Serological and histological evaluation of species-specific immunocontraceptive vaccine antigens based on zona pellucida 3 in the small Indian mongoose (Herpestes auropunctatus)

Abstract

The small Indian mongoose (Herpestes auropunctatus) was introduced to Japanese islands and has impacted on the island's biodiversity. Population control has been attempted through capturing but its efficiency has rapidly declined. Therefore, new additional control methods are required. Our focus has been on the immunocontraceptive vaccines, which act in an especially species-specific manner. The amino-acid sequence of the mongoose ovum zona pellucida protein 3 (ZP3) was decoded and two types of synthetic peptides (A and B) were produced. In this study, these peptides were administered to mongooses (each n=3) and the sera were collected to verify immunogenicity using ELISA and IHC. Treated mongoose sera showed an increasing of antibody titer according to immunizations and the antigen-antibody reactions against the endogenous mongoose ZP. In addition, IHC revealed that immune sera absorbed with each peptide showed a marked reduction in reactivity, which indicated the specificity of induced antibodies. These reactions were marked in peptide A treated mongoose sera, and the antibody titer of one of them lasted for at least 21 weeks. These results indicated that peptide A was a potential antigen, inducing autoantibody generation. Moreover, immunized rabbit antibodies recognized mongoose ZP species-specifically. However, the induction of robust immune memory was not observed. Also, the actual sterility effects of peptides remain unknown, it should be verified as a next step. In any case, this study verified synthetic peptides we developed are useful as the antigen candidates for immunocontraception of mongooses.

Keywords: fertility control; immunocontraception; invasive alien species; mongoose; zona pellucida.

Fig. 1.

Antibody titers (absorbance at 450–655 nm; mean ± SD) in mongoose sera following immunization with peptide A (a) and peptide B (b). Horizontal lines mean the cut-off value calculated from each control on same microplate: cut-off value=mean antibody titer of control + 3 SD. Arrows indicate the times of immunization.

Fig. 2.

Durability of antibody titers (absorbance at 450–655 nm; mean ± SD) in mongoose following immunization with peptide A (A-1) and peptide B (B-2) and degree of immune memory against reimmunization with each peptides. The cut-off value was calculated from control on each peptide A (horizontal solid line) and peptide B (horizontal broken line): cut-off value=mean antibody titer of each control + 3 SD. Arrows indicate the times of immunization.

Fig. 3.

Immunoreactivity of immunized mongoose sera with synthetic peptides against normal mongoose ovary in IHC. a: A-1 antiserum, b: A-2 antiserum, c: A-3 antiserum, d: B-1 antiserum, e: B-2 antiserum, f: B-3 antiserum, g: C-1 serum (control), and h: HE-stained normal mongoose ovary. Arrowheads: zona pellucida. Bars=50 µm.

Fig. 4.

Immunoreactivity of the A-2 mongoose antiserum against zona pellucida of various stages of follicles. PF: primary follicle, SF: secondary follicle, TF: tertiary follicle, GF: Graafian follicle, and AF: atretic follicle. Arrowheads: zona pellucida. Bar=100 µm.

Fig. 5.

Immunoreactivity of immunized mongoose sera with synthetic peptides and absorbed sera with each peptide against normal mongoose ovary in IHC. a: A-2 antiserum, b: A-2 antiserum absorbed with peptide A, c: B-1 antiserum, and d: B-1 antiserum absorbed with peptide B. Arrowheads: zona pellucida. Bars=50 µm.

Fig. 6.

Immunoreactivity of immunized rabbit sera (a-2) with synthetic peptides A against ovary of various animal species in IHC. a: small Indian mongoose, b: Japanese marten, c: cat, d: dog, e: Japanese wild boar, and f: rabbit. Arrowheads: zona pellucida. Bars=50 µm.