatomines tested positive for flagellates identified as T. cruzi TCI (3 specimens), T. cruzi TCI, and T. rangeli (1 specimen), and one with T. cruzi TCV. Two tabanids were infected with T. theileri. CONCLUSIONS: These findings may benefit vector control strategies.

Trypanosoma evansi (Kinetoplastida: Trypanosomatidae) in capybaras ( Hydrochoerus hydrochaeris ): prevalence, effect and sexual selection / Trypanosoma evansi(Kinetoplastida: Trypanosomatidae) en capibaras (Hydrochoerus hydrochaeris):prevalencia, efecto y selección sexual

AbstractParasites play a crucial role in the ecology of animals. They also appear to be important in mechanisms underlying sexual selection processes. In this article we study the prevalence, effect and potential role in sexual selection of the protozoon Trypanosoma evansi in capybaras, Hydrochoerus hydrochaeris. We collected our samples from the annual capybara cull of a ranch in Venezuela, using the volume of the snout scent gland as an indicator of dominance; the residuals of body weight as indicators of condition; and the residuals of the spleen mass as indicators of immune function. Overall prevalence was 30.9 % (N= 97) with no difference between males and females, and no relation between infection with T. evansi and condition. However, we found that infected animals had larger spleens (residuals), indicating an immunological cost of the infection. Furthermore, males with larger snout scent glands (more dominant) were less likely to be infected than males with smaller glands (less dominant), suggesting that by choosing males with a large glands, females may be using the gland as an indicator of health, which is consistent with the "good genes" view of sexual selection. Rev. Biol. Trop. 65 (1): 229-237. Epub 2017 March 01.

ResumenLos parásitos juegan un papel crucial en la ecología de todos los animales. También parecen ser importantes en los mecanismos subyacentes a la selección sexual. En este artículo estudiamos la prevalencia, el efecto y el papel potencial en procesos de selección sexual del protozoario Trypanosoma evansi sobre el capibara (chigüire o carpincho), Hydrochoerus hydrochaeris. Recolectamos las muestras en una finca ganadera en Venezuela donde se lleva a cabo la matanza anual de capibaras para aprovechar su carne. Usamos el volumen de la glándula del hocico (el "morrillo") como indicador de dominancia; los residuales del peso como indicadores de condición física; y los residuales del peso del bazo como indicadores de la función inmunológica. La prevalencia total fue de 30.9 % (N= 97) y no encontramos diferencia entre machos y hembras ni tampoco detectamos correlación entre estado de infección y condición física. Sin embargo, encontramos que los animales infectados tenían el bazo inflamado, lo que indica un costo inmunológico de la infección. Además los machos con morrillos más grandes (más dominantes) tendían a estar menos infectados que los machos con morrillos más pequeños (subordinados), lo cual sugiere que al escoger machos con morrillos grandes, las hembras pueden estar escogiendo machos saludables, lo cual es consistente con la visión "buenos genes" de la selección sexual.

Tripanosomiasis bovina en ganadería lechera de trópico alto: primer informe de Haematobia irritans como principal vector de T. vivax y T. evansi en Colombia

La tripanosomiasis bovina es una enfermedad hemoparasitaria transmitida en Latinoamérica principalmente por moscas picadoras de la familia Tabanidae. El objetivo del estudio fue evaluar la infección por Trypanosoma vivax y Trypanosoma evansi en ganadería bovina especializada en producción de leche en una hacienda y sus potenciales vectores. Se realizó un estudio parasitológico y entomológico directo por técnicas de microscopia y reacción en cadena de la polimerasa (PCR) con dos marcadores moleculares para diferenciar especies de Trypanosoma en muestras de sangre de bovinos y moscas. La frecuencia de infección por Trypanosoma vivax y Trypanosoma evansi en bovinos fue de 3,6 y 0 %, respectivamente. La caracterización de vectores muestra a Haematobia irritans como la mosca más frecuente en la zona de estudio (97,1 %), seguida de Stomoxys calcitrans (2,8 %). No se identificaron tabánidos. Se encontró T. vivax y T. evansi en probóscide y toráx-abdomen de las moscas picadoras Haematobia irritans y Stomoxys calcitrans, lo que representa un comportamiento epizoótico atípico al que sucede en países de Suramérica. Por su alta densidad poblacional, se sugiere la mosca Haematobia irritans como el principal potencial vector.

Bovine trypanosomiasis is a hemoparasitic disease transmitted in Latin America mainly by biting flies of the family Tabanidae. The study aimed to evaluate infection by Trypanosoma vivax and Trypanosoma evansi in cattle specialized in milk production on a farm and their potential vectors. A direct parasitological and entomological study was performed using microscopy techniques and polymerase chain reaction (PCR) with two molecular markers to differentiate Trypanosoma species in blood samples of cattle and flies. Infection frequency with Trypanosoma vivax and Trypanosoma evansi in cattle was 3.6 and 0%, respectively. Characterization of vectors shows Haematobia irritans as the most frequent fly in the study area (97.1%), followed by Stomoxys calcitrans (2.8%). No horseflies were identified. T. vivax and T. evansi were found in proboscis and thorax-abdomen of biting flies Haematobia irritans and Stomoxys calcitrans, representing an epizootic behavior, atypical in South American countries. Due to its high population density, it is suggested that the Haematobia irritans fly is the main potential vector.

A tripanossomíase bovina é uma doença causada por hemoparasitas e transmitida na América Latina principalmente por moscas picadoras da família Tabanidae. O objetivo do estudo foi avaliar a infecção por Trypanosoma vivax e Trypanosoma evansi em gado bovino especializada em produção de leite em uma fazenda e seus potenciais vectores. Se realizou um estudo parasitológico e entomológico direto por técnicas de microscopia e reação em cadeia da polimerase (PCR) com dois marcadores moleculares para diferenciar espécies de Typanosoma em amostras de sangue de bovinos e moscas. A frequência de infecção por Trypanosoma vivax e Trypanosoma evansi em bovinos foi de 3,6 e 0 %, respectivamente. A caracterização de vetores mostra a Haematobia irritans como a mosca mais frequente na zona de estudo (97,1 %), seguida de Stomoxys calcitrans (2,8 %). Não se identificaram tabanídeos. Se encontrou T. vivax e T. evansi em proboscídea e tórax-abdômen das moscas picadoras Haematobia irritans e Stomoxys calcitrans, o que representa um comportamento epizoótico atípico ao que sucede em países da América do Sul. Por sua alta densidade populacional, se sugere a mosca Haematobia irritans como o principal potencial vetor.

Tripanosomiasis bovina en ganadería lechera de trópico alto: primer informe de Haematobia irritans como principal vector de T. vivax y T. evansi en Colombia / Bovine trypanosomiasis in dairy farming in the high tropics: First report of Haematobia irritans as the main vector for T. vivax and T. evansi in Colombia / Tripanossomíase bovina em gado leiteiro de trópico alto: primeiro relatório de Haematobia irritans como principal vetor de T. vivax e T. evansi na Colômbia

Resumen: La tripanosomiasis bovina es una enfermedad hemoparasitaria transmitida en Latinoamérica principalmente por moscas picadoras de la familia Tabanidae. El objetivo del estudio fue evaluar la infección por Trypanosoma vivax y Trypanosoma evansi en ganadería bovina especializada en producción de leche en una hacienda y sus potenciales vectores. Se realizó un estudio parasitológico y entomológico directo por técnicas de microscopia y reacción en cadena de la polimerasa (PCR) con dos marcadores moleculares para diferenciar especies de Trypanosoma en muestras de sangre de bovinos y moscas. La frecuencia de infección por Trypanosoma vivax y Trypanosoma evansi en bovinos fue de 3,6 y 0 %, respectivamente. La caracterización de vectores muestra a Haematobia irritans como la mosca más frecuente en la zona de estudio (97,1 %), seguida de Stomoxys calcitrans (2,8 %). No se identificaron tabánidos. Se encontró T. vivax y T. evansi en probóscide y toráx-abdomen de las moscas picadoras Haematobia irritans y Stomoxys calcitrans, lo que representa un comportamiento epizoótico atípico al que sucede en países de Suramérica. Por su alta densidad poblacional, se sugiere la mosca Haematobia irritans como el principal potencial vector.

Abstract: Bovine trypanosomiasis is a hemoparasitic disease transmitted in Latin America mainly by biting flies of the family Tabanidae. The study aimed to evaluate infection by Trypanosoma vivax and Trypanosoma evansi in cattle specialized in milk production on a farm and their potential vectors. A direct parasitological and entomological study was performed using microscopy techniques and polymerase chain reaction (PCR) with two molecular markers to differentiate Trypanosoma species in blood samples of cattle and flies. Infection frequency with Trypanosoma vivax and Trypanosoma evansi in cattle was 3.6 and 0%, respectively. Characterization of vectors shows Haematobia irritans as the most frequent fly in the study area (97.1%), followed by Stomoxys calcitrans (2.8%). No horseflies were identified. T. vivax and T. evansi were found in proboscis and thorax-abdomen of biting flies Haematobia irritans and Stomoxys calcitrans, representing an epizootic behavior, atypical in South American countries. Due to its high population density, it is suggested that the Haematobia irritans fly is the main potential vector.

Resumo: A tripanossomíase bovina é uma doença causada por hemoparasitas e transmitida na América Latina principalmente por moscas picadoras da família Tabanidae. O objetivo do estudo foi avaliar a infecção por Trypanosoma vivax e Trypanosoma evansi em gado bovino especializada em produção de leite em uma fazenda e seus potenciais vectores. Se realizou um estudo parasitológico e entomológico direto por técnicas de microscopia e reação em cadeia da polimerase (PCR) com dois marcadores moleculares para diferenciar espécies de Typanosoma em amostras de sangue de bovinos e moscas. A frequência de infecção por Trypanosoma vivax e Trypanosoma evansi em bovinos foi de 3,6 e 0 %, respectivamente. A caracterização de vetores mostra a Haematobia irritans como a mosca mais frequente na zona de estudo (97,1 %), seguida de Stomoxys calcitrans (2,8 %). Não se identificaram tabanídeos. Se encontrou T. vivax e T. evansi em proboscídea e tórax-abdômen das moscas picadoras Haematobia irritans e Stomoxys calcitrans, o que representa um comportamento epizoótico atípico ao que sucede em países da América do Sul. Por sua alta densidade populacional, se sugere a mosca Haematobia irritans como o principal potencial vetor.

Molecular detection of Trypanosoma evansi (Kinetoplastida: Trypanosomatidae) in procyonids (Carnivora: Procyonidae) in Eastern Amazon, Brazil / Detecção molecular de Trypanosoma evansi (Kinetoplastida: Trypanosomatidae) em procionídeos (Carnivora: Procyonidae) na Amazônia Oriental, Brasil

ABSTRACT: The present study aimed to diagnose the natural infection of captive and free-living procyonids with Trypanosoma evansi in the states of Amapá and Pará, Brazil. From February 2012 to August 2013, whole blood samples and blood smears were obtained from 45 free-living procyonids and from nine procyonids kept in captivity in wild life refuges and zoobotanical parks in the states of Amapá and Pará. Whole blood samples were collected and kept at -20ºC for the detection of T. evansi DNA by PCR using the RoTat 1.2 forward and RoTat 1.2 reverse primers. In addition, the blood smears were processed and examined for the presence of trypomastigote forms of T. evansi. T. evansi DNA was detected in 18.52% (10/54) of the procyonids, namely, in captive crab-eating raccoons and captive and free-living coatis in Pará State. No trypomastigote forms were observed in the blood smears. DNA from T. evansi was detected in P. cancrivorus and N. nasua in Pará State, being this the first such report in P. cancrivorus.

RESUMO: O objetivo do presente trabalho foi realizar o diagnóstico da infecção natural por Trypanosoma evansi em procionídeos de vida livre e de cativeiro dos estados do Amapá e Pará, Brasil. Durante o período de fevereiro de 2012 a agosto de 2013, amostras de sangue total e esfregaços sanguíneos foram obtidos de 45 procionídeos de vida livre e de nove mantidos em cativeiro em mantenedores e Parques Zoobotânicos dos estados do Amapá e Pará. As amostras de sangue total foram coletadas e mantidas a -20ºC para pesquisa de DNA de T. evansi pela PCR utilizando-se os iniciadores RoTat 1.2 forward e RoTat 1.2 reverse. Os esfregaços sanguíneos também foram processados e examinados para a pesquisa de formas tripomastigotas do agente. DNA de T. evansi foi detectado em 18,52% (10/54) dos procionídeos, ocorrendo em mãos-peladas de cativeiro e quatis de vida livre e de cativeiro no estado do Pará. Não foram observadas formas tripomastigotas nos esfregaços sanguíneos. DNA de T. evansi foi detectado em P. cancrivorus e N. nasua no estado do Pará, sendo este o primeiro relato em P. cancrivorus.

Achillea fragrantissima, rich in flavonoids and tannins, potentiates the activity of diminazine aceturate against Trypanosoma evansi in rats

OBJECTIVE@#To evaluate activity of methanol extract of Achillea fragrantissima (meth) (A. fragrantissima) alone or in combination with diminazine aceturate (DA) against Trypanosoma evansi (T. evansi) in experimentally infected rats.@*METHODS@#Sixty adult male Wister albino rats were divided equally into 6 groups (A-F). Rats in groups A-E were experimentally infected with T. evansi and those in group F were uninfected. The groups were treated respectively as follows: group A-with 3.5 mg/kg DA; group B- with 1000 mg/kg meth A. fragrantissima; group C-3.5 mg/kg DA plus 500 mg/kg meth A. fragrantissima; group D-3.5 mg/kg DA plus 1000 mg/kg meth A. fragrantissima. Group E was left untreated. Parasitemia, survivability, packed cell volume, hemoglobin concentration, total leucocytes count, lymphocyte count, and serum malondialdehyde and reduced glutathione (GSH) levels were estimated. Phytochemical screening of meth A. fragrantissima was also performed.@*RESULTS@#The phytochemical analysis of the meth A. fragrantissima indicated a higher content from polyphenolic tannins and non tannins and flavonoids. The efficacy percentage against trypanosomiasis in groups A - E was respectively as follows 80, 40, 90, 100, 0. The administration of meth-A. fragrantissima (1000 mg/kg b.wt.) produced a moderate efficacy against trypanosomiasis. Untreated rats in group E died between 25 and 30 d post infection. The rats given DA and meth A. fragrantissima combinations (C and D) showed faster and higher recovery rates than the uninfected control and groups A and B. The initial reduction in packed cell volume, hemoglobin, total leucocytes count, increases in serum malondialdehyde and decreases in GSH levels were reversed by the treatments.@*CONCLUSION@#The administration of the methanol extracts of A. fragrantissima and DA combination therapy was more effective than each product alone in the treatment of rats infected with T. evansi and further studies are required to isolate more active ingredients.

Achillea fragrantissima, rich in flavonoids and tannins, potentiates the activity of diminazine aceturate against Trypanosoma evansi in rats

Objective: To evaluate activity of methanol extract of Achillea fragrantissima (meth) (A. fragrantissima) alone or in combination with diminazine aceturate (DA) against Trypanosoma evansi (T. evansi) in experimentally infected rats. Methods: Sixty adult male Wister albino rats were divided equally into 6 groups (A-F). Rats in groups A-E were experimentally infected with T. evansi and those in group F were uninfected. The groups were treated respectively as follows: group A-with 3.5 mg/kg DA; group B- with 1. 000 mg/kg meth A. fragrantissima; group C-3.5 mg/kg DA plus 500 mg/kg meth A. fragrantissima; group D-3.5 mg/kg DA plus 1. 000 mg/kg meth A. fragrantissima. Group E was left untreated. Parasitemia, survivability, packed cell volume, hemoglobin concentration, total leucocytes count, lymphocyte count, and serum malondialdehyde and reduced glutathione (GSH) levels were estimated. Phytochemical screening of meth A. fragrantissima was also performed. Results: The phytochemical analysis of the meth A. fragrantissima indicated a higher content from polyphenolic tannins and non tannins and flavonoids. The efficacy percentage against trypanosomiasis in groups A - E was respectively as follows 80, 40, 90, 100, 0. The administration of meth-A. fragrantissima (1. 000 mg/kg b.wt.) produced a moderate efficacy against trypanosomiasis. Untreated rats in group E died between 25 and 30 d post infection. The rats given DA and meth A. fragrantissima combinations (C and D) showed faster and higher recovery rates than the uninfected control and groups A and B. The initial reduction in packed cell volume, hemoglobin, total leucocytes count, increases in serum malondialdehyde and decreases in GSH levels were reversed by the treatments. Conclusion: The administration of the methanol extracts of A. fragrantissima and DA combination therapy was more effective than each product alone in the treatment of rats infected with T. evansi and further studies are required to isolate more active ingredients.

Oxidative Stress in the Heart of Rats Infected with Trypanosoma evansi

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.

Development of an enzyme immunoassay using recombinant invariant surface glycoprotein (rISG) 75 for serodiagnosis of bovine trypanosomosis.

Trypanosomosis or surra is caused by the haemoflagellate parasite, Trypanosoma evansi and is an important disease of animals, including domestic and wild herbivores and carnivores, in tropical countries. The invariant surface glycoproteins (ISGs) are blood stream stage specific and are uniformly distributed over the entire surface of the trypanosomes. In the present study, the extracellular domain (ED) region of ISG-75 from T. evansi, consisting of 1320 nt, encoding a polypeptide of 440 amino acids, has been heterologously expressed in Escherichia coli. Further, the immunoreactivity of recombinant ISG-75 (rISG-75) was characterized in immunoblot and ELISA using T. evansi hyper immune sera raised in experimental animals. The protein was found immunoreactive when compared with a panel of antigens (VSG RoTat 1.2 and whole cell lysate) using bovine serum samples from field. The diagnostic potential of rISG-75 was evaluated in ELISA with large number of bovine field serum samples. The optimum sensitivity and specificity were 98.47 and 99.1, respectively. The present finding showed that the expressed protein has potential use in the serodiagnosis of trypanosomosis.

Hematological and serum biochemical aspects associated with a camel (Camelus dromedarius) naturally infected by Trypanosoma evansi with severe parasitemia in Semnan, Iran

Objective: To determine the presence of Trypanosoma evansi (T. evansi) and the effect of trypanosomosis on hemato-biochemical profile of dromedary camels in Semnan, Iran, which has not been reported yet. Methods:To perform this project, blood samples were collected by venipuncture into plain and EDTA-K2-containing vacutainer tubes from 21 dromedary camels (12 males and 9 females) aged 3-18 years, from 4 different regions of Semnan. Results: Microscopic examination of stained thin blood smears revealed the presence of T. evansi in one of the samples. However, it should be noted that this sample showed a very high parasitemia (more than 5 trypomastigote were visible per microscopic field with MGG, 1 000×). This heavy parasitemia was associated with an 18-year-old female camel that showed symptoms of corneal opacity, intense emaciation and pale mucous membranes. Comparison of hematologyical and serum biochemical profiles between the camel infected by T. evansi and uninfected camels indicated anemia, leukocytosis, hyperproteinemia, hypoalbuminemia, hyperglobulinemia, reduction A/G ratio, increasedα1,βand globulins and decreased ofα2 globulins and increased the concentration of gamma-glutamyl transferase enzyme. Conclusions: Results of the present study revealed that trypanosomosis was present in dromedary camels of Semnan, Iran (infection rate is 4.76%) and hemato-biochemical parameters were markedly affected by camel trypanosomosis.

Hematological and serum biochemical aspects associated with a camel (Camelus dromedarius) naturally infected by Trypanosoma evansi with severe parasitemia in Semnan, Iran

Objective: To determine the presence of Trypanosoma evansi (T. evansi) and the effect of trypanosomosis on hemato-biochemical profile of dromedary camels in Semnan, Iran, which has not been reported yet. Methods: To perform this project, blood samples were collected by venipuncture into plain and EDTA-K2-containing vacutainer tubes from 21 dromedary camels (12 males and 9 females) aged 3-18 years, from 4 different regions of Semnan. Results: Microscopic examination of stained thin blood smears revealed the presence of T. evansi in one of the samples. However, it should be noted that this sample showed a very high parasitemia (more than 5 trypomastigote were visible per microscopic field with MGG, 1 000×). This heavy parasitemia was associated with an 18-year-old female camel that showed symptoms of corneal opacity, intense emaciation and pale mucous membranes. Comparison of hematologyical and serum biochemical profiles between the camel infected by T. evansi and uninfected camels indicated anemia, leukocytosis, hyperproteinemia, hypoalbuminemia, hyperglobulinemia, reduction A/G ratio, increased α

In Vitro Trypanocidal Activity of Macela (Achyrocline satureioides) Extracts against Trypanosoma evansi

The aim of this study was to verify the trypanocidal effectiveness of aqueous, methanolic, and ethanolic extracts of Achyrocline satureioides against Trypanosoma evansi in vitro. A. satureioides extracts, known as macela, were used on trypomastigotes at different concentrations (1, 5, 10, 50, 100, 500, and 1,000 microg/ml) and exposure times (0, 1, 3, 6, and 9 hr). A dose-dependent effect was observed when the 3 extracts were tested. The concentrations of 1, 5, and 10 microg/ml were not able to kill trypomastigotes until 3 hr after exposure, and the highest concentrations (500 and 1,000 microg/ml) were able to kill all trypomastigotes after 1 hr. When the time of exposure was increased up to 9 hr, the concentrations at 50 and 100 microg/ml were 100% effective to 3 extracts. The chemical analysis of the extracts revealed the presence of flavonoids, a trypanocidal compound already described. Based on the results, we can conclude that the A. satureioides extracts exhibit trypanocidal effects.

Saponins-rich fraction of Calotropis procera leaves elicit no antitrypanosomal activity in a rat model

Objective:To examine the in vitro and in vivo anti-Trypanosoma evansi (T. evansi ) activity of saponins-rich fraction of Calotropis procera (cpsf) leaves as well as the effect of the fraction on the parasite-induced anemia. Methods:A 60-minutes time course experiment was conducted with various concentrations of the fraction using a 96-well microtiter plate technique, and subsequently used to treat experimentally T. evansi infected rats at 100 and 200 mg/kg body weight. Index of anemia was analyzed in all animals during the experiment. Results:The cpsf did not demonstrate an in vitro antitrypanosomal activity. Further, the cpsf treatments did not significantly (P>0.05) keep the parasites lower than the infected untreated groups. At the end of the experiment, all T. evansi infected rats developed anemia whose severity was not significantly (P>0.05) ameliorated by the cpsf treatment. Conclusions:It was concluded that saponins derived from Calotropis procera leaves could not elicit in vitro and in vivo activities against T. evansi.

Apoptotic-like activity of staurosporine in axenic cultures of Trypanosoma evansi / Actividad proapoptótica de la estaurosporina en cultivos axénicos de Trypanosoma evansi

Trypanosoma evansi is a blood protozoan parasite of the genus Trypanosoma which is responsible for surra (Trypanosomosis) in domestic and wild animals. This study addressed apoptotic-like features in Trypanosoma evansi in vitro. The mechanism of parasite death was investigated using staurosporine as an inducing agent. We evaluated its effects through several cytoplasmic features of apoptosis, including cell shrinkage, phosphatidylserine exposure, maintenance of plasma membrane integrity, and mitochondrial trans-membrane potential. For access to these features we have used the flow cytometry and fluorescence microscopy with cultures in the stationary phase and adjusted to a density of 10(6) cells/mL. The apoptotic effect of staurosporine in T. evansi was evaluated at 20 nM final concentration. There was an increase of phosphatidylserine exposure, whereas mitochondrial potential was decreased. Moreover, no evidence of cell permeability increasing with staurosporine was observed in this study, suggesting the absence of a necrotic process. Additional studies are needed to elucidate the possible pathways associated with this form of cell death in this hemoparasite.

Trypanosoma evansi es un hemoparásito, el cual es el agente causal de la surra (tripanosomiasis) en mamíferos, perteneciente al orden Kinetoplastidae. Este estudio se oriento a caracterizar la muerte celular similar a apoptosis en cultivos in vitro de Trypanosoma evansi a través del uso del inductor esturosporina. Este efecto se evaluó a través de diversos aspectos fenotípicos de la apoptosis: el encogimiento celular, la exposición de fosfatidilserina, el mantenimiento de la integridad de la membrana plasmática y el potencial de membrana mitocondrial. Para evaluar estas características se utilizaron técnicas de citometría de flujo y microscopía de fluorescencia con cultivos en fase estacionaria ajustados a una densidad de 10(6) células/mL. El efecto apoptótico de la estaurosporina en Trypanosoma evansi fue evaluado a una concentración de 20 nM. Se evidenció un aumento de la exposición a fosfatidilserina, mientras que el potencial mitocondrial disminuyó. Por otra parte, no hay evidencias de aumento de la permeabilidad celular con estaurosporina, sugiriendo la ausencia de un proceso necrótico. Estudios adicionales son necesarios para dilucidar las posibles vías asociadas con esta forma de muerte celular en este hemoparásito.

Trypanocidal activity of human plasma on Trypanosoma evansi in mice / Atividade tripanocida do plasma humano sobre Trypanosoma evansi em camundongos

This study aimed to test an alternative protocol with human plasma to control Trypanosoma evansi infection in mice. Plasma from an apparently 27-year-old healthy male, blood type A+, was used in the study. A concentration of 100 mg.dL-1 apolipoprotein L1 (APOL1) was detected in the plasma. Forty mice were divided into four groups with 10 animals each. Group A comprised uninfected animals. Mice from groups B, C and D were inoculated with a T. evansi isolate. Group B was used as a positive control. At three days post-infection (DPI), the mice were administered intraperitoneally with human plasma. A single dose of 0.2 mL plasma was given to those in group C. The mice from group D were administered five doses of 0.2 mL plasma with a 24 hours interval between the doses. Group B showed high increasing parasitemia that led to their death within 5 DPI. Both treatments eliminated parasites from the blood and increased the longevity of animals. An efficacy of 50 (group C) and 80% (group D) of human plasma trypanocidal activity was found using PCR. This therapeutic success was likely achieved in the group D due to their higher levels of APOL1 compared with group C.

Este estudo teve como objetivo testar um protocolo alternativo com plasma humano para controlar a infecção por Trypanosoma evansi em camundongos. O plasma foi oriundo de um homem aparentemente saudável, com idade entre 27 anos e tipo de sangue A+. Foi detectada uma concentração de 100 mg.dL -1 de apolipoproteína L1 (APOL1) no plasma. Quarenta camundongos foram divididos em quatro grupos, contendo dez animais cada. Grupo A, composto de animais não infectados. Os roedores dos grupos B, C e D foram inoculados intraperitonealmente com um isolado de T. evansi. O Grupo B foi usado como um controle positivo. Três dias pós-infecção (DPI), os camundongos foram tratados com plasma humano. Uma dose única de 0,2 mL de plasma foi administrada nos roedores do grupo C. Os ratos do grupo D receberam cinco doses de 0,2 mL de plasma em intervalos de 24 horas. Os ratos do grupo B apresentaram parasitemia crescente, o que ocasionou a morte dos animais em 5 DPI. Ambos os tratamentos foram capazes de eliminar o parasito do sangue e aumentar a longevidade dos animais. O método da PCR detectou uma eficácia de 50% (grupo C) e 80% (grupo D) no tratamento com plasma humano. Este sucesso terapêutico obtido nos animais do grupo D provavelmente foi por receber maiores níveis de APOL1, comparado ao grupo C.

Morfometría ultraestructural de la glándula adrenal de ratones infectados experimentalmente con dos aislados venezolanos de trypanosoma evansi / Ultrastructural morphometry of the adrenal gland of mice experimentally infected with two Venezuelan isolates of trypanosoma evansi

Este trabajo describe cualitativamente y analiza cuantitativamente las variaciones ultraestructurales de la glándula adrenal de ratones durante el desarrollo de infecciones murinas experimentales por dos aislados venezolanos de Trypanosoma evansi, uno proveniente de asno y otro de caballo. Las modificaciones submicroscópicas observadas incluyeron alteraciones en el retículo endoplasmático liso, mitocondrias, gotas lipídicas, aparato de Golgi y núcleo. También se apreciaron alteraciones en el número de vesículas de epinefrina, norepinefrina, gránulos de lipofucsina y presencia singular de figuras mielínicas. Estos cambios ultraestructurales estarían asociados a problemas metabólicos que inducirían la muerte de los animales infectados. El estudio cuantitativo demostró diferencias significativas en las características y las magnitudes del daño causado por ambos aislados, siendo el tripanosoma proveniente de caballo más patogénico. El análisis multivariante de los cambios submicroscópicos discriminó la acción patogénica en la glándula adrenal de tripanosomas de la misma especie que provienen de hábitats diferentes (Equus asinus y E. caballus) y de zonas geográficas distintas (estado Apure y estado Guárico).

This work describes qualitatively and analyzes quantitatively the ultrastructural variations of the mice adrenal gland during the development of murine experimental infections by two Venezuelan isolates of Trypanosoma evansi, one de rived from a donkey and another from a horse. The submicroscopic modifications included modifications in the smooth endoplasmic reticulum, mitochondria, lipid droplets, Golgi apparatus, and nucleus. Alterations in the number of epinephrine, norepinephrine vesicles, and lipofuscin granules and singular myelin-like figures were also observed. Such ultrastructural changes would be associated to metabolic problems including the infected animal’s death. The quantitative study demonstrated significant differences in the characteristics and magnitude of the damage caused by both isolates, being more pathogenic the horse derived trypanosome. The multivariate analysis of the submicroscopic changes discriminated the pathogenic action of trypanosomes of the same species coming from different habitats (Equus asinus and E. caballus) and different geographic zones (Apure state and Guarico state) in the adrenal gland.

Expresión de HSP60 en infecciones murinas experimentales por Trypanosoma evansi / HSP60 expression in experimental murine infections by Trypanosoma evansi

La inducción de la síntesis de proteínas de choque térmico ha sido relacionada con la defensa del organismo frente a infecciones diversas. Este trabajo examina la acumulación de Hsp60 en Mus musculus infectados experimentalmente con un aislado equinos de Trypanosoma evansi, agente etiológico de la “derrengadera” en los Llanos venezolanos. Ratones NMRI (♀; 20 gr de peso corporal), normales e inmunosuprimidos (ciclofosfamida; 250 mg/kg) fueron inoculados intradermicamente con 1 tripomastigote/gr de peso corporal. La acumulación de Hsp60 se determinó mediante Western blot con el uso de un anticuerpo monoclonal en muestras de plasma obtenidas cada cuatro días post-inoculación. Los niveles de parasitemia también fueron registrados. Se observa acumulación diferencial y variable de Hsp60 que se correlaciona la parasitemia y la condición inmune del hospedador. Los resultados sugieren un control inmunológico en la regulación de la expresión de Hsp60, cuyos patrones de expresión dependen de la condición fisiológica del hospedador y los niveles de parasitemia.

Heat shock protein induction has been related to organism defense in diverse kinds of infection. This investigation examines Hsp60 accumulation in Mus musculus infected ex - perimentally with Trypanosoma evansi isolated from horse. This parasite is the causal agent of the “derrengadera” in Venezuelan planes. Normal and immunosupressed (cyclophosphamide( 250 mg/kg).) NMRI mice (♂; 20 gr body weight) were intra-dermal inoculated with 1 trypomastigote/ gr body weight; control groups were injected with distillated water in the same conditions. Hsp60 accumulation was analyzed by Western blot with a monoclonal antibody in plasma samples obtained each four days post-inoculation. Parasitemia levels were also registered. We observed a differential accumulation of Hsp60, which correlates with pa - rasitemia and immune conditions of the host. These results suggest an immunological control of Hsp60 expression that depends on the physiological condition of the host and parasitemia.

Susceptibility of Mice to Trypanosoma evansi Treated with Human Plasma Containing Different Concentrations of Apolipoprotein L-1

The aim of this study was to test the susceptibility of mice to Trypanosoma evansi treated with human plasma containing different concentrations of apolipoprotein L-1 (APOL1). For this experiment, a strain of T. evansi and human plasma (plasmas 1, 2, and 3) from 3 adult males clinically healthy were used. In vivo test used 50 mice divided in 5 groups (A to E) with 10 animals in each group. Animals of groups B to E were infected, and then treated with 0.2 ml of human plasma in the following outline: negative control (A), positive control (B), treatment with plasma 1 (C), treatment with plasma 2 (D), and treatment with plasma 3 (E). Mice treated with human plasma showed an increase in longevity of 40.9+/-0.3 (C), 20+/-9.0 (D) and 35.6+/-9.3 (E) days compared to the control group (B) which was 4.3+/-0.5 days. The number of surviving mice and free of the parasite (blood smear and PCR negative) at the end of the experiment was 90%, 0%, and 60% for groups C, D, and E, respectively. The quantification of APOL1 was performed due to the large difference in the treatments that differed in the source plasma. In plasmas 1, 2, and 3 was detected the concentration of 194, 99, and 115 mg/dl of APOL1, respectively. However, we believe that this difference in the treatment efficiency is related to the level of APOL1 in plasmas.

Antigenic characterization of Trypanosoma evansi using sera from experimentally and naturally infected bovines, equines, dogs, and coatis / Caracterização antigênica do Trypanosoma evansi usando soros de bovinos, equinos, cães e quatis experimentalmente e naturalmente infectados

The present research investigated the presence of T. evansi antibodies in animals from the subregion of Nhecolandia, in the Pantanal Sul-mato-grossense, by means of an enzyme linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody test (IFAT), and the pattern of polypeptide recognition by sera from experimentally and naturally infected hosts using Western blotting. Serum samples were obtained from bovines (n = 102), horses (n = 98), and dogs (n = 55), and from 32 free-ranging coatis (Nasua nasua). None of the bovines were found positive, while sera from 16 dogs (29 percent) and 23 horses (23.4 percent) were positive by ELISA. Sera from 8 coatis (25 percent) were found positive using IFAT. Western blotting revealed major polypeptides of T. evansi with molecular weight ranging from 74 to 38 kDa. The polypeptides of 66, 48-46, and 38 kDa were identified by sera from experimentally infected bovines, donkeys, dogs, and coatis. The 48-46 and 38 kDa bands were mainly recognized in chronic phase of infection. The antigen with apparent molecular weight of 66 kDa, revealed by antibodies from all experimental animals, was also recognized in sera of horses and dogs from the Pantanal. The 48-46 kDa polypeptide was identified by antibodies from all naturally infected animals and must be further evaluated for use in specific diagnosis of T. evansi infection.

O trabalho de pesquisa investigou a presença de anticorpos anti- T. evansi em animais da sub-região da Nhecolândia, no Pantanal sul-mato-grossense, pelo ensaio imunoenzimático (ELISA) e a reação de imunofluorescência indireta (RIFI). O reconhecimento de polipeptídeos de T. evansi foi realizado pela técnica de "Western blotting", utilizando soros de animais experimentalmente e naturalmente infectados. As amostras de soro foram obtidas de bovinos (n = 102), cavalos (n = 98) e cães (n = 55) e de 32 quatis de vida livre (Nasua nasua) do pantanal mato-grossense. Todos os soros dos bovinos foram negativos, enquanto soros de 16 cães (29 por cento) e 23 cavalos (23,4 por cento) foram positivos pelo ELISA. Soros de oito quatis (25 por cento) foram positivos pela RIFI. Pelo "Western-blotting" foi possível revelar polipeptídeos de T. evansi, com peso molecular variando de 74 a 38 kDa. Os polipeptídeos de 66, 48-46 e 38 kDa foram identificados por soros de bovinos, cavalos, cães e quatis experimentalmente infectados. As bandas de 48-46 e 38 kDa foram reconhecidas principalmente na fase crônica da infecção. O antígeno com peso molecular aparente de 66 kDa, revelado por anticorpos de todos os animais experimentais, também foi reconhecido por soros de cavalos e cães do Pantanal. O polipeptídeo de 48-46 kDa foi identificado por anticorpos de todos os animais naturalmente infectados, devendo ser avaliado para o diagnóstico específico da infecção pelo T. evansi.