Abstract
Immunoassays are among the frontline methods used for disease diagnosis and surveillance. Despite this, there are no immunoassays developed for the Chinese soft-shelled turtle (Pelodiscus sinensis), which has expanded into large scale commercial production in several Asian countries. One of the critical factors delaying the development of immunoassays is the lack of characterized soft-shelled turtle immunoglobulins. Herein, we used mass spectrometry together with the ProtQuest software to identify the soft-shelled turtle IgM heavy chain in serum, which again was used to produce a polyclonal anti-turtle-IgM in rabbits. Thereafter, the polyclonal anti-turtle-IgM was used as a secondary antibody in an indirect ELISA to evaluate antibody responses of soft-shelled turtles injected with the bovine serum albumin (BSA) model antigen. Our findings show that only turtle immunized with a water-in-oil BSA plus ISA 763A VG adjuvant (SEPPIC, France) emulsion had antibodies detected at 42 days post vaccination (dpv) while turtles injected with phosphate buffered saline (PBS) only as well as turtle injected with BSA dissolved in PBS had no significant antibody levels detected in serum throughout the study period. In summary, our findings show that rabbit polyclonal anti-turtle-IgM produced can be used in ELISA to measure serum antibody responses in immunized soft-shelled turtles. Future studies should explore its application in other immunoassays needed for the disease diagnosis and vaccine development for soft-shelled turtles.
Highlights
The Chinese soft-shelled turtle (Pelodiscus sinensis), formerly known as Trionyx sinensis, is widely cultured in China, Japan and Vietnam and more recently it has expanded to Korea, Malaysia, Singapore, Thailand, and Philippines [1]
We have identified the soft‐shelled turtle immunoglobulin M (IgM) heavy chain protein by mass spectrometry and excised it to produce a rabbit polyclonal anti‐IgM antibody for use in ELISA
Grey [22] showed that painted turtles (Chrysemus pincta) injected with 25 mg bovine serum albumin (BSA) reared at 22 to 25 ◦ C had antibodies detected at six weeks post vaccination in the group injected with BSA plus adjuvant, which is in line with our findings that the soft-shelled turtle cultured at 25 ◦ C in the present study developed antibodies in the BSA-adjuvant group being the same temperature range (22 to 25 ◦ C) and duration of six weeks post vaccination for antibodies increase in both Grey’s [22] and the present study
Summary
The Chinese soft-shelled turtle (Pelodiscus sinensis), formerly known as Trionyx sinensis, is widely cultured in China, Japan and Vietnam and more recently it has expanded to Korea, Malaysia, Singapore, Thailand, and Philippines [1]. Its production has increased exponentially from about 5000 tons in 1994 to more than. 300,000 tons in 2016 because of its high reproductive potential, pharmaceutical value, wide consumer acceptance and high demand as food for human consumption [3]. Expansion has been hampered from increasing disease problems. Bacterial species identified to cause diseases in soft-shelled turtles include. Viruses identified to cause diseases in soft shelled turtles include the Soft-shelled turtle iridovirus (STIV) [10], Trionyx sinensis hemorrhagic syndrome virus (TSHSV) [11] and turtle
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