Iranian Journal of Medical Microbiology

In the summer of 2017, 50 pieces of Koi fish (with an average weight of 25 ± 2 grams and a mean length of 24 ± 3 cm) were transferred from one of the concrete ponds of the Koi Fish Farming and Breeding Center in Shahriar city to the Razef Research Center, Tehran Science and Research Branch, Islamic Azad University, Tehran, Iran. Then, the fish were randomly divided into five tanks (170 L) (10 fish per tank). After a few days after the transfer, all the fish developed similar macroscopic symptoms, including darkening of the skin, abdominal petechiae, skin lesions, corneal opacity and anophthalmia, lethargy, immobility, imba-lance, anorexia, and seating on the floor of the aquar-ium. After observing these clinical signs, we took the necessary measures (including autopsy for obtaining samples from the skin and internal organs for patho-logical and microbial examination). As this study was a case report, seven fish presenting these clinical signs were randomly caught from each tank (a total of 35 fish). After anesthesia by clove powder, sampling was performed to obtain specimens from the skin and internal organs and identify the pathogenic agent. All the histological and microbial examinations were conducted under completely sterile conditions.
Liver, gill, and kidney samples were initially washed with the physiological serum and then completely homogenized using a tissue homogenizer. Afterward, the specimens were cultured on Brain Heart Infusion Agar (BHI Agar) medium and incubated at 28°C for two days. After this step, the grown colonies were re-cultured on BHI Agar medium; the motile, oxidase-positive, and glucose fermenter bacteria suspected as Aeromonas spp., underwent further biochemical and physiological analyses. The confirmatory tests at this step included: H₂S production, urease test, citrate reduction, indole production, nitrate reduction, gro-wth on MacConkey medium, and the fermentation of carbohydrates (glucose, inositol, sucrose, sorbitol, arabinose, and mannitol) (16).
For histological examinations, gill, liver and kidney samples were initially fixed in formalin buffer 10% for 48 hours. Then the fixed samples were placed in para-ffin after dewatering and clearing with xylene. Tissue sections with a diameter of 5 µm were prepared using a microtome device and stained by hematoxylin-Eosin (H&E). The prepared histological slides were examined by optical microscope (Olympus BX51; Olympus, Tokyo, Japan) to evaluate pathological features (17).
The results of the microbial tests showed that 48% of septicemia cases (24 samples) were caused by A. hydrophila (Figure 1). In autopsy specimens, in addi-tion to petechiae and skin darkening and ulcerative lesions, the internal organs were pale, and the liver was enlarged with observable petechiae on its surface (Figure 2). In the microscopic examination of the gills, we observed collapsed and swollen secondary lame-llae, the swelling, and shortening of lamellae and their complete destruction, as well as the edema of gill filaments. In addition, the epithelial detachment was observed along with the detachment of the lamellar epithelium outer layer. Congestion was evident in the primary and secondary lamellae (Figure 3 A and B). In the kidney, the infiltration of inflammatory cells (neut-rophils) was evident in vessels and renal parenchyma (Figure 3C). The most prominent changes in the liver were irregular hepatic fibers, nuclear pyknosis, and cytoplasmic vacuolation. In addition, varying degrees of pancreatic cell degeneration were evident in some parts (Figure 3D).

 
 
Figure 1. The identification of Aeromonas hydrophila bacterium in the studied samples
 
 
Figure 2. The darkening of the skin (A), ulcerative skin lesions (B), petechiae on the skin surface (the white arrow) (C), the enlargement of the liver, pale internal organs, and petechiae on the liver surface (the black arrow) (D).


Figure 3. Gill, kidney, and liver histopathological examination. NO: normal gill blades, Ne: acute necrosis, Ca: gill cartilage, black arrow: the necrosis of epithelial and mucosal cells, white arrow: infiltration of inflammatory cells, arrow tip: the necrosis of hepatocytes, star: pancreatic cell degeneration. A: Gills (40X), B: Gills (400X), C: Kidney, D: Liver, H&E staining. 

The present study identified A. hydrophila bacteria in Koi fish, which is known as one of the most critical lethal pathogens in various carp fish (9). Aeromonas bacteria are a part of the water's natural flora, as well as fish. Still, they can become opportunistic pathogens causing super-acute epidemics (e.g., Aeromonas hydr-ophila pathotype) under certain conditions, including environmental stresses. This event can cause consi-derable casualties in the aquaculture industry (18, 19), often occurring within a few hours of the disease onset (20). In the present study, it can be said that transport stress and reduced immune function of fish could have been contributors to the outbreak. This is in line with the notion that Aeromonas bacteria are opportunistic pathogens that can only cause disease in immunocompromised fish (21). In Iran, several studies have investigated A. hydrophila infection in various fish, including goldfish (22), angelfish (23), and common carp (24).
In the autopsy examination of the obtained samples, wounds and bleeding spots were evident on the body's surface, as well as the abdomen and inter-nal organs. These findings were similar to the symp-toms observed in the study of Stratev et al. in 2015 (11). Many studies have shown that chronic infection with A. hydrophila is associated with ulcerative skin lesions and local bleeding and inflammation (10).
In our study, histological examinations showed multiple tissue damages in the gills, liver, and kidneys. These included necrosis, inflammation, and hemorr-hage in the gills, inflammation in the kidney, necrosis, and nuclear pyknosis in hepatocytes. In several studies examining the histological features of A. hydrophila infection, similar findings have been reported (11, 25-29). In this regard, degenerative histological changes due to A. hydrophila infection have been reported in organs such as the gills, kidneys, and liver (10). Also, in line with the present study in which tissue damages in the gills were prominent, Al Yahya et al. (2018) (10) and Cookiyaei et al. (2012) (30) also reported necrosis in gill blades and hemorrhage in gills due to A. hydrophila infection. One of the significant issues in the histopathological features of A. hydrophila infec-tion is that most tissue damage due to this bacterium has been observed in the liver and kidneys (11). In fact, the liver can be considered the target organ of A. hydrophila (10). In the histological evaluation of the kidney, similar to the study of Sellegounder et al. (2018), infiltration of inflammatory cells was observed (31). In a study considering Nile tilapia, bleeding foci were observed in the kidney due to a bacterial infection (32). In the examination of the liver and pancreas tissues, we noticed vacuolation and nuclear pyknosis, as well as the necrosis of hepatocytes and pancreatic cells, which were similar to the results of other studies (10, 31, 33). In a study, Afifi et al. (2000) attributed these changes to the toxins and extra-cellular compounds released by A. hydrophila, inclu-ding hemolysin, proteases, and elastase, in the liver, leading to severe tissue damage and necrosis (34).

The results of the present study showed that freshwater fish are always prone to Aeromonas spp. infections. According to our research, pathological changes in the gills, liver, and kidneys can be suitable biomarkers for assessing the pathogenicity of A. hydrophila in Koi fish. In addition, Aeromonas species can be a threat to public health, especially for those who are in contact with sick fish. Therefore, the proper management of water quality and food health and the quarantine of new fish can play a significant role in preventing and controlling A. hydrophila infection in fish and avoiding its transmission to humans.

We sincerely thank all the colleagues at Razaf Research Center who assisted us in conducting this research.

Conflicts of Interest

The authors declared no conflicts of interest.