A new species of Loricaria (Loricariidae: Loricariinae) from the upper Amazon River basin, Colombia

A new species of Loricaria is described from the upper Amazon River basin, Colombia. The new species is distinguished from its congeners primarily by having the dorsal portion of head with uniform black or dark brown coloration extending to three or four plates posterior to dorsal fin base, or with two longitudinal bands from tip of the snout to origin of dorsal fin; abdominal plates tightly joined and completely covering the median abdominal space and pectoral girdle; and pectoral and dorsal fins totally black or dark brown, without bands, spots, or blotches. The new species is further distinguished by plate counts, and body measurements. An analysis of genetic distances using the cytochrome oxidase c subunit 1 marker of the mitochondrial genome showed a clear differentiation between the new species and Loricaria cataphracta (5.8-7.6%), L. nickeriensis (5.7-6.1%), and L. simillima (2.7-7.0%). Species delimitation analyses were carried out, which further supported the new species as a divergent lineage within the genus. Fish species diversity of the upper Amazon River basin and taxonomic issues related to L. simillima are included as part of the discussion.(AU)

Se describe una nueva especie de Loricaria de la cuenca alta del río Amazonas, Colombia. La nueva especie se distingue de sus congéneres principalmente por presentar la parte dorsal de la cabeza con un color uniforme negro o marrón oscuro que se extiende a tres o cuatro placas posteriores a la base de la aleta dorsal, o con dos franjas longitudinales desde la punta del hocico hasta el origen de la aleta dorsal; placas abdominales unidas y cubriendo completamente la porción central del abdomen y la cintura pectoral; y aletas pectorales y dorsal completamente negras o marrón oscuro, sin bandas ni manchas. La nueva especie se distingue además por conteos de placas y medidas corporales. Un análisis de distancias genéticas utilizando el marcador de la subunidad 1 del citocromo oxidasa c del genoma mitocondrial mostró una clara diferenciación entre la nueva especie y Loricariacataphracta (5,8-7,6%), L. nickeriensis (5,7-6,1%) y L. simillima (2,7-7,0%). Adicionalmente se realizaron análisis de delimitación de especies, lo que mostró información adicional para reconocer la nueva especie como un linaje divergente dentro del género. La diversidad de especies de peces en la parte superior del río Amazonas y cuestiones taxonómicas relacionadas con L. simillima se incluyen como parte de la discusión.(AU)

Genetic differentiation through dispersal and isolation in two freshwater fish species from coastal basins of Northeastern Brazil

The coastal basins in Northeastern Brazil used in this study make up two different ecoregions for freshwater fishes (Amazonas estuary and coastal drainages, and Parnaiba) and two areas of endemism for Characiformes (Maranhão and Parnaíba), and exhibits a diversified yet poorly explored freshwater fish fauna. The population structure and biogeography of two migratory freshwater fish species that are commercially exploited from Maranhão and Parnaíba regions were herein analyzed. Molecular sequence data and statistical analyses were used to estimate haplotypes networks and lineage divergence times and correlated with hydrographic history of drainage and paleodrainages of the region. A total of 171 sequences was produced for both species, Schizodon dissimilis (coI, n = 70) and Prochilodus lacustris (D-loop, n = 101). All analyses identified the presence of three genetically delimited groups of S. dissimilis and six groups of P. lacustris. The lineage time analyses indicate diversification among these species within the past 1 million year. The results indicate the influence of geodispersal in the formation of the ichthyofauna in the studied area through headwater stream capture events and reticulated connections between the mouths of rivers along the coastal plain due to eustatic sea level fluctuations.(AU)

As bacias costeiras do nordeste do Brasil usadas neste estudo compõem duas ecorregiões diferentes para peixes de água doce (Estuário do Amazonas e drenagens costeiras e Parnaíba) e duas áreas de endemismo para Characiformes (Maranhão e Parnaíba), exibindo uma diversificada e ainda pouco explorada fauna de peixes de água doce. A estrutura populacional e biogeografia de duas espécies migradoras de peixes de água doce exploradas comercialmente nas regiões do Maranhão e Parnaíba foram analisadas. Dados de sequências moleculares e análises estatísticas foram utilizados para estimar redes de haplótipos e tempos de divergência entre linhagens, e foram correlacionados com a história hidrográfica das drenagens e paleodrenagens da região. Um total de 171 sequências foram geradas para ambas espécies, Schizodon dissimilis (coI, n = 70) e Prochilodus lacustris (D-loop, n = 101). Todas análises identificaram a presença de três grupos geneticamente delimitados para S. dissimilis e seis grupos para P. lacustris. A análise de tempo de divergência das linhagens indicou uma diversificação entre estas espécies nos últimos 1 Ma. Os resultados indicam influência da geodispersão na formação da ictiofauna do Maranhão, devido eventos de capturas de cabeceira e conexões reticuladas entre as fozes dos rios ao longo da planície costeira devido às flutuações eustáticas do nível do mar.(AU)

Genetic differentiation through dispersal and isolation in two freshwater fish species from coastal basins of Northeastern Brazil

The coastal basins in Northeastern Brazil used in this study make up two different ecoregions for freshwater fishes (Amazonas estuary and coastal drainages, and Parnaiba) and two areas of endemism for Characiformes (Maranhão and Parnaíba), and exhibits a diversified yet poorly explored freshwater fish fauna. The population structure and biogeography of two migratory freshwater fish species that are commercially exploited from Maranhão and Parnaíba regions were herein analyzed. Molecular sequence data and statistical analyses were used to estimate haplotypes networks and lineage divergence times and correlated with hydrographic history of drainage and paleodrainages of the region. A total of 171 sequences was produced for both species, Schizodon dissimilis (coI, n = 70) and Prochilodus lacustris (D-loop, n = 101). All analyses identified the presence of three genetically delimited groups of S. dissimilis and six groups of P. lacustris. The lineage time analyses indicate diversification among these species within the past 1 million year. The results indicate the influence of geodispersal in the formation of the ichthyofauna in the studied area through headwater stream capture events and reticulated connections between the mouths of rivers along the coastal plain due to eustatic sea level fluctuations.(AU)

As bacias costeiras do nordeste do Brasil usadas neste estudo compõem duas ecorregiões diferentes para peixes de água doce (Estuário do Amazonas e drenagens costeiras e Parnaíba) e duas áreas de endemismo para Characiformes (Maranhão e Parnaíba), exibindo uma diversificada e ainda pouco explorada fauna de peixes de água doce. A estrutura populacional e biogeografia de duas espécies migradoras de peixes de água doce exploradas comercialmente nas regiões do Maranhão e Parnaíba foram analisadas. Dados de sequências moleculares e análises estatísticas foram utilizados para estimar redes de haplótipos e tempos de divergência entre linhagens, e foram correlacionados com a história hidrográfica das drenagens e paleodrenagens da região. Um total de 171 sequências foram geradas para ambas espécies, Schizodon dissimilis (coI, n = 70) e Prochilodus lacustris (D-loop, n = 101). Todas análises identificaram a presença de três grupos geneticamente delimitados para S. dissimilis e seis grupos para P. lacustris. A análise de tempo de divergência das linhagens indicou uma diversificação entre estas espécies nos últimos 1 Ma. Os resultados indicam influência da geodispersão na formação da ictiofauna do Maranhão, devido eventos de capturas de cabeceira e conexões reticuladas entre as fozes dos rios ao longo da planície costeira devido às flutuações eustáticas do nível do mar.(AU)

Progressive histiocytosis of non-epitheliotropic dendritic cells in a feline

Background: Histiocytic tumors in felines are nodules that commonly develop on limbs and head extremities. They can be divided into many subtypes including cutaneous histiocytoma, histiocytic sarcoma, reactive fibrohistiocytic nodule, Langerhans cell histiocytosis, and progressive feline dendritic cell. Despite the same origin, they have behaviors that differ from each other, thus it is important to confirm diagnosis with histopathological and immunohistochemical tests, because early identification can facilitate prognosis and treatment. In this study, we describe the pathological and immunohistochemical characteristics, enabling differentiation feline neoplasms derived from histiocytes. Case: A 5-year-old, crossbreed, male, feline presented with a nodulation at the base of the left ear. The mass was slow growing, partially alopecic, with no other changes associated with tumor development. The nodule was round and circumscribed, movable, with an elevated surface. He was referred for surgery and an elliptical sample around the tumor was carefully dissected. Routine histopathological evaluation was performed with hematoxylin and eosin (HE), as well as immunohistochemistry. Histopathology showed circumscribed proliferation of histiocytic cells, with abundant and eosinophilic cytoplasm. The proliferative cells were large and rounded, extending from the superficial dermis and basement membrane to the deep dermis. At the extremities, some cells had visible vacuoles. Mitotic activity ranged from 3 to 4 mitoses per field in 40x magnification. Immunohistochemistry showed positive staining for histocompatibility complex MCII and lysozyme antibodies, marking histiocytic cells. Labeling was positive for CD20 in cells of lymphoid lineage B and negative for E-cadherin. Histiocytic cells did not invade the epidermis; hence, proliferation was classified as nonepitheliotropic. These methods contribute to the literature regarding the...

Progressive histiocytosis of non-epitheliotropic dendritic cells in a feline

Background: Histiocytic tumors in felines are nodules that commonly develop on limbs and head extremities. They can be divided into many subtypes including cutaneous histiocytoma, histiocytic sarcoma, reactive fibrohistiocytic nodule, Langerhans cell histiocytosis, and progressive feline dendritic cell. Despite the same origin, they have behaviors that differ from each other, thus it is important to confirm diagnosis with histopathological and immunohistochemical tests, because early identification can facilitate prognosis and treatment. In this study, we describe the pathological and immunohistochemical characteristics, enabling differentiation feline neoplasms derived from histiocytes. Case: A 5-year-old, crossbreed, male, feline presented with a nodulation at the base of the left ear. The mass was slow growing, partially alopecic, with no other changes associated with tumor development. The nodule was round and circumscribed, movable, with an elevated surface. He was referred for surgery and an elliptical sample around the tumor was carefully dissected. Routine histopathological evaluation was performed with hematoxylin and eosin (HE), as well as immunohistochemistry. Histopathology showed circumscribed proliferation of histiocytic cells, with abundant and eosinophilic cytoplasm. The proliferative cells were large and rounded, extending from the superficial dermis and basement membrane to the deep dermis. At the extremities, some cells had visible vacuoles. Mitotic activity ranged from 3 to 4 mitoses per field in 40x magnification. Immunohistochemistry showed positive staining for histocompatibility complex MCII and lysozyme antibodies, marking histiocytic cells. Labeling was positive for CD20 in cells of lymphoid lineage B and negative for E-cadherin. Histiocytic cells did not invade the epidermis; hence, proliferation was classified as nonepitheliotropic. These methods contribute to the literature regarding the...(AU)

Fibrin biopolymer as scaffold candidate to treat bone defects in rats

Background:Bone tissue repair remains a challenge in tissue engineering. Currently, new materials are being applied and often integrated with live cells and biological scaffolds. The fibrin biopolymer (FBP) proposed in this study has hemostatic, sealant, adhesive, scaffolding and drug-delivery properties. The regenerative potential of an association of FBP, biphasic calcium phosphate (BCP) and mesenchymal stem cells (MSCs) was evaluated in defects of rat femurs.Methods:Adult male Wistar rats were submitted to a 5-mm defect in the femur. This was filled with the following materials and/or associations: BPC; FBP and BCP; FBP and MSCs; and BCP, FBP and MSCs. Bone defect without filling was defined as the control group. Thirty and sixty days after the procedure, animals were euthanatized and subjected to computed tomography, scanning electron microscopy and qualitative and quantitative histological analysis.Results:It was shown that FBP is a suitable scaffold for bone defects due to the formation of a stable clot that facilitates the handling and optimizes the surgical procedures, allowing also cell adhesion and proliferation. The association between the materials was biocompatible. Progressive deposition of bone matrix was higher in the group treated with FBP and MSCs. Differentiation of mesenchymal stem cells into osteogenic lineage was not necessary to stimulate bone formation.Conclusions:FBP proved to be an excellent scaffold candidate for bone repair therapies due to application ease and biocompatibility with synthetic calcium-based materials. The satisfactory results obtained by the association of FBP with MSCs may provide a more effective and less costly new approach for bone tissue engineering.(AU)

Fibrin biopolymer as scaffold candidate to treat bone defects in rats

Bone tissue repair remains a challenge in tissue engineering. Currently, new materials are being applied and often integrated with live cells and biological scaffolds. The fibrin biopolymer (FBP) proposed in this study has hemostatic, sealant, adhesive, scaffolding and drug-delivery properties. The regenerative potential of an association of FBP, biphasic calcium phosphate (BCP) and mesenchymal stem cells (MSCs) was evaluated in defects of rat femurs. Methods: Adult male Wistar rats were submitted to a 5-mm defect in the femur. This was filled with the following materials and/or associations: BPC; FBP and BCP; FBP and MSCs; and BCP, FBP and MSCs. Bone defect without filling was defined as the control group. Thirty and sixty days after the procedure, animals were euthanatized and subjected to computed tomography, scanning electron microscopy and qualitative and quantitative histological analysis. Results: It was shown that FBP is a suitable scaffold for bone defects due to the formation of a stable clot that facilitates the handling and optimizes the surgical procedures, allowing also cell adhesion and proliferation. The association between the materials was biocompatible. Progressive deposition of bone matrix was higher in the group treated with FBP and MSCs. Differentiation of mesenchymal stem cells into osteogenic lineage was not necessary to stimulate bone formation. Conclusions: FBP proved to be an excellent scaffold candidate for bone repair therapies due to application ease and biocompatibility with synthetic calcium-based materials. The satisfactory results obtained by the association of FBP with MSCs may provide a more effective and less costly new approach for bone tissue engineering.(AU)

The differentiation potential of adipose tissue-derived mesenchymal stem cells into cell lineage related to male germ cells / Potencial de diferenciação de células-tronco mesenquimais derivadas do tecido adiposo em células da linhagem germinativa masculina

The adipose tissue is a reliable source of Mesenchymal stem cells (MSCs) showing a higher plasticity and transdifferentiation potential into multilineage cells. In the present study, adipose tissue-derived mesenchymal stem cells (AT-MSCs) were isolated from mice omentum and epididymis fat depots. The AT-MSCs were initially compared based on stem cell surface markers and on the mesodermal trilineage differentiation potential. Additionally, AT-MSCs, from both sources, were cultured with differentiation media containing retinoic acid (RA) and/or testicular cell-conditioned medium (TCC). The AT-MSCs expressed mesenchymal surface markers and differentiated into adipogenic, chondrogenic and osteogenic lineages. Only omentum-derived AT-MSCs expressed one important gene marker related to male germ cell lineages, after the differentiation treatment with RA. These findings reaffirm the importance of adipose tissue as a source of multipotent stromal-stem cells, as well as, MSCs source regarding differentiation purpose.(AU)

O tecido adiposo é uma fonte apropriada de células-tronco mesenquimais (MSCs), as quais demonstram ampla plasticidade com capacidade de transdiferenciar em diversas linhagens. No presente estudo, as células-tronco mesenquimais derivadas do tecido adiposo (AT-MSC) foram isoladas de tecido adiposo localizado nas regiões próximas ao omento e testículos de camundongos. Primeiramente, as AT-MSCs foram comparadas com base na expressão de marcadores antigênicos de superfície e no potencial de diferenciação nas três linhagens mesodérmicas. Além disso, AT-MSC, de ambas as fontes, foram cultivadas com meio de diferenciação contendo ácido retinóico (RA) e / ou meio condicionado testicular (TCC). As AT-MSCs expressaram marcadores de superfície mesenquimais e diferenciaram nas linhagens adipogênica, condrogênica e osteogênica. Após o tratamento com RA, somente as AT-MSCs isoladas do tecido adiposo depositado na região do omento expressaram um único importante marcador relacionado às células da linhagem germinativa masculina. Estes resultados reafirmam a importância do tecido adiposo como fonte de células-tronco estromais-multipotentes, bem como, uma fonte de MSCs para estudos de diferenciação.(AU)

The differentiation potential of adipose tissue-derived mesenchymal stem cells into cell lineage related to male germ cells / Potencial de diferenciação de células-tronco mesenquimais derivadas do tecido adiposo em células da linhagem germinativa masculina

The adipose tissue is a reliable source of Mesenchymal stem cells (MSCs) showing a higher plasticity and transdifferentiation potential into multilineage cells. In the present study, adipose tissue-derived mesenchymal stem cells (AT-MSCs) were isolated from mice omentum and epididymis fat depots. The AT-MSCs were initially compared based on stem cell surface markers and on the mesodermal trilineage differentiation potential. Additionally, AT-MSCs, from both sources, were cultured with differentiation media containing retinoic acid (RA) and/or testicular cell-conditioned medium (TCC). The AT-MSCs expressed mesenchymal surface markers and differentiated into adipogenic, chondrogenic and osteogenic lineages. Only omentum-derived AT-MSCs expressed one important gene marker related to male germ cell lineages, after the differentiation treatment with RA. These findings reaffirm the importance of adipose tissue as a source of multipotent stromal-stem cells, as well as, MSCs source regarding differentiation purpose.(AU)

O tecido adiposo é uma fonte apropriada de células-tronco mesenquimais (MSCs), as quais demonstram ampla plasticidade com capacidade de transdiferenciar em diversas linhagens. No presente estudo, as células-tronco mesenquimais derivadas do tecido adiposo (AT-MSC) foram isoladas de tecido adiposo localizado nas regiões próximas ao omento e testículos de camundongos. Primeiramente, as AT-MSCs foram comparadas com base na expressão de marcadores antigênicos de superfície e no potencial de diferenciação nas três linhagens mesodérmicas. Além disso, AT-MSC, de ambas as fontes, foram cultivadas com meio de diferenciação contendo ácido retinóico (RA) e / ou meio condicionado testicular (TCC). As AT-MSCs expressaram marcadores de superfície mesenquimais e diferenciaram nas linhagens adipogênica, condrogênica e osteogênica. Após o tratamento com RA, somente as AT-MSCs isoladas do tecido adiposo depositado na região do omento expressaram um único importante marcador relacionado às células da linhagem germinativa masculina. Estes resultados reafirmam a importância do tecido adiposo como fonte de células-tronco estromais-multipotentes, bem como, uma fonte de MSCs para estudos de diferenciação.(AU)

Isolation, culture and characterization of multipotent mesenchymal stem cells from goat umbilical cord blood

ABSTRACT: Mesenchymal stem cells (MSC) reside in small numbers in many adult tissues and organs, and play an active role in the homeostasis of these sites. Goat derived multipotent MSC have been established from bone marrow, adipose tissues and amniotic fluid. Umbilical cord blood (UCB) is considered an important source of these cells. However, the MSC isolation from the goat UCB has not been demonstrated. Therefore, the aim of the present study was to isolate, culture and characterize goat umbilical cord blood derived mesenchymal stem cells. MSC were isolated from UCB by Ficoll-Paque density centrifugation and cultured in DMEM supplemented with 10% or 20% FBS. FACS analysis was performed and induction lineage differentiation was made to characterize these cells. They exhibited two different populations in flow cytometry, and revealed the positive expression of CD90, CD44 and CD105, but negative staining for CD34 in larger cells, and positive stained for CD90 and CD105, but negative for CD44 and CD34 in the smaller cells. MSC from goat UCB showed capability to differentiate into chondrocytes and osteoblasts when incubated with specific differentiation medium. Present study established that goat mesenchymal stem cells can be derived successfully from umbilical cord blood.

RESUMO: As células tronco mesenquimais (MSC) residem em pequenas quantidades em muitos tecidos e órgãos adultos, desempenhando um papel ativo na homeostase destes locais. O isolamento de MSC já foi demonstrado em amostras de medula óssea, tecido adiposo e fluido amniótico de cabras. O sangue de cordão umbilical é considerado uma fonte importante desse tipo de células. No entanto, até o presente momento, não foi demonstrado o isolamento de MSC provenientes do sangue de cordão umbilical de cabras. Dessa forma, o objetivo do presente estudo foi isolar, cultivar e caracterizar células tronco mesenquimais provenientes do sangue do cordão umbilical caprino. As MSC foram isoladas utilizando o gradiente de densidade Ficoll-Paque e cultivadas em DMEM suplementado com 10% ou 20% de FBS. A caracterização desse tipo celular foi realizada através de análise por citometria de fluxo e diferenciação em linhagens celulares mesodermais. A analise no citômetro de fluxo demonstrou a presença de duas populações distintas, um grupo com células maiores e outro com células menores; observando expressão positiva de CD90, CD44 e CD105, e negativa para CD34 nas células maiores; enquanto que as menores foram positivas para CD90 e CD105, mas negativas para CD44 e CD34. As células isoladas demonstraram capacidade de se diferenciar em condrócitos e osteoblastos quando incubadas com meio de diferenciação específico. O presente estudo demonstrou que células tronco mesenquimais podem ser obtidas com sucesso do sangue do cordão umbilical caprino.

Goat umbilical cord cells are permissive to small ruminant lentivirus infection in vitro

Small ruminant lentiviruses isolated from peripheral blood leukocytes and target organs can be propagated in vitro in fibroblasts derived from goat synovial membrane cells. These cells are obtained from tissues collected from embryos or fetuses and are necessary for the establishment of the fibroblast primary culture. A new alternative type of host cells, derived from goat umbilical cord, was isolated and characterized phenotypically with its main purpose being to obtain cell monolayers that could be used for the diagnosis and isolation of small ruminant lentiviruses in cell culture. To accomplish this goal, cells were isolated from umbilical cords; characterized phenotypically by flow cytometry analysis; differentiate into osteogenic, chondrogenic and adipogenic lineage; and submitted to viral challenge. The proliferation of goat umbilical cord cells was fast and cell monolayers formed after 15 days. These cells exhibited morphology, immunophenotype, growth characteristics, and lineage differentiation potential similar to mesenchymal stem cells of other origins. The goat umbilical cord derived cells stained positive for vimentin and CD90, but negative for cytokeratin, CD34 and CD105 markers. Syncytia and cell lysis were observed in cell monolayers infected by CAEV-Cork and MVV-K1514, showing that the cells are permissive to small ruminant lentivirus infection in vitro. These data demonstrate the proliferative competence of cells derived from goat umbilical cords and provide a sound basis for future research to standardize this cell lineage.(AU)

Isolation, culture and characterization of multipotent mesenchymal stem cells from goat umbilical cord blood / Isolamento, cultura e caracterização de células tronco mesenquimais multipotentes provenientes do sangue do cordão umbilical caprino

Mesenchymal stem cells (MSC) reside in small numbers in many adult tissues and organs, and play an active role in the homeostasis of these sites. Goat derived multipotent MSC have been established from bone marrow, adipose tissues and amniotic fluid. Umbilical cord blood (UCB) is considered an important source of these cells. However, the MSC isolation from the goat UCB has not been demonstrated. Therefore, the aim of the present study was to isolate, culture and characterize goat umbilical cord blood derived mesenchymal stem cells. MSC were isolated from UCB by Ficoll-Paque density centrifugation and cultured in DMEM supplemented with 10% or 20% FBS. FACS analysis was performed and induction lineage differentiation was made to characterize these cells. They exhibited two different populations in flow cytometry, and revealed the positive expression of CD90, CD44 and CD105, but negative staining for CD34 in larger cells, and positive stained for CD90 and CD105, but negative for CD44 and CD34 in the smaller cells. MSC from goat UCB showed capability to differentiate into chondrocytes and osteoblasts when incubated with specific differentiation medium. Present study established that goat mesenchymal stem cells can be derived successfully from umbilical cord blood.(AU)

As células tronco mesenquimais (MSC) residem em pequenas quantidades em muitos tecidos e órgãos adultos, desempenhando um papel ativo na homeostase destes locais. O isolamento de MSC já foi demonstrado em amostras de medula óssea, tecido adiposo e fluido amniótico de cabras. O sangue de cordão umbilical é considerado uma fonte importante desse tipo de células. No entanto, até o presente momento, não foi demonstrado o isolamento de MSC provenientes do sangue de cordão umbilical de cabras. Dessa forma, o objetivo do presente estudo foi isolar, cultivar e caracterizar células tronco mesenquimais provenientes do sangue do cordão umbilical caprino. As MSC foram isoladas utilizando o gradiente de densidade Ficoll-Paque e cultivadas em DMEM suplementado com 10% ou 20% de FBS. A caracterização desse tipo celular foi realizada através de análise por citometria de fluxo e diferenciação em linhagens celulares mesodermais. A analise no citômetro de fluxo demonstrou a presença de duas populações distintas, um grupo com células maiores e outro com células menores; observando expressão positiva de CD90, CD44 e CD105, e negativa para CD34 nas células maiores; enquanto que as menores foram positivas para CD90 e CD105, mas negativas para CD44 e CD34. As células isoladas demonstraram capacidade de se diferenciar em condrócitos e osteoblastos quando incubadas com meio de diferenciação específico. O presente estudo demonstrou que células tronco mesenquimais podem ser obtidas com sucesso do sangue do cordão umbilical caprino.(AU)

Isolation, culture and characterization of multipotent mesenchymal stem cells from goat umbilical cord blood / Isolamento, cultura e caracterização de células tronco mesenquimais multipotentes provenientes do sangue do cordão umbilical caprino

Mesenchymal stem cells (MSC) reside in small numbers in many adult tissues and organs, and play an active role in the homeostasis of these sites. Goat derived multipotent MSC have been established from bone marrow, adipose tissues and amniotic fluid. Umbilical cord blood (UCB) is considered an important source of these cells. However, the MSC isolation from the goat UCB has not been demonstrated. Therefore, the aim of the present study was to isolate, culture and characterize goat umbilical cord blood derived mesenchymal stem cells. MSC were isolated from UCB by Ficoll-Paque density centrifugation and cultured in DMEM supplemented with 10% or 20% FBS. FACS analysis was performed and induction lineage differentiation was made to characterize these cells. They exhibited two different populations in flow cytometry, and revealed the positive expression of CD90, CD44 and CD105, but negative staining for CD34 in larger cells, and positive stained for CD90 and CD105, but negative for CD44 and CD34 in the smaller cells. MSC from goat UCB showed capability to differentiate into chondrocytes and osteoblasts when incubated with specific differentiation medium. Present study established that goat mesenchymal stem cells can be derived successfully from umbilical cord blood.(AU)

As células tronco mesenquimais (MSC) residem em pequenas quantidades em muitos tecidos e órgãos adultos, desempenhando um papel ativo na homeostase destes locais. O isolamento de MSC já foi demonstrado em amostras de medula óssea, tecido adiposo e fluido amniótico de cabras. O sangue de cordão umbilical é considerado uma fonte importante desse tipo de células. No entanto, até o presente momento, não foi demonstrado o isolamento de MSC provenientes do sangue de cordão umbilical de cabras. Dessa forma, o objetivo do presente estudo foi isolar, cultivar e caracterizar células tronco mesenquimais provenientes do sangue do cordão umbilical caprino. As MSC foram isoladas utilizando o gradiente de densidade Ficoll-Paque e cultivadas em DMEM suplementado com 10% ou 20% de FBS. A caracterização desse tipo celular foi realizada através de análise por citometria de fluxo e diferenciação em linhagens celulares mesodermais. A analise no citômetro de fluxo demonstrou a presença de duas populações distintas, um grupo com células maiores e outro com células menores; observando expressão positiva de CD90, CD44 e CD105, e negativa para CD34 nas células maiores; enquanto que as menores foram positivas para CD90 e CD105, mas negativas para CD44 e CD34. As células isoladas demonstraram capacidade de se diferenciar em condrócitos e osteoblastos quando incubadas com meio de diferenciação específico. O presente estudo demonstrou que células tronco mesenquimais podem ser obtidas com sucesso do sangue do cordão umbilical caprino.(AU)

Culture, characterization and differentiation of neural precursors from the central nervous system of guinea pigs (Cavia porcellus Linnaeus, 1758) / Cultura, caracterização e diferenciação de precursores neurais do sistema nervoso central de porquinho-da-índia (Cavia porcellus Linnaeus, 1758)

Potentially neurogenic areas were initially identified by incorporation of bromodeoxyuridine (BrdU) in cells underlying the subventricular zone (SVZ) of the lateral ventricles wall, hippocampus and olfactory bulbs of newborn guinea pigs. Neural precursors from the SVZ were cultured in suspension, generating neurospheres (NSFs), which, upon dissociation were able to generate new NSFs. Upon culture in the absence of growth factors, cells dissociated from NSFs displayed evidence for neural differentiation, giving rise to cells from neural lineage. Flow cytometry analysis for of NSFs-derived cells after differentiation revealed approximately 13.3% nestin positive, 5.5% Beta-III-tubulin positive, 9% GFAP positive and 7.8% mGalC positive. Functional assays by measurement of calcium influx upon gamma butiric amino acid (GABA) and glutamate stimuli, revealed stimulation in differentiated cells, an indicator of neuronal differentiation. The ability of guinea pig SVZ cells to originate functional neurons in vitro is promising for research and towards a future use of neural stem cells in the therapy of neurological disorders.(AU)

Áreas potencialmente neurogênicas foram identificadas por incorporação de bromodeoxiuridina (BrdU) na zona subventricular (SVZ) dos ventrículos laterais, hipocampo e bulbos olfatórios de cobaias neonatos. Precursores neurais provenientes da SVZ foram cultivados em suspensão, resultando na geração de neuroesferas (NSFs), que quando dissociadas foram capazes de proliferar e gerar novas NSFs. Quando cultivadas na ausência de fatores de crescimento, as células provenientes de NSFs dissociadas apresentaram evidências de diferenciação neuronal, dando origem a células da linhagem neural. Citometria de fluxo em células das NSFs após a diferenciação revelou aproximadamente 13,3% positivas para nestina, 5,5% positivas para Beta-III-tubulina, 9% positivas para GFAP e 7,8% positivas para mGalC. Testes de funcionalidade pela mensuração de influxo de cálcio após estímulo com ácido gama amino butírico (GABA) e glutamato revelaram a estimulação de células diferenciadas, um indicador de função neuronal. A capacidade de células da SVZ de fetos de cobaias originarem células neurais funcionais in vitro é promissora para a pesquisa e eventual uso terapêutico de células tronco em disordens do sistema nervoso.(AU)

Culture, characterization and differentiation of neural precursors from the central nervous system of guinea pigs (Cavia porcellus Linnaeus, 1758) / Cultura, caracterização e diferenciação de precursores neurais do sistema nervoso central de porquinho-da-índia (Cavia porcellus Linnaeus, 1758)

Potentially neurogenic areas were initially identified by incorporation of bromodeoxyuridine (BrdU) in cells underlying the subventricular zone (SVZ) of the lateral ventricles wall, hippocampus and olfactory bulbs of newborn guinea pigs. Neural precursors from the SVZ were cultured in suspension, generating neurospheres (NSFs), which, upon dissociation were able to generate new NSFs. Upon culture in the absence of growth factors, cells dissociated from NSFs displayed evidence for neural differentiation, giving rise to cells from neural lineage. Flow cytometry analysis for of NSFs-derived cells after differentiation revealed approximately 13.3% nestin positive, 5.5% Beta-III-tubulin positive, 9% GFAP positive and 7.8% mGalC positive. Functional assays by measurement of calcium influx upon gamma butiric amino acid (GABA) and glutamate stimuli, revealed stimulation in differentiated cells, an indicator of neuronal differentiation. The ability of guinea pig SVZ cells to originate functional neurons in vitro is promising for research and towards a future use of neural stem cells in the therapy of neurological disorders.(AU)

Áreas potencialmente neurogênicas foram identificadas por incorporação de bromodeoxiuridina (BrdU) na zona subventricular (SVZ) dos ventrículos laterais, hipocampo e bulbos olfatórios de cobaias neonatos. Precursores neurais provenientes da SVZ foram cultivados em suspensão, resultando na geração de neuroesferas (NSFs), que quando dissociadas foram capazes de proliferar e gerar novas NSFs. Quando cultivadas na ausência de fatores de crescimento, as células provenientes de NSFs dissociadas apresentaram evidências de diferenciação neuronal, dando origem a células da linhagem neural. Citometria de fluxo em células das NSFs após a diferenciação revelou aproximadamente 13,3% positivas para nestina, 5,5% positivas para Beta-III-tubulina, 9% positivas para GFAP e 7,8% positivas para mGalC. Testes de funcionalidade pela mensuração de influxo de cálcio após estímulo com ácido gama amino butírico (GABA) e glutamato revelaram a estimulação de células diferenciadas, um indicador de função neuronal. A capacidade de células da SVZ de fetos de cobaias originarem células neurais funcionais in vitro é promissora para a pesquisa e eventual uso terapêutico de células tronco em disordens do sistema nervoso.(AU)

Expressão de genes associados ao crescimento muscular em tilápia do Nilo (Oreochromis Niloticus) melhoradas

O processo de crescimento muscular envolve a ativação, proliferação e diferenciação de mioblastos, ocorrendo por dois mecanismos: a hiperplasia e hipertrofia que são reguladas por uma série de proteínas, como os fatores de regulação miogênica (MRFs) e a miostatina (Mstn). Neste sentido, os objetivos deste trabalho foram avaliar em tilápia do Nilo (Oreochromis niloticus), dentro de um Programa de Melhoramento Genético a expressão de genes que controlam o crescimento muscular, e caracterizar os mecanismos de crescimento hipertrófico e hiperplásico da musculatura branca através da morfometria das fibras musculares. Os animais são provenientes da décima geração do Programa de Melhoramento da Linhagem GIFT- TILAMAX/UEM, da Universidade Estadual de Maringá (PMGT-UEM), onde foram coletadas amostras da musculatura branca dorsal dos 16 peixes adultos de tilápia do Nilo (Oreochromis Niloticus), separadas em dois grupos: animais de alto valor genético (AVG) e animais de baixo valor genético (BVG). Foram realizadas análises de peso e comprimento ao peso final, análises de expressão gênica para os genes relacionados ao crescimento muscular -MyoD, miogenina (Myog) e miostatina (Mstn) e análises de morfologia e morfometria das fibras musculares. Os animais de alto valor genético apresentaram valores de peso e comprimento maiores comparados aos animais de baixo valor genético. Para as análises de expressão gênica, os resultados não foram significativos para os genes MyoD e miogenina para ambos os grupos. Entretanto, para o gene da miostatina o resultado foi significativo, e houve maior expressão para o tratamento com baixo valor genético. A análise morfométrica das fibras musculares mostrou valores significativos para as classes de diâmetros entre 20 e 50µm e maiores que 50µm, porém não foi significativo para a classe de diâmetro das fibras menores que 20 µm, demostrando que o intenso crescimento muscular hipertrófico. Como conclusão, os animais de AVG apresentaram maior desempenho como resultado de maior ganho de massa muscular por hipertrofia, como consequência de menor expressão de miostatina em relação ao BVG. Salienta-se que o presente trabalho é o primeiro estudo com tilápias do Nilo melhoradas e que é de grande interesse futuros estudos relacionados ao crescimento muscular, principalmente em peixes de água doce, de rápido crescimento.

The muscle growth process involves the activation, proliferation and differentiation of myoblasts, occurring by two mechanisms: hyperplasia and hypertrophy, which are regulated by a series of proteins, such as myogenic regulation factors (MRFs) and myostatin (Mstn). In this sense, the objectives of this work were to evaluate in Nile tilapia (Oreochromis Niloticus), from a Genetic Improvement Program the expression of genes that control muscle growth, and to characterize the mechanisms of hypertrophic and hyperplastic growth of white musculature through morphometry muscle fibers. The animals come from the tenth generation of the GIFT-TILAMAX / UEM Lineage Improvement Program, of the State University of Maringá (PMGTUEM), where samples of the dorsal white musculature of 16 adult Nile tilapia fish (Oreochromis Niloticus) were collected and separated into two groups: animals of high genetic value (AVG) and animals of low genetic value (BVG). Analyzes of weight and length at final weight, analysis of gene expression for genes related to muscle growth -MyoD, myogenin (Myog) and myostatin (Mstn) and analysis of morphology and morphometry of muscle fibers were carried out. The animals with high genetic value had higher weight and length values compared to the animals with low genetic value. For gene expression analyzes, the results were not significant for MyoD and myogenin genes in both groups. However, for the myostatin gene the result was significant, where there was greater expression for treatment with low genetic value. The morphometric analysis of the muscle fibers showed significant values for classes of diameters between 20 and 50µm and greater than 50µm, however it was not significant for fibers class of diameter smaller than 20 µm, demonstrating that the intense hypertrophic muscle growth. As a conclusion, the AVG animals showed higher performance as a result of greater muscle mass gain due to hypertrophy and as a consequence of a lower expression of myostatin compared to BVG. We emphasize that the present work is the first study with improved Nile tilapia and that it is of great interest for future studies related to muscle growth, especially in fast-growing freshwater fish.

DESENVOLVIMENTO DE MARCADORES MICROSSATÉLITES E AVALIAÇÃO DA DIVERSIDADE GENÉTICA DO PEIXE ORNAMENTAL AMAZÔNICO Pterophyllum scalare

O acará-bandeira (Pterophyllum scalare) é um peixe de água doce que está distribuído em diversas regiões da América do Sul, estando presente na bacia do rio Amazonas, no Brasil, Colômbia, Guianas e Peru. Ele apresenta elevada demanda de mercado, estando entre uma das espécies ornamentais mais importantes. Devido a sua importância biológica, econômica e as cressentes pressões humanas sobre seu habitat, nenhum primer microssátelite foi desenvolvido para pesquisas da sua estrutura genética. O objetivo do presente trabalho foi desenvolver primers espécie- específicos para o P. scalare e analisar a estrutura genética de uma população selvagem e três estoques comerciais. Foram coletadas amostras da nadadeira caudal de 24 indivíduos do rio Amazonas, na reagião de Macapá - AP, 24 da linhagem comum e 24 da linhagem marmorato e 24 da linhagem palhaço, todas de criadores de Vieras - MG. Foram indentificados oito loci polimórficos e 66 alelos com 4,250 a 4,625 alelos por locus. O conteúdo da informação polomórifica variou entre 0,031 a 0,827. Observou-se alta diferenciação genética par a par entre a população serlvagem e os estoques e moderada entre somente os estoques . Foi observado desvio no equilibrio de Hardy-Weinberg na população e nos estoques. As analises de estrutura populacional não demonstrou sobreposição entre a população e os estoquese demonstrou a existencia de duas subpopulações (selvagem) e as três (comercias). Os primers desenvolvidos servem como ferramenta para as análises de diversidade e estrutura genética e estudos de conservação do P. scalare.

The angelfish (Pterophyllum scalare) is a freshwater fish that is distributed in varius regions of South America and is present in the Amazon River basin, in Brazil, Colombia, Guyana and Peru. It has high market demand, being among one of the most important ornamental species. Due to its biological and economic importance and the increasing human pressures on its habitat, no microsatellite primer has been developed for research into its genetic structure. The objective of the present work was to develop species-specific primers for P. scalare and to analyze the genetic structure of one wild population and three commercial stocks. Samples of the caudal fin were collected from 24 individuals from the Amazon River, in the region of Macapá - AP, 24 from the common lineage, 24 from the marmorato lineage and 24 from the clown lineage, all from fish farms from Vieras - MG. Eight polymorphic loci and 66 alleles were identified with 4,250 to 4,625 alleles per locus. The content of the polomorphic information ranged from 0.031 to 0.827. It was observed high pairwise genetic differentiation between population and stocks and moderate between only stocks. Hardy-Weinberg equilibrium deviation in population and stocks was observed. Population structure analyzes showed no overlap between population and the stocking showed two subpopulations (wild) and the three (commercial). The developed primers serve as a tool for the analysis of diversity and genetic structure and conservation studies of P. scalare. Key-words: Genetic conservation. Molecular marker. Ornamental aquaculture. Population structure. SSR.

IMUNOFENOTIPAGEM COMPARADA DOS PLASMOCITOMAS EXTRAMEDULARES, HISTIOCITOMAS E LINFOMAS CUTÂNEOS EM CÃES

Plasmocitomas extramedulares caninos (PEC), sobretudo cutâneos, são frequentes, e representam cerca de 1,5 a 2,3% de todas as neoplasias de pele em cães, entretanto há diversos aspectos não esclarecidos ou francamente obscuros em relação aos PEC, sobretudo no que se refere à epidemiologia, às variações morfológicas, à patogênese e à etiologia. Atualmente, descrevem-se seis diferentes tipos histológicos de PEC em cães, evidência que tal neoplasia apresenta grande variação morfológica. Por outro lado, muitos PEC apresentam características morfológicas muito semelhantes às de neoplasias malignas de origem histiocítica, com pleomorfismo nuclear acentuado e multinucleação. Todavia, o espectro celular abrangido pelo termo histiocítico é genérico e inclui tanto células dendríticas, quanto células da linhagem de macrófagos. As células dendríticas se originam na medula óssea a partir de células-tronco hematopoiéticas pluripotentes e dão origem a duas linhagens de células: dendríticas mieloides e dendríticas linfóides. Cabe investigar se os PEC irão apresentar algum padrão diferente na imunomarcação ou na clonalidade através da PCR para detecção de rearranjo de genes receptores de antígeno (PARR). Esse estudo tem como objetivo caracterizar os PEC e histiocitomas caninos mediante a expressão de marcadores celulares e determinação da clonalidade através da técnica de PCR para PARR. Este método será empregado para diferenciação de origem não linfoide versus linfoide dos tumores. As características imunofenotípicas serão verificadas em 100 PEC e 30 histiocitomas cutâneos por anticorpos anti: CD79a, CD20, CD3 e MUM-1. Também serão testados o anticorpo CD138 como possível alternativa ao MUM-1, tendo em vista que este último de acordo com um trabalho recente possui reação cruzada com histiocitomas e o CD123 para a hipótese de possível origem dendrítica linfoide dos PECs. Com isso espera-se acurar o protocolo de imunohistoquímica utilizado para o diagnóstico de PEC, além de padronizar e validar a técnica de PCR para estudo da clonalidade em neoplasias plasmocíticas e testar a hipótese linfoide versus não linfoide de alguns PEC (com estudo da clonalidade e imunomarcação para células dendríticas plasmocitoides com CD 123), o que pode, em parte, desvendar a diferença de comportamento dos PECs, principalmente em relação ao mieloma múltiplo.

Canine Extramedullary Plasmacytomas (CEP), mainly cutaneous, are frequent and represent about 1.5 to 2.3% of all skin neoplasms in dogs, however there are several aspects not clear in relation to CEP, especially with regard to epidemiology, morphological variations, pathogenesis and etiology. Currently, six different histological types of CEP are described in dogs, indicating that such neoplasia presents great morphological variation. On the other hand, many CEPs present morphological characteristics very similar to malignant neoplasms of histiocytic origin, with marked nuclear pleomorphism and multinucleation. However, the cellular spectrum encompassed by the histiocytic term is generic and includes both dendritic cells and macrophage lineage cells. Dendritic cells originate in the bone marrow from pluripotent hematopoietic stem cells and give rise to two cell lines: dendritic myeloid and dendritic lymphoid. It is relevant investigating whether CEPs will exhibit any different pattern in the immunostaining or clonality by PCR for the detection of antigen receptor gene rearrangement (PARR). This study aims to characterize CEPs and canine histiocytomas by expression of cell markers and determination of clonality by PCR technique to PARR. This method will be used for differentiation of non-lymphoid versus lymphoid origin of tumors. Immunophenotypic characteristics will be verified in 100 CEPs and 30 cutaneous histiocytomas by anti-CD79a, CD20, CD3 and MUM-1 antibodies. CD138 antibody will also be tested as a possible alternative to MUM-1, since in according to recent literature these antibody to present cross-reaction with canine histiocytomas. The CD123 for the hypothesis of possible lymphoid dendritic origin of the CEPs. Thus, the immunohistochemical protocol used for the diagnosis of CEPs is expected to be acured, as well as to standardize and validate the PCR technique for the study of clonality in CEPs and to test the lymphoid versus non-lymphoid hypothesis of some CEPs (with a clonality study and immunostaining for plasmacytoid dendritic cells with CD 123), which may, in part, reveal the difference in behavior of CEPs, especially in relation to multiple myeloma.

Transcriptoma e caracterização do perfil proteômico de células-tronco mesenquimais do tecido adiposo de mini-horses (Equus ferus caballus).

As células-tronco mesenquimais extraídas da medula óssea ou tecido adiposo são utilizadas em abordagens terapêuticas, em vista das suas propriedades anti-inflamatórias, imunomoduladoras e regenerativas. Estas células são consideradas uma nova alternativa para o tratamento de enfermidades em diversas espécies, incluindo as dos equinos. Dessa forma, com o intuito de investigar a possível utilização das células estromais de mini-horses em tratamentos de cavalos, nesse estudo foi realizada a colheita, isolamento, expansão, caracterização imunofenotípica e diferenciação em duas linhagens mesenquimais: osteogênica e adipogênica de células estromais de mini-horses (Equus ferus caballus), objetivando a formação de um banco celular. Foi ainda realizada a análise transcricional comparativa dessas células, com as células estromais oriundas de cavalos (Equus caballus), produzidas e caracterizadas nas mesmas condições. Os parâmetros analisados foram o perfil de genes relacionados à angiogênese, regulação negativa da proliferação de células T, regulação negativa da ativação de células T, proliferação negativa de linfócitos reguladores, regulação negativa da proliferação de células mononucleares, regulação negativa da adesão célula-célula de leucócitos, regulação negativa da proliferação de leucócitos, regulação negativa da adesão célula-leucócito, regulação negativa da ativação de linfócitos. Também foram pesquisados os principais genes up e down regulados comparando mini-horses aos cavalos. Para formação do banco celular foram realizadas avaliações que comprovaram as características de células estromais nas células obtidas dos mini-horses, a avaliação imunofenotípica por citometria de fluxo demonstrou expressão alta dos marcadores CD90 (99,83 ± 0,28) e esperada do CD105 (38,66 ± 18,47) e expressão baixa dos marcadores CD34 (3,55 ± 1,14) e MHC II (4,21 ± 2,03). O potencial de diferenciação em linhagens adipogênicas in vitro foi observado após o 14º dia através da visualização da deposição de gotículas lipídicas no citoplasma, e a linhagem osteogênica foi identificada após o 21° dia pela coloração positiva da matriz de cálcio extracelular. Ao final da avaliação transcricional, observamos um perfil de expressão gênica diferente entre os grupos, sugerindo que as diferenças entre os animais estudados se expande a mutação do gene Aggrecan que determina o nanismo. Apesar disso, os resultados indicam que as células estromais de mini-horse podem ser utilizadas como uma alternativa para o tratamento de lesões equinas com a vantagem de apresentarem uma maior expressão de genes relacionados à imunomodulação.

Mesenchymal stem cells extracted from bone marrow or adipose tissue are used in therapeutic approaches in view of their anti-inflammatory, immunomodulatory and regenerative properties. These cells are considered a new alternative for the treatment of diseases in several species. In order to investigate the possible use of mini-horses stromal cells in horse treatments, in this study was performed the collection, isolation, expansion, immunophenotypic characterization and differentiation in two mesenchymal lines were performed: osteogenic and adipogenic stromal cell mini-horses (Equus ferus caballus), aiming the formation of a cellular bank. We also performed the comparative transcriptional analysis of these cells, with stromal cells from horses (Equus caballus), produced and characterized under the same conditions. The parameters analyzed were the profile of genes related to angiogenesis, negative regulation of T cell proliferation, negative regulation of T cell activation, negative proliferation of regulatory lymphocytes, negative regulation of mononuclear cell proliferation, negative regulation of cell-cell adhesion leukocytes, negative regulation of leukocyte proliferation, negative regulation of cell-leukocyte adhesion, negative regulation of lymphocyte activation. We also searched the top 10 genes up and down regulated comparing mini-horses to horses. For cell bank formation tests were performed that demonstrated the characteristics of stromal cells in the cells obtained from the mini-horses, the immunophenotypic evaluation by flow cytometry showed high CD90 (99.83 ± 0.28) and expected CD105 ( 38.66 ± 18.47) and low expression of the CD34 (3.55 ± 1.14) and MHC II (4.21 ± 2.03) markers. The potential for differentiation in in vitro adipogenic lines was observed after day 14 by visualization of the deposition of lipid droplets in the cytoplasm, and the osteogenic lineage was identified after 21 ° day by the positive staining of the extracellular calcium matrix. At the end of the transcriptional evaluation, we observed a different gene expression profile between the groups, suggesting that the differences between the studied animals expands the Aggrecan gene mutation that determines the dwarfism. However, the results strongly indicate that ASCs from mini-horses can be used as an alternative to treat horses diseases, with the vantages of presenting a higher expression of immunomodulatory genes.

DESENVOLVIMENTO E VALIDAÇÃO DE ENSAIO DE RT-PCR MULTIPLEX PARA DETECÇÃO E DIFERENCIAÇÃO DOS SUBTIPOS DO VÍRUS INFLUENZA A (H1/H3, N1/N2) CIRCULANTES EM SUÍNOS NO BRASIL

A influenza é uma doença respiratória viral aguda que afeta várias espécies de aves e mamíferos, incluindo o homem. A doença em suínos é causada pelo vírus influenza A (IAV), tendo um impacto econômico significativo nos rebanhos suínos afetados, além de representar uma ameaça à população humana devido ao seu potencial pandêmico e zoonótico. Muito embora os subtipos virais globalmente prevalentes em suínos sejam o H1N1, H1N2 e H3N2, estes são geneticamente e antigenicamente distintos, de acordo com a região geográfica de origem. Estudos recentes realizados com isolados do IAV de suínos confirmaram que as linhagens virais detectadas no Brasil são restritas ao território brasileiro, não sendo encontrados vírus semelhantes em suínos de outros países. Desta forma, um teste de diagnóstico rápido, que seja capaz de detectar e diferenciar o subtipo do IAV em suínos é importante para o monitoramento e controle da infecção em rebanhos suínos. Além disto, no Brasil não existem testes rápidos disponíveis comercialmente, como RT-qPCR para subtipar o IAV em amostras clínicas de suínos. Assim, este estudo teve como objetivo desenvolver e validar dois ensaios de RT-qPCR multiplex (um para a hemaglutinina viral H1pdm, H1hu, H3; e outro para a neuraminidase viral N1pdm, N1hu, N2) para a detecção e diferenciação dos subtipos H1N1, H1N2 e H3N2 do IAV circulantes em suínos no Brasil. A especificidade analítica da técnica foi de 100%, na correta identificação do subtipo e linhagem viral em 85 IAVs previamente caracterizados por sequenciamento genômico. O ensaio apresentou 100% de especificidade diagnóstica em 50 amostras negativas para o IAV e positivas para outros patógenos de suínos. O limite de detecção da RT-qPCR variou entre 5,09x103 e 5,09x101 cópias de RNA viral/L, conforme o gene avaliado. Posteriormente, 73 amostras clínicas de suínos positivas para o IAV por RT-PCR (gene da matriz) foram testadas. A RT-qPCR multiplex identificou o subtipo e a linhagem viral em 74% das amostras clínicas analisadas, sendo que o subtipo H3N2 de origem humana (46,3%) foi o mais detectado, seguido pelo H1N1 de origen pandêmica (33,3%), H1N2 de origem humana (11,1%) e HxN1pdm (3,7%). Além disto, coinfecções (3,7%) e vírus que sofreram rearranjo gênico (1,9%) foram detectados. Portanto, o ensaio de RT-qPCR multiplex desenvolvido e validado neste estudo mostrou-se um método rápido, muito sensível e específico para a identificação dos subtipos e linhagens do IAV em amostras de suínos. A técnica descrita é economicamente viável quando comparada a outros métodos, como o sequenciamento genômico, e uma vez implementada em laboratórios de diagnóstico, poderá fornecer informações sobre a prevalência dos subtipos virais em suínos, contribuindo para o monitoramento e vigilância da influenza em rebanhos suínos.

Influenza is an acute viral respiratory disease that affects several species of birds and mammals, including humans. The disease in pigs is caused by influenza A virus (IAV), which may has a significant economic impact on the affected swine herds, posing a threat to the human population due to its pandemic and zoonotic potential. Although the prevalent virus subtypes in swine herds worldwide are H1N1, H1N2 and H3N2, these are genetically and antigenically distinct, according to the geographic region of origin. Recent studies with IAV isolates from swine confirmed that the viral lineages detected in Brazil are restricted to the Brazilian territory, and no similar viruses were found in swine from other countries. In this way, a rapid diagnostic test, able to detect and differentiate the IAV subtype in pigs is important for the monitoring and control of the infection in swine herds. In addition, in Brazil there are no rapid tests commercially available, such as RT-qPCR, to subtype IAV in swine clinical samples. This study aimed to develop and validate two multiplex RT-qPCR assays (one for viral hemagglutinin H1pdm, H1hu, H3; and another for viral neuraminidase N1pdm, N1hu, N2) for the detection and differentiation H1N1, H1N2 and H3N2 IAV subtypes circulating in swine in Brazil. The analytical specificity of the technique was 100%, identifying the correct viral subtype and lineage in 85 IAVs previously characterized by genomic sequencing. The assay presented 100% of diagnostic specificity in 50 negative samples for IAV and positive for other swine pathogens. The limit of detection of the RT-qPCR ranged from 5.09x103 to 5.09x101 copies of viral RNA/L, according to the gene evaluated. Afterwards, 73 swine clinical samples positive for IAV by RT-PCR (matrix gene) were tested. The multiplex RT-qPCR identified the viral subtype and lineage in 74% of the clinical samples analyzed, which the human-origin H3N2 (46.3%) subtype was the most detected, followed by the pandemic-origin H1N1 (33.3%), human-origin H1N2 (11.1%) and HxN1pdm (3.7%). In addition, co-infections (3.7%) and reassortant viruses (1.9%) were detected. Therefore, the multiplex RT-qPCR assay developed and validated in this study showed to be a rapid, very sensitive and specific method for IAV subtyping and lineages identification in swine samples. The technique described is cost-effective when compared to other methods, such as genomic sequencing, and once implemented in diagnostic laboratories, may provide information on the prevalence of viral subtypes in pigs, contributing to the monitoring and surveillance of influenza in swine herds.