Department of Parasitology and Animal Diseases, Veterinary Research Institute, National Research Centre, El Buhouth Street, Dokki, Cairo, Egypt.
Department of Animal Reproduction and AI, Veterinary Research Institute, National Research Centre, El Buhouth Street, Dokki, Cairo, Egypt.
BMC Vet Res. 2023 Oct 19;19(1):216. doi: 10.1186/s12917-023-03699-w.
Cryptosporidium parvum is a protozoan parasite of medical and veterinary importance that causes neonatal diarrhea in many vertebrate hosts. In this study, we evaluated the efficacy of an affinity-purified antigen as a C. parvum vaccine candidate using ileal and liver tissues of experimentally infected neonatal mice by immunohistochemical profiling and immune scoring of CD4, CD8, Caspase-3, and nuclear factor kappa B (NF-κB). This vaccine was prepared from the C. parvum oocysts antigen using immune affinity chromatography with cyanogen bromide-activated Sepharose-4B beads.
Thirty neonatal mice were divided into three groups (10 mice/group): (1) non-immunized non-infected, (2) non-immunized infected (using gastric tubes with a single dose of 1 × 10 of C. parvum oocysts in 250 µl PBS solution 1 h before a meal) and (3) immunized (twice with 40 µg/kg of purified C. parvum antigen at 2-week intervals and then infected with 1 × 10 C. parvum oocysts simultaneously with the second group). After euthanizing the animals on the 10th day, post-infection, their ileal and liver tissues were collected and prepared for immunohistochemistry (IHC) staining to detect CD4, CD8+, Caspase-3, and NF-κB levels, which are indicators for T helper cells, cytotoxic T cells, apoptosis, and inflammation, respectively.
The IHC results showed that CD4, CD8, Caspase-3, and NF-κB expression varied significantly (P < 0.001) in both organs in all the groups. We also recorded high CD4 levels and low CD8 expression in the non-immunized non-infected mice tissues, while the opposite was observed in the non-immunized infected mice tissues. In the immunized infected mice, the CD4 level was higher than CD8 + in both organs. While the Caspase-3 levels were higher in the ileal tissue of non-immunized infected than immunized infected mice ileal tissues, the reverse was seen in the liver tissues of both groups. Furthermore, NF-κB expression was higher in the liver tissues of non-immunized infected mice than in immunized infected mice tissues. Therefore, the IHC results and immune-scoring program revealed a significant difference (P < 0.001) in the CD4, CD8+, Caspase-3, and NF-κB expression levels in both ileal and liver tissues of all mice groups, which might be necessary for immunomodulation in these tissues.
The improvement observed in the immunized infected mice suggests that this vaccine candidate might protect against cryptosporidiosis.
微小隐孢子虫是一种医学和兽医重要的原生动物寄生虫，会导致许多脊椎动物宿主的新生儿腹泻。在这项研究中，我们使用免疫组织化学分析和 CD4、CD8、半胱天冬酶-3 和核因子 kappa B（NF-κB）的免疫评分，评估了一种亲和纯化抗原作为微小隐孢子虫疫苗候选物的功效，该抗原使用溴化氰活化的 Sepharose-4B 珠通过免疫亲和层析从微小隐孢子虫卵囊抗原中制备。
将 30 只新生小鼠分为三组（每组 10 只）：（1）未免疫未感染、（2）未免疫感染（在餐前 1 小时使用含有 1×10 的微小隐孢子虫卵囊的胃管单次给药，体积为 250 µl PBS 溶液）和（3）免疫（两次，间隔 2 周给予 40 µg/kg 纯化微小隐孢子虫抗原，然后与第二组同时感染 1×10 微小隐孢子虫卵囊）。在感染后第 10 天处死动物后，收集其回肠和肝脏组织，进行免疫组织化学（IHC）染色，以检测 CD4、CD8+、半胱天冬酶-3 和 NF-κB 水平，分别是辅助性 T 细胞、细胞毒性 T 细胞、细胞凋亡和炎症的指标。
IHC 结果显示，各组所有器官的 CD4、CD8、半胱天冬酶-3 和 NF-κB 表达均有显著差异（P<0.001）。我们还记录到未免疫未感染小鼠组织中 CD4 水平较高，CD8 表达较低，而未免疫感染小鼠组织中则相反。在免疫感染的小鼠中，CD4 水平在两个器官中均高于 CD8+。而回肠组织中未免疫感染的小鼠的半胱天冬酶-3 水平高于免疫感染的小鼠，而在两组的肝脏组织中则相反。此外，未免疫感染的小鼠肝脏组织中的 NF-κB 表达高于免疫感染的小鼠。因此，IHC 结果和免疫评分程序显示，所有小鼠组的回肠和肝脏组织中 CD4、CD8+、半胱天冬酶-3 和 NF-κB 表达水平存在显著差异（P<0.001），这可能对这些组织中的免疫调节很重要。
免疫感染的小鼠的改善表明，该疫苗候选物可能对隐孢子虫病具有保护作用。
