A Cryptosporidium parvum vaccine candidate effect on immunohistochemical profiling of CD4, CD8, Caspase-3 and NF-κB in mice.

Abstract

BACKGROUND: 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-&#x3ba;B). This vaccine was prepared from the C. parvum oocysts antigen using immune affinity chromatography with cyanogen bromide-activated Sepharose-4B beads. METHODS: 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&#x2009;&#xd7;&#x2009;10of C. parvum oocysts in 250&#xa0;&#xb5;l PBS solution 1&#xa0;h before a meal) and (3) immunized (twice with 40&#xa0;&#xb5;g/kg of purified C. parvum antigen at 2-week intervals and then infected with 1&#x2009;&#xd7;&#x2009;10&#xa0;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-&#x3ba;B levels, which are indicators for T helper cells, cytotoxic T cells, apoptosis, and inflammation, respectively. RESULTS: The IHC results showed that CD4, CD8, Caspase-3, and NF-&#x3ba;B expression varied significantly (P&#x2009;<&#x2009;0.001) in both organs in all the groups. We also recorded high CD4levels and low CD8expression 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 CD4level was higher than CD8&#x2009;+&#x2009;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-&#x3ba;B 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&#x2009;<&#x2009;0.001) in the CD4, CD8+, Caspase-3, and NF-&#x3ba;B expression levels in both ileal and liver tissues of all mice groups, which might be necessary for immunomodulation in these tissues. CONCLUSIONS: The improvement observed in the immunized infected mice suggests that this vaccine candidate might protect against cryptosporidiosis.