CHARACTERISTIC OF CRYPTOSPORIDIUM APICAL ANTIGENS USING CHICKEN MONOCLONAL ANTIBODIES AGAINST Eimeria
M. Matsubayashi1, K. Sasai2*, I. Kimata3, H. Matsuda4,
H. S. Lillehoj5, M. Iseki6, H. Tani2, E. Baba2
1. Department of Food and Nutrition, Osaka Yuhigaoka
Gakuen Junior College, Osaka 543-0073, Japan.
2. Department of Veterinary Internal Medicine, Division of Veterinary Science,
Graduate School of Life and Environmental Sciences, Osaka
Prefecture University, Osaka 599-8531, Japan.
3. Department of Protozoal Diseases, Graduate
School of Medicine, Osaka City University, Osaka 545-8585, Japan.
4. Laboratory of Immunobiology, Department of Molecular and Applied Biosciences,
Graduate School of Biosphere Sciences, Hiroshima University,
Higashi-Hiroshima 739-8528, Japan. 5. U.S.
Department of Agriculture, Animal Parasitic Diseases Laboratory, Animal and
Natural Resources Institute, BARC-East, 20705, Beltsville, MD, USA. 6.
Department of Parasitology,
Graduate School of Medical Science, Kanazawa University, Kanazawa 920-8640,
Japan.
* Corresponding author
Kazumi Sasai
E-mail: ksasai@vet.osakafu-u.ac.jp
Cryptosporidium is a coccidian parasite that causes
diarrhea in human and other animals. Although Cryptosporidium represents a
pathogen of importance to the health of humans and animals, there are no
effective drugs and vaccines. In the previous study, we have developed chicken
monoclonal antibodies (mAbs) against Eimeria acervulina and demonstrated their
use in the identification of potential vaccine antigens for avian coccidiosis.
Furthermore, we found that these antibodies cross reacted with Cryptosporidium
parasites. In the present study, we characterized the antigen of Cryptosporidium,
which was recognized by one of these chicken mAbs, 6D-12-G10. In indirect
immunofluorescent analysis, the chicken mAb 6D-12-G10 showed intense staining on
the apical region of C. parvum and C. muris sporozoites, and merozoites of C.
parvum. In western blot analysis, 6D-12-G10 antibody identified a 48-kDa
molecular weight band of C. parvum and C. muris soluble antigens. Furthermore,
the mAb 6D-12-G10 significantly inhibited the invasion of C. parvum sporozoites
into HCT-8 cells in vitro. Immunoelectron microscopy revealed that the antigen
recognized by this mAb was located only at the apical surface membrane of the
invasive stages of C. parvum. Additionally, we compared the antigen specificity
of mAb 6D-12-G10 with several anti-cytoskeletal (actin, myosin, a-, ß-, ?-tubulin)
antibodies, and the reactivity of mAb 6D-12-G10 was clearly different from those
of anti-cytoskeletal antibodies. These results indicate that the target antigen
recognized by mAb 6D-12-G10 is a good vaccine candidate for Cryptosporidium
infection.