Molecular Characterization and Prevalence of Bovine Cryptosporidium Isolates in the Afyonkarahisar Region

Abstract

Cryptosporidiosis is a zoonotic protozoan disease that affects humans and animals. This disease, which causes diarrhea, dehydration, weight loss, stunted growth, and death, especially in newborn calves, leads to economic losses. Early diagnosis of disease on cattle farms, and even taking preventive measures against it, is of great importance. This study aimed to identify the cryptosporidiosis species and prevalence in diarrheic cattle in the Afyonkarahisar region, where cattle farming is quite intensive. For this reason, a total of 188 fecal samples were obtained rectally from 152 diarrheic bovine calves (0-6 months) and 36 diarrheic cattle (≥ 6 months) from the farms. The specimens were analyzed for Cryptosporidium oocysts by the modified acid-fast staining (mAF) procedure. Whereas the frequency of bovine cryptosporidiosis was 9.2% (14/152) in diarrheic calves, no Cryptosporidium oocysts were detected in the feces of diarrheic cattle older than six months. In the 14 identified Cryptosporidium infections, 13.6% were in calves aged 0 to 3 months, whereas 3.1% were in calves aged 3 to 6 months. The sequencing from the 14 isolates at the 18S ribosomal RNA (rRNA) loci indicated the detection of two Cryptosporidium species: C. parvum (64.2%) and C. bovis (35.7%). These findings highlight that C. parvum, one of the most important factors causing zoonoses, is the primary cause of neonatal cryptosporidial diarrhea in calves, and that more comprehensive epidemiological studies are needed to determine the epidemiology and etiology of cryptosporidiosis in this region.

Keywords

• 18S rRNA
• Afyonkarahisar
• Calves
• Cattle
• Cryptosporidium spp.
• DNA Sequencing

Kaynakça

1. 1. Buchanan R, Wieckowski P, Matechou E, Katzer F, Tsaousis AD, Farré M. Global prevalence of Cryptosporidium infections in cattle: A meta-analysis. Curr Res Parasitol Vector Borne Dis. 2025;100264.
2. 2. Weldemariam DT, Awoke MG, Aklilu MA. Review on bovine cryptosporidiosis, its associated risk factors and diagnostics methods. Sm Trop Med J. 2024;6:1-8.
3. 3. Huang J, Chen M, He Y, Chen H, Huang M, Li N, Guo Y. Cryptosporidium equi n. sp.(Apicomplexa: Cryptosporidiidae): biological and genetic characterisations. Int J Parasitol. 2023;53(10):545-554.
4. 4. Ryan UM, Feng Y, Fayer R, et al. Taxonomy and molecular epidemiology of Cryptosporidium and Giardia–a 50 year perspective (1971–2021). Int J Parasitol. 2021;51(13-14):1099-1119.
5. 5. Fayer R, Santin M, Trout JM. Prevalence of Cryptosporidium species and genotypes in mature dairy cattle on farms in eastern United States compared with younger cattle from the same locations. Vet Parasitol. 2007;145:260-266.
6. 6. Fayer R, Santín M,Trout, JM. Cryptosporidium ryanae n. sp.(Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus). Vet Parasitol. 2008;156(3-4):191-198.
7. 7. Widmer G, Carmena D, Kváč M, et al. Update on Cryptosporidium spp.: Highlights from the seventh international Giardia and Cryptosporidium conference. Parasite. 2020;27:14.
8. 8. Santín M, Trout JM, Fayer R. A Longitudinal study of cryptosporidiosis in dairy cattle from birth to 2 years of age. Vet Parasitol. 2008;155(1-2):15-23.

9. 9. Imre K, Lobo LM, Matos O, Popescu C, Genchi C, Dărăbuş G. Molecular characterisation of Cryptosporidium isolates from pre-weaned calves in Romania: Is there an actual risk of zoonotic infections? Vet Parasitol. 2011;181(2-4):321-324.
10. 10. Roblin M, Canniere E, Barbier A, et al. Study of the economic impact of cryptosporidiosis in calves after implementing good practices to manage the disease on dairy farms in Belgium, France, and the Netherlands. CRPVBD. 2023;4:100149.
11. 11. Razakandrainibe R, Diawara EHI, Costa D, et al. Common occurrence of Cryptosporidium hominis in asymptomatic and symptomatic calves in France. PLoS Negl Trop Dis. 2018;12:e0006355.
12. 12. Reif JS, Wimmer L, Smith JA, Dargatz DA, Cheney JM. Human cryptosporidiosis associated with an epizootic in calves. Am J Public Health. 1989;79(11):1528-1530.
13. 13. Karakavuk M, Can H, Can Ş, Karakavuk T, Döşkaya M, Döşkaya AD. Development of a “Rapid‑Crypto Colorimetric LAMP Test” to detect cryptosporidiosis in feces of newborns calves. Acta Parasitol. 2024;69:691-699.
14. 14. Oğuz EF, Kilinç OÖ, Ayan A, et al. Cryptosporidiosis in cattle and buffaloes in Türkiye- molecular analysis and public health significance. Acta Sci Vet. 2025;53:1995
15. 15. Arslan MÖ, Gıcık Y, Erdoğan HM, Sarı, B. Prevalence of Cryptosporidium spp. oocysts in diarrhoeic calves in Kars province, Türkiye. Turk J Vet Anim Sci. 2001;25(2):161-164.
16. 16. Çitil M, Arslan MÖ, Güneş V, Erdoğan HM. Neonatal buzağı ishallerinde Cryptosporidium ve Eimeria enfeksiyonlarının rolü. Kafkas Üniv Vet Fak Derg. 2004;10(1):59-64.
17. 17. Sungur T, Kar S, Güven E, Aktaş M, Karaer Z, Vatansever Z. Cryptosporidium spp’nin dışkıdan Nested PCR ve carbol fuchsin boyama yöntemi ile teşhis edilmesi. Türkiye Parazitol Derg. 2008;32(4):305-308.
18. 18. Sarı B. Aktaş MS, Arslan MÖ. Erzurum yöresinde buzağılarda Cryptosporidium türlerinin prevalansı. Türkiye Parazitol Derg. 2008;32(2):116-119.
19. 19. Ekinci Ö, Sevinç F, Coşkun A, Işık N, Sevinç N. İshalli buzağılarda cryptosporidiosisin yaygınlığı. Eurasian J Vet Sci. 2011;27(2):123-126.
20. 20. Çelik ÖY, Şahin T, Çelik BA. Prevalence and molecular characterization of Cryptosporidium spp. in calves in the Siirt Province, Türkiye. Acta Veterinaria Brno. 2023;92(3):243-250.
21. 21. Yıldırım A, Adanır R, Inci A, et al. Prevalence and genotyping of bovine Cryptosporidium species in the Mediterranean and Central Anatolia Region of Türkiye. Comp Immunol Microbiol Infect Dis. 2020;69:101425.
22. 22. Sayın İpek DN, Sarı B. Occurrence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in lambs and calves in Southeastern Anatolia, Türkiye. Egyptian J Vet Sci. 2024;55(2):599-606.
23. 23. Şimşek A, İnci A, Yıldırım A, Çiloğlu A, Bişkin Z, Düzlü Ö. Nevşehir yöresindeki yeni doğan ishalli buzağılarda Cryptosporidiosis’in Real Time PCR ve Nested PCR yöntemleri ile saptanması. Erciyes Üniv Vet Fak Derg. 2012;9(2):79-87.
24. 24. Güven E, Avcioglu H, Balkaya I, Hayırlı A, Kar S, Karaer Z. Prevalence of cryptosporidiosis and molecular characterization of Cryptosporidium spp. in calves in Erzurum. Kafkas Univ Vet Fak Derg. 2013;19(6):969-974.
25. 25. Kabir MHB, Ceylan O, Ceylan C, et al. Molecular detection of genotypes and subtypes of Cryptosporidium infection in diarrheic calves, lambs, and goat kids from Türkiye. Parasitol Int. 2020;79:102163.
26. 26. İpek DNS. Diyarbakır yöresinde ishalli buzağılarda Cryptosporidium spp. yaygınlığı ve moleküler karakterizasyonu. Dicle Üniv Vet Fak Derg. 2022;15(2):9-13.
27. 27. Özçelik S, Poyraz Ö, Kalkan K, Malatyalı E, Değerli S. Hayvancılıkla uğraşanlarda ve sığırlarda Cryptosporidium spp. yaygınlığının ELISA ile araştırılması. Kafkas Univ Vet Fak Derg. 2012;18(Suppl-A):A61-A62
28. 28. Gündüz N, Arslan, MÖ. Kars yöresinde buzağılarda cryptosporidium enfeksiyonları prevalansının asit fast boyama (mAF) ve ELISA yöntemleriyle belirlenmesi. Türkiye Parazitol Derg. 2017:41;5-8.
29. 29. Turgay N. Özel Boyama Yöntemleri. Korkmaz M, Ok ÜZ. (Eds) Parazitolojide laboratuvar. Türkiye Parazitoloji Derneği. Yayın No: 2011;23:37-38.
30. 30. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, Noordaa JVJ. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990;28(3):495-503.
31. 31. Kalendar R, Lee D, Schulman AH. FastPCR software for PCR primer and probe design and repeat search. Genes Genomes Genomics. 2009;3:1-14.
32. 32. Hall TA. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In: Nucleic acids symposium series. 1999;95-98.
33. 33. Saitou N, M Nei. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4(4):406-25.
34. 34. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547-1549.
35. 35. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985;39(4):783-791.
36. 36. Innan H, Stephan W. The coalescent in an exponentially growing metapopulation and its application to Arabidopsis thaliana. Genetics. 2000;155:2015-2019.
37. 37. McVean GA. A genealogical interpretation of linkage disequilibrium. Genetics. 2002;162(2):987-991.
38. 38. Tajima F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics. 1989;123(3):585-595.
39. 39. Nei M, Kumar S. Molecular evolution and phylogenetics. Oxford university press. 2000.
40. 40. Ouakli N, Belkhiri A, Lucio A, et al. Cryptosporidium-associated diarrhoea in neonatal calves in Algeria. Vet Parasitol. 2018;12:78-84.
41. 41. Lichtmannsperger K, Harl J, Freudenthaler K. Cryptosporidium parvum, Cryptosporidium ryanae, and Cryptosporidium bovis in samples from calves in Austria. Parasitol Res. 2020;119:4291-4295.
42. 42. Aboelsoued D, Megeed KNA. Diagnosis and control of cryptosporidiosis in farm animals. J Parasit Dis. 2022;46(4):1133-1146.
43. 43. Guy RA, Yanta CA, Bauman CA. Molecular identification of Cryptosporidium species in Canadian post-weaned calves and adult dairy cattle. Vet Parasitol. 2022;34:100777.
44. 44. Mammeri M, Chevillot A, Chenafi I, et al. Molecular characterization of Cryptosporidium Isolates from diarrheal dairy calves in France. Vet Parasitol: Reg Stud Rep. 2019;18:100323.
45. 45. Alali F, Abbas I, Jawad M, Hijjawi N. Cryptosporidium infection in humans and animals from Iraq: A review. Acta Trop. 2021;220:105946.
46. 46. Paul S, Chandra D, Tewari AK, et al. Comparative evaluation and economic assessment of coprological diagnostic methods and PCR for detection of Cryptosporidium spp. in bovines. Vet Parasitol. 2009;164(2-4):291-295.
47. 47. Zhao L, Wang M, Wang L, et al. Prevalence and molecular characterization of Cryptosporidium spp. in dairy and beef cattle in Shanxi, China. Parasitol Res. 2024;123:8.
48. 48. Bamaiyi PH, Redhuan NEM. Prevalence and risk factors for cryptosporidiosis: A global, emerging, neglected zoonosis. Asian Biomed. 2016;10:309-325. 49. Alba P, Garro C, Florin-Christensen M. Prevalence, risk factors and molecular epidemiology of neonatal cryptosporidiosis in calves: The Argentine perspective. Curr Res Parasitol Vector Borne Dis. 2023;4:100147. 50. Certad G, Gantois N, Merlin S, et al. Frequency and molecular identification of Cryptosporidium in adult Prim’Holstein dairy cattle farms in the North of France. Microorganisms. 2024;12(2):335.