Bell-Sakyi Lesley, Palomar Ana M, Bradford Emma L, Shkap Varda
The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK.
The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK; CIBIR, C/ Piqueras, 98, Logroño 26006, Spain.
Vet Microbiol. 2015 Sep 30;179(3-4):270-6. doi: 10.1016/j.vetmic.2015.07.008. Epub 2015 Jul 9.
Anaplasma centrale has been used in cattle as a live blood vaccine against the more pathogenic Anaplasma marginale for over 100 years. While A. marginale can be propagated in vitro in tick cell lines, facilitating studies on antigen production, immunisation and vector-pathogen interaction, to date there has been no in vitro culture system for A. centrale. In the present study, 25 cell lines derived from 13 ixodid tick species were inoculated with the Israeli vaccine strain of A. centrale and monitored for at least 12 weeks by microscopic examination of Giemsa-stained cytocentrifuge smears. Infection of 19 tick cell lines was subsequently attempted by transfer of cell-free supernate from vaccine-inoculated tick cells. In two separate experiments, rickettsial inclusions were detected in cultures of the Rhipicephalus appendiculatus cell line RAE25 28-32 days following inoculation with the vaccine. Presence of A. centrale in the RAE25 cells was confirmed by PCR assays targeting the 16S rRNA, groEL and msp4 genes; sequenced PCR products were 100% identical to published sequences of the respective genes in the Israeli vaccine strain of A. centrale. A. centrale was taken through three subcultures in RAE25 cells over a 30 week period. In a single experiment, the Dermacentor variabilis cell line DVE1 was also detectably infected with A. centrale 11 weeks after inoculation with the vaccine. Availability of an in vitro culture system for A. centrale in tick cells opens up the possibility of generating a safer and more ethical vaccine for bovine anaplasmosis.
中央无形体作为一种活血液疫苗用于牛群，以预防致病性更强的边缘无形体，已有100多年历史。虽然边缘无形体可在蜱细胞系中进行体外培养，便于开展抗原生产、免疫和媒介-病原体相互作用的研究，但迄今为止，尚无中央无形体的体外培养系统。在本研究中，用中央无形体以色列疫苗株接种了源自13种硬蜱的25种细胞系，并通过吉姆萨染色细胞离心涂片的显微镜检查至少监测12周。随后，通过转移接种疫苗的蜱细胞的无细胞上清液，尝试感染19种蜱细胞系。在两个独立实验中，接种疫苗后28 - 32天，在血红扇头蜱细胞系RAE25的培养物中检测到立克次氏体包涵体。通过针对16S rRNA、groEL和msp4基因的PCR检测，证实RAE25细胞中存在中央无形体；测序的PCR产物与中央无形体以色列疫苗株中相应基因的已发表序列100%相同。在30周内，中央无形体在RAE25细胞中传代培养了三代。在一次实验中，接种疫苗11周后，变异革蜱细胞系DVE1也被检测到感染了中央无形体。蜱细胞中中央无形体体外培养系统的建立，为牛无浆体病开发更安全、更符合伦理的疫苗开辟了可能性。
