We have previously shown that the subunit 1 of Leishmania amazonensis RPA (LaRPA-1) alone binds the G-rich telomeric strand and is structurally different from other RPA-1. It is analogous to telomere end-binding proteins described in model eukaryotes whose homologues were not identified in the protozoan´s genome. Here we show that LaRPA-1 is involved with damage response and telomere protection although it lacks the RPA1N domain involved with the binding with multiple checkpoint proteins. We induced DNA double-strand breaks (DSBs) in Leishmania using phleomycin. Damage was confirmed by TUNEL-positive nuclei and triggered a G1/S cell cycle arrest that was accompanied by nuclear accumulation of LaRPA-1 and RAD51 in the S phase of hydroxyurea-synchronized parasites. DSBs also increased the levels of RAD51 in non-synchronized parasites and of LaRPA-1 and RAD51 in the S phase of synchronized cells. More LaRPA-1 appeared immunoprecipitating telomeres in vivo and associated in a complex containing RAD51, although this interaction needs more investigation. RAD51 apparently co-localized with few telomeric clusters but it did not immunoprecipitate telomeric DNA. These findings suggest that LaRPA-1 and RAD51 work together in response to DNA DSBs and at telomeres, upon damage, LaRPA-1 works probably to prevent loss of single-stranded DNA and to assume a capping function.

The purpose of the present work was a comparative study of diagnostic methods for Canine Visceral Leishmaniasis (CVL) using serological methods, enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT), histochemical (HE) and immunohistochemical (IMHC) tests using spleen, lymph node and liver canine tissues. In addition, Polymerase Chain Reaction (PCR) was done in blood and in tissues in order to compare and confirm no conclusive and negative diagnosis by the methods above. For this study, 34 dogs were divided according to clinical signs in asymptomatic, oligosymptomatic and polisymptomatic Leishmania-infected dogs euthanized by Zoonotic Disease Control Center (CCZ) from Ilha Solteira, SP, Brazil. The positivism indexes of ELISA, IMHC, IFAT and HE were 65.0, 62.0, 56.0 and 56.0%, respectively with the highest numbers of positive dogs in polisymptomatic (92.0%) followed by oligosymptomatic (57.0%) and asymptomatic dogs (12.5%). Furthermore, PCR confirmed the positive results and detected DNA in tissues from 100% of negative dogs and 89.0% suspects raising the animal positivism index up to 97.0%. In conclusion, PCR was the most sensitive and a valuable method for a definitive CVL diagnosis.

BackgroundTelomeres are specialized structures at the end of chromosomes essential for maintaining genome stability and cell viability. The importance of telomeric proteins for telomere maintenance has increased our interest in the identification of homologues within the genus Leishmania. The mammalian TRF1 and TRF2 proteins, for example, bind double-stranded telomeres via a Myb-like DNA-binding domain and are involved with telomere length regulation and chromosome end protection. In addition, TRF2 can modulate the activity of several enzymes and influence the conformation of telomeric DNA. In this work, we identified and characterized a Leishmania protein (LaTRF) homologous to both mammalian TRF1 and TRF2.ResultsLaTRF was cloned using a PCR-based strategy. ClustalW and bl2seq sequence analysis showed that LaTRF shared sequence identity with the Trypanosoma brucei TRF (TbTRF) protein and had the same degree of sequence similarities with the dimerization (TRFH) and the canonical DNA-binding Myb-like domains of both mammalian TRFs. LaTRF was predicted to be an 82.5 kDa protein, indicating that it is double the size of the trypanosome TRF homologues. Western blot and indirect immunofluorescence combined with fluorescence in situ hybridization showed that LaTRF, similarly to hTRF2, is a nuclear protein that also associates with parasite telomeres. Native and full length LaTRF and a mutant bearing the putative Myb-like domain expressed in bacteria bound double-stranded telomeric DNA in vitro. Chromatin immunoprecipitation showed that LaTRF interacted specifically with telomeres in vivo.ConclusionThe nuclear localization of LaTRF, its association and co-localization with parasite telomeres and its high identity with TbTRF protein, support the hypothesis that LaTRF is a Leishmania telomeric protein.

ResumoO objetivo da presente pesquisa foi avaliar comparativamente os métodos diagnósticos da Leishmaniose Visceral Canina (LVC), utilizando-se o ensaio imunoenzimático indireto (ELISA), a reação de imunofluorescência indireta (RIFI), a histoquímica (HE) e a imunoistoquímica (IMIQ) em tecidos de órgãos, como o baço, linfonodo e fígado. Além disso, a Reação em Cadeia pela Polimerase (PCR) das amostras de sangue e dos tecidos foi utilizada para comparar e confirmar os diagnósticos negativos e não conclusivos pelos métodos acima. Para esse estudo, foram utilizados 34 cães com diferentes sintomas da LVC, classificados em polissintomáticos, oligossintomáticos e assintomáticos e eutanasiados no Centro de Controle de Zoonoses (CCZ) de Ilha Solteira, SP. Os índices de positividade para os testes ELISA, IMIQ, RIFI e HE foram de 65,0, 62,0, 56,0 e 56,0%, respectivamente, sendo a maior positividade detectada nos cães polissintomáticos (92,0%), seguida pelos oligossintomáticos (57,0%) e assintomáticos (12,5%). A PCR confirmou os resultados positivos pelas outras técnicas e ainda detectou DNA do parasita nos tecidos de 100% dos cães negativos e em 89,0% dos suspeitos, elevando para 97,0% a positividade. Em conclusão, a PCR demonstrou ser o método mais sensível e preciso para o diagnóstico definitivo da LVC.Palavras-chave: Leishmania (L.) chagasi, imunoistoquímica, ELISA, RIFI, PCR. AbstractThe purpose of the present work was a comparative study of diagnostic methods for Canine Visceral Leishmaniasis (CVL) using serological methods, enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT), histochemical (HE) and immunohistochemical (IMHC) tests using spleen, lymph node and liver canine tissues. In addition, Polymerase Chain Reaction (PCR) was done in blood and in tissues in order to compare and confirm no conclusive and negative diagnosis by the methods above. For this study, 34 dogs were divided according to clinical signs in asymptomatic, oligosymptomatic and polisymptomatic Leishmania-infected dogs euthanized by Zoonotic Disease Control Center (CCZ) from Ilha Solteira, SP, Brazil. The positivism indexes of ELISA, IMHC, IFAT and HE were 65.0, 62.0, 56.0 and 56.0%, respectively with the highest numbers of positive dogs in polisymptomatic (92.0%) followed by oligosymptomatic (57.0%) and asymptomatic dogs (12.5%). Furthermore, PCR confirmed the positive results and detected DNA in tissues from 100% of negative dogs and 89.0% suspects raising the animal positivism index up to 97.0%. In conclusion, PCR was the most sensitive and a valuable method for a definitive CVL diagnosis.

SUMMARYIn Brazil, domestic dogs are branded as the primary reservoir for zoonotic visceral leishmaniasis, due to the clear positive correlation observed between human and canine infection rates. This study aimed to carry out a serological survey of canine visceral leishmaniasis (CVL) in dogs housed at a public kennel in the municipality of Juiz de Fora, Minas Gerais State, Brazil, using the immunochromatographic TR DPP ® CVL rapid test. Additionally, conventional and/or real time PCR assay was used to detect and confirm L. infantum infection in the DPP positive dogs only. Of the 400 dogs studied, most did not present clinical signs for CVL (p < 0.05), and fifteen (3.8%) were seropositive in the DPP test. There was no statistically significant difference between the DPP seropositive dogs and the clinical signs of the disease (p > 0.05). Both conventional and real time PCR tests confirmed L. infantum infection in nine (75.0%) of the twelve DPP seropositive dogs that remained alive during the follow-up period. This study is the first seroepidemiologic survey of CVL held in the city of Juiz de Fora, and the results reinforce the idea that this disease is currently in a process of expansion and urbanization in Brazil. Furthermore, this study highlights the use of the DPP test as an alternative for diagnosing CVL in large and mid-sized cities, due to its ease of implementation. KEYWORDS:Canine visceral leishmaniasis; Serological diagnosis; Immunochromatographic diagnosis; DPP; PCR diagnosis. INTRODUCTIONVisceral leishmaniasis (VL) is a potentially fatal protozoan vectorborne disease caused by the Leishmania donovani complex, and represents a serious risk to public health. This disease is endemic in 88 countries with approximately 0.2 to 0.4 million cases each year in Europe, South America, Africa and Asia (ALVAR et al., 2012). Anthroponotic transmission caused by L. donovani is present in the Indian subcontinent and in Central Africa, and zoonotic transmission caused by L. infantum is present in the Americas, Mediterranean basin, Middle East and Central Asia, and parts of Africa (HARHAY et al., 2011, PALATNIK-DE-SOUSA & DAY, 2011.Among the countries of the Americas, Brazil accounts for the highest number of human VL cases and is the third largest VL focus globally (ALVAR et al., 2012;BELO et al., 2013). Despite having presented a typical rural and wild pattern of VL until the 80's (WERNECK, 2008), current data show that the numbers have increased considerably, thus creating a new epidemiological profile, due to the spread of the disease to urban centers of the North, South and West regions of the country (JERONIMO et al., 2004;ROMERO & BOELAERT, 2010;HARHAY et al., 2011). In Brazil, domestic dogs are the main reservoir, and thus play an important role in the epidemiology of the disease, while foxes and other wild animals play a role in sylvatic transmission (DEANE, 1956;QUINNELL et al., 1997;ROMERO & BOELAERT, 2010). There is a clear positive correlation between human and canine infection rates, so that diagnosing dog...

This study was about a semi-quantitative analysis of T lymphocytes (CD4 + and CD8 + , FoxP3 + regulatory T cells), and macrophages in the gut wall of dogs naturally infected with Leishmania infantum. Thirteen dogs were divided into three groups: group 1 (G1, n=5), dogs with canine visceral leishmaniasis (CVL) and infected with L. infantum amastigotes in the intestine; group 2 (G2, n=5), dogs with CVL but without intestinal amastigotes; and group 3 (G3, n=3), uninfected dogs (control group). There was no significant difference (p ≥ 0.05) on CD4 + and Treg cell numbers among the groups, whereas the levels of CD8 + T cells and macrophages were significantly higher in dogs from G1 group than in G2 and G3 (p ≤ 0.05), especially in intestinal segments with high parasite burden. Parasite burden correlated positively with levels of CD8 + T cells and macrophages (p ≤ 0.05), but was inversely correlated to levels of CD4 + T lymphocytes and FoxP3 + Treg cells. In conclusion, in the intestine of dogs with CVL, the increase of CD8 + T cells and macrophages population associated with high parasite burdens, but no changes of CD4 + T cells and FoxP3 + Treg cells suggest a possible immunoregulation by the parasite not dependent on Treg cells.Keywords: T lymphocytes, macrophages, canine visceral leishmaniasis, intestinal tract. ResumoEste estudo foi uma análise semi-quantitativa de linfócitos T (CD4 + , CD8 + e regulatórios -Treg FoxP3 + ) e macrófagos na parede intestinal de cães naturalmente infectados com Leishmania infantum. Treze cães foram divididos em três grupos: grupo 1 (G1, n=5) continha cães com leishmaniose visceral canina (LVC) e com amastigotas intestinais; grupo 2 (G2, n=5) continha cães com LVC, mas sem amastigotas intestinais e o grupo 3 (G3, n=3) continha cães não infectados (grupo controle). Verificou-se que não houve diferença significativa (p ≤ 0.05) no número de células CD4 + e de Treg entre os grupos, mas o número de células T CD8 + e macrófagos foi significativamente superior nos cães do grupo G1 em relação ao G2 e ao G3 (p ≤ 0,05), especialmente nos segmentos intestinais com altas cargas parasitárias. As altas cargas parasitarias correlacionaram positivamente com os números de CD8 + e macrófagos (p ≤ 0,05), mas negativamente com as células CD4 + e Treg. Em conclusão, no intestino dos cães com LVC, o aumento das populações de células T CD8 + e de macrófagos associado a altas cargas parasitárias, mas nenhuma alteração de células T CD4 + e células Treg FoxP3 + sugerem uma possível imunorregulação pelo parasita não dependente de células Treg.Palavras-chave: Linfócito T, macrófagos, leishmaniose visceral canina, trato intestinal.

Introdução: A dengue é uma arbovirose que ocorre em áreas tropicais e subtropicais e é transmitida pela picada do mosquito Aedes aegypti. O vírus da dengue pertence à família Flaviviridae e apresenta manifestações clínicas variadas, como náuseas ou vômitos, cefaleias, febre entre 39 e 40ºC, mialgias, equimoses na pele, sangramento gengival e ou intensa dor abdominal. Objetivo: Este trabalho tem como objetivo realizar uma análise comparativa e descritiva de números de casos de dengue no Brasil, no estado de São Paulo e no município de Jaú anterior e durante à pandemia de COVID 19 no Brasil. Material e métodos: Este é um estudo descritivo, onde foram utilizados dados de Boletins Epidemiológicos da Secretaria de Vigilância em Saúde – Ministério da Saúde dos anos de 2019 e 2020; Centro de Vigilância Epidemiológica ´´Prof. Alexandre Vranjac``, Sistema de Informação de Agravos de Notificação – SINAN. Resultados: Segundo o Boletim Epidemiológico 02, de janeiro de 2020, os números de casos de dengue em 2019 no Brasil, contabilizaram 1.544.987 casos prováveis, isto é, os casos notificados exceto os descartados. Ao comparar esses dados com o Boletim Epidemiológico 51, de dezembro de 2020 (979.764 casos prováveis) nota-se uma redução de aproximadamente 63,41 %. Além disso, observou-se reduções à nível estadual e municipal. No estado de São Paulo, segundo dados do SINAN, em 2019, foram notificados 718.314 casos da doença, dos quais, 400.856 foram confirmados, já no ano de, 2020, o estado registrou uma queda de cerca de 55,8 % no número de casos confirmados, sendo 384.815 casos notificados, dos quais 185.849 foram confirmados. A queda desses agravos também ocorreu no município de Jaú, em 2019 foram registrados 1876 casos confirmados, e no ano de 2020 esses números reduziram em 48,45 % (909 casos). Conclusão: Diante disso, acredita- se que essa redução nos números de casos de dengue pode estar relacionada ao efeito da pandemia de COVID19, visto que, muitos indivíduos, com receio de se infectarem, deixaram de buscar atendimento nas unidades básicas de saúde, o que pode ter gerado a subnotificação desses casos, conforme também sinaliza os dados do Ministério da Saúde.

Visceral leishmaniasis (VL) is a disease caused by the protozoa Leishmania infantum and can cause an inflammatory reaction in the gastrointestinal tract, however the role of granulocytic cells (neutrophils, eosinophils, and mast cells) in the intestine of dogs infected is not fully understood. We performed a quantitative analysis these cells in the intestinal wall of dogs with canine visceral leishmaniasis (CVL). Twenty dogs were assigned to one of three groups: group 1 (G1, n=8), dogs with CVL and L. infantum amastigotes in the intestine; group 2 (G2, n=9), dogs with CVL but without intestinal amastigotes; and group 3 (G3, n=3), uninfected dogs (control group). Granulocytic cells were counted in the crypt-villus unit (mucosa), submucosa, and muscle layer of the intestinal mucosa. Cell counts were higher in the intestinal wall of dogs from G2 followed by G1 and G3 (p≤0.05). In G1, there was a low inverse correlation between parasite burden of the small intestine and granulocyte counts (r= -0.1, p≤0.01). However, in G2 dogs, mast cell and eosinophil numbers showed positive correlation (r=0.85, p≤0.01). The granulocytic cell hyperplasia observed in the intestine of L. infantum-infected dogs suggests that these cells may be involved in the cell-mediated immune response for parasite elimination.