Development of a Multiplex Fluorescence in Situ Hybridization Assay to Identify Coinfections in Young-of-the-Year Smallmouth Bass

Abstract

Histopathological assessments of young-of-the-year (age-0) Smallmouth Bass Micropterus dolomieu in the Susquehanna River drainage identified a high prevalence of the myxozoan Myxobolus inornatus. This myxozoan infects the connective tissue of the muscle below the skin but is sometimes observed in the esophagus and buccal cavity. In some instances, shallow infections cause breaks in the skin, which could increase the chance of opportunistic bacterial infections. Several microbial pathogens, including Flavobacterium columnare, Aeromonas spp., and Largemouth Bass virus, have also been cultured from clinically diseased young of year. A multiplex fluorescence in situ hybridization (FISH) assay was developed to determine potential colocalization of M. inornatus, Flavobacterium spp., and Aeromonas spp. infections. With FISH, 75% of age-0 Smallmouth Bass exhibited M. inornatus infections, 10% had Aeromonas spp. infections, and 5% had Flavobacterium spp. infections, while 3% had coinfections with both bacterial species and M. inornatus. The results of the multiplex FISH assay revealed a low occurrence of coinfections of Flavobacterium spp. and/or Aeromonas spp. with M. inornatus in randomly sampled individuals.

FIGURE 1

Collection sites (green circles) for young‐of‐the‐year Smallmouth Bass within the Susquehanna River drainage in 2014.

FIGURE 2

Cross sections of young‐of‐the‐year Smallmouth Bass (hematoxylin and eosin stain) chosen to analyze with FISH, showing (A) an area of inflammation (oval) that may be associated with M. inornatus and (D) a higher magnification depicting a vessel with red blood cells (solid arrowhead), small mononuclear cells (thin arrowhead), vacuolated fibroblasts in dermis (thin arrow), and hypertrophied epithelial cells (thick arrow) next to an area of necrosis. Panel (B) shows a skin lesion with inflammation (thick arrow) spreading into the subepidermal layers and surrounding muscle and focal areas of erosion (thin arrows), and panel (E) shows a higher magnification depicting necrosis (thin arrow), hypertrophied epithelial cells (thick arrow), and small mononuclear cells (arrowhead). Panel (C) shows multiple Myxobolus inornatus plasmodia (thin arrows), with (F) a higher magnification depicting an erosion of the epidermis (arrowhead), vacuolated fibroblasts in the dermis (thin arrow), and small mononuclear cells (thick arrow) surrounding the plasmodium. The scale bar = 20 μm for panels (A), (B), and (C); the scale bar = 5 μm for panels (D), (E), and (F).

FIGURE 3

Various types of gross lesions observed on young‐of‐the‐year Smallmouth Bass in the Susquehanna River drainage. Panel (A) shows spreading skin erosions with necrotic ulceration exposing the underlying dermis (arrows) and a pale white cyst at the base of the caudal fin (circle) caused by M. inornatus. In panel (B), the top fish displays a more advanced spreading skin ulcer with epidermal, dermal, and muscle necrosis and degeneration and hemorrhaging (bold arrow) and the bottom fish displays focal areas of erosion (thin arrows). Panel (C) shows an early stage spreading lesion (thin arrow) and a reddened area of erosion at the base of caudal fin (bold arrow).

FIGURE 4

Fluorescence in situ hybridization (FISH) of samples from young‐of‐the‐year Smallmouth Bass tissues, showing (A) Myxobolus inornatus plasmodium (arrow) infecting the connective tissue of the muscle (scale bar = 50 μm), (B) Flavobacterium spp. (filamentous bacteria; arrows) in the skin (scale bar = 10 μm), (C) Aeromonas spp. (rod‐shaped bacteria; arrows) in the gut (scale bar = 5 μm), and (D) a coinfection of loose spores of M. inornatus (circles) and Flavobacterium spp. (filamentous bacteria; arrows) infecting the connective tissue and soft cartilage around the bone in the caudal peduncle (scale bar = 10 μm).