In vitro antiviral effect of ethanolic extracts from Azadirachta indica and Melia azedarach against goat lentivirus in colostrum and milk.

This study aimed to evaluate, in vitro, the use of leaf extracts of Azadirachta indica (A. indica) and Melia azedarach (M. azedarach) as antivirals against caprine lentivirus (CLV) in colostrum and milk of goat nannies. These were collected from eight individuals and infected with the standard strain of CLV. Samples were then subdivided into aliquots and treated with 150 µg/mL of crude extract, and with ethyl acetate and methanol fractions for 30, 60, and 90 min. Next, somatic cells from colostrum and milk were co-cultured with cells from the ovine third eyelid. After this step, viral titers of the supernatants collected from treatments with greater efficacy in co-culture were assessed. The organic ethyl acetate fractions of both plants at 90 min possibly inhibited the viral activity of CLV by up to a thousandfold in colostrum. In milk, this inhibition was up to 800 times for the respective Meliaceae. In conclusion, the ethanolic fraction of ethyl acetate from both plants demonstrated efficacy against CLV in samples from colostrum and milk when subjected to treatment, which was more effective in colostrum.

Detection and isolation of small ruminant lentivirus in the amniotic fluid of goats.

The objective of this study was to verify the presence of small ruminant lentivirus in the amniotic fluid of goats using molecular tests and viral isolation by cocultivation in the amniotic fluid of naturally infected goats. The study analyzed eight goats: seven were small ruminant lentivirus-positive and one was negative. The amniotic fluid was collected from each of the eight animals during cesarean section at 147 days of pregnancy. Cocultivation was undertaken using secondary goat nictitating membrane cell cultures obtained by explant from a small ruminant lentivirus-negative calf followed by trypsinization and sub-cultivation of the cells for 63 days. During this period, five supernatant collections were performed for DNA extraction and subsequent nested polymerase chain reaction. DNA was extracted from the amniotic fluid after 3 h of cellular sedimentation, from which a sample of 600 µL was taken from the sediment and another 600 µL sample from the supernatant. After DNA extraction, nested polymerase chain reaction was performed. Of the eight goats, 62.5 % (05/08) were small ruminant lentivirus-positive, with 43.75 % (07/16) of the total samples positive when considering the two repetitions (supernatant and cell sediment). Moreover, positivity was confirmed by small ruminant lentivirus pro-viral DNA amplification in the cell supernatant throughout the cocultivation period. Small ruminant lentivirus were present in the amniotic fluid samples from the naturally infected goats indicating an intrauterine transmission route. Moreover, this biological fluid can be adopted for the diagnosis of these lentiviruse because it is an important risk factor related to intrauterine transmission.

A panel of protein candidates for comprehensive study of Caprine Arthritis Encephalitis (CAE) infection.

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.

C. pseudotuberculosis Phop confers virulence and may be targeted by natural compounds.

The bacterial two-component system (TCS) regulates genes that are crucial for virulence in several pathogens. One of such TCS, the PhoPR system, consisting of a transmembrane sensory histidine kinase protein (PhoR) and an intracellular response regulator protein (PhoP), has been reported to have a major role in mycobacterial pathogenesis. We knocked out the phoP in C. pseudotuberculosis, the causal organism of caseous lymphadenitis (CLA), and using a combination of in vitro and in vivo mouse system, we showed for the first time, that the PhoP of C. pseudotuberculosis plays an important role in the virulence and pathogenicity of this bacterium. Furthermore, we modeled the PhoP of C. pseudotuberculosis and our docking results showed that several natural compounds including Rhein, an anthraquinone from Rheum undulatum, and some drug-like molecules may target PhoP to inhibit the TCS of C. pseudotuberculosis, and therefore may facilitate a remarkable attenuation of bacterial pathogenicity being the CLA. Experiments are currently underway to validate these in silico docking results.