Some standard content:
National Standard of the People's Republic of China
Freshwater fish quarantine methods Part 1
The quarantine methods of freshwater fish-Part 11 Subject content and scope of application
GB/T 15805.1—1995
This standard specifies the quarantine methods for freshwater fish infectious pancreatic necrosis (IPN), infectious hematopoietic necrosis (IHN), viral hemorrhagic septicemia (VHS), spotted forktail crucian carp virus disease (CCVD), spring carp virus disease (SVC), scabies, vibriosis and vertigo. This standard applies to the quarantine of foreign freshwater fish imported from ports, and also to the quarantine of the same disease between regions in my country. 2 Equipment and instruments
Except for special requirements according to various disciplines, the equipment and instruments used in quarantine refer to the equipment and instruments commonly used in fish disease laboratories. 3 Infectious Pancreatic Necrosis (IPN) The pathogen of this disease is a double-stranded RNA virus.
This disease is prevalent in America, Europe and Asia. The main infected objects are salmonid fish such as rainbow trout and American trout within 6 months of age. Fish over 6 months old can be asymptomatic carriers of the virus, and fish eggs can also carry the virus. 3.1 Clinical examination
3.1.1 Activity
The diseased fish have disordered swimming and often swim vertically and then sink to the bottom and die. The time from the beginning of the rotation to death is 1 to 2 hours. In the breeding pond with water flow, fish that have lost the ability to swim can be seen sticking to the screen of the drain with the water flow. 3.1.2 External examination
The body color of the diseased fish is dark, the eyeballs are protruding, and the abdomen is swollen. There is bleeding on the abdominal wall and the base of the fins. The diseased fish often discharge thread-like mucous feces from the anus.
3.1.3 Autopsy
Except for pyloric bleeding, the liver and spleen are significantly discolored, there is usually no food in the digestive tract, and the stomach and the front of the intestine are swollen with milky white mucus. 3.2 Laboratory Test
3.2.1 Virus Isolation
3.2.1.1 Cell Preparation
a. Equipment and Instruments
Sterile room, clean bench, constant temperature incubator, low temperature refrigerator (30℃), liquid nitrogen tank, Saybolt funnel and 0.3, 0.45μm mixed cellulose ester microporous filter membrane, cell culture bottle, white rubber stopper, etc. b. Culture Medium and Reagents
Cell Growth Fluid, Trypsin-EDTA (ethylenediaminetetraacetic acid) Mixed Digestive Fluid, Calf Serum, L-Glutamine, Sodium Bicarbonate, Alcohol. The preparation method of cell growth fluid, etc. is shown in Appendix A (Supplement). Cell preparation before inoculation
Approved by the State Administration of Technical Supervision on December 8, 1995, and implemented on July 1, 1996
GB/T15805.1--1995
Infectious pancreatic necrosis virus is isolated from CHSE-214 and RTG-2 cells. The optimal culture temperatures for CHSE-214 and RTG-2 are 21℃ and 20℃, respectively. Select cells that grow well and form a monolayer, pour out the growth solution and add an appropriate amount of trypsin-EDTA mixed digestion solution for digestion, then add growth solution, and divide the digested cells in the original bottle into 2 or 3 culture bottles at a seeding rate of 1:2 or 1:3, and continue to culture at a suitable temperature. When the monolayer density reaches about 80%, it can be used for virus inoculation. 3.2.1.2 Sample inoculation
a. Equipment and instruments
Same as the provisions of 3.2.1.1 a, with the addition of a refrigerated centrifuge and a 1,2 mL pipette. b. Commonly used solutions
Cell maintenance medium, phosphate buffered saline [preparation method see Appendix A (Supplement)]. c. Sampling
For symptomatic fish, take more than 10 samples for virus isolation. For asymptomatic fish, take more than 60 samples for virus isolation. For fish eggs, take 60 to 100 samples for virus isolation. For a small number of imported and exported adult fish, draw 1 to 2 mL of blood from the tail artery, and use the plasma for virus isolation and the serum for antibody detection. d. Sample processing
For larger fish, take the kidneys, liver and spleen. For fry and juvenile fish, take the whole body of the fish. If the fish has yolk sac, remove the yolk sac. For fish eggs, take the whole egg. Weigh the above-mentioned samples to be tested, add phosphate buffered saline (PBS) to homogenize, and dilute to 10-1, centrifuge for 30 minutes (4000r/min), discard the precipitate, sterilize the supernatant through a 0.45μm microporous filter membrane, and then dilute 10 times with PBS to form the sample. After the blood sample is placed at room temperature for 1 hour, it is placed at 4℃ for more than 12 hours to precipitate the serum. Then centrifuge at low speed, and the precipitate is used as the sample to be tested. The sample solution is prepared according to the above method, and the serum is used for neutralization test. e. Inoculation of cells
Pour out the culture medium in the cell bottle, and then inoculate the sample solution. The inoculation amount is 10-1 of the maintenance solution. After adsorption for 1 hour, add cell maintenance solution for culture, and set up a control at the same time.
3.2.1.3 Virus culture
The culture temperature of infectious pancreatic necrosis disease (IPNV) is 15-20℃. During the culture process, observe the changes in cells every day and observe for 7 days.
3.2.1.4 Result observation
If the inoculated sample contains virus, obvious cytopathic effect (CPE) will appear after 2-5 days of culture, with the main characteristics of cell disintegration and cell monolayer destruction. Collect the positive cell suspension, freeze and thaw once, centrifuge at low speed to remove cell debris, and use the supernatant for virus identification. If CPE is still not seen 7 days after inoculation, it is judged as a negative result. If suspicious CPE occurs within ? d, the bottle of cell culture fluid needs to be inoculated again as inoculation material to determine whether CPE exists.
3.2.2 Virus identification
3.2.2.1 Neutralization test [(Fixed serum dosage-dilution virus method) This test is used to identify whether the sample with CPE in cell culture is IPNV. 3.2.2.1.1 Test preparation
Cells RTG-2 cells are cultured in 96-well microplates. a
The virus is a standard IPNV strain, which is passaged to make a virus suspension. b.
Standard antiserum The standard serum against IPNV is inactivated at 56℃ for 60min before use. c.
d. Freeze and thaw the sample to be tested (cell culture with CPE) once, and remove the cell debris before use. 3.2.2.1.2 Operation steps
3.2.2.1.2.1 Use Hanks solution to make a series of dilutions of the sample to be tested, so that the dilution ranges from 10-1 to 10-3, and the content of each tube is 0.5mL. 3.2.2.1.2.2 Take two rows of test tubes, take out 0.2mL of the samples to be tested from the above dilutions and put them into the two rows of test tubes, then add 0.2mL of anti-IPNV standard serum to the first row of tubes, and add 0.2mL of maintenance solution to the second row of tubes, and mix them thoroughly. 3.2.2.1.2.3 Incubate at 30℃ for 60min.
3.2.2.1.2.4 After incubation, inoculate the samples from the first and second rows of tubes into a 96-well microtiter cell culture plate that has grown a monolayer of RTG-2 cells, 4 wells for each dilution, 0.1mL per well. 3.2.2.1.2.5 Dilute the anti-IPNV standard serum in multiple ratios (1:8 to 1:64) and inoculate it into the above culture plate, 4 wells for each dilution, 0.05mL per well, as a standard antiserum control. Inoculate 8 wells with standard IPNV, 0.05 mL per well, as a virus control, and the remaining 8 wells as normal cell controls.
3.2.2.1.2.6 Add maintenance solution to each well to a total of 0.2 mL3.2.2.1.2.7 Place in a 20°C incubator for culture, observe the lesions daily, and record the results (fill in according to Table 1). Table 1 Neutralization test 1 result record sheet
Test sample-standard serum
3.2.2.1.3 Result determination
Test sample-maintenance solution
Standard serum
IPNV control
Cell control
According to the Reed and Muench methods, calculate the cell half-lethal dose (TCID5o) of the test sample-standard serum and the test sample-maintenance solution, respectively. The antilogarithm of the difference in the titer index between the two is the neutralization index. If the normal cells and standard serum control are negative, and the standard virus control is positive, the result is determined according to Table 2.
Neutralization index
10～50
3.2.2.2 Neutralization test I (fixed disease-dilution serum method) This test is used to identify whether fish serum contains neutralizing antibodies against IPNV. 3.2.2.2.1 Test preparation
RTG-2 cells are cultured on a 96-well microplate virus
Result determination
Negative (the subject to be tested is not virus-carrying)
Standard IPNV, diluted to 50TCID5/0 with Hanks solution before use.1mL. The serum to be tested and normal serum (negative serum) are diluted with an equal amount of Hanks solution (containing 200 IU of penicillin and 489
GB/T15805.1—1995
200 mg of streptomycin per mL), inactivated at 45°C for 30 min, and used after cooling. 3.2.2.2.2 Operation steps
3.2.2.2.2.1 The inactivated serum to be tested is serially diluted with Hanks solution, with the dilution ranging from 1:4 to 1:256, and the content of each tube is 0.2mL.
3.2.2.2.2.2 Add 0.2mL (i.e., the same amount of serum to be tested) of 50TCIDso/0.1mL IPNV suspension to each of the above tubes, and shake well to mix.
3.2.2.2.2.3 Incubate at 30℃ for 60 min. 3.2.2.2.2.4 Take out and inoculate in 96-well micro-cell culture plates covered with RTG-2 cell monolayers, 4 wells for each dilution, 0.1 mL per well
3.2.2.2.2.5 Set up normal serum control according to the above steps; dilute the standard IPNV suspension 10 times continuously (10-1～10-7) and inoculate the above culture plates, 4 wells for each dilution, 0.1 mL per well, as a control for the toxicity titer of the virus suspension; 12 wells in row H of the 96-well micro-cell culture plate are used as normal cell controls.
3.2.2.2.2.6
Add cell maintenance solution to each well to a total of 0.2 mL. 3.2.2.2.2.7 Place in a 20℃ incubator for culture, observe the lesions daily, and record the results (fill in according to Table 3). Table 3 Neutralization test I result record
1:16
1:128
G1:256
H cell control
Blood to be tested
3.2.2.2.3 Result judgment
Normal serum control
Virus titer control
According to the lesions, the antibody titer in the serum is calculated by Reed-Muench method or Karber method (that is, the highest serum dilution that can protect 50% of the cell culture wells from lesions). Serum neutralizing antibody titer greater than or equal to 1:16 is positive, and less than 1:16 is negative. 3.3 Comprehensive judgment
Necrosis.
If the above clinical symptoms appear, CPE appears after cell culture, and the neutralization test is positive, it can be judged that the patient has infectious pancreatitis b. If there are no clinical symptoms, but CPE appears in cell culture and is positive in neutralization test, it is determined to be an IPNV carrier. If the neutralization test is suspicious, it is determined to be suspicious. c.
d. If there are no clinical symptoms, no CPE appears in cell culture, but the anti-IPNV neutralizing antibody in the fish serum is positive, it is determined that the patient has suffered from infectious pancreatic necrosis in the recent period.
e. If there are only clinical symptoms, or CPE only appears in cell culture, but cannot be neutralized by anti-IPN neutralizing antibody, it cannot be said that the patient has IPN and needs to be further examined for other diseases. 490
GB/T15805.1—1995
4 Infectious Hematopoietic Neorosis (IHN) The pathogen of this disease is a rhabdovirus.
This disease is prevalent in the United States, Canada and Japan. It mainly harms the juveniles of salmonids such as rainbow trout and red-spotted salmon. One-year-old fish may also be infected. When the water temperature is above 15℃, the fish are asymptomatic and carry the virus. The fish eggs may also carry the virus. 4.1 Clinical examination
4.1.1 Activity
The sick fish initially swims slowly and floats weakly with the water. Then it swims swayingly with the current. Although it moves like a spasm, it soon floats up, swims sideways and often swims rapidly, and soon dies. The frantic running movement seen during this period is a characteristic of this disease. 4.1.2 External examination
The body color of the sick fish is dark, and there is usually a semi-transparent feces dragging from the anus, which is also a characteristic of this disease. The abdomen is swollen with ascites and the eyeballs are protruding. Anemia, the gills are significantly faded. There is often bleeding from the base of the pectoral fin, the dorsal fin to the front, and the trunk muscles near the anus, and there are also bleeding spots on the oral wall. The yolk sac of fry with yolk is bleeding, filled with serous fluid and swollen. 4.1.3 Autopsy
The spleen and kidney are necrotic, and there are often bleeding spots in the fat tissue around the spleen and pyloric sac, peritoneum, brain and pericardium, and there is also bleeding in the intestine. 4.2 Laboratory Test
4.2.1 Virus Isolation
4.2.1.1 Cell Preparation
Same as 3.2.1.1, except that the infectious hematopoietic necrosis virus (IHNV) can be isolated by CHSE-214 cells, and the most suitable cell for virus isolation is FHM, and the culture temperature is 28℃. 4.2.1.2 Sample Inoculation
Same as 3.2.1.2.
4.2.1.3 Virus Culture
The culture temperature of IHNV is 15℃. During the culture process, observe the changes of cells every day, and the observation ends at 7 days. 4.2.1.4 Result observation
If the inoculated sample contains virus, CPE will appear after 3-5 days, and the cells will aggregate into clusters, like grapes, and then disintegrate. If CPE appears, freeze and thaw once, centrifuge at low speed to remove cell debris, and use the supernatant for disease identification. If there is no CPE within 7 days, it is a negative result.
If suspected CPE phenomenon appears after 7 days, the bottle of culture fluid will be used as inoculation material and inoculated again to determine whether CPE exists. 4.2.2 Virus identification
Same as 3.2.2, except that IHN standard virus and IHN standard antiserum are used. FHM or CHSE cells are used, and the virus culture temperature is 15℃.
4.3 Comprehensive judgment
Same as 3.3.
5 Viral hemorrhagic septicemia (VHS) The pathogen of this disease is rhabdovirus.
This disease is endemic in Europe.
It mainly harms rainbow trout juveniles with a total length of about 5 cm and edible fish with a weight of about 200-300g. There are three types of symptoms: the acute type is seen in the early stage of the epidemic, the symptoms develop quickly, and the mortality rate is high. The chronic type can be seen after the early stage of the epidemic, the symptoms develop slowly, the bleeding in various parts of the fish body is not as obvious as the acute type, and the mortality rate is low. The neurological type is seen in the late stage of the epidemic. 5.1 Clinical examination
5.1.1 Activity
GB/T 15805.1—1995
In the late stage of the epidemic, the diseased fish sometimes twisted and swam in the water, sometimes swam sideways, and sometimes suddenly swam forward violently. 5.1.2 External examination
The body color of the diseased fish is dark, the eyeballs are protruding, the abdomen is swollen, anemia, and bleeding in the gills, fin bases, eyes and eye sockets. 5.1.3 Autopsy
Bleeding in muscles, visceral fat tissue, and intestines is prominent in the acute type. No specific lesions can be seen in the neurological type. 5.2 Laboratory tests
5.2.1 Virus isolation
5.2.1.1 Cell preparation
Same as 3.2.1.1. VHS is isolated using RTG-2 cells. 5.2.1.2 Sample inoculation
Same as 3.2.1.2. However, serum samples are generally not taken for antibody testing, because the neutralizing antibodies against VHSV in fish serum are complement-dependent and cannot be detected in the absence of complement. 5.2.1.3 Virus culture
Cultivate at 15°C and observe cell changes every day during the culture process. The observation ends after 7 days. 5.2.1.4 Result observation
If the inoculated material contains virus, RTG-2 cells will show CPE after 2-4 days under the condition of pH 7.6, the cells will shorten and then become spherical, and soon necrotize and fall off the bottle wall.
If CPE appears, freeze and thaw once, centrifuge at low speed to remove cell debris, and use the supernatant for virus identification. If there is no CPE within 7 days, it is a negative result.
If suspected CPE appears within 7 days, the bottle of cell culture fluid will be used as inoculation material for another inoculation to determine whether there is CPE. 5.2.2 Virus identification
For VHS identification, only samples that have CPE in cell culture are identified as VHS virus, and the method is the same as 3.2.2.1. The antiserum is changed to standard anti-VHS serum. RTG-2 cells are used, and the virus culture temperature is 15℃. 5.3 Comprehensive judgment
a. If the above clinical symptoms occur, CPE appears in cell culture, and the neutralization test is positive, the patient can be diagnosed with viral hemorrhagic sepsis.
b. If there are no clinical symptoms, but CPE appears in cell culture and the neutralization test is positive, the patient is determined to be a VHS virus carrier. c. If the neutralization test is suspicious, it is determined to be suspicious. d. If there are only clinical symptoms, or CPE can only appear in tissue culture,However, those who cannot be neutralized by anti-VHS serum cannot be said to have VHS and need to be further examined for other diseases. 6 Channel Catfish Virus Disease (CCVD) The pathogen of this disease belongs to herpes virus.
This disease is prevalent in the United States.
It is an acute infectious disease for channel catfish under 8 months old, with a mortality rate of up to 100%. Fish over 8 months old are in a state of being infected.
6.1 Clinical examination
6.1.1 Activity
The sick fish swims abnormally, spinning and twitching, and soon sinks to the bottom of the water, then floats vertically on the water surface and eventually dies. 6.1.2 External examination
The sick fish has protruding eyes and significantly faded gills. There is bleeding on the skin and fins. In the late stage of the disease, the abdomen of the fish is generally swollen and the anus is protruding. 6.1.3 Autopsy
GB/T15805.1—1995
The stomach is dilated and contains mucous fluid. Bleeding can be seen in the muscles, kidneys, liver, spleen, etc. 6.2 Laboratory Test
6.2.1 Virus Isolation
6.2.1.1 Cell Preparation
Same as 3.2.1.1, except that CCVD virus is isolated using BB cells, and the optimal culture temperature is 25-30℃. 6.2.1.2 Sample Inoculation
Same as 3.2.1.2.
6.2.1.3 Virus Culture
CCVD virus culture temperature is 25℃, and cell changes are observed daily during the culture process, and the observation ends at 7 days. 6.2.1.4 Result observation wwW.bzxz.Net
If the inoculated material contains virus, CPE will appear within 1-3 days, syncytia will begin to appear, and then the cells will disintegrate. The supernatant of cells with CPE can be used for virus identification.
No CPE within 7 days is a negative result.
If suspected CPE occurs within 7 days, the bottle of cell culture fluid will be used as inoculation material for another inoculation to determine whether CPE exists. 6.2.2 Virus identification
Same as 3.2.2. Use CCVD standard virus and CCVD standard antiserum. Use BB cells, and the virus culture temperature is 25°C. 6.3 Comprehensive judgment
Same as 3.3.
7 Spring Viremia of Carp (SVC) The pathogen of this disease is rhabdovirus.
This disease is prevalent in Europe.
It harms carp over one year old.
7.1 Clinical examination
7.1.1 Activity
The sick fish gathered at the water inlet. Although they breathed slowly, they soon sank to the bottom of the water. 7.1.2 External examination
The sick fish had a dark body color, bulging eyes, swollen abdomen, and red and swollen anus. There were bleeding spots on the gills. 7.1.3 Autopsy
The body cavity was opened and ascites was found. The intestine was severely inflamed. Almost all internal organs had bleeding spots, with the wall being the most common site. The muscles were also red due to bleeding. The spleen was significantly enlarged. Symptoms of jaundice were also seen. In addition, edema of the internal organs was also seen. 7.2 Laboratory tests
7.2.1 Virus isolation
7.2.1.1 Cell preparation
Same as 3.2.1.1. SVC virus was isolated using FHM cells. 7.2.1.2 Sample inoculation
Same as 3.2.1.2. However, fish eggs are generally not taken for virus isolation. 7.2.1.3 Virus culture
The optimal culture temperature for SVC virus is 20-22°C. During the culture process, observe the changes in cells every day and the observation ends after 7 days. 7.2.1.4 Result observation
If the inoculated material contains virus, CPE may appear within 2-4 days, characterized by cell rounding, followed by collapse and detachment. Others are the same as 3.2.1.4.
7.2.2 Virus identification
GB/T15805.1—1995
Same as 3.2.2. Use SVC standard virus and anti-SVC standard antiserum. Use FHM cells and the virus culture temperature is 20-22°C. 7.3 Comprehensive judgment
Same as 3.3.
8 Furunculosis
The pathogen of this disease is Aeromonas salmonicida. This disease is prevalent in Europe, the United States, Canada, Japan and other countries, and its prevalence is relatively wide. It mainly harms old salmonids. It is generally divided into three types: acute type, in which the diseased fish dies immediately and has no external symptoms; subacute type, in which the disease progresses slowly and forms infected lesions (furunculosis) in the trunk muscles; chronic type, in which the diseased fish is in a long-term carrier state. 8.1 Clinical examination
8.1.1 Activity
The diseased fish swims alone and moves slowly.
8.1.2 External examination
The body color of the diseased fish is dark, and there are bulges or ulcers on the trunk. The base of the fins is congested, and there are usually bleeding spots on the gills. 8.1.3 Autopsy
The intestines are congested and inflamed, the kidneys are softened and swollen, the spleen is also swollen, mostly light red, and sometimes dark red. The liver is generally faded and has increased fat. 8.2 Laboratory Test
8.2.1 Bacterial Test
8.2.1.1 Principle of Bacterial Test
Aeromonas salmonicida grows on PBG medium and forms yellow colonies; it grows well on FA medium and produces brown water-soluble pigments; the bacteria are non-motile; most strains have negative VP reactions. These characteristics can be used to check for the presence of Aeromonas salmonicida. 8.2.1.2 Preparation
a. Equipment and instruments
Microscope (with oil immersion lens, dark field), incubator, inoculation loop, culture dish, etc. b. Culture medium and reagents
PBG culture medium, 2% agar, FA culture slant, glucose protein water, Gram staining solution I, I, I, V, VP reagent L. See Appendix B (Supplement) for preparation method].
8.2.1.3 Inspection
8.2.1.3.1 Use a scalpel to cut open the suspected sore part of the trunk (no need to cut when necrotic tissue is exposed). Use a clean slide to press against the lesion, remove the slide and add 1 to 2 drops of sterile water to the slide, cover with a cover slip, and use a 400-600 times dark field microscope to observe whether the bacteria move and the morphology of the bacteria.
8.2.1.3.2 Use an awl or dissecting needle to pierce the skull and destroy the cerebellum, making the fish lose its ability to move. Then use an alcohol cotton ball to clean and disinfect the body surface of the fish to be inspected. Use sterile scissors to cut the abdomen 0.5 cm in front of the anus. Then cut from this point along one side to the back and gradually turn to the head to form an arc, open the abdominal cavity and expose the kidney.
8.2.1.3.3 Take 0.5g of kidney and put it into sterile culture III. Use a sterile glass rod to grind the kidney. Then pour 15mL of PBG culture medium at 45-48℃ and mix it evenly. After it solidifies and dries slightly, add 45℃ 2% agar to cover it. After the agar solidifies, turn the culture blood upside down and culture it in a 25℃ incubator for 48 hours.
8.2.1.3.4 Use an inoculation loop to pierce the agar layer on the surface of the PBG culture dish. Pick up the colony and inoculate it on the FA slant, place it in a 22℃ constant temperature box and culture it for 72 hours, and observe whether there is water-soluble brown pigment. When there are more than 3 yellow colonies in each culture, select 3, and when there are less than 3, all of them are inoculated on the FA slant, and each colony is inoculated on one slant. 8.2.1.3.5 Select a clean glass slide with a thickness of 1.0-1.2mm, drop 1-2 drops of sterile water on the glass slide, use an inoculation loop to pick up a small amount of bacteria cultured on the FA slant for 13-24 hours and smear it in the water droplets, cover the glass slide and observe whether it moves under a 400-600 times dark field microscope.
GB/T15805.1—1995
Take another clean glass slide, drop 1-2 drops of sterile water, use an inoculation loop to pick up a small amount of bacterial moss on the same slant and smear it, and fix it three times on the flame after drying. Stain with Gram staining solution I for 1min and solution I for 2min. Decolorize solution II until the alcohol just stops showing purple color, and stain with solution V for 1 to 2 minutes. Use an oil immersion lens to observe the staining characteristics and morphology. 8.2.1.3.6 V-P test. Use an inoculation loop to pick a small amount of bacterial moss cultured for 18 to 24 hours from the FA slant and inoculate it into the glucose protein aged water. After culturing at 25°C for 72 hours, add the VP reagent in the same amount as the culture medium, return to 25°C for 6 hours, and observe. 8.2.1.3.7 Inspection items are exempted from inspection according to the following rules. If there is no visible sore lesion on the trunk, do not perform the inspection of 8.2.1.3.1. In 8.2.1.3.1, if non-motile short rods are detected, do not perform the inspections of 8.2.1.3.2 to 8.2.1.3.6. In the inspection of 8.2.1.3.3, if there are no colonies or no yellow colonies on the PBG culture blood, do not perform the inspections of 8.2.1.3.4 to 8.2.1.3.6.
8.2.1.4 Observation of results
Observation of bacterial movement and morphology. The water on the slide for observing bacterial movement must not dry up. In the dark field of view, the range of bacterial activity is a.
If the bacteria exceed 1/4 of the visual field, it is called motile. Brownian motion of the bacteria in place cannot be called motile or motile. After Gram staining, red bacteria under the microscope are Gram-negative bacteria. Bacteria that are purple are positive bacteria. When the color of the bacteria is difficult to distinguish, the staining characteristics should be rechecked. The bacteria of the salmon-killing Aeromonas spp. that are about 0.8~1.2μm×1.0~1.8μm are called short rods.
b. The yellow colonies that appear on the PBG. culture dish are large and small, and they are all treated as yellow colonies. When transferring to the FA slope, the smaller colonies are selected. When no colonies appear or they are not yellow colonies, it is determined that there is no sore. c. When brown pigments on the FA slopes penetrate into the culture medium, it is the production of water-soluble brown pigments. Only the bacterial lawn has pigments but does not penetrate into the culture medium, which is not considered to be the production of water-soluble pigments. d. VP test, observe 6 hours after adding VP reagent, red is positive and yellow is negative. 8.3 Comprehensive judgment
a. Clinical examination reveals obvious lesions such as redness, swelling, bulge or local softening, tissue necrosis, etc. in one part of the fish trunk (a few fish may have multiple parts), and non-motile short rods are detected, which can be judged as ulcer disease. b. Whenever bacteria are detected in the kidneys, the bacteria appear as yellow colonies on the PBG culture blood, the bacteria are Gram-negative short rods, non-motile, produce water-soluble brown pigments on the FA. slope, and the VP test is negative, it can be judged as ulcer disease regardless of whether there are visible symptoms to the naked eye. 9 Vibrio disease (Vibriosis)
The pathogen of this disease is mostly Vibrio anguillarum. This disease is prevalent in Europe, the United States, Canada, Japan and other countries. etc.
Vibrio bacteria have a certain pathogenicity to freshwater fish, brackish water fish and marine fish, and harm many kinds of fish, such as salmonids, sweetfish and eels. 9.1 Clinical examination
9.1.1 Activity
The diseased fish are slow to move and swim slowly at the edge of the pool and on the water surface. 9.1.2 External examination
The symptoms vary according to the type of fish. a. Iris: A relatively large ulcer similar to sores is formed under the skin or muscle of any part of the body. The shape is irregular and accompanied by bleeding. There are often bleeding spots in the muscles of other places. The eyeball is protruding, and the gills, eyeballs and fins are also bleeding. The anus is red, and mucus mixed with blood often flows out.
b. Eel: The skin is punctately bleeding and red. The abdomen, lower jaw and fins are most obvious, especially the pectoral fins, which are often necrotic and eroded. c. Ayu: A translucent white spot appears in the muscle of any part of the trunk, and the ulcerated part is accompanied by bleeding. 9.1.3 Autopsy
GB/T15805.1---1995
Autopsy of fish infected with Vibrio infection may show all or part of the following symptoms. Intestine inflammation, congestion or bleeding of the intestinal tract, yellow-brown mucus inside, and visible ulcers on the intestinal wall. Liver congestion, swelling, bleeding spots or spots. 9.2 Laboratory test
9.2.1 Bacterial examination
9.2.1.1 Preparation
Equipment and instruments
Same as a in 8.2. 1.2.
b. Culture medium and reagents
TYE culture medium, sugar fermentation culture medium, Gram staining solution, 1% bromothymol blue alcohol solution, 1% α-naphthol alcohol solution, 1% hydrochloric acid dimethyl paraphenylenediamine aqueous solution, Vibriostatin 0/129 test paper, 3% to 10% hydrogen peroxide solution [For preparation method, see Appendix C (Supplementary)].
9.2.1.2 Inspection
9.2.1.2.1 Sampling: When the ulcer on the body surface of the diseased fish has penetrated into the dermis, it is generally directly sampled from the affected part with an inoculation loop and streaked on the TYE flat III. When there is no ulcer on the body surface or the ulcer is superficial, the abdominal cavity can be opened with aseptic operation to cut a piece of liver or kidney tissue onto the TYE flat blood, and the tissue block can be pressed down with an inoculation loop and then streaked on the flat blood. 9.2.1.2.2 Invert the streaked plate, incubate at 25℃ for 24 hours, and then select a single colony and transfer it to the TYE slant. 9.2.1.2.3 Take the bacterial moss on the TYE slant that has been incubated at 25℃ for 18-24 hours and perform the following tests: a. Observation of bacterial moss, staining characteristics and morphology. For specific operations, see 8.2.1.3.5.
b. Sugar fermentation test. Use an inoculation loop to pick up a small amount of bacterial moss and inoculate it into the sugar fermentation medium. Incubate at 25℃ and observe after 24 hours. c. Oxidase test. Place a piece of filter paper in a clean culture dish, drop a small amount of 1% dimethyl paraphenylenediamine aqueous solution to wet the filter paper, then drop about the same amount of 1% α-naphthol solution, and use a platinum inoculation loop or a glass rod or a wooden stick (do not use an iron-containing chromium-molybdenum wire inoculation loop) to pick up a small amount of bacterial moss and smear it on the filter paper.
d. Catalase test. Use an inoculation loop to pick up a loop of bacterial moss and apply it on a clean glass slide, then add a drop of 3% to 10% hydrogen peroxide solution.
e. Use an inoculation loop to pick up a loop of bacterial moss and apply it evenly on TYE Ping III, then stick a small round test paper that covers Vibriostatin 0/129 on the center of the applied area, turn it upside down and observe it at 25℃ for 18 to 24 hours. 9.2.1.3 Result Observation
a. Carefully observe the color, shape, gloss, bacterial movement, bacterial morphology and Gram staining characteristics of the bacterial colonies on TYE Ping III, and make a record.
b. After 24 hours of sugar fermentation culture, the culture medium changes from blue to yellow, which is positive, indicating that glucose is used. If there is gas in the small cannula, it is gas production, and if the small cannula is filled with culture medium without gas, it is no gas production. If the bacterial moss on the filter paper changes to blue within 10 seconds after smearing, it is positive; if it changes to blue within 10 to 60 seconds, it is a slow reaction; if it turns blue after more than 60 seconds, it is negative.
If small bubbles emerge after adding hydrogen peroxide, it is catalase positive; if no bubbles emerge, it is negative. d. If there is no bacterial growth around the test paper, it is sensitive (i.e. positive); if there is bacterial growth, it is insensitive. 9.3 Comprehensive judgment
a. If the fish in the school have ulcers on the skin, bleeding spots or spots on the body surface, and the autopsy finds inflammation of the intestine or congestion in the liver, it should be considered as vibriosis.
b. All bacteria detected from the ulcer on the body surface or the liver and kidney with the following characteristics are judged as orphan disease. On TYE flat III, the colony is grayish white to light yellow brown, translucent, round and raised, with smooth and shiny edges. The bacteria are Gram-negative short rods, straight or slightly curved, fermenting glucose, but not producing gas. Oxidase positive, catalase positive, sensitive to Vibriostatin 0/129. 496
10 Whirling Disease GB/T 15805.11995
The pathogen of this disease is Myxosoma cerebralis. This disease is prevalent in Europe, the United States, Canada, New Zealand, South America and some African countries, and the prevalence range is very wide. It is mainly a disease of salmonids.
10. 1 Clinical symptoms
2-8-month-old sick fish have their tails exposed above the water and swim wildly in a rotating motion, with a rotation angle of 180-360°. The tail is black. In the late stage of whirling disease, the gill cover bones atrophy, the gills are exposed, the mandible is deformed, the spine is curved, and the skull behind the eyes is severely sunken. This fish still carries the pathogen three years later.
10.2 Parasite inspection
10.2.1 Sampling
Choose fish with clinical symptoms for parasite inspection. If there are no symptoms, sample inspection can be carried out according to the number of fish. For less than 1,000 fish, 4 fish should be inspected. For more than 1,000 fish, 5 to 10 fish should be inspected.
10.2.2 Inspection method
The spore size of brain myxoma is 6.5 to 7 μm × 7.5 to 8 μm. The shell surface is oval to round. There are two pear-shaped polar capsules. There are two round embryonic nuclei in the spore. There is no iodine-loving vacuole. Cysts are formed about three months after infection. 10.2.2.1 Cut the head longitudinally and observe whether there are cysts (milky white, 1 to 2 mm in diameter) in the cartilage tissue of the ear area. If there are cysts, take a small piece of cysts and place them on a slide. Crush them and examine them under a microscope at 450 to 600 times. A large number of brain myxoma spores can be seen. 10.2.2.2 If no cysts are found, the cartilage tissue is cut open from the otolith area and the contents are scraped for smear microscopy. Both the left and right otolith areas need to be examined.
10.2.2.3 If no spores are found in the above two examinations, the fish head can be cut off and cut longitudinally. Place it in a high-speed blender or mortar, add 20ml of distilled water, stir thoroughly, take 2 drops of water, and place it on a slide for microscopic examination. 10.2.2.4 Salmon and trout may be infected with vertigo 3 days after hatching. For diseased fish that have not formed cysts, the vegetative bodies of brain myxoma can be observed through the pathological sections of the head cartilage tissue. The pathological sections are stained with hematoxylin and eosin according to the conventional sectioning method. 10.3 Comprehensive judgment
If the above clinical symptoms appear and the spores or vegetative bodies of brain myxoma are examined, it is judged to be vertigo. a.
b. If the above clinical symptoms do not appear, but the trophozoites or spores of Myxozoa cerebralis are detected, it is judged as vertigo. 497
A1 Cell growth medium
GB/T 15805.1--1995
Appendix A
Preparation method of cell growth medium, etc.
(Supplement)
Weigh 9.4g EagleMEM\Nissui\ and dissolve it in 1000mL double distilled water. Sterilize it with high pressure steam at 103.421MPa for 15min. After cooling, add 0.3g of filter-sterilized glutamine in a sterile room, adjust the pH value to 7.2~～7.4 with 10mL of 7.5% sodium bicarbonate, and then divide it for use. Add 10% calf serum before use. Note: ① Japan Pharmaceutical Co., Ltd.
A2 Cell maintenance medium
Cell growth medium containing 3% to 5% calf serum is used as the cell maintenance medium. A3 Pancreatin-EDTA mixed digestion solution
Sodium chloride (Nacl, analytical grade)
Potassium chloride (KCl, analytical grade)
Potassium dihydrogen phosphate (KH, PO4, analytical grade)
Disodium hydrogen phosphate (Na2HPO4, analytical grade) Ethylenediaminetetraacetic acid (EDTA, analytical grade) Pancreatin (analytical grade)
Double distilled water
Filter and sterilize, then divide into portions for use.
A4 Phosphate buffered saline (PBS)
Sodium chloride (NaCl, analytical grade)
Potassium chloride (KC1, analytical grade)
Calcium chloride (CaCl2, analytical grade)
Magnesium chloride (MgClz·6HO, analytical grade) Disodium hydrogen phosphate (NazHPO,·2H2O, analytical grade) Potassium dihydrogen phosphate (KH,PO4 analytical grade)
Double distilled water
1000 ml
103.421MPa, 15min autoclave, or filter sterilize, store in ordinary refrigerator for later use. A57.5% Sodium bicarbonate
Weigh 7.5g of sodium bicarbonate (analytical grade or chemical grade) and dissolve it in 100mL of double distilled water, then seal and autoclave (103.421MPa, 15 min) or filter sterilize.
Appendix B
Preparation method of sore bacteria culture medium and other solutions (supplement)
B1PBG culture medium
Protein Chen (biochemical reagent)
Glycogen (biochemical reagent)
Sodium azide (analytical grade)
Meat extract (biochemical reagent)
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.