Department of Food Hygiene and Veterinary Public Health, Faculty of Veterinary Medicine, Banat's University of Agricultural Sciences and Veterinary Medicine Timişoara, Calea Aradului no. 119, 300645 Timişoara, Romania.
Vet Parasitol. 2013 Jan 16;191(1-2):119-22. doi: 10.1016/j.vetpar.2012.08.020. Epub 2012 Aug 30.
This study was undertaken to investigate the occurrence and public health significance of Cryptosporidium species/genotypes and subtypes in a newborn lambs. A total of 175 diarrheic fecal samples from lambs (younger than 21 days) were collected in seven sheep flocks located in western Romania, and were microscopically examined for the presence of Cryptosporidium oocysts after staining with modified Ziehl-Neelsen technique. Twenty-four (13.7%) fecal samples were tested Cryptosporidium positive by microscopy and were subjected for molecular characterization. All positive samples were successfully amplified through a nested polymerase chain reaction (PCR) of the small subunit (SSU) rRNA gene (18S). Cryptosporidium species were determined by restriction fragment length polymorphism (RFLP) analysis of the secondary PCR products using the conventional SspI and VspI restriction enzymes. The identified species were: Cryptosporidium parvum (20/24), C. ubiquitum (2/24) and C. xiaoi (2/24), respectively. PCR-RFLP results for C. ubiquitum and C. xiaoi isolates were confirmed by DNA sequencing. Subsequently, subtyping of seven randomly selected C. parvum isolates, based on sequence analysis of the GP60 gene, revealed the presence of five different subtypes (IIaA17G1R1, IIaA16G1R1, IIdA20G1, IIdA24G1 and IIdA22G2R1) belonging in two zoonotic subtype families (IIa and IId). These findings may suggest the potential role of the newborn lambs as a source for human cryptosporidiosis. This is the first published report about the presence of C. ubiquitum and C. xiaoi in lambs from Romania.
本研究旨在调查罗马尼亚西部七个绵羊养殖场中新生羔羊中隐孢子虫种类/基因型和亚型的发生情况及其公共卫生意义。采集了 175 份来自 21 日龄以下羔羊的腹泻粪便样本，通过改良的齐尔-尼尔森染色技术，在显微镜下检查隐孢子虫卵囊的存在。24 份（13.7%）粪便样本经显微镜检查呈隐孢子虫阳性，并进行分子特征分析。所有阳性样本均通过小亚基（SSU）rRNA 基因（18S）的嵌套聚合酶链反应（PCR）成功扩增。通过使用常规的 SspI 和 VspI 限制性内切酶对二级 PCR 产物进行限制性片段长度多态性（RFLP）分析，确定隐孢子虫种类。鉴定的种类为：微小隐孢子虫（20/24）、C. ubiquitum（2/24）和 C. xiaoi（2/24）。C. ubiquitum 和 C. xiaoi 分离株的 PCR-RFLP 结果通过 DNA 测序得到证实。随后，基于 GP60 基因序列分析，对 7 个随机选择的微小隐孢子虫分离株进行了亚分型，发现存在 5 种不同的亚型（IIaA17G1R1、IIaA16G1R1、IIdA20G1、IIdA24G1 和 IIdA22G2R1），属于两个人畜共患亚型家族（IIa 和 IId）。这些发现可能表明新生羔羊可能成为人类隐孢子虫病的传染源。这是罗马尼亚首次报道 C. ubiquitum 和 C. xiaoi 在羔羊中的存在。
