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.

Non-agreement between 2 serologic techniques for detecting antibody to Brucella ovis in naturally infected sheep.

Brucella ovis is the causative agent of ovine brucellosis, which is an important infectious disease in sheep farming worldwide and is responsible for economic losses because of its negative effect on the reproductive system of rams and ewes. Serologic tests are the main tools for detection of infection; however, these tests commonly yield a high frequency of false-negative results. We compared 2 serologic tests, agar gel immunodiffusion (AGID) and ELISA, for the detection of anti-B. ovis antibodies in naturally infected sheep. Of the 728 serum samples analyzed, 0.3% were positive by AGID and 9.2% by ELISA. Positive results were obtained for different animals and flocks. There was no statistical difference between the detection frequency of the 2 methods (p = 0.674), and the kappa test indicated low concordance (κ = 0.005). The lack of agreement between results obtained using AGID and ELISA, associated with the absence of clinical signs, makes it difficult to detect ovine brucellosis efficiently, and demonstrates the need for effective tests for the definitive detection of B. ovis infection.

Characterization and frequency of foot injuries and foot-related lameness in a sheep herd with prophylactic measures for foot-related diseases / Caracterização e frequência de lesões podais e da claudicação em rebanho ovino com medidas profiláticas de doenças podais

Foot lesions in sheep herds are responsible for great economic losses, because pain during locomotion interferes with feeding and behavior, leading productivity losses. The objective of this work was to describe the main foot disease found in sheep herd that uses prophylactic measures against foot diseases. A total of 346 ewes of different ages, with or without lameness, were assessed for the presence of foot lesions in all limbs, digits and adjacent structures. Among all the 2768 digits evaluated, 103 (29.76%) had lesions, representing 1.04 lesion per animal. 41.75% (43/103) had only one lesion, 39.81% (41/103) had two lesions, 16 (15.53%) had three lesions, and only 2.91% (3/103) had four or more lesions. Hind limbs were the most affected and the most frequent diseases were white line disease (40.05%), interdigital dermatitis (33.70%) and footrot (12.15%). It was concluded that foot disease in sheep is frequent and constitute a health problem for herds, even on farms that use strategies to prevent it. Therefore, it is essential to classify the lesions, as well as the adoption of effective prophylactic and therapeutic measures.

Lesões podais em ovinos são responsáveis por grandes perdas econômicas, pois a dor durante a locomoção interfere na alimentação e no comportamento, levando à perda de produtividade. O objetivo deste trabalho foi descrever as principais doenças podais encontradas em rebanho ovino, que utiliza medidas profiláticas contra as doenças podais. Foram examinadas 346 ovelhas quanto à presença de lesões podais em todos os membros, dígitos e estruturas adjacentes. Entre os 2.768 dígitos avaliados, 103 (29,76%) apresentavam lesões, representando 1,04 lesões por animal. 41,75% (43/103) apresentavam apenas uma lesão, 39,81% (41/103) tinham duas lesões, 16 (15,53%) tinham três lesões, e apenas três animais apresentavam quatro ou mais lesões. Os membros posteriores foram os mais afetados e as doenças mais frequentes foram a doença da linha branca (40,05%), dermatite interdigital (33,70%) e footrot (12,15%). Pode-se concluir que as lesões podais em ovinos são frequentes e constituem um problema sanitário para os rebanhos, mesmo em propriedade que realiza medidas profiláticas. Portanto, é fundamental classificar as lesões, assim como adotar as medidas profiláticas e terapêuticas eficazes.

Histophilus somni-associated syndromes in sheep from Southern Brazil

Histophilus somni is a Gram-negative bacterium that is associated with a disease complex (termed histophilosis) that can produce several clinical syndromes predominantly in cattle, but also in sheep. Histophilosis is well described in North America, Canada, and in some European countries. In Brazil, histophilosis has been described in cattle with respiratory, reproductive, and systemic disease, with only one case described in sheep. This report describes the occurrence of Histophilus somni-associated disease in sheep from Southern Brazil. Eight sheep with different clinical manifestations from five farms were investigated by a combination of pathological and molecular diagnostic methods to identify additional cases of histophilosis in sheep from Brazil. The principal pathological lesions were thrombotic meningoencephalitis, fibrinous bronchopneumonia, pulmonary abscesses, and necrotizing myocarditis. The main clinical syndromes associated with H. somni were thrombotic meningoencephalitis (n = 4), septicemia (n = 4), bronchopneumonia (n = 4), and myocarditis (n = 3). H. somni DNA was amplified from multiple tissues of all sheep with clinical syndromes of histophilosis; sequencing confirmed the PCR results. Further, PCR assays to detect Pasteurella multocida and Mannheimia haemolytica were negative. These findings confirmed the participation of H. somni in the clinical syndromes investigated during this study, and adds to the previous report of histophilosis in sheep from Brazil.(AU)

Histophilus somni-associated syndromes in sheep from Southern Brazil

Abstract Histophilus somni is a Gram-negative bacterium that is associated with a disease complex (termed histophilosis) that can produce several clinical syndromes predominantly in cattle, but also in sheep. Histophilosis is well described in North America, Canada, and in some European countries. In Brazil, histophilosis has been described in cattle with respiratory, reproductive, and systemic disease, with only one case described in sheep. This report describes the occurrence of Histophilus somni-associated disease in sheep from Southern Brazil. Eight sheep with different clinical manifestations from five farms were investigated by a combination of pathological and molecular diagnostic methods to identify additional cases of histophilosis in sheep from Brazil. The principal pathological lesions were thrombotic meningoencephalitis, fibrinous bronchopneumonia, pulmonary abscesses, and necrotizing myocarditis. The main clinical syndromes associated with H. somni were thrombotic meningoencephalitis (n = 4), septicemia (n = 4), bronchopneumonia (n = 4), and myocarditis (n = 3). H. somni DNA was amplified from multiple tissues of all sheep with clinical syndromes of histophilosis; sequencing confirmed the PCR results. Further, PCR assays to detect Pasteurella multocida and Mannheimia haemolytica were negative. These findings confirmed the participation of H. somni in the clinical syndromes investigated during this study, and adds to the previous report of histophilosis in sheep from Brazil.