Proceedings of the Third World Fisheries Congress: Feeding the World with Fish in the Next Millenium—The Balance between Production and Environment

Impacts of the Abiotic and Biotic Factors on the Immune Response of Soft-Shelled Turtle

Xianle Yang, Yanli Zhao


In China, with the expansion of commercially farmed soft-shelled turtle Trionyx sinensis, losses from disease have been significant. To prevent and cure turtle diseases, the use of drugs traditionally is limited (Zhang et al. 1996). However, vaccination has become an effective promising approach (Yang 1990; Yang et al. 1995).

Environmental factors such as water temperature, water quality, and sunshine greatly influence the immune response of fish and other aquatic animals (Sarot and Perlmutter 1976; Yamaguchi et al. 1981; Horne et al. 1982; Bly and Clem 1992; Yang and Zhuo 1997). The immunogen and its dose cannot be neglected because they are essential for provoking the immune system of fish and determining the response level (Manning 1982; Dorson 1984).

The soft-shelled turtle is an aquatic reptile. Although its immune system is more advanced than that of the fish (Borysenko and Cooper 1972; Yang 1990), whether immunogens and the environment have the same influence on turtles as on fish remains to be determined. Furthermore, the effects of in vivo factors such as weight, age, and nutrition work have not been studied.

The present study examines the in vitro and in vivo factors that influence the immune response of soft-shelled turtle. Our results will not only help to achieve the optimum immune regime, but will also enrich the foundational theory of aquatic animal immunology.

Bacterial pathogens (strains T3, 19C-4-2, 142G, SL63, Tio63, and Fii32) were isolated from soft-shelled turtles, which were caught and identified with Aeromonas hydrophila (isolated from our laboratory; identification and characteristics are reported elsewhere). Pathogen samples were cultured for 24–48 h in a shaker (1,500 rpm), inactivated with 0.4% of formalin for 48 h at 28°C, then examined for safety by measuring the remaining virulence of the strain to make bacterial vaccine. They were stored at 4°C until use.

Healthy turtles were inoculated with 0.3 mL of the bacterin by intramuscular injection in a hind leg. Except for the test of immunogen quality and quantity, bacterin T3 was used and the dose was 60 × 108 colony-forming units (CFU)/mL. At 30 d postvaccination, some of the animals were cut in the neck and blood sampled to assay the serum indirect agglutination titer (IAT) by erythrocyte indirect agglutination (indicated by the reciprocal value of highest antiserum dilution that made the sensitized erythrocyte agglutination greater than 2+). The others were challenged by infection with the bacterial strain corresponding to the immunogen (3 × 108 CFU/mL) to calculate the percent relative protection (PRP).

Several groups of turtles were vaccinated with bacterin T3, SL63, 19C-4-2, 142G, and T3 in different doses to study the different immunogenicity and the influence of differently dosed immunogens (set up corresponding to control groups at the same time).

The tests were designed with different conditions for water temperature, water quality (well-aerated clean water and contaminated inferior water), and illumination (enough sunshine or no sunshine). The vaccinated groups were injected with bacterin T3, and the corresponding control groups were injected with an equal volume of phosphate-buffered saline (PBS).