Human proinsulin production in the milk of transgenic cattle.

BACKGROUND: The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk. METHODS AND RESULTS: Pseudo-lentivirus containing the bovine ß-casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced. Recombinant protein in milk was evaluated using western blotting and mass spectrometry. One transgenic cow was generated, and in milk analysis, two bands were observed in western blotting with a molecular mass corresponding to the proinsulin and insulin. The mass spectrometry analysis showed the presence of human insulin more than proinsulin in the milk, and it identified proteases in the transgenic milk that could convert proinsulin into insulin and insulin-degrading enzyme that could degrade the recombinant protein. CONCLUSION: The methodologies used for generating the transgenic cow allowed the detection of the production of recombinant protein in the milk at low relative expression compared to milk proteins, using mass spectrometry, which was efficient for detecting recombinant protein with low expression in milk. Milk proteases could act on protein processing converting recombinant protein to functional protein. On the other hand, some milk proteases could act in degrading the recombinant protein.

In vivo evaluation of the antiretroviral activity of Melia azedarach against small ruminant lentiviruses in goat colostrum and milk.

This study aimed to evaluate in vivo the use of the extract from the leaves of Melia azedarach in the ethyl acetate fraction at a concentration of 150 µg/mL as an antiretroviral treatment against small ruminant lentiviruses (SRLV) in goat colostrum, and milk with a 90-min action. Two groups of six kids were treated with the extract. One group received three supplies of colostrum from does naturally positive for SRLV, treated with the ethyl acetate fraction of M. azedarach (EAF-MA) for three days, while the other group consumed milk from does also carrying the virus with the respective extract twice a day for five days. After undergoing treatment, all animals began to receive thermized milk until weaning (60 days) and were monitored for six months using nested polymerase chain reaction (nPCR) and western blot (WB) tests. The study revealed cumulative percentages of positive animals in WB or nPCR in the milk group of 66.66% on the seventh day, 83.33% in the following week, and 100% at 120 days, while the colostrum group showed values of 66.66% at 14 days, 83.33% at 90 days, and 100% at 120 days. Variation and intermittency were observed in viral detection, but all animals tested positive in WB or nPCR at some point. A potential delay in infection was observed, which was more significant in the colostrum group. The need for the combination of serological and molecular tests for a more efficient detection of the disease is also emphasized.

OPTO-BLUE: An Integrated Bidirectional Optogenetic Lentiviral Platform for Controlled Light-Induced Gene Expression.

Regulated systems for transgene expression are useful tools in basic research and a promising platform in biomedicine due to their regulated transgene expression by an inducer. The emergence of optogenetics expression systems enabled the construction of light-switchable systems, enhancing the spatial and temporal resolution of a transgene. The LightOn system is an optogenetic tool that regulates the expression of a gene of interest using blue light as an inducer. This system is based on a photosensitive protein (GAVPO), which dimerizes and binds to the UASG sequence in response to blue light, triggering the expression of a downstream transgene. Previously, we adapted the LightOn system to a dual lentiviral vector system for neurons. Here, we continue the optimization and assemble all components of the LightOn system into a single lentiviral plasmid, the OPTO-BLUE system. For functional validation, we used enhanced green fluorescent protein (EGFP) as an expression reporter (OPTO-BLUE-EGFP) and evaluated the efficiency of EGFP expression by transfection and transduction in HEK293-T cells exposed to continuous blue-light illumination. Altogether, these results prove that the optimized OPTO-BLUE system allows the light-controlled expression of a reporter protein according to a specific time and light intensity. Likewise, this system should provide an important molecular tool to modulate gene expression of any protein by blue light.

Multiplex assay for the simultaneous detection of antibodies against small ruminant lentivirus, Mycobacterium avium subsp. paratuberculosis, and Brucella melitensis in goats.

Background and Aim: Brucellosis, paratuberculosis (PTb), and infections caused by small ruminant lentivirus (SRLV), formerly known as caprine arthritis encephalitis virus (CAEV), adversely affect goat production systems. Nonetheless, commonly used diagnostic tests can only determine one analyte at a time, increasing disease surveillance costs, and limiting their routine use. This study aimed to design and validate a multiplex assay for antibody detection against these three diseases simultaneously. Materials and Methods: Two recombinant proteins from the SRLV (p16 and gp38), the native hapten of Brucella melitensis, and the paratuberculosis-protoplasmic antigen 3 from Mycobacterium avium subsp. paratuberculosis (MAP) were used to devise and assess a multiplex assay. Conditions for the Luminex® multiplex test were established and validated by sensitivity, specificity, repeatability, and reproducibility parameters. Cut-off points for each antigen were also established. Results: The 3-plex assay had high sensitivity (84%) and specificity (95%). The maximum coefficients of variation were 23.8% and 20.5% for negative and positive control samples, respectively. The p16 and gp38 SRLV antigens are 97% and 95%, similar to the CAEV sequence found in GenBank, respectively. Conclusion: The multiplex test can be effectively used for the simultaneous detection of antibodies against SRLV, MAP and B. melitensis in goats.

Stable expression of shRNA for the control of recombinant adenovirus replication

Preventing the replication of adenovirus could have practical uses, such as controlling infection with wild-type virus or in applications involving recombinant vectors. Mainly transient methods have been used to inhibit adenovirus replication, including siRNA or drugs. Here, we tested whether stable expression of shRNA designed to target hexon, Iva2, or pol can inhibit the replication of a recombinant adenoviral vector, Ad-LacZ (serotype 5, E1/E3 deleted), in 293T cells. Significant knockdown correlating with reduced Ad-LacZ replication was achieved only when hexon was targeted. Cell sorting and isolation of cellular clones further accentuated knockdown of the hexon transcript, reduced protein levels by more than 90%, and diminished adenovirus production. As visualized by transmission electron microscopy, the cellular clone expressing the hexon-specific shRNA yielded 89.2% fewer particles compared to the parental 293T cells. Full scale production followed by purification revealed a 90.2% reduction in Ad-LacZ biological titer. These results support the notion that stable expression of shRNA can be used as a means to control adenovirus replication.

Padronização de um ELISA indireto a partir da utilização de uma proteína de superfície do vírus da anemia infecciosa equina como antígeno para o diagnóstico dessa enfermidade / Standardization of an indirect ELISA using a surface protein of the equine infectious anemia virus as an antigen for the diagnosis of this disease

A anemia infecciosa equina é uma importante enfermidade que acomete os equídeos em todo o mundo, se apresentando de forma aguda, crônica e assintomática causando grandes prejuízos para a economia tanto para criadores que vivem do trabalho desses animais quantos aos criadores que investem no melhoramento das raças, impedindo o acesso ao mercado tanto nacional quanto internacional. O Ministério da Agricultura, Pecuária e Abastecimento considera o IDGA como teste oficial para diagnóstico dessa enfermidade, porém essa técnica é demorada e muita vez acaba sendo subjetiva, dependendo da experiencia particular de cada Laboratorista. Além de não conseguir detectar animais no início da infecção. Logo, a necessidade de se buscar novas técnicas como o ELISA indireto que aperfeiçoem o tempo de análise dos resultados, facilita a automação e obtém resultados confiáveis. O estudo realizado teve como objetivo padronizar uma técnica de ELISA indireto utilizando uma proteína de envelope viral GP90 como antígeno para diagnóstico da anemia infecciosa equina. Avaliando o desempenho do teste a partir da sensibilidade, especificidade e valores preditivos positivo e negativo. Os valores obtidos foram: 91,11%, 93,33%, 91,11% e 93,33% respectivamente. Concluiu-se que o teste apresenta bom desempenho, além da possibilidade de detectar amimais positivos no início da infecção.

Equine infectious anemia is an important disease that affects horses all over the world, presenting in an acute, chronic and asymptomatic way, causing great damage to the economy, both for breeders who live off the work of these animals and for breeders who invest in the improvement of breeds, preventing access to both national and international markets. The Ministry of Agriculture, Livestock and Food Supply considers AGID to be the official test for diagnosing this disease, but this technique takes time and often ends up being subjective, depending on the particular experience of each laboratory worker. In addition to not being able to detect animals at the beginning of the infection. Therefore, the need to seek new techniques such as indirect ELISA that improve the time of analysis of results, facilitate automation and obtain reliable results. The aim of this study was to standardize an indirect ELISA technique using a GP90 viral envelope protein as an antigen for the diagnosis of equine infectious anemia. Evaluating test performance based on sensitivity, specificity and positive and negative predictive values. The values obtained were 91.11%, 93.33%, 91.11 and 93.33 respectively. It was concluded that the test performs well, in addition to the possibility of detecting positive animals at the beginning of the infection.

Chimeric Antigen by the Fusion of SARS-CoV-2 Receptor Binding Domain with the Extracellular Domain of Human CD154: A Promising Improved Vaccine Candidate.

COVID-19 is a respiratory viral disease caused by a new coronavirus called SARS-CoV-2. This disease has spread rapidly worldwide with a high rate of morbidity and mortality. The receptor-binding domain (RBD) of protein spike (S) mediates the attachment of the virus to the host's cellular receptor. The RBD domain constitutes a very attractive target for subunit vaccine development due to its ability to induce a neutralizing antibody response against the virus. With the aim of boosting the immunogenicity of RBD, it was fused to the extracellular domain of CD154, an immune system modulator molecule. To obtain the chimeric protein, stable transduction of HEK-293 was carried out with recombinant lentivirus and polyclonal populations and cell clones were obtained. RBD-CD was purified from culture supernatant and further characterized by several techniques. RBD-CD immunogenicity evaluated in mice and non-human primates (NHP) indicated that recombinant protein was able to induce a specific and high IgG response after two doses. NHP sera also neutralize SARS-CoV-2 infection of Vero E6 cells. RBD-CD could improve the current vaccines against COVID-19, based in the enhancement of the host humoral and cellular response. Further experiments are necessary to confirm the utility of RBD-CD as a prophylactic vaccine and/or booster purpose.

Genetic and antigenic characterization of Brazilian SRLV strains: Natural small ruminant interspecies transmission from mixed herds.

Cross-species transmission events and mixed infection of small ruminant lentiviruses (SRLVs) were studied in seven goats and two sheep from three small ruminant mixed flocks from Northeast and Southeast Brazil. Genetic and antigenic analyses with gag/env genes and ELISA multiepitope SU1/SU5 recombinant antigens were carried out, respectively. The genetic analysis of gag and env sequences showed high viral diversity in both species, MVV-like (subtype A1) and CAEV-like B1 in goats, and CAEV-like (subtype B1) in sheep, revealing SRLV interspecies transmission from sheep to goats and vice versa in Brazilian farms. Two Brazilian caprine lentiviruses were segregated in two new genetic clades based on gag analyses, which suggests a new classification into heterogenic genotype A. Furthermore, goat isolates were grouped into subtype A1 and B1 clusters. Cross-reactive antibodies were detected in goats using ELISA with a recombinant antigen carrying SU1 and SU5 immunodominant epitopes; the results showed anti-CAEV and MVV antibodies in goats and anti-CAEV antibodies in sheep. This result can be associated with the high divergence in the V4 region due to SRLV variability. All results confirm cross-species infection of SRLV in Brazilian mixed herds.

An outbreak of visna-maedi in a flock of sheep in Southern Brazil.

Visna-maedi is a multisystemic and progressive inflammatory disease caused by a non-oncogenic retrovirus (Visna-maedi virus, VMV). An outbreak of visna-maedi occurred in Southern Brazil in sheep with clinical signs of blindness and stumbling gait. At post-mortem examination, all animals had similar lesions, including heavy non-collapsed lungs and multifocal yellow areas in the cerebral white matter, affecting mainly the periventricular region. These lesions corresponded histologically to lymphocytic interstitial pneumonia and histiocytic periventricular encephalitis surrounding areas of necrosis, in addition to significant demyelination in the brain. Serology was performed in all the sheep from the flock and 14% were seropositive for VMV. The presence of VMV was confirmed through PCR and partial sequencing of the 5'LTR. Sequencing demonstrated that the virus had 89.7 to 90.0% of nucleotide identity with VMV strains reported in the USA. This is the first description of clinical disease related to VMV in Brazil leading to economic losses. This study calls for the need to implement control measures to prevent the spread of small ruminant lentiviruses in Brazil.

A combined antitumor strategy of separately transduced mesenchymal stem cells with soluble TRAIL and IFNß produces a synergistic activity in the reduction of lymphoma and mice survival enlargement.

As the understanding of cancer grows, new therapies have been proposed to improve the well-known limitations of current therapies, whose efficiency relies mostly on early detection, surgery and chemotherapy. Mesenchymal stem cells (MSCs) have been introduced as a promissory and effective therapy. This fact is due to several useful features of MSCs, such as their accessibility and easy culture and expansion in vitro, and their remarkable ability for 'homing' towards tumors, allowing MSCs to exert their anticancer effects directly into tumors. Additionally, MSCs offer the practicability of being genetically engineered to carry anticancer genes, increasing their specificity and efficacy for fighting tumors. In the present study, the antitumoral efficacy and post-implant survival of mice bearing lymphomas implanted intratumorally were determined using mouse bone marrow-derived (BM)-MSCs transduced with soluble TRAIL (sTRAIL), full length TRAIL (flTRAIL), or interferon ß (IFNß), naïve BM-MSCs, or combinations of these. The percentage of surviving mice was determined once all not-implanted mice succumbed. It was found that the percentage of surviving mice implanted with the combination of MSCs-sTRAIL and MSCs-IFN-ß was 62.5%. Lymphoma model achieved 100% fatality in the non-treated group by day 41. On the other hand, the percentage of surviving mice implanted with MSCs-sTRAIL was 50% and with MSCs-INFß 25%. All the aforementioned differences were statistically significant (P<0.05). In conclusion, all implants exhibited tumor size reduction, growth delay, or apparent tumor clearance. MSCs proved to be effective anti-lymphoma agents; additionally, the combination of soluble TRAIL and IFN-ß resulted in the most effective antitumor and life enlarging treatment, showing an additive antitumoral effect compared with individual treatments.

Boosting antitumor response with PSMA-targeted immunomodulatory VLPs, harboring costimulatory TNFSF ligands and GM-CSF cytokine.

Therapeutic strategies based on immunomodulation have improved cancer therapy. Most approaches target co-stimulatory pathways or the inhibition of immunosuppressive mechanisms, to enhance immune response and overcome the immune tolerance of tumors. Here, we propose a novel platform to deliver targeted immunomodulatory signaling, enhancing antitumor response. The platform is based on virus-like particles derived from lentiviral capsids. These particles may be engineered to harbor multifunctional ligands on the surface that drive tropism to the tumor site and deliver immunomodulatory signaling, boosting the antitumor response. We generated virus-like particles harboring a PSMA-ligand, TNFSF co-stimulatory ligands 4-1BBL or OX40L, and a membrane-anchored GM-CSF cytokine. The virus-like particles are driven to PSMA-expressing tumors and deliver immunomodulatory signaling from the TNFSF surface ligands and the anchored GM-CSF, inducing T cell proliferation, inhibition of regulatory T cells, and potentiating elimination of tumor cells. The PSMA-targeted particles harboring immunomodulators enhanced antitumor activity in immunocompetent challenged mice and may be explored as a potential tool for cancer immunotherapy.

Lentiviral transduction of neonatal rat ventricular myocytes preserves ultrastructural features of genetically modified cells.

Preserving morphological features that are important for cell function and structure is a critical parameter for in vitro experiments with rat cardiomyocytes. Lentiviral vectors are commonly used as gene transfer tool because of its high flexibility, efficiency to deliver expression cassettes and versatility of transducing quiescent cells. The tropism of the recombinant viral particle can be determined depending on the virus envelope, which shows a specific binding to cell surface receptors on the target cell. The combination of promoter arrangement and viral envelope must be optimized to achieve a greater transduction efficiency and a higher transgene expression. In this study we explored the optimization of promoters and heterologous envelopes to transduce primary culture of neonatal rat ventricular myocytes. Our results suggest a robust expression driven by the cytomegalovirus promoter, and high efficiency transduction mediated by VSV-G envelope with no apparent compromising ultrastructural features of genetically modified cells.

Optimization of the Light-On system in a lentiviral platform to a light-controlled expression of genes in neurons

BACKGROUND: Molecular brain therapies require the development of molecular switches to control gene expression in a limited and regulated manner in time and space. Light-switchable gene systems allow precise control of gene expression with an enhanced spatio-temporal resolution compared to chemical inducers. In this work, we adapted the existing light-switchable Light-On system into a lentiviral platform, which consists of two modules: (i) one for the expression of the blue light-switchable transactivator GAVPO and (ii) a second module containing an inducible-UAS promoter (UAS) modulated by a light-activated GAVPO. RESULTS: In the HEK293-T cell line transfected with this lentiviral plasmids system, the expression of the reporter mCherry increased between 4 to 5 fold after light induction. A time expression analysis after light induction during 24 h revealed that mRNA levels continuously increased up to 9 h, while protein levels increased throughout the experiment. Finally, transduction of cultured rat hippocampal neurons with this dual Light-On lentiviral system showed that CDNF, a potential therapeutic trophic factor, was induced only in cells exposed to blue light. CONCLUSIONS: In conclusion, the optimized lentiviral platform of the Light-On system provides an efficient way to control gene expression in neurons, suggesting that this platform could potentially be used in biomedical and neuroscience research, and eventually in brain therapies for neurodegenerative diseases.

Screening and selection strategy for the establishment of biosimilar to trastuzumab-expressing CHO-K1 cell lines.

The high prices of biopharmaceuticals or biologics used in the treatment of many diseases limit the access of patients to these novel therapies. One example is the monoclonal antibody trastuzumab, successfully used for breast cancer treatment. An economic alternative is the generation of biosimilars to these expensive biopharmaceuticals. Since antibody therapies may require large doses over a long period of time, robust platforms and strategies for cell line development are essential for the generation of recombinant cell lines with higher levels of expression. Here, we obtained trastuzumab-expressing CHO-K1 cells through a screening and selection strategy that combined the use of host cells pre-adapted to protein-free media and suspension culture and lentiviral vectors. The results demonstrated that the early screening strategy obtained recombinant CHO-K1 cell populations with higher enrichment of IgG-expressing cells. Moreover, the measurement of intracellular heavy chain polypeptide by flow cytometry was a useful metric to characterize the homogeneity of cell population, and our results suggest this could be used to predict the expression levels of monoclonal antibodies in early stages of cell line development. Additionally, we propose an approach using 25 cm2 T-flasks in suspension and shaking culture conditions as a screening tool to identify high producing cell lines. Finally, trastuzumab-expressing CHO-K1 clones were generated and characterized by batch culture, and preliminary results related to HER2-recognition capacity were successful. Further optimization of elements such as gene optimization, vector selection, type of amplification/selection system, cell culture media composition, in combination with this strategy will allow obtaining high producing clones.

Cochlea cell-specific marker expression upon in vitro Hes1 knockdown

NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.

Bases para um programa de controle da artrite encefalite caprina em rebanho leiteiro / [Bases for a program of control of caprine arthritis encephalite in dairy flock]

Objetivou-se avaliar um programa de controle da artrite encefalite caprina (AEC), por meio de testes diagnósticos sensíveis, separação de mãe e cria após o parto e medidas de manejo, com o intuito de formar rebanho livre do vírus. Utilizou-se um total de 47 cabritos da raça Saanen, mantidos isoladamente até o resultado dos primeiros testes de reação em cadeia de polimerase nested (PCR nested) e Western Blotting (WB), com base na coleta de sangue no momento do nascimento (M0). No PCR nested, quatro animais foram positivos, no M0, e foram eutanasiados. Posteriormente, os demais 43 cabritos foram submetidos à coleta de sangue aos 60 (M60) e 270 (M270) dias de vida para realização de novos testes de WB e PCR nested, que não detectaram animais positivos. Pode-se afirmar que a metodologia adotada neste estudo foi efetiva no controle da doença, nas fases de aleitamento e pós-aleitamento, e que a combinação do sistema de manejo, a fim de propiciar diminuição de risco de transmissão horizontal, com técnicas de diagnóstico mais apuradas, como o WB e a PCR nested, é relevante para elaboração de plano estratégico de controle da enfermidade.(AU)

We aimed to evaluate a program to control Caprine Arthritis Encephalitis (CAE), using diagnostic tests, separation of the mother and postpartum and other management measures, in order to form a free flock of the virus. We used a total of 47 Saanengoats in isolation until the results of the first nested Polymerase Chain Reaction (nested PCR) and Western Blotting (WB) tests, based on blood collection at the time of birth (M0). In the nested PCR, 4 animals were positive, at M0, and were eliminated. Later, the other 43goats were submitted to blood collection at 60 (M60) and 270 (M270) days of life to perform new tests of WB and nested PCR, which did not detect positive animals. We can affirm that the methodology adopted in this study was effective in the control of the disease, in the phase of breastfeeding and post-breastfeeding, and that the combination of the management system, which allows a reduction of risk of horizontal transmission, with more accurate diagnostic techniques, such as WB and nested PCR, is relevant for the elaboration of a strategic plan for the disease control.(AU)

Seropositivity for Maedi-Visna virus in sheep in Porto Acre city - Western Amazon, Brazil / Soropositividade para o vírus Maedi-Visna em ovinos da cidade de Porto Acre - Amazônia Ocidental, Brasil

Lentivirosis of small ruminants (LVPR) are chronic and degenerative infectious diseases, caused by Lentivirus, associated with numerous losses such as: drop in meat and milk production, predisposition to secondary infections, expenses with veterinary assistance and, even, early disposal of animals. In the northern region of Brazil, the epidemiological situation is poorly understood. Thus, this study aimed to determine the seropositivity of sheep for Lentivirus in Porto Acre city, Western Amazon, Brazil. 122 blood samples from sheep were collected and as a diagnostic method, agarose gel immunodiffusion was used, using the p28 protein of the capsid as antigen. The seropositivity of the sheep to the test was 8.2% (10/122). In 80% (4/5) of the investigated properties, the presence of seropositive animals was detected. It is worth noting that the acquisition of small ruminants from other states likely represented a risk to sheep health in the municipality of Porto Acre, Western Amazon, Brazil. It is concluded that there is a need for more systematic investigations on the prevalence of LVPR in the state of Acre.(AU)

As lentiviroses de pequenos ruminantes (LVPR) são enfermidades infecciosas crônicas e degenerativas, causadas por Lentivírus, associadas a inúmeros prejuízos como: queda na produção de carne e leite, predisposição a infecções secundárias, gastos com assistência veterinária e, até mesmo, descarte precoce dos animais. Na região norte do Brasil, a situação epidemiológica é pouco elucidada. Objetivou-se, assim, por meio deste estudo, determinar a soropositividade de ovinos para Lentivírus no município de Porto Acre, Amazônia Ocidental, Brasil. Foram coletadas 122 amostras de sangue de ovinos e como método diagnóstico foi empregada a imunodifusão em gel de agarose, utilizando a proteína p28 do capsídeo como antígeno. A soropositividade dos ovinos ao teste foi de 8,2% (10/122). Em 80% (4/5) das propriedades investigadas, detectou-se a presença de animais soropositivos. É válido ressaltar ainda que a aquisição de pequenos ruminantes advindos de outros estados provavelmente representou um risco à sanidade ovina no município de Porto Acre, Amazônia Ocidental, Brasil. Conclui-se que existe a necessidade de mais investigações sistemáticas sobre a prevalência de LVPR no estado do Acre.(AU)

Short communication: Genetic parameter estimates for caprine arthritis encephalitis in dairy goats.

Caprine arthritis encephalitis (CAE) is a chronic disease caused by a retrovirus from the Lentivirus genus. No effective vaccines or treatments exist, and therefore genetic selection for CAE resistance might be a feasible alternative. To our best knowledge, no other studies have investigated the genetic architecture of CAE resistance in dairy goats. In this context, this study was designed to estimate genetic parameters for CAE infection in Alpine and Saanen goats using a Bayesian threshold model. A total of 542 adult goats (and >3-generation pedigree), which were group-housed in a population with high CAE prevalence, were tested based on a serological infection assessment test (negative = 1 or positive = 2) and used for this study. Genetic parameters were estimated using the BLUPF90 family programs. There was considerable genetic variability for CAE resistance, and pedigree-based heritability was significantly different from zero (0.026 < heritability < 0.128). Our findings indicate that the prevalence of CAE in goat herds can be reduced or eliminated through direct genetic selection for CAE resistance in addition to proper management strategies.

Seropositivity for Maedi-Visna virus in sheep in Porto Acre city - Western Amazon, Brazil / Soropositividade para o vírus Maedi-Visna em ovinos da cidade de Porto Acre - Amazônia Ocidental, Brasil

Lentivirosis of small ruminants (LVPR) are chronic and degenerative infectious diseases, caused by Lentivirus, associated with numerous losses such as: drop in meat and milk production, predisposition to secondary infections, expenses with veterinary assistance and, even, early disposal of animals. In the northern region of Brazil, the epidemiological situation is poorly understood. Thus, this study aimed to determine the seropositivity of sheep for Lentivirus in Porto Acre city, Western Amazon, Brazil. 122 blood samples from sheep were collected and as a diagnostic method, agarose gel immunodiffusion was used, using the p28 protein of the capsid as antigen. The seropositivity of the sheep to the test was 8.2% (10/122). In 80% (4/5) of the investigated properties, the presence of seropositive animals was detected. It is worth noting that the acquisition of small ruminants from other states likely represented a risk to sheep health in the municipality of Porto Acre, Western Amazon, Brazil. It is concluded that there is a need for more systematic investigations on the prevalence of LVPR in the state of Acre.

As lentiviroses de pequenos ruminantes (LVPR) são enfermidades infecciosas crônicas e degenerativas, causadas por Lentivírus, associadas a inúmeros prejuízos como: queda na produção de carne e leite, predisposição a infecções secundárias, gastos com assistência veterinária e, até mesmo, descarte precoce dos animais. Na região norte do Brasil, a situação epidemiológica é pouco elucidada. Objetivou-se, assim, por meio deste estudo, determinar a soropositividade de ovinos para Lentivírus no município de Porto Acre, Amazônia Ocidental, Brasil. Foram coletadas 122 amostras de sangue de ovinos e como método diagnóstico foi empregada a imunodifusão em gel de agarose, utilizando a proteína p28 do capsídeo como antígeno. A soropositividade dos ovinos ao teste foi de 8,2% (10/122). Em 80% (4/5) das propriedades investigadas, detectou-se a presença de animais soropositivos. É válido ressaltar ainda que a aquisição de pequenos ruminantes advindos de outros estados provavelmente representou um risco à sanidade ovina no município de Porto Acre, Amazônia Ocidental, Brasil. Conclui-se que existe a necessidade de mais investigações sistemáticas sobre a prevalência de LVPR no estado do Acre.

Co-transfection of hepatocyte growth factor and truncated TGF-ß type II receptor inhibit scar formation

Wound scarring remains a major challenge for plastic surgeons. Transforming growth factor (TGF)-β plays a key role in the process of scar formation. Previous studies have demonstrated that truncated TGF-β type II receptor (t-TGF-βRII) is unable to continue signal transduction but is still capable of binding to TGF-β, thereby blocking the TGF-β signaling pathway. Hepatocyte growth factor (HGF) is a multifunctional growth factor that promotes tissue regeneration and wound healing. Theoretically, the combination of HGF and t-TGF-βRII would be expected to exert a synergistic effect on promoting wound healing and reducing collagen formation. In the present study, lentivirus-mediated transfection of the two genes (t-TGF-βRII/HGF) into fibroblasts in vitro and in a rat model in vivo was used. The results demonstrated that the expression of t-TGF-βRII and HGF in NIH-3T3 cells was successfully induced. The expression of both molecules significantly reduced collagen I and III expression, and also inhibited fibroblast proliferation. Furthermore, histological examination and scar quantification revealed less scarring in the experimental wound in a rat model. Moreover, on macroscopic inspection, the experimental wound exhibited less visible scarring compared with the control. Therefore, the present study demonstrated that the combination gene therapy of t-TGF-βRII and HGF promoted wound healing, with less scarring and more epithelial tissue formation, not only by suppressing the overgrowth of collagen due to its antifibrotic effect, but also by promoting tissue regeneration.