Ohashi N, Unver A, Zhi N, Rikihisa Y
Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210-1093, USA.
J Clin Microbiol. 1998 Sep;36(9):2671-80. doi: 10.1128/JCM.36.9.2671-2680.1998.
A 30-kDa major outer membrane protein of Ehrlichia canis, the agent of canine ehrlichiosis, is the major antigen recognized by both naturally and experimentally infected dog sera. The protein cross-reacts with a serum against a recombinant 28-kDa protein (rP28), one of the outer membrane proteins of a gene (omp-1) family of Ehrlichia chaffeensis. Two DNA fragments of E. canis were amplified by PCR with two primer pairs based on the sequences of E. chaffeensis omp-1 genes, cloned, and sequenced. Each fragment contained a partial 30-kDa protein gene of E. canis. Genomic Southern blot analysis with the partial gene probes revealed the presence of multiple copies of these genes in the E. canis genome. Three copies of the entire gene (p30, p30-1, and p30a) were cloned and sequenced from the E. canis genomic DNA. The open reading frames of the two copies (p30 and p30-1) were tandemly arranged with an intergenic space. The three copies were similar but not identical and contained a semivariable region and three hypervariable regions in the protein molecules. The following genes homologous to three E. canis 30-kDa protein genes and the E. chaffeensis omp-1 family were identified in the closely related rickettsiae: wsp from Wolbachia sp. , p44 from the agent of human granulocytic ehrlichiosis, msp-2 and msp-4 from Anaplasma marginale, and map-1 from Cowdria ruminantium. Phylogenetic analysis among the three E. canis 30-kDa proteins and the major surface proteins of the rickettsiae revealed that these proteins are divided into four clusters and the two E. canis 30-kDa proteins are closely related but that the third 30-kDa protein is not. The p30 gene was expressed as a fusion protein, and the antibody to the recombinant protein (rP30) was raised in a mouse. The antibody reacted with rP30 and a 30-kDa protein of purified E. canis. Twenty-nine indirect fluorescent antibody (IFA)-positive dog plasma specimens strongly recognized the rP30 of E. canis. To evaluate whether the rP30 is a suitable antigen for serodiagnosis of canine ehrlichiosis, the immunoreactions between rP30 and the whole purified E. canis antigen were compared in the dot immunoblot assay. Dot reactions of both antigens with IFA-positive dog plasma specimens were clearly distinguishable by the naked eye from those with IFA-negative plasma specimens. By densitometry with a total of 42 IFA-positive and -negative plasma specimens, both antigens produced results similar in sensitivity and specificity. These findings suggest that the rP30 antigen provides a simple, consistent, and rapid serodiagnosis for canine ehrlichiosis. Cloning of multigenes encoding the 30-kDa major outer membrane proteins of E. canis will greatly facilitate understanding pathogenesis and immunologic study of canine ehrlichosis and provide a useful tool for phylogenetic analysis.
犬埃立克体病的病原体犬埃立克体的一种30 kDa主要外膜蛋白，是自然感染和实验感染犬血清识别的主要抗原。该蛋白与抗重组28 kDa蛋白（rP28）的血清发生交叉反应，rP28是恰菲埃立克体基因（omp-1）家族的外膜蛋白之一。基于恰菲埃立克体omp-1基因序列，用两对引物通过PCR扩增犬埃立克体的两个DNA片段，进行克隆和测序。每个片段都包含犬埃立克体部分30 kDa蛋白基因。用部分基因探针进行基因组Southern印迹分析表明，这些基因在犬埃立克体基因组中存在多个拷贝。从犬埃立克体基因组DNA中克隆并测序了整个基因的三个拷贝（p30、p30-1和p30a）。两个拷贝（p30和p30-1）的开放阅读框串联排列，中间有基因间隔区。这三个拷贝相似但不相同，在蛋白质分子中包含一个半可变区和三个高可变区。在密切相关的立克次体中鉴定出了与三个犬埃立克体30 kDa蛋白基因和恰菲埃立克体omp-1家族同源的以下基因：来自沃尔巴克氏体属的wsp、来自人粒细胞埃立克体病病原体的p44、来自边缘无形体的msp-2和msp-4，以及来自反刍兽考德里氏体的map-1。对三个犬埃立克体30 kDa蛋白和立克次体主要表面蛋白进行系统发育分析表明，这些蛋白分为四个簇，两个犬埃立克体30 kDa蛋白密切相关，但第三个30 kDa蛋白并非如此。p30基因表达为融合蛋白，并在小鼠中制备了针对重组蛋白（rP30）的抗体。该抗体与rP30和纯化的犬埃立克体30 kDa蛋白发生反应。29份间接荧光抗体（IFA）阳性犬血浆标本强烈识别犬埃立克体的rP30。为了评估rP30是否是犬埃立克体病血清诊断的合适抗原，在斑点免疫印迹试验中比较了rP30与整个纯化的犬埃立克体抗原之间的免疫反应。两种抗原与IFA阳性犬血浆标本的斑点反应用肉眼可明显区别于与IFA阴性血浆标本的反应。通过对总共42份IFA阳性和阴性血浆标本进行光密度测定，两种抗原产生的敏感性和特异性结果相似。这些发现表明，rP30抗原为犬埃立克体病提供了一种简单、一致且快速的血清诊断方法。克隆编码犬埃立克体30 kDa主要外膜蛋白的多基因将极大地促进对犬埃立克体病发病机制和免疫学研究的理解，并为系统发育分析提供有用工具。
