Summary of the invention
For the single and unfavorable problem of drug effect of medicine that solves above treatment infectious bursal disease, the invention provides the preparation method of a kind of infectious bursal disease specific transfer factor (hereinafter to be referred as the specific chicken transfer factor), the specific chicken transfer factor combining yolk antibody of preparation is to improve clinical efficacy.In Shandong Province in 2003 12 chicken house clinical trials treatment, obtained good effect.Prove that this scheme can control the chicken bursa state of an illness effectively and play good therapeutical effect.
The present invention is achieved by the following measures:
A kind of preparation method of infectious bursal disease specific transfer factor may further comprise the steps:
(1) adopt 10 dosage of infectious bursal disease live vaccine B87 strain to carry out immunity to the healthy poultry of every plumage part, each dosage viral level is not less than 1000ELD
50, after 7 days, 20 dosage intramuscular injection of the sinister strain of reuse infectious bursal disease live vaccine B38 booster immunization, each dosage viral level is not less than 2000ELD
50, use the immunity of infectious bursal disease oil-emulsion inactivated vaccine 0.5mL subcutaneous injection simultaneously, viral level is 10
7.2TCID
50/ 0.1mL after 7 days, re-uses the sinister strain of same dose injection infectious bursal disease live vaccine B38 and strengthens once immunity, and the infectious bursal disease antibody horizontal reaches 2 in the blood when the agar gel diffusion test method detects
4After above, cut open extremely animal and take fresh spleen, kidney or fabricius bursa, tissue is pulverized, after the homogenate at subzero 20 ℃ of-30 ℃ of multigelations more than 8 times, freeze 16h at every turn, melt 8h, a circulation needs 24h altogether, and 4000rpm is centrifugal, gets supernatant, molecular sieve filtration is got relative molecular mass less than 5000 daltonian components;
(2) relative molecular mass that obtains is less than 5000 daltonian components, and being concentrated into solid content is 5mg/mL, adds 0.2mg stabilizing agent EDTA-Na among every mL
2With the compound thermal protecting agent of 0.3mg, degerming behind the mixing, both;
Described compound thermal protecting agent is a mannitol: lactose: chitosan: glycerol: glycine: sorbitol was by weight 2: 3: 2: mixing in 3: 1: 2 obtains.
Content of peptides 〉=2.0mg/mL in the infectious bursal disease specific transfer factor that makes.Nucleic acid, aminoacid and the vitamin, the trace element that also contain trace in the infectious bursal disease specific transfer factor.Because of the content of peptides height, be easy to detect, be the composition of national quality control standard.
It is the novel biochemical para-immunity reinforcing agent that main component is developed that this product adopts with the specific chicken transfer factor.This product dosage is little, biological activity is high, can effectively improve the immunity of poultry, can improve simultaneously the immune level of vaccine, eliminate systemic immune response most possibly.And this product is biogenetic products, have no side effect, noresidue, and be a kind of green veterinary drug of high effect nontoxic.Be applicable to the man poultry infectious bursal disease of control; Be applicable to that immunity of livestock is low, improve immunity; Be applicable to the virosis high-incidence season, reduce infection rate and mortality rate; Be applicable to that immune effect of vaccine is undesirable, can make that the vaccine immunity level is neat, antibody is high, potent antibodies is held time length; Be applicable to stress, the respiratory tract disease that the minimizing vaccine causes etc.
Usage and consumption: mix drink, oral, eye dripping or spraying.Every 10mL is used for 1000 plumage poultry; 1000 kilograms of domestic animals.
Effect duration: preserved 2 years-20 ℃ of freezing preservations 5 years for 4 ℃.Room temperature was preserved 6 months.
Beneficial effect of the present invention:
1, for example the levamisole hydrochloride activity is high, effect is rapid, dosage lacks, noresidue than the chemical medicine series products;
2, rapider, easy to use than the effect of Chinese medicine class, be convenient to the transportation.
3, small-molecule substance, the use approach is many, and drinking-water, oral, eye dripping, collunarium, injection etc. all can.
The specific embodiment
For a better understanding of the present invention, further specify below in conjunction with specific embodiment.
Embodiment 1:
(1) adopt 10 dosage of infectious bursal disease live vaccine B87 strain to carry out immunity to the healthy poultry of every plumage part, each dosage viral level is not less than 1000ELD
50, after 7 days, 20 dosage intramuscular injection of the sinister strain of reuse infectious bursal disease live vaccine B38 booster immunization, each dosage viral level is not less than 2000ELD
50, use the immunity of infectious bursal disease oil-emulsion inactivated vaccine 0.5mL subcutaneous injection simultaneously, viral level is 10
7.2TCID
50/ 0.1mL after 7 days, re-uses the sinister strain of same dose injection infectious bursal disease live vaccine B38 and strengthens once immunity, measures antibody horizontal after 10 days, and the infectious bursal disease antibody horizontal reaches 2 in the agar gel diffusion test method detection blood
4More than, cut open extremely animal and take fresh spleen, kidney or fabricius bursa, tissue is pulverized, after the homogenate at subzero 20 ℃ of-30 ℃ of multigelations more than 8 times, freeze 16h at every turn, melt 8h, a circulation needs 24h altogether, and 4000rpm is centrifugal, gets supernatant, molecular sieve filtration is got relative molecular mass less than 5000 daltonian components;
(2) relative molecular mass that obtains is less than 5000 daltonian components, and being concentrated into solid content is 5mg/mL, adds 0.2mg stabilizing agent EDTA-Na among every mL
2With the compound thermal protecting agent of 0.3mg, degerming behind the mixing, both.Content of peptides is 2.21mg/mL in the infectious bursal disease specific transfer factor that makes.
Compound thermal protecting agent is a mannitol: lactose: chitosan: glycerol: glycine: sorbitol was by weight 2: 3: 2: mixing in 3: 1: 2 obtains.
Zoopery
One, the infectious bursal disease specific transfer factor promotes the experiment of chickling cellular immunization
1 test material
1.1 experimental animal: 200 of 3 age in days healthy chicks.
1.2 test drug: infectious bursal disease specific transfer factor solution, specification is the 5mL/ bottle, contains infectious bursal disease specific transfer factor 5mg.
1.3 feedstuff: chickling perfect compound feed.
1.4 chemical reagent: lymphocyte separation medium, 2.5% glutaraldehyde, paramagenta solution, 4% sodium nitrite solution, six azo paramagenta solution, acetic acid naphthalene ester solution, phosphate buffer, C.I. 42590 dyeing liquor etc., all available from units concerned or preparation voluntarily.
1.5 key instrument: blood counting chamber, six figure place classification counters, binocular biological microscope etc.
2 test methods
2.1 test grouping: will test with chicken and be divided into A, B, C, D, five groups of E, 40 every group at random.A, B are test group, C, D matched group, and the E group is the blank group.0.05 milliliter of infectious bursal disease specific transfer factor solution of every chicken injection of A group, B organizes every infectious bursal disease specific transfer factor solution that chicken is oral 0.05 milliliter, C organizes oral 0.05 milliliter normal saline, the normal saline that the injection of D group is 0.05 milliliter, the E group is not done any processing.
Infectious bursal disease specific transfer factor and normal saline that 2.2 test method: A, B, C, D, five group of 7 age in days immunity of E used the present invention to prepare respectively according to grouping in preceding 12 hours, cell counting is carried out in blood sampling during respectively at the 14th day, 21 days, and asks its meansigma methods.Concrete grammar is referring to relevant immunological testing method (ANAE method).
3 result of the tests: T, bone-marrow-derived lymphocyte count results see Table 1.As can be seen from the table, the peripheral blood TC value of 14 Japanese instar chicklings, the average T C value of A, B, C, D, E group is respectively 56.167%, 53.468%, 34.167%, 35.123%, 32.333%.Handle by statistics and show that each test group TC value is compared with matched group and improved significantly (t>1.96, P<0.05).
The peripheral blood TC value of 21 Japanese instar chicklings, the average T C value of A, B, C, D, E group is respectively 65.333%, 61.512%, 38.5433%, 39.586%, 36.127%.Showing by statistics that each test group TC value is compared with matched group improves highly significant (t>2.57, P<0.01).
T, bone-marrow-derived lymphocyte ration statistics table (ANAE staining) in table 1 chicken blood
Annotate: this result records during for repeated trials, and it is that 6 what take is that the new multipole of randomized block experiment is checked that test repeats.Numeral back mark letter representation significance test result: all have a same letter person, and difference is not remarkable; Capitalization is represented 0.01 significant level; Lower case is represented 0.05 significant level.
4 conclusions and discussion
4.1 this result of the test shows, two test group and matched group T lymphocyte percentage significant difference (P<0.05) prove that infectious bursal disease specific transfer factor solution has tangible raising effect to the T cell value.Contain bioactive little peptide in the infectious bursal disease transfer factor solution, in entering body, it can improve functions of immune system, and a kind of as immunocyte of T lymphocyte, also obtain raising to a certain degree, therefore test group T cell percentage is than the matched group height, and significant difference.
4.2 injection group and oral group of T cell proportion differ not remarkable, prove that injection is identical with the effect of oral infectious bursal disease specific transfer factor solution.Because this solution contains little peptide, it is comparatively fast acted on body by the speed that body absorbs by injecting pathway, so the injection group can promote the T cell proportion to raise.Little peptide molecular weight less (being lower than 10000 dalton), can be not by decomposition such as trypsin, Chymotrypsin, pepsin, DNA enzyme and RNA enzymes, therefore meeting can be fast by intestinal absorption in the time of in it enters body, enter blood circulation, thereby the immune system of acting on, so oral group of T cell proportion also can raise.Injection is all very fast with the speed that oral route absorbs effective ingredient, therefore shows as both clinically and promotes that the effect of immunity is suitable, and difference is not remarkable, so this immunostimulant use is comparatively convenient.
4.3 infectious bursal disease specific transfer factor solution has than high bioactivity, its mechanism of action and chemical classes immunostimulant (for example levamisole hydrochloride etc.) exist than big-difference.The infectious bursal disease transfer factor solution is the material of normal presence in the animal body, its concentration is lower under the normal condition, after concentration improves, its biological activity performance is quite remarkable, can within a short period of time the intensity and the speed of human body immunity improving responsing reaction, therefore the biological activity advantage is very remarkable than traditional chemical para-immunity reinforcing agent, is the biological immunopotentiator that has very much development prospect.
Two, the infectious bursal disease specific transfer factor is to the treatment experiment of infectious bursal disease
1 materials and methods
1.1 material
1.1.1 the infectious bursal disease specific transfer factor, this research department's preparation, freezing preservation.
1.1.2 the chicken transfer factor, this research department's preparation, freezing preservation.
1.1.3 the infectious bursal disease yolk antibody is purchased in market, freezing preservation.
1.1.4 supply the selection of examination chicken to supply the examination chicken to select second day chicken group's of natural occurrence commodity fowl disease chicken respectively, select disease chicken, dead chicken at random, dissection and laboratory diagnosis all meet the chicken group of bursal disease pathological change feature for supplying the examination chicken.Be divided into 3 groups, every group is 500, and the feeding and management environmental condition is similar.
1.2 method
1.2.1 infectious bursal disease specific transfer factor and chicken transfer factor are taken out, and aseptic subpackaged according to the 5mg/mL concentration dilution, 4 ℃ of preservations are standby.
1.2.2 yolk antibody is taken out, and after thawing, 4 ℃ of preservations are standby, the special-purpose yolk antibody injection of chicken that used yolk antibody is sold for Beijing side letter animal pharmaceutcal corporation, Ltd.
1.2.2 bursal disease diagnosis: cut open the inspection variation according to clinical symptoms and pathology, and, can confirm that this disease is infectious bursal disease in conjunction with laboratory diagnosis.
1.2.3 therapeutic test: test divides 3 groups, and the 1st group is that yolk antibody mixes with infectious bursal disease specific transfer factor volume ratio at 1: 1, and the 2nd group is that yolk antibody+chicken transfer factor group volume ratio is mixed at 1: 1, and the 3rd group is the yolk antibody group.Preceding 2 groups all with behind two kinds of component mixings, only all adopt chest muscle injection 2mL/ after placing room temperature.The 3rd group yolk antibody placed and only adopt chest muscle injection 2mL/ after the room temperature.After the injection, each group all adopts identical measure, suits the remedy to the case.
2 results
As can be seen from the test results, each test group has good therapeutical effect to infectious bursal disease (IBD), just can produce effects at second day of medication, just controlled the state of an illness in the 3rd day, sick chicken stops death, and wherein yolk antibody+infectious bursal disease specific transfer factor group survival rate reaches 98.4%, yolk antibody+chicken transfer factor group survival rate reaches 76.0%, yolk antibody group survival rate is 74.2%, and significant difference (P<0.05) sees Table 2.
The therapeutic effect of table 2 pair infectious bursal disease
Group |
Test chicken |
The morbidity age in days |
The death toll in back 5 days of falling ill |
The survival number |
Cure rate |
The 1st group |
500 plumages |
24 days |
8 plumages |
492 |
98.4% |
The 2nd group |
500 plumages |
24 days |
120 plumages |
380 |
76.0% |
The 3rd group |
500 plumages |
24 days |
129 plumages |
371 |
74.2% |
3 clinical expansion therapeutic effect
According to result of the test, adopt yolk antibody+specific chicken transfer factor scheme that the clinical chicken bursal disease that breaks out is carried out expanding test, treat 20153 according to statistics, 19646 of therapy rehabilitations, cure rate reaches more than 97%.Just can control death in second day after the medication, just can take a turn for the better in the 3rd day, can recover appetite gradually after the 4th day, substantially can rehabilitation after the week.
4 analyze and discuss
4.1 infectious bursal disease is a kind of inhibitive ability of immunity, height contagious disease, is perplexing poultry husbandry for many years, brings mortality harm to poultry husbandry always.Chickling mainly relies on fabricius bursa to produce antibody, when fabricius bursa is subjected to encroaching on, the ability of body generation immune antibody goes down or disappears, and cause immunosuppressant, young chicken group is descended to the immune response of vaccination, other virosis of the easy accompanying infection of ill chicken or bacterial disease as newcastle disease, Marek, colibacillosis, salmonellosis and coccidiosis etc., cause enormous economic loss to poultry husbandry.So, must strengthen prevention and control at ordinary times to this disease.
4.2 according to the morbific reason of primary disease is because tangible material is dissolved, got rid of to infringement kidney and fabricius bursa so matter of utmost importance is weak from alleviating kidney,, the mediation urinary tract, and control death is set about.Because the infectious bursal disease specific transfer factor not only has regeneration and repair to pathological tissue, can also improve the omnibearing immunity of body, so have the sick chicken death of rapid control behind the use said preparation, characteristics such as the course of disease is short, recovery is fast fully demonstrate the advantage that biogenetic products are prevented and treated Animal diseases.
4.3 the infectious bursal disease specific transfer factor has the enhancing human body immunity effect, and the symptom of bursal disease is had selectivity and effect targetedly.Pharmaceutical research proves, the infectious bursal disease specific transfer factor is a kind of immunologic active material, can improve the immunogen performance, phagocytic function with enhancing body reticuloendothelial system, in addition, can also improve the weight of immune organ, improve total white blood cells and lymphocyte number, strengthen antibody forming capacity.Therefore, the excellent popularization using value is arranged in production reality.
The bin stability experiment
The stability of product is to guarantee primary condition, especially polypeptide amino acid series products safe and effective for medication, and effective content descends comparatively fast in water, so stability test is an important topic of this subject study.Influence the factor of preparation stability, except that factors such as its prescription, technology, also have factors such as temperature, light, humidity.Therefore, this product having been carried out influence factor's test, accelerated test, room temperature keeps sample for a long time and investigates test.
1.1 sample: select to meet the sample of three batch samples of infectious bursal disease specific transfer factor solution quality standard as stability; Reference substance infectious bursal disease specific transfer factor is employed to be mannitol: lactose: the glycine weight proportion is 2: 3: 1 a thermal protecting agent.
1.2 period of storage and condition
1) influence factor's test:
A. high temperature and humidity test: above-mentioned sample and reference substance are placed 40 ± 2 ℃ of the mid-temperature of commercially available back respectively, following 3 months of relative humidity 75% condition, each month detected once.
B. exposure experiments to light: above-mentioned sample and reference substance are positioned over respectively in the lighting box, illuminance 4500 ± 500Lx is set, temperature is following 10 days of 25 ℃ of conditions, detects once respectively at sampling in the 1st day, 3 days, 5 days, 10 days.
2) long-term experiment: above-mentioned sample and reference substance are placed 25 ± 2 ℃ of the mid-temperature of commercially available back respectively, following 8 months of relative humidity 60% condition, respectively at 0,2,4,6,7,8 month pick test once.
1.3 testing index and method:
Inspection item: outward appearance; Folin phenol method is measured content of peptides.
1.4, result of the test:
1) influence factor's result of the test:
Table 3 is stored statistical table as a result under hot and humid experiment condition
Table 4 is stored statistical table as a result under the illumination experiment condition
2) long-term test results
Table 5 is long term store statistical table as a result under the room temperature experiment condition
1.5 conclusion (of pressure testing):
This product is tested through the influence factor; accelerated test; the room temperature investigation experimental observation that keeps sample for a long time; at steady quality; aspects such as appearance character all are better than reference substance; after using 1: 1 mixing of six months infectious bursal disease specific transfer factor volume ratio of yolk antibody+storage at normal temperature; only adopt chest muscle injection 2mL/; be used for the treatment of infectious bursal disease; just can produce effects at second day of medication; just controlled the state of an illness in the 3rd day; sick chicken stops death; survival rate still reaches 94.4%, and as seen, thermal protecting agent used in the present invention plays a good role to the stable aspect of whole system.