Background and Aim: Cryptosporidium is recognized to infect several mammalian species as well as humans, causing substantial economic losses and serious public health concern. Infected animals can be a source of environmental contamination and human infections. In general, the occurrence of Cryptosporidium species in animals and human in Sudan and zoonotic importance is not well documented. This study aimed to identify Cryptosporidium spp. infecting different animal species and humans and to compare between different isolates obtained.

Materials and Methods: To provide molecular information about Cryptosporidium in animals and humans, both modified Ziehl-Neelsen (MZN) specific stain and molecular assay were used. Concentration techniques followed by three protocols of DNA extraction were carried out. After microscopic screening of 263 fecal samples (goats [n=197], cattle [n=12], sheep [n=12], and human [n=42]), 61 positive and 30 negative, randomly selected samples were used in nested polymerase chain reaction (PCR) targeting part of the 18S RNA.

Results: Nested PCR amplification confirmed 91.8% (56/61) of microscopic-positive samples. 8.2% (5/61) of negative samples by PCR (positive by microscopy) were considered false negatives. Sequencing followed by alignment of the 14 isolates indicated that all samples were identical (100%) and belonged to Cryptosporidium parvum.

Conclusion: MZN staining procedure is reliable for the routine diagnosis of Cryptosporidium; cetyltrimethylammonium bromide extraction buffer and nested PCR targeting 18S rRNA gene are reliable and useful in epidemiological studies of this parasite.

Keywords: Cryptosporidium parvum, nested polymerase chain reaction, staining techniques, Sudan, zoonotic.

1. Fayer, R. (2008) General biology. In: Fayer, R. and Xiao, L., editors. Cryptosporidium and Cryptosporidiosis. 2nd ed. CRC Press, Boca Raton-London New York. p1-42.

2. Sharbatkhori, M., Mojarad, E.N., Taghipour, N., Pagheh, A.S. and Mesgarian, F. (2015) Prevalence and genetic characterization of Cryptosporidium spp. In diarrheic children from Gonbad Kavoos city, Iran. Iran. J. Parasitol., 10(3): 441. [PubMed]

3. Shafieyan, H., Alborzi, A., Hamidinejat, H., Tabandeh, M.R. and Hajikolaei, M.R.H. (2016) Prevalence of Cryptosporidium spp. In ruminants of Lorestan province, Iran. J. Parasit. Dis., 40(4): 1165-1169. [Crossref] [PubMed] [PMC]

4. Li, P., Cai, J., Cai, M.W.W., Li, C., Lei, M., Xu, H., Feng, L., Ma, J., Feng, Y. and Xiao, L. (2016) Distribution of Cryptosporidium species in Tibetan sheep and yaks in Qinghai, China. Vet. Parasitol., 215: 58-62. [Crossref] [PubMed]

5. Romero-Salas, D., Alvarado-Esquivel, C., Cruz-Romero, A., Aguilar-Dominguez, M., Ibarra Priego, N., Merino-Charrez, J.O., Perez de Leon, A.A. and Hernandez-Tinoco, J. (2016) Prevalence of Cryptosporidium in small ruminants from Veracruz, Mexico. BMC Vet. Res., 12: 14-19. [Crossref] [PubMed] [PMC]

6. Taylan-Ozkan, A., Yasa-Duru, S., Usluca, S., Lysen, C., Ye, J., Roellig, D.M., Feng, Y. and Xiao, L. (2016) Cryptosporidium species and Cryptosporidium parvum subtypes in dairy calves and goat kids reared under traditional farming systems in Turkey. Exp. Parasitol., 170: 16-20. [Crossref] [PubMed]

7. Kaupke, A., Michalski, M.M. and Rzezutka, A. (2017) Diversity of Cryptosporidium species occurring in sheep and goat breeds reared in Poland. Parasitol. Res., 116(3): 871-879. [Crossref] [PubMed] [PMC]

8. Adam, K.Y., Gameel, A.A. and Ismail, A.A. (2017) Detection of Cryptosporidium species copro antigen in goats in Khartoum state, Sudan. Sudan J. Vet. Res., 33: 21-26.

9. Noordeen, F., Rajapakse, R.P.V., Horadagoda, N.U., Abdul-Careem, M.F. and Arulkanthan, A. (2012) Cryptosporidium, an important enteric pathogen in goats. A review. Small Rumin. Res., 106: 77-82. [Crossref]

10. OIE (2018) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals Chapter 2.9.4. Cryptosporidiosis. p.1193http://www.oie.int/standard-setting/terrestrial-manual/access-online. Last accessed on 11-12-2018.

11. Anderson, B.C., Donndelinger, T., Wilkins, R.M. and Smith, J. (1982) Cryptosporidiosis in a veterinary student. J. Am. Vet. Med. Assoc., 180(4): 408-409. [PubMed]

12. Current, W.L. (1983) Human cryptosporidiosis. N. Engl. J. Med., 309: 1326-1327.

13. Current, W.L., Reese, N.C., Ernst, J.V., Bailey, W.S., Heyman, M.B. and Weinstein, W.M. (1983) Human cryptosporidiosis in immunocompetent and immunodeficient persons. Studies of an outbreak and experimental transmission. N. Engl. J. Med., 308(21): 1252-1257. [Crossref]

14. Fayer, R. and Ungar, B.L.P. (1986) Cryptosporidium spp. and cryptosporidiosis. Microbiol. Rev., 50(4): 458-483. [PubMed] [PMC]

15. Ma, P. (1986) Cryptosporidium biology and diagnosis. Adv. Exp. Med. Biol., 202: 135-152. [Crossref] [PubMed]

16. Robertson, L.J., Gjerde, B.K. and Furuseth, H.E. (2010) The zoonotic potential of Giardia and Cryptosporidium in Norwegian sheep: A longitudinal investigation of 6 flocks of lambs. Vet. Parasitol., 171(1-2): 140-145. [Crossref] [PubMed]

17. Wang, R., Li, G., Cui, B., Huang, J., Cui, Z., Zhang, S., Dong, H., Yue, D., Zhang, L., Ning, C. and Wang, M. (2014) Prevalence, molecular characterization and zoonotic potential of Cryptosporidium spp. In goats in Henan and Chongqing, China. Exp. Parasitol., 142: 11-16. [Crossref] [PubMed]

18. Morgan-Ryan, U.M., Fall, A., Ward, L.A., Hijjawi, N., Sulaiman, I., Fayer, R., Thompson, R.C., Olson, M., Lal, A. and Xiao, L. (2002) Cryptosporidium hominus n.sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens. J. Eukaryot. Microbiol., 49(6): 433-440. [Crossref] [PubMed]

19. Xiao, L. and Ryan, U.M. (2004) Cryptosporidiosis: An update in molecular epidemiology. Curr. Opin. Infect. Dis., 17(5): 483-490. [Crossref]

20. Xiao, L. and Feng, Y. (2007) Zoonotic cryptosporidiosis. FEMS Immunol. Med. Microbiol., 52(3): 309-323. [Crossref] [PubMed]

21. Kosek, M., Alcantara, C., Lima, A.A. and Guerrant, R.L. (2001) Cryptosporidiosis: An update. Lancet Infect. Dis., 1(4): 262-269. [Crossref]

22. Mackay, I.M. (2004) Real-time PCR in the microbiology laboratory. Clin. Microbiol. Infect., 10(3): 190-212. [Crossref]

23. Zhao, X., Duszynski, D.W. and Loker, E.S. (2001) A simple method of DNA extraction for Eimeria species. J. Microbiol. Methods, 44(2): 131-137. [Crossref]

24. Wasser, S.K., Houston, S., Koehler, G.M., Cadd, G.G. and Fain, S.R. (1997) Techniques for application of faecal DNA methods to field of Ursids. Mol. Ecol., 6(11): 1091-1097. [Crossref]

25. Baird, D.R. (2005) Microscopy in bacteriology application and sample preparation. In: Crocker, J. and Burnett, D., editors. Science of Laboratory Diagnosis. 2nd ed. John Wiley and Sons Ltd., USA. p123-128.

26. Schrader, C., Schielke, A., Ellerbroek, L. and Johne, R. (2012) PCR inhibitors occurrence, properties and removal. J. Appl. Microbiol., 113(5): 1014-1026. [Crossref] [PubMed]

27. Oikarinen, S., Tauriainen, S., Viskari, H., Simell, O. and Knip, M. (2009) PCR inhibition in stool samples in relation to age of infants. J. Clin. Virol., 44(3): 211-221. [Crossref] [PubMed]

28. Current, W.L. and Reese, N.C. (1986) A comparison of endogenous development of three isolates of Cryptosporidium in suckling mice. J. Protozool., 33(1): 98-108. [Crossref]

29. Elwin, K., Chalmers, R.M., Roberts, R., Guy, E.C. and Casemore, D.P. (2001) Modification of a rapid method for the identification of gene-specific polymorphisms in Cryptosporidium parvum and its application to clinical and epidemiological investigations. Appl. Environ. Microbiol., 67(12): 5581-5584. [Crossref] [PubMed] [PMC]

30. Ryley, J.F., Meade, R., Hazelhurst, J. and Robinson, T.E. (1976) Methods in coccidiosis research: Separation of oocysts from faeces. Parasitology, 73(3): 311-326. [Crossref]

31. Abbaszadegan, M.R., Velayati, A., Tavasoli, A. and Dadkhah, E. (2007) Rapid DNA extraction protocol from stool, suitable for molecular genetic diagnosis of colon cancer. Iran. Biomed. J., 11(3): 203-208. [PubMed]

32. Kuzehkanan, A.B., Rezaeian, M., Zeraati, H., Mohebali, M., Meamar, A.R., Babaei, Z., Kashi, L., Heydarnezhadi, M. and Rezaie, S.A. (2011) Sensitive and specific PCR based method for identification of Cryptosporidium Sp. Using new primers from 18S ribosomal RNA. Iran. J. Parasitol., 6(4): 1-7.

33. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol., 30(12): 2725-2729. [Crossref] [PubMed] [PMC]

34. Saitou, N. and Nei, M. (1987) The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol., 4(4): 406-425. [PubMed]

35. Rzhetsky, A. and Nei, M. (1992) A simple method for estimating and testing minimum evolution trees. Mol. Biol. Evol., 9(5): 945-967.

36. Dopazo, J. (1994) Estimating errors and confidence intervals for branch lengths in phylogenetic trees by a bootstrap approach. J. Mol. Evol., 38(3): 300-304. [Crossref]

37. Hawash, Y. (2014) DNA extraction from protozoan oocysts/cysts in faeces for diagnostic PCR. Korean J. Parasitol., 52(3): 263-271. [Crossref] [PubMed] [PMC]

38. Morgan, U.M., Pallant, L., Dwyer, B.W., Forbes, D.A., Rich, G. and Thompson, C.A. (1998) Comparison of PCR and microscopy for detection of Cryptosporidium parvum in human fecal specimens: Clinical trial. J. Clin. Microbiol., 36(4): 995-998. [PubMed] [PMC]

39. Tahvildar-Biderouni, F. and Salehi N. (2014) Detection of Cryptosporidium infection by modified ziehl-neelsen and PCR methods in children with diarrheal samples in pediatric hospitals in Tehran. Gastroenterol. Hepatol. Bed Bench, 7(2): 125-130. [PubMed] [PMC]

40. Gawad, S.S.A., Ismail, M.A.M., Imam, N.F.A., Eassa, A.H.A. and Sarea, E.Y. (2018) Cryptosporidium in diarrheic patients in Egypt. Korean J. Parasitol., 56(2): 113-119. [PubMed] [PMC]

41. Paul, S., Chandra, D., Tewari, A.K., Banerjee, P.S., Ray, D.D. and Boral, R. (2009) Comparative evaluation and economic assessment of coprological diagnostic methods and PCR for detection of Cryptosporidium spp. In bovines. Vet. Parasitol., 164(2-4): 291-295. [Crossref] [PubMed]

42. Adam, K.Y. (2017) Prevalence and Molecular Characterization of Cryptosporidium Species in Goats in Khartoum State and Pathogenicity of Cryptosporidium parvum in Goat Kids. Ph.D. Thesis, (Parasitology) Faculty of Veterinary Medicine, Sudan University of Science and Technology.

43. Musa, H.A., Salim, G.A., Ismael, A.A., Elfadil, A. and Kafi, S.K. (2011) Diarrhea due to Cryptosporidium parvum immunocompromised and immunocompetent patients in Khartoum state. Sudan J. M. S., 6(1): 39-42. [Crossref]

44. Sim, S., Yu, J., Lee, Y., Lee, J., Jeong, H., Mohamed, A.S. and Hong, S. (2015) Prevalence of Cryptosporidium infection among inhabitants of 2 rural areas in White Nile state, Sudan. Korean J. Parasitol., 53(6): 745-747. [Crossref] [PubMed] [PMC]