Controlling Important Pathogens in Zebrafish (Danio rerio) : Assessing Cryopreservation Survival of Bacteria and Parasites and Clinical Sensitivity of Mycobacterial qPCR Assays Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/vq27zt37x

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  • Zebrafish (Danio rerio) are one of the most commonly used animal models in biomedical research. Zebrafish resource facilities, like the Zebrafish International Resource Center (ZIRC) in Eugene, Oregon, are the main providers and keepers of numerous zebrafish wild-type, mutant, and transgenic lines. Although ZIRC maintains live zebrafish at various life stages, sperm cryopreservation allows them to maintain the vast array of zebrafish lines that they receive from outside facilities. Hence, there is a concern about the potential of vertical transmission of pathogens capable of surviving the freezing and thawing process.My first study was to determine whether zebrafish pathogens are capable of surviving the sperm cryopreservation process used by ZIRC (i.e., the ZIRC method). I assessed the survival of two strains of Mycobacterium chelonae (H1E1 and H1E2), one strain of Mycobacterium marinum (OSU 214), one strain of Edwardsiella ictaluri, Pseudocapillaria tomentosa eggs and Pseudoloma neurophilia spores, which are allpathogens of concern in zebrafish research facilities. These pathogens were also frozen and thawed without cryopreservant, and the pathogens were frozen at either -80oC or - 20oC with only a small amount of Phosphate Buffered Saline (PBS).Each bacterial species survived both freezing and thawing methods, however the samples subjected to the ZIRC method had the higher percentages of bacterial survival compared to the freezing without cryopreservant samples. The mycobacteria had higher survival rates compared to Gram-negative E. ictaluri in both freezing methods. E. ictaluri exhibited a 1-2 log decrease in concentration following the freezing without cryopreservant.For the P. tomentosa eggs, survival was based on larvation. Eggs were examined at Day 0 (immediately after collecting or thawing) and at Day 7 (a week after being collected or thawed). No larvation was observed on Day 7 with eggs processed by the ZIRC method or simple freezing (-80oC in 1X PBS and no cryopreservant). In contrast, the positive controls, kept at 28oC, showed 80-93% larvation at Day 7. Most of the eggs observed in either freezing method were unlarvated and intact, however some, exhibited signs of internal deformation of the egg contents.In 2014, our lab conducted a similar cryopreservation study on the ZIRC cryopreservation method in place at that time. In that study P. neurophilia spores were tested for their ability to survive cryopreservation. I repeated this study in 2017 using the 2017 ZIRC cryopreservation protocol. In both experiments, two fluorescent stains, SYTOX and Fungi-Fluor, and presence of a spore vacuole were used to determine spore viability. SYTOX green is a fluorescent nucleic acid stain, and cells are scored as dead when the dye enters the cells and results in green fluorescence. P. neurophilia spores alsocontain a long, coiled, polar filament or tube that is thought to aid in infecting hosts cells when extruded. Spores are scored as alive if they are stained with Fungi-Fluor, exposed to ultra violet light, and then expel their polar tubes. Presence of a vacuole observed by light microscopy indicated that spores were alive.The 2014 and 2017 experiments yield very similar results, and some spores were able to survive the ZIRC cryopreservation method. Spore survival varied depending on the fluorescent stain used. Samples stained with SYTOX yielded higher percentages of survival than those stained with Fungi-Fluor or quantified using vacuole presence. Nevertheless, in both the 2014 and 2017 experiments, about 10% of the spores were scored as alive using the more conservative Fungi-Fluor and vacuole presence tests following the ZIRC cryopreservation method. Very few spores were scored as alive following freezing without cryopreservant with any method.The second study I conducted entailed working with the Oregon State University Veterinary Diagnostics Laboratory (OSU VDL) to evaluate the clinical sensitivity of their real-time quantitative PCR (qPCR) assays for three Mycobacterium spp: M. chelonae, M. marinum, and M. haemophilum. To test the clinical sensitivity of these assays, we spiked actual zebrafish tissue samples with known concentrations of diluted bacterial samples and determined the lowest detectable bacterial concentration. For this study M. chelonae (H1E2) and M. marinum (OSU 214) were diluted and spiked into minced zebrafish tissue. These samples were then assayed using qPCR. M. haemophilum was not used in this study due to difficulties with culturing. For M. chelonae, 61,000 colony forming units (CFUs) mL and 437 CFUs per PCR reaction was the lowest detectableconcentration. On the other hand, 3,700 CFU mL and 27 CFUs per PCR reaction was the lowest detectable concentration for M. marinum.In this thesis research, I showed that M. chelonae, M. marinum, E. ictaluri, and P. neurophilia spores can survive the ZIRC cryopreservation method and in some cases freezing without a cryopreservant, but P. tomentosa eggs did not survive freezing. Given these results, I recommend that zebrafish and fish facilities that implement sperm cryopreservation consider testing sperm samples prior to freezing them or using them for in vitro fertilization. We also determined the clinical sensitivity of the Mycobacterium qPCR assay used by the OSU VDL. Although this assay can identify Mycobacterium species in fish tissue, specifically M. chelonae and M. marinum, it showed rather moderate sensitivity. I therefore recommend these tests for species identification of mycobacteria in fish in which mycobacteria are first detected by other methods (e.g., acid fast staining) rather than for screening zebrafish for the presence of bacteria in fish with no other indications of infection.
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