Genotyping of South American clinical isolates of Pythium insidiosum based on single nucleotide polymorphism-based multiplex PCR / Genotipagem de isolados clínicos de Pythium insidiosum da América do Sul utilizando polimorfismos de nucleotídeo único baseado em PCR multiplex

We aimed to genotype the South American clinical isolates of Pythium insidiosum using the single nucleotide polymorphisms (SNP) of the ribosomal DNA sequences (rDNA). Previously, an SNP-based multiplex-PCR was able to distinguish three different clades of P. insidiosum isolates. Thus, we used this assay to evaluate South American clinical isolates of P. insidiosum (n=32), standard strains from Costa Rica (n=4), Thailand (n=3), Japan (n=1), and India (n=1), a standard strain of Pythium aphanidermatum, and Brazilian environmental isolates of Pythium torulosum, Pythium rhizo-oryzae and Pythium pachycaule voucher (n=3). It was possible to allocate each American P. insidiosum isolate to clade I, the isolates of India, Japan, and Thailand to clade II, and the Thai isolate to clade III. P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher isolates were not amplified. For the first time, a P. insidiosum isolate from Uruguay, South America, was included in molecular analyzes. By SNP-based multiplex-PCR, it was possible to perform the identification and genotyping of the South American isolates of P. insidiosum, demonstrating similar genetic characteristics of these isolates.(AU)

O objetivo deste estudo foi genotipar isolados clínicos de Pythium insidiosum da América do Sul utilizando polimorfismos de nucleotídeo único (SNP) de sequências de rDNA. Anteriormente, um multiplex-PCR baseado em SNP foi capaz de distinguir P. insidiosum em três diferentes clados. Dessa forma, utilizamos este método para avaliar isolados clínicos de P. insidiosum da América do Sul (n=32), cepas padrão da Costa Rica (n=4), Tailândia (n=3), Japão (n=1) e Índia (n=1), uma cepa padrão de Pythium aphanidermatum e isolados ambientais brasileiros de Pythium torulosum; Pythium rhizo-oryzae e Pythium pachycaule voucher (n=3). Os isolados analisados foram alocados aos clados: I (americanos), II (isolados da Índia, Japão e Tailândia), e III (um isolado tailandês). P. aphanidermatum, P.torulosum, P.rhizo-oryzae e P.pachycaule voucher não foram amplificados. Pela primeira vez, um isolado de P. insidiosum do Uruguai foi incluído em análises moleculares. Através da multiplex-PCR baseada em SNP, foi possível realizar a identificação e genotipagem dos isolados sul-americanos de P. insidiosum, demonstrando características genéticas semelhantes entre esses isolados.(AU)

Isolation and molecular characterization of symbiotic fungus from Acromyrmex ambiguus and Acromyrmex heyeri ants of Rio Grande do Sul State, Brazil / Isolamento e caracterização molecular do fungo simbionte de formigas-cortadeiras (Acromyrmex heyeri e Acromyrmex ambiguus) do Rio Grande do Sul, Brasil

Leaf-cutting ants of the genera Atta and Acromyrmex determine serious agricultural problems and live on symbiosis with Leucoagaricus gongylophorus. The aim of this study is to identify morphological and molecularly, as well as to verify the genotypic variability of the symbiotic fungus cultivated by A. heyeri and A. ambiguus from three different regions of the state of Rio Grande do Sul, Brazil. Fungus gardens were collected and fragments of mycelia were grown in selective medium. Total DNA was extracted and amplification of the ITS region was performed by PCR using universal primers. After DNA sequencing, the chromatograms were assembled and phylogenetic analyzes were performed by the Neighbor-Joining method. A total of six isolates of L. gongylophorus were obtained and their identities were confirmed by molecular analyses. Phylogenetic analysis of the ITS region showed a tree with two distinct groups regarding the fungus isolates from A. heiyeri and A. ambiguous. In this study, it was verified that A. heyeri and A. ambiguous, cultivate the same fungus. Additionally, the molecular marker used in this study showed variations in L. gongylophorus, evidencing two distinct branches in the phylogenetic tree, according to the ant species that cultivate L. gongylophorus. However, other studies involving the inclusion of a great number of isolates of L. gongylophorus, as well as the use of other molecular markers to validate the possible variations in the phylogenetic relationship of this symbiotic fungus are required.(AU)

Formigas-cortadeiras dos gêneros Atta e Acromyrmex causam elevados prejuízos à agricultura e são dependentes obrigatórias da simbiose com Leucoagaricus gongylophorus. O objetivo deste estudo foi identificar morfologicamente e molecularmente, bem como verificar a variabilidade genotípica do fungo simbionte,cultivado por Acromyrmex heyeri e Acromyrmex ambiguus em três regiões do RS, Brasil. Jardins de fungo foram coletados do interior de formigueiros e fragmentos de micélio foram cultivados em meio de cultura seletivo. O DNA total foi extraído e a amplificação da região ITS foi realizada por PCR, utilizando primers universais. Após sequenciamento, os cromatogramas foram montados e as análises filogenéticas foram realizadas pelo método de Neighbor-Joining. Dos jardins de fungo, obtiveram-se seis isolados de L. gongylophorus, confirmados por análise molecular. A análise filogenética da região ITS mostrou uma árvore com dois grupos distintosem relação aos isolados do fungo oriundos de ninhos de A. heyeri e A. ambiguus. Neste estudo, evidenciou-se que as espécies de formigas A. heyeri e A. ambiguus cultivam o mesmo fungo. Entretanto, o marcador molecular estudado evidenciou variações de L. gongylophorus que permitiram formar duas ramificações diferentes na árvore filogenética relacionada à espécie de formiga que o cultiva. Todavia, para validar as possíveis variações nas relações filogenéticas deste fungo simbionte, é necessária a inclusão de um maior número de isolados de L. gongylophorus, bem como o emprego de outros marcadores moleculares.(AU)

Pythium insidiosum: morphological and molecular identification of Brazilian isolates / Pythium insidiosum: identificação morfológica e molecular de isolados brasileiros

Pythium insidiosum is an oomycete belonging to the kingdom Stramenipila and it is the etiologic agent of pythiosis. Pythiosis is a life-threatening infectious disease characterized by the development of chronic lesions on cutaneous and subcutaneous, intestinal, and bone tissues in humans and many species of animals. The identification of P. insidiosum is important in order to implement a rapid and definitive diagnosis and an effective treatment. This study reports the identification of 54 isolates of P. insidiosum of horses, dogs and sheep that presented suspicious clinical lesions of pythiosis from different regions in Brazil, by using morphological and molecular assays. Throughout the PCR it was possible to confirm the identity of all Brazilian isolates as being P. insidiosum.(AU)

Pythium insidiosum é um oomiceto pertencente ao Reino Stramenopila e agente etiológico da pitiose, uma doença infecciosa com riscos de morte. A pitiose é caracterizada pelo desenvolvimento de lesões crônicas sobre os tecidos cutâneos, subcutâneas, intestinal e ósseo em humanos e muitas espécies de animais. A identificação de P. insidiosum é importante, a fim de se obter um diagnóstico rápido e definitivo, bem como um tratamento eficaz. Este estudo relata a identificação de 54 isolados de P. insidiosum de cavalos, cães e ovelhas que apresentavam lesões compatíveis e suspeita clínicas de pitiose, provenientes de diferentes regiões do Brasil, através de métodos morfológicos e moleculares. Através da PCR foi possível confirmar a identidade de todos os isolados brasileiros como sendo P. insidiosum.(AU)

Activities of the enzymes NTPDase, 5'-nucleotidase and adenosine deaminase in platelets of rats experimentally infected with Leptospira interrogans serovar icterohaemorrhagiae

Background: Leptospirosis, a spirochetal zoonotic disease caused by different serovars of Leptospira interrogans, is increasingly recognized as an important cause of hemorrhagic fever. Although the haemorrhagic potential of leptospirosis was noted by Weil (1886) as early as 1886, its pathophysiology is still not clearly elucidated, particularly regarding the cause and mechanisms of bleeding. Studies with ectonucleoside triphosphate diphosphohydrolase (NTPDase; EC 3.6.1.5; CD39), 5'-nucleotidase (EC 3.1.3.5; CD73) and adenosine deaminase (ADA) have demonstrated the involvement of these enzymes in thromboregulation mechanisms, and altered enzymatic activities have been reported in many diseases. Since leptospirosis is a disease increasingly recognized as an important cause of hemorrhagic fever, the aim of this study was to evaluate these enzymes activities and parameters of platelet aggregation in platelets from rats experimentally infected with Leptospira interrogans serovar icterohaemorrhagiae during different periods of experimental infection. Materials, Methods & Results: For this purpose, thirty-six adult male rats were divided into two groups: A, as uninfected control (subgroups A1, A2 and A3); and B, infected (subgroups B1, B2 and B3). Group B was inoculated intraperitoneally (Day 0) with 2 x 108 organisms per rat. Blood samples were collected on days 05 (A1 and B1), 10 (A2 and B2) and 20 (A3 and B3) post-inoculation (PI). In the infected group, platelet count had a decrease on day 10 PI and prothrombin time (PT) had an increase on day 5 PI. In the same group, platelet aggregation decreased (P < 0.01) day 10 PI. The hydrolysis of ATP in platelets was also decreased (P < 0.05) on day 10 PI, when compared to the control group. By the other side, ADP hydrolysis was increased (P < 0.05) on days 5 and 10 PI. 5'-nucleotidase activity was significantly increased on day 5 (P < 0.01) and 20 (P < 0.05) PI. Results of adenosine deamination into inosine by ADA in platelets showed a signifi cant (P < 0.01) increase on days 5 and 10 PI in the infected group. Discussion: Studies with NTPDase, 5'nucleotidase and ADA have demonstrated the involvement of these enzymes in the thromboregulation mechanisms, and altered enzymatic activities have been reported in many diseases. It has been established that extracellular adenosine nucleotides and adenosine are versatile signaling molecules known to participate in an array of platelet functions. For example, the nucleotide ADP is the main promoter of platelet aggregation, while adenosine can act as a vasodilator and an inhibitor of platelet aggregation. In addition, high concentrations of ATP have been shown to inhibit ADP-induced aggregation in vitro, while low concentrations of ATP can significantly enhance platelet aggregation. In our experimental study the coagulation cascade was activated, since when the activities of NTPDase, 5'-nucleotidase and ADA were analyzed is possible to suggest that levels of ATP were decreased, unlike of ADP and AMP levels, supposedly increased during determinate periods of our experiment. Adenosine levels were also enhanced due to the higher levels of its precursors. This cascade activation may be a mechanism of bleeding prevention front to leptospires infection, especially the ones caused by serovar icterohaemorrhagiae.

Leptospirose bovina: aumento na incidência da Leptospira interogans sorovar butembo no rebanho do estado de Santa Catarina, Brasil / Bovine leptospirosis: increased incidence of Leptospira interogans serovar butembo in the herd of the State of Santa Catarina, Brazil

Os problemas reprodutivos são as principais manifestações clínicas da leptospirose crônica em fêmeas bovinas, sendo freqüentemente os únicos sinais observados no rebanho. Leptospira interogans sorovar hardjo é o mais predominante e importante porque compromete o desempenho reprodutivo dos rebanhos acometidos, porém levantamentos sorológicos no Brasil têm revelado resultados variados quanto à ocorrência dos sorovares nesta espécie animal. O sorovar butembo, normalmente quando encontrado, está presente em pequena parcela dos resultados positivos, porém, nosso levantamento apontou um aumento na prevalência deste sorovar nas amostras recebidas e processadas no período de Janeiro de 2005 a Dezembro de 2008. Desta maneira o objetivo deste trabalho foi relatar um aumento na detecção do variante sorológico butembo em soros bovinos provenientes do estado de Santa Catarina, nas amostras analisadas pelo Laboratório de leptospirose (Lablepto) da Universidade Federal de Santa Maria (UFSM).

Reproductive problems are the main clinical manifestations of leptospirosis in cows and it is often the only signs observed in the herd. Leptospira Interogans serovar hardjo is the most prevalent and important because it affects the reproductive performance of herds, but serological surveys in Brazil have shown mixed results regarding the occurrence of serovars in animal species. The serovar butembo, usually when found, is present in small proportion of positive results, however, our survey indicated an increase in the prevalence of this serovar in the samples received and processed from January 2005 to December 2008. Thus, the objective of this study was to report an increase in the detection of serological variant butembo in bovine serum from the state of Santa Catarina, in the samples analyzed by the Laboratory of leptospirosis (Lablepto) from Universidade Federal de Santa Maria (UFSM).