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 caprine arthrite encephalite (CAE) is a disease that affects especially dairy goat. The virus shows compartmentalization features, that allows it to hide at certain times during the course of the disease, making it difficult to control. The present study was conducted to identify the major seminal plasma protein profile of goats infected by CAE and its associations with seroconversion using Western blotting. Two groups containing five males each, were used in this experiment. The first group was composed by seropositive animals and the control by seronegative confirmed by Western blotting and PCR. The semen was collected through artificial vagina and after that, two-dimensional electrophoresis and MALDI-TOF MS were used. Seventy-five spots were identified in the goat seminal plasma gels, equivalent to 13 different proteins with more expression. The similar proteins found in both groups and related to reproduction were spermadhesin Z13-like, bodhesin and bodhesin-2, Lipocalin, protein PDC-109-like, and albumin. In infected goats, proteases such as arisulfatase A have been identified, whose function probably is related to metabolism control of sulfatides, involved to virus control. The other ones were bifunctional ATP-dependent dihydroxyacetone kinase/FAD-AMP lyase, cathepsin F isoform X1, disintegrin and metalloproteinase domain-containing protein 2-like isoform X1, clusterin, carbonic anhydrase 2, electron transfer flavoprotein subunit beta, and epididymal secretory glutathione peroxidase. The results of this study show the reaction of the innate immune system against chronic infection of goats by CAE.
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
Produção de antígeno e separação da proteína p28 por microfiltragem seriada para sorodiagnóstico da artrite encefalite caprina por ensaio imunoenzimático
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