Evidence for oral ingestion as the principal route ofantigen entry in bath-immunized fish

This study re-examines the route of antigen entry in bath-immunized fish by making use of a 150 kDa toxin molecule (from Clostridium botulinum type E) which acted as both prototype antigen and marker for its own entry, through its neurotoxic effects. Initially, toxin uptake was established as a function of dose by seeding serial toxin dilutions into small aquaria containing 4–10 cm goldfish (Carassius auratus). The possibility that gills or lateral lines were principal toxin entry points was ruled out by plugging fish oesophagi with a rubber-like, dental-impression compound. Toxin uptake was reduced nearly sixfold by this procedure. Water ingestion rates were measured by exposing groups of 8–10 cm fish for varying time intervals to a constant flow of water containing 0·2% toxic supernatant from a botulinum culture. The potency of this supernatant was determined simultaneously in additional fish by intragastric inoculation. The measured ingestion rate in 8–10 cm fish was at least 0·37 ml h−1 (with an additional 0·18 ml h−1 entering fish either by other routes or leaking past the oesophageal plugs). Therefore, if goldfish were immersed for 2 min in a bath containing 2 × 1010 bacteria ml−1, their gastrointestinal tract would accumulate at least 2·5 × 108 bacteria.