Application of recombinant Cryptosporidium parvum P23 for isolation and prevention.

Cryptosporidium parvum is a coccidian protozoan that causes diarrhea in immunocompromised humans and newborn animals. Billions of oocysts of C. parvum can be released from the infected calves and can contaminate the environment. The severity of the disease depends on the immunological status of the individual. Oocysts of Cryptosporidium are extremely resistant to many environmental stresses, and no effective disinfectant and curative agent against this organism is available. In our study, recombinant C. parvum P23 was prepared for application in the isolation and prevention of cryptosporidiosis. P23 is a glycoprotein that belongs to a family of protein of 23-27 kDa and is believed to be expressed in the different life stages of the parasite. Immunostaining analysis using the indirect fluorescent antibody test showed that P23 could be recognized on the surface of the oocysts. The antibody prepared in rabbit against P23 was bound to Sepharose 4B and used for the isolation of oocysts. The results showed that the prepared column was able to bind specifically only the oocysts. The effect of specific recombinant C. parvum IgY antibody against infection with C. parvum was examined in a mouse model. For this aim, purified egg yolk antibody prepared from immunized hens was used to analyze the protective effect of recombinant P23 specific antibody in immunosuppressed adult mice. The results showed more than 70% reduction in oocyst shedding after challenge with 1 × 10(4) oocysts. These results support previous studies of other investigators regarding the protective effect of P23 as an antigen against C. parvum infection and showed that it could be possible to design a passive immunization strategy against C. parvum based on the anti-P23 yolk antibody in animals and immunosuppressed humans.

Recombinant Cryptosporidium parvum p23 as a target for the detection of Cryptosporidium-specific antibody in calf sera.

Cryptosporidium parvum is a widely distributed coccidian parasite and causes enteric disease in humans and animals. In addition to being a cause of life-threatening disease in immunodeficient people, mostly AIDS patients, C. parvum has been reported as a common serious primary cause of outbreaks of diarrhea in newborn calves, especially newborn ruminants (De Graaf et al. in Int J Parasitol 29:1269-1287, 1999). To obtain the recombinant P23 protein, we isolated the mRNA from oocyst of C. parvum and amplified the cDNA of P23 gene by reverse transcriptase PCR. Sequencing of cDNA showed 100% homology to the known P23 sequences. The double strand P23-cDNA was then cloned in pGEX-5X-2 expression vector. Western blot analysis of recombinant P23 showed that it could be recognized by the positive C. parvum serum. Since P23 is an immunodominant surface glycoprotein expressed in the early phase of infection (Jakobi and Petry in Microbes Infect 8:2186-2194, 2006) and the immunogenic epitopes are also found in the residual chain of amino acid sequence of this glycoprotein, the recombinant P23 was used for the screening of 437 serum samples collected from calves (#264) and cattle (#173). The dot blot analysis showed that from 264 calf and 173 cattle sera, 33% and 37% sera were positive, respectively. Due to the simple handling and equipment, dot blot analysis with P23 could be recommended for calves screening against cryptosporidiosis.