Articles

Isolation of Small Number of Cryptosporidium parvum Oocyst Using Immunochromatography

Abstract

Background: Cryptosporidium parvum causes severe gastroenteritis in immunocompromised human and new borne animals. The organism can be transmitted through water. Since small number of C. parvum is infectious, the aim of the present study was to develop a chromatography method for the isolation of C. parvum oocyst in samples with limited number of oocysts.

Methods: Antibody was prepared against whole antigen from C. parvum oocysts, the achieved Ab bound to the sepharose 4B and used for the isolation of oocysts. Antibody against P23 bound to the sepharose 4B, used also for the isolation of C. parvum oocyst. In comparison to these both methods, 2 traditional methods (Salt floatation and 55% sucrose floatation) were also performed.

Results: Both chromatography methods could bind oocysts with capacity depends on the column size. The isolated oocysts were free of bacteria. Our results showed that the traditional methods are useful for the isolation of oocysts from feces, in its smear stained with ziehl-nelsen, at least 3 oocyts are detectable in each microscopic field under 1000 X magnification. In contrast to the chromatography methods, the bacterial contamination was always observed in oocysts isolated with traditional methods.

Conclusion: Immunochromatography could be used for the successful isolation of C. parvum oocysts from the samples containing limited number of oocysts.

Fayer R, Xia L. Cryptosporidium and cryptosporidiosis. 2nd ed. CRC, New York; 2008.

Anderson BC. Patterns of shedding of cryptosporidial oocysts in Idaho calves. J Am Vet Med Assoc. 1981; 178 (9):982–4.

De Graaf DC, Vanopdenbosch E, Ortega- Mora LM, Abbassi H, Peeters JE. A review of the importance of cryptosporidiosis in farm animals. Int J Parasitol. 1999; 29(8):1269–87.

Fricker CR, Crabb JH. Water-borne cryptosporidiosis: detection methods and treatment options. Adv Parasitol. 1998; 40: 241– 78.

Smith HV, Rose JB. Waterborne cryptosporidiosis. Parasitol Today. 1990; 6 (1):8–12.

Blewett DA, Wright SE, Casemore DP, Booth NE, Jones CE. Infective dose size studies on Cryptosporidium parvum using gnotobiotic lambs. Water Sci Technol. 1993; 27(3):61–4.

Pereira SJ, Ramirez NE, Xiao L, Ward LA. Pathogenesis of human and bovine Cryptosporidium parvum in gnotobiotic pigs. J Infect Dis. 2002; 186 (5):715–8.

Anonymous . Method 1622: Cryptosporidium in Water by Filtration/IMS/FA EPA-821-R-99-001. US Environmental Protection Agency, Washington, DC. 1999a

Anonymous. Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA EPA- 821-R-99-006. US Environmental Protection Agency, Washington, DC. 1999b.

Manouchehri Naeini K, Asadi M and Hashemzade Chaleshtori M. Detection and Molecular Characterization of Cryptosporidium species in Recreational Waters of Chaharmahal va Bakhtiyari Province of Iran using nested- PCR-RFLP. Iran J Parasitol. 2011; 6(1):20-27.

Mahmoudi M, Ashrafi K, Abedinzadeh H, Tahvildar-Bideruni F, Haghighi A, Bandehpour M, Taghipour Lailabadi N, Kazemi B. Development of sensitive detection of Cryptosporidium and giardia from surface water in iran. Iran J Parasitol. 2011; 6(3):43-51.

McNabb SJ, Hensel DM, Welch DF, Heijbel H, Mckee GL, Istre GR. Comparison of Sedimentation and Flotation Techniques for Identification of Cryptosporidium sp. Oocysts in a Large Outbreak of Human Diarrhea. J Clin Microbiol. 1985; 22 (4):587-89.

Petry F, Robinson HA, McDonald V. Murine infection model for maintenance and amplification of Cryptosporidium parvum oocysts. J Clin Microbiol. 1995; 33(7):1922–4.

Waldman E, Tzipori S, Forsyth JR. Separation of Cryptosporidium species oocysts from feces by using a percoll discontinuous density gradient. J Clin Microbiol. 1986; 23(1): 199-200.

Arrowood MJ, Sterling CR. Isolation of Cryptosporidium Oocysts and Sporozoites Using Discontinuous Sucrose and Isopycnic Percoll Gradients. J Parasitol. 1987; 73(2): 314-19.

Truong Q, Ferrari BC. Quantitative and qualitative comparisons of Cryptosporidium faecal purification procedures for the isolation of oocysts suitable for proteomic analysis. Int J Parasitol. 2006; 36(7):811–9.

Henriksen SVA, Poglenz JF. Staining of Cryptosporidium by a modified ZiehlNeelsen technique. Acta Vet Scand. 1981; 22: 594 -96.

Winter G, Andrew AG, Williams LK, Slade MB. Characterization of a major sporozoite surface glycoprotein of Cryptosporidium parvum. Funct Integr Genomics. 2000; 1 (3): 207-17

Shayan P, Asghari Z, Ebrahimzadeh E, Omidian Z, Zarghami F. Presenting an Appropriate Method for Isolation of Bacteria Free Cryptosporidium parvum Oocysts. J VM. 2012; 8 ( 2): 75-90.

Sadr-Shirazi N , Shayan P, Eckert B, Ebrahimzadeh E, Amininia N . Cloning, and Molecular Characterization of Polymorphic Iranian Isolate Theileria annulata Surface Protein (Tasp). Iran J Parasitol: 2012; 7(2) :29-39.

Omidian Z, Ebrahimzadeh E, Shahbazi P, Asghari Z, Parviz Shayan. Application of recombinant Cryptosporidium parvum P23 for isolation and prevention. Parasitol Res.2013; DOI 10.1007/s00436-013-3648-0

Harp JA, Goff JP. Protection of calves with a vaccine against Cryptosporidium parvum. J Parasitol. 1995; 81(1):54–7.

Jenkins MC, O’Brien C, Trout J, Guidry A, Fayer R. Hyperimmune bovine colostrum specific for recombinant Cryptosporidium parvum antigen confers partial protection against cryptosporidiosis in immunosuppressed adult mice. Vaccine.1998; 17(19):2453–60.

Reynolds DT, Slade RB, Sykes NJ, Jonas A, Fricker CR. Detection of Cryptosporidium oocysts in water: techniques for generating precise recovery data. J Appl Microbiol.1999; 87(6):804–13.

Deng MQ, Lam KM, Cliver DO. Immunomagnetic separation of Cryptosporidium parvum oocysts using MACS Micro Beads and high gradient separation columns. J Microbiol Methods. 2000; 40 (1) 11–17.

Fontaine M, Guillot E. An immunomagnetic separation–real-time PCR method for quantification of Cryptosporidium parvum in water samples. J Microbiol Methods. 2003; 54(1) : 29– 36.

Boulter-Bitzer JI, Lee H, Trevors J T. Molecular targets for detection and immunotherapy in Cryptosporidium parvum .Biotechnol Adv. 2007; 25 (1):13-44.

Shayan P, Ebrahimzadeh E, Mokhber- Dezfouli MR, Rahbari S. Recombinant Cryptosporidium parvum p23 as a target for the detection of Cryptosporidium-specific antibody in calf sera. Parasitol Res. 2008; 103:1207–11.

O'Brien CN, Jenkins MC. A Rapid Method for Producing Highly Purified Cryptosporidium parvum oocysts. J Parasitol. 2007; 93 (2): 434-36.

Hijjawi NS, Meloni BP, Morgan UM, Thompson RCA. Complete development and longterm maintenance of Cryptosporidium parvum human and cattle genotypes in cell culture. Int J Parasitol. 2001; 31(10):1048–1055.

Arrowood MJ, Sterling CR . Isolation of Cryptosporidium oocysts and sporozoites using discontinuous

sucrose and isopycnic Percoll gradients. J Parasitol. 1987; 73 (2):314–19.

Truong Q. and Ferrari BC. Quantitative and qualitative comparisons of Cryptosporidium faecal purification procedures for the isolation of oocysts suitable for proteomic analysis. Int J Parasitol. 2006; 36 (7): 811-19.

Koompapong K, Sutthikornchai Ch and Sukthana Y. Cryptosporidium Oocyst Detection in Water Samples:Floatation Technique Enhanced with ImmunofluorescenceIs as Effective as Immunomagnetic Separation Method. Korean J Parasitol. 2009; 47(4): 353-57.

Files
IssueVol 9 No 4 (2014) QRcode
SectionArticles
Keywords
Chromatography Cryptosporidium parvum Flotation Oocyst

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
1.
Ebrahimzade E, Shayan P, Asghari Z, Jafari S, Omidian Z. Isolation of Small Number of Cryptosporidium parvum Oocyst Using Immunochromatography. Iran J Parasitol. 1;9(4):482-490.