Introduction
Silver rasbora (Rasbora argyrotaenia Bleeker, 1849) is one of the freshwater consumption fish from the Cyprinidae family with highly economical values. The demand for this fish has enormously increased, reaching 80% annually [1]. A bacterial disease that frequently infected Cyprinid fish was motile Aeromonads septicaemia (MAS) caused by Aeromonas hydrophila [2]. Aeromonas hydrophila infection caused high economic losses in fish culture and high fish mortality until 100% in several days [3], including in several cultured fishes in India [4], China [5], and ornamental fish industry in Malaysia [6]. Early detection is needed to confirm fish infected with A. hydrophila, such as the clinical symptoms it causes.
Fish infected with A. hydrophila present several clinical signs [7], including tail rot, hemorrhage in the fin and skin, septicemia, fin erosion, depigmentation in the skin, and exophthalmia [8]. Internally, infected fish show deposition of blood in the abdominal cavity; edema in the swim bladder and renal; and hemorrhage in the liver, renal, and lymph [9]. In addition, some can be detected from histopathology in liver and renal tissues including hemorrhage, atrophy, degeneration, and necrosis [10]. It is caused by extracellular products/toxins secreted by A. hydrophila, such as hemolysin, aerolysin, protease, and lecithinase enzymes [11]. This toxin could disrupt the cell membrane and inactivate the erythrocyte, which was assisted by other enzymes [12].
Kidney and liver tissue could be used as indicators to demonstrate the tissue changes caused by A. hydrophila toxins through histopathology examination. The liver is a metabolism organ that could be a detoxification organ, and the renal is a hemopoetic organ in fish. Therefore, this organ was targeted for A. hydrophila infection [13]. Furthermore, hematology profiles can be used as indicators of infected fish with A. hydrophila [14]. Previous studies reported that experimental infection of silver rasbora with Streptococcus agalactiae affected the hematology profile [15] and pathological changes [16]. Limited information about bacterial infection diseases in silver rasbora mainly caused by A. hydrophila encourages this study to analyze clinicopathological changes and hematology profiles in silver rasbora infected by A. hydrophila. The data obtained were used for the early detection of MAS disease in silver rasbora.
Materials and Methods
Ethical approval
The research was conducted and approved by the examiner committee of the Fisheries and Marine Faculty, Universitas Airlangga (Letter of Assignment from Dean of Fisheries and Marine Faculty, Universitas Airlangga, 53/UN3.1.16/KP/2022). The fish were well-cared for throughout the study. Furthermore, feeding methods and water quality monitoring were done in accordance with Indonesian National Standard (SNI) 7733:2018.
Study period and location
This study was conducted in January–February 2022 in the Teaching Farm, School of Health and Life Sciences, Universitas Airlangga, Indonesia.
Animals
Four hundred silver rasbora (R. argyrotaenia) (6 ± 0.1 cm and 3.6 ± 0.2 gm) from local farmers were immersed in 30 ppm NaCl solution for 5 min and acclimatized for 7 days.
Bacterial preparation
Aeromonas hydrophila was cultured in a selective medium (M-Aeromonas selective medium) (Himedia, India) and biochemically tested, including Gram staining, catalase test, oxidase test, motility test, and oxidative/fermentative test, to confirm the pure isolate bacteria referred to Barrow and Feltham [17]. Aeromonas hydrophila was diluted in NaCl 0.9% solution until the bacterial densities desired were 1010 CFU ml−1 based on McFarland standard number 4 (0.1 × 1010 CFU ml−1), and diluted according to the treatment.
Experimental fish and design
This study was conducted using a complete randomized design with five treatments in quadruplicate. The treatments were an intramuscular injection of 0.1 ml A. hydrophila with densities 104 CFUml−1 (P1), 106 CFUml−1 (P2), 108 CFUml−1 (P3), and 1010 CFUml−1 (P4), respectively, and NaCl 0.9% solution for the negative control (P0). Post infection with A. hydrophila, silver rasbora was reared in a glass aquarium (40 × 30 × 20 cm) with a water volume of 200 l (densities 1 tail l−1) and aerated using an air blower for 48 h (Resun LP 100). During the rearing period, fish were dietary with commercial feed (30% protein content) twice daily, with ad satiation methods. Water quality parameters (temperature, pH, dissolved oxygen, and ammonia) were maintained optimally with syphoning and 50% water replacement daily.
Clinicopathological observation
Fish were maintained for 28 h post-infection (hpi) until the clinicopathological signs included behavioral changes, external lesions, and histopathology changes after infection. The organs for histopathology observation were collected from the liver and kidney and taken at different periods consisting of 3 (P4), 9 (P3), 20 (P2), and 28 hpi (P1 and P0). The sample organs were obtained in sterile glass bottles, fixed in 10% neutral buffered formalin, and processed with a standard histological method based on Slaoui and Fiette [18].
Blood sampling
Blood sampling was conducted during similar periods with histopathology preparation. Fish blood was taken after being stunned with ice from artery caudals with a 1 ml spuit (Terumo, Japan) that was already given with an anticoagulant (Na-citrate 3%). Hematological observation consists of the erythrocyte; leucocytes; hemoglobin and percentages of neutrophils, monocytes, and lymphocytes was calculated based on Witeska et al. [19].
Data analysis
Data on survival rate (SR) (percentage of fish alive from a total of fish used) [20] and histopathology organ were analyzed using one-way analysis of variance (p < 0.05). Furthermore, if a significant value was detected, further tests would be carried out using Duncan multiple range test and Mann-Whitney test (p < 0.05), respectively. The clinicopathological alteration from infected fish consisting of external findings and hematological parameters was analyzed descriptively and compared with a normal value from the literature.
Result
Survival rate
The SR (Fig. 1) in P0 (negative control) shows the highest value (100%) and the lowest (10%) in P4 (infection of 1010 CFUml−1 A. hydrophila) compared with other treatments. In the treatment of P1 (infection of 104 CFUml−1 A. hydrophila) and P2 ((infection of 106 CFUml−1 A. hydrophila) were no significant differences. However, all treatments showed statistically different from the control (p < 0.05).
Gross clinical sign
Infected fish exhibit passive and irregular swimming (crashing at the bottom and floating on the surface), as well as congregating around aeration. External lesions (Fig. 2) show hemorrhage in the abdomen (Fig. 2A), operculum (Fig. 2C), and anus (Fig. 2D and F), scaling (Fig. 2B), and skin ulceration (Fig. 2E).
Histopathological alteration
Several clinical abnormalities, including lipid degradation and necrosis, were discovered in the kidney (Fig. 3) and liver (Fig. 4). The percentage of every cell damaged in internal organs was highest at P4 (1010 CFUml−1) and lowest at P0 (negative control), as shown in Table 1. The Mann-Whitney test revealed that the normal cell at P0 (negative control) differed significantly from the other treatments. Nevertheless, the total number of damaged cells was statistically different between treatments, with the largest in P4 (1010 CFUml−1). The results demonstrated a linear correlation between bacterial density and cell damage in the fish liver and kidney.
Figure 1.
The SR in silver rasbora after being infected with A. hydrophila at 28 hpi. P1 (infection of A. hydrophila 104 CFUml−1), C=P2 (infection of A. hydrophila 106 CFUml−1), D=P3 (infection of A. hydrophila 108 CFUml−1), and E=P4 (infection of A. hydrophila 1010 CFUml−1).
Figure 2.
Clinical signs from infected fish with A. hydrophila. A=ventral hemorrhage, B=scaling, C=hemorrhage in abdomen and operculum, D=hemorrhage in anal, E=ulceration, and F=hemorrhage in abdomen.
Figure 3.
Histopathological section of kidney with HE staining. (1) P0 (negative control), (2) P1 (infection of A. hydrophila 104 CFU ml−1), (3) P2 (106 CFU ml−1), (4) P3 (108 CFU ml−1), and (5) P4 (1010 CFU ml−1). T=Tubule, D=Degeneration, N=Necrosis.
Figure 4.
Histopathological section of liver with HE staining. (1) P0 (negative control), (2) P1 (infection of A. hydrophila 104 CFU ml−1), (3) P2 (106 CFU ml−1), (4) P3 (108 CFU ml−1), and (5) P4 (1010 CFU ml−1). H=Hepatocyte, N=Necrosis, D=Degeneration.
Hematological profile
The hematological profile (Table 2) displayed the number of erythrocytes, leucocytes, and hemoglobin levels declined from P4 (infection of A. hydrophila 1010 CFUml−1) to P0 (negative control). Total erythrocyte and leucocyte; hemoglobin levels from infected fish were lower than the normal range. In contrast, the percentage of neutrophils, monocytes, and lymphocytes rose from P0 (negative control) to P4 (infection with 1010 A. hydrophila CFUml−1). The percentage of neutrophils and lymphocytes was greater than the optimum range.
Discussion
Aeromonas hydrophila is a Gram-negative, rod-shaped bacteria found in various natural aquatic environments. The bacterium is primarily harmful to poikilothermy animals especially fish, turtles, snakes, and amphibians [21]. Several regions reported that A. hydrophila as a primary pathogen caused significant mortality in freshwater fish farming including in Nile tilapia (Oreochromis niloticus) in Egypt [22]; channel catfish (Ictalurus punctatus) in the United States [23], and freshwater cultured Murray cod (Maccullochella peelii) from Shanghai, China [24]. Our finding demonstrated that the mortality of silver rasbora was increasing linearly with bacterial density. The highest value (100%) presented in P0 (negative control), whereas the lowest (10%) was at P4 (infection of A. hydrophila 1010 CFUml−1). It was caused by the high amounts of virulence factors (enterotoxin, adhesins, aerolysin, cholinesterase, hemolysin, hemagglutinin, and protease) that were lethal to fish [25]. Zhao et al. [26] claimed that A. hydrophila is highly virulent, especially when present in large quantities or concentrations. It also indicates that A. hydrophila was the primary bacterial pathogen for many fish and aquatic organisms, causing high mortality and economic losses, and representing a risk to public health and environmental safety [27].
Table 1.
Average percentage of histology alteration in organ of silver rasbora during A. hydrophila infection.
| Treatment | Kidney | Liver | ||||
|---|---|---|---|---|---|---|
| Normal | Degeneration | Necrosis | Normal | Degeneration | Necrosis | |
| P0 | 92 ± 2 1a | 6 ± 1.8b | 2 ± 1.1c | 93 ± 0.9a | 5 ± 0.8b | 2 ± 0.9c |
| P1 | 65 ± 6 1a | 21 ± 5.2b | 14 ± 3.5b | 68 ± 10.5a | 22 ± 8.3b | 10 ± 3.1b |
| P2 | 39 ± 7 3a | 33 ± 6.2a | 28 ± 12a | 31 ± 10.6a | 40 ± 11.9a | 29 ± 9.9a |
| P3 | 25 ± 7 9b | 30 ± 7.4b | 45 ± 3.1a | 20 ± 4.9b | 46 ± 8.5a | 34 ± 10.4a |
| P4 | 13 ± 3 1b | 37 ± 10.7a | 50 ± 9.5a | 8 ± 2.1c | 21 ± 4.2b | 71 ± 5.9a |
P0: negative control, P1: A. hydrophila 104 CFU ml−1, P2: 106 CFU ml−1, P3: 108 CFU ml−1, P4: 1010 CFU ml−1. Average ± standard deviation.
a,b,cShowing significancy of the treatment (p < 0.05).
Table 2.
Hematology profile of silver rasbora experimentally infected with A. hydrophila. (average ± standard deviation).
| Parameters | Treatment | Normal range | ||||
|---|---|---|---|---|---|---|
| P0 | P1 | P2 | P3 | P4 | ||
| Erythrocyte (×104 cell ml−1) | 22.54 ± 3.11 | 16.26 ± 2.65 | 10.68 ± 2.87 | 8.95 ± 1.97 | 4.52 ± 1.65 | 20.80 ± 3.6 |
| Leucocyte (×104 cell ml−1) | 6.29 ± 1.43 | 5.48 ± 2.95 | 5.40 ± 1.76 | 5.35 ± 0.89 | 5.16 ± 0.95 | 6.11 ± 3.5 |
| Hemoglobin (gm%) | 5.05 ± 0.87 | 5.2 ± 2.41 | 4.75 ± 1.83 | 3.95 ± 0.54 | 3.6 ± 1.32 | 5 ± 2.06 |
| Neutrophil (%) | 36.5 ± 1.0 | 39 ± 1.5 | 40 ± 1.0 | 40.5 ± 2.0 | 41.5 ± 1.5 | 33.62 ± 3.5 |
| Lymphocyte (%) | 33.5 ± 0.5 | 31 ± 1.5 | 31 ± 2.0 | 30.5 ± 2.0 | 29 ± 1.0 | 31.4 ± 3.26 |
| Monocyte (%) | 26 ± 1.5 | 28 ± 1.5 | 29 ± 3.0 | 29.5 ± 2.5 | 33 ± 3.0 | 25.12 ± 6.1 |
(P0): negative control, (P1): A. hydrophila 104 CFU ml−1, (P2): 106 CFU ml−1, (P3): 108 CFU ml−1, (P4): 1010 CFU ml−1.
External clinical signs displayed passive and abnormal swimming and hemorrhage in the fin and skin. Another study also reported similar results in several aquatic species including red hybrid tilapia (O. niloticus × Oreochromis mossambicus) [28]; Chinese soft-shelled turtle (Trionyx sinensis) [29] and Siberian sturgeon (Acipenser baerii) [30]. Hemolysin activity could lysis the red blood cells and cause hemorrhage in the skin and fin of infected fish [31]. Furthermore, lytic erythrocytes release many nutrients and ions, which can promote bacterial growth. Erythrocytolysin reduces the number of erythrocytes and causes oxygen deficiency, leading to organism dysfunction and even death [32].
In severe cases, histological lesions in the kidney (Fig. 3) and liver (Fig. 4) revealed numerous cell damage, such as degeneration and necrosis. Vacuoles or rounded empty areas containing lipid globules indicated cell degeneration; thus, the nucleus shrank and was pushed to the edge [33]. It is caused by cytolytic enterotoxin generated by A. hydrophila, which reduces hepatocyte ability to effectively metabolize lipids, resulting in lipid deposits and injury to renal tissue as a hematopoietic organ [34]. Furthermore, Ahangarzadeh et al. [32] revealed that not only do A. hydrophila hemolysin toxins have hemolysis activity, but they also have cytolytic action which causes cell damage in infected fish.
Necrosis is irreversible cell destruction in tissue caused by pathogen toxins [35]. The previous study also found similar findings in gold fish (Carassius auratus) [36]; stripped catfish (Pangasius hypophthalmus) [37], and Chinese sucker (Myxocyprinus asiaticus) [38]. It is related to several toxins and enzymes secreted by A. hydrophila and diffused to the cell including serine proteases, elastase, and lipase [26]. Furthermore, two enzymes (proteases and lecithinase) may be the primary enzymes responsible for cell necrosis [39]. Long-term toxicity exposure leads to more severe cell and tissue damage [40].
Hematology profiles, such as total erythrocyte, leucocyte, and hemoglobin levels, can indicate disease infection in fish [14]. The average total erythrocytes count from the Cyprinid range is 20.80 ± 3.6 × 104 cellml−1 [41]. All treatments revealed a decrease in total erythrocytes after A. hydrophila infection. This bacterium is thought to release various toxins, including aerolysin and hemolysin. Aerolisin toxin can secrete monomers, be influenced by proteolytic modifications, and release propeptides. It is activated after attaching to receptors on the cell surface and causing cellular reactions such as erythrocyte vacuolization [42]. Furthermore, aerolysin is a primary virulent factor secreted by A. hydrophila that can bind to specific gly-cophosphatidylinositol-anchored proteins on the surface of erythrocytes and cause significant damage to fish erythrocytes by forming pores in the cell membrane, then exhibiting strong hemolytic activity and triggering deep wound infection [31]. Hemoglobin levels were lower in all treatments post A. hydrophila infection compared to the normal range (5 ± 2.06 gm%) [41]. It has also been caused by a decrease in the total number of erythrocytes, which has a significant positive relationship with hemoglobin levels [43]. Several previous study also revealed the similar finding, including in red crucian carp (C. auratus red var) [31]; snakehead murrel (Channa striata) [44]; and zebrafish (Danio rerio) [45] after infection with A. hydrophila.
After infection with A. hydrophila, the number of leucocyte demonstrated a decreasing value in all treatments compared with the average value in Cyprinid fish (6.11 ± 3.5 ×104 cell ml−1) [41]. It implied that leucocyte cells had moved to the tissue. Leukocyte cells were found in the blood vessels of the fish. When there is inflammation in the body’s tissues, leukocyte cells move to the inflamed tissue by penetrating the capillary walls. They participate in intracellular and extracellular systems that produce antibacterial compounds in response to infections [46].
Neutrophil is a type of leucocyte cell that rapidly migrates from a blood artery to the infected cell and contains vacuole and lysozyme to inactivate the pathogen. Neutrophils can cause significant collateral damage while destroying pathogenic germs [47]. The percentage of neutrophils in experimental fish after being infected by A. hydrophila show increased to the peak in the P4 treatment (injection of 1010 CFUml−1 A. hydrophila), which is almost 42%, higher than the normal value (33.62% ± 3.5%) [48]. This condition indicates that neutrophil cells were stimulated to eliminate the infection of A. hydrophila in silver rasbora. When infected, they rapidly recruit from tissues such as the head kidney, eventually accounting for up to 50% of total blood leukocytes. Neutrophils contribute significantly to antimicrobial defenses via mechanisms such as respiratory burst, phagocytosis, and the production of neutrophil extracellular traps and other toxic components. Neutrophils have unique granules that store bactericidal chemicals that kill microorganisms intracellularly or extracellularly via exocytosis [49]. Furthermore, Abarike et al. [50] stated that neutrophils are thought to be important in controlling and neutralizing infection at wound sites, as well as cleaning debris. They are also likely to have other immunomodulatory effects that contribute directly to regeneration processes.
The greatest monocyte percentage found in the P4 treatment (injection of 1010 CFUml−1 A. hydrophila) was about 33% more than expected (25.12% ± 6.1%) [48]. It showed that the immune system was eliminating the pathogen. Monocytes have been demonstrated to respond to inflammatory chemokines by migrating to the site of inflammation and exerting antimicrobial defenses. Monocytes migrate to tissues, where they differentiate into macrophages and are regulated by chemokines (e.g., CCL1 and CXCL12) [49]. Several roles played by macrophages during pathogen infection include phagocytosis, tryptophan degradation, respiratory burst, and nitric oxide responses [51]. After infection with A. hydrophila, silver rasbora showed a lower lymphocyte percentage compared with normal conditions (31.4% ± 3.26%) [48]. It was believed that lymphocyte cells move to tissues to eliminate pathogens. Lymphocytes stimulate innate immunological and regulatory functions, which help initiate and resolve acute inflammation. These cells were concentrated in the mucosal tissues of the intestine and gills demonstrating cytotoxic activity [49]. Fish lymphocytes are divided into T cells and B cells that produce three types of immunoglobulins (IgM, IgT, and IgD) to provide cellular immunity [52]. Other studies reported similar result in ornamental koi carp (Cyprinus carpio) [53]; golden mahseer (Tor putitora) [54]; and C. carpio var. Communis [55].
Conclusion
Infection with A. hydrophila caused many clinicopathological alterations in silver rasbora (R. argyrotaenia), including behavioral changes, external lesions, and histological changes in the kidney and liver (cell degeneration and necrosis). The hematological profile revealed an increase in neutrophil and monocyte percentages while decreasing erythrocyte, hemoglobin level, leucocyte, and lymphocyte percentages. Silver rasbora infected with 1010 CFUml−1 of A. hydrophila had the highest mortality rate, significant changes in hematological profiles, and severe histolopathological alterations. Further study was needed to determine whether herbal medicine might be used to prevent A. hydrophila infection in silver rasbora.
Acknowledgment
The authors would like to thank the support of the Teaching Farm, School of Health and Life Sciences Universitas Airlangga for all equipment. The authors also thank the anonymous reviewer for their valuable comments to revise the paper.
