Ezzaty Mirhashemi Marzieh, Zintl Annetta, Grant Tim, Lucy Frances E, Mulcahy Grace, De Waal Theo
School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland; School of Public Health and Population Science, University College Dublin, Belfield, Dublin 4, Ireland.
School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
Exp Parasitol. 2015 Apr-May;151-152:14-20. doi: 10.1016/j.exppara.2015.01.018. Epub 2015 Feb 3.
While a large number of laboratory methods for the detection of Cryptosporidium oocysts in faecal samples are now available, their efficacy for identifying asymptomatic cases of cryptosporidiosis is poorly understood. This study was carried out to determine a reliable screening test for epidemiological studies in livestock. In addition, three molecular tests were compared to identify Cryptosporidium species responsible for the infection in cattle, sheep and horses. A variety of diagnostic tests including microscopic (Kinyoun's staining), immunological (Direct Fluorescence Antibody tests or DFAT), enzyme-linked immunosorbent assay (ELISA), and molecular methods (nested PCR) were compared to assess their ability to detect Cryptosporidium in cattle, horse and sheep faecal samples. The results indicate that the sensitivity and specificity of each test is highly dependent on the input samples; while Kinyoun's and DFAT proved to be reliable screening tools for cattle samples, DFAT and PCR analysis (targeted at the 18S rRNA gene fragment) were more sensitive for screening sheep and horse samples. Finally different PCR primer sets targetedat the same region resulted in the preferential amplification of certain Cryptosporidium species when multiple species were present in the sample. Therefore, for identification of Cryptosporidium spp. in the event of asymptomatic cryptosporidiosis, the combination of different 18S rRNA nested PCR primer sets is recommended for further epidemiological applications and also tracking the sources of infection.
虽然现在有大量用于检测粪便样本中隐孢子虫卵囊的实验室方法，但对于它们在识别隐孢子虫病无症状病例方面的功效却知之甚少。开展这项研究是为了确定一种用于家畜流行病学研究的可靠筛查试验。此外，还比较了三种分子检测方法，以鉴定导致牛、羊和马感染的隐孢子虫种类。对包括显微镜检查（金胺染色）、免疫学方法（直接荧光抗体试验或DFAT）、酶联免疫吸附测定（ELISA）和分子方法（巢式PCR）在内的多种诊断测试进行了比较，以评估它们检测牛、马和羊粪便样本中隐孢子虫的能力。结果表明，每种检测方法的敏感性和特异性高度依赖于输入样本；虽然金胺染色和DFAT被证明是牛样本可靠的筛查工具，但DFAT和PCR分析（针对18S rRNA基因片段）对羊和马样本的筛查更敏感。最后，当样本中存在多种隐孢子虫物种时，针对同一区域的不同PCR引物集会优先扩增某些隐孢子虫物种。因此，对于无症状隐孢子虫病情况下隐孢子虫种类的鉴定，建议将不同的18S rRNA巢式PCR引物集组合用于进一步的流行病学应用以及追踪感染源。
