Development Of Microarray For Detection Of Fish Pathogenic Bacteria

The bacterial fish diseases break out frequently and cause great economic loss in aquaculture,therefore the precise and quick detection of pathogens is important for the efficient cure of the disease and the sustainable development of fish farming. Hence a novel method to parallel detection of multiple fish pathogens is urgently needed. Immunochip technique has the advantages of strong specificity, high sensitivity, simple sample handling, and high through-put analysis with minimal sample consumption, and has shown the vast prospect on the detection of pathogenic bacteria in medicine. However, it has not been applied in the fish pathogen detection. In this paper, two kinds of immunochip were developed including the antibody microarray detecting fish pathogenic bacteria and the antigen microarray detecting the antibody of pathogenic bacteria in blood serum of fish.6 strains of fish pathogenic bacteria, including Streptococcus iniae, Edwardsiella tarda, Vibrio anguillarum, Aeromonas salmonicida, Psedomonas fluorescens and Mycobacterium marinum, were used in this paper. After confirmed their pathogenicity, the outer membrane protein (OMP) of four G- bacteria were extracted, and the specific bands were cut. The polyclonal antibody (from rabbit) of 6 strains of bacteria and the specific bands of OMP were prepared. By using ELISA, Western-blot and Dot-blot, the immune reactions of 6 pathogenic bacteria and the developed rabbit anti-serum were analyzed. Then the antibody microarray which used rabbit anti-serum as capture antibody to detect pathogenic bacteria in fish was constructed, the optimum concentration of capture antibody added on the chip, the time of bacterial incubation, the dilute times of enzyme label antibody were determined by experiments. Subsequently each strain of bacteria was detected using the antibody microarray. On the other hand, the antigen microarray which detected the antibody of pathogenic bacteria in fish blood serum was constructed with the specific bacterial antigen. Both immunochips were applied to the diagnosis of pathogen in artificial infected Japanese flounder(Paralichthys olivaceus), and naturally diseased puffer fish (Takifugu rubripes).The detail results are as follows.(1)The LD50 of 6 strains of bacteria were 7.94×105,6.31×105, 1×106,7.94×105, 1.26×107,2.51×107 cfu each fish respectively. All of them have strong virulence and could be as the material for further research.(2) The OMP from the bacteria of E.tarda, V. anguillarum, A. salmonicida, P. fluorescens were extracted.35kD OMP from E. tarda,38kD OMP from V. anguillarum,42kD OMP from A. salmonicida,34kD and 22kD OMP from P. fluorescens were cut respectively to produce the rabbit polyclonal antibodies, and the polyclonal antibodies against bacteria were prepared as well. ELISA, Western-blot and Dot-blot were used to analyze the immune reaction between 6 strains of pathogenic bacteria and the rabbit anti-serum. The results show that most of the anti-serums are suitable to be utilized in the detection of immune-chip, except the anti-serum against 34kD OMP of P. fluorescens, which displayed strong cross reaction with other bacteria.(3)The optimum concentration of capture antibody on the antibody microarray is 1.5 mg/mL for M. marinum, and 0.5 mg/mL for other 5 strains of bacteria. The incubation time is 1 h for S. iniae, V.anguillarum and P. fluorescens, and 1.5h for E. tarda, A. salmonicida and M. marinum. The dilution times of the enzyme labeled antibody are as follow:2000 times of dilution for the labeled anti-serum of S. iniae, 6000 times for labeled anti-serum of E. tarda,and 4000 times for the labeled anti-serum of V. anguillarum, A. salmonicida, P. fluorescens and M. marinum.(4) When detecting the single strain of bacteria with the single enzyme labeled antibody,1010-103 cfu/mL of S.iniae and V. anguillarum,1010-104 cfu/mL of E.tarda, 109-103 cfu/mL of A. salmonicida,P. fluorescens,and M. marinum could be detected. When the capture antibody replaced by the anti-serum against OMP protein of different bacteria,1010-103 cfu/mL concentration of S.iniae,1010-104 cfu/mL of E. tarda,109-105 cfu/mL of V. anguillarum and P. fluorescens,109-103 cfu/mL of A. salmonicida and M. marinum could be detected,and no cross reaction. The detection results of several different bacteria at the same time with single detection antibody showed that 1010-106 cfu/mL of S. iniae and M. marinum could be detected,109-105 cfu/mL of E. tarda and V.anguillarum,109-106 cfu/mL of A. salmonicida and 1010-107 cfu/mL of P. fluorescens could be detected as well. When using the mixed enzyme labeled antibody to detect a single strain of bacteria, the results revealed that 1010-104cfu/mL of S. iniae,E. tarda and A. salmonicida,and 1010-103cfu/mL of V. anguillarum, P. fluorescens and M. marinum was detectable.(5) The bacterial antigen components, including M-like protein, capsular polysaccharide, and extracellular products of S. iniae, OMP, extracellular products, and flagellum protein of E. tarda, V.anguillarum, A. salmonicida, and P. fluorescens, cell wall-associated proteins of M. marinum, were extracted respectively. Liquid from 6 kinds of broken bacteria were prepared as well, and the dilution concentration of liquid is 1 mg/mL. The samples were added onto the matrix in the form of 3 X 3,the specific antigens were screened depending on whether they could react with the antiserum of Japanese flounder immunized by corresponding bacteria.The detection result of the antibody level in Japanese flounder with antigen microarray revealed that the color of the spot on microarray gradually deepened after further immunization. The antigen microarray showed the deepest red purple color on the fourth week after flounder immunized with S. iniae, indicating that the concentration of antibody reached the maximum level. The antibodies of flounder immunized with other bacteria didn't reach the maximum level until the fifth week, then decreased gradually with time extend, which are consistent with the detection results of ELISA.The purified specific antigens from 6 kinds of bacteria were added onto the same matrix to construct the antigen microarray, and diagnosis the pathogenic bacteria by detecting the antibodies in blood serum of fish. The results indicated that the anti-serum of Japanese flounder, produced by injecting with sterile PBS solution, showed no any reaction except for the spot of positive control, which appear the dark magenta inside. For the flounder anti-serum immunized by S. iniae, all of the spot of antigens from S. iniae were present dark magenta, suggesting the infection of S. iniae. However, no reaction or only part reaction were observed between the flounder anti-serum and antigens from other bacteria. Similar results were obtained for antiserum of flounder immunized with other five kinds of bacteria, suggesting that the antigen microarray constructed can lead to an accurate diagnosis.(6) The antibody microarray was applied in the diagnosis of pathogenic bacteria in Japanese flounder artificially infected with S. iniae or E. tarda, and the bacteria could be detected accurately and specifically. Additionally, the mixed liquid of V. anguillarum, P. fluorescens and M. marinum were injected to the flounder, when detected with the single detection antibody, the corresponding pathogenic bacteria could be detected; when detected with the multiple detection antibody,3 strains of bacteria could be detected at the same time. For diagnosis of the pathogens in naturally diseased puffer fish, the antibody microarray showed the positive signals of S. iniae, V. anguillarum, A. salmonicida and P. fluorescens, suggesting a combined infection. The antigen microarray was used to detect the antibody of pathogen in the blood serum of Japanese flounder infected with S. iniae or E. tarda, and all antigen components from S. iniae or E. tarda on the chip were stained deeper red. The results to detect the diseased puffer fish and health flounder indicated negative reaction. The similar results were obtained to the same samples by ELISA.In conclusion, this paper developed the immunochips for diagnosis of 6 kinds of pathogenic bacteria in cultured fish, and applied in the detection of pathogens and antibody in fish. This study indicated that the constructed antibody microarray could be used to detect multiple bacteria parallelly, rapidly and specifically with minimal sample consumption, and it will have extensive prospect in high-throughput diagnosis of pathogenic bacteria in aquatic animals.The antigen microarray constructed with the purified specific antigens could detect the antibodies against various pathogens on one slide, suggesting it was a suitable format for the serodiagnosis of bacterial disease in fish.