Delayed-type hypersensitivity (DTH) has been used in human and veterinary medicine as a skin testing for evaluating in vivo cell-mediated immune responses (CMIR). Whereas CMIR is a key process to control intracellular pathogens, its value at identifying cattle exposed to the abortigenic intracellular coccidian parasite Neospora caninum is unknown. In this work, we have evaluated a DTH skin testing in cattle exposed to N. caninum and still seronegative. Female calves were experimentally sensitized by subcutaneous (SC) inoculation with live tachyzoites of N. caninum (NC-Argentina LP1) in sterile phosphate-buffered saline (PBS) (group A; n: 8) whereas other calveswere mock-sensitized with PBS (group B; n: 6). Two DTH skin tests were performed by intradermal inoculation with a soluble lysate of N. caninum tachyzoites (NC-Argentina LP1) in the neck region at 60d and 960 d after sensitization. Skinfold thickness at the intradermal inoculation site was measured at 0, 24, 48 h post each DTH skin test and skin biopsies taken for microscopic evaluation. Specific N. caninum antibodies kinetics was evaluated all throughthe experiment. We found that whereas N. caninum specific antibodies remained below the ELISA cut-off, a distinctive skinfold thickness increase was detected in sensitized animals (group A) at the DTH skin test site, showing induration, swelling and inflammatory infiltration. Mock sensitized animals (group B) showed no skinfold thickness growth and lacked specific antibody response. Thus, N. caninum DTH skin testing could be a useful diagnostic tool for the detection of CMIR during N. caninum infection in non-humoral responders.

迟发型超敏反应（DTH）已在人类和兽医学中用作皮肤测试，用于评估体内细胞介导的免疫反应（CMIR）。尽管CMIR是控制细胞内病原体的关键过程，但其在鉴定暴露于流产性细胞内球虫寄生虫新孢子虫的牛中的价值尚不清楚。在这项工作中，我们评估了暴露于犬新孢子虫但仍呈血清阴性的牛的DTH皮肤测试。用犬新孢子虫的活速殖子通过皮下（SC）接种对雌性小牛进行实验致敏（NC-阿根廷LP1）在无菌磷酸盐缓冲盐水（PBS）中（A组； n：8），而其他犊牛则用PBS进行模拟致敏（B组； n：6）。在致敏后60天和960天，通过在颈部颈部的犬新孢子虫速殖子（NC-Argentina LP1）的可溶性裂解物皮内接种进行了两次DTH皮肤测试。在每次DTH皮肤测试后0、24、48小时测量皮内接种部位的皮褶厚度，并进行皮肤活检以进行显微镜评估。在整个实验过程中都评估了犬新孢子虫的特异性抗体动力学。我们发现而犬新孢子虫特异性抗体保持在ELISA临界值以下，在DTH皮肤测试部位的致敏动物（A组）中检测到明显的皮褶厚度增加，显示出硬结，肿胀和炎性浸润。模拟致敏动物（B组）未出现皮褶厚度增加，并且缺乏特异性抗体反应。因此，犬新孢子虫DTH皮肤测试可能是检测非体液性应答者犬新孢子虫感染期间CMIR的有用诊断工具。