Department of Parasitology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt.
PLoS One. 2020 May 20;15(5):e0233453. doi: 10.1371/journal.pone.0233453. eCollection 2020.
Toxocara vitulorum is an Ascarid nematode infecting the small intestine of buffalo and cattle particularly neonate calves, with the postnatal route through milk is the main infection source. However, little is known about shedding rates and the optimum detection methods of T. vitulorum larvae in the milk of the infected bovine hosts. In this study, we aimed to evaluate the use of two methods, microscopy and PCR, and their detection limits both under the experimental and natural infection situations. In doing this, T. vitulorum eggs extracted from naturally occurring adult female worms were successfully subjected to experimental embryonation, and larvae were implemented in experimental infection of milk in ascending infection doses of 0, 1, 5, 10, 20, 50 larvae/2-ml milk samples. With the except of negative control, microscopy-based examination detected larvae in all samples, albeit with means, ranges, and the total number of larvae were detected in exponential rates relative to larvae densities in milk samples. PCR technique corresponded well to microscopy in detecting genomic DNA of T. vitulorum larvae in all milk samples down to a single larva/sample. On the other hand, and by applying the same methodology approach on 50 naturally-occurring bovine colostrum/milk samples, 13 (26%) and 20 (40%) samples were tested positive for T. vitulorum infection by microscopy and the PCR-based detection, respectively. Of these, 11 out of 26 buffalo samples (42.30%) and 2 out of 24 cow samples (8.33%) were tested positive by microscopy, while 16 (61.54%) and 3 (12.50%) of buffalo and cow samples were tested positive by PCR, respectively. By applying the Agreement Coefficient, substantial agreement (0.77) between molecular and microscopy detection was detected from all tested samples. In conclusion, larvae of T. vitulorum were unequivocally detected by microscopy and molecular methods in milk samples both under the experimental and natural field situations. Nevertheless, slightly higher rates by PCR than microscopy were obtained when detecting naturally-infected milk samples. To the best of our knowledge, this is the first in situ detection of larvae of T. vitulorum in the milk of the naturally infected animals.
牛弓首蛔虫是一种寄生在水牛和牛小肠内的蛔虫，尤其感染新生小牛，其出生后途径主要通过乳汁感染。然而，对于牛弓首蛔虫幼虫在感染牛宿主乳汁中的脱落率和最佳检测方法知之甚少。在本研究中，我们旨在评估两种方法，显微镜检查和 PCR 及其检测限，包括在实验和自然感染情况下的检测限。为此，我们成功地从自然发生的成年雌虫中提取牛弓首蛔虫卵，并将其用于实验胚胎发生，然后在上升剂量的实验感染乳中实施实验感染，剂量为 0、1、5、10、20、50 条幼虫/2 毫升乳样。除阴性对照外，基于显微镜检查的检测方法在所有样本中均检测到幼虫，尽管幼虫数量的平均值、范围和总数均呈指数增长，与乳样中的幼虫密度相对应。PCR 技术与显微镜检查一样，在所有乳样中均能很好地检测到牛弓首蛔虫幼虫的基因组 DNA，检测下限低至单个幼虫/样本。另一方面，通过应用相同的方法学方法，对 50 份自然发生的牛初乳/乳样进行检测，显微镜检查和基于 PCR 的检测分别有 13 份（26%）和 20 份（40%）样本检测出牛弓首蛔虫感染阳性。其中，26 份水牛样本中有 11 份（42.30%），24 份牛样本中有 2 份（8.33%）通过显微镜检查呈阳性，而 16 份（61.54%）和 3 份（12.50%）水牛和牛样本通过 PCR 检查呈阳性。通过应用一致性系数，从所有测试样本中检测到分子和显微镜检测之间存在高度一致性（0.77）。总之，牛弓首蛔虫幼虫在实验和自然现场条件下的乳样中均通过显微镜和分子方法明确检测到。然而，在检测自然感染乳样时，PCR 方法的检出率略高于显微镜检查。据我们所知，这是首次在自然感染动物的乳中检测到牛弓首蛔虫幼虫。
