Protective immunity toLeucocytozoon caulleryi in chickens by a killed vaccine

Doi

Eiguchi

et al. 2002

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...

supporting

confidence: 78%

Section: Chickens That Recovered From the Primary Infection Withmentioning

confidence: 99%

mentioning

confidence: 99%

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

Doi

Eiguchi

et al. 2002

“…Morii et al [18,19] have reported that chickens that have recovered from a primary infection with L. caulleryi sporozoites are resistant to reinfection, and that the acquired immunity to L. caulleryi in chickens is strongly expressed against second-generation schizonts (2GS) of L. caulleryi [20,24]. Recently, Gotanda et al established two monoclonal antibodies ( MAbs ) , named MAb R1 and MAb M5, that recognize 2GS antigen of L. caulleryi [3].…”

mentioning

confidence: 99%

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

Itoh

2002

ABSTRACT. Protective effects of recombinant R7 (rR7) vaccine against Leucocytozoon caulleryi in chickens were studied. After injection of oil-adjuvanted rR7 vaccine into chickens, antibody titers against second-generation schizonts (2GS) antigen of L. caulleryi (anti-2GS antibody) rapidly rose in all the immunized chickens, reached to a peak value 2 weeks after injection, and the titers persisted through 4 or 5 months after injection. Chickens having high levels of the anti-2GS antibody titers (≥ 102,400 ) at pre-challenge completely protected against sporozoites challenge of L. caulleryi. After the challenge inoculation, relatively high parasitemia of L. caulleryi was observed in all the inadequately immunized chickens having low levels of the antibody titers (≤ 3,200 ) at pre-challenge, although some of them seemed to be clinically normal. Correlation of protective effects in the immunized chickens was observed between both prevention of appearance of clinical signs and parasitemia after parasites challenge and anti-2GS antibody titers of the chickens at pre-challenge. The present study shows that chicken leucocytozoonosis can be prevented by vaccination, and humoral immunity may play an important role in the control of chicken leucocytozoonosis. KEY WORDS: humoral immunity, Leucocytozoon caulleryi, recombinant vaccine, second-generation schizont.

“…In the previous studies for immunological prevention against leucocytozoonosis, vaccines that used inactivated 2GS of L. caulleryi [5,17] , or vaccine that is made with live sporozoites [16,18], and etc. have been tried [20] .…”

Section: Discussionmentioning

confidence: 99%

Field Efficacy of Recombinant R7 Vaccine against Chicken Leucocytozoonosis

Ito

2004

ABSTRACT. Effectiveness of vaccine that used recombinant R7 protein (rR7) as antigen that is derived from second-generation schizont (2GS) of Leucocytozoon caulleryi was verified under a field condition against chicken leucocytozoonosis. Chickens reared in a poultry farm where the chickens are attacked by leucocytozoonosis in every year were inoculated with oil-adjuvanted rR7 vaccine (O-rR7), and the immunized chickens were found to have production of antibodies against 2GS at a high level by one shot. Leucocytozoonosis was observed at post-injection. During the epidemic period of leucocytozoonosis, the unique clinical signs of the disease such as discharge of green feces and anemia, and also parasitemia were observed, however, compared to chickens in control group, those in O-rR7 vaccinated group had significantly slight symptoms (P<0.05). In addition to this, immunized chickens had better result of egg produc tion than the unvaccinated chickens did, and the maximum difference of egg production rate, 22%, was observed at the peak of the disease. In conclusion, it is verified that O-rR7 vaccine has efficacy against leucocytozoonosis under field condition, and this vaccine can be put into practical use. KEY WORDS: field trial, Leucocytoon caulleryi, leucocytozoonosis, recombinant R7 vaccine.