Immune prevention Grass Carp hemorrhage
Grass carp hemorrhage is a serious damage of viral diseases, the use of antibiotics and drug is uncontrollable, and the use of grass carp hemorrhage inactivated vaccine prevention of hemorrhage of grass carp is currently the only the most effective method.
1. Papyrus hemorrhage Histoplasma de live vaccine of grass carp hemorrhage inactivated vaccine research began in early 1973. The results show that the vaccine either in interior or is the production of fish immunity test, either by injection or soaking method, can make the grass carp was immune, immune protection Lida above 95%. Inactivated vaccine, live in more than 20 DEG C, 4 days after immunization of the immunity, and at 10 DEG C need 30 days have immunity, in 3 to 9 DEG C to 33 days to have immunity, with after the death of the vaccination with live fish can be maintained more than 1 year without hemorrhage.
According to the 1978 to 1984 statistics, in Hubei, Hunan, Jiangxi, Jiangsu, Zhejiang and other provinces to promote immunization, injection pond with a total area of about 9 million mu, immune species of 65 million tail, survival rate from 30% to 80%.
(1) Grass Carp hemorrhagic disease inactivated vaccine
1) the key to produce a vaccine is to produce a vaccine. Toxic species can be obtained from natural infected fish and artificial infection. Collect the virus, we must choose the typical symptoms of hemorrhagic disease of fish. Natural infection of fish from the onset of fishponds, choose a significant bleeding symptoms of the diseased fish immediately placed in ice bottle, back to the room diseased fish tissues were collected. Artificially infected fish need to be carried out in the indoor artificial infection.
Micrograms of tissue preparation of artificial infection method, is a virus suspension, the step is to take a fresh diseased fish tissues (if the diseased fish tissues is preserved in glycerin, must with sterile water washed three times, final reoccupy saline irrigation 1 times, so as to avoid glycerol did not wash clean and cause side effects), after weighing per gram of tissue and 0.85% normal saline 10 ml with mortar or homogenizer were homogenized, 10% concentration of suspension, and then to 3000 rpm / min speed centrifugal 30 points, taking the supernatant, according to 800 IU / ml (or 8OO ml) adds the penicillin, streptomycin, room temperature placed 2 hours for sterilization in the refrigerator to save stand-by.
Suspension will make a good virus liquid of healthy grass carp species by intraperitoneal injection of artificial infection, each tail injection of the virus suspension was 0.3 ~ 0.5 ml, the fish injected into the tank water temperature 25 to 28 DEG C, feeding was observed in 15 days. When the water temperature is less than 25 C, the temperature is 25 ~ 28 C, because the temperature is the most suitable for the reproduction of the virus. After infection, 5 to 6 days.
Of artificial infection experiment and querulous disease of fish material, must choose the healthy grass carp species (generally refers to has not sent a hemorrhage of the year or 1 winter age fish) had hemorrhage and surviving fish, has produced the immunity, it is not easy to re infection, will affect the immune system and injection infection effect. Indoor tests, the fish must be in the indoor arm 7 ~ 20 days, not the incidence of available,
Diseased fish tissue collection method to get the fresh hemorrhagic disease open the fish belly, collect fish diseases of liver, spleen, kidney, if fish peeled, hyperemia of the muscles can also collection, after weighing, people a 40 ~ – 30 DEG C low-temperature refrigerator preservation, or into the 50% phosphate buffered glycerol, set 4 to 8 the refrigerator to save. The collection and preservation of fish tissue is regarded as hemorrhagic disease virus hemorrhage.
50% phosphate buffered glycerol with first with disodium hydrogen phosphate solution (weigh g 358.24 dissolved in 1000 ml of distilled water), with potassium dihydrogen phosphate (weigh 136.16 grams dissolved in 1000 ml of distilled water), as the original glycerol pH value to determine the different amounts of hydrogen phosphate and disodium phosphate acid two hydrogen potassium into pH 7.4 50% glycerin solution. PH values of glycerol can be detected by test paper.
The pH value of glycerol at 5.5 or more time:
Hydrogen phosphate 17.5 ml solution two ml
7.5 ml of potassium dihydrogen phosphate
Distilled water 475 ml
Glycerol (pH 5.5) 500 ml
The pH value of glycerol at 5.5
Hydrogen phosphate 40 ml solution two ml
10 ml solution of potassium phosphate.
Distilled water 450 ml
Glycerol (pH 5.5) 500 ml
With good, with 0.68 MPa sterilization 30 points.
2) it is very important to keep the virus infection and antigenic change. There are three ways to save the virus: 1, low temperature preservation. The infected organs of hemorrhagic disease of fish block 50% phosphate buffered glycerol under 4T in the refrigerator can be preserved for 2 to 3 months, good & to reach more than half a year, glycerin plays a role of infectious virus particles in the protection. When the material is infected, the Hanks medium containing 5% calf serum was added to 50% to 15% sterile glycerol. 2, frozen preservation method. The frozen storage of the virus can be carried out in the refrigerator (-20 ~ C), or the ultra low temperature trough (-90 ~ C, -40), or liquid nitrogen container (-180 C), at present, the majority of the use of a 20 degree C of frozen, 3, freeze drying method. To freeze dryer.
Be crushed) 3 Histoplasma inactivated vaccine preparation extraction have significant bleeding symptoms of disease fish liver, spleen, kidney and muscle tissue or with 50% phosphate buffered glycerol preservation of diseased fish tissue, the latter with sterile distilled water rinse 3 times, final reoccupy saline irrigation 1 times, and then weighed and cut into pieces, on a scale of 1 gram of tissue and 0.85% normal saline 10 ml or 100 ml of homogenate, and then 3 000 rpm / min speed centrifugal 30 points, taking the supernatant, per ml of virus suspension with penicillin 800 international units and streptomycin 800 mcg. Finally, add 10% formalin solution, so that the final concentration of 0.1% after shaking release 32t constant temperature water bath inactivated 72 hours. 2 times a day, in the process of inactivation. After inactivation, sampling and testing for safety and efficacy. The qualified vaccine, 4 ~ 8 DEG C refrigerator stand-by.
4) safety and efficacy test from the death of disease, to year healthy grass carp for by intraperitoneal injection, each tail injection of 0.2 ~ 0.5 ml, breeding in the water at 26 to 28 DEG C it in a tank of water, continuous observation of 15 days, if there is no hemorrhage of dead fish found that the vaccine is safe, and on the other with 10% fresh or glycerol preservation of diseased fish tissues into virus suspension, each tail according to 0.2 ~ 0.5 ml by intraperitoneal injection attack and not by immunized control group, continuous observation of 15 days. If all control group bleeding disease, mortality in more than 70%, the fish disease symptoms and natural occurrence of fish diseases symptoms, and immune group protection, still alive and healthy, explanation the vaccine is effective.
(2) the use of inactivated vaccine against hemorrhagic disease
Four) the immune pathway is currently used in the fish’s immune pathway to have 1 kinds of injection, oral, spray and immersion, but usually uses the method of injection and immersion.
The injection of immune reliability, good effect, but by the end of injection, waste of human and time, the vaccine injection into the abdominal cavity, intramuscular and subcutaneous injection of three. Grass carp hemorrhage death live disease is by intraperitoneal injection method, the amount of vaccine as fish size and decide, small fish less, fish mostly note, general control in 0.2 ~ 0.5 ml / tail. Using 5 ml syringe is appropriate, needle size according to the fish body size and vaccine suspended particle size and viscosity and, general 3.3 centimeters or so species with a 4 needle, 6.7 ~ 10 cm species with needle No.5, 20 ~ 23.3 cm species with 6 or 7, needle.
The method is simple and easy to implement, and is not limited by the size of the fish, which is beneficial to the immune system of a large number of fish, but it needs a large container and a large number of vaccines.
At present in domestic application in production of pond of grass carp hemorrhage death live soaking vaccine immunization with vaccine plus scopolamine immersion immune method. With the concentration of 0.5% of the vaccine, and adding 1/100000 tanguticus, at 20 to 25 DEG C temperature under oxygen for 3 hours. The survival rate was 78.3% to 92.2%, and the survival rate of the immunized fish was 20.5% ~ 35%.. The sperm was soaked by the vaccine. The vaccine was carried to the eggs in the egg. The immune effect of the 73%. can be obtained from the 5 to June.
2) using the vaccine to pay attention to the matter 1, the health of the body of the grass carp, if inoculated with a poisonous grass carp, after immunization, the disease will be more fierce. 2, in the preparation of vaccines, we must choose a strong virulence. The diseased fish tissue must be fresh, not smelly corruption organization. 3, the preparation of a good vaccine, must be carried out in the indoor safety and efficacy test. If not for the safety test applied to production, will bring to production losses, because did not destroy the good live vaccine to grass carp, not only can not prevent the hemorrhage and promote the diseased grass carp. 4, before using the vaccine, be sure to fully mix the concentrated vaccine, and then according to the concentration of 1:100 dilution. If there is a large group of floc or vaccine particles in shake loose, that has long time bacteria, such a vaccine can not be used. 5, before injection shall be 5000 times of trichlorfon water solution to fish for disinfection and anesthesia can kill ectoparasites, can be reduced by struggling fish injured. 6, the immune system, can be used in Shanghai Normal University affiliated factory of the metal continuous syringe, also can be used to 5 ml syringe needle, needle as the size of the fish body. Garden day out or dilute the vaccine must be used, if not finished, should immediately set aside 4 ~ 8’C refrigerator to save. Must note in the field to avoid direct sunlight and heat, so as to avoid vaccine metamorphism. In the process of injection, the operation should be careful, so as to avoid the injury of fish body.