Comparison of the Immunogenicity of Different Proportions of Chitosan Adjuvant in an Attenuated Neospora caninum Vaccine in BALB/c Mice

Document Type : Research Article

Authors

Seyed Reza Hosseini ¹

marzieh kefayat ²

Milad Hamzehali Tehrani ³

Amir Hosein Norouzi ²

¹ Department of Veterinary, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.

² Department of Pathobiology, Faculty of Veterinary, Karaj Branch, Islamic Azad University, Karaj, Iran.

³ Department of Pathobiology, Faculty of Veterinary, Science and Research Branch, Islamic Azad University, Tehran, Iran.

10.22067/ijvst.2025.94043.1567

Abstract

Neosporosis is a neuromuscular infectious disease in dogs caused by the protozoan N. caninum. This disease is globally distributed; canines are the definitive hosts, while a wide range of animals serve as intermediate hosts. This study aimed to compare the immunogenicity of different proportions of chitosan adjuvant in an attenuated N. caninum vaccine in BALB/c mice. Twenty-eight BALB/c mice were divided into four groups (n = 7 per group). Groups 1, 2, and 3 were subcutaneously immunized with attenuated tachyzoites of N. caninum strain Nc-1 combined with micronized chitosan adjuvant at concentrations of 10%, 50%, and 90%, respectively. Group 4 served as the negative control and did not receive any vaccine formulation or adjuvant. All groups were monitored daily over a three-week period. Following sample collection, antibody levels were quantified using ELISA, and cellular immune responses were evaluated by measuring IFN-γ levels with a commercial assay kit. According to ELISA results, the highest antibody levels were observed in the group immunized with the attenuated N. caninum strain containing 90% chitosan adjuvant, showing significantly higher antibody levels compared to the control and other groups (p < 0.05). Similarly, IFN-γ analysis revealed that the strongest IFN-γ-associated immune response was observed in the group immunized with 90% chitosan adjuvant, with significant differences compared to the control and other groups (p < 0.05).Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test for multiple comparisons. Based on these findings and the considerable effect of chitosan adjuvant on both antibody levels and IFN-γ-associated cellular immunity, these findings support further investigation of chitosan-containing formulations as potential adjuvants for Neospora vaccine development.

Keywords

Neospora caninum

Chitosan

Vaccine adjuvant

Immunogenicity

BALB/c mice

IFN-&‌‌‌gamma

Subjects

• Parasitology

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References

1-    Cabrera A, Berna L, Lopez L, Faral-Tello P, Arevalo AP, Crispo M, et al. New insights into phenotype and genotype relationships in Neospora caninum. Front Vet Sci. 2023;10:1214971. Doi:10.3389/fvets.2023.1214971.
2-    2-Reichel MP, Ellis JT. Control options for Neospora caninum infections in cattle--current state of knowledge. N Z Vet J. 2002 Jun;50(3):86-92. Doi: 10.1080/00480169.2002.36288. 
3-    Singla AK, Chawla M. Chitosan: Some pharmaceutical and biological aspects—an update. J Pharm Pharmacol. 2001;53(8):1047-67. Doi:10.1211/0022357011776441.
4-    Tuo W, Feng X, Cao L, Vinyard B, Dubey JP, Fetterer R, et al. Vaccination with Neospora caninum-cyclophilin and -profilin confers partial protection against experimental neosporosis-induced abortion in sheep. Vaccine. 2021;39(32):4534-44. Doi:10.1016/j.vaccine.2021.06.032.
5-    Dubey JP. Neosporosis in cattle. Vet Clin North Am Food Anim Pract. 2005;21(2):473-83. Doi:i:10.1016/j.cvfa.2005.03.004
6-    Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry. 1971;8(9):871-74. Doi:10.1016/0019-2791(71)90454-X.
7-    Aguilar JC, Rodríguez EG. Vaccine adjuvants revisited. Vaccine. 2007;25(19):3752-3762. Doi:10.1016/j.vaccine.2007.01.111. 
8-    Guo J, Sun X, Yin H, Wang T, Li Y, Zhou C, et al. Chitosan microsphere used as an effective system to deliver a linked antigenic peptides vaccine protect mice against acute and chronic toxoplasmosis. Front Cell Infect Microbiol. 2018;8:163. Doi: 10.3389/fcimb.2018.00163.
9-    Rauw F, Gardin Y, Palya V, Anbari S, Gonze M, Lemaire S, et al. The positive adjuvant effect of chitosan on antigen-specific cell-mediated immunity after vaccination. Vaccine. 2010;28(6):1607-1614. Doi: 10.1016/j.vetimm.2009.10.028.