Small ruminant lentiviruses (SRLV) have high genetic variability which results in different viral strains around the world. This create a challenge to design sensible primers for molecular diagnosis in different regions. This work proposes a protocol of duplex nested-PCR for the precise diagnosis of SRLV. The technique was designed and tested with the control strains CAEV Co and MVV 1514. Then, field strains were submitted to the same protocol of duplex nested-PCR. Blood samples of sheep and goats were tested with AGID and nested PCR with specific primers for pol, gag and LTR. The AGID results showed low detection capacity of positive animals, while the nested PCR demonstrated a greater capacity of virus detection. Results demonstrated that LTR-PCR was more efficient in detecting positive sheep samples, whereas gag-PCR allowed a good detection of samples of positive goats and positive sheep. In addition, pol-PCR was more efficient with goat samples than for sheep. Duplex nested PCR performed with standard virus samples and field strains demonstrated that the technique is more efficient for the detection of multiple pro-viral DNA sequences. This study demonstrated a successful duplex nested PCR assay allowing a more accurate diagnosis of SRLV.

RESUMO.-[Isolamento, cultura e caracterização de cé-lulas tronco mesenquimais multipotentes provenientes do sangue do cordão umbilical caprino.] 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 aná-lise 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. 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.

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.

Background: Small ruminant lentiviruses (SRLV) are characterized by a high degree of genetic variability related to your replication process, resulting in several strains in different geographic regions. The Polymerase Chain Reaction (PCR) is very successful in the detection of proviral DNA of SRLV, however, due to the high variability of the lentivirus genome, the efficiency and sensibility of PCR depends mainly on the specificity of the primers designed and the choice of the amplified target viral region. The aim of this study was to compare detection of Maedi Visna Virus (MVV) from bronco alveolar lavage samples of sheep by Nested PCR using primers for the gag and LTR genes.Materials, Methods & Results: Samples of sheep bronchoalveolar lavage (n = 58) from slaughterhouse in the Metropolitan Region of Fortaleza were previously tested by nested PCR using primers for region gag. Thereafter, these samples were tested by nested PCR with primers designed for the LTR region. Both tests were conducted using thermocycler (Biocycler®) under the following conditions: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing of primers at 56°C for 1 min and extension of DNA at 72°C for 45 s with a final extension at 72 for 7 min. The first and second round were performed under the same conditions. Every amplification was performed using a positive control MVV-K1514 and water RNA/DNA free with a negative control. After the amplification, the PCR products were separated by agarose gel electrophoresis at 1% stained with ethidium bromide in TBE buffer. The tests revelead only 1 sample (P1) was detected exclusively for the primer of gag gene, while 8 samples were positive only for the test performed with primers of the LTR region, 5 samples were positive for both sets of primers tested and 30 samples were negative for all tests. The test with the LTR gene demonstrated 37.93% (22/58) positives of Maedi Visna in the samples studied.Discussion: In recent years, with advances in molecular biology techniques, some PCR protocols have been developed for the diagnosis of SRLV. However, these viruses exhibit a high instability and mutation rate becomes very difficult to use the same primers in different geographic regions. In this study, comparing the MVV detection capability by nested PCR with differents primer sets was possible to demonstrate that primers LTR gene were more efficient in detecting positive animals when compared with the primers designed for the gag region. In all tests, only the animal (P1) was positive for the nested PCR performed with the primers for the gag gene, not being detected by the LTR gene. Some studies suggest success in the detection of MVV using primers for the gag gene. However, for more efficient detection of MVV in sheep samples, many studies have shown that the choice of primers for the LTR region is more accurate, since these primers have better MVV detection capability even when it has a large range of circulating virus strains. it is known that the genetic diversity of SRLV generate difficulties in carrying out molecular tests, since the molecular diagnostic tests depend on factors such as the percentage of identity of nucleotides of the viral populations circulating in the herds and the sequences used for testing. In this study it is possible to conclude that the effective control of lentiviruses diagnostic methods should be chosen properly in order to be applied in disease control programs.

The production performance of a livestock herd can be compromised by various diseases. In sheep, maedi-visna (MV) infections, which have a chronic nature, are caused by a virus (maedi-visna virus (MVV)) belonging to the genus Lentivirus of the Retroviridae family. The infection can cause significant economic losses and has considerable health impacts on sheep breeding in production systems. Due to the importance of this disease in sheep flocks, the objective was to conduct a serosurvey of MVV in the states of Ceará (CE), Rio Grande do Norte (RN), Paraíba (PB), and Sergipe (SE). A total of, 3332 serum samples were collected in the four states, 1011 in CE, 931 in RN, 459 in PB, and 931 in SE, with the number of samples proportional to the actual herd size of each state. The samples were analyzed using the agar gel microimmunodiffusion test (AGID). Reproducers were revaluated using western blotting (WB). In addition to this serological survey, we administered an investigative questionnaire to identify possible risk factors that facilitate the introduction and spread of diseases (location, category, sex, breed type, creation system, production, herd size, and association with goats). After analysis of the sera using the AGID test, there was zero prevalence. Revaluating breeders by WB revealed a 5.5% prevalence of MV in the four states studied, with prevalences for the states of CE, RN, Paraiba, and SE of 2.3% (2/88), 10.4% (8/77), 3.6% (1/28), and 4.7% (2/42), respectively, corresponding to 13 breeders containing antibodies to the virus. These findings emphasized that the choice of diagnostic tests is extremely important for the early detection of seropositive animals and thus the prevention of the spread of the virus among herds in the region.

RESUMOAvaliou-se a influência do vírus da CAE nas características físico-químicas de amostras de leite de 54 cabras, sem predileção racial, distribuindo-as em dois grupos: cabras positivas e negativas para o teste de imunodifusão em gel de agarose. As amostras de leite foram submetidas à análise ultrassônica para obtenção de parâmetros físico-químicos -gordura, extrato seco, proteínas, lactose e densidade; realização de microbiologia -bactérias mesófilas (UCF/mL). Foram coletadas amostras de tecido mamário para exame histopatológico e imunohistoquímica. Não houve diferença significativa das características avaliadas entre os dois grupos; no microbiológico, não houve relação direta da presença de mesófilas associada à infecção pelo CAEV. Na histopatologia, observaram-se áreas com infiltração celular de monócitos, polimorfonucleares, plasmócitos, fibrose, ausência de morfologia normal do parênquima mamário, denotando processo inflamatório crônico; e foi confirmada a presença do vírus na glândula pela imunohistoquímica. Os resultados não mostraram relação direta da incidência da CAE como fator negativo no desenvolvimento do rebanho.Palavras-chave: CAE, lentivírus, análise físico-química, leite ABSTRACT Aiming to evaluate the influence of CAE viruses in the chemical and physical characteristics of milk

RESUMO -O crescimento e desenvolvimento do rebanho caprino no Nordeste são observados com o aumento na produção da pecuária do Brasil. Esse aumento é reflexo, a princípio, das maiores exigências do mercado consumidor por produtos de melhor qualidade obtidos a partir de rebanhos de alto padrão zootécnico. As pesquisas atuais ilustram a necessidade de dispor de biomarcadores que auxiliem a indicação do potencial reprodutivo dos animais, uma vez que isso não pode ser expresso apenas com o exame andrológico. A avaliação da expressão das proteínas, tomando-as como biomarcadores, é análise potencial uma vez que estas, dentre os constituintes do plasma seminal, são encontradas em maior quantidade na forma de complexos associados, desempenhando papel crucial em todos os processos relacionados à capacidade fecundante dos espermatozoides. Essa análise é realizada por métodos de separação e detecção simultânea de proteínas utilizando técnicas como a eletroforese bidimensional (2DE) ou cromatografias, acoplados a métodos cada vez mais eficientes e sensíveis de identificação e quantificação de níveis de expressão de proteínas por espectrometria de massas. O objetivo desta revisão é abordar sobre a técnica de eletroforese bidimensional associada à espectrometria de massa como ferramenta na análise da expressão de proteínas dentro do campo da proteômica.Palavras-Chave: proteínas, plasma seminal, eletroforese 2DE, espectrometria de massa.ABSTRACT -The growth and development of goat herds in the northeast are being observed due to the increase in livestock production in Brazil. This increase reflects demands of the consumer market for high quality product, which are obtained from flocks of high standard zootechnics. Current research illustrates the need for biomarkers that indicate an animal´s reproductive potential, since this cannot be expressed solely with andrologic evaluation. For this reason, the expression of the proteins as biomarkers is a potential analysis. The proteins from the seminal plasma constituents are found in larger amounts in the form of associated complexes, playing a crucial role in all processes related to the fertilizing capacity of sperm. This analysis is performed by separation methods and simultaneous detection of proteins using techniques such as two-dimensional electrophoresis (2DE) or chromatography. These techniques are coupled with methods to identify and quantify expression levels of proteins by mass spectrometry that are increasingly efficient and sensitive. The aim of this review was to discuss the technique of two-dimensional electrophoresis combined with mass spectrometry as a tool in the analysis of protein expression within the field of proteomics.

___________________________________________________________________________________________________ Resumo:As pesquisas com células-tronco datam da década de 60, por meio de estudos pioneiros com o transplante de medula em camundongos. Com a demonstração de sua capacidade de autorrenovação e diferenciação, a comunidade científica despertou para o grande potencial destas células para pesquisas na área da saúde. Novos paradigmas são estabelecidos a cada avanço. O fato, é que as células-tronco têm se tornado a grande esperança no tratamento de enfermidades e desenvolvimento de terapias regenerativas, tanto na área humana quanto veterinária, sendo um valioso achado da comunidade científica. Nesse contexto, a proposta dessa revisão é abordar de forma concisa os conceitos, avanços e perspectivas das pesquisas com células-tronco. Termos para indexação:Células-tronco, Terapêutica, Biotecnologia. Abstract:Research on stem cells dating from the 1960s through pioneering studies with bone marrow transplantation in mice. With the demonstration of its ability to self-renewal and differentiation, the scientific community has woken up to the great potential of these cells for research in healthcare. New paradigms are established at each advance. The fact is that the stem cells have become the great hope for the treatment of diseases and development of regenerative therapies, both in human to veterinary area as being a valuable finding of the scientific community. In this context, the purpose of this review is to address concisely the concepts, progress and prospects of research on stem cells.