Indirect ELISA (iELISA) standardization for the diagnosis of bovine enzootic leukosis / Padronização de um ELISA indireto (iELISA) para diagnóstico da leucose enzoótica bovina

Enzootic bovine leukosis (EBL) is an infectious disease caused by bovine leukemia virus (BLV) that affects cattle worldwide. Agar gel immunodiffusion (AGID) was the reference test for EBL diagnosis for many years, but enzyme-linked immunosorbent assay (ELISA) showed higher sensitivity, was faster to perform, and resulted in an objective reading. However, the importation of ELISA kits is lengthy and expensive, and currently, no AGID kits are available in Brazil. The aim of this work was to standardize an indirect ELISA (iELISA) for EBL diagnosis using BLV antigens produced in Tadarida brasiliensis lung (Tb1Lu) cells, which are Bovine viral diarrhea virus (BVDV) free, unlike fetal lamb kidney (FLK) cells, currently used for this purpose. Following standardization, iELISA results were compared with those obtained by AGID and the commercial Chekit Leucose-Serum ELISA. Compared to AGID, iELISA had 94,44% sensitivity, 75.68% specificity, 79.10% positive predictive value (PPV) and 93.30% negative predictive value (NPV), with 84% concordance and a Kappa index of 0.699. Compared to the Chekit Leucose-Serum ELISA, iELISA showed 92.60% sensitivity, 87.09% specificity, 90.27% PPV and 90,00% NPV, with 90.27% concordance and a Kappa index of 0.801. Taking into account the high agreement with the traditional tests and the absence of non-specific reactions with BVDV, the developed assay could be used as diagnostic method to control EBL in Brazil.(AU)

A leucose enzoótica bovina (LEB) é uma doença infecciosa natural dos bovinos com distribuição mundial causada pelo "bovine leukemia virus" (BLV). A imunodifusão em gel de ágar (IDGA) foi considerada por muitos anos o teste de eleição, porém ensaios imunoenzimáticos (ELISA) apresentam sensibilidade mais elevada e leitura mais rápida e objetiva. No entanto, a importação de kits de ELISA é um processo dispendioso e demorado, e atualmente não há kits de IDGA comercialmente disponíveis no Brasil. Desta forma, o objetivo deste trabalho foi padronizar um ELISA indireto (iELISA) para diagnóstico da LEB utilizando antígenos produzidos a partir do cultivo do BLV em linhagem celular Tadarida brasiliensis "lung" (Tb1Lu) livre de "bovine viral diarrhea virus" (BVDV), diferentemente do que acontece com as linhagens "fetal lamb kidney" (FLK) atualmente utilizadas na produção desses antígenos para uso em ensaios sorológicos. Após a padronização do iELISA, os resultados foram comparados com aqueles obtidos por IDGA e pelo ELISA comercial "Chekit Leucose-Serum". Comparado ao IDGA, o iELISA apresentou 94,44% de sensibilidade, 75,68% de especificidade, valor preditivo positivo (VPP) de 79,1% e valor preditivo negativo (VPN) de 93,3%, com concordância entre os testes de 84% e o índice Kappa 0,699. Quando comparado ao ELISA "Chekit Leucose-Serum", o iELISA apresentou sensibilidade de 92,6%, especificidade de 87,09%, VPP de 90,27% e VPN de 90%, com concordância de 90,27% e o índice Kappa 0,801. Portanto, devido à alta concordância com os testes tradicionais e ausência da ocorrência de reações inespecíficas com BVDV, o ensaio desenvolvido pode ser utilizado como ferramenta diagnóstica para o controle da LEB no Brasil.(AU)

Indirect ELISA (iELISA) standardization for the diagnosis of bovine enzootic leukosis / Padronização de um ELISA indireto (iELISA) para diagnóstico da leucose enzoótica bovina

Enzootic bovine leukosis (EBL) is an infectious disease caused by bovine leukemia virus (BLV) that affects cattle worldwide. Agar gel immunodiffusion (AGID) was the reference test for EBL diagnosis for many years, but enzyme-linked immunosorbent assay (ELISA) showed higher sensitivity, was faster to perform, and resulted in an objective reading. However, the importation of ELISA kits is lengthy and expensive, and currently, no AGID kits are available in Brazil. The aim of this work was to standardize an indirect ELISA (iELISA) for EBL diagnosis using BLV antigens produced in Tadarida brasiliensis lung (Tb1Lu) cells, which are Bovine viral diarrhea virus (BVDV) free, unlike fetal lamb kidney (FLK) cells, currently used for this purpose. Following standardization, iELISA results were compared with those obtained by AGID and the commercial Chekit Leucose-Serum ELISA. Compared to AGID, iELISA had 94,44% sensitivity, 75.68% specificity, 79.10% positive predictive value (PPV) and 93.30% negative predictive value (NPV), with 84% concordance and a Kappa index of 0.699. Compared to the Chekit Leucose-Serum ELISA, iELISA showed 92.60% sensitivity, 87.09% specificity, 90.27% PPV and 90,00% NPV, with 90.27% concordance and a Kappa index of 0.801. Taking into account the high agreement with the traditional tests and the absence of non-specific reactions with BVDV, the developed assay could be used as diagnostic method to control EBL in Brazil.(AU)

A leucose enzoótica bovina (LEB) é uma doença infecciosa natural dos bovinos com distribuição mundial causada pelo "bovine leukemia virus" (BLV). A imunodifusão em gel de ágar (IDGA) foi considerada por muitos anos o teste de eleição, porém ensaios imunoenzimáticos (ELISA) apresentam sensibilidade mais elevada e leitura mais rápida e objetiva. No entanto, a importação de kits de ELISA é um processo dispendioso e demorado, e atualmente não há kits de IDGA comercialmente disponíveis no Brasil. Desta forma, o objetivo deste trabalho foi padronizar um ELISA indireto (iELISA) para diagnóstico da LEB utilizando antígenos produzidos a partir do cultivo do BLV em linhagem celular Tadarida brasiliensis "lung" (Tb1Lu) livre de "bovine viral diarrhea virus" (BVDV), diferentemente do que acontece com as linhagens "fetal lamb kidney" (FLK) atualmente utilizadas na produção desses antígenos para uso em ensaios sorológicos. Após a padronização do iELISA, os resultados foram comparados com aqueles obtidos por IDGA e pelo ELISA comercial "Chekit Leucose-Serum". Comparado ao IDGA, o iELISA apresentou 94,44% de sensibilidade, 75,68% de especificidade, valor preditivo positivo (VPP) de 79,1% e valor preditivo negativo (VPN) de 93,3%, com concordância entre os testes de 84% e o índice Kappa 0,699. Quando comparado ao ELISA "Chekit Leucose-Serum", o iELISA apresentou sensibilidade de 92,6%, especificidade de 87,09%, VPP de 90,27% e VPN de 90%, com concordância de 90,27% e o índice Kappa 0,801. Portanto, devido à alta concordância com os testes tradicionais e ausência da ocorrência de reações inespecíficas com BVDV, o ensaio desenvolvido pode ser utilizado como ferramenta diagnóstica para o controle da LEB no Brasil.(AU)

Geographic variation in ectoparasitic mites diversity in Tadarida Brasiliensis (Chiroptera, Molossidae) / Variação geográfica na diversidade de ácaros ectoparasitos em Tadarida Brasiliensis (Chiroptera, Molossidae)

Tadarida brasiliensis (Geoffroy, 1824), the Brazilian free-tailed bat, is an insectivorous bat that occurs from southern United States of America to southern South America. In this study we present the first data on diversity of ectoparasitic mites of T. brasiliensis in Brazil. A compilation and analysis of the studies of mite diversity conducted in different points the geographic distribution this bat species are provided. The mites were collected from March 2010 to November 2011 on 160 T. brasiliensis adult bats captured in southern Brazil. Four species of mites have been found: Chiroptonyssus robustipes (Ewing, 1925), Ewingana longa (Ewing, 1938), Ewingana inaequalis (Radford, 1948), and specimens of Cheyletidae. Chiroptonyssus robustipes was the most prevalent species (100%), followed by E. longa (20%), E. inaequalis (10%), and specimens of Cheyletidae (1.25%). The data currently available show that C. robustipes parasitizes T. brasiliensis throughout its region of occurrence, and this mite is highly prevalent and abundant. The two species of Ewingana accompany the geographical distribution of T. brasiliensis, but with much lower prevalence and abundance.(AU)

Tadarida brasiliensis (Geoffroy, 1824) é um morcego insetívoro que ocorre desde o sul dos Estados Unidos até o sul da América do Sul. Neste estudo são apresentados os primeiros dados sobre diversidade de ácaros ectoparasitos de T. brasiliensis no Brasil e é disponibilizada uma compilação e análise dos estudos de diversidade de ácaros realizados nos diferentes pontos da distribuição geográfica desta espécie de morcego. Os ácaros foram coletados de 160 espécimes adultos de T. brasiliensis capturados no extremo sul do Brasil, entre março de 2010 e novembro de 2011. Quatro espécies de ácaros foram coletadas: Chiroptonyssus robustipes (Ewing, 1925), Ewingana longa (Ewing, 1938), Ewingana inaequalis (Radford, 1948) e espécimes de Cheyletidae. Chiroptonyssus robustipes foi a espécie mais prevalente (100%), seguido de E. longa (20%), E. inaequalis (10%) e exemplares de Cheyletidae (1,25%). Os dados disponíveis atualmente demonstram que C. robustipes parasita T. brasiliensis em toda sua região de ocorrência e que este ácaro é altamente prevalente e abundante. As duas espécies de Ewingana acompanham a distribuição geográfica de T. brasiliensis, porém com prevalências e abundâncias muito menores.(AU)

Geographic variation in ectoparasitic mites diversity in Tadarida Brasiliensis (Chiroptera, Molossidae) / Variação geográfica na diversidade de ácaros ectoparasitos em Tadarida Brasiliensis (Chiroptera, Molossidae)

Tadarida brasiliensis (Geoffroy, 1824), the Brazilian free-tailed bat, is an insectivorous bat that occurs from southern United States of America to southern South America. In this study we present the first data on diversity of ectoparasitic mites of T. brasiliensis in Brazil. A compilation and analysis of the studies of mite diversity conducted in different points the geographic distribution this bat species are provided. The mites were collected from March 2010 to November 2011 on 160 T. brasiliensis adult bats captured in southern Brazil. Four species of mites have been found: Chiroptonyssus robustipes (Ewing, 1925), Ewingana longa (Ewing, 1938), Ewingana inaequalis (Radford, 1948), and specimens of Cheyletidae. Chiroptonyssus robustipes was the most prevalent species (100%), followed by E. longa (20%), E. inaequalis (10%), and specimens of Cheyletidae (1.25%). The data currently available show that C. robustipes parasitizes T. brasiliensis throughout its region of occurrence, and this mite is highly prevalent and abundant. The two species of Ewingana accompany the geographical distribution of T. brasiliensis, but with much lower prevalence and abundance.

Tadarida brasiliensis (Geoffroy, 1824) é um morcego insetívoro que ocorre desde o sul dos Estados Unidos até o sul da América do Sul. Neste estudo são apresentados os primeiros dados sobre diversidade de ácaros ectoparasitos de T. brasiliensis no Brasil e é disponibilizada uma compilação e análise dos estudos de diversidade de ácaros realizados nos diferentes pontos da distribuição geográfica desta espécie de morcego. Os ácaros foram coletados de 160 espécimes adultos de T. brasiliensis capturados no extremo sul do Brasil, entre março de 2010 e novembro de 2011. Quatro espécies de ácaros foram coletadas: Chiroptonyssus robustipes (Ewing, 1925), Ewingana longa (Ewing, 1938), Ewingana inaequalis (Radford, 1948) e espécimes de Cheyletidae. Chiroptonyssus robustipes foi a espécie mais prevalente (100%), seguido de E. longa (20%), E. inaequalis (10%) e exemplares de Cheyletidae (1,25%). Os dados disponíveis atualmente demonstram que C. robustipes parasita T. brasiliensis em toda sua região de ocorrência e que este ácaro é altamente prevalente e abundante. As duas espécies de Ewingana acompanham a distribuição geográfica de T. brasiliensis, porém com prevalências e abundâncias muito menores.

Pneumocystis spp. and Histoplasma capsulatum in bats lungs in Southern and Midwestern regions of Brazil / Pneumocystis spp. e Histoplasma capsulatum detectados em pulmões de morcegos das regiões Sul e Centro-Oeste do Brasil

Background: Histoplasma capsulatum and Pneumocystis spp. may cause a host infection through the respiratory airway, mainlyaffecting the pulmonary tissue. These fungal pathogens affect a wide range of mammalian species, including humans and bats.The co-infection of bats with both organisms above has never been studied in Brazil. The aim of the present research was todetect the presence of the H. capsulatum and Pneumocystis spp. in lung samples of bat species from two states of Brazil. For thispurpose, a highly sensitive nested PCR was used with specific molecular markers for each pathogen.Materials, Methods & Results: Two hundred and forty-nine bats were captured between 2007 and 2009 in caves, forests, andurban areas of Mato Grosso (MT) and Rio Grande do Sul (RS), located respectively in the Mid-Western and Southern regions. Thebats were captured following the guidelines of the rabies control manual for herbivores, standardized by the Ministry of the Agriculture. Detection of Pneumocystis spp. DNA was based upon nested PCR, which amplified a portion of the mitochondrial smallsubunit (mtSSU) of the rRNA gene, whereas the H. capsulatum DNA was amplified employing the Hcp 100 locus. Amplificationproducts were sequenced to confirm fungal presence in bat lungs. The amplifications results for H. capsulatum and Pneumocystisspp. were positive in 63 [25.3%, IC95% (20.1%-31.25%)] and 95 [(38,2%, IC95% (32.1%-44.52%)] samples, respectively. Thegreatest occurrence of Histoplasma capsulatum was observed in Desmodus rotundus (20.6%), Tadarida brasiliensis (20.6%),Histiotus velatus (19.0%) and Molossus molossus (11.1%), with the detection in the other species being lower than 7.9%, amongthe 24 studied bat species. For Pneumocystis spp., the detection was higher in Tadarida brasiliensis (23.1%), Desmodus rotundus(18.%), Histiotus velatus (14.7%), and Molossus molossus (11,6%), being lower than 5.3% in the other species. A co-infection...(AU)

Pneumocystis spp. and Histoplasma capsulatum in bats lungs in Southern and Midwestern regions of Brazil / Pneumocystis spp. e Histoplasma capsulatum detectados em pulmões de morcegos das regiões Sul e Centro-Oeste do Brasil

Background: Histoplasma capsulatum and Pneumocystis spp. may cause a host infection through the respiratory airway, mainlyaffecting the pulmonary tissue. These fungal pathogens affect a wide range of mammalian species, including humans and bats.The co-infection of bats with both organisms above has never been studied in Brazil. The aim of the present research was todetect the presence of the H. capsulatum and Pneumocystis spp. in lung samples of bat species from two states of Brazil. For thispurpose, a highly sensitive nested PCR was used with specific molecular markers for each pathogen.Materials, Methods & Results: Two hundred and forty-nine bats were captured between 2007 and 2009 in caves, forests, andurban areas of Mato Grosso (MT) and Rio Grande do Sul (RS), located respectively in the Mid-Western and Southern regions. Thebats were captured following the guidelines of the rabies control manual for herbivores, standardized by the Ministry of the Agriculture. Detection of Pneumocystis spp. DNA was based upon nested PCR, which amplified a portion of the mitochondrial smallsubunit (mtSSU) of the rRNA gene, whereas the H. capsulatum DNA was amplified employing the Hcp 100 locus. Amplificationproducts were sequenced to confirm fungal presence in bat lungs. The amplifications results for H. capsulatum and Pneumocystisspp. were positive in 63 [25.3%, IC95% (20.1%-31.25%)] and 95 [(38,2%, IC95% (32.1%-44.52%)] samples, respectively. Thegreatest occurrence of Histoplasma capsulatum was observed in Desmodus rotundus (20.6%), Tadarida brasiliensis (20.6%),Histiotus velatus (19.0%) and Molossus molossus (11.1%), with the detection in the other species being lower than 7.9%, amongthe 24 studied bat species. For Pneumocystis spp., the detection was higher in Tadarida brasiliensis (23.1%), Desmodus rotundus(18.%), Histiotus velatus (14.7%), and Molossus molossus (11,6%), being lower than 5.3% in the other species. A co-infection...

Molecular detection of Neorickettsia risticii in Brazilian free-tailed bats (Tadarida brasiliensis) from Buenos Aires, Argentina

Neorickettsia risticii is the causative agent of Potomac Horse Fever, a severe febrile disease affecting horses, transmitted by trematodes species with a complex life cycle. A total of 30 insectivorous bats (Brazilian free-tailed bat Tadarida brasiliensis) were analyzed by PCR for presence of genus Anaplasma, Ehrlichia, Neorickettsia and Rickettsia. Three samples showed positive reactions for genus Anaplasma, Ehrlichia and Neorickettsia, and the sequences were 99.67% identical to Neorickettsia risticii. The role of bats in the life cycle of N. risticii has yet to be elucidated; however bats may be reservoirs for this bacterium. To our knowledge, this is the first evidence of N. risticii in Argentina.(AU)

ESTUDO METAGENÔMICO PARA MONITORAMENTO DE VÍRUS EM AMOSTRAS DE MORCEGOS DO PROGRAMA DA RAIVA DO RIO GRANDE DO SUL

Morcegos são reservatórios naturais de uma grande variedade de vírus de interesse a saúde pública, incluindo vírus zoonóticos que podem causar patologias graves aos humanos. Dados epidemiológicos demonstram que o morcego é a espécie animal responsável pela maioria dos casos de raiva humana no Brasil nas últimas duas décadas. A mudança no perfil epidemiológico desta doença, que anteriormente era predominantemente transmitida por cães, gerou um aumento nas amostras de morcegos enviadas aos órgãos públicos para a vigilância do vírus rábico. No presente estudo, amostras obtidas de morcegos urbanos da espécie Tadarida brasiliensis monitorados no âmbito do Programa de Controle e Profilaxia da Raiva do Estado do Rio Grande do Sul foram analisadas por abordagem de metagenômica, com objetivo de identificar outros vírus, que representassem risco à saúde pública ou não, colaborando para a melhor compreensão da diversidade viral destes animais. Foram encontradas sequências relacionadas a 04 famílias virais que infectam uma ampla variedade de hospedeiros: Anelloviridae, Circoviridae, Papillomaviridae e Poxviridae. Estes achados auxiliam no melhor conhecimento sobre os vírus carreados por morcegos desta região e demonstram que o uso da metagenômica como instrumento de vigilância amplia a gama de vírus de interesse que podem ser monitorados nestas amostras.

Bats are natural reservoirs for a wide variety of viruses of public health concern, including zoonotic viruses that can cause severe diseases in humans. Epidemiological data revealed that bats are the animal species responsible for most cases of human rabies in Brazil in the last two decades. This change in the rabies epidemiological profile, which was previously predominantly transmitted by dogs, generated an increase in the number of bats samples sent to public organs for surveillance of rabies virus. In the present study, samples obtained from urban bats from the species Brazilian free-tailed bat, monitored under the Rio Grande do Sul State Rabies Control and Prophylaxis Program were analyzed by a metagenomic approach with the objective of identifying other viruses, that pose as a risk to public health or not, providing a better understanding of bats viral diversity locally and in the general way. Sequences related to 04 viral families that infect a wide variety of hosts have been found: Anelloviridae, Circoviridae, Papillomaviridae e Poxviridae. These findings improves the knowledge about the viruses carried by these animals and demonstrate that the use of metagenomics as a surveillance tool expand the range of viruses of public health concern investigated in these samples.

Estudo da mcrobiota fúngica gastrintestinal de morcegos (Mammalia, Chiroptera) da região noroeste do estado de São Paulo: potencial zoonótico / Study of gastrointestinal fungal flora of bats (Mammalia, Chiroptera) of the northwest region of São Paulo state: zoonotic potential

Os morcegos são hospedeiros de uma rica diversidade de microrganismos. Muitos trabalhos apontam uma estreita ligaçãocom o crescimento e a disseminação dos microrganismos na natureza e qual a implicação dos quirópteros no ciclo epidemiológico.

Bats are hosts of a rich diversity of microorganisms. Many studies indicate a close link between bats and fungi with pathogenic potential, especially for living in environments such as caves, caverns and hollow trees, favorable to the maintenance and spread of fungi. The objective was to study the gastrointestinal mycoflora of bats. Of the 98 samples belonging to 11 species of bats coming from 15 studied cities, 20% of the species were Carollia perspicillata, 19% Artibeus lituratus, 17% Molossus rufus, 13% Glossophaga soricina, 9% Nyctinomops macrotis, 8% Molossus molossus, 7% Desmodus rotundus, 2% Lasiurus ega and 1% Eptesicus furinalis, Myotis nigricans and Tadarida brasiliensis. The genus Aspergillus sp. was isolated from 29% of the samples, followed by 6% Microsporum sp. and Penicillium sp. 4% Trichophyton sp. and zygomycetes and 2% Fusarium sp. Of yeast species, 14% were from Rhodotorula sp., 10% Candida sp. and 2% Cryptococcus sp., 22% of isolates remained unidentified. All 82 cultures of organs were negative for Histoplasma capsulatum. There was a statistically significant association between the results of microbiological culture and bat species (p < 0.05). We conclude that the bats can act as disperser agents of fungi with pathogenic potential, although other studies should be performed to establish strategies to identify the main factors correlated with the growth and spread of microorganisms in nature and implication of bats in the epidemiological cycle.(AU)

Real-time PCR and nested-PCR assays for detection of Pneumocystis sp. in lung tissues of bats

Background: Pneumocystis constitutes a highly diversified biological group, with numerous species, which are strongly host-specific and well adapted to live inside the lungs of a diverse range of mammals. The detection of DNA from Pneumocystis in clinical specimens by PCR assays is leading to important advances in pneumonia caused by Pneumocystis and its epidemiology. The aim of this study was to analyze two different diagnostic methods, real-time PCR (qPCR) using primers based in the Major Surface Glycoprotein (MSG) of Pneumocystis sp. and conventional nested PCR using primers designed to the small subunit of mitochondrial rRNA (mtSSU rRNA) for detection of Pneumocystis DNA in lung tissue from bats. Materials, Methods & Results: Bats (195 samples) were captured (2007-2009) in caves, forests, and urban areas, were obtained from the Program of Rabies Control of two states in Brazil: Mato Grosso and Rio Grande do Sul, located respectively in the Mid-Western and Southern regions of the country approximately 2000 km apart. Lung tissue (250 mg) was finely minced, homogenized with crushing and DNA extraction was carried out with commercial kit. DNA samples the lung tissue of bats were analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial small subunit rRNA (mtSSU rRNA) and Taqman probe and primers for qPCR were selected based on the Major Surface Glycoprotein (MSG) of Pneumocystis sp. Chi-square (P < 0.001 was considered signifi cant) and the McNemar's test was used to analyze nested PCR and qPCR as methods of detection of Pneumocystis sp. and the Kappa was calculated by Win Episcope 2.0. To assess the sensitivity and specificity of the qPCR assay, a nested PCR assay was considered as the reference method. The positivity was 36.4% in the nested PCR and 24.1% using the qPCR. Concordance was obtained in 68.2% of the samples (133/195). It was demonstrated that there was no statistically significant difference between the techniques used and, both tests proved to be specific for the detection of Pneumocystis species. Specificity was 71% for the nested PCR and 84.6% for the qPCR. Pneumocystis was detected (71/195) by the nested PCR assay in 14 species:. Tadarida brasiliensis, Histiotus velatus, Desmodus rotundus, Molossus molossus, Glossophaga soricina, Nyctinomops laticaudatus, Promops nasutus, Artibeus sp., Eptesocus furinalus, Lasurus blossevillii, Molossus currentium, Molossus rufus, Myotis levis and Nyctinomops macrotis. Discussion: This study detected the DNA from Pneumocystis through the nested PCR and qPCR assays, and the frequency found is comparable to that obtained in a previous study, which used the nested PCR in Central American, South American and European countries. Pneumocystis sp. was observed in a high number of different bat species (14) in two Brazilian States (RS and MT). The qPCR showed a higher specificity in comparison to the nested PCR. The literature has similar findings to the results obtained by this research, employing the same tests and genes. The nested PCR and qPCR assays are indicated in the diagnosis of Pneumocystis sp. in bats and it is important to highlight that a better diagnostic precision is achieved with the association of both tests. Additionally, this study was the first to detect Pneumocystis sp. in the lungs of bats using qPCR.

METAVIROMA DE MORCEGOS TADARIDA BRASILIENSIS

Tadarida brasiliensis é uma espécie de morcego típica das Américas adaptada a áreas urbanas, facilitando o contato com e a disseminação de diversos agentes virais. Dentre estes, os Coronavirus (CoV) se destacam para vigilância sanitária e epidemiológica, pois nas últimas décadas surgiram estirpes altamente patogênicas que evoluíram a partir dos morcegos. Os objetivos deste trabalho foram realizar a metagenômica viral (metaviroma) e estabelecer as relações filogenéticas dos Coronavírus identificados. Foram utilizados 10 swabs anais e 10 swabs traqueais de 10 morcegos da espécie T. brasiliensis coletados em 2011 no Bosque dos Jequitibás, Campinas, Estado de São Paulo, Brasil. As amostras foram submetidas à Next-Gen Sequencing (NGS) usando a plataforma Illumina HiSeq 2500, obtendo-se 345.409.110 reads paired-ends das quais 76,47% apresentaram Q 30. Foram utilizadas duas plataformas distintas para montagem dos contigs e scaffolds, MetaVelvet e Metavir 2, cujos resultados foram semelhantes. A busca por similaridade BLAST foi conduzida a partir de diferentes bancos de dados. Quando submetidas ao UniRef90, obteve-se 97 matches com sequências de origem viral de grande interesse para vigilância epidemiológica, sendo 2 para Coronavírus. Utilizando apenas bancos de dados específicos para sequências de origem viral, observou-se 827 matches, dos quais 6 com Alphacoronavirus não classificados. Aproximadamente 85% das sequências apresentaram similaridade com vírus de DNA fita dupla enquanto que os 15% foram divididos entre vírus de RNA fita simples (5%), Orthoretrovirinae (4%), vírus de RNA fita dupla (2%), fagos não classificados (2%), vírus de DNA fita simples (0.8%) e 0.2% das sequências apresentaram similaridade com Muska hytrovirus. As análises filogenéticas realizadas apenas para os matches de Coronavírus incluíram sequências representativas de todos os gêneros alfa, beta, gama e DeltaCoV e foram realizadas utilizando métodos de máxima verossimilhança (ML) e neighbor-joining. Foi também identificado sequências de estreita relação filogenéticas com AlphaCoV-like, Appalachian Ridge Cov.2, Porcine Epidemic Diarrea Virus (PEDV), HCoV-NL63, Bat Coronavírus 1B, agentes de importância na clínica médica humana e veterinária. Deste modo, pode-se concluir que a abordagem metagenômica realizada com NGS representa uma metodologia com grande potencial discriminatório ou de identificação em estudos epidemiológicos. Considerando-se o impacto zoonótico de muitos CoV, os resultados deste trabalho contribuem fortemente para uma melhor compreensão da eco-epidemiologia molecular na evolução desses agentes virais.

Tadarida brasiliensis is a bats' typical species of the Americas adapted to urban areas, enabling contact and spread of several viral agents. Among them, coronavirus (CoV) stand out for sanitary and epidemiological surveillance, once in recent decades emerge highly pathogenic strains that evolved from bats. The goals of this study were to perform viral metagenomics (metavirome) and establish the phylogenetic relationships of the identified coronavirus. We used 10 anal and 10 tracheal swabs 10 of T. brasiliensis bats specimens collected in 2011 Jequitibás Wood, Campinas, São Paulo, Brazil. Samples were subjected to Next Gen-Sequencing (NGS) using the Illumina platform HiSeq 2500, yielding 345 409 110 reads paired ends of which 76.47% had Q 30. We used two separate platforms for assembly of contigs and scaffolds, MetaVelvet and Metavir 2, which showed similar results. The BLAST similarity search was conducted from different databases. When subjected to UniRef90, it obtained 97 matches with sequences of viral origin of great interest for health surveillance, including 2 coronavirus. Using only specific databases for viral sequences, there was 827 matches, of which 6 with Alphacoronavirus unclassified. About 85% of sequences showed similarity to dsDNA virus no RNA stage while 15% were divided in ssRNA virus (5%), Orthoretrovirinae (4%), dsRNA viruses (2%), Unclassified phage (2%), ssDNA virus (0.8%) and 0.2% of the sequences showed similarity to Muska hytrovirus. Phylogenetic analyzes only for coronavirus matches included representative sequences of all genuses alpha, beta, gamma and deltaCoV and were being performed using maximum likelihood methods (ML) and neighbor-joining (NJ). We identify sequences phylogenetically related AlphaCoV-like, Appalachian Ridge Cov.2, Porcine Epidemic Diarrhea Virus (PEDV), HCoV-NL63, Bat Coronavirus 1B, viruses of importance in human and veterinary medicine. Thus, we conclude that metagenomic approach performed with NGS represents a powerful methodology for epidemiological studies. Considering the zoonotic impact of many CoV, our results contribute greatly to a better understanding of the molecular eco-epidemiology in the evolution of these viral agents.

First record of albinism in Tadarida brasiliensis (Chiroptera: Molossidae) in South America and new records of leucism in central Argentina

Pigmentary disorders such as albinism and leucism are rare in bats. These phenomena are caused by genetic mutations resulting in complete absence of pigmentation in eyes, skin and fur (albinism) or partial or total deficiency of pigment in all the body or parts (leucism). While monitoring a maternal colony of Tadarida brasiliensis located in Rosario, central Argentina, one albino specimen and other 12 with different kinds of leucism were recorded. The albino T. brasiliensis represents the first record for Argentina and South America. Shapes and location of these pigmentary disorders were described in leucitic individuals and compared with previous records from other authors. Here we report a new type of leucism not described until now. These records constitute the first description for South America of many individuals with different leucitic patterns in the same colony. Possible ecological implications for these atypical patterns are discussed. Keywords: Bats; fur patterns; pigmentary disorders; Brazilian free-tailed bat.

First record of albinism in Tadarida brasiliensis (Chiroptera: Molossidae) in South America and new records of leucism in central Argentina

Pigmentary disorders such as albinism and leucism are rare in bats. These phenomena are caused by genetic mutations resulting in complete absence of pigmentation in eyes, skin and fur (albinism) or partial or total deficiency of pigment in all the body or parts (leucism). While monitoring a maternal colony of Tadarida brasiliensis located in Rosario, central Argentina, one albino specimen and other 12 with different kinds of leucism were recorded. The albino T. brasiliensis represents the first record for Argentina and South America. Shapes and location of these pigmentary disorders were described in leucitic individuals and compared with previous records from other authors. Here we report a new type of leucism not described until now. These records constitute the first description for South America of many individuals with different leucitic patterns in the same colony. Possible ecological implications for these atypical patterns are discussed. Keywords: Bats; fur patterns; pigmentary disorders; Brazilian free-tailed bat.(AU)

Canine rabies in Rio Grande do Sul caused by an insectivorous bat rabies virus variant

Rabies is caused by rabies virus (RV), a RNA virus member of the Lyssavirus genus, family Rhabdoviridae. The aim of this study was to determine antigenic characteristics of a rabies virus isolate (RV183-07) recovered from a stray bitch that died of rabies and to infer the most likely source of contamination, since no urban rabies has been reported in the area in more than 20 years. The virus was identified by direct immunofluorescence and multiplied by one passage in mice. The antigenic profile of the isolate was determined with a panel of monoclonal antibodies to lyssavirus antigens on infected brain tissues. A fragment of the viral genome corresponding to the nucleoprotein (N) gene was submitted to reverse transcription/polymerase chain reaction and the amplicon obtained was subjected to restriction enzyme analysis. A 303 base pair fragment of the N gene was cloned, sequenced and compared to other RV sequences available at Genbank. The isolate RV183-07 displayed antigenic and genomic characteristics of rabies virus variants whose natural reservoir is the non-hematophagous bat Tadarida brasiliensis. Therefore, the most likely source of contamination of the bitch was an incidental contact with an infected bat of that species, common inhabitants of the area. In view of that, the status of urban rabies-free of the area was not compromised.

Detection of Pneumocystis in lungs of bats from Brazil by PCR amplification / Detecção de Pneumocystis em pulmões de morcegos no Brasil por Nested-PCR

Pneumocystis has been isolated from a wide range of unrelated mammalian hosts, including humans, domestic and wild animals. It has been demonstrated that the genome of Pneumocystis of one host differs markedly from that of other hosts. Also, variation in the chromosome and DNA sequence of Pneumocystis within a single host species has been observed. Since information about the occurrence and nature of infections in wild animals is still limited, the objective of this work was to detect the presence of Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. The bats, captured in caves and in urban areas, were obtained from the Program of Rabies Control of two States in Brazil, Mato Grosso and Rio Grande do Sul, located in the Mid-Western and Southern regions of the country, respectively. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA). Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10R-RI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25), Desmodus rotundus (20), and Nyctinomops laticaudatus (19). Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3 percent = 5/19), Tadarida brasiliensis (24 percent = 6/25), and Desmodus rotundus (20 percent = 4/20). Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1 percent), Artibeus fimbriatus (1/1, 100 percent), Sturnira lilium (1/3, 33.3 percent), Myotis levis (2/3, 66.7 percent)and Diphylla ecaudata (1/2, 50 percent). PCR products which could indicate the presence of Pneumocystis (21.56 percent) were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified). This is the ...(AU)

Pneumocystis tem sido isolado de uma grande variedade de hospedeiros mamíferos, incluindo humanos, animais domésticos e selvagens. Tem se demonstrado que o genoma do Pneumocystis de um hospedeiro difere marcadamente do de outros, assim como há variação no cromossomo e na seqüência de DNA dentro de uma única espécie de hospedeiro. Sabendo que a informação da ocorrência e natureza da infecção em animais silvestres ainda é limitada, o objetivo do trabalho foi detectar, por Nested-PCR, a presença de Pneumocystis sp. em pulmões de diferentes espécies de morcegos de dois estados do Brasil. Estes mamíferos voadores foram capturados em cavernas, áreas florestadas, de campo e urbanas pelo Programa de Controle da Raiva do Mato Grosso (região Centro-Oeste) e do Instituto de Pesquisas Veterinárias Desidério Finamor (RS) e Instituto Sauver no Rio Grande do Sul (região Sul). Os DNAs foram extraídos de 102 pulmões e realizado Nested-PCR utilizando os primers pAZ102H-pAZ102E e pAZ102X/R1-pAZY/R1 para amplificação do gene mtLSU-rRNA, e pAZ102 10F-RI - pAZ102 10R-RI e pAZ102 13-RI - pAZ14-RI para amplificação do gene mtSSU-rRNA. As espécies mais freqüentes foram Tadarida brasiliensis (25), Desmodus rotundus (20) e Nyctinomops laticaudatus (19). Pneumocystis foi detectado com maior prevalência nas Nyctinomops laticaudatus (26,3 por cento = 5/19), Tadarida brasiliensis (24 por cento = 6/25) e Desmodus rotundus (20 por cento = 4/20). Além destas espécies, Pneumocystis foi também detectado nos pulmões de Molossus molossus (1/11, 9,1 por cento), Artibeus fimbriatus (1/1, 100 por cento), Sturnira lilium (1/3, 33 por cento), Myotis levis (2/3, 66,7 por cento)e Diphylla ecaudata (1/2, 50 por cento). Os produtos de PCR indicaram a presença de Pneumocystis (21.56 por cento) em amostras obtidas de 8 das 16 espécies classificadas para ambos os estados (cinco morcegos não foram classificados). Este é o primeiro registro de detecção de Pneumocystis em morcegos no Brasil.(AU)

Bat predation by Barn Owls (Tyto alba) in central-eastern Argentina

We report the predation of bats by Barn owls (Tyto alba) in Entre Ríos province (~33.2º S-33.7º S), central-eastern Argentina. We studied pellet samples from 87 localities, registering 12 species of bats of two families. The best-represented taxa in the samples were the molossids Tadarida brasiliensis (40.7%), Molossus molossus (8.7%), the vespertilionids Myotis spp. (19.67%) and Eptesicus furinalis (9.2%). Molossops temminckii was documented for the first time in the province. The average weight of bats consumed by Barn Owl ranged from ~4 g (Myotis spp.) to ~68 g (Eumops perotis). In terms of frequency, bats represented between 0.04 and 5.11% of the owls diet. Our results, together with literature records, suggest that bats tend to be captured more regularly in tropical to subtropical areas than in temperate to cold ones, and that they represent a minor item in the diet of Barn Owls.

Bat predation by Barn Owls (Tyto alba) in central-eastern Argentina

We report the predation of bats by Barn owls (Tyto alba) in Entre Ríos province (~33.2º S-33.7º S), central-eastern Argentina. We studied pellet samples from 87 localities, registering 12 species of bats of two families. The best-represented taxa in the samples were the molossids Tadarida brasiliensis (40.7%), Molossus molossus (8.7%), the vespertilionids Myotis spp. (19.67%) and Eptesicus furinalis (9.2%). Molossops temminckii was documented for the first time in the province. The average weight of bats consumed by Barn Owl ranged from ~4 g (Myotis spp.) to ~68 g (Eumops perotis). In terms of frequency, bats represented between 0.04 and 5.11% of the owls diet. Our results, together with literature records, suggest that bats tend to be captured more regularly in tropical to subtropical areas than in temperate to cold ones, and that they represent a minor item in the diet of Barn Owls.(AU)

Wing morphology, flights speeds and insights into niche structure in Caribbean bats from Dominica

We compiled data from six species in four families of Caribbean bats collected on the island of Dominica in order to explore the relation between wing morphology and flight speed. An 18.3 m flight tunnel was constructed in second growth rain forest using a concrete structure as a frame and large tarps to enclose the perimeter, leaving one end open. Bats were caught using mist nets in areas of both mature and second growth rain forest and disturbed habitats. Bats were moved to the tunnel area where morphological measurements were recorded and each bat was photographed with the wing extended. Wing area and wing span were measured using the program ImageJ. These data in combination with other morphological measurements were used to calculate aspect ratio and wing loading, which were each compared to flight speed. To measure flight speed, a bat was released in the flight tunnel and times were recorded at 6.1 m, 12.2 m, and 18.3 m. The findings reveal that flight speed shows little relation to aspect ratio, with the exception of Tadarida brasiliensis, and that flight speed increases as a function of wing loading. The comparison of aspect ratio to relative wing loading demonstrates strong ecomorphological segregation based on foraging mode.

Leucism in live species of bats from México

Leucism is a partial hypopigmentary congenital disorder previously recorded in México in seven bat specimens of six species. In August 2009, in the state of Hidalgo, we caught one Sturnira ludovici and one Artibeus watershousii was caught in April 2010, in Guerrero. Leucism has not been frequently reported and it may be more common than suggested by pubblished results. In order to evalute this supposition, we cheked the mammal collection of the Escuela Nacional de Ciencias Biológicas (Instituto Politécnico Nacional) to look for other evidences of leucism in bats. We found siz more specimens, three Artibeus jamaicensis and three Tadarida brasiliensis with this condition. These specimens raise to 16 the records of bats of nine species with leucism in México.

Leucism in live species of bats from México

Leucism is a partial hypopigmentary congenital disorder previously recorded in México in seven bat specimens of six species. In August 2009, in the state of Hidalgo, we caught one Sturnira ludovici and one Artibeus watershousii was caught in April 2010, in Guerrero. Leucism has not been frequently reported and it may be more common than suggested by pubblished results. In order to evalute this supposition, we cheked the mammal collection of the Escuela Nacional de Ciencias Biológicas (Instituto Politécnico Nacional) to look for other evidences of leucism in bats. We found siz more specimens, three Artibeus jamaicensis and three Tadarida brasiliensis with this condition. These specimens raise to 16 the records of bats of nine species with leucism in México.(AU)