Effect of formalin-fixation and paraffin-embedded tissue storage times on RNAscope in situ hybridization signal amplification.

RNAscope in situ hybridization (ISH) detects target RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. Protocols suggest that prolonged FFPE storage and formalin fixation may impact signal detection, potentially limiting the utility of RNAscope ISH in retrospective studies. To develop parameters for RNAscope use with archived specimens, we evaluated the effect of formalin-fixation time by measuring the signal of a reference gene (16srRNA) in selected tissues fixed in 10% neutral-buffered formalin for 1, 2, 3, 5, 7, 10, 14, 21, 28, 60, 90, 180, and 270 d. The signal intensity and percent area of signal decreased after 180 d. Tissues had detectable signal at 180 d but not at 270 d of formalin fixation. To assess target detection in paraffin blocks, we qualitatively compared the signal of canine distemper virus (CDV) antigen via immunohistochemistry and CDV RNA via RNAscope ISH in replicate sections from blocks stored at room temperature for 6 mo, 1, 3, 6, 8, 11, 13, and 15 y; RNA was detected in FFPE tissues stored for up to 15 y. Our results demonstrate that RNAscope ISH can detect targets in tissues with prolonged paraffin storage intervals and formalin-fixation times.

Parasitism and branchitis in various fish species from 4 Cook County, Illinois inland freshwater lakes, 5-year survey, 2017-2021.

We investigated disease trends of concern for fish or public health in a 5-y (2017-2021) prospective survey of fish in Cook County, IL, inland lakes. Lesions were assessed in relation to fish species, lake type and location, season and collection year, and lake water chemistry parameters. Fish included bullheads (n = 98), common carp (n = 99), bluegill (n = 114), and largemouth bass (n = 118). Annually, fish collection and point-source water sampling were conducted in spring, summer, and fall from both seepage and impoundment lakes. Examinations included autopsy, wet-mount cytologic assessment for ectoparasites, and histopathology. No lesions of public health concern were detected. The most common abnormalities were branchitis (261 of 429; 60.8%) and endoparasitism (312 of 429; 72.7%). Branchitis was mild in most cases (189 of 261; 72.4%) and concurrent with branchial parasitism in 175 of 261 (67%) cases. Monogeneans were the most common gill parasites but did not influence branchitis severity (Kruskal-Wallis, p = 0.484). Using generalized ordered logistic regression, predictors of branchitis severity included fish species (p < 0.001), the interaction of lake or location and alkalinity (p < 0.001), and water temperature or season (p < 0.001). Endoparasites included tissue larval trematodes (metacercaria), nematodes, and cestodes (plerocercoids), enteric acanthocephalans, gastric trematodes, renal myxosporidia, biliary and gall bladder myxosporidia, enteric cestodes, and tissue microsporidia. Using generalized ordered logistic regression, variables influencing endoparasitism severity included species (p < 0.001), year (p < 0.001), chloride level (p = 0.009), and the interaction of year and chloride level (p < 0.001). Our results suggested overall good health of fish in sampled Cook County inland lakes and provide a foundation for continued monitoring of ecosystem and public health in the urban environment.

Pathologic impacts of contaminants in freshwater fish of Cook County IL.

Mercury (Hg) and polychlorinated biphenyls (PCBs) are widespread environmental toxicants in urban environments with negative impacts to fish health. The present study evaluated the potential association between muscle tissue contaminant (total Hg and total PCB) concentrations and indicators of health in benthic and predatory fish collected from four Forest Preserves of Cook County lakes in the Chicago metropolitan area. Common carp (carp; Cyprinus carpio) and largemouth bass (LMB; Micropterus salmoides) were sampled three times a year (spring, summer, fall) during 2019 and 2020. Water quality analyses (temperature, color, turbidity, dissolved oxygen, total alkalinity, pH, chloride, nitrate, phosphate, ammonium, and pH) were performed concurrently with fish collections. Tissue (skin-on fillet) contaminant concentrations were compared between lake types and fish species and assessed for any relationship with fish morphometric data and pathologic lesions. Main health indicator endpoints included muscle lipid content, parasite burden, and pathologic lesions. Mean total PCB concentrations were greater in carp (203.1 ± 152 µg/kg, wet weight), and mean Hg concentrations were greater in LMB (0.11 ± 0.1 mg/kg, wet weight). In most fish, concentrations of both toxicants surpassed the EPA's lowest threshold to restrict fish consumption for sensitive cohorts (0.029 mg/kg for Hg and 1.5 µg/kg for PCBs). In both species, Hg positively correlated with splenic pigmented macrophage aggregate area (P < 0.001). In carp, Hg also positively correlated with hepatocellular pigmentation (P < 0.01). Mercury correlated with standard length in both species (LMB: P < 0.001, carp: P = 0.95), but polychlorinated biphenyls only correlated with standard length in carp (P < 0.001). No association was found between intraspecific contaminant concentrations and parasite burden, year, or lake type, though differences were noted among individual lakes. The contaminant burden appeared well-tolerated with only mild Hg-associated and no appreciable PCB-associated lesions. However, possible effects on reproduction or behavior were not fully assessed, and future studies are warranted.