Characterization of Monoclonal Antibodies against the Second-Generation Schizonts of Leucocytozoon caulleryi.

Abstract

ABSTRACT. Monoclonal antibodies (MAbs), R1 and M5, were established against the second-generation schizont of Leucocytozoon caulleryi (L. caulleryi). Both antibodies reacted to membrane and internal structure proteins of the second-generation schizont by immunofluorescence microscopy. Molecular weight of the second-generation schizont (2GS) antigen was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. At least 40 protein bands were detected in 2GS antigen by SDS-PAGE under reduced condition and ranged from 10 to 270 kDa. MAb R1 reacted to polypeptides of 150-268 kDa in 2GS antigen, whereas MAb M5 did with that of 66 kDa. Injection with a protein of 2GS antigen fractionated by affinity chromatography using MAbs R1 and M5 protected chickens against challenge with sporozoites of L. caulleryi. These results suggest that MAbs, R1 and M5, recognize 2GS antigen of L. caulleryi.KEY WORDS: Leucocytozoon caulleryi, monoclonal antibody, second-generation schizont antigen.J. Vet. Med. Sci. 64(3): 281-283, 2002 Chickens that recovered from the primary infection with Leucocytozoon caulleryi (L. caulleryi) become solidly immune to reinfection, and thus vaccination has been considered as an effective method to prevent the infection. Proteins demonstrating antigenic and immunogenic properties against L. caulleryi have been reported to reside in the second-generation schizonts (2GS) [3,14,15]. Similar proteins designated as the serum-soluble antigen (SSA) have likewise been noted from the second-generation schizonts [6,16]. In the present study, two different monoclonal antibodies (MAbs), designated as MAb R1 and MAb M5, were established in mice injected with 2GS antigen. Some properties of the MAbs were analyzed.Three-week-old specific-pathogen-free chickens (Line M, Nisseiken Co., Ltd.) and free of both the antigen and antibody against L. caulleryi were used in the preparation of 2GS antigen. The Shizuoka strain [11][12][13] of L. caulleryi used was kindly supplied by Dr. T. Morii in 1989 and has been maintained by cyclic transmission in chickens and its vector, Culicoides arakawae (C. arakawae), which likewise have been reared in our laboratory.Experimental exposure of C. arakawae to L. caulleryi, and the preparation of sporozoites for inoculation into chickens and collection of the second-generation schizonts from the lungs, kidneys, thymus and Bursa of Fabricius of chickens followed those earlier described by Morii [9]. Thirteen days after infection of chickens with L. caulleryi, the second-generation schizonts were collected and suspended in phosphate-buffered saline (PBS, pH 7.0) at a concentration of 1:1. The suspension was homogenized, filtered, sonicated for 5 min, and centrifuged at 14,000 × g for 15 min, and the resulting supernatant was used as a 2GS antigen in this study. Concentration of protein in 2GS antigen was determined using BCA Kit (Bio-Rad Laboratories, U. S. A.).Six-week-old mice (BALB/c) were intraperitoneally immunized with 2GS antigen (120 µg of p...

The Correlation of Protective Effects and Antibody Production in Immunized Chickens with Recombinant R7 Vaccine against Leucocytozoon caulleryi.

“…Concentration of rR7 protein was determined by BCA Protein Assay Kit (Pierce Chemical, Rockford, IL). Antigen titration of rR7 protein was done by enzyme-linked immunosorbent assay (ELISA) following the method described in a previous paper [ 23 ] , except for using MAb R1 [3] as an antibody. Two-folds serial dilutions of rR7 protein were made, and the samples were assayed by ELISA.…”

“…Amount of the inactivated rR7 protein for preparation of aluminium-hydroxyde [Al(OH) 3 ] gel-adjuvanted rR7 vaccine was adjusted to 70 µg/ml, 100,000 AU/ml, with PBS. Al(OH) 3 gel was added to a final concentration of 0.6 mg/ ml.…”

“…Six chickens in group 1 received 0.25 ml/chicken (10,000 AU/dose) of oil-adjuvanted rR7 vaccine at 5 weeks of age. Six chickens in group 2 were injected 0.5 ml/dose (10,000 AU/dose) of Al(OH) 3 gel-adjuvanted rR7 vaccine twice at 5 and 7 weeks of age. The remaining 6 chickens were kept as a challenge control.…”

“…Recently, Gotanda et al established two monoclonal antibodies ( MAbs ) , named MAb R1 and MAb M5, that recognize 2GS antigen of L. caulleryi [3]. Chickens injected with the fractionated antigen purified by affinity chromatography using the MAbs protected against L. caulleryi sporozoites challenge.…”

Field Efficacy of Recombinant R7 Vaccine against Chicken Leucocytozoonosis

“…Gotanda et al established monoclonal antibody that recognize immunogenic antigen of 2GS [3]. Recently, the gene encoding an immunogenical protein of 2GS, named gene R7, has been cloned and vaccine with recombinant R7 protein (rR7) have protective effects strongly against sporozoite of L. caulleryi challenge [8] .…”