Molecular identification of hemotropic mycoplasmas (Hemoplasmas) in wild mammals

Orientador : Prof. Dr. Rafael Felipe da Costa VieiraCoorientador : Prof. Dr. Alexandre Welker BiondoTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Agrárias, Programa de Pós-Graduação em Ciências Veterinárias. Defesa: Curitiba, 09/12/2016Inclui referências : f. 15-19;45-54;62-64;67-70;72-86;97-101;105-108Área de concentração : Ciências veterináriasResumo: Os micoplasmas hemotrópicos (hemoplasmas) são bactérias distribuídas mundialmente que afetam animais domésticos e animais selvagens além dos seres humanos. Eles ainda permanecem incultiváveis in vitro. Os hemoplasmas tem sido descritos como potenciais causadores de anemia hemolítica em mamíferos domésticos e selvagens. O objetivo deste estudo foi detectar por intermédio de métodos moleculares a presença de micoplasmas hemotrópicos em mamíferos selvagens nativos e exóticos. Esta tese de doutorado apresenta três artigos. O primeiro artigo é de revisão sistemática e meta-análise sobre a detecção molecular de hemoplasmas em mamíferos selvagens, utilizando-se os artigos indexados no MEDLINE e SCIELO, no periodo de dezembro de 1967 a outubro 2016. Um total de 45/1235 artigos (3,64%) relacionaram-se à identificação molecular de hemoplasmas em mamíferos selvagens, e 78 espécies de mamíferos selvagens foram relatadas como infectadas. A metanálise foi realizada utilizando um modelo de efeitos aleatórios para comparação dos dados de prevalência disponíveis para mamíferos selvagens em cativeiro e em vida livre e entre as ordens. Uma árvore filogenética com base em sequencias do gene 16S rRNA foi construída, comparada e discutida. Hemoplasmas estão distribuídos em mamíferos selvagens em todo o mundo, com prevalencia de 29,92% (IC 24,53 - 33,74) para todos os animais, sendo 31,00% (IC 24,97 - 37,76, I² p<0,001) para animais em vida livre e 22,33% (IC 17,20 - 28,47, I² p<0,001) para animais em cativeiro. O segundo artigo refere-se a pesquisa de micoplasmas hemotrópicos em morcegos, sendo este o primeiro estudo com micoplasmas hemotrópicos em morcegos no Brasil. Foram colhidas amostras de sangue (n = 10) de oito morcegos hematófagos: seis machos, morcego-vampiro comum (Desmodus rotundus, família Phyllostomidae), dois machos, morcego-vampiro de patas peludas (Diphylla ecaudata, família Phyllostomidae); e duas fêmeas não-hematófagas, morcego-de-Pallas (Molossus sp., Família Molossidae), na região de Curitiba, Estado do Paraná, sul do Brasil. Para a anestesia, as gaiola com os morcegos foram colocadas dentro de um recipiente de plástico e o isoflurano foi infundido com uma máquina com oxigênio. A manutenção da sedação foi realizada utilizando máscara de inalação. A punção intracardíaca foi realizada para se obter o sangue, sequencialmente, os morcegos foram submetidos a eutanasia com dose letal de cloreto de potássio intracardíaco. Os esfregaços sanguíneos de dois Desmodus rotundus, preparados imediatamente após a colheita de sangue, foram corados com May-Grünwald-Giemsa e examinados por microscopia de luz com ampliação de 1000x para a presença de hemoplasma. O DNA foi extraído de 200 ?L de sangue utilizando um kit comercialmente disponível de acordo com as instruções do fabricante. A PCR para o gene desidrogenase gliceraldeído-3-fosfato (GAPDH), foi realizada para garantir a extração bem sucedida do DNA. Em seguida, as amostras foram rastreadas por PCR pan-hemoplasma convencional direcionando para as regiões 16S rDNA específicas para hemoplasmas. Utilizando-se iniciadores universais para o 16S rRNA (Referência), amostras de 2 morcegos da espécie Desmodus rotundus que testaram positivo para Mycoplasma sp na primeira reação de PCR, foram amplificados. Os produtos de PCR de 745 pb foram purificados a partir do gel de agarose a 1,5% e sequenciados. As sequências nucleotídicas dos isolados de hemoplasmas de morcegos foram submetidas à base de dados GenBank sob o número de acesso KX722541. Foi construída uma árvore filogenética baseada em sequências de genes 16S rRNA. Em geral, 8/10 (80,0%) morcegos testaram positivos para Mycoplasma sp. incluindo 5/6 (83,3%) Desmodus rotundus, 2/2 (100%) Diphylla ecaudata e 1/2 (50,0%) Molossus sp. As análises da seqüência parcial do gene 16S rRNA identificaram potencialmente uma nova espécie de hemoplasma infectando morcegos na região de Curitiba, Estado do Paraná, Sul do Brasil. No terceiro artigo, o objetivo do estudo foi aplicar um protocolo PCR de Mycoplasma ovis em 12 aoudads (Ammotragus lervia) de cativeiro do Zoológico de Curitiba, sul do Brasil. Foi utilizado um total de 12 amostras de sangue com EDTA, previamente pesquisadas para outros patógenos. O DNA foi extraído e um protocolo para PCR do gene desidrogenase gliceraldeído-3-fosfato (GAPDH) foi realizado em todas as amostras para garantir DNA amplificável. Em seguida, todas as amostras foram testadas e resultaram negativas em protocolo de PCR específico para a detecção e amplificação de M. ovis. Em anexo estão dois artigos, o primeiro anexo trata-se de uma revisão sobre patógenos em aoudads, com artigos publicados entre setembro de 1959 e outubro de 2016, identificados por meio de busca informatizada nas bases de dados eletrônicas PubMed e SciELO. Alguns patógenos detectados em aoudads, como Mycobacterium tuberculosis e Toxoplasma gondii, também podem infectar animais domésticos e seres humanos. O segundo anexo refere-se a pesquisa de Plasmodium sp. em cervídeos no Brasil. Foi avaliado um rebanho cativo de 22 veado-bororó (Mazama nana), quatro veado-mateiro (Mazama americana) e seis cervo-do-Pantanal (Blastocerus dichotomus) do Sul do Brasil; utilizando microscopia de luz e abordagens moleculares. Os testes microscópico e molecular utilizados não indicaram a presença do parasita nas amostras. Palavras-chave: Micoplasma Hemotrópico, Hemoplasma, Mamíferos Selvagens, PCR, Patógenos, Plasmodium sp.Abstract: Hemotropic mycoplasmas (hemoplasmas) are worldwide distributed bacteria affecting domestic and wildlife animals besides human beings. They still remain uncultivated in vitro. Hemoplasms have been described as potential causes of hemolytic anemia in domestic and wild mammals. The objective of this study was to detect by molecular methods the presence of hemotropic mycoplasmas in native and exotic wild mammals. This doctoral thesis presents three articles. The first article is of systematic review and meta-analysis on the molecular detection of hemoplasms in wild mammals, using articles indexed in MEDLINE and SCIELO, from December 1967 to October 2016. A total of 45/1235 articles (3.64%) related to molecular identification of hemoplasmas in wild mammals, and 78 wild mammal species were reported to be infected. The meta-analysis was performed using a random-effects model to compare the prevalence data available for wild mammals in capitivity and in free ranging between orders. A phylogenetic tree based on 16S rRNA gene sequences was constructed, compared and discussed. Hemoplasmas are distributed in wild mammals throughout the world, with prevalence of 29.92% (CI 24.53 - 33.74) for all reported animals: 31.00% (CI 24.97 - 37.76, I² p < 0.001) for wild animals and 22.33% (CI 17.20 - 28.47, I² p < 0.001) for captive animals. The second article refers to the research of hemotropic mycoplasmas in bats, being this the first study with hemotropic mycoplasmas in bats in Brazil. Blood samples (n=10) were taken from eight hematophagous bats: six males common vampire bat (Desmodus rotundus; Family Phyllostomidae), two males hairy-legged vampire bat (Diphylla ecaudata; Family Phyllostomidae); and two no-hematophagous females Pallas's mastiff bat (Molossus sp.; Family Molossidae), at Curitiba's region, Parana State, southern Brazil. For anesthesia, bat cages were put inside a plastic container and isoflurane was infused with a machine with oxygen. Sedation maintenance was performed using inhalation mask. Intracardiac puncture was performed to obtain blood, sequentially, the bats were euthanized with lethal dose of intracardiac potassium chloride. Blood smears of two Desmodus rotundus, prepared immediately after blood collection, were stained with May-Grünwald-Giemsa and examined using light microscopy at 1,000x magnification for the presence of hemoplasma. DNA was extracted from 200 ?L blood using a commercially available kit according to the manufacturer's instructions. A PCR for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase GAPDH), was performed to ensure successful DNA extraction. Thereafter, samples were screened by conventional pan-hemoplasma PCR targeting the 16S rDNA regions specific for hemoplasmas. 'Candidatus M. haemovis'-positive goat blood sample and nuclease-free water were used as positive and negative control, respectively. Using universal primers for the 16S rRNA (Reference), samples from two bats of the species Desmodus rotundus that tested positive for Mycoplasma sp in the first PCR reaction, were amplified. The PCR products of 745 bp were purified from the 1.5% agarose gel and sequenced. The nucleotide sequences of the hemoplasmas isolates from bats were submitted to the GenBank database under the accession number KX722541. A phylogenetic tree based on 16S rRNA gene sequences was constructed. In overall, 8/10 (80.0%) bats tested positive to Mycoplasma sp. including 5/6 (83.3%) Desmodus rotundus, 2/2 (100%) Diphylla ecaudata and 1/2 (50.0%) Molossus sp. The analyses of the partial sequence of 16S rRNA gene have identified a potentially novel hemoplasma species infecting bats at Curitiba's region, Parana State, Southern Brazil. In the third article, the objective of the study was to apply a PCR protocol of Mycoplasma ovis in 12 captive Barbary sheep (Ammotragus lervia) at Curitiba Zoo, in southern Brazil. A total of 12 blood samples with EDTA, previously searched for other pathogens were used. DNA extracted and a PCR protocol for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was performed on all samples to ensure amplifiable DNA. Subsequently, all the samples were tested and found to be negative using a specific PCR protocol for M. ovis detection and amplification. In the supplement are two articles, the first supplement is a review of pathogens in aoudads, with articles published between September 1959 and October 2016, identified through a computerized search in the electronic databases PubMed and SciELO. Some pathogens detected in aoudads, such as Mycobacterium tuberculosis and Toxoplasma gondii, can also infect domestic animals and humans. The second supplement refers to the research of Plasmodium sp. in cervidae in Brazil. A captive herd of 22 deer-bororó (Mazama nana), four deer-mateiro (Mazama americana) and six marsh deer (Blastocerus dichotomus) from southern Brazil were evaluated using light microscopy and molecular approaches. Microscopic and molecular analyses were both negative for parasite presence. Keywords: Hemotropic Mycoplasma, Hemoplasma, Wild Mammals, PCR, Pathogens, Plasmodium sp

ANÁLISE DE MICROORGANISMOS PATOGÊNICOS PARA AVALIAÇÃO DA BALNEABILIDADE

Para an&aacute;lises de qualidade de &aacute;gua, tem se utilizado as bact&eacute;rias do grupo coliforme como indicadores de contamina&ccedil;&atilde;o, supondo-se que haja uma rela&ccedil;&atilde;o entre a presen&ccedil;a destas e a ocorr&ecirc;ncia de microorganismos patog&ecirc;nicos. Mas esta rela&ccedil;&atilde;o deve ser verificada, pois se n&atilde;o existir realmente, pode estar mascarando os riscos &agrave; que a popula&ccedil;&atilde;o est&aacute; exposta. Este estudo teve como objetivo realizar an&aacute;lise de Salmonella sp., Cryptosporidium sp., Giardia sp. e Acanthamoeba sp. em 3 pontos do rio Tamandu&aacute;, no munic&iacute;pio de Foz do Igua&ccedil;u-PR, bem como, determinar a balneabilidade, relacionando-se esta classifica&ccedil;&atilde;o com a ocorr&ecirc;ncia dos pat&oacute;genos. Os pontos analisados foram classificados como pr&oacute;prios para o banho, na categoria excelente, por&eacute;m, houve a presen&ccedil;a de patog&ecirc;nicos em todos os pontos. A positividade para Cryptosporidium sp. e Acanthamoeba sp. esteve entre 80% e 100%, j&aacute; para Salmonella sp. variou de 0 a 80% e para Giardia ficou entre 0 e 20%. Foi poss&iacute;vel perceber que a classifica&ccedil;&atilde;o com base em coliformes n&atilde;o condiz com a presen&ccedil;a de microorganismos patog&ecirc;nicos, sendo que a popula&ccedil;&atilde;o pode estar exposta a riscos de sa&uacute;de se a qualidade for determinada somente atrav&eacute;s da quantifica&ccedil;&atilde;o de bact&eacute;rias do grupo coliforme

Detection of Plasmodium sp. in capybara

In the present study, we have microscopically and molecularly surveyed blood samples from 11 captive capybaras (Hydrochaeris hydrochaeris) from the Sanctuary Zoo for Plasmodium sp. infection. One animal presented positive on blood smear by light microscopy. Polymerase chain reaction was carried out accordingly using a nested genus specific protocol, which uses oligonucleotides from conserved sequences flanking a variable sequence region in the small subunit ribosomal RNA (ssrRNA) of all Plasmodium organisms. This revealed three positive animals. Products from two samples were purified and sequenced. The results showed less than 1% divergence between the two capybara sequences. When compared with GenBank sequences, a 55% similarity was obtained to Toxoplasma gondii and a higher similarity (73– 77.2%) was found to ssrRNAs from Plasmodium species that infect reptile, avian, rodents, and human beings. The most similar Plasmodium sequence was from Plasmodium mexicanum that infects lizards of North America, where around 78% identity was found. This work is the first report of Plasmodium in capybaras, and due to the low similarity with other Plasmodium species, we suggest it is a new species, which, in the future could be denominated ''Plasmodium hydrochaeri''

Pathology and first report of natural infections of the eye trematode Philophthalmus lachrymosus Braun, 1902 (Digenea, Philophthalmidae) in a non-human mammalian host

The avian eye trematode Philophthalmus lachrymosus Braun, 1902 is for the first time referred naturally occurring in a non-human mammalian host. Previously, natural infections with P. lachrymosus and other species of Philophthalmus have been occasionally reported from man, with few data on experimental infections of non-human mammals. Results presented here are related to the report of two cases of philophthalmosis due to natural infections of wild Brazilian capybaras, Hydrochaeris hydrochaeris L., 1766 with P. lachrymosus and associated pathology. Clinical signs, gross and microscopic lesions as well as new morphometric data on the parasite are presented

First molecular screening of Plasmodium species in ungulates from Southern Brazil

Abstract Objective Despite malaria epidemiology has been extensively studied in primates, few studies were conducted in ungulates. After half a century without descriptions of Plasmodium spp. in deer since its first identification, recent research has rediscovered Plasmodium on ungulates in Africa, Asia, North America and South America, including Central Brazil. Here, a captive herd was evaluated in southern Brazil using light microscopy and PCR. DNA samples were tested for fragment amplification of two Plasmodium spp. genes: mitochondrial cytochrome b and small subunit ribosomal RNA. Results All analyses were negative. However, the tests were performed on samples that were collected at a single time point, and parasitemia may fluctuate over the parasite’s life cycle. Thus, the possibility of occult infection cannot be ruled out. Despite the negative results of all of the methods applied, it cannot be categorically stated that these animals are free from Plasmodium sp. infection. Further monitoring and/or multiple sequential sampling may improve the success rate of detecting parasites. Moreover, although this survey of Plasmodium represents the first molecular study on ungulate malaria parasites from Southern Brazil, further analysis of samples from different ungulate species is important for characterizing the epidemiology of Plasmodium of these mammals in this region

Ophidascaris durissus sp. nov. (Nematoda Ascarididae) parasitizing Crotalus durissus Linnaeus (Ophidia, Viperidae) in Brazil

The species Ophidascaris durissus sp. nov. is proposed with basis on specimens recovered from the rattlesnake Crotalus durissus L., 1758 (type host) captured in Foz do Iguaçu, Brazil (type locality). By the lack of interlabia, the new species can be compared only to O. natricis Yamaguti, 1935 from Japan and O. freitasi Hoa & Lien, 1970, from Vietnam. However, O. durissus sp. nov. differs from O. natricis mainly by the absence of internal lip papillae, location of the vulvar aperture and length of the spicules; from O. freitasi mostly by the greater number of pre-cloacal and distribution of post-cloacal papillae

Interfacial stress and container failure during freezing of bulk protein solutions can be prevented by local heating

Lisboa 2020_Project 17653_CryocubeBottles and carboys are used for frozen storage and transport of biopharmaceutical formulations under a wide range of conditions. The quality of freezing and thawing in these systems has been questioned due to the formation of heterogeneous ice structures and deformation of containers. This work shows that during freezing of bulk protein solutions, the liquid at the air-liquid interface freezes first, forming an ice crust and enclosing the liquid phase. As the enclosed liquid freezes, internal pressure rises, pushing the liquid phase through the porous ice crust towards the air interface, leading to interfacial stress and protein aggregation. The aggregation of bovine serum albumin was more intense in the foam-like ice mound that was formed at the top, where bubbles were entrapped. This was characterized experimentally with the assistance of magnetic resonance imaging (MRI). An isothermal cover is proposed to prevent the early freezing of the liquid at the air interface, attenuating substantially interfacial stress to proteins and releasing hydrostatic pressure, preserving the shape and integrity of the containers.info:eu-repo/semantics/publishedVersio

Use of a Mycoplasma suis-PCR protocol for screening a population of captive peccaries (Tayassu tajacu and Tayassu pecari) Uso de um protocolo de PCR para a detecção de Mycoplasma suis para avaliação de uma população de catetos e queixadas de cativeiro (Tayassu tajacu and Tayassu pecari)

Mycoplasma suis is a hemotropic bacteria of red blood cells and the causative agent of swine eperythrozoonosis. Diagnosis of infection may be reached by direct examination of blood smears; however, the use of polymerase chain reaction (PCR) of the 16S RNA gene of M. suis improves the sensitivity and specificity of detection. The aim of this study was to screen peccaries (Tayassu tajacu and T. pecari) for M. suis infection using a specific conventional PCR. A total of 28 blood samples from captive collared and white-lipped peccaries were collected, DNA extracted and a specific M. suis PCR assay performed. All samples were negatives by both blood smear examination and PCR testing. To verify the presence of amplifiable DNA, PCR for beta-actin gene was performed in all samples. This study was part of an active surveillance program, which is crucial for monitoring animal health status, particularly in wildlife species.<br>Mycoplasma suis é uma bactéria hemotrópica dos eritrócitos e é o agente causador da eperitrozoonose suína. O diagnóstico da infecção pode ser realizado pelo exame direto de esfregaços sanguíneos; entretanto, o uso da reação em cadeia da polimerase (PCR) baseada no gene 16S RNA de M. suis aumenta a sensibilidade e especificidade da detecção. O objetivo deste estudo foi avaliar catetos e queixadas (Tayassu tajacu e T. pecari) para a infecção por M. suis, utilizando PCR convencional específico. Um total de 28 amostras de sangue de catetos e queixadas de cativeiro foram coletadas, o DNA foi extraído e a PCR específica para a detecção de M. suis realizada. Todas as amostras foram negativas pelo esfregaço sanguíneo e PCR. Para verificar a presença de DNA amplificável, PCR para o gene da beta actina foi realizada em todas as amostras. Este estudo foi parte de um programa de vigilância ativa, o qual é crucial para o monitoramento do estado de saúde animal, particularmente em espécies selvagens