Le Maréchal C, Jan G, Even S, McCulloch J A, Azevedo V, Thiéry R, Vautor E, Le Loir Y
INRA, UMR1253 STLO, 85 Rue de Saint Brieuc, 35042 Rennes Cedex, France.
J Microbiol Methods. 2009 Oct;79(1):131-6. doi: 10.1016/j.mimet.2009.08.017. Epub 2009 Sep 4.
Staphylococcus aureus is a major agent of mastitis in ruminants worldwide. So far, efficient measures for its prophylaxis (including vaccination) have proven to be unsuccessful and there is a need for a better understanding of the host response to udder infection by S. aureus. Serological proteome analysis (SERPA) is a promising technique that can be used to identify S. aureus immuno-dominant determinants providing that bacterial culture conditions used to grow S. aureus strains for protein sample preparation mimic the context of mastitis. A S. aureus strain was used in experimental mastitis to generate sheep serum used to determine the best growth conditions for SERPA. Sera collected in the field from different ewes suffering from mastitis by S. aureus were used to confirm experimental observations. Three different culture media (BHI, whey and iron-depleted RPMI) were tested. The influence of aeration and growth phase on protein production was also evaluated by immuno-detection of protein samples prepared from cultures grown in different conditions and obtained from different culture fractions (supernatant, cell wall, and total lysates). Our results showed that culturing in iron-depleted RPMI with (secreted proteins, prepared from stationary phase) or without aeration (cell wall proteins, prepared from early stationary phase, and total proteins, prepared from exponential phase) is the condition that best mimics growth in vivo during mastitis and this in vitro growth condition is to be used henceforth in experiments involving SERPA.
金黄色葡萄球菌是全球反刍动物乳腺炎的主要病原体。到目前为止，其预防措施（包括疫苗接种）已被证明是不成功的，因此需要更好地了解宿主对金黄色葡萄球菌引起的乳房感染的反应。血清学蛋白质组分析（SERPA）是一种很有前景的技术，只要用于培养金黄色葡萄球菌菌株以制备蛋白质样品的细菌培养条件模拟乳腺炎的环境，就可用于鉴定金黄色葡萄球菌的免疫优势决定簇。在实验性乳腺炎中使用一株金黄色葡萄球菌来制备绵羊血清，以确定SERPA的最佳生长条件。从现场采集的患有金黄色葡萄球菌引起的乳腺炎的不同母羊的血清用于证实实验观察结果。测试了三种不同的培养基（脑心浸液培养基、乳清和缺铁的RPMI培养基）。还通过对在不同条件下培养并从不同培养部分（上清液、细胞壁和总裂解物）获得的培养物制备的蛋白质样品进行免疫检测，评估了通气和生长阶段对蛋白质产生的影响。我们的结果表明，在缺铁的RPMI培养基中培养（从稳定期制备的分泌蛋白）或不通气（从早期稳定期制备的细胞壁蛋白和从对数期制备的总蛋白）是最能模拟乳腺炎期间体内生长的条件，今后在涉及SERPA的实验中应使用这种体外生长条件。
