Instituto de Agrobiotecnología (IdAB, CSIC-Gobierno de Navarra), Mutilva, Navarra, Spain.
Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
Transbound Emerg Dis. 2019 Jan;66(1):505-516. doi: 10.1111/tbed.13053. Epub 2018 Nov 26.
Brucellosis is a worldwide zoonosis causing important economic loss and a public health problem. Small ruminants are the preferred hosts of Brucella melitensis and thus the main source of human infections. Effective control of sheep and goat brucellosis has been achieved in several countries through vaccination with the live-attenuated B. melitensis Rev1 vaccine. However, Rev1 induces a long-lasting serological response that hinders the differentiation between infected and vaccinated animals. A Rev1::gfp strain expressing constitutively the Green Fluorescent Protein (GFP) was built by stable insertion of a mini-Tn7-gfp in the glmS-recG non-codifying chromosomal region. An associated indirect ELISA-GFP was developed to identify anti-GFP antibodies in vaccinated animals. The resulting Rev1::gfp kept the biological properties of the Rev1 reference strain, including residual virulence and efficacy in mice, and was readily distinguished from Rev1 and other Brucella field strains by direct visualization under ultraviolet illumination, fluorescence microscopy and a multiplex PCR-GFP. The Rev1::gfp strain did not elicit anti-GFP antibodies itself in lambs but when applied in combination with recombinant GFP induced an intense and long-lasting (>9 months) anti-GFP serological response readily detectable by the ELISA-GFP. Overall, our results confirm that Rev1 GFP-tagging can be a suitable alternative for identifying vaccinated sheep in infected contexts.
布鲁氏菌病是一种世界性的动物传染病，给经济造成重大损失，并成为公共卫生问题。小反刍动物是绵羊附睾种布鲁氏菌的首选宿主，因此也是人类感染的主要来源。在一些国家，通过接种减毒活疫苗布鲁氏菌 Rev1 疫苗，已经实现了对绵羊和山羊布鲁氏菌病的有效控制。然而，Rev1 会引起长期的血清学反应，从而阻碍了对感染动物和接种疫苗动物的区分。本研究通过在 glmS-recG 非编码染色体区域的稳定插入 mini-Tn7-gfp，构建了一个稳定表达绿色荧光蛋白 (GFP) 的 Rev1::gfp 菌株。建立了一种相关的间接 ELISA-GFP 方法，用于检测接种疫苗动物中的抗 GFP 抗体。与 Rev1 参考株相比，Rev1::gfp 菌株保持了生物学特性，包括在小鼠中的残余毒力和效力，并且可以通过直接在紫外光下观察、荧光显微镜观察和多重 PCR-GFP 很容易地从 Rev1 菌株和其他布鲁氏菌田间分离株中区分出来。该 Rev1::gfp 菌株本身不会在羔羊中引发抗 GFP 抗体，但当与重组 GFP 联合应用时，会引发强烈且持久（>9 个月）的抗 GFP 血清学反应，通过 ELISA-GFP 很容易检测到。总的来说，我们的研究结果证实，Rev1 GFP 标记可以作为在感染背景下识别接种疫苗绵羊的一种合适替代方法。
