莫桑比克马罗梅乌保护区感染非洲水牛（非洲野水牛）的蜱传无形体科病原体的分子诊断及遗传多样性
Molecular diagnosis and genetic diversity of tick-borne Anaplasmataceae agents infecting the African buffalo Syncerus caffer from Marromeu Reserve in Mozambique.
作者信息
Machado Rosangela Zacarias, Teixeira Marta Maria Geraldes, Rodrigues Adriana Carlos, André Marcos Rogério, Gonçalves Luiz Ricardo, Barbosa da Silva Jenevaldo, Pereira Carlos Lopes
机构信息
Department of Veterinary Pathology, School of Agrarian and Veterinary Sciences (FCAV/UNESP), Jaboticabal, SP, Brazil.
Institute of Biomedical Sciences, University of São Paulo (ICB/USP), São Paulo, SP, Brazil.
出版信息
Parasit Vectors. 2016 Aug 17;9:454. doi: 10.1186/s13071-016-1715-y.
BACKGROUND
Tick-borne diseases (TBDs) are very important in relation to domestic ruminants, but their occurrence among wild ruminants, mainly in the African buffalo Syncerus caffer, remains little known.
METHODS
Molecular diagnostic methods were applied to detect Anaplasma marginale, Anaplasma centrale, Anaplasma phagocytophilum, Ehrlichia ruminantium and Ehrlichia chaffeensis in 97 blood samples of African buffalo captured at the Marromeu Reserve in Mozambique. Molecular detection of agents belonging to the family Anaplasmataceae were based on conventional and qPCR assays based on msp5, groEL, 16S rRNA, msp2, pCS20 and vlpt genes. Phylogenetic reconstruction of new Anaplasma isolates detected in African buffalo was evaluated based on msp5, groEL and 16S rRNA genes.
RESULTS
All the animals evaluated were negative for specific PCR assays for A. phagocytophilum, E. ruminantium and E. chaffeensis, but 70 animals were positive for A. marginale, showing 2.69 × 10(0) up to 2.00 × 10(5) msp1β copies/μl. This result overcomes the conventional PCR for A. marginale based on msp5 gene that detected only 65 positive samples. Sequencing and phylogenetic analyses were performed for selected positive samples based on the genes msp5, groEL and 16S rRNA. Trees inferred using different methods separated the 29 msp5 sequences from buffalo in two distinct groups, assigned to A. centrale and A. marginale. The groEL sequences determined for African buffalo samples revealed to be more heterogeneous and inferred trees could not assign them to any species of Anaplasma despite being more related to A. marginale and A. centrale. The highly conserved 16S rRNA gene sequences suggested a close relationship of the new 16 sequences with A. centrale/A. marginale, A. platys and A. phagocytophilum.
CONCLUSIONS
Our analysis suggests that different species of Anaplasma are simultaneously present in the African buffalo. To the best of our knowledge, this is the first study that diagnosed Anaplasma spp. in the African buffalo and inferred the taxonomic status of new isolates with different gene sequences. The small fragment of msp5 sequences revealed to be a good target for phylogenetic positioning of new Anaplasma spp. isolates.
背景
蜱传疾病（TBDs）对家养反刍动物非常重要，但它们在野生反刍动物中的发生情况，主要是在非洲水牛（非洲野水牛）中，仍然鲜为人知。
方法
应用分子诊断方法检测莫桑比克马罗梅乌保护区捕获的97份非洲水牛血液样本中的边缘无形体、中央无形体、嗜吞噬细胞无形体、反刍兽埃立克体和恰菲埃立克体。基于msp5、groEL、16S rRNA、msp2、pCS20和vlpt基因，通过传统和定量PCR检测无形体科病原体。基于msp5、groEL和16S rRNA基因对非洲水牛中检测到的新无形体分离株进行系统发育重建评估。
结果
所有评估动物的嗜吞噬细胞无形体、反刍兽埃立克体和恰菲埃立克体特异性PCR检测均为阴性，但70只动物的边缘无形体检测呈阳性，每微升血液中msp1β拷贝数在2.69×10(0)至2.00×10(5)之间。这一结果超过了基于msp5基因检测边缘无形体的传统PCR，传统PCR仅检测到65份阳性样本。基于msp5、groEL和16S rRNA基因对选定的阳性样本进行测序和系统发育分析。使用不同方法推断的系统发育树将水牛的29个msp5序列分为两个不同的组，分别归为中央无形体和边缘无形体。非洲水牛样本的groEL序列显示出更高的异质性，尽管与边缘无形体和中央无形体关系更密切，但推断的系统发育树无法将它们归为任何无形体物种。高度保守的16S rRNA基因序列表明，新的16个序列与中央无形体/边缘无形体、血小板无形体和嗜吞噬细胞无形体关系密切。
结论
我们的分析表明，非洲水牛中同时存在不同种类的无形体。据我们所知，这是首次在非洲水牛中诊断无形体属并推断不同基因序列新分离株分类地位的研究。msp5序列的小片段被证明是新无形体属分离株系统发育定位的良好靶点。
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