SN/T 5189-2020Technical specifications for quarantine of catfish intestinal sepsis (English PDF)

4.1 Group inspection

Diseased fish ponds are often of poor water quality. The diseased fish swim alone along the edge of the pond, are slow to respond, decrease their food intake, or do not eat. In the early stage of illness, the disease Fish pectoral fins often have damages with a diameter of 3 mm to 5 mm, such as needle-like wounds on the outside, and penetrate deep into the muscles. Some sick fish have their heads up and their tails up the bottom is swimming vertically, and some diseased fish sometimes swim in a convulsive spiral shape, and then die.

4.2 Individual examination

Due to differences in catfish specifications and individual immunity, the clinical symptoms of catfish intestinal sepsis are not the same, mainly showing acute and chronic Type in two forms.

Acute type, also known as intestinal sepsis type, is the most common type with high mortality, mainly manifested as sepsis and enteritis. Fading on the surface of sick fish Spots, swelling of the abdomen, some skin ulcers on the head and body, prominent eyeballs on one or both sides, and a bit of bleeding or bleeding spots on the muscles. Gill silk green White with bleeding spots, ecchymosis and bleeding on the skin of the operculum, abdomen, jaw and fin base, while some have white spots, red and swollen anus, in the later stage, the diseased fish showed generalized edema.

The chronic type is also known as the cranial perforation type. The initial infection site of the bacteria is the olfactory sac of the root of the nose, and then migrate to the brain through the olfactory organs to form flesh.

Swelling inflammation. Later, the side of the head and dorsal skull ulcerated to form a deep hole, until the entire brain tissue was exposed, forming a "saddle"-like lesion;

As the disease progresses, ulcers form in the center of the lesion, which manifests as a "cephalic perforation type" disease.

4.3 Anatomy

Open the abdominal cavity of the diseased fish, there is a lot of blood or clear fluid in it, and the ascites flowing out is not easy to coagulate. Liver edema, brittle texture, bleeding spots and Gray-white necrosis; spleen and kidney swelling and bleeding; gastric enlargement; intestinal dilatation, intestinal mucosa edema, congestion, inflammation, intestinal cavity full of gas Body or pale yellow watery liquid; sometimes accompanied by the appearance of the intestine. The brain tissue forms granulomas, and the muscles have diffuse granulomatous inflammation.

4.4 Judgment by clinical examination

The above symptoms appear in the group examination and individual examination, combined with the results of the necropsy, it can be preliminarily judged as suspected catfish intestinal sepsis.

Further laboratory diagnosis.

5.1 Equipment

5.1.1 Optical microscope.

5.1.2 Incubator (28 °C ± 1 °C and 37 °C ± 1 °C).

5.1.3 Water bath.

5.1.4 High-speed centrifuge.

5.1.5 Micro adjustable pipette.

5.1.6 Thermal cycler.

5.1.7 Electrophoresis instrument.

5.1.8 Ultraviolet gel imaging system.

5.1.9 Alcohol lamp.

5.2 Materials and reagents

5.2.1 Water should meet the requirements of Grade 1 water specified in GB/T 6682.

5.2.2 For blood agar plates, see Appendix A.1 for the preparation method.

5.2.3 Lu's alkaline methylene blue dye solution, the preparation method is shown in Appendix A.2.

5.2.4 Crystal Violet Staining Solution, the preparation method is shown in Appendix A.3.

5.2.5 Gram iodine solution, the preparation method is shown in Appendix A.4.

5.2.6 Safranine counterstain solution, the preparation method is shown in Appendix A.5.

5.2.7 Semi-solid agar tube. See Appendix A.6 for the preparation method.

5.2.8 Tetramethyl-p-phenylenediamine 1% aqueous solution, the preparation method is shown in Appendix A.7.

5.2.9 Hugh-Leifson medium. See Appendix A.8 for the preparation method.

5.2.10 Phosphate buffered saline (PBS), see Appendix A.9 for the preparation method.

5.2.11 The preparation method of TE buffer (pH 8.0) is shown in Appendix A.10.

5.2.12 Sodium dodecyl sulfate (SDS).

5.2.13 Proteinase K.

5.2.14 Sodium chloride (NaCl).

5.2.15 Cetyltriethylammonium bromide (CTAB).

5.2.16 Chloroform.

5.2.17 Isoamyl alcohol.

5.2.18 Water saturated phenol.

5.2.19 70% ethanol.

5.2.20 Taq DNA polymerase.

SN/T 1589-2020

5.2.21 Deoxynucleotide triphosphate dNTPs (including dCTP, dGTP, dATP, dTTP).

5.2.22 Primer. 10 µmoL/L. Designed according to the sequence of the Eipase gene (Eip) of the Edwardsiella catfish, and the amplified length the degree is 276 bp.

Upstream primer ESC-CM3.5'-TCA TCA CAT CAT CTA GCG ATT-3' Downstream primer ESC-CM4.5'-CTT TAC ACA GAT AGG CGA TAC-3'

5.2.23 Agarose (electrophoresis pure).

5.2.24 TAE electrophoresis buffer, the preparation method is shown in Appendix A.11.

5.2.25 Loading buffer, see Appendix A.12 for the preparation method.

5.2.26 DNA relative molecular mass standard marker, it is recommended to use a 100 bp DNA ladder.

5.2.27 DNA purification and recovery kit.

5.2.28 Edwardsiella spp., the number of the bacterial species collection is ATCC 33202.

5.3 Collection of samples

The collection and transportation of samples shall be carried out in accordance with the regulations of SC/T 7103.

5.4 Isolation and identification of bacteria

5.4.1 Sample processing

Asymptomatic fish can be aseptically collected from internal organs such as intestines, kidneys, liver and brain as samples, or directly collected from lesion tissues as samples.

Samples, the samples were washed with sterile saline and mashed and then separated and cultured. For fish with clinical symptoms on the body surface, sterile A cotton swab or a sterilized inoculation loop is inserted into the tissue deep within the lesion for sampling.

5.4.2 Separation of bacteria

The samples were streaked and inoculated on the blood agar plate according to the conventional method (see Appendix A.1 for the preparation method), and incubated at 28 °C ± 1 °C for 24 h ~ 48 h. pick Take a single dominant colony that is round, off-white, neat, moist, and translucent, and streaked on the blood agar plate for separation and purification.

5.4.3 Staining and morphological observation

5.4.3.1 Methylene blue staining of diseased smears

Take the kidney, liver, brain and other tissues of the diseased fish for smears, fix them on the flame of an alcohol lamp, and cool them with Lu's alkaline methylene blue stain (prepared for the method, see Appendix A.2) Stain for 1 min~3 min, wash with water, and check with microscopy after drying.

5.4.3.2 Gram stain of culture smear

After the culture is smeared, it is fixed on the flame of an alcohol lamp, and crystal violet staining solution is added dropwise (see Appendix A.3 for the preparation method), stained for 1 min and then washed with water.

Then add gram iodine solution dropwise (see Appendix A.4 for the preparation method), and wash with water after acting for 1 min. Add 95% ethanol dropwise to decolorize for about 30 s; or decolorize 95% After ethanol is dripped over the entire smear, pour away immediately, and then use 95% ethanol to drip the entire smear and decolorize for 10 s. Wash with water and add sahuang re-staining solution (with for the preparation method, see Appendix A.5) Counterstaining for 1 min. Rinse with water and check with microscope after drying.

5.4.3.3 Bacterial morphology

Edwardsiella spp. Gram staining is negative, rod-shaped, mostly singly, without spores, and flagella.

5.4.4 Physiological and biochemical tests

5.4.4.1 Exercise test

The isolated bacteria were punctured and inoculated into two tubes of semi-solid agar (see Appendix A.6 for the preparation method), and set them at 28 °C±1 °C and 37 °C±1 °C respectively.

SN/T 1589-2020

Cultivate for 24 h. If the isolate spreads from the puncture line to the surroundings, its edge is cloudy, which is positive for motility; if the isolate only grows in the piercing the edge of the thorn line is very clear, and it is motility-negative.

5.4.4.2 Oxidase test

Pipette a 1% aqueous solution of tetramethyl-p-phenylenediamine (see Appendix A.7 for the preparation method) and drop it on the bacterial colony. The colony is rose red or dark Purple is masculine, and colorless is feminine.

5.4.4.3 Glucose oxidation and fermentation (O/F) test

Pick the isolates, puncture and streak on Hugh-Leifson medium (see Appendix A.8 for the preparation method), and cultivate at 28 °C ± 1 °C 18 h~24 h, if both the open tube and the medium in the sealed tube produce acid (yellow), the isolate is fermented (F), if the open tube turns yellow If the color of the sealed tube does not change, it is oxidized (O).

5.4.4.4 Other biochemical tests

Observe the isolates that meet the requirements of 5.4.3.3 by microscopy, when the following conditions are met. the exercise test is positive at 28 °C and at 37 °C it is negative; the oxidase test is negative; the O/F test is a fermentation type, and the isolate can be identified as Enterobacteriaceae. Enterobacteriaceae isolates can be According to SC/T 7014 and SC/T 7214, the biochemical characteristics of Edwardsiella spp. are tested (see Appendix B).

5.4.4.5 Judgment

The isolate meets both the characteristics of Enterobacteriaceae and the results of Appendix B, and the isolate is identified as Edwardsiella serrata. If it is part of Standards should be further identified by PCR.

5.4.5 Polymerase Chain Reaction (PCR)

5.4.5.1 DNA extraction

You can take the brain, liver, spleen or kidney of fish, add PBS buffer at 1.10 (W/V) (see Appendix A.9 for the preparation method), After homogenization as a sample, centrifuge at 12 000 r/min for 1 min, and pour the supernatant; it can also be picked from ordinary nutrient agar plates and cultured purely.

The suspicious single colony was used as a sample, suspended in 1 mL of PBS buffer, centrifuged at 12,000 r/min for 1 min, and the supernatant was decanted. In precipitation Add 567 µL of TE buffer (pH 8.0) (see Appendix A.10 for the preparation method), after mixing, add 30 µL of 10% SDS and 3 µL Proteinase K (20 mg/µL), mix well. After warming the bath at 56 °C for 1 h, add 100 µL of NaCl (5 mol/L) and mix well. Add 80 µL CTAB/NaCl solution (10% CTAB and 0.7 mol/L NaCl), mix well. After incubating at 65 °C for 10 min, add equal volume of chloroform/isoamyl Alcohol (24.1 by volume), mix well, centrifuge at 12,000 r/min for 10 min, take the supernatant, and add an equal volume of phenol/chloroform/isoamyl alcohol (body the product ratio is 25.24.1), mix well, centrifuge at 12,000 r/min for 10 min, take the supernatant and add 0.6 times the volume of isopropanol, mix gently, 12,000 r/min Centrifuge for 10 min, take the precipitate, wash twice with 70% ethanol, dry, and add 100 µL of TE buffer (pH 8.0) to dissolve it. Can also be used Equivalent commercial DNA extraction test kit.

5.4.5.2 PCR amplification

The PCR reaction system includes 2.5 µL of 10×PCR buffer, 0.5 µL of 2.5 mmol/L dNTP, and 10 µmol/L upstream and downstream primers.

Each of 0.5 µL, 5 µL of template DNA, 0.5 µL of 5U/µL of Taq DNA polymerase, add water to 25 µL. The PCR reaction conditions are.

Pre-denaturation at 94 °C for 5 min, then denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 45 s, a total of 35 cycles; 72 °C supplement Extend for 10 min and keep warm at 4 °C. The standard E. coli strain was used as a positive control, E. coli was used as a negative control, and water was used as an empty control.

White control. An equivalent commercial PCR amplification test kit can also be used.

5.4.5.3 Agarose gel electrophoresis

Use TAE running buffer (see Appendix A.11 for the preparation method) to prepare a 2% agarose gel. Put the gel in the horizontal electrophoresis tank so that SN/T 1589-2020 the running buffer just flooded the gel surface. Put 6 µL sample and 1 µ...