We know that it is not easy to cultivate the industry, and seek the prosperity of the aquatic industry. Not misleading, not exaggerating, not hyping, not headlined the party. With the context of breeders, let you enjoy a fine product in a short period of time!
Yellowtail (Pelteobagrus fulvidraco Richardson) is a relatively widely distributed small fish with relatively high economic value in China's main freshwater waters, and is well received by consumers because of its tender meat, delicious taste and few intermuscular spines. In recent years, in some parts of Jiangsu, Zhejiang, Guangdong and Sichuan Provinces, the scale and intensification of artificial cultivation of yellow jaw fish have been increasing, and some farmers have also obtained relatively good economic benefits due to the success of yellow jaw fish farming. With the rapid expansion of the scale of yellow jaw fish farming and the great increase in the degree of intensification, the harm of various diseases to yellow jaw fish is also becoming more and more serious, of which bacterial diseases are a type of disease that is more seriously harmful to the culture of yellow jaw fish. The pathogens of bacterial diseases reported in China mainly belong to some species of aeromonas, such as aeromonas subsppunctata, which can cause gills, enteritis, printing disease, hemorrhagic edema and other symptoms in yellowjabed fish.
Since May 2012, we have conducted a preliminary study of the pathogenic bacteria of yellow jawfish that appear in captivity rearing in Yancheng, Jiangsu Province, and Linghu Lake, Zhejiang Province. Yellow jaw fish with typical diseases were collected from diseased ponds, bacteria were isolated from ascites, liver, kidneys, etc., and the results were obtained with colonies with highly consistent morphology on the culture medium, and the results of artificial infection with healthy yellow jaw fish confirmed that the isolated strain was the pathogen that caused "split head disease" in yellow jaw fish. After the use of classical physiological and biochemical index determination and 16SrDNA homology and phylogenetic analysis, the pathogenic bacteria that cause "split head disease" in yellow jaw fish were determined to be Edwardsiella ictaluri. The findings are reported below.
1 Materials and methods
1.1 Experimental materials
Sick yellowtail with typical symptoms of "split head disease" (Fig. 1) was harvested from ponds raised in some farms in Yancheng District of Jiangsu Province and Linghu District of Zhejiang Province from May to October 2008. The body length of these diseased yellow jawfish ranges from 3.0 to 13.0 cm, and the healthy yellow jaw fish is taken from the yellow jaw fish culture pond of Dafeng Farm of Dahao Aquatic Products (Yancheng) Co., Ltd. in Yancheng, Jiangsu Province, with a body length of about (5.5±1.6) cm.
1.2 Pathogenic bacteria isolation
Yellow jaw fish with typical "split head disease" disease, after disinfection of the body surface with 70.0% alcohol, under sterile conditions, according to the method of sterile operation, sampled from its liver, kidneys, gills, ulcerated skin and ascites and other lesions, inoculated on BHI (Difco) agar plates, incubated in a 36 °C incubator for 72 h, picked up dominant colonies with roughly the same morphological characteristics for isolation, purification 3 times, transferred to test tube inclined medium, and stored under low temperature conditions.
1.3 Artificial poison attack
Healthy, lively, trauma-free yellowtail with an average body length of (6.3±0.6) cm was selected as the test fish. 30 yellowtails are raised in plastic aquariums of 1.0 m× 1.0 m × 0.5 m per day, fed an artificial compound feed equivalent to 2.0% of the fish's body weight (special compound feed for yellow jaw fish produced by Yancheng Yuda Feed Co., Ltd.). After domestication for 10 days, the yellow jaw fish to be tested to adapt to the environment and confirm that there are no symptoms of disease, the trial is started. During the test period, the average daily feeding water was about 50.0%, continuous oxygenation and timely cleanup. Four experimental groups and two control groups were set up, with 30 yellowtails per group. Five strains isolated from diseased yellowtail and purified culture were used in BHI medium for 72 h, and the colonies were washed off with sterilized saline and diluted to a concentration of 3.2× l07cfu/mL, which was used as a venom solution.
Two methods are used to attack the virus by live bacteria, one is the injection method. That is, after directly injecting 0.1 mL of live bacterial liquid with a concentration of 3.2 × l07cfu/mL at the base of the pectoral fin of each yellowtail, it was placed in an aquarium for further rearing observation (Figure 4). The bacterial solution prepared by each strain was used in 2 parallel groups as the injection test group, and the other group of yellow jaw fish was injected with the same dose of sterilized saline as the control group of the injection method.
The other is the soaking method to attack the poison, that is, first add an appropriate amount of live bacteria liquid to the glass beaker, so that the concentration of bacteria in the water reaches 3.2×l05cfu/mL, and then the yellow jaw fish that scratches the surface of the body with a steel wire ball is placed in the glass beaker for more than 1.0 hours to ensure that the test fish can fully contact the pathogenic bacteria (Figure 5). After soaking, the yellowtail is put back into the plastic aquarium and continues to be reared and observed.
After different methods of live bacteria attack, the yellow jaw fish is recorded in the process of rearing and observation for 15 days, and the yellow jaw fish with the same typical "split head disease" symptoms as natural conditions is taken, and the pathogens are re-isolated and identified from the ascites, gills, liver and ulcerated skin.
1.4 Determination of physiological and biochemical indicators
For the determination of physiological and biochemical characteristics of pathogenic bacteria and related classification and identification, references are made to "Common Identification Methods for General Bacteria" (Bacterial Classification Group, Beijing Institute of Microbiology, Chinese Academy of Sciences, 1978) and "Berger Bacterial Identification Manual (Ninth Edition)" (Buchanan and Gibbons, 1994).
1.5 16SrDNA sequence homology analysis and construction of phylogenetic tree
1.5.1 Preparation of template DNA
Single colonies were selected and suspended in 50.0 μL of deionized water, heated in a 100.0 °C water bath for 5.0 min, and centrifuged at a speed of 12,000 r/min for 20 min at 4 °C, and the supernatant was used as the template DNA.
1.5.2 PCR amplification
Using the bacterial 16SrDNA broad-spectrum primer, forward 27F:5'-AGAGTTTGATC (C/A)TGGCTCAG-3' (corresponding to the 8-27 bp position of Escherichia coli 16SrDNA), reverse 1492R:5'-TACGG(C/T)TACCTT GTTACGACTT-3'(corresponding to E. COLI 16S rDNA at position 1492-1510 bp) for PCR amplification. The PCR reaction system (100 μL) contains 10.0 μL of 10.0 × PCR buffer (with Mg2), 2.0 μL of l0.0 mmo/L 4×dNTP, 5.0 μL of 10.0 μmol/L forward and reverse primers each, 1.0 μL of Taq DNA polymerase (5U), 10.0 μL of template. The PCR reaction conditions were: denaturation at 94°C for 4 min, equilibrium at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 1min and 40s for 30 cycles, and incubation at 72°C for 6min. PCR amplification products are purified and sequenced by Shanghai Bioengineering Technology Co., Ltd.
1.5.3 Sequence Analysis
The measured strain 16SrDNA sequence was entered into the GenBank database for Blast comparison, and together with the similar sequences obtained from the GenBank database, the Clonex1.8 software was used to perform multiple sequence matching arrangements to generate MEGA format files. Using MEGA 3.0 software, a phylogenetic tree is built using neighbour-joining. Confidence assessment of the phylogenetic tree was performed by Bootstrapping, and the self-development dataset was 1,000 times.
1.5.4 Nucleic acid sequence
The relevant strains used to construct the phylogenetic tree and their sequence access numbers in the GenBank nucleotide sequence database are shown in Table 1.
2 Results
2.1 Symptoms and death status of yellow jaw fish after the artificial live bacteria attack
After the yellow jaw fish has been artificially infected with isolated strains in different ways, the number of deaths and the time of initiation of death are different, and the detailed results are shown in Table 2.
As shown in Table 2, it can be seen from the number of infected yellow jaw fish deaths and the time when they start to die that the virulence of isolated strains is different. The earliest death of the yellow jaw fish after injection occurred at the 48th hour after the injection of the strain, and the mortality rate of the immersion attack was relatively low, while the mortality rate of the injected attack group was relatively high, and the mortality rate of the experimental group reached 100.0%. The symptoms of the test fish before death are similar to those of diseased fish under natural conditions, mainly showing hyperemia on the body surface and head. The control group had normal yellow jawfish activity and did not die. Infected bacteria were isolated again from the gills, liver, kidneys, ascites and ulcerated skin of the diseased fish, and the test results were in line with Koch's law.
Under the test conditions, the diseased fish with symptoms of the disease have reduced feeding, slow swimming, often swimming alone or vertically suspended on the surface of the water, and have symptoms of non-stop rotation; the body surface (including fins, fin bases, head, gill caps, mouth, jaw, ventral surface and other body surfaces) have different degrees of bleeding; abdominal swelling, ascites; liver congestion or bleeding, or due to blood loss is pale, accompanied by back and head bleeding, skin ulceration, mortality is also very high.
2.2 Identification of physiological and biochemical characteristics of pathogenic bacteria
Dominant strains were isolated from the ascites, liver, kidneys, gills, ulcerated skin, etc. of yellow jaw fish with typical diseases, and the colony morphology, size, and color were consistent on the BHI agar plate: the colonies were round, neatly edged, off-white, and moist. The results of the physiological and biochemical characteristics analysis of these five strains are shown in Table 3.
From the results shown in Table 3, it can be seen that the results of gram staining, flagella staining and other photocopic observations and physiological and biochemical index determination results show that oxidase negative, arginine bihydrolyticase negative, urease negative, H2S positive, indole positive, lactose hydrolysis negative, sucrose hydrolysis negative, rhamnose hydrolysis negative, contact enzyme positive, lysine decarboxylase negative, citrine negative, gelatin hydrolysis negative, ornithine decarboxylase positive, catalase negative, V.P experiment negative, methyl red test positive, Malondiic acid use negative, horse chestnut glycoside hydrolysis negative, L-arabinose hydrolysis negative, D-mannose utilization positive, sorbitol hydrolysis negative, maltose hydrolysis positive and glucose fermentation acid production, gas production; after 16S rDNA sequence analysis found that all their indicators are consistent, so take one of them HSY-12-02 (isolated and purified from the kidney) as the representative strain, follow-up experiments.
2.3 Phylogenetic analysis of 16S rDNA sequences
PCR results of HSY-12-02 strains were obtained from the kidneys of diseased yellowtail with Eduardo catfish (Chinese Microbial Culture Center) as a control (Figure 6). The homologous sequence search results in GenBank show that the l6S rDNA sequences of HSY-12-02 and Edvardinae catfish bacteria are naturally clustered, and more than 10 results with the highest similarity to experimental bacteria belong to Edwardsiella, of which the homology with Edwardiana catfish is the highest, reaching 99.0%. In the search results, 9 reference strains were selected, and their l6S rDNA sequences were downloaded from Genbank to construct a phylogenetic tree; 2 standard strains of the same species in the phylogenetic tree were closely related and clustered into 1 branch, and the experimental strains and Eduardella catfish naturally converged into one, indicating that they were most closely related, so we preliminarily identified them as Edwardsiella ictaluri.
3 Discussion
There are three main methods for identifying pathogenic bacteria causing fish diseases after isolation, namely, the traditional physiological and biochemical index determination method, the method identified by the automatic identification system, and the method of identification by molecular biology means (Guo Mingyuan et al., 2006). These methods have their own characteristics and shortcomings, the traditional physiological and biochemical experiments because the operation process is more cumbersome, time-consuming and affected by subjective factors are relatively large; 16SrDNA phylogenetic tree analysis by comparing the homology of bacterial ribosomal RNA gene fragments to achieve taxonomic identification of bacteria, reliability is relatively strong, however, so far there are still a variety of bacteria Complete 16SrDNA sequence has not been determined, therefore, to the results of the newly identified bacteria Accuracy may also have a certain impact, It is best to have the results of biochemical characteristics as evidence (Fan Wenhui et al., 2005). In this study, on the basis of using physiological and biochemical indicators to determine the characteristics of its strains, it was also identified by molecular biology methods, and the two methods verified each other, and the results obtained should be relatively accurate.
According to the results of existing studies, Edwardiana catfish has a relatively special parasitic state, that is, when the edemaciella entering the fish body is engulfed by phagocytes, it is not killed by the cells, but can be carried into the liver and kidney sinuses with the phagocytes, and can also proliferate in the phagocytes, thereby destroying these phagocytes. The spread of Edwardian salmonella can infect the endothelial cells around the blood-like sinuses and form a tiny lesion there, where it gradually enlarges (Wakabayashi and Egusa, 1972; Wakabayashi and Egusa, 1973). Compared with other fish that develop Edward's disease, the most typical endemic symptom after the development of Edwardellosis in yellow jaws is redness of the head (this symptom is similar to the diseased spotted forktail catfish), the abdomen is enlarged and a large amount of blood-red ascites, and bleeding spots or blood spots appear on the surface of the body.
In addition, Edwardia can infect thermotropic animals such as snakes, lizards, turtles, crocodiles, and thermostatic animals such as birds, skunks, pigs, horses, and rabbits, and it is believed that Edwardia is one of the members of the normal intestinal flora of snakes (Sakazaki, 1965).
(Authors: Chen Fuchang, Song Gangjie, Sun Liwei, Wu Qiangqiang, He Dengping, Zhang Jialin College of Fisheries, Huazhong Agricultural University, Suzhou Tongwei Special Feed Co., Ltd.)
Aquatic practitioners, conspire for the prosperity of the fishery! Please pay attention to my headline number @ Fisherman Liu Wenjun, for more wonderful content, please log on to the southwest fishery network and fish farming first-line public number, this article original if you need to reprint please indicate the source, if there are different opinions or content supplements, please share private messages or messages or comments! #Aquaculture##Fish Disease##Fish##Aquatic##鱼药 #