Graduate Thesis Or Dissertation
 

Expanding Diagnostic Assays for Pseudoloma neurophilia & Description of the Progression of Infection in Adult Zebrafish Populations

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/9w0329983

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  • Pseudoloma neurophilia is the most common pathogen reported in zebrafish (Danio rerio) research facilities and is an important threat to the zebrafish model. This microsporidian parasite can cause clinical disease, but more importantly is a causative agent of non-protocol induced variation in research. Studies utilizing infected zebrafish, could potentially confound their research findings, as this parasite can cause clinical disease and significant changes to the behavior, immune system, fecundity, and overall physiology of the fish. Therefore, there is growing investment in diagnostics for zebrafish research facilities, to ensure embryos and fish are specific-pathogen free. Assays for prevalent zebrafish pathogens are continually expanding, however for P. neurophilia, the only diagnostic methods available are through lethal sampling, often requiring euthanasia of a large portion of a given population for accurate estimates of prevalence. Thus, I optimized nonlethal assays to detect Pseudoloma neurophilia in tank water adapting a previously developed qPCR assay that was adapted to the digital PCR (dPCR) platform. I coupled the assay with the use of sonication to improve spore disruption in water samples, resulting in an eDNA assay with a detection limit in water lower than all previously reported assays (77.5 spores/ L). Prevalence data and occupancy modeling supported our observations, revealing that samples collected in static conditions were more informative for diagnostics than samples from flow-through conditions, with prevalence of positive samples at 80% or higher compared to 47%, respectively. Utilizing the array of diagnostics assays available for the detection of P. neurophilia (histology, whole body analysis by qPCR, and eDNA analysis of tank water), I then compare the application of these assays and tracked the progression of infection over a 10-month period. I found that detection of the parasite occurs as early as 4 days post exposure (dpe) by whole-body qPCR, while detection by histological analysis did not occur until 92 dpe. Moreover, I highlight that there are positive associations with the days post exposure and the sensitivity of the assays, as PCR is much more sensitive than histology, however the probability of detection for both assays, increases over time. I then investigated modifying our eDNA assay, by employing the use of different equipment, namely an upgrade to the sonication equipment and a different PCR machine, to detect the parasite more reliably in the water. Thus, I also describe a modified protocol for extraction and quantification of parasite DNA from the environment for nonlethal diagnosis of P. neurophilia in adult zebrafish populations, which has a detection limit of 4.5 spores/L. Using this modified eDNA assay, I evaluated previously collected water samples from the 10-mon longitudinal analysis, targeting times near initial infection. The parasite was detectable in the water after initial exposure until week 4 post exposure (pe), when the parasite was undetectable until 7 weeks pe. After that time, the parasite was sporadically detected in the water for the 10-month study, highlighting the important dynamics of infection and subsequently, our ability to detect the parasite in the environment. I showed that the parasite is frequently in the environment in low concentrations (presumably as spores), until infections become prominent in fish, and are likely only shed in high concentrations during spawning and chronic states of infection. Thus, I elucidated the mystery of early infection and progression of infection within a population, enhancing our knowledge about the dynamics of infection. Using water samples from the Zebrafish International Resource Center, Eugene Oregon, I also validated the clinical relevance on the assay, revealing an association, again, with the age of the fish and the amount of parasite in the water. The integration of this assay will significantly compliment surveillance and control efforts in zebrafish research facilities. Coupling all diagnostics assays will aid in control efforts to eradicate the pathogen from zebrafish research facilities.
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  • Pending Publication
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  • 2021-12-30 to 2022-04-02

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