Pseudoloma neurophilia in the Zebrafish (Danio rerio) : Consequences of Infection on Neurobehavioral and Biomedical Research using a Burgeoning Model Organism Public Deposited


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  • Since its inception as a laboratory animal in the early 1970s, the zebrafish has proven itself a rising star in the world of comparative biomedical sciences due to its short generation time, ease of care, external fertilization, and transparent larvae. In a very few decades, the zebrafish has been utilized as a model organism for as many experimental topics and modalities as its rodent counterparts, which have been in use for centuries. Whereas the rodent world has had time to develop biosecurity protocols and specific pathogen free lines, the zebrafish community has had little time to cleanse the animals of contaminating infectious organisms from their tenure as ornamental fish in the pet trade. Initially, because they were used primarily for developmental genetics studies, the need for biosecurity and elimination of infectious disease was not viewed as particularly important. However, as zebrafish have been used as model organisms in sensitive studies of biomedical and neurobehavioral phenomena, the scientific community must evaluate the potential for subclinical infections to alter experimental data. Pseudoloma neurophilia, a microsporidian endoparasite, is currently one of the most commonly diagnosed infections in zebrafish facilities worldwide. Because of its tropism for the central nervous system, and because of the opportunistic nature of microsporidia, it is vital to understand the potential effects that infection can have on behavior and studies involving immunosuppression. Armed with this knowledge, researchers will be able to make informed decisions regarding biosecurity and husbandry protocols in order to mitigate the effects of infection-associated, non-protocol induced variation. The hypothesis of this dissertation is that infection of zebrafish by Pseudoloma neurophilia causes non-protocol induced variation in neurobehavioral and biomedical experiments. In order to explore experimental protocols most likely to be influenced by P. neurophilia, I first performed a retrospective study with the intention of identifying the most common features of neuronal and muscular infections. Five hundred fifty-nine zebrafish infected with P. neurophilia submitted to ZIRC (Zebrafish International Resource Center, Eugene, OR) from 86 laboratories between the years 2000 and 2013 were examined via histopathology. Parasite clusters (PCs) occurred in distinct axonal swellings, frequently with no associated inflammation. Inflammation was observed in viable cell bodies distant from PCs. Multiple PCs occasionally occurred within a single axon, suggesting axonal transport. PCs occurred most frequently in the spinal cord ventral white matter (40.3% of all PCs) and the spinal nerve roots (25.6%). Within the rhombencephalon, PCs were most common in the primary descending white matter tracts. Within the rhombencephalon gray matter, PCs occurred most frequently in the reticular formation and the griseum centrale (61% and 39%, respectively). High numbers of PCs within brain and spinal cord structures mediating startle responses and anxiety suggest that related behaviors could be altered by neural microsporidiosis. Infection could, therefore, introduce unacceptable variation in studies utilizing these behaviors. I chose a commonly utilized neurobehavioral testing protocol that involved motor activity and anxiety-associated responses since it appeared to be the most likely experimental protocol to be influenced by P. neurophilia infection: The progressive tap test for startle response habituation. Fish infected via cohabitation were tested for startle response habituation in parallel with controls in a device that administered ten taps over 10 min along with taps at 18 and 60 min to evaluate habituation extinction. After testing, fish were euthanized and evaluated for infection via histopathology. Infected fish had a significantly smaller reduction in startle velocity during habituation compared to uninfected tankmates and controls. Habituation was eliminated in infected and control fish at 18 min, whereas exposed negative fish retained partial habituation at 18 min. Infection was also associated with enhanced capture evasion: Despite the absence of external symptoms, infected fish tended to be caught later than uninfected fish netted from the same tank. The combination of decreased overall habituation, early extinction of habituation compared to uninfected cohorts, and enhanced netting evasion indicates that P. neurophilia infection is associated with a behavioral phenotype distinct from that of controls and uninfected cohorts. In order to demonstrate a causative link between infection and behavior change, and to evaluate another common neurobehavioral experimental protocol, we performed a shoaling test, which examines social behavior, before and after infection. Tanks containing 10 fish each were divided into 6 control and 6 experimental shoals and recorded prior to exposure. Over 123 days, control fish were exposed to water housing uninfected fish and experimental fish were exposed to water housing infected fish. Shoals were re-recorded following exposure and infection status was determined via histopathology. There were no significant differences in mean interfish distance and percent of top-dwelling fish between control and experimental shoals prior to exposure. Following the exposure period, shoals exposed to and infected by P. neurophilia showed a significantly reduced mean interfish distance compared to controls. The percentage of top-dwelling fish was also reduced in infected shoals, although this difference was not statistically significant. This study supports the fact that P. neurophilia infection causes altered behavior in zebrafish and it should act as a warning to neurobehavioral researchers to use parasite-free fish in their research. Because of the opportunistic nature of microsporidial infections as demonstrated by fatal Enterocytozoon bieneusii and Encephalitozoon intestinalis infections in human AIDS patients, we decided to evaluate the effects of immunosuppressive gamma irradiation protocols on zebrafish infected with P. neurophilia. In this study we exposed zebrafish to combinations of P. neurophilia infection and gamma irradiation in order to explore the interaction between this immunosuppressive experimental modality and a normally subclinical infection. Zebrafish infected with P. neurophilia and exposed to gamma irradiation exhibited higher mortality, increased parasite loads, and increased incidences of myositis and extraneural parasite infections than fish exposed either to P. neurophilia or gamma irradiation alone.
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