Oral immunization of the African clawed frog (Xenopus laevis) upregulates the mucosal immunoglobulin IgX

Abstract

The frog Xenopus laevis is a model species for developmental biology but is also of significant interest to comparative immunologists. Amphibians are the oldest group of organisms in which both the B lymphocytes of some species undergo immunoglobulin (Ig) class switch recombination and also have a dedicated mucosal Ig isotype. The purpose of this study was to test the hypothesis that frog IgX would be produced in response to oral immunization. In order to facilitate studies of humoral, and especially mucosal immunity, in this model species, we developed a gavage technique for oral immunization. The result of this oral administration of antigen to frogs was assayed by the induction of the mucosal antibody isotype, IgX, in plasma by enzyme linked immunosorbant assay (ELISA), and a significant IgX upregulation was detected compared to frogs receiving systemic immunization into the coelom. These data are consistent with the view that IgX is the functional analog of mammalian IgA and mandate further studies of the relationship between IgX and IgA. Additionally, the gavage technique should be adaptable for functional studies of gut-associated immunology in other small aquatic vertebrates.

Figure 1. Optimized oral gavage in Xenopus laevis

A. Euthanized frog used in determination of best placement of 1.5 inch steel feeding needle. The ball of the needle can be seen through the stomach. Red arrow is pointing to the gavage needle terminus. B. Incomplete anesthesia, improper needle size or material, and failure to properly negotiate pharynx all can result in immunogen leakage and regurgitation, as shown by the yellow inoculant on this frog. C. Demonstration of proper gavage technique. After teasing jaw open at corner of mouth, needle is moved to the rostrum and gently inserted through pharynx and into the stomach until the hilt touches the frog’s mouth.

Figure 2. IgX induction by oral immunization

ELISA for total IgX in frog serum was measured by absorbance at an optical density of 450 nm. Results from several dilutions of frog serum show a titratable effect of the IgX serum concentration in the ELISA. The far right bars are assay controls where PBS instead of frog serum is bound to plate before primary and secondary antibodies. Shaded bars represent orally immunized (p.o.), intracoelomicly immunized (i.c.) or non-immunized control frogs. Asterisks mark statistically significant (p ≤ 0.05) comparisons between points linked by bars.