Abstract
Botulinum neurotoxins (BoNT) are the most potent known toxins. The mouse LD50 assay is the gold standard for testing BoNT potency, but is not sensitive enough to detect the extremely low levels of neurotoxin that may be present in the serum of sensitive animal species that are showing the effects of BoNT toxicity, such as channel catfish affected by visceral toxicosis of catfish. Since zebrafish are an important animal model for diverse biomedical and basic research, they are readily available and have defined genetic lines that facilitate reproducibility. This makes them attractive for use as an alternative bioassay organism. The utility of zebrafish as a bioassay model organism for BoNT was investigated. The 96 h median immobilizing doses of BoNT/A, BoNT/C, BoNT/E, and BoNT/F for adult male Tübingen strain zebrafish (0.32 g mean weight) at 25 °C were 16.31, 124.6, 4.7, and 0.61 picograms (pg)/fish, respectively. These findings support the use of the zebrafish-based bioassays for evaluating the presence of BoNT/A, BoNT/E, and BoNT/F. Evaluating the basis of the relatively high resistance of zebrafish to BoNT/C and the extreme sensitivity to BoNT/F may reveal unique functional patterns to the action of these neurotoxins.
Highlights
Clostridium botulinum is a Gram-positive, spore-forming, anaerobic, and rod-shaped bacterium that produces extremely potent neurotoxins
100% immobilization was observed at the highest dose
Our experiments showed similar behavior in zebrafish; the fish injected with Botulinum neurotoxins (BoNT)/F in lower doses such as 0.61 and 0.3 pg appeared to have quicker recovery compared to Botulinum neurotoxin A (BoNT/A)- and BoNT/E-injected fish
Summary
Clostridium botulinum is a Gram-positive, spore-forming, anaerobic, and rod-shaped bacterium that produces extremely potent neurotoxins Their ability to effect neuromuscular paralysis has made them useful for treatment of muscle hyperactivity, blepharospasm, strabismus, and cosmetic defects [1]. Mouse bioassays are traditionally used to test the activity or concentration of BoNTs. There are several other diagnostic methods available to detect the causative agents of botulism outbreaks, such as polymerase chain reaction (PCR) for C. botulinum, anaerobic bacterial culture, and the enzyme-linked immunosorbent assay (ELISA). There are several other diagnostic methods available to detect the causative agents of botulism outbreaks, such as polymerase chain reaction (PCR) for C. botulinum, anaerobic bacterial culture, and the enzyme-linked immunosorbent assay (ELISA) These assays, including the mouse bioassay, failed to detect BoNT/E in sera of catfish affected with visceral toxicosis of catfish (VTC) [3]. Possible reasons these assays did not work include the presence of the toxin in the affected fish in the absence of the C. botulinum organism (in the case of the PCR assay), the Toxins 2016, 8, 132; doi:10.3390/toxins8050132 www.mdpi.com/journal/toxins
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