Characterization of Histopathological and Ultrastructural Changes in Channel Catfish Experimentally Infected with Virulent Aeromonas hydrophila

Abstract

A highly virulent clonal population of Aeromonas hydrophila (vAh) has been the cause of recent motile Aeromonas septicemia epizootic in channel catfish (Ictalurus punctatus) farms in the Southeastern United States. The pathology of the disease caused by vAh has not been studied well yet. Thus, our aim was to determine histopathological and ultrastructural changes in channel catfish following vAh challenge. To accomplish this, catfish fingerlings were challenged with vAh (strain ML09-119) by bath. Six fish per each time point were collected at 1, 3, 5, 6, 24, and 48 h for light microscopy, and six fish were collected at 48 h for transmission electron microscopy (TEM). The first pathological lesions were detected in the spleen and stomach at 1 h post-challenge (HPC) while intestine, gills, kidney, and liver lesions were observed at 24 and 48 HPC. Histopathological examination revealed degenerative changes, necrosis, extensive edema, and inflammation in internal organs. The TEM showed severe tissue destruction with multiple bacterial cells secreting outer membrane vesicles, especially in spleen and gills and far number in the stomach. Degenerated bacterial cells were observed in the intestinal lumen and the phagosomes of phagocytic kidney cells. We identified, for the first time, degranulate eosinophilic granular cells, and dendritic cells like (DC-like) cells in the necrotic intestinal epithelium. These findings suggest that vAh rapidly proliferated and spread through the catfish organs following bath challenge.

Keywords: DC-like cells; electron microscopy; eosinophilic granular cells; histopathology; vAh.

FIGURE 1

Photomicrograph of control and infected catfish spleen. (A) Photomicrograph of control spleen showed normal architecture with ellipsoids (E) beside mixed red and white pulp (P). (B) Infected spleen showed mild splenitis with an accumulation of proteinaceous substance around ellipsoid arterioles (arrow) at 5 HPC, 100×. (C) Moderate splenitis with numerous macrophages and lymphocytes aggregated around ellipsoids (arrow) in addition to hemosiderosis (H) at 24 HPC, 400×. (D) Severe splenitis with diffuse fibrinoid necrosis of ellipsoidal sheath (thick arrow) and aggregation of inflammatory cells (thin arrow) at 48 HPC, 400×, H&E.

FIGURE 2

Photomicrograph of control and infected catfish stomach. (A) Control stomach showing its normal organization, mucosal epithelium (ME) with a gastric gland (GG) in its lamina propria, submucosa (SM), muscularis (MM) and serosa (S), 100×, H&E. (B) Infected stomach showed extensive edema (E) and hemorrhage (thick arrow) of submucosa with necrosis of its gastric glands (thin arrow), 100×, H&E.

FIGURE 3

Histopathological changes in the catfish intestine. (A) Photomicrograph of control intestine showed its characteristic layers: mucosal epithelium (ME), submucosa (SM), muscularis (MM), and serosa (S). (B,C) Intestine of infected catfish showed epithelial necrosis with sloughed necrotic debris in its lumen (arrows) 100×, 400×. (D) Multifocal aggregation of bacteria in the intestinal lumen (B), 200×, Giemsa stain.

FIGURE 4

Photomicrograph of control and infected catfish gills. (A) Control gills showed normal primary lamellae (PL) and secondary lamellae (SL), 200×, H&E. (B) Infected gills showed thickening (thin arrow) and clubbing of the primary and secondary lamellae (thick arrow) with moderate numbers of lymphocytic infiltration (white arrow) at 24 HPC 200×, H&E.

FIGURE 5

Photomicrograph of control and infected catfish posterior kidney. (A) Normal organization of posterior kidney with many renal tubules (arrow) and area of hematopoietic tissue in between (H). (B) Infected posterior kidney showing hyaline droplet accumulation in its tubular epithelium (arrow) at 24 HPC 400×, H&E. (C) Diffuse necrosis of renal tubule (thin arrow) beside separation of renal tubular epithelium from its basement membrane with karyolysis of its nucleus (thick arrow) at 48 HPC, 400×, H&E. (D) Severe interstitial nephritis replaced the renal tubules (arrow) at 48 HPC 400×, H&E.

FIGURE 6

Photomicrograph control and infected anterior kidney. (A) Normal arrangement of the anterior kidney with normal hematopoietic aggregation (H). (B) Infected head kidney showing moderate pronephritis (left arrow) with some cells appear necrotic (arrowhead) and others showed mitotic figures (M) at 48 HPC 400×, H&E.

FIGURE 7

Photomicrograph of control and infected catfish liver. (A) The liver is showing the normal organization of polygonal hepatic cells and blood capillary. (B) Infected liver showed focal area of lymphocytic aggregation (thin arrow) migrate from dilated blood vessels (thick arrow) toward necrotic hepatic cells at 48 HPC, 400×, H&E.

FIGURE 8

Transmission electron microscopy of control and infected spleen at 48 HPC. (A) Normal spleen with normal ellipsoids (E) surrounded by reticular cells (R) and mixed pulp and characterized by the presence of multiple erythrocytes (ER) and lymphocyte (L). (B) Infected spleen with the loss of its cellularity and complete lysis of the remaining cells. The cell nucleus had pyknosis with clumping of the chromatin (arrow) beside extracellular bacteria (B). (C) Outer membrane vesicles linked to the bacterial cell membrane (arrow). (D) Bulges around the bacterial envelope (black arrow) and chain of vesicles released near the site of its origination (white arrow). Bar = 4 μm (A), 1 μm (B), and 200 nm (C,D).

FIGURE 9

Transmission electron microscopy of control and infected catfish stomach at 48 HPC. (A) The normal structure of stomach with columnar epithelial cells (Ep), many granules concentrated on the apical part of epithelial cells (G), normal nucleus (Nu), and lumen contains secretion (Lu). (B) Severe damage to gastric gland with complete loss of its normal structure (arrow) beside neutrophilic infiltrations (N). (C) Extensive edematous fluid widely separated collagen bundles and necrosis of cellular structure of connective tissue (thick arrow) beside bacterial cell (thin arrow). (D) Edematous fluid (arrow) containing leukocyte (Le) accumulated around blood vessels (BV). Bar = 4 μm (A), 2 μm (B,D) and 1 μm (C).

FIGURE 10

Transmission electron microscopy of control and infected catfish intestine at 48 HPC. (A) Intestine of control fish with normal microvilli (MV) tightly packed to enterocyte surface, numerous goblet cells (G) and enterocyte with several spherical mitochondria (M) and normal nucleus scattered inside the enterocyte. (B) Infected intestine with many damaged bacterial cells inside its lumen (arrow). (C) Intestine of infected catfish showed up to four vesicular chains attached to intestinal microvillus with little change in enterocyte structure (arrow). (D) Severe enterocyte damage with the complete deterioration of its microvilli and cytoplasmic organelles beside detached enterocyte filled its lumen (arrow). (E,F) Degranulate eosinophilic granular cells (EGC) invaded the necrotic enterocyte characterized by electron-lucent multivesicular granules accumulated in its peripheral cytoplasm beside few number of electron-dense granules (arrow). (G,H) Dendritic cells like (DC-Like) invaded the intestinal epithelium (arrow) characterized by birbeck like granules (black arrow) associated with electron dense lysosome and phagocytic particles (white arrow). Bar = 2 μm (A,G), 200 nm (B), 1 μm (C–E), 500 nm (F) and 600 nm (H).

FIGURE 11

Transmission electron microscopy of control and infected catfish gills at 48 HPC. (A) Control gills showing normal primary lamellae epithelium with different cell types: mucous cells (M) and pavement cells with micro fridge (arrow). (B) Gills of the infected group showing diffuse divided bacteria (thin arrow) admixed with sloughed necrotic lamellar epithelial lining (thick arrow). (C) Control gills showing cartilaginous portion (Ca) and blood cells (BC) inside the primary lamellar structures. (D) Necrosis of interstitial connective tissue surrounding cartilaginous portion (arrow) beside a bacterial (B) attached to lamellar epithelium completely destructed. (E) Multiple dividing bacteria showing secretion of many OMV chains attached to its envelope (arrow). (F) Bacteria attached to the remnant tissue of gill lamellae with the secretion of OMVs (arrow). Bar = 4 μm (A–D), 1 μm (E), 500 and 200 nm (F).

FIGURE 12

Transmission electron microscopy of control and infected catfish anterior kidney at 48 HPC. (A) The normal structure of anterior kidney with a different type of cells including lymphocyte (Ly), and granulocytic cells (arrow) in the hematopoietic tissue. (B) Infected anterior kidney showing degenerative cells with dilated cytoplasmic organelles (thick arrow) beside hyperplastic cells had mitotic figures (thin arrow). (C) Three necrotic cells with complete lysis of its cytoplasmic organelles and clumped chromatin of its nucleus (thin arrows) beside degenerative granulocytic cells showed mitochondrial dilation (thick arrow). (D) Severe necrosis of multiple granulocytic cell types (arrows) beside infiltrated neutrophil with its characteristic granules inside the cytoplasm (Ne). (E) Anterior kidney phagocytic cells showed disruption of its plasma membrane, cytoplasmic and nuclear structural damage (arrow) beside degenerated bacteria inside its phagosome (B). (F) Phagocytic cell with multiple degenerated bacteria (B) inside its phagosome (arrow) (B). Bar = 4 μm (A), 2 μm (C,D), 1 μm (B,E), and 800 nm (F).