Survival of Bacterial and Parasitic Pathogens from Zebrafish (Danio rerio) After Cryopreservation and Thawing

Abstract

Cryopreservation is a common method used to preserve the sperm of various animal species, and it is widely used with zebrafish (Danio rerio). As with other animals, there is a possibility of paternal pathogen transmission through sperm. We evaluated the ability of five common and important pathogens of zebrafish to survive cryopreservation as used with zebrafish sperm and freezing without cryopreservant. We evaluated Mycobacterium chelonae, Mycobacterium marinum, and Edwardsiella ictaluri, each originally isolated from zebrafish, eggs of Pseuodocapillaria tomentosa, and spores of Pseudoloma neurophilia. Each mycobacterial isolate showed relatively minimal reduction in survival after freezing and thawing, particularly when subjected to cryopreservation. E. ictaluri also showed survival after cryopreservation, but exhibited a several log reduction after freezing at -80°C without cryopreservant. With P. neurophilia, two separate experiments conducted 3 years apart yielded very similar results, showing some, but reduced, survival of spores by using three different viability assays: SYTOX stain, Fungi-Fluor stain, and presence of a spore vacuole. Eggs of P. tomentosa showed no survival based on larvation of eggs when subjected to either freezing method. Given that four of the five pathogens exhibited survival after cryopreservation, we recommend that sperm samples or donor male zebrafish fish be tested for pathogens when sperm are to be stored by using cryopreservation.

Rederivation of a mutant line (prop 1) of zebrafish Danio rerio infected with Pseudoloma neurophilia using in vitro fertilization with eggs from pathogen‐free wild‐type (AB) females and sperm from prop 1 males

“…neurophilia has been detected in testis and sperm (Murray et al., 2011). The spores can also survive cryopreservation as used with zebrafish sperm (Norris et al., 2018). We further confirmed maternal transmission by observing the infection derived from infected prop 1 females.…”

Review of diseases and health management in zebrafish Danio rerio (Hamilton 1822) in research facilities

“…Eggs in the environment are killed by high levels of chlorine, iodine, UV, desiccation and heat (Kent, Watral, Villegas, & Gaulke, 2019). Eggs of P. tomentosa do not survive freezing, neither by cryopreservation typically used for preserving zebrafish sperm nor by freezing without cryopreservant (Norris, Watral, & Kent, 2018).…”