Development of a multiplex real-time PCR surveillance assay for monitoring the health status of Ecuadorian amphibians at risk of extinction

Chytrid fungi and viruses within the genus Ranavirus have been associated with mass mortality events and declines in amphibian populations worldwide. The fungus Batrachochytrium dendrobatidis (Bd) was reported in Ecuador; however, other chytrid fungi like Batrachochytrium salamandrivorans (Bsal) or ranaviruses have not been described in the country so far. To prevent the introduction of pathogens into amphibian populations under conservation programs and to implement a successful disease surveillance program, the development of a sensitive and specific diagnostic assay was required. We describe here the optimization of one TaqMan probe-based multiplex quantitative polymerase chain reaction (qPCR) assay that enables the simultaneous detection of Bsal and ranavirus, and one monoplex TaqMan qPCR assay for the detection of Bd. Standard curves, with a high linear correlation (r2 > 0.995), were generated using a synthetic genome template (gBlocks®) containing the target sequences from all three pathogens. Different samples from skin, liver, kidney, spleen, and lung from six different amphibian species were tested, and both qPCR assays showed highly reproducible and reliable results. To our knowledge, this method is the first multiplex qPCR system developed in Ecuador for identifying amphibian pathogens and represents a valuable tool for the early detection of these pathogens and for infection and co-infection monitoring in future epidemiological surveillance of amphibian species at risk of extinction

Notes on the hematology of free-living Phrynops geoffroanus (Testudines: Chelidae) in polluted rivers of Southeastern Brazil

Phrynops geoffroanus (Schweigger, 1812) is the freshwater turtle with the widest geographical distribution in South America. During 2006, physical examination and hematological evaluation were performed on free-ranging turtles from two polluted rivers, the Piracicaba River (n = 51) and its tributary Piracicamirim (n = 42), in southeastern Brazil. Red blood cell and thrombocyte counts, mean corpuscular volume and mean corpuscular hemoglobin levels differed in turtles from the two water courses. Although free-ranging turtles showed ectoparasites and boat propeler lesions, animals apparently had no signs of clinical disease. In spite of our results, further monitoring of the demography and health status of Phrynops geoffroanus in anthropogenically altered environments is recommended.FAPESP [05/00210-9]CNPqCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

Epizootic amebiasis outbreak in wild black howler monkeys (Alouatta caraya) in a wildlife facility during captivity prior to translocation - Recommendations to wildlife management programs

The capture and quarantine of non-human primates could be necessary in some circumstances including those required under environmental permit. Mortality is undesirable for wildlife management programs and could be related to opportunistic pathogens, for example, deaths due to intestinal protozoa infection outbreaks as described here. Parasitological, necroscopic, microbiological, and molecular tests were used in the diagnosis of severe necrotic enteritis leading to death of three female and two male black howler monkeys (Alouatta caraya) captured and held in quarantine prior to translocation. Parasitological tests showed the presence of cysts of Entamoeba histolytica/dispar (5/5), Entamoeba coli (5/5) and Giardia duodenalis (1/5). Necroscopic assessment revealed areas of severe multifocal necrosis in the intestinal mucosa and submucosa. Histopathological examination revealed the presence of structures morphologically compatible to Entamoeba spp. in all individuals examined. Furthermore, G. duodenalis (1/5) was demonstrated by the nested PCR technique. During temporary captivity of non-human primates in management programs, proper handling protocols, including fast or immediate destination, are suggested in order to mitigate the negative effects of stress and decrease the risk of infections.Óbitos por infecções causadas por protozoários intestinais são indesejáveis para programas de manejo de fauna, exigidos no âmbito do licenciamento ambiental e que demandam a captura e quarentena de primatas não humanos. Exames coproparasitológicos, necroscópicos, microbiológicos e moleculares foram utilizados para a confirmação do diagnóstico de uma severa enterite necrótica que levou a óbito três fêmeas e dois machos de Alouatta (A.) caraya capturados e mantidos em quarentena prévia à translocação. Exames coproparasitológicos revelaram a presença de cistos de Entamoeba (E.) histolytica/dispar (5/5), Entamoeba (E.) coli (5/5) e Giardia (G.) duodenalis (1/5). A avaliação necroscópica revelou áreas de necrose multifocal severa na mucosa e submucosa intestinal. A análise microscópica revelou a presença de estruturas morfologicamente compatíveis com trofozoítos do gênero Entamoeba spp. em todos os indivíduos examinados. Além disso, G. duodenalis (1/5) foi demonstrada pela técnica de Nested PCR. Sugere-se que quando for necessário o cativeiro temporário de primatas, deverão ser adotados protocolos de manejo adequados buscando a destinação imediata dos animais, de moto a mitigar os efeitos negativos do estresse e reduzir o risco da ocorrência de infecções

Valores normales de producción de lágrimas y presión intra ocular en psittácidos Ara spp. y Amazona spp. en cautiverio de los zoológicos Jaime Duque y la Fundación Zoológico Santacruz

En la presente investigación se determinaron los valores normales de presión intra ocular y producción lagrimal en psittácidos del género Ara spp. y Amazona spp. Se tomaron 38 individuos del género Ara pertenecientes a las especies Ara ararauna, Ara macao, Ara chloroptera, Ara militaris y Ara ambigua y, 2 híbridos; mientras que del género Amazona fueron 42 psittácidos de las especies Amazona amazonica, Amazona ochrocephala, Amazona farinosa y Amazona automnalis. Todos los individuos estaban en cautiverio. Las mediciones se realizaron en el Zoológico del Parque Jaime Duque ubicado en el municipio de Tocancipá, a 43 kilómetros de Bogotá y a 2600 m.s.n.m, con una temperatura media de 14 °C y, en la Fundación Zoológico Santacruz ubicada en el municipio de San Antonio del Tequendama, a 45 kilómetros de Bogotá y 1882 m.s.n.m. de altitud y con una temperatura media de 18.8° C. Se realizó un examen clínico general; la toma de presión intra ocular con un tonómetro de aplanamiento portátil, tonómetro de Perkins MK2 ® y la prueba de Schirmer con tiras estandarizadas estériles (Ital - químic ®). Ambos ojos fueron evaluados con cada prueba. Se demostró diferencias en la producción lagrimal y presión intra ocular entre ambos géneros de psittácidos. El intervalo de confianza de producción lagrimal en psiitácidos del género Ara spp. fue 8.39 ± 0.64 mm/min; mientras que, para el género Amazona spp. fue 5.95 ± 0.38 mm de lágrimas por minuto. La presión intra ocular en especímenes Ara spp. fue de 13.18 ± 0.42 mm Hg. y las aves del género Amazona spp. mostraron un intervalo de confianza de 12.71 ± 0.35 mm Hg. de presión intra ocular. Los resultados más representativos correspondieron a Ara ararauna (13.26 ± 0.59 mm Hg.; 8.48 ± 1.03 mm/min), Ara macao (13.20 ± 0.76 mm Hg.; 8.30 ± 1.03 mm/min), Amazona amazonica (12.59 ± 0.76 mm Hg.; 5.23 ± 0.52 mm/min), y Amazona ochrocephala (12.67 ± 0.40 mm Hg.; 6.03 ± 0.38 mm/min), por aportar la mayoría de individuos al estudio realizado

Flow cytometry of blood leucocytes of Phrynops geoffroanus (Schweigger, 1812) from polluted environments: isolation and stimulation methodology

O quelônio Phrynops geoffroanus aparece freqüentemente associado a cursos d\'água poluídos, sem que, no entanto, sejam conhecidos aspectos que descrevam sua atividade imune celular frente situações adversas (efeitos antrópicos). A citometria de fluxo permite mensurar características estruturais e imunológicas de células em suspensão submetidas a um fluxo contínuo, sendo capaz de analisar inúmeros tipos celulares. Neste trabalho foi desenvolvida metodologia para estudar e avaliar a função celular de leucócitos sangüíneos do Phrynops geoffroanus através da fagocitose e burst oxidativo espontâneo e induzido utilizando a citometria de fluxo. Para definir a metodologia foram utilizadas 86 amostras sangüíneas de Phrynops geoffroanus encontrados em ambientes antrópicos do rio Piracicaba, ribeirão Piracicamirim e Fundação Parque Zoológico de São Paulo como grupo de comparação. Estes resultados foram complementados pelo perfil hematológico. O processamento incluiu transporte e conservação das amostras em RPMI 1640 Gibco® para posterior utilização de Ficoll-PaqueTM PLUS como agente separador entre leucócitos e hemácias, utilizando-se centrifugações refrigeradas (18°C) com acelerações e desacelerações graduais. Amostras com porcentagens de viabilidade inferiores a 90 % não foram utilizadas na realização dos estímulos: Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate para induzir fagocitose e miristato acetato de phorbol (PMA) e Saccharomyces cerevisiae (Zymosan) na indução do burst oxidativo. Condições ambientais, espécie específica e da metodologia da citometria de fluxo determinaram uma melhor resposta para realização da fagocitose em comparação com o burst oxidativo dos leucócitos aqui avaliados.The freshwater turtle Phrynops geoffroanus is frequently associated with polluted waters; however, the aspects that describe the cellular immune activity against adverse situations (anthropogenic effects) are still unknown. Flow cytometry allows us to measure and analyze the characteristics of the suspended cells when submitted to a continuous flow; it is capable of analyzing a number of different types of cells. In this study, it designed a methodology in order to study and evaluate the cellular function of blood leucocytes, through phagocytosis and spontaneous or induced oxidative bursts, using flow cytometry. This methodology, was designed with 86 blood samples, collected from Phrynops geoffroanus in anthropogenic environments of the river Piracicaba and its tributary Piracicamirim and the Sao Paulo Zoological Park Foundation, as a comparison group, all in Sao Paulo State, Brazil. These results were complemented with the blood profile. The samples were stored in RPMI 1640 Gibco®, medium for transportation. Initially, it was used Ficoll-PaqueTM PLUS, as a separator of leucocytes and red blood cells. Later the samples were centrifuged at 18°C, with gradual break and speed. Samples with viability percentage under 90% were not used. The agents used for stimulation were Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate to induce phagocytosis and phorbol miristate-acetate (PMA) and Saccharomyces cerevisiae (Zymosan) to induce oxidative burst. Environmental, species-specific and cytometric methodology conditions determined a better response for the occurrence of phagocytosis in comparison with oxidative burst of the leucocytes analyzed

Cause of death of wildlife from a rescue area: conservation implications

No Brasil, no transcorrer da última década tem-se aumentado a produção de energia renovável. Dessa forma, empreendimentos visando produção e distribuição de energia tem sido desenvolvidos, exemplo disto são as Pequenas Centrais Hidrelétricas (PCHs). Perda e fragmentação de habitat são consideradas fatores negativos para a conservação in situ, sendo que podem colocar em perigo de extinção numerosas espécies de vertebrados terrestres. Durante a construção de hidrelétricas, esses dois eventos podem ocorrer em um espaço de tempo curto e abranger pequenas ou grandes extensões de habitat natural dessas espécies. Dentro do âmbito ambiental, essas obras demandam a criação e desenvolvimento de programas de monitoramento e conservação, visando diminuir o impacto sobre as populações silvestres que ocorrem na área diretamente afetada pelo empreendimento, resultando no óbito de numerosos indivíduos durante o desenvolvimento das etapas desse programa. Sendo assim, visando entender quais são os fatores que podem levar à perda de um animal silvestre nesses programas se projetou acompanhar entre 2008 e 2010 as etapas do programa de monitoramento e conservação e as do centro de triagem de animais selvagens (CETAS) da PCH Anhanguera, que foi construída no nordeste do Estado de São Paulo. Vertebrados terrestres e aves que vieram a óbito foram submetidos à necropsia e amostras dos principais órgãos e lesões representativas foram fixadas em formalina 10% e posteriormente processadas para avaliação histopatológica. Ao final do exame necroscópico, laudo macroscópico foi elaborado e quando possível foi determinada a causa de morte. Havendo impossibilidade da determinação da causa de morte, resultados dos exames complementares foram aguardados. Foram avaliados 486 animais, entre anfíbios (81), aves (12), mamíferos (164) e répteis (229) em três momentos: monitoramento, resgate na supressão da vegetação e centro de triagem de animais silvestres. As causas de morte estiveram associadas a processos não infecciosos (64,82%), seguido por causa indeterminada (22,43%), eutanásia (9,26%), suspeita de infeccioso (2,26%) e processos infecciosos (1,23%). Acidentes por objetos contundentes durante supressão que causaram trauma foram relevantes em répteis durante a supressão (χ2= 23.3490; ρ= 0.0000). Ataques por contactantes ou predação e acidentes (afogamento e hipotermia) acontecidos nas armadilhas de captura foram significativas causas de morte para mamíferos durante o monitoramento (χ2= 25.5620; ρ= 0.0003). Para anfíbios, as eutanásias induzidas na supressão em decorrência de injúrias graves em tecidos moles e de tecido muscular esquelético foram a causa de morte que mais afetou esse grupo (χ2= 8.2437; ρ= 0.0041). A conservação da fauna durante um programa faunístico de um empreendimento está associada ao manejo adequado dessas populações em campo e centros de triagem. Aumento da captura prévia nas áreas a serem suprimidas, das vistorias nas armadilhas de captura e propensão por uma destinação oportuna dos indivíduos capturados facilitaria a sobrevivência da fauna acometida pelo empreendimento.In the course of the last decade Brazil has increased the production of renewable energy. Thus, projects aiming energy production and distribution have been developed such as Small Hydro Power Stations (SHPS). Loss and fragmentation of habitat are considered negative factors for in situ conservation, and can put many endangered species of terrestrial vertebrates. During the construction of dams, these two events may occur in a short space of time and cover small or large tracts of natural habitat of these species. Within an environmental framework these enterprise create and develop conservation programs in order to reduce the impact on wild populations that occur in the influence area project. Commonly, many individuals may die during the stages of the program. In order to understand what are the factors that can lead to loss of wild animals in these programs, monitoring, rescue and triage center activities, of the program of the SHP Anhanguera, were accompanied between 2008 and 2010. Dam and reservoir were built in the northeastern of São Paulo State, Brazil. Post-mortem examinations were performed in terrestrial vertebrates and birds that died. Samples of major organs were collected and representative lesions were fixed in 10% formalin and later processed to pathological evaluation. At the end of the necropsy, macroscopic report was prepared and, if possible, it was determined the cause of death. Were not possible laboratory tests were awaited. It was evaluated 486 animals, including amphibians (81), birds (12), mammals (164) and reptiles (229) in three phases: monitoring, rescue in forest clearing and triage center. Causes of death were associated with non-infectious processes (64.82%), followed by unknown causes (22.43%), euthanasia (9.26%), suspected infection (2.26%) and infectious processes (1,23%). Accidents caused by blunt objects that resulted in trauma during removal forest were relevant in reptiles (χ2 = 23.3490, ρ = 0.0000). Attacks by predators or contacted and accidents (drowning and hypothermia) that occurred in the traps capture were significant causes of death of mammals during monitoring (χ2 = 25.5620, ρ = 0.0003). For amphibians, euthanasia induced due to severe soft tissue and skeletal muscle injuries in the suppression, were the cause of death that most affected this group (χ2 = 8.2437, ρ = 0.0041). The conservation of wildlife fauna during a conservation program of a hydroelectric enterprise is linked to the appropriate management of these populations in the affected areas and triage centers. Increase of previous captures in the areas of vegetal suppression, check of traps capture and opportune destination of captured animals facilitate the survival of wildlife affected by the project

Radiographic and histological evidence of metabolic bone disease in gliding leaf frogs (Agalychnis spurrelli)

Bone alterations due to metabolic bone disease in captive animal populations can have a negative impact on repopulation and research initiatives. This investigation has the purpose of describing the principal radiographic and anatomopathological findings present in nine gliding leaf frogs (Agalychnis spurrelli) kept in captivity with alterations in their spines and long bones. The observed histopathological findings were in the canalis vertebralis, paraspinal muscle and long bones, and included deformed bones with alteration of the adjacent tissues, alterations in the ossification process, bone degeneration and resorption, decreased number of osteocytes and deposition of osteoid and fibrous material in the compact bone tissue. Additionally, the spinal cord showed compressed white matter, chronic meningitis in the duramater, alteration in the number of glial cells and loss of delimitation between the gray and white matter. Radiographical changes were found mainly in the long bones and included moth-eaten osteolysis, solid periosteal reaction, bone deformities, cortical tunneling and inflammation of adjacent soft tissues. Also, pathological fractures of the femur and urostyle were observed together with spinal column deviations with increased bone density

Epizootic amebiasis outbreak in wild black howler monkeys (Alouatta caraya) in a wildlife facility during captivity prior to translocation - Recommendations to wildlife management programs

The capture and quarantine of non-human primates could be necessary in some circumstances including those required under environmental permit. Mortality is undesirable for wildlife management programs and could be related to opportunistic pathogens, for example, deaths due to intestinal protozoa infection outbreaks as described here. Parasitological, necroscopic, microbiological, and molecular tests were used in the diagnosis of severe necrotic enteritis leading to death of three female and two male black howler monkeys (Alouatta caraya) captured and held in quarantine prior to translocation. Parasitological tests showed the presence of cysts of Entamoeba histolytica/dispar (5/5), Entamoeba coli (5/5) and Giardia duodenalis (1/5). Necroscopic assessment revealed areas of severe multifocal necrosis in the intestinal mucosa and submucosa. Histopathological examination revealed the presence of structures morphologically compatible to Entamoeba spp. in all individuals examined. Furthermore, G. duodenalis (1/5) was demonstrated by the nested PCR technique. During temporary captivity of non-human primates in management programs, proper handling protocols, including fast or immediate destination, are suggested in order to mitigate the negative effects of stress and decrease the risk of infections