Immune prevention and treatment of bacterial diseases

Immune prevention and treatment of bacterial diseases
In early 1942, Duff (Duff) began to cold water of silver carp and trout to fire frog Aeromonas bacteria immunity test, in our country the earliest is R. (1962) with the spot gas single cell bacteria brother shis control grass carp enteritis. From the 20th century? In the 1960s to now, prevention and control of bacterial fish disease vaccine has been a hot spot in the research of, but due to various reasons, the majority of domestic vaccine is still in research or production trial, has not yet reached the production of large-scale application stage.
The general process of vaccine preparation
1, we need to have the vaccine preparation of pathogenic strains of virulent, good antigenicity. Can a pathogenic strain, also can be more than two kinds of pathogenic strains combination preparation vaccine be Tuesday vaccine, triple vaccine,. 2, and train a large number of pathogenic bacteria. The suitable medium of solid medium or liquid medium was used to enlarge the culture medium at the optimum temperature to obtain the most bacteria. 3, the bacteria can be collected, and the culture of solid culture medium can be washed by sterile physiological saline, and then the bacteria can be collected directly by centrifugal precipitation. A certain concentration of bacterial suspension made with saline, which become viable han. Toxoid is bacterial exotoxins by 0,3% – 0.4% Faure Marin made. 4, will kill the live vaccine become inactivated vaccine. Destroy methods generally have o.1% ~ 0.5% formalin, or add 0.5% phenol, or plus 8% chloroform, placed in 25t 24 hours to inactivate, also can be used in heating method (60 ~ 651.2 hours or so) inactivation. The purpose of the inactivation is to kill the bacteria and to keep the complete disturbance as well as possible. Throw in the formalin test is more sensitive, with O, Wei% formalin killing live vaccine, if not remove formalin can cause the death of white crucian carp. 5, after the preparation of inactivated vaccine, to do sterility test, safety test, test titer test, can be used.

Cell culture Grass Carp hemorrhage inactivated vaccine (the cell vaccine)

Cell culture Grass Carp hemorrhage inactivated vaccine (the cell vaccine)
Grass carp hemorrhagic tissue plasma inactivated vaccine, can enhance the immune ability of grass carp hemorrhage has popularized and applied in the whole country and achieved obvious effect. However, the production of this vaccine is derived from the diseased fish tissues, which is not stable, the yield is limited, and is not convenient for the factory production. Therefore, is currently being developed a kind of efficient, safe, easy to industrialized production of hemorrhage of grass carp, Ctenopharyngodon idella cell cultivation inactivated vaccine, will soon be published.
Cell death of live vaccine prepared first through cell culture screening strong virulence and immunity high grass carp hemorrhagic virus strains (strains currently used have ZV-8802.GCHV a 854), selection of GCHV sensitive cells (such as grass kiss end tissue strains ZC 7901, grass carp kidney cell line GCK a 84, grass carp CIK cells). With proper training method, the strain to adsorption method. Cell, until the cells just lesions appear, the virus cell with Hanks’ solution after washing, with 0.02% EDTA (EDTA) digestive fluid cell, then 10% calf serum 199 liquid made cell suspension, then add an appropriate amount of normal cells, to 25 DEG C under static culture, according to the method of continuous passage. To the tenth generation, the use of rotating bottle culture method to expand the cultivation. After the expansion of culture, the culture of 15 to 25 generation of virus was collected, which was used to prepare the inactivated vaccine. Inactivation methods by adding 0.1% formalin, 37 degrees C killed 48 hours to live or to 4 DEG C inactivation of 17 days and prepared cell inactivated vaccine to a safety test, and determination of the immune protection rate

Immune prevention Grass Carp hemorrhage

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.

Immunity and disease prevention technology

Immunity and disease prevention technology
Immunity, simply to mobilize the body’s own defense against pathogen invasion, propagation and spread, to maintain the internal environment of the spike set, to achieve the process of living organisms. Immune prevention is the use of artificial immunity method enhanced biological law of immunity in order to achieve the purpose of preventing disease, artificial immune is artificial made vaccine, Miao Pu, tumor vaccine, poison cable or cellular immune preparation and vaccination to fish, the fish itself defense ability of corresponding diseases. Made with pathogen pick preparation raw said Miao Pu, virus, made of rickettsial antigen preparations known as (some of the two referred to as vaccine) vaccine, made by tumor tissue known as tumor vaccine. Bacterial exotoxin after 0.3% – 4% formalin treatment, toxicity and disappear immunogenicity still retain is called toxoid. Cellular immune agents are dry, transfer factor, etc..
Prevention and control of fish diseases by artificial immune method. Than the use of drugs for prevention and treatment, has the following advantages: 1, fish with most drugs have different degrees of injury and poison, easy to cause the water environmental pollution and drug toxicity and immune prevention methods on the gold does not have this situation. 2, the fish farms often long-term use of antibiotics, the aquaculture water resistant strains increased significantly, the efficacy was significantly decreased, the drugs increase, immune prevention method does not produce resistance in the isolates. 3, a large area of water fish, the use of drugs cost is too high, and the immune prevention is simple, economic. 4, some diseases, such as virus diseases, tumor, robe worm disease, prevention and control of drug is difficult to bear fruit, but also need to study the immune prevention methods,

Cut off the source of infection disease

Cut off the source of infection disease
Control the spread of pathogens of fish diseases, reduce or inhibit the growth rate of the reproduction of the pathogen and drug prevention measures is very important, especially on fish, bait, tools disinfection is not neglect.
1, fish body disinfection
Disinfection of fish body mainly through the bath method, will fish body is arranged in the higher concentration solution in, after a short time of bath, killing fish pathogens. The bath container available barrel, plastic tub, cabin, water tanks, etc., if the number of fish, batch no bubble or close cage for soaking container. Soaking drug before it is best to check out the fish pathogen, and then select the drug, according to the fish body size and physical strength and Tianzhu, JuShui flexible grasp the concentration. In the process of immersion, the fish should be regularly checked for the tolerance of the fish, so that the time of immersion is easy to master. There are several drugs for the disinfection of fish body;
Salt: concentration 2% ~ 4%. Soak 5 to 10 points, the main prevention baizui disease, gill rot disease, killing some protozoa, three generations of insect, Dactylogyrus etc..
Copper sulfate: concentration of 8 mg / kg, no washing 20 points, mainly to prevent fish wave Bodo disease, Trichodina disease, kill parasites on the body surface of the protozoan pathogens.
Bleach: concentration of 10 ~ 20 mg / kg, soak 10 minutes or so, can prevent fine Han skin disease and gill disease.
Trichlorfon: concentration of 10 mg / kg, dipped 15 points, can kill some protozoa, three generations of insect, Dactylogyrusis etc..
2 food disinfection
Commonly used food and field disinfection method has suspension method and sprinkling method two kinds.
Suspension method: the drug is placed in a container with a micro hole, suspended in the food field, so that it can be dissolved in water, and can achieve the purpose of disinfection. Suspension for drug and food disinfection with field bleaching powder, sulfuric acid, pin dipterex. The container is hanging baskets, bags, plastic bags, a hollow foam block material. After suspected bagged liquid material, need to tip in bag nimble holes, hole size and number should be shellfish live Zhang Yue, the drug in more than 5 hours the dissolving as well, and to in suspension to surrounding reaches a certain concentration as the basis. Foam blocks cut inside after the drug is placed in the central chamber, and then have the rope hanging in the food field. Hang number tender to fish to into the feeding site for the degree, in the water and fish cage culture in the suspension feeding grounds of the disinfection effect is also very good.
Test method: medicine every 1 to 2 weeks, with chlorine preparation, such as bleaching powder 250 grams of water in the food field around it 1 times, better disinfection effect.
3 disinfection
Fish with a variety of tools, especially tools used in the pond onset, such as nets, cask, wooden ladle, fishing, waterproof clothing etc., tend to become a medium for the spread of fish diseases, small tools disinfection can be used 10 mg / kg CuSO4 or higher concentrations of potassium permanganate soak for more than 10 points, large tool can be in the sun drying in the reoccupy after. Disinfection should be complete, such as the attachment to the fish on the tool of the small melon worm, encountered a bad environment can form a cell capsule, and in the cell capsule continue to split, with the tools to bring people to the environment, thousands of larvae will be broken out, so that the small melon worm disease.
4 water disinfection
After a period of time, the organic matter and suspended substance increase, the water quality is gradually deteriorated, and the pathogen is gradually increasing. Therefore the fish disease epidemic season regular disinfection of aquaculture water, killing in water or fish pathogens, effective measures of prevention. Water disinfection is in the entire culture water body in the drug. Effective disinfection drugs commonly used is the lime, the water depth of 1 meters per acre with lime 25 kg. There are many excellent disinfectant, such as bleaching powder (1 mg / kg), bleaching powder concentrate (0.1 ~ 0.2 mg / kg) and trichloro isocyanuric acid (0.3 mg / kg) and sodium dichloroisocyanurate (0.3 mg / kg), chloramine T (2 mg / kg) and the sterilization effect is good; copper sulfate, ferrous sulfate mixture (5:2.0.7 milligrams (mg) / kg), trichlorfon (crystal content of 90%. 0.2 mg / kg), miechongling (0.5 mg / kg), good insecticidal effect.
5 food disinfection
Fish pathogens often with bait into the bait, so delivery must be clean and fresh, after disinfection. Plant food, such as water plants can be used 6 mg / kg bleach solution diffuse foam 20 to 30 points. The land plants collected under the sun can not be disinfected. Animal feed, such as snail, fish and shrimp and merchandise bait mix a small amount of CTC or Oxytetracycline Residue (according to the amount of feed 5 valves mixing ratio) can be fed. The fish manure must be rotten, and add 120 grams for every 500 kilograms of bleach disinfection treatment can be put on the big pool.