Interactions of highly and low virulent Flavobacterium columnare isolates with gill tissue in carp and rainbow trout

Abstract

The interactions of Flavobacterium columnare isolates of different virulence with the gills of carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum) were investigated. Both fish species were exposed to different high (HV) or low virulence (LV) isolates and sacrificed at seven predetermined times post-challenge. Histopathological and ultrastructural examination of carp and rainbow trout inoculated with the HV-isolate disclosed bacterial invasion and concomitant destruction of the gill tissue, gradually spreading from the filament tips towards the base, with outer membrane vesicles surrounding most bacterial cells. In carp, 5-10% of the fish inoculated with the LV-isolate became moribund and their gill tissue displayed the same features as described for the HV-isolate, albeit to a lesser degree. The bacterial numbers retrieved from the gill tissue were significantly higher for HV- compared to LV-isolate challenged carp and rainbow trout. TUNEL-stained and caspase-3-immunostained gill sections demonstrated significantly higher apoptotic cell counts in carp and rainbow trout challenged with the HV-isolate compared to control animals. Periodic acid-Schiff/alcian blue staining demonstrated a significantly higher total gill goblet cell count for HV- and LV-isolate challenged compared to control carp. Moreover, bacterial clusters were embedded in a neutral matrix while being encased by acid mucins, resembling biofilm formation. Eosinophilic granular cell counts were significantly higher in the HV-isolate compared to LV-isolate inoculated and control carp. The present data indicate a high colonization capacity, and the destructive and apoptotic-promoting features of the HV-isolate, and point towards important dynamic host mucin-F. columnare interactions warranting further research.

Figure 1

Gill section of a carp challenged with the HV-isolate at SP 2. The bacterial cells (arrows) are clustered focally in an eosinophilic matrix encompassing the tips of the gill filaments (F) and extending to the middle of the gill filaments. L = gill lamella (H&E, bar = 200 μm).

Figure 2

Gill section of a carp challenged with the HV-isolate at SP 2. The bacterial cells are surrounded by a PAS-positive matrix (thin arrows, magenta) enveloped by AB-positive mucins (thick arrow, blue). F = gill filament; L = gill lamella (PAS/AB, bar = 75 μm).

Figure 3

Carp gill after inoculation with HV-isolate at SP 2. The gill filament (F) and gill lamellae (L) are covered by long, slender bacterial cells (B), clustering in between mucus (M) and cell debris (SEM, bar = 5 μm).

Figure 4

Carp gill after inoculation with the HV-isolate at SP 3. Bacterial cells present a knurled outer membrane (long arrows), and are regularly surrounded by outer membrane vesicles (short arrows) (TEM, bar = 400 nm).

Figure 5

Gill section of a carp challenged with the HV-isolate at SP 7. Large clumps of bacterial micro-colonies (thick arrows) embedded in an eosinophilic matrix are discerned. Areas of complete architectural loss (asterisk) are noted in close proximity of bacterial clumps. Overall oedema (thin arrows) and total lamellar (L) and filament (F) fusion are apparent (H&E, bar = 50 μm).

Figure 6

Gill of a carp inoculated with the LV-isolate at SP 2. A cluster of bacterial cells (long arrows) is separated from the lamellar epithelium (L) (short arrows) by a translucent layer (asterisk) (TEM, bar = 1.5 μm).

Figure 7

Prevalence (in%) of PAS-positive (A), AB-positive (B) and EG (C) cells in carp. The values are calculated per sampling point, for fish belonging to a certain virulence group sampled. Significantly higher odds were found for PAS-positive and AB-positive cells counted in the gills after inoculation with the HV-isolate (blue) compared to the control animals, and for the AB-positive cells in the gill tissue of the LV-isolate (red) challenged fish compared to the control animals (green). Significantly higher odds were encountered for the EGC counted in the gills of fish challenged with the HV-isolate compared to the control fish, and for the HV-isolate compared to the LV-isolate inoculated fish.

Figure 8

Gill bacterial titres (log CFU/g) after challenge with the HV- and LV-isolate in carp. The bacterial titres of fish inoculated with the HV-isolate (blue) remain high during the course of the experiments and show a significant higher difference compared to the fish infected with the LV-isolate (red). The bacterial titres retrieved from the latter carp follow a same trend compared to the HV-isolate and remain high until the end of the experiment.

Figure 9

Gill tissue of a trout following exposure to HV-isolate at SP 1. Localized necrosis (long arrows) is observed in a gill lamella (L) while the neighbouring lamella remains intact (short arrow), apart from slight oedema (white arrows) of the epithelium on the sides where bacteria are present (TEM, bar = 10 μm).

Figure 10

Gill bacterial titres (log CFU/g) after challenge with the HV- and LV-isolate in trout. The bacterial titres of fish inoculated with the HV-isolate (blue) remain high during the course of the experiments. The bacterial titres retrieved from fish infected with the LV-isolate (red) show progressively a substantially decreasing trend.