Investigation of In Vitro Effectiveness of Polymeric Nanoparticles Containing Clinoptilolit on Cryptosporidium Parvum

Abstract

Cryptosporidium species, which do not have a definite prevention and treatment protocol, are pathogenic protozoans that cause diarrhoea in humans and many animal species. This study aimed to demonstrate the in vitro efficacy of clinoptilolite polymer nanoparticle, which has known antidiarrheal effects. DNA isolation was made for species identification of Cryptosporidium oocysts obtained from faeces samples from naturally infected calves and lambs. RFPL analysis was performed in typing. For this purpose, nested PCR and SrpI, VspI and MboII enzymes were used. The coverslip surface was covered with 80% CaCo-2 cells and infected with 1x109 C. parvum. Nanoparticles containing 250, 500, 750 and 1000 μg/mL clinoptilolite were applied to infected cells. Percent of infection rate was calculated by counting under a fluorescent microscope following incubation. While the infection rate was 23.46% in the water-treated control cell group, the percentage infection rates in the clinoptilolite-containing nanoparticle treated group were respectively 15.60%, 8.13%, 10.33% and 13.46%. Inhibition percentages were determined as 33.54%, 65.56%, 55.99% and 42.66%, respectively. As a result, it was observed that the nanoparticle containing clinoptilolite had anticriptocidal activity in infection with C. parvum in Caco-2 cells. In addition, it was observed that the efficacy was dose-dependent, and the IC50 value was the most appropriate value at 750 and 1000 μg/mL doses.

Keywords

• Cryptosporidium parvum
• clinoptilolite
• nanoparticle

Supporting Institution

ADÜ-BAP

Project Number

VTF-17026

References

1. Adkins, P. R. (2022). Cryptosporidiosis. Veterinary Clinics: Food Animal Practice, 38(1), 121-131. https://doi.org/10.1016/j.cvfa.2021.11.009
2. Al-Mathal, E.M., Alsalem, A.A. (2013). Pomegranate (Punica granatum) peel is effective in a murine model of experimental Cryptosporidium parvum ultrastructural studies of the ileum. Experimental Parasitology,134(4),482–94. https://doi.org/10.1016/j.exppara.2013.05.004
3. Alcala-Canto, Y., Gutierrez-Olvera, L., Gutierrez-Olvera, C., & Sumano- Lopez, H. (2011) Effects of clinoptilolite on Eimeria spp. İnfection in sheep, Small Ruminant Research, 100(2),184- 188. https://doi.org/10.1016/j.smallrumres.2011.05.014
4. Ay, C. D., Voyvoda, H., Ulutas, P. A., Karagenc, T., & Ulutas, B. (2021). Prophylactic and therapeutic efficacy of clinoptilolite against Cryptosporidium parvum in experimentally challenged neonatal lambs. Veterinary Parasitology, 299, 109574. https://doi.org/10.1016/j.vetpar.2021.109574
5. Aysul, N., Ulutaş, B., Ünlü, H., Hoşgör, M., Atasoy, A., & Karagenç, T. (2009). Aydın ilinde ishalli buzağılarda bulunan Cryptosporidium türlerinin moleküler karakterizasyonu. 16. Ulusal Parazitoloji Kongresi, Program ve Özet Kitabı, 208.
6. Cameron, P., Gaiser, B. K., Bhandari, B., Bartley, P. M., Katzer, F., & Bridle, H. (2016). Silver nanoparticles decrease the viability of Cryptosporidium parvum oocysts. Applied and Environmental Microbiology, 82(2), 431-437. https://doi.org/:10.1128/AEM.02806-15
7. de Graaf, D. C., Spano, F., Petry, F., Sagodira, S., & Bonnin, A. (1999). Speculation on whether a vaccine against cryptosporidiosis is a reality or fantasy. International Journal for Parasitology, 29(8), 1289-1306. https://doi.org/10.1016/S0020-7519(99)00082-X
8. Deligiannis, K., Lainas, T., Arsenos, G., Papadopoulos, E., Fortomaris, P., Kufidis, D., Stamaradis, C. & Zygoyiannis, D. (2005). The effect of feeding clinoptilolite on food intake and performance of growing lambs infected or not with gastrointestinal nematodes. Livestock Production Science, 96(2-3), 195-203. https://doi.org/10.1016/j.livprodsci.2005.01.011

9. Divers, T.J., & Peek, S.F. (2008). Rebhun’s Diseases of Dairy Cattle (2nd ed.). St. Elsevier Inc.
10. Fallah, M., Sedighi, F., Abbasalipourkabi, R., Maghsood, A. (2017). Comparison of the therapeutic effects of anti-Cryptosporidium nano nitazoxanide (NZT) with free form of this drug in neonatal rats, European Congress of Clinical Microbiology And Infectious Diseases, 7c, 24, Vienna, Austria.
11. Feng, Y., Ortega, Y., He, G., Das, P., Xu, M., Zhang, X., Fayer, R., Gatei, W, Cama,V., & Xiao, L. (2007). Wide geographic distribution of Cryptosporidium bovis and the deer-like genotype in bovines. Veterinary Parasitology, 144(1-2), 1-9. https://doi.org/10.1016/j.vetpar.2006.10.001
12. Haghi, M. M., Khorshidvand, Z., Khazaei, S., Foroughi-Parvar, F., Sarmadian, H., Barati, N., & Ghasemikhah, R. (2020). Cryptosporidium animal species in Iran: a systematic review and meta-analysis. Tropical Medicine and Health, 48, 1-15. https://doi.org/10.1186/s41182-020-00278-9
13. Hamnes, I. S., Gjerde, B., & Robertson, L. (2006). Prevalence of Giardia and Cryptosporidium in dairy calves in three areas of Norway. Veterinary Parasitology, 140(3-4), 204-216. https://doi.org/10.1016/j.vetpar.2006.03.024
14. Lorenzo, M. L., Ares-Mazas, E., & De Maturana, I. V. M. (1993). Detection of oocysts and IgG antibodies to Cryptosporidium parvum in asymptomatic adult cattle. Veterinary Parasitology, 47(1-2), 9-15.https://doi.org/10.1016/0304-4017(93)90171-I
15. Papaioannou, D., Katsoulos, P. D., Panousis, N., & Karatzias, H. (2005). The role of natural and synthetic zeolites as feed additives on the prevention and/or the treatment of certain farm animal diseases: A review. Microporous and mesoporous Materials, 84(1-3), 161-170. https://doi.org/10.1016/j.micromeso.2005.05.030
16. Ranjbar, R., Mirhendi, H., Izadi, M., Behrouz, B., & Mohammadi, M.R. (2018). Molecular identification of cryptosporidium spp. in Iranian dogs using seminested PCR: a first report. Vector Borne Zoonotic Disease. 18(2):96–100. https://doi.org/10.1089/vbz.2017.2136
17. Sadeghi, A. A., & Shawrang, P. (2008). Effects of natural zeolite clinoptilolite on passive immunity and diarrhea in newborn Holstein calves. Livestock Science, 113(2-3), 307-310. https://doi.org/10.1016/j.livsci.2007.08.010
18. Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: a laboratory manual (2nd ed.). Cold Spring Harbor Laboratory press.
19. Santın, M., Trout, J. M., Xiao, L., Zhou, L., Greiner, E., & Fayer, R. (2004). Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves. Veterinary Parasitology, 122(2), 103- 117. https://doi.org/10.1016/j.vetpar.2004.03.30
20. Schnyder, M., Kohler, L., Hemphill, A., & Deplazes, P. (2009). Prophylactic and therapeutic efficacy of nitazoxanide against Cryptosporidium parvum in experimentally challenged neonatal. Veterinary Parasitology, 169(1-2), 149-154. https://doi.org/10.1016/j.vetpar.2008.10.094
21. Šverko, V., Sobočanec, S., Balog, T., Colić, M., & Marotti, T. (2004). Natural micronised clinoptilolite and clinoptilolite mixtures with Urtica dioica L. extract as possible antioxidants. Food Technology and Biotechnology, 42(3), 189-192.
22. Viel, H., Rocques, H., Martin, J., & Chartier, C. (2007). Efficacy of nitazoxanide against experimental cryptosporidiosis in goat neonates. Parasitology Research, 102(1), 163-166. https://doi.org/10.1007/s00436-007-0744-z
23. Vrzgula, L., Prosbova, M., Blazovsky, J., Jacobi, U., Schubert, T., & Kovac, G. (1988) The effect of feeding natural zeolite on indices of the internal environment of calves in the postnatal period. Occurrence, Properties and Utilization of Natural Zeolites, 747-752, ed. Akademiai Kiado, Budapest, Hungary.
24. Xiao, L., Herd, R. P., & McClure, K. E. (1994). Periparturient rise in the excretion of Giardia sp. cysts and Cryptosporidium parvum oocysts as a source of infection for lambs. The Journal of Parasitology, 80 (1), 55-59. https://doi.org/10.2307/3283345
25. Zambriski, J. A., Nydam, D. V., Bowman, D. D., Bellosa, M. L., Burton, A. J., Linden, T. C., & Mohammed, H. O. (2013). Description of fecal shedding of Cryptosporidium parvum oocysts in experimentally challenged dairy calves. Parasitology Research, 112, 1247-1254. https://doi.org/10.1007/s00436-012-3258-2.