Allsopp M, Visser E S, du Plessis J L, Vogel S W, Allsopp B A
Onderstepoort Veterinary Institute, South Africa.
Vet Parasitol. 1997 Aug;71(4):283-300. doi: 10.1016/s0304-4017(97)00012-5.
Cowdria ruminantium is a rickettsial parasite which causes heartwater, a economically important disease of domestic and wild ruminants in tropical and subtropical Africa and parts of the Caribbean. Because existing diagnostic methods are unreliable, we investigated the small-subunit ribosomal RNA (srRNA) gene from heartwater-infected material to characterise the organisms present and to develop specific oligonucleotide probes for polymerase chain reaction (PCR) based diagnosis. DNA was obtained from ticks and ruminants from heartwater-free and heartwater-endemic areas from Cowdria in tissue culture. PCR was carried out using primers designed to amplify only rickettsial srRNA genes, the target region being the highly variable V1 loop. Amplicons were cloned and sequenced; 51% were C. ruminantium sequences corresponding to four genotypes, two of which were identical to previously reported C. ruminantium sequences while the other two were new. The four different Cowdria genotypes can be correlated with different phenotypes. Tissue-culture samples yielded only Cowdria genotype sequences, but an extraordinary heterogeneity of 16S sequences was obtained from field samples. In addition to Cowdria genotypes we found sequences from previously unknown Ehrlichia spp., sequences showing homology to other Rickettsiales and a variety of Pseudomonadaceae. One Ehrlichia sequence was phylogenetically closely related to Ehrlichia platys (Group II Ehrlichia) and one to Ehrlichia canis (Group III Ehrlichia). This latter sequence was from an isolate (Germishuys) made from a naturally infected sheep which, from brain smear examination and pathology, appeared to be suffering from heartwater; nevertheless no Cowdria genotype sequences were found in this isolate. In addition no Cowdria sequences were obtained from uninfected ticks. Complete 16S rRNA gene sequences were determined for two C. ruminantium genotypes and for two previously uncharacterised heartwater-associated Ehrlichia spp. Sequenced difference within the V1 loop were sufficient for the derivation of four Cowdria genotype-specific oligonucleotide probes. Four further probes were designed; one for the detection of any Cowdria genotype, one for the detection of any Group II Ehrlichia sp., one for any Group III Ehrlichia sp. and one for all Pseudomonadaceae. All the probes were specific except that for the Cowdria (Ball 3) genotype. The high prevalence (96%) in field samples of pseudomonad-like 16S sequences was the result of environmental contamination. The probes were used to screen DNA from goats in an area free of both Amblyomma ticks and clinical heartwater. A substantial proportion (42%) gave positive reactions for the apparently apathogenic Cowdria (Omatjenne), indicating that this genotype is relatively common.
反刍动物考德里氏体是一种立克次氏体寄生虫，可引发心水病，这是一种在热带和亚热带非洲以及加勒比部分地区对家养和野生反刍动物具有重要经济影响的疾病。由于现有的诊断方法不可靠，我们对来自感染心水病材料的小亚基核糖体RNA（srRNA）基因进行了研究，以鉴定其中存在的生物体，并开发用于基于聚合酶链反应（PCR）诊断的特异性寡核苷酸探针。从组织培养中的考德里氏体获取无心水病地区和心水病流行地区的蜱及反刍动物的DNA。使用设计用于仅扩增立克次氏体srRNA基因的引物进行PCR，目标区域是高度可变的V1环。对扩增子进行克隆和测序；51%为反刍动物考德里氏体序列，对应四种基因型，其中两种与先前报道的反刍动物考德里氏体序列相同，另外两种是新的。四种不同的考德里氏体基因型可与不同表型相关联。组织培养样本仅产生考德里氏体基因型序列，但从野外样本中获得了16S序列的异常异质性。除了考德里氏体基因型外，我们还发现了来自先前未知的埃立克体属物种的序列、与其他立克次氏体目显示同源性的序列以及各种假单胞菌科的序列。一个埃立克体序列在系统发育上与血小板埃立克体（埃立克体属第二组）密切相关，另一个与犬埃立克体（埃立克体属第三组）密切相关。后一个序列来自从一只自然感染绵羊分离出（Germishuys株）的样本，从脑涂片检查和病理学来看，该绵羊似乎患有心水病；然而，在该分离物中未发现考德里氏体基因型序列。此外，从未感染的蜱中未获得考德里氏体序列。确定了两种反刍动物考德里氏体基因型以及两种先前未鉴定的心水病相关埃立克体属物种的完整16S rRNA基因序列。V1环内的测序差异足以推导四种考德里氏体基因型特异性寡核苷酸探针。又设计了四种探针；一种用于检测任何考德里氏体基因型，一种用于检测任何第二组埃立克体属物种，一种用于检测任何第三组埃立克体属物种，一种用于检测所有假单胞菌科。除了针对考德里氏体（Ball 3）基因型的探针外，所有探针都是特异性的。野外样本中假单胞菌样16S序列的高流行率（96%）是环境污染的结果。这些探针用于筛选来自一个既没有安氏蜱也没有临床心水病的地区的山羊的DNA。相当大比例（42%）的样本对明显无致病性的考德里氏体（奥马杰内株）呈阳性反应，表明该基因型相对常见。
