INTRODUCTION:

Fishery resources owned by Indonesia is very diverse and has many potential uses, including aquaculture, freshwater ponds or brackish ponds and that leads to economic progress in Indonesia. One source is the mainstay of economic growth of aquaculture, which is realized through the cultivation of competitive systems. Fish farming activities are man's attempt to manage the factors of cultivation, pest and disease organisms. An indicator of success in the cultivation of fish is the health of fish associated with the maintenance of the environment and durability against pathogenic bacteria.¹

One of the common bacteria found in aquatic ecosystems and has a role as a microbial flora to water organisms in a stable environment is Aeromonas hydrophila. Aeromonas hydrophila is bacteria that causing a dangerous disease- in freshwater fish farming. The bacteria are common in carp is one of the leading commodity and can infect freshwater fish in all sizes which can cause death by up to 80%, resulting in huge losses in the cultivation of freshwater fish. Aeromonas hydrophila is abundant in freshwater environments, especially with high organic matter content. This bacterial infection is oppotunistic, which is an infection that usually does not cause disease in animals with normal immune systems, but can attack animals with poor immune systems. Aeromonas hydrophila infection can be recognized for their external wounds (ulcers), mucus dries up, there are patches of bleeding in a latero-ventral region of the body and fins and scales chipped.²

The human can also be contracted Aeromonas hydrophila when consuming a sick fish undercooked, infected through cuts, or not washing hands after holding the fish sick. Humans are contracting the disease will experience diarrhea and abdominal pain.³ Minnagati et al (2000)⁴ also reported that these bacteria are able to infect humans and cause the manifestation in the skin, soft tissues and even cause diarrhea.

Research by Vivekanandhan et. al. (2002)⁵ conducted Aeromonas hydrophila in India stating that the attacked fish tilapia are already resistant to amoxicillin, ampicillin, lincomycin, novobiocin, oxacillin, penicillin, and rifampicin. While the results of research by Yuliani and Herupradoto (2014)⁶ showed that 10% of the fish and shrimp samples of contaminated bacteria Aeromonas hydrophilla and of the hydrophilla Aeromonas bacteria isolated 100% indicating the resistant to the antibiotic Novobiocin, Penicillin G, and Oxacillin.

Aeromonas hydrophila has the Outer Membrane Protein (OMP) which is a protein in the cell wall of gram-negative bacteria that associated with virulence and immunogenic properties.⁷Outer Membrane Protein (OMP) bacteria play an important role in virulence for OMP is an outermost surface layer of cells that are also involved in the induction of immune factors in defense. OMP is located in the outermost layer of bacteria that are important for the host immune response and as a target for drug therapy. More recently, much attention about OMP as a potentially very important in vaccine components.⁸

The research about Outer Membrane Protein (OMP) has been studied in an attempt to obtain a reliable antigen both for diagnosis and for vaccines. In this study, the authors wanted to do research on the reactivity of the Outer Membrane Protein (OMP) Aeromonas hydrophila in New Zealand rabbits with Immunohistochemistry techniques to investigate the interaction of antibodies with antigens on kidney, spleen, and liver of New Zealand rabbit microscopically. In addition, this study aims to determine the effectiveness of immunohistochemical techniques in detecting the Aeromonas hydrophila on histopathology preparations. Based on this background, the problem can be formulated as follows: How the interaction of antibodies with antigens on rabbit kidney tissue histopathology New Zealand using Immunohistochemistry techniques?

MATERIALS AND METHODS:

Bacteria Culture of Aeromonas hydrophila

Suspension of Aeromonas hydrophila in this study was obtained from a previously studied. The suspension of bacteria cultured in media TSA (Trypticase Soy Agar) subsequently identified in media urea agar, Simmon citrate, SIM (Sulfite Motility Indol) and blood agar.

Extraction of Outer Membrane Protein Aeromonas hydrophila

The extraction was done by Sonication technique. A. hydrophila washed with PBS by centrifugation at 8,000 rpm for 15 minutes. Pellet A. hydrophila was dissolved with 1ml PBS then disrupted by ultrasonication at 25 Hz for 4x4 min with 2 min intervals. Unbroken cells were removed by centrifugation at 8000 rpm for 15 minutes. The supernatant containing the Outer Membrane Protein was transferred into a new tube and centrifuged again at 13.000 rpm, 30 min, 4^oC, the pellet was dissolved in 2 ml sarcosyl 1% and added PBS as much and incubated 37C for 20 minutes. Homogenized with vortex for 1 minute. Then centrifuged at 4^oC at 20,000 rpm for 20 min. the pellets obtained are OMP and then stored at -20^oC for the protein analysis material.⁹

Immunization At the Rabbit using Specific Protein A. hydrophilla:

Immunization of rabbits is conducted using specific proteins that have been successful in isolation through the previous phase and injected sub cutan to the New Zealand strain rabbit sex male and 5 months old. As a control is a rabbit without immunization. Six Rabbits were immunized using a mixture of antigen with Freund's complete adjuvant (1:1). Second immunization carried out after 14 days with a mixture of antigen in incomplete Freund's adjuvant (1:1). The third immunization is done after the next 7 days in the manner and the same material as the second immunization. Immunization fourth and so on until the sixth immunization is done after the next 7 days using the same antigen with a second immunization, injected subcutaneously. On the 49th day after the first immunization or seven days after the last immunization, rabbit serum was taken by V. auricular. Rabbit serum is used to primary antibody for immunohistochemistry method.

Sampling Method:

Tissues (Kidney, Liver, and Spleen) sampling is done on 7^th day after infection. Rabbits in euthanasia by using chloroform and then laparotomy for taking the tissues, the tissues immediately placed on a jar/pot containing formalin neutral buffer solution (BNF) and then made preparations rabbit tissues histopathology.

Immunohistochemistry Method:

At first, clearing and rehydration done in stages and then washed 3 times with destillate water (DW) respectively 5 minutes and then drained of fluid around the network. After that, the tissue pieces circled in pen and then soaked in 3% H2O2 in DW 15minutes at room temperature. Then preparations were rinsed 3times with DW and 3 times with PBS every 5minutes.

The next step preparations were incubated with 10% normal goat serum 30minutes at room temperature and then rinsed 3times with PBS every 5 minutes. Then preparations were incubated with primary antibodies and then rinsed 3 times with PBS every 5 minutes, after which it was incubated with secondary antibody for 30 minutes at room temperature. At the same time, 10ml avidin and biotin 10ml incubated in 1ml of PBS. Furthermore, preparations were rinsed 3 times with PBS each 5minutes then incubated with a mixture of Strepvidin Horseradish Peroxidase (SA-HRP) 30 minutes at room temperature and then rinsed 3 times with PBS every 5minutes and incubated with a solution of DAB. The next step is done counterstaining with hematoxylin for 2 minutes and then dehydrated, further preparations were closed with a cover glass.

Analysis of The Result

The results will be obtained in this study will be presented in the form of descriptive from spleen, kidney, and liver histopathology tissues that showed the bond between antigens with antibodies with immunohistochemical techniques that visualized with a brownish color.

RESULT AND DISCUSSION:

Aeromonas hydrophila is gram-negative bacilli, which have isolated from fresh water, brackish water, tap water, soil, and non fecal organic materials.

The results are obtained in this study in the form of spleen (Figure 1), liver (Figure 2) and kidney (Figure 3) histopathology tissue images that showed the bond between antigens with antibodies with immunohistochemical techniques are visualized with a brownish color.

Figure 1. Antigen-antibody expression (red arrow) of Aeromonas hydrophilla on spleen. Control (A); Antigen-antibody (B). magnification 400x

The result of this study in line with Kamano et al (2003)¹⁰ that spectrum Aeromonas hydrophila includes acute gastroenteritis, cholecystitis, cholangitis, liver abscess, pneumonia, pneumonia, empyema, meningitis, septic arthritis, osteomyelitis, endocarditis, myonecrosis and necrotizing fasciitis. Using immunohistochemical methods we can detect Aeromonas infection before manifestation in many organs.

Figure 2. Antigen-antibody expression (red arrow) of Aeromonas hydrophila on the liver. Control (A); Antigen-antibody (B). magnification 400x

In rabbit, histopathologic preparations can detect Aeromonas hydrophila infection by immunohistochemical methods. The presence of Aeromonas hydrophila in rabbit histopathologic tissue is visualized by brownish color. The brown color is the result of the reaction between the antigen binding to the primary antibody and the secondary antibody of Strep Avidin Horseradish Peroxidase (SA-HRP) and Diamino Benzidine substrate (DAB).

Figure 3. Antigen-antibody expression (red arrow) of Aeromonas hydrophila on the kidney. Control (A); Antigen-antibody (B). magnification 400x

Immunohistochemistry is a process for the detection of antibodies (proteins, carbohydrates, etc.) on the cells of the network with the principle of antibodies binding reaction to antigens on the network. Immunohistochemical name taken from the name "immune" to indicate that the basic principle in this process is the use of antibodies and "histo" indicates tissue microscopically. Immunohistochemistry is often used to measure and identify the processes of cell proliferation and apoptosis antibody-producing cells. Immunohistochemistry is also often used for basic research in order to understand the distribution and location of biomarkers or protein expressed in a wide variety of tissues in the body.¹¹

The histopathologic preparations of the spleen, liver and kidneys in rabbits can be detected the presence of bacteria Aeromonas hydrophila with immunohistochemical methods. To visualize the result of interaction between antigens and antibodies can be done in various ways, which means most often used is the conjugation of antibodies with an enzyme such as peroxidase. The presence of Aeromonas hydrophila in spleen, liver, and kidneys rabbit histopathology tissue can be visualized by brownish color. The brown color is the result of the interaction between the antigen binding to the primary antibody (serum anti-OMP) and the secondary antibody of Strep Avidin Horseradish Peroxidase (SA-HRP) and Diamino Benzidine (DAB). It also can be used are like fluorescent or fluorophore rhodamine. To study the morphology of cells, the cells in the tissues between cells were fixed and then localized and visualized by electron microscopy or light microscopy.¹²

Immunohistochemistry is a combination of histology or cytology and immunology. Immunohistochemistry is a method of staining substances or active ingredients in the network by using the basic principles of immunology that is binding the active ingredient (antigen) in the active site specific by an anti active ingredient (antibodies). Results of antigen and antibody can be identified on the specimen when the antibodies are bound by a marker (marker) in the form fluoresin, enzymes, material particles, or isotopes that can be visualized, thus marking the presence of the active ingredients in the network.¹³ The active ingredient may be proteins, carbohydrates, nucleic acids, fats, and other natural materials and synthetic materials.^14,15

CONCLUSION:

The results will be obtained in this study is the primary antibody can detect the presence of Aeromonas hydrophila bacteria in the spleen, liver, and kidney via immunohistochemical methods and visualized with a brownish color.

ACKNOWLEDGMENT:

The author would like thanks to the Directorate General of Higher Education on Giving Fund and the Rector of the University of Airlangga that facilitated that Research Activities.

CONFLICT Of INTEREST:

All authors declare no conflict of interest.

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Received on 23.04.2019 Modified on 25.05.2019

Research J. Pharm. and Tech. 2019; 12(11):5189-5192.

DOI: 10.5958/0974-360X.2019.00898.9