The detection, and identification of Cryptosporidium species by nested polymerase chain reaction (nested-PCR) and restriction fragment length polymorphism (RFLP) of calves in Shahroud town

Subject Areas : Journal of Comparative Pathobiology

مصطفی Meshkat ¹ , , B Shemshadi ² , , K. Amini ³

1 - Department of Pathobiology, Faculty of Veterinary Medicine, Science and Research Branch, Islamic Azad University, Tehran, Iran
2 - Department of Pathobiology, Faculty of Veterinary Medicine, Science and Research Branch, Islamic Azad University, Tehran, Iran
3 - Department of Microbiology, Faculty of Basic Sciences, Saveh Branch, Islamic Azad University, Saveh, Iran

Received: 2021-11-03 Accepted : 2021-11-03 Published : 2021-05-22

Keywords: Cryptosporidiosis, claves, RFLP, shahroud,

Abstract :

Cryptosporidium species belong to Apicomplexa phylum and are opportunistic protozoans that infect the gastrointestinal and respiratory systems of some animals and humans. This study was conducted to investigate the prevalence, detection, and identification of Cryptosporidium species by nested PCR and restriction fragment length polymorphism (RFLP) in calves in Shahroud town.  A total number of 256 calves was collected from fecal samples of pre-weaned calves (≤2 months) in Shahroud town, and positive samples were stained by Ziehl-Neelsen (ZN) staining method. Specific primers were used to investigate the Nested-PCR and sub-species were investigated by RFLP method. The microscopic results showed that 27 samples (10.54 %) of the samples were positive. The results for nested-PCR showed that out of samples, 92.59 % and 7.41 % of samples were positive for C. parvum and C. Andersoi, respectively. The results showed that 25 samples were affected by VSP enzyme in regions of 104 and 628 bp that belong C. parvum bovine, and genotype A gene sub-species. The samples were affected by Ssp I enzyme and the results showed bands in regions of 385 and 448 bp that show C. muris/C. andersoni sub-species. The results for dde enzyme showed bands in regions of 156, 186, and 224 bp that confirm C. muris sub-specie. The different species and sub-species were identified by different methods and can help to control of the cryptosporidiosis.

References:

1. Cho Y-i, Yoon K-J. An overview of calf diarrhea-infectious etiology, diagnosis, and intervention. J Vet Sci. 2014;15(1):1-17.

2. Tyzzer E. Cryptosporidium parvum (sp. nov.), a coccidium found in the small intestine of the common mouse. Arch Protistenkd. 1912;26:394-412.

3. Lindsay DS, Upton SJ, Owens DS, Morgan UM, Mead JR, Blagburn BL. Cryptosporidium andersoni n. sp.(Apicomplexa: Cryptosporiidae) from cattle, Bos taurus. J Eukaryot Microbiol. 2000;47(1):91-5.

4. Ralston B, Thompson R, Pethick D, McAllister T, Olson M. Cryptosporidium andersoni in Western Australian feedlot cattle. Aust Vet J. 2010;88(11):458-60.

5. Zambriski JA, Nydam DV, Bowman DD, Bellosa ML, Burton AJ, Linden TC, et al. Description of fecal shedding of Cryptosporidium parvum oocysts in experimentally challenged dairy calves. Parasitol Res. 2013;112(3):1247-54.

6. Chalmers RM, Davies AP. Minireview: clinical cryptosporidiosis. Exp Parasitol. 2010;124(1):138-46.

7. Chalmers R, Giles M. Zoonotic cryptosporidiosis in the UK–challenges for control. J Appl Microbiol. 2010;109(5):1487-97.

8. Wells B, Shaw H, Hotchkiss E, Gilray J, Ayton R, Green J, et al. Prevalence, species identification and genotyping Cryptosporidium from livestock and deer in a catchment in the Cairngorms with a history of a contaminated public water supply. Parasites & vectors. 2015;8(1):66.

9. Chalmers RM, Katzer F. Looking for Cryptosporidium: the application of advances in detection and diagnosis. Trends in parasitology. 2013;29(5):237-51.

10. Thomson S, Innes EA, Jonsson NN, Katzer F. A multiplex PCR test to identify four common cattle-adapted Cryptosporidium species. Parasitology Open. 2016;2.

11. Smith R, Clifton-Hadley F, Cheney T, Giles M. Prevalence and molecular typing of Cryptosporidium in dairy cattle in England and Wales and examination of potential on-farm transmission routes. Vet Parasitol. 2014;204(3-4):111-9.

12. Rekha K, Puttalakshmamma G, D’Souza PE. Comparison of different diagnostic techniques for the detection of cryptosporidiosis in bovines. Veterinary world. 2016;9(2):211.

13. Xiao L, Fayer R, Ryan U, Upton SJ. Cryptosporidium taxonomy: recent advances and implications for public health. Clin Microbiol Rev. 2004;17(1):72-97.

14. Xiao L, Lal AA, Jiang J. Detection and differentiation of Cryptosporidium oocysts in water by PCR-RFLP. Public Health Microbiology: Springer; 2004. p. 163-76.

15. Ranjbar-Bahadon S, Toni S. Infection to Cryptosporidium in diarrheic calves: a provincial study in southern Khorasan. Journal of Veterinary Research. 2013;68(1):13-9.

16. Changizi E, Salimi-Bejestani M, Vayeghan A. The Cryptosporidium ryanae infection commence in Iranian cattle. Journal of Veterinary Research. 2012;67(2):127-33.

17. Utaaker KS, Chaudhary S, Bajwa RS, Robertson LJ. Prevalence and zoonotic potential of intestinal protozoans in bovines in Northern India. Veterinary Parasitology: Regional Studies and Reports. 2018;13:92-7.

18. Tahvildar Bidrooni F, Dalimi Asl A, Kazemi D B. Using a 1055 bp fragment of 18s rRNA for differentiation of human and cattle Cryptosporidiosis. Research in Medicine. 2008;32(1):5-10.

19. Mohammadi Ghaleh Bin B, Fallah E, Asgharzadeh M, Kazemi A. Detection of Cryptosporidium species in water resources of Ardabil Province by PCR, RFLP. Journal of Ardabil University of Medical Sciences. 2007;7(2):177-83.