Macroscopic and microscopic analysis of Aeromonas hydrophila infection in Nile tilapia (Oreochromis niloticus) in traditional fish farms in Surabaya

Introduction

Tilapia was first cultivated in Kenya in 1924. It is one of the most popular fish to be widely cultivated, and it contributes to improving local livelihoods, especially in developing countries (1). More than 140 countries use Tilapia for commercial trade and fish farming (2). Tilapia cultivation reaches 6 million tons (MT), placing Tilapia in second place as the world's most widely cultivated freshwater fish after carp (3). There are around 70 species of Tilapia that have been identified globally, and Nile tilapia (Oreochromis niloticus L.) is the most widely cultivated species, so various efforts are needed to ensure the quality and quantity of Tilapia needed by the community (4). In line with the development of cultivation businesses, several disturbing problems, such as pests and diseases, hinder the development of cultivation businesses. Various diseases are the main problem in the cultivation process because of various negative impacts, such as decreased production, decreased water quality and even total death. This disease can be caused by several pathogenic agents such as viruses, parasites or bacteria (5). One of the bacterial diseases that often becomes an obstacle in tilapia cultivation is Aeromonas hydrophila (A. hydrophila). Aeromonas is a gram-negative bacterium that belongs to the Aeromonadaceae family of Gammaproteobacteria, motile bacilli or coccobacilli rods, non-spore-forming with rounded ends that size 1–3.5 μm (6). Haemolysin, aerolysin, cytosine, gelatinase, enterotoxin and antimicrobial peptides have been identified as virulence factors in A. hydrophila. The habitat of these bacteria is often found in freshwater, aquatic plants and fish bodies, and it is known as Motile Aeromonas Septicemia (7) or Hemorrhagic Septicemia. This disease is common in tropical fish such as Nile tilapia, gourami, and goldfish. Infected fish usually have bruises on their bodies but can also show other signs, such as protrusion of the eyeballs (exophthalmia) and bleeding in various parts of the body. Another condition is a red mouth, bloated abdomen, blood on the exterior surface and around the anal scale sloughing, surface lesions and septicemia (8). Infected fish with open wounds can spread this disease to other fish, and fish that appear healthy but carry this disease (sub clinical carriers) may be present and will release the bacteria in their feces into the aquatic environment. Aeromonas bacteria are opportunistic human pathogens that can cause septicemia, wound infections, and gastroenteritis, especially in children (9). Like other enteropathogenic bacteria, the pathogenicity of Aeromonas bacteria is always associated with mechanisms for producing toxins, such as cytotoxins and enterotoxins (including those with hemolytic activity) and attachment to host tissue. Apart from toxins, Aeromonas bacteria also produce other extracellular fluids such as protease, amylase, chitinase, lipase and nuclease (10).

This study aims to be a comprehensive analysis whose results can provide insight into the effects of Aeromonas hydrophila bacterial infection on Tilapia, which affects aspects of blood results and macroscopic and microscopic tilapia organ tissue. These findings also contribute to a better understanding of the etiology and pathogenesis of the observed clinical manifestations, guiding future research and intervention strategies in fish health management.

Materials and methods

Study period and location

This research was carried out from April to May 2024. The research used the biosurvey method with fish sampling locations at Kayoon Fish Market, Gunungsari and Bratang, Surabaya, East Java. Necropsy and preparation for sampling organs, microbiology and hematology analysis at the Pathology, Microbiology and Clinical Pathology Laboratory of the University of Wijaya Kusuma Surabaya. Histopathology preparations were made at the Integrated Research Laboratory, Faculty of Dentistry, Gajah Mada University.

Taking of sample

A sampling of Tilapia fish in freshwater weighing 950-1000 g, and physically, there are abnormal lesions around its body. The method used to examine Tilapia with protocol number F-19 consists of necropsy and blood sampling. Macroscopic and microscopic organ examinations (histopathology) were carried out. Microbiological examination consists of Tryptone Soya Agar (TSA) media or Mac Conkey Agar (MCA) media, gram staining, catalase test, then continued with biochemical tests including Triple Sugar Iron Agar (TSIA), Simmon's Citrate Agar (SCA), Urease, Sulfide Indole Motility (SIM) (11), and clinical pathology examination with hematology analysis which includes observing the number of erythrocytes/red blood cells (RBC), leukocytes/white blood cells (WBC), hemoglobin, hematocrit/packed cell volume (PCV), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), initial and final Total Plasma Protein (TPP), and differential calculations.

Making histopathology preparations

Organ samples were fixed with 10% BNF (Buffered Neutral Formalin) for 24-48 hours and then processed for histology preparations by rehydrating using graded alcohol, embedding in paraffin, and cutting to a thickness of 5 microns. Then, the samples were painted using Haematoxylin and Eosin (HE). The parameters observed were inflammatory cell infiltration, hemorrhage, necrosis, and abnormalities in the organ (12).

Results

Pathology examination

Tilapia fish with protocol number F-19 (because of the category of fish) have a body weight of 1 kg with anamnesis; the fish aquarium is cleaned once a week. Nine fish are in one large aquarium, but they must be more specific about the material and size. They are given pellet food. The clinical symptoms are that the fish looks weak, swims to the surface has reddish spots (hemorrhage) on the surface of the body, and the fish's eyes become bulging (exophthalmia) and yellowish (icterus). The results of a macroscopic examination of the organs (anatomical pathology) show that there is an ecchymosis hemorrhage on the abdomen and pectoral fin of the fish, and the gills look pale and ruptured. The liver organ is pale yellowish (icterus), there is linear hemorrhage, a rubbery consistency, and the incision area is wet. Meanwhile, his tail experienced linear hemorrhage (Figure 1).

Figure 1: Results of Macroscopic Examination of Tilapia. (a) Abdominal ecchymoses hemorrhage (blue arrow). (b) Ruptured gills (yellow arrow). (c) Liver. (d) Tail.

Based on macroscopic observations, there were changes in the gills, liver and tail, so we continued with histopathological observations. The following is a histopathological description of the gills, liver and tail skin of the F-19 Tilapia fish. There is also a microscopic examination of gills experiencing rupture in the secondary lamella, telangiectasis, erosion of the primary lamella, hemorrhage, necrosis, and accompanied by fluid and inflammatory cell infiltration. The microscopic examination of the liver is congested in the hepatic portal vein and synocytic congestion, hemorrhage, inflammatory cell infiltration and hemosiderosis. Meanwhile, microscopically, the tail skin experienced inflammatory cell infiltration (Figure 2).

Figure 2: (a) & (b) Gills histopathology. (c) Liver histopathology. (d) Microscopic view of the tail of the skin. (HE stain 40-100×).

Microbiology examination

In diagnosing the cases of Aeromonas hydrophila, a swab is carried out on the eye, kidney and liver organs that are experiencing abnormalities. Then, the swab results are cultured on cultural media, such as for sample screening. The culture media was then incubated at 37℃ for colonies to appear when examined on Tryptone Soya Agar (TSA) media. Aeromonas hydrophila is a gram-negative bacterium, has a short rod morphology with varying sizes (width: 0.8-1.0 microns; length: 1.0-3.5 microns), does not have spores, bacteria are motile because they have one flagellum (monotrichous flagella), which comes out from one of their poles. The surface morphology of the colony is slightly prominent, round, shiny, and creamy, with entire colony edges and a diameter of 1-3 mm (Table 1).

Table 1: Biochemical Test Results

In the subsequent examination, gram staining was carried out after bacterial colonies had formed for 24 hours; then, to see the morphology of the colonies formed, gram staining was carried out. This test aims to determine the nature of the bacteria being tested. The nature of gram-positive bacteria is characterized by purple-coloured bacterial cells, and the gram-negative bacteria are red/pink-coloured. The primary characterization of A. hydrophila is gram-negative, rod-shaped, motile, non-capsulated and non-sporulated. Can appear singly or in pairs with or without short chains. After gram staining identifies the sample, biochemical tests are continued. The media used for the biochemical tests are the catalase test, Triple Sugar Iron Agar (TSIA), Simmon's Citrate Agar (SCA), Urease, Sulfide Indol Methility (SIM), and Methyl Voges Proskauer (MR—VP), which will be incubated for 24 hours. After 24 hours, the changes observed were observed (Figures 3 and 4).

Figure 3: (a) Preparation of gram staining      (b) Microscopic examination with gram staining.

Figure 4: Biochemical tests (a) TSIA, (b) SCA, (c) Urease, (d) SIM, (e) MR, (f) VP.

Haematology examination

Based on the results of blood tests in the clinical pathology laboratory, it was discovered that Tilapia with protocol number F-19 was anemic, macrocytic, hypochromic, hypoproteinemic, hyperfibrinogenemic, neutrophilia, lymphocytopenia, monocytopenia and basophilia. On blood smear or hematology examination, there are abnormalities in erythrocytes such as echinocytes, teardrops and sickle cells (Table 2).

Table 2: Tilapia hematology examination results

Discussion

Histopathological examination showed gill rupture in the secondary lamella. If there is a rupture or damage to the lamella, it will disrupt blood circulation and gas exchange. Ion exchange in the lamellae can transfer 60-80% of oxygen from water into the blood (13). If damage like this occurs over time, it will disrupt the circulation system, which can cause the fish to lack oxygen supply, which in turn will cause a lethal effect on the fish due to disruption of the fish's respiratory system (13). Histopathological examination of the gill organs also found telangiectasis. Telengiectasis is the dilatation or widening of blood vessels in fish's gills. In other conditions, it was dilated postcapillary venules in the papillary and superficial reticular dermis (14). Telengiectasis is a condition where the capillaries in the gills become swollen. This condition can be caused by various factors, including infection with the Aeromonas hydrophila bacteria. In A. hydrophila infection, the bacteria invade the gill epithelial cells (15). These bacteria then release toxic substances that cause damage to epithelial cells. The injury to these epithelial cells causes swelling of the capillaries. Thus, swelling of these capillaries disrupts blood flow to the gills. This can cause hypoxia, a condition where the gills do not get enough oxygen. Hypoxia is a significant threat because it can cause fish to die (15).

Histopathological examination of the gill organs also revealed necrosis and erosion of the gills. As a result of the hypoxic condition or other toxins, the mitochondria in the cells are damaged so that they cannot be repaired, and swelling of the cell organelles (in Yunani known as”Oncosis”), rupture of the plasma membrane and then necrosis occurs (16). Necrosis is irreversible or irreversible cell death. Necrosis can occur in cells in all body tissues, including fish. Necrosis is often associated with inflammatory reactions. Necrosis can be caused by hypoxia, physical factors, chemical agents, immunology and infectious or microbial agents such as A. hydrophila (17). The histopathology of the gills showed necrosis and was characterized by dead and destroyed gill cells. These dead gill cells will look pale or white and have an irregular shape. Apart from that, A. hydrophila bacteria can also be seen in the infection of gill cells. In severe infections, necrosis can cause extensive damage to the gills. This can cause the fish to have difficulty breathing and eventually die. Aeromonas hydrophila bacteria are Gram-negative bacteria that can cause various diseases in fish, including gill necrosis. These bacteria can enter the fish's body through wounds or injured skin. After entering the fish body, these bacteria will attack the gill cells and cause them to die. Necrosis The microscopic picture of erosion on the gills of fish infected with A. hydrophila bacteria shows damage to the gill tissue. This damage can include cell detachment, white blood cell infiltration, and ulcer formation. Erosion of gills infected with A. hydrophila bacteria can cause various health problems in fish. Infected fish can experience difficulty breathing, loss of appetite, and death (18)

Echymoses hemorrhage of the abdomen and microscopic examination of the liver showed congestion, hemorrhage and hemosiderosis. The area around the liver portal vein also experiences hemorrhage characterized by an accumulation of red blood cells (erythrocytes) coming out of the lumen of the blood vessels. Hemorrhage is an outcome of blood channel disruption and is a symptom of severe physical impairment (19). The occurrence of congestion and hemorrhage in the liver indicates that the blood vessels in the liver are damaged. Congestion can be interpreted as a condition where red blood cells (erythrocytes) collect in the lumen of blood vessels. Other hemorrhage, vacuolization, patchy degeneration, blood congestion, and hypertrophy nucleus can also occur in the liver because the liver is an organ directly affected by toxic materials or other infectious agents (19). Apart from that, the histopathology of the liver also experiences hemosiderosis, a condition characterized by a buildup of hemosiderin in the fish liver. Hemosiderin is a protein that contains iron, and a buildup of hemosiderin in the liver can cause liver damage. Microscopically, fish liver hemosiderosis is characterized by hemosiderin pigment in the liver cells. This pigment can be seen as black or brown spots in the cytoplasm of liver cells (20).

On microscopic examination, the fishtail organ shows inflammatory cell infiltration, which can be caused by various factors, such as bacterial, viral or parasitic infections. Infection can cause histological changes such as hepatocyte vacuolization, glycogen depletion, inflammation, changes in the shape of sinusoidal vessels, and neoplasms, which can be interpreted as a response to environmental stress or other pathological processes (21). On microscopic observation, inflammatory cell infiltration is characterized by a collection of inflammatory cells in the organ tissue. The inflammatory cells most often found in this case are neutrophils, which are white blood cells that play a role in fighting infection. In the examination carried out on the case of A. hydrophila tilapia fish with protocol number F-19, the symptoms were red spots on the body surface, rotting fins and tail, peeling scales and protruding eyes. These symptoms were similar to the research (22).

The results of hematology test, the erythrocytes (RBC), 0.51 x 10/µL, are within the normal range. Erythrocytes in Tilapia infected with A. hydrophila bacteria experienced a significant decrease in erythrocytes. Usually, a decrease in erythrocytes is also followed by a decrease in hematocrit (23,24). The erythrocyte value of Tilapia infected with Aeromonas hydrophila bacteria ranges from 0.02-3 x 106 cells/mm3. This decrease in erythrocytes is caused by hemolysis, which breaks down red blood cells. Hemolysis can be caused by toxins produced by the bacteria A. hydrophila.

The catalase test results show a positive reaction as indicated by bubbles (gas) forming in the tube or glass object. Bacteria can produce catalase and oxidase enzymes in the catalase and oxidase test. A positive sign is obtained because the bacteria can produce catalase and oxidase enzymes (25), and the genus Aeromonas has gram-negative characteristics, fermentative, oxidase-positive and catalase-positive (26). The TSIA (Tryple Sugar Iron Agar) test results include sucrose, lactose and glucose. The working principle of this test is to detect bacteria that can ferment lactose, sucrose and glucose. In the TSIA test results, the Alk/Alk and H2S (-) results were obtained. The TSIA test results on Aeromonas bacteria can be observed based on changes in the colour and shape of the bacterial colony. The colour changes that occur in TSIA media can indicate the ability of bacteria to ferment carbohydrates and produce acid. Interpretation of TSIA test results on Aeromonas bacteria with red colonies indicates that the bacteria are positive for fermenting glucose and producing acid (27).

The SCA test showed the colour change from green to blue. SCA is a slant agar medium with a solid surface, making it easy to determine the significance of growth. Because the use of citrate requires oxygen, this slant pattern can increase the amount of growth. The increase in pH of the medium after the decomposition of citrate can be detected by the colour change of BTB (Brom Timol Blue) that co-occurs (28). The result of the SCA biochemical test was positive. The SCA test results showed that the fish samples were positive. This is indicated by a change in the colour of the SCA media from green to blue. The colour change of the SCA media from green to blue is caused by the formation of citric acid from the citrate metabolism by bacteria. The results of this study indicate that the bacteria Aeromonas. contained in tilapia fish can utilize citrate as a carbon and energy source. This shows that Aeromonas bacteria can cause disease in fish.

The urease test shows negative results because positivity is indicated by a change in the colour of the media from yellow to pink (29). A negative urease test result indicates that the bacterial isolate does not produce the urease enzyme, which breaks down urea into ammonia and carbon dioxide. Negative results on the urease test indicate that the bacterial isolate cannot break down urea (30).

In the Sulfide Indole Motility Test (SIM), the results showed a red ring after being dropped on the Kovacs reagent, showing a positive result where the bacteria formed indole and there was no motility (31). Biochemically tested Aeromonas hydrophilia showed positive results. The motility test is carried out to determine the movement of a microorganism. Motility is seen by the spread of germ growth at the puncture site or the appearance of cloudy media. The results of this study indicate that the SIM test is an effective method for identifying Aeromonas hydrophila bacteria in fish. Other research results support this by showing that the SIM test has high sensitivity and specificity for identifying Aeromonas hydrophila bacteria (32).

A positive Methyl Red (Mr) test result indicates a change to red after methyl red is dropped. The research results show that the Aeromonas bacteria isolate is positive in the MR test. MR media containing Aeromonas bacterial isolates changed colour to red after incubation for 24 hours. This colour change is caused by the production of acetic acid by Aeromonas bacteria from glucose fermentation. A positive result indicates that there is no mixed fermentation in the isolate. The bacteria ferment glucose and produce various acidic substances (33,34)

The results of the Voges Proskauer (VP) biochemical test after planting bacteria and incubating them for 24 hours in an incubator and adding 10% KOH reagent and Alpanapthol showed no change, the results of the VP test on Aeromonas bacteria found in fish showed negative results. The bacteria cannot ferment glucose by producing acetic acid and pyruvic acid. These results are consistent with the results of other studies, which show that Aeromonas hydrophila, one of the most commonly found Aeromonas species, has negative VP test results (35).

The results also showed low hemoglobin (Hb). Erythrocytes produce hemoglobin during the maturation process. Heme synthesis occurs in mitochondria, while globin is produced in ribosomes (36). Other factors that can influence Hb concentration include the adequacy of iron in the body. Iron is needed to produce hemoglobin, so anemia due to iron deficiency will cause the formation of smaller erythrocytes with a low Hb content. Iron is an important substance for the formation of hemoglobin (Hb). The function of iron as a medium for transporting, storing, and utilizing oxygen as hemoglobin, myoglobin, or cytochrome is to meet the needs of hemoglobin formation (37). Hemoglobin levels also affect erythrocyte and hematocrit levels. The relationship between hemoglobin and hematocrit is that erythrocytes contain Hb, which binds oxygen and is used for the catabolism process to produce energy. The fewer erythrocyte cells, the lower the hemoglobin levels in the blood (38). In fish, A. hydrophila bacteria produce hemolysin, a toxin that can break down red blood cells. Hemolysin damages the red blood cell membrane, releasing hemoglobin and entering the blood plasma. Low hemoglobin levels in fish blood can cause fish to experience hypoxia, a condition of lack of oxygen. Hypoxia can cause various health problems in fish, such as reduced growth, decreased appetite, and even death (39).

The decrease in PCV values ​​in Tilapia infected with A. hydrophila is thought to be caused by several factors, namely, hemolysis in red blood cells, decreased production of red blood cells by the bone marrow and increased capillary permeability due to infection with the Aeromonas hydrophila bacteria (40). The increased MCV calculation results (431.37 fL) show that the volume of erythrocytes or large erythrocyte size will affect the viscosity of blood fluid, thus disrupting the activity and smooth circulation of blood (41). The normal average MCV value is 80-100 fL. Mean cell volume (MCV) is the average volume of red blood cells. It is calculated from hematocrit (HCT) and erythrocyte (RBC) count. Low MCV indicates microcytic (small RBC size), but high MCV indicates macrocytic (large RBC size) (42). The increase in the MCV value of 431.37 fL from the normal value. So, we can interpret it as indicating macrocytic anemia. It is caused by excessive production of red blood cells, accumulation of immature red blood cells and changes in red blood cell metabolism (43). At the same time, MCHC is decreased in fish with A. hydrophila bacteria and is usually caused by iron or protein deficiency. This is because A. hydrophila is a facultative anaerobic bacterium. These bacteria can grow in both aerobic and anaerobic conditions. Under aerobic conditions, these bacteria will use oxygen for respiration. However, under anaerobic conditions, these bacteria will use iron or protein as a substitute for oxygen (44).

WBCs, known as leukocytes, are a heterogeneous population of cells, including lymphocytes, monocytes and granulocytes consisting of neutrophils, eosinophils and basophils. In the differential counting calculation, F-19 tilapia experienced an increase in lymphocytes and a decrease in eosinophils. Neutrophils decrease in fish infected with Aeromonas hydrophila bacteria because these bacteria releases toxins that can damage white blood cells, including neutrophils. Neutrophils are white blood cells that play an important role in the fish's immune system. The main function of neutrophils is to fight bacterial infections (45). A decrease in the number of lymphocytes will cause fish to become more susceptible to infection. Fish infected with A. hydrophila will usually experience symptoms such as skin wounds, bleeding, and death (46). A decrease in monocytes in fish infected with A. hydrophila bacteria can be an early indicator of infection. A decreased number of monocytes can cause fish to be more susceptible to secondary infection and death. The bone marrow produces eosinophils in fish and then circulates in the fish in the blood. When fish are exposed to parasites, eosinophils will move to the site of infection and release granules containing antiparasitic substances. Eosinophils have been traditionally associated with initiating and propagating inflammatory responses, particularly in allergic diseases and helminth infections (47). Basophils increase in fish infected with A. hydrophila bacteria. This is an immune response from the fish body to fight the bacterial infection.

Reticulocytes are young red blood cells originating from the bone marrow's normoblast maturation process. Reticulocytes will enter the peripheral blood circulation and survive for approximately 24 hours before finally maturing into erythrocytes. The increase in A. hydrophila bacterial reticulocytes in Tilapia is caused by the fish's immune response. Reticulocytes are young erythrocytes that still contain a nucleus. An increase in the number of reticulocytes is a sign of the fish body's nonspecific immune response to bacterial infections (48). Thrombocytopenia can occur in the bacteremia phase, and long-term disorders of bone marrow reproduction can cause platelet deficiency. Anemia can cause a decrease in platelet production when the bone marrow no longer produces enough red blood cells for the body. Factors that cause internal and external hemorrhage can also cause thrombocytopenia (49-51).

Conclusion

Nile Tilapia is from traditional fish farms in Surabaya, which are infected by A. hydrophila bacteria. Comprehensive pathological analysis is important for examining infected fish. Through microbiological examination to find and grow bacteria in the media, gram staining and biochemical tests prove the presence of these bacteria. During macroscopic and microscopic examinations, there were abnormal lesions such as necrosis, hemorrhage, and inflammation of the fish's gills, liver, and tail skin. In addition, the hematology results showed macrocytic anemia, hypochromia, hyperfibrinogenemia, neutropenia, lymphocytopenia, monocytopenia and basophilia.

Acknowledgment

The authors thank to the University of Wijaya Kusuma Surabaya for supporting this research.

Conflict of interest

There is no conflict of interest.