Kooyman F N J, van Doorn D C K, Geurden T, Wagenaar J A
Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands.
Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands.
Vet Parasitol. 2016 Jan 30;216:59-65. doi: 10.1016/j.vetpar.2015.12.009. Epub 2015 Dec 17.
Cyathostomins are the most prevalent horse nematodes worldwide and over 50 species are described. The eggs and the infective larvae (L3) can easily be obtained or cultured from infected horses, but cannot be differentiated morphologically at species level. A reverse line blot (RLB) method based on the hybridization of a PCR fragment with a species specific probe, has previously been developed for the differentiation of individual eggs and/or L3s, but is too labor intensive for large scale studies. In the present study a RLB method on multiple pooled L3s for the semi-quantitative differentiation of cyathostomin larval cultures was developed and validated. First, the probability of the presence of a certain species within a pool was calculated as function of the frequency and the number of L3s within a pool. Ten L3s per pool were found to be optimal. Next, the probability, the chance of occurrence was calculated when 4 pools per culture were used. The probability distributions for 0, 1, 2, 3 or 4 positive pools were transformed into the corresponding median frequency of the cumulative probability: 0.014, 0.04, 0.08, 0.16 and 0.59, respectively. Based on these calculated probabilities, RLB on 10 L3s per pool and 4 pools per sample was validated by estimating the cross-hybridization, precision and accuracy in 3 groups of horses. First, absence of cross-hybridization was confirmed by differentiation of the same L3s (160 L3s from the 4 horses from group 1) in the RLB on individual as well as on pooled L3s. Cross-hybridization was excluded for 9 of the most common cyathostomins. Next, the precision and accuracy were determined by the differentiation of 10 replicates of 3 cultures from 3 horses from group 2 (1200 L3s). The coefficient of variation (CV) was between 0 and 0.90 and the accuracy was between 0.42 and 1.73. A Monte Carlo simulation based on the observed scores and associated probability distributions gave similar results as the use of a fixed median frequency. The LPGs obtained from 276 larval culture counts from a larger cohort (23 horses, group 3) were not significantly different from the LPGs obtained from summation of the LPG per species found by RLB on pooled L3s. The RLB on pooled L3s was found therefore an useful semi-quantitative method for the differentiation of the most common cyathostomin L3, with a workload of approximately one tenth of that of the RLB on individual L3s.
圆线虫是全球最常见的马线虫，已描述的种类超过50种。卵和感染性幼虫（L3）很容易从感染的马匹中获取或培养，但在物种水平上无法通过形态学进行区分。此前已开发出一种基于PCR片段与物种特异性探针杂交的反向线杂交（RLB）方法，用于区分单个卵和/或L3，但对于大规模研究来说劳动强度太大。在本研究中，开发并验证了一种基于多个混合L3的RLB方法，用于圆线虫幼虫培养物的半定量区分。首先，计算池中某一特定物种存在的概率，作为池中L3频率和数量的函数。发现每个池10个L3是最佳的。接下来，计算当每个培养物使用4个池时出现的概率。将0、1、2、3或4个阳性池的概率分布转换为相应的累积概率中位数频率：分别为0.014、0.04、0.08、0.16和0.59。基于这些计算出的概率，通过估计3组马匹中的交叉杂交、精密度和准确性，对每个池10个L3和每个样本4个池的RLB进行了验证。首先，通过在单个L3和混合L3的RLB中区分相同的L3（来自第1组4匹马的160个L3），确认不存在交叉杂交。排除了9种最常见圆线虫的交叉杂交。接下来，通过区分来自第2组3匹马的3种培养物的10个重复样本（1200个L3）来确定精密度和准确性。变异系数（CV）在0至0.90之间，准确性在0.42至1.73之间。基于观察到的分数和相关概率分布的蒙特卡罗模拟给出了与使用固定中位数频率相似的结果。从更大队列（23匹马，第3组）的276次幼虫培养计数中获得的LPG与通过对混合L3的RLB发现的每个物种的LPG总和获得的LPG没有显著差异。因此，发现混合L3的RLB是一种用于区分最常见圆线虫L3的有用半定量方法，其工作量约为单个L3的RLB的十分之一。
