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Anisakis haemoglobin is a main antigen inducing strong and prolonged immunoreactions in rats


Anisakis simplex larvae are well known to cause gastrointestinal and allergic manifestations after ingestion of parasitized raw or undercooked seafood. The antibody recognition dynamics against the components of Anisakis larval antigen after primary and re-infection with Anisakis live larvae remain unclear. For this study, immunoblot analyses of serum IgG, IgE, and IgM against Anisakis larval somatic extract were performed in rats that had been orally inoculated with A. simplex live larvae. Multiple antigen fractions were recognized after primary infection. Their reaction was enhanced after re-infection. Antibody recognition was observed for 12 weeks after re-infection. The fraction of approximately 35 kDa contained a main antigen that induced strong and prolonged immunoreactions in IgG and IgE. The antibody reaction to this fraction appeared to be enhanced after inoculation of larval homogenates. This fraction was heat tolerant with boiling for 30 min. The fraction was spotted by immunoblotting after two-dimensional electrophoresis and was identified as Anisakis haemoglobin (Ani s 13) using mass spectrometry analysis. The amino acid sequences of haemoglobin mRNAs from two A. simplex sensu stricto and one Anisakis pegreffii were identified by RACE-PCR. They differed from those of two isolates of Pseudoterranova decipiens and A. pegreffii. Results of this study show that Anisakis haemoglobin, which is known to be a major allergen of A. simplex, induces strong and prolonged immunoreaction in rats. This report is the first to show the amino acid sequence variation of Anisakis haemoglobin mRNA between A. simplex sensu stricto and A. pegreffii.

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This study was supported by a Grant-in-Aid for Scientific Research (C) (26460818) from the Japan Society for the Promotion of Sciences (JSPS).

Author information

Correspondence to Niichiro Abe.

Ethics declarations

All animals were kept in an animal room of the Osaka City Institute of Public Health and Environmental Sciences according to the guidelines of the Experimental Animal Committee of this institution for laboratory animals.

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Figure S1

Immunoblotting with pool sera IgG (A), IgE (B), and IgM (C) from rats orally inoculated with live Anisakis larvae and its homogenates. Lane 1, negative control sera. Lanes 2, 3, and 4 respectively show sera at 2, 6, and 8 weeks after primary inoculation. Lanes 5, 6, 7, 8, 9, and 10 were obtained respectively at 1, 4, 6, 8, 10, and 12 weeks after re-inoculation with live larvae. Lanes 11 and 12 were obtained respectively at 1 and 2 weeks after inoculation with larval homogenates. Approximately 35 kDa, 14 kDa and smeary 130 kDa fractions are indicated respectively by an arrow, arrowhead and dot arrow. Molecular mass markers (10–250 kDa, Precision Plus Protein Standards All Blue; Bio-Rad Laboratories) were incorporated into each electrophoresis run. (PPTX 1920 kb)

Figure S2

Immunoblotting with pool sera IgG for heat-treated somatic extract (lane 1, 30 min in boiling water; lane 2, 90 min at 115 °C; lane 3, 20 min at 121 °C). Pool sera obtained at 2 weeks after inoculation of larval homogenate was used for each experiment. The arrow and arrowhead respectively indicate approximately 35 kDa and 14 kDa fractions. (PPTX 2819 kb)

Figure S3

Two-dimensional electrophoresis (2D) (A) and 2D-immunoblotting (B). 2D-page gel was stained with CBB and visible approximately 35 kDa spot are indicated respectively by the arrow and dashed arrow. The spot indicated by an arrow showed a strong positive reaction in comparison to the spot indicated by the dashed arrow. (PPTX 16888 kb)

Figure S4

Amino acid alignments of the present Anisakis haemoglobin (LC209224-LC209226) with those of P. decipiens (AAA29796) and A. pegreffii (AFY98826). Alignment of sequences was conducted using the MUSCLE program. The amino acid sequences were deduced. The hydrophobic leader portion and the C-terminal tail are boxed in black, whereas hydrophobic hem-binding regions are boxed in red. The two homologous domains in the haemoglobin protein are shown by dashed (domain 1) and dotted (domain 2) lines. The B10 tyrosine, E7 distal glutamine, and F8 proximal histidine, are marked with boxes. The amino acid differences in the epitopes 1, 3 and 4 among A. simplex sibling species are shown by arrows. (PPTX 79 kb)

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Abe, N., Teramoto, I. Anisakis haemoglobin is a main antigen inducing strong and prolonged immunoreactions in rats. Parasitol Res 116, 2035–2039 (2017). https://doi.org/10.1007/s00436-017-5475-1

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  • Anisakis simplex
  • Anisakis haemoglobin
  • Immunoblotting
  • Two-dimensional electrophoresis
  • Mass spectrometry
  • Rat