CLONING AND EXPRESSION OF A DNA SEQUENCE ENCODING A 41 KDA CRYPTOSPORIDIUM PARVUM OOCYST WALL PROTEIN

Authors: Jenkins, Mark; Trout, James; Murphy, Charles; HARP, JAMES - NADC, AMES, IA; Higgins, James; Wergin, William; Fayer, Ronald

Submitted to: Clinical and Diagnostic Laboratory Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/27/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: Cryptosporidiosis is an intestinal parasitic disease of humans and a variety of animals caused by protozoa in the genus Cryptosporidium. The disease is marked by severe diarrhea, fatigue, and weight loss due to reduced absorptive capacity of the intestine. Although there are at least 8 different species of Cryptosporidium, only one, C. Parvum, is known to infect humans and calves. At present, there is no approved drug for treating cryptosporidiosis nor is there any cost-effective disinfectant for removing the parasite from the water or food. The most effective management technique is thus preventing infection, by identifying the parasite in contaminated water and exposing the water to boiling temperature. The difficulty is that no test is available for detecting only the species of Cryptosporidium that infects humans and calves - C. parvum. The present study describes the cloning and expression of a DNA sequence from C. parvum that is specific for this parasite. The DNA sequence expressed in Escherichia coli produces a recombinant protein that is useful for serodiagnosis of cryptosporidiosis and may be useful for producing a method for detecting C. parvum in water.

Technical Abstract: This study was conducted to produce a recombinant species-specific oocyst wall protein of Cryptosporidium parvum. Antigens unique to C. parvum were identified by gradient SDS-PAGE/immunoblotting of oocyst protein from several different Cryptosporidium species. Antiserum was then prepared against a 41 kDa antigen unique to C. parvum and used to identify a recombinant DNA clone, designated rCP41. Expression of CP41 mRNA in C. parvum oocysts was confirmed by RT-PCR. Although the CP41 sequence was shown by PCR to be present in the genome of C. baileyi, CP41 mRNA was not detected by RT-PCR in this species. Immunofluorescence staining (IFA) with antisera against recombinant CP41 detected native CP41 antigen on the surface of C. parvum oocysts, but failed to detect CP41 on C. baileyi oocysts. Immunoelectron microscopy (IEM) demonstrated that native CP41 was distributed unevenly on the C. parvum oocyst surface and associated with amorphous oocyst wall material. In ELISA, purified rCP41 performed as well as native C. parvum oocyst protein in measuring the serological response of young calves and adult cows to experimental and natural of C. parvum infection. These results indicate that rCP41 antigen may have immunodiagnostic potential for cryptosporidiosis.