Evaluation of a vaccine candidate isolated from Cryptosporidium parvum oocyst in mice

Abstract

Background and aim: Cryptosporidiosis is a leading cause of diarrheal disease worldwide and is an animal and public health burden. This study aimed to evaluate the protective potential of affinity-purified Cryptosporidium parvum oocyst antigen as a vaccine candidate according to fecal oocyst shedding, humoral and cellular immune responses, histopathological changes, and the number of parasite developmental stages in ileal and hepatic tissues.

Materials and methods: We isolated oocysts from naturally infected buffalo calves and identified them molecularly as C. parvum isolates (GenBank: ON730707 and ON730708) by targeting the Cryptosporidium oocyst wall protein gene. We propagated the C. parvum oocysts in mice. In addition, we prepared crude antigen from the isolated oocysts by purification using cyanogen bromide-activated Sepharose-4B affinity chromatography coupled with rabbit hyperimmune serum. Then, we divided 81 parasite-free mice into three groups: (1) non-vaccinated non-infected mice, (2) mice orally infected with 1 × 10⁵ C. parvum oocysts on week 4 of the experiment, and (3) mice immunized twice with 40 μg/kg of the purified fraction at 2-week intervals. Then, we challenged the vaccinated group with C. parvum oocysts after 2 weeks, and the positive control group was infected at the same time.

Results: We observed a prolonged prepatent period and decreased oocyst shedding in the vaccinated infected mice compared with the non-vaccinated infected mice (t < 0.001). The vaccinated mice had significantly higher immunoglobulin G levels than those in the other two groups at all examined weeks. In addition, the production of cytokines interferon-gamma, interleukin (IL)-10, IL-12, and IL-15 was activated post-vaccination. After the challenge, all tested cytokines were significantly increased (p < 0.001) in the two infected groups compared with the non-vaccinated non-infected group, with the highest levels in the vaccinated infected group. Vaccinated infected mice exhibited significantly fewer pathological lesions in the ileum and liver than non-vaccinated infected mice, which showed prominent histopathological lesions. Endogenous developmental stages of C. parvum indicated that the ileum was more parasitized than the liver and that vaccination resulted in a lower number of oocysts in ileal and hepatic tissues (p < 0.05).

Conclusion: Our prepared affinity-purified vaccine candidate could be promising in protecting against cryptosporidiosis.

Keywords: Cryptosporidium parvum; affinity chromatography; cytokines; enzyme-linked immunosorbent assay; histopathology; polymerase chain reaction; vaccine.

Figure-1

Phylogenetic analysis using the maximum likelihood method based on Cryptosporidium oocyst wall protein gene for Cryptosporidium spp. The new obtained sequences in this study are highlighted (black dots). There were a total of 495 positions in the final dataset. The scale bar represents a 2% nucleotide sequence divergence.

Figure-2

Immunoglobulin G antibody levels measured by enzyme-linked immunosorbent assay in vaccinated and non-vaccinated mice sera. Bars represent standard error.

Figure-3

Photomicrographs displaying the histopathological changes in ileal tissue sections between studied mice groups; (a, d and g) Ileal sections of non-infected non-vaccinated group presented the normal histological structure of ileum as follows: lining epithelium of simple columnar absorptive (thin arrow) with goblet cells in between (arrowhead), lamina propria of dense irregular connective tissue (cube), simple tubular intestinal gland (circle), and muscular layer organized as inner circular and outer longitudinal of smooth muscle cells (thick arrow). (b, e and h) Ileal sections of Cryptosporidium parvum-infected group highlighted severe degeneration of ileal tissue as shortening of villi, sloughing and erosion of lining epithelium, aggregated Cryptosporidium parvum developmental stages (arrows), as well as infiltration of inflammatory cells (arrowhead). (c, f and i) Ileal sections of vaccinated infected mice group revealed less alterations in tissue structure as most villi appeared in normal architecture except some (c) with sloughing and erosions of epithelial cells, (f) vacuolations in lamina propria, (i) and few Cryptosporidium parvum developmental stages. (Hematoxylin and Eosin Stain, Magnification Power; 100×, 400×, 100×, respectively, Scale bar; 200 μm, 50 μm, 20 μm, respectively).

Figure-4

Photomicrographs demonstrated the histopathological alterations in liver tissue sections between experimented mice groups; (a and d) Liver sections of non-infected non-vaccinated group detailed the normal hepatic plates composition: (a) Central vein area with its endothelial lining of simple squamous epithelium (arrowhead), hepatocytes organized in regular cords (arrow), (d) nucleus of hepatocytes presented centrally in a light vesicular form (arrow). Notice hepatic sinusoids between cords (arrowhead). (b and e) Liver sections of Cryptosporidium parvum-infected group highlighted intense destructive hepatic tissue with severe manifestation of Cryptosporidium parvum developmental stages (arrows) and inflammatory cells (arrowhead) alongside necrotic areas (cube). (c and f) Liver sections of vaccinated infected mice group exposed few changes in hepatic tissue structure, (c) vacuolations (arrowhead) and aggregated inflammatory cells in hepatic sinusoids (arrow), and (f) limited necrotic areas (cube) and few Cryptosporidium parvum developmental stages. (Hematoxylin and Eosin Stain, Magnification Power; 100×, 400×, respectively, Scale bar; 200).