BACKGROUND Trypanosoma cruzi shows an exuberant genetic diversity. Currently, seven phylogenetic lineages, called discrete typing units (DTUs), are recognised: TcI-TcVI and Tcbat. Despite advances in studies on T. cruzi and its populations, there is no consensus regarding its heterogeneity. OBJECTIVES This study aimed to perform molecular characterisation of T. cruzi strains, isolated in the state of São Paulo, to identify the DTUs involved and evaluate their genetic diversity. METHODS T. cruzi strains were isolated from biological samples of chronic chagasic patients, marsupials and triatomines through culture techniques and subjected to molecular characterisation using the fluorescent fragment length barcoding (FFLB) technique. Subsequently, the results were correlated with complementary information to enable better discrimination between the identified DTUs. FINDINGS It was possible to identify TcI in two humans and two triatomines; TcII/VI in 19 humans, two marsupials and one triatomine; and TcIII in one human host, an individual that also presented a result for TcI, which indicated the possibility of a mixed infection. Regarding the strains characterised by the TcII/VI profile, the correlation with complementary information allowed to suggest that, in general, these parasite populations indeed correspond to the TcII genotype. MAIN CONCLUSIONS The TcII/VI profile, associated with domestic cycles and patients with chronic Chagas disease, was the most prevalent among the identified DTUs. Furthermore, the correlation of the study results with complementary information made it possible to suggest that TcII is the predominant lineage of this work.

Resumo Em bovinos, infecções por Trypanosoma vivax geram sinais clínicos inespecíficos que, associados a intervalos aparasitêmicos, faz com que o diagnóstico da enfermidade seja desafiador. No Brasil, somente aceturato de diaminazeno e cloridrato de isometamidum (ISM) estão disponíveis para o tratamento da tripanossomose bovina. Este trabalho teve como objetivo acompanhar bovinos leiteiros naturalmente infectados por T. vivax, após o tratamento com ISM por meio de técnicas moleculares e sorológica. Foram utilizados 30 bovinos naturalmente infectados com T. vivax, sendo estes tratados com duas aplicações de ISM, na dosagem de 1,0 mg/kg por via intramuscular profunda, nos dias 0 e 150. Foram avaliadas, para diagnóstico de T. vivax, amostras de sangue acrescido de EDTA e soro, colhidas nos 0, 7, 15, 30, 60, 90, 120, 150, 180, 210 e 240 dias após os tratamentos pela reação em cadeia da polimerase (PCR), amplificação circular isotérmica do DNA (LAMP) e ensaio de imunoabsorção enzimático (ELISA). Verificou-se a presença de animais com persistência na detecção de DNA de T. vivax pela PCR e LAMP, bem como detecção contínua de anticorpos IgG anti-T. vivax pelo método de ELISA, sugerindo a presença de resistência de T. vivax ao ISM. A combinação dos testes LAMP e ELISA pode evitar falsos diagnósticos da eliminação do parasita nos bovinos tratados, contribuindo para um melhor controle da doença. Este é o primeiro experimento que demonstra infecção persistente do T. vivax em rebanho naturalmente infectado, tratado com ISM, e evidencia possível resistência ao quimioterápico no Brasil.

Abstract Trypanosoma vivax infections cause nonspecific clinical signs in cattle associated with aparasitemic intervals, making disease diagnosis a challenge. In Brazil, diminazene aceturate and isometamidium chloride (ISM) are available to treat bovine trypanosomosis. The objective of this study was to follow-up, by molecular and serological techniques, dairy cattle naturally infected by T. vivax after ISM treatment. Thirty cattle naturally infected with T. vivax received two applications of ISM, at a dosage of 1.0 mg/kg intramuscularly, on days 0 and 150. For T. vivax diagnosis, EDTA-blood and serum samples were evaluated on 0, 7, 15, 30, 60, 90, 120, 150, 180, 210, and 240 days after treatment PCR, Loop-mediated isothermal amplification (LAMP) and ELISA. Animals with persistent detection of T. vivax DNA by both PCR and LAMP were found and continuous detection of anti-T. vivax IgG antibodies by ELISA, suggesting the presence of T. vivax resistance to ISM. The combination of LAMP and ELISA tests can prevent misdiagnosis of the parasite clearance in treated cattle, contributing to better disease control. This is the first experiment that demonstrates the persistence infection of T. vivax under ISM treatment in a natural infected herd and evidence of ISM chemotherapy-resistant T. vivax in Brazil.

Resumo A tripanossomíase, causada por Trypanosoma vivax, é responsável por grandes perdas econômicas na bovinocultura da África e da América do Sul. Durante seu ciclo de vida, o parasita pode apresentar diferentes características morfológicas, metabólicas e fisiológicas em função das interações que ele encontra em cada ponto do seu ciclo. Embora o T. vivax seja reportado, frequentemente, na circulação dos seus hospedeiros mamíferos, o protozoário tem a capacidade de migrar para os tecidos desses indivíduos. Entretanto, essa característica é pobremente conhecida. Neste contexto, o objetivo foi verificar a presença, assim como as alterações causadas pelo T. vivax nos diferentes tecidos de caprinos experimentalmente infectados. Apesar dos animais não terem sido perfundidos antes da coleta dos tecidos, utilizando-se diferentes abordagens, foi evidenciada a presença do T. vivax em diferentes amostras teciduais, incluindo no tecido adiposo e pele dos animais infectados. Além disso, foi observada reação inflamatória mononuclear, caracterizada majoritariamente por infiltrado de linfócitos, plasmócitos e macrófagos. Os resultados evidenciam a possibilidade de que, assim como outros tripanossomatídeos, T. vivax pode usar esses tecidos durante o seu ciclo de vida. São necessários futuros estudos, objetivando elucidar o período em que o T. vivax permanece ativo nesses sítios, se ele utiliza esses locais como refúgio das drogas tripanocidas, e se ele é capaz de recolonizar a circulação sanguínea.

Abstract Trypanosomiasis, caused by Trypanosoma vivax, is responsible for great economic losses among livestock in Africa and South America. During the life cycle of these parasites, they may present different morphological, metabolic and physiological characteristics depending on the interactions that are encountered at each point of their life cycle. Although T. vivax is frequently reported in the circulation of its mammalian hosts, it has the ability to migrate to the tissues of these individuals. However, this characteristic is poorly understood. In this context, we aimed to investigate the presence of T. vivax and the changes caused in different tissues of experimentally infected goats. Despite the animals were not perfused before tissues collection, using different approaches, we demonstrated its presence in different samples, including in the adipose tissue and skin of infected animals. In addition, a mononuclear inflammatory reaction, mostly characterized by an infiltrate of lymphocytes, plasma cells and macrophages were observed. The results highlight the possibility that, like other trypanosomatids, T. vivax may use these tissues during its life cycle. Future studies aiming to elucidate the length of time for which T. vivax remains active in these sites, and whether it uses these sites as a refuge from trypanocidal drugs, and whether it is capable of recolonizing the blood circulation, are much needed.

Abstract: INTRODUCTION: To characterize Trypanosoma cruzi (TcI) isolated from a Panstrongylus megistus specimen found in one of the biggest metropolitan areas of Latin America, the relationship between the TcI group of T. cruzi and the transmission cycle in the urban environment was studied. METHODS: The T. cruzi strain, Pm, was isolated in a culture medium from the evolutionary forms present in the hindgut of a live male specimen of P. megistus found in the Jabaquara subway in São Paulo City. The sample from the triatomine showed trypomastigote forms of Trypanosomatidae, which were inoculated in the peritoneum of Balb/c mice. The sample was then inoculated in Liver Infusion Tryptose medium and J774 cells for the molecular identification and characterization of the parasite. The Pm strain of T. cruzi was identified by isolation in axenic culture medium, and based on the morphology, cell infection, growth kinetics, and molecular characterization. RESULTS: After isolation, the protozoan was identified as T. cruzi. No parasites were detected in the peripheral blood of the animal, which can be a characteristic inherent to the strain of T. cruzi that was isolated. Cell invasion assays were performed in triplicate in the J774 cell line to confirm the invasive ability of the Pm strain and revealed amastigote forms of the parasite within macrophages. CONCLUSIONS: Our biological and molecular characterizations helped understand parasite-host interactions and their evolutionary history in context of the associations between vectors, ecotopes, hosts, and groups of the parasite.

Esta é a primeira descrição de um surto de Trypanosomavivax ocorrido no Estado de São Paulo, no município de Lins. Animais acometidos apresentaram febre, icterícia, diminuição da produção de leite, perda de peso, diarreia profusa, abortos, anemia, leucocitose e hiperfibrigenemia. Foram registrados 31 óbitos de vacas e bezerros em 1.080 bovinos no total. Três vacas apresentaram sintomatologia nervosa, como dismetria, ataxia e fraqueza muscular, além de ptialismo, aumento de linfonodos e edema submandibular. Hemoparasitas flagelados foram observados em esfregaços sanguíneos, e a espécie de tripanossomo foi diagnosticada como T.vivax por PCR. A cepa de T. vivax mostrou ser resistente ao tratamento com aceturato de diaminozeno e a infecção disseminou rapidamente no rebanho. Pelo ELISA, observou-se que 98,36% (599) das amostras de soro colhidas apresentaram títulos positivos para IgG anti-T.vivax. O surto ocorreu em condições de baixa precipitação pluviométrica, fato que indica que outros fatores estavam envolvidos na ocorrência desse surto, como a ausência de tabanídeos e a grande presença de Haematobia irritans e Stomoxys calcitrans, cujo aumento populacional pode ser devido ao uso de resíduos de usinas de açúcar e álcool nos canaviais que circundavam a granja leiteira.

This is the first description of a Trypanosoma vivax outbreak in the state of São Paulo (municipality of Lins). Fever, jaundice, decreased milk production, weight loss, profuse diarrhea, abortion, anemia, leukocytosis and hyperfibrinogenemia were observed in the affected animals. Thirty-one cows and calves died out of a total of 1080 in the herd. Three cows showed neurological symptoms like dysmetria, ataxia, muscle weakness, ptyalism, lymph node enlargement and submandibular edema. Flagellated hemoparasites were observed in blood smears. The species was diagnosed as T. vivax by means of PCR. This T.vivax strain showed resistance to diaminazene aceturate and the infection spread quickly at the herd. From the ELISA test, 599 serum samples (98.36%) were positive for anti-T.vivax IgG antibodies. This outbreak occurred during a very dry period, which indicates that other factors were involved in the outbreak, such as absence of tabanids and large populations of Haematobia irritans and Stomoxys calcitrans. The increases in these populations may have been due to the use of biosolid waste from sugar and ethanol plants in the sugarcane plantations surrounding the dairy farm.

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.

This study aimed at reporting the occurrence of Trypanosoma vivax in southern Brazil. The protozoan was diagnosed through peripheral blood smear evaluation of a bovine and confirmed by the evaluation of the trypomastigote forms morphology and by the Polimerase Chain Reaction (PCR). The animal showed clinical signs similar to the nervous form of the infection by T. vivax. Negative results for T. vivax were found in other bovines grazing in the same paddock. This is the first report of T. vivax in the state of Rio Grande do Sul and in the southern region of Brazil.