Clinic for Birds, Reptiles, Amphibians and Fish, Faculty of Veterinary Medicine, Justus Liebig University, Giessen, Germany.
Department of Veterinary Pathology, Freie Universitaet Berlin, Germany.
PLoS One. 2019 Feb 19;14(2):e0210790. doi: 10.1371/journal.pone.0210790. eCollection 2019.
Tortoise Picornavirus (ToPV) commonly known as Virus "X" was recently discovered in juvenile European tortoises suffering from soft carapace and plastron as well as kidney disease. Therefore, this virus was a potential candidate to be a causative agent for these disease patterns. Spur thighed tortoises (Testudo graeca) seemed to be more susceptible to establish clinical symptoms than other European species like T. hermanni. Thus this trial investigated the role of ToPV in the described syndrome. Two groups of juvenile European tortoises (T. graeca and T.hermanni) each of 10 animals, were cloacally, oronasally and intracoelomically inoculated with an infectious dose (~ 2000 TICD) of a ToPV strain isolated from a diseased T. graeca. A control group of two animals of each species received non-infected cell culture supernatant. The tortoises were examined daily and pharyngeal and cloacal swabs for detection of ToPV-RNA by RT-PCR were taken from each animal every six days for a period of 6 months. At the end of the study the remaining animals were euthanised and dissected. Bacteriological and parasitological tests were performed and organ samples of all tortoises were investigated by RT-PCR for the presence of ToPV and histopathology. Animals that were euthanised at the end of the experiment, were examined for presence of specific anti-ToPV antibodies. Several animals in both inoculated groups showed retarded growth and a light shell weakness, in comparison to the control animals. Three animals were euthanised during the trial, showing reduced weight gain, retarded growth, severe shell weakness and apathy, in parallel to clinical observations in naturally infected animals. In all inoculated animals of both species an intermittent virus shedding, starting from 18 days post inoculation (d.p.i.), till 164 d.p.i. was detected, while the control animals remained negative. The virus was successfully reisolated in terrapene heart cell culture in 16 of 20 inoculated animals of both species. Histopathology of most inoculated animals revealed a lack of bone remodeling and vacuolisation in kidney tubuli which supports the described pathogenesis of nephropathy and osteodystrophy. Anti- ToPV antibody titres ranged from 1:2 to >1:256 in 13 of 20 animals, whereas all control animals were seronegative. The study proofed the Henle Koch`s postulates of ToPV as causative agent for shell dystrophy and kidney disease in both testudo species. The proposed species specific sensitivity towards clinical disease was not observed.
龟微病毒（ToPV）通常被称为“病毒 X”，最近在患有软壳和腹甲以及肾脏疾病的幼年欧洲龟中被发现。因此，这种病毒是引起这些疾病模式的潜在候选因素。靴脚陆龟（Testudo graeca）似乎比其他欧洲物种（如 T. hermanni）更容易出现临床症状。因此，本试验研究了 ToPV 在所述综合征中的作用。两组幼年欧洲龟（T. graeca 和 T. hermanni）各 10 只，通过感染性剂量（约 2000 TICD）的 ToPV 株，经泄殖腔、口鼻和腹腔内接种。每组两个动物的对照组接受非感染细胞培养上清液。每天检查龟，每六天从每只动物采集咽和泄殖腔拭子，通过 RT-PCR 检测 ToPV-RNA。在研究结束时，剩余的动物被安乐死并解剖。进行细菌学和寄生虫学检测，并用 RT-PCR 检测所有龟的器官样本是否存在 ToPV 和组织病理学。在实验结束时安乐死的动物检查是否存在特定的抗 ToPV 抗体。与对照组动物相比，两组接种动物中的几只有生长迟缓，壳变软的现象。在试验过程中，有三只动物被安乐死，体重增加减少，生长迟缓，壳严重变软和无精打采，与自然感染动物的临床观察平行。在两种动物的所有接种动物中，从接种后 18 天（dpi）开始，到 164 dpi 间歇性检测到病毒脱落，而对照组动物保持阴性。在两种动物的 20 只接种动物中的 16 只中成功地在 Terrapene 心脏细胞培养物中重新分离出病毒。大多数接种动物的组织病理学显示肾小管中缺乏骨重塑和空泡化，这支持肾病和骨营养不良的所述发病机制。在 20 只动物中的 13 只中，抗 ToPV 抗体滴度范围为 1:2 至 >1:256，而所有对照组动物均为阴性。该研究证明了 ToPV 作为两种 Testudo 物种壳营养不良和肾病的病原体的 Henle Koch 假设。没有观察到针对临床疾病的特定物种敏感性。
