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Resistance-breaking (RB) isolates of citrus tristeza virus (CTV) can replicate and move systemically in Poncirus trifoliata, a rootstock widely used for management of decline caused by CTV and other purposes. In Uruguay, severe CTV isolates are prevalent, and an RB isolate (designated as RB-UY1) was identified. In order to predict the implications of this genotype circulating in citrus crops grafted on trifoliate rootstocks, the aim of this work was to determine the biological and molecular characteristics of this isolate, the efficiency of its transmission by Toxoptera citricida, and its effects on plant growth performance of P. trifoliata. Our results show that RB-UY1 can be classified as a mild isolate, that it is phylogenetically associated with the RB1 group, and that it is efficiently transmitted by T. citrida. They also suggest that the RB-UY1 isolate should not affect the performance of citrus crops grafted on trifoliate rootstocks, although some growth parameters of P. trifoliata seedlings were affected four years after inoculation.

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The possibility of a Zika virus epidemic resurgence requires studies to understand its mechanisms of pathogenicity. Here, we describe the isolation of the Zika virus from breast milk (Rio-BM1) and compare its genetic and virological properties with two other isolates (Rio-U1 and Rio-S1) obtained during the same epidemic period. Complete genomic analysis of these three viral isolates showed that they carry characteristics of the American isolates and belong to the Asian genotype. Furthermore, we detected eight non-synonymous single nucleotide variants and multiple nucleotide polymorphisms that reflect phenotypic changes. The new isolate, Rio-BM1, showed the lowest replication rates in mammalian cells, induced lower cell death rates, was more susceptible to treatment with type I IFN, and was less pathogenic than Rio-U1 in a murine model. In conclusion, the present study shows evidence that the isolate Rio-BM1 is more attenuated than Rio-U1, probably due to the impact of genetic alterations in the modulation of virulence. The results obtained in our in vitro model were consistent with the pathogenicity observed in the animal model, indicating that this method can be used to assess the virulence level of other isolates or to predict the pathogenicity of reverse genetic constructs containing other polymorphisms.

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Cell lines are valuable tools to safeguard genetic material from species threatened with extinction that is mainly due to human action. In this scenario, the puma constitutes a species whose population is being rapidly reduced in the ecosystems it inhabits. For the first time, we characterized puma skin-derived cell lines and assessed these cells after extended culture (experiment 1) and cryopreservation (experiment 2). Initially, we identified and characterized four dermal fibroblast lines using morphology, ultrastructure, and immunofluorescence assays. Moreover, we evaluated the effects of culture time (1st, 3rd, and 10th passages) and cryopreservation on their morphology, ultrastructure, viability, metabolism, proliferative activity, reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis. The cells showed a typical spindle-shaped morphology with centrally located oval nuclei. The cells were identified as fibroblasts by staining for vimentin. In vitro culture after the 1st, 3rd, and 10th passages did not alter most of the evaluated parameters. Cells in the 3rd and 10th passages showed a reduction in ROS levels (p < 0.05). The ultrastructure revealed morphological damage in the prolongments, and nuclei of cells derived from the 3rd and 10th passages. Moreover, cryopreservation resulted in a reduction in ΔΨm compared with that of noncryopreserved cells, suggesting that the optimization of cryopreservation methods for puma fibroblasts is essential. In conclusion, we found that viable fibroblasts could be obtained from puma skin, with slight changes after the 10th passage in in vitro culture and cryopreservation. This is the first report on the development of cell lines derived from pumas.

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This study describes the morphological, biochemical, and molecular differences among Trypanosoma dionisii isolates from hemocultures of hematophagous (Desmodus rotundus; n = 2) and insectivorous (Lonchorhina aurita; n = 1) bats from the Atlantic Rainforest of Rio de Janeiro, Brazil. Fusiform epimastigotes from the hematophagous isolates were elongated, whereas those of the insectivorous isolate were stumpy, reflected in statistically evident differences in the cell body and flagellum lengths. In the hemocultures, a higher percentage of trypomastigote forms (60%) was observed in the hematophagous bat isolates than that in the isolate from the insectivorous bat (4%), which demonstrated globular morphology. Three molecular DNA regions were analyzed: V7V8 (18S rDNA), glycosomal glyceraldehyde 3-phosphate dehydrogenase gene, and mitochondrial cytochrome b gene. The samples were also subjected to multilocus enzyme electrophoresis and random amplified polymorphic DNA analysis. All isolates were identified as T. dionisii by phylogenetic analysis. These sequences were clustered into two separate subgroups with high bootstrap values according to the feeding habits of the bats from which the parasites were isolated. However, other T. dionisii samples from bats with different feeding habits were found in the same branch. These results support the separation of the three isolates into two subgroups, demonstrating that different subpopulations of T. dionisii circulate among bats.

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BACKGROUND: Biobanking of cell lines is a promising tool of support for wildlife conservation. In particular, the ability to preserve fibroblast cell lines derived from collared peccaries is of significance as these wild mammals are unique to the Americas and play a large role in maintaining the ecosystem. We identified collared peccary fibroblasts by immunofluorescence and evaluated their morphology, growth and adherence capacity. Further, we monitored the viability and metabolic activity of the fibroblasts to determine the effects of passage number and cryopreservation on establishment of cell lines. METHODS: Skin biopsies were collected from the peripheral ear region from five adult animals in captivity. Initially, cells were isolated from fragments and cultured in the Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 2% antibiotic-antimycotic solution under a controlled atmosphere (38.5 °C, 5% CO2). We evaluated the maintenance of primary cells for morphology, adherence capacity of explants, explants in subconfluence, cell growth and absence of contamination. Moreover, we identified the fibroblast cells by immunofluorescence. Additionally, to evaluate the influence of the number of passages (first, third and tenth passage) and cryopreservation on establishment of cell lines, fibroblasts were analysed for the viability, metabolic activity, population doubling time (PDT), levels of reactive oxygen species (ROS), and mitochondrial membrane potential (ΔΨm). RESULTS: All explants (20/20) adhered to the dish in 2.4 days ± 0.5 with growth around the explants in 4.6 days ± 0.7, and subconfluence was observed within 7.8 days ± 1.0. Moreover, by morphology and immunocytochemistry analyses, cells were identified as fibroblasts which presented oval nuclei, a fusiform shape and positive vimentin staining. No contamination was observed after culture without antibiotics and antifungals for 30 days. While there was no difference observed for cell viability after the passages (first vs. third: P = 0.98; first vs. tenth: P = 0.76; third vs. tenth: P = 0.85), metabolic activity was found to be reduced in the tenth passage (23.2 ± 12.1%) when compared to that in the first and third passage (100.0 ± 24.4%, P = 0.006). Moreover, the cryopreservation did not influence the viability (P = 0.11), metabolic activity (P = 0.77), or PDT (P = 0.11). Nevertheless, a greater ΔΨm (P = 0.0001) was observed for the cryopreserved cells (2.12 ± 0.14) when compared to that in the non-cryopreserved cells (1.00 ± 0.05). Additionally, the cryopreserved cells showed greater levels of intracellular ROS after thawing (1.69 ± 0.38 vs. 1.00 ± 0.22, P = 0.04). CONCLUSIONS: This study is the first report on isolation, characterization and cryopreservation of fibroblasts from collared peccaries. We showed that adherent cultures were efficient for obtaining fibroblasts, which can be used as donor cells for nuclei for species cloning and other applications.

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In this research project, synthesis and characterization of ionic liquids and their subsequent utilization as facilitators of transdermal delivery of human insulin was pursued. Choline geranate and choline oleate ionic liquids (and their deep eutectic solvents) were produced and characterized by nuclear magnetic resonance (1H NMR), water content, oxidative stability, cytotoxicity and genotoxicity assays, and ability to promote transdermal protein permeation. The results gathered clearly suggest that all ionic liquids were able to promote/facilitate transdermal permeation of insulin, although to various extents. In particular, choline geranate 1:2 combined with its virtually nil cyto- and geno-toxicity was chosen to be incorporated in a biopolymeric formulation making it a suitable facilitator aiming at transdermal delivery of insulin.

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Ticks are one of the most important groups of blood-eating parasitic arthropods in domestic, wild and human animals. They belong to the subclass Acari of the class Arachnida, order Ixodida. It is a group with about 900 described species, distributed in three families: Argasidae, Nuttalliedae and Ixodidae. The hematophagy process of ticks is different from other hematophagous animals, as they use their chelicerae to destroy small peripheral vessels and thus keep a quantity of blood in puddles to obtain food. This blood is kept liquid due to the secretion of the salivary glands of innumerable molecules that interfere in the host's physiological and immunological response. The tick O. brasiliensis is aggressive to humans, being considered in its areas of occurrence, as a public health problem, its bite can cause pain, intense inflammatory reaction at the location of the bite and in some people fever and malaise. Cases of symptoms of paralysis have also been reported in highly parasitized dogs, which may consider that this tick is capable of inducing in the host a reaction called toxicosis related to the bite, defined as reactions caused by toxic substances that are secreted by the tick salivary glands. In this work, we carried out a preliminary in vivo characterization of the activity of the salivary gland extract (EGS) on the effect of toxicity and evaluated the development of paw edema in mice, in addition to the potential for inhibiting blood coagulation from global coagulation assays ( Prothrombin Time - PT and Activated Partial Thromboplastin Time - APTT), the study of clot formation through the thromboelastógramo in the presence of EGS was also carried out. In the doses used by us it was not possible to verify any evidence of toxicity in the animals, as for the formation of local reaction measured by the edema of mouse paw it can be observed that there was a slight edema formation, although not significant. Still in the coagulation tests, there was little inhibition in both PT and APTT. By the elastography test, it was observed that there was a weak formation of fibrin networks, which may suggest that fibrinogen is involved in the fluidity of blood during hematophagy. Although these studies are preliminary, the results may serve as a basis for new information on the regulatory mechanisms of hemostasis, physiological mechanisms of ticks and future practical applications.

Os carrapatos constituem um dos mais importantes grupos de artrópodes parasitas hematófagos de animais domésticos, silvestres e do homem. Pertencem à subclasse Acari da classe Arachnida, ordem Ixodida. É um grupo com cerca de 900 espécies descritas, distribuídas em três famílias: Argasidae, Nuttalliedae e Ixodidae. O processo de hematofagia dos carrapatos é diferente de outros animais hematófagos, pois utilizam suas quelíceras para a destruição dos pequenos vasos periféricos e assim manter uma quantidade de sangue em poças para a obtenção de alimento. Esse sangue é mantido líquido devido à secreção das glândulas salivares de inúmeras moléculas que interferem na resposta fisiológica e imunológica do hospedeiro. O carrapato O. brasiliensis é agressivo aos humanos, sendo considerado em suas áreas de ocorrência, como um problema de saúde públicas, sua picada pode causar dor, intensa reação inflamatória no local da picada e em algumas pessoas febre e mal estar. Também foram relatados casos de sintomas de paralisia em cães altamente parasitados, podendo considerar que este carrapato é capaz de induzir no hospedeiro uma reação denominada de toxicose relacionada à picada, definida como reações causadas por substâncias tóxicas que são secretadas pelas glândulas salivares de carrapatos. Neste trabalho realizamos uma caracterização preliminar in vivo da atividade do extrato das glândulas salivares (EGS) sobre o efeito da toxicidade e avaliamos o desenvolvimento do edema de pata em camundongos, além do potencial de inibição da coagulação sanguínea a partir de ensaios globais da coagulação (Tempo de Protrombina – TP e Tempo de Tromboplastina Parcial Ativada - TTPa), também foi realizado o estudo da formação do coágulo através do tromboelastógramo em presença do EGS. Nas doses por nós utilizadas não foi possível constatar qualquer indício de toxicidade nos animais, quanto a formação de reação local medida pelo edema de pata de camundongo pode-se observar que houve uma leve formação de edema, embora pouco significativo. Ainda nos ensaios de coagulação houve pequena inibição tanto no TP como no TTPA. Pelo teste de elastografia observou-se que houve fraca formação das redes de fibrina, podendo sugerir que o fibrinogênio esteja envolvido na fluidez do sangue durante a hematofagia. Apesar de estes estudos serem preliminares, os resultados poderão servir de base para novas informações sobre os mecanismos regulatórios da hemostasia, mecanismos fisiológicos dos carrapatos e futuras aplicações práticas.

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Introduction The biological diversity of Trypanosoma cruzi strains plays an important role in the clinical and epidemiological features of Chagas disease. Methods Eight T. cruzi strains isolated from children living in a Chagas disease vector-controlled area of Jequitinhonha Valley, State of Minas Gerais, Brazil, were genetically and biologically characterized. Results The characterizations demonstrated that all of the strains belonged to T. cruzi II, and showed high infectivity and a variable mean maximum peak of parasitemia. Six strains displayed low parasitemia, and two displayed moderate parasitemia. Later peaks of parasitemia and a predominance of intermediate and large trypomastigotes in all T. cruzi strains were observed. The mean pre-patent period was relatively short (4.2±0.25 to 13.7±3.08 days), whereas the patent period ranged from 3.3±1.08 to 34.5±3.52 days. Mortality was observed only in animals infected with strain 806 (62.5%). Histopathological analysis of the heart showed that strains 501 and 806 caused inflammation, but fibrosis was observed only in animals infected with strain 806. Conclusions The results indicate the presence of an association between the biological behavior in mice and the genetic characteristics of the parasites. The study also confirmed general data from Brazil where T. cruzi II lineage is the most prevalent in the domiciliary cycle and generally has low virulence, with some strains capable of inducing inflammatory processes and fibrosis. .

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INTRODUCTION: For a long time, the importance of Chagas disease in Mexico, where many regarded it as an exotic malady, was questioned. Considering the great genetic diversity among isolates of Trypanosoma cruzi, the importance of this biological characterization, and the paucity of information on the clinical and biological aspects of Chagas disease in Mexico, this study aimed to identify the molecular and biological characterization of Trypanosoma cruzi isolates from different endemic areas of this country, especially of the State of Jalisco. METHODS: Eight Mexican Trypanosoma cruzi strains were biologically and genetically characterized (PCR specific for Trypanosoma cruzi, multiplex-PCR, amplification of space no transcript of the genes of the mini-exon, amplification of polymorphic regions of the mini-exon, classification by amplification of intergenic regions of the spliced leader genes, RAPD - (random amplified polymorphic DNA). RESULTS: Two profiles of parasitaemia were observed, patent (peak parasitaemia of 4.6×10(6) to 10(7) parasites/mL) and subpatent. In addition, all isolates were able to infect 100 percent of the animals. The isolates mainly displayed tropism for striated (cardiac and skeletal) muscle. PCR amplification of the mini-exon gene classified the eight strains as TcI. The RAPD technique revealed intraspecies variation among isolates, distinguishing strains isolated from humans and triatomines and according to geographic origin. CONCLUSIONS: The Mexican T. cruzi strains are myotrophic and belong to group TcI.

INTRODUÇÃO: Durante muito tempo, foi questionada a importância da doença de Chagas no México onde muitos a consideravam um padecimento exótico. Considerando a grande diversidade genética existente, entre os isolados de Trypanosoma cruzi, a importância da caracterização biológica desses e o escasso número de informações sobre os aspectos clínicos e biológicos da doença de Chagas no México, o objetivo deste trabalho foi realizar a caracterização biológica e molecular de isolados de Trypanosoma cruzi originários de diferentes áreas endêmicas deste país, principalmente do Estado de Jalisco. MÉTODOS: Oito cepas mexicanas de Trypanosoma cruzi foram caracterizadas biologicamente e geneticamente (PCR específica para Trypanosoma cruzi, PCR-multiplex, amplificação do espaço não transcrito dos genes do mini-exon, amplificação das regiões polimórficas do gene do mini-exon, classificação pela amplificação de regiões intergênicas dos genes do spliced leader, RAPD - random amplified polymorphic DNA). RESULTADOS: Foram observados dois tipos de parasitemia: patente com picos máximos de parasitemia entre 4,6x10(6) e 10(7) parasitas/mL e subpatente. Além disso, todos os isolados foram capazes de infectar 100 por cento dos animais. Observou-se tropismo predominante pelo músculo estriado (cardíaco e esquelético). As técnicas de PCR do gene do mini-éxon classificaram as oito cepas como TcI e a técnica de RAPD mostrou variação intra-especifica das mesmas, separando as cepas isoladas de humanos daquelas de triatomíneos e por origem geográfica. CONCLUSÕES: As cepas mexicanas de Trypanosoma cruzi são miotrópicas e correspondem ao TcI.

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This study was carried out during 2002/2003, aiming to determine the prevalence of virulent Newcastle disease virus strains (NDV) in Brazilian commercial poultry farms. Clinical samples were obtained from the Southeastern, Southern and Central-Western regions, which comprise the main area of the Brazilian poultry production. Serum samples and tracheal and cloacal swabs of 23,745 broiler chickens from 1,583 flocks, including both vaccinated chickens and those with no vaccination information, were tested for NDV using a diagnostic ELISA kit. The seropositivity was 39.1 percent, and the isolation percentage by flock varied from 1.0 to 7.6 percent, and by region from 6.5 to 58.4 percent. Higher isolation rates (74.3-83.3 percent) were obtained after three passages in embryonated chicken eggs. All isolates preliminarily identified as NDV were characterized as nonpathogenic strains, as their Intracerebral Pathogenicity Index (ICPI) was below 0.7. Based on results of this study, Brazil can claim a virulent NDV-free status for commercial flocks.

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