Summary of the invention
In order to the problem that the medicine solving above treatment Nephropathogenic infectious bronchitis is single and drug effect is undesirable, the invention provides the preparation method of a kind of Nephropathogenic infectious bronchitis specific transfer factor (hereinafter referred to as specific chicken transfer factor), the specific chicken transfer factor of preparation combines composite treatment of suiting the medicine to the illness to improve clinical efficacy.More than 30, Shandong Province chicken house clinical trial treatment in 2006, achieve good effect.Prove that this scheme can effectively control the Nephropathogenic infectious bronchitis state of an illness and play good therapeutical effect.
The present invention is achieved by the following measures:
A kind of Nephropathogenic infectious bronchitis specific transfer factor compound thermal protective agent, is mixed by the raw material of following weight proportion:
Mannitol, maltose, chitosan, histidine, magnesium sulfate are by weight 2:3:1:1:3.
The application of described Nephropathogenic infectious bronchitis specific transfer factor compound thermal protective agent in the preparation of infectious bronchitis of chicken specific transfer factor, comprises the following steps:
(1) adopt infectious bronchitis of chicken live vaccine 28/86 strain 10 multiple dose to carry out immunity to the healthy poultry of every plumage part, each doses of virus content is not less than 1500ELD
50, after 7 days, then use Nephropathogenic infectious bronchitis live vaccine W93 strain 20 multiple dose intramuscular injection booster immunization, each doses of virus content is not less than 2000ELD
50, use the immunity of Nephropathogenic infectious bronchitis oil-emulsion inactivated vaccine 1.5mL subcutaneous injection, viral level is 10 simultaneously
3.8tCID
50/ 0.1mL, after 10 days, then use Nephropathogenic infectious bronchitis oil-emulsion inactivated vaccine 1.5mL subcutaneous injection immune strengthening once, viral level is not less than 10
3.8tCID
50/ 0.1mL, antibody horizontal is measured after 10-15 days, reach after more than 16 when AGP method detects antibody horizontal, cut open and kill animal and take fresh spleen, kidney or fabricius bursa, tissue is pulverized, after homogenate at subzero 20 DEG C of-30 DEG C of multigelations more than 8 times, freeze 16h at every turn, melt 8h mono-circulation and need 24h altogether, 4000rpm is centrifugal, get supernatant, molecular sieve filtration, get molecular weight and be less than 3000 dalton's parts;
(2) molecular weight obtained is less than in 3000 daltonian parts; being concentrated into moisture is 99wt%; 0.2mg stabilizing agent potassium sorbate and 0.3mg Nephropathogenic infectious bronchitis specific transfer factor according to claim 1 compound thermal protective agent compound thermal protective agent is added in every mL; degerming after mixing, to obtain final product.
This product adopts the novel biochemical para-immunity reinforcing agent developed for main component with specific chicken transfer factor.This product dosage is little, biological activity is high, effectively can improve the immunity of poultry, can improve the immune level of vaccine simultaneously, eliminate systemic immune response most possibly.And this product is biogenetic products, has no side effect, noresidue, it is a kind of green veterinary drug of high effect nontoxic.Be applicable to prevent and treat Nephropathogenic infectious bronchitis; Be applicable to immunity of livestock low, improve immunity; Be applicable to the virosis high-incidence season, reduce infection rate and mortality rate; Be applicable to immune effect of vaccine undesirable, can make that vaccine immunity level is neat, antibody is high, potent antibodies holds time length; Be applicable to stress, reduce the respiratory tract disease etc. that vaccine causes.
Usage and consumption: mixed to drink, oral, eye dripping or spraying.Every 10ml is used for 1000 plumage poultry.
Effect duration: preserve 2 years for 4 DEG C ,-20 DEG C of freezen protective 5 years.Room temperature preserves 6 months.
Beneficial effect of the present invention:
1, Nephropathogenic infectious bronchitis specific transfer factor compound thermal protective agent of the present invention, good to Nephropathogenic infectious bronchitis specific transfer factor protected effect, long storage periods only has little loss of effective components;
2, Nephropathogenic infectious bronchitis transfer factor solution is a type cytokines preparation, biological activity is high, there is wide spectrum enhancing immunity, alleviate Disease Clinical symptom, improve the effects such as immune effect of vaccine, effect higher than chemical medicine series products such as levamisole hydrochloride activity rapidly, few, the noresidue of dosage; Rapider, easy to use than the effect of Chinese medicine class, be convenient to transport, be small-molecule substance, use approach many, drinking-water, oral, eye dripping, collunarium, injection etc.
Detailed description of the invention
For a better understanding of the present invention, further illustrate below in conjunction with specific embodiment.
embodiment 1:
A kind of Nephropathogenic infectious bronchitis specific transfer factor compound thermal protective agent, is mixed by the raw material of following weight proportion:
Mannitol, maltose, chitosan, histidine, magnesium sulfate are by weight 2:3:1:1:3.
The application of Nephropathogenic infectious bronchitis specific transfer factor compound thermal protective agent in the preparation of infectious bronchitis of chicken specific transfer factor, comprises the following steps:
(1) adopt infectious bronchitis of chicken live vaccine 28/86 strain 10 multiple dose to carry out immunity to the healthy poultry of every plumage part, each doses of virus content is not less than 1500ELD
50, after 7 days, then use Nephropathogenic infectious bronchitis live vaccine W93 strain 20 multiple dose intramuscular injection booster immunization, each doses of virus content is not less than 2000ELD
50, use the immunity of Nephropathogenic infectious bronchitis oil-emulsion inactivated vaccine 1.5mL subcutaneous injection, viral level is 10 simultaneously
3.8tCID
50/ 0.1mL, after 10 days, then use Nephropathogenic infectious bronchitis oil-emulsion inactivated vaccine 1.5mL subcutaneous injection immune strengthening once, viral level is not less than 10
3.8tCID
50/ 0.1mL, antibody horizontal is measured after 10-15 days, reach after more than 16 when AGP method detects antibody horizontal, cut open and kill animal and take fresh spleen, kidney or fabricius bursa, tissue is pulverized, after homogenate at subzero 20 DEG C of-30 DEG C of multigelations more than 8 times, freeze 16h at every turn, melt 8h mono-circulation and need 24h altogether, 4000rpm is centrifugal, get supernatant, molecular sieve filtration, get molecular weight and be less than 3000 dalton's parts;
(2) molecular weight obtained is less than in 3000 daltonian parts; being concentrated into moisture is 99wt%; 0.2mg stabilizing agent potassium sorbate and 0.3mg Nephropathogenic infectious bronchitis specific transfer factor compound thermal protective agent compound thermal protective agent is added in every mL, degerming after mixing, to obtain final product.4 DEG C of cold preservations or-20 DEG C of freezen protective.
zoopery
one, Nephropathogenic infectious bronchitis specific transfer factor promotes chickling cell immunization experiments
1 test material
1.1 experimental animals: 3 age in days healthy chicks 200.
1.2 test drugs: Nephropathogenic infectious bronchitis specific transfer factor solution, specification is 10ml/ bottle, containing Nephropathogenic infectious bronchitis specific transfer factor 50mg.
1.3 feedstuffs: chickling perfect compound feed.
1.4 chemical reagent: lymphocyte separation medium, 2.5% glutaraldehyde, paramagenta solution, 4% sodium nitrite solution, six azo paramagenta solution, naphthylacetate solution, phosphate buffer, C.I. 42590 dyeing liquor etc., all purchased from units concerned or prepare voluntarily.
1.5 key instruments: blood counting chamber, six figure place classification counters, binocular biological microscope etc.
2 test methods
2.1 test groupings: test is divided into A, B, C, D, E five groups at random with chicken, often organizes 40.A, B are test group, C, D matched group, and E group is blank group.A group every chicken injects 0.05 milliliter of Nephropathogenic infectious bronchitis specific transfer factor solution, the Nephropathogenic infectious bronchitis specific transfer factor solution of oral 0.05 milliliter of B group every chicken, the normal saline that C group is oral 0.05 milliliter, the normal saline of D group injection 0.05 milliliter, E group does not do any process.
2.2 test methods: the Nephropathogenic infectious bronchitis specific transfer factor that A, B, C, D, E five group of 7 age in days immunity uses the present invention to prepare according to dividing into groups in first 12 hours respectively and normal saline, carry out cell counting respectively at blood sampling when the 14th day, 21 days, and ask its meansigma methods.Concrete grammar is see relevant immunological testing method (ANAE method).
3 result of the tests: T, bone-marrow-derived lymphocyte count results are in 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 57.189%, 53.368%, 34.337%, 35.201%, 33.327%.Show through statistical disposition, each test group TC value improves significantly (t > 1.96, P < 0.05) compared with matched group.
The peripheral blood TC value of 21 Japanese instar chicklings, the average T C value of A, B, C, D, E group is respectively 66.115%, 61.489%, 38.372%, 39.481%, 36.236%.Show that each test group TC value improves highly significant (t > 2.57, P < 0.01) compared with matched group through statistics.
T, bone-marrow-derived lymphocyte ration statistics table (ANAE staining) in table 1 chicken blood
Note: record when this result is repeated trials, test repeats to be that 6 what take is the new multipole inspection of randomized block experiment.Mark letter representation significance test result after numeral: all have same letter person, and difference is not remarkable; Capitalization represents 0.01 significant level; Lower case represents 0.05 significant level.
4 conclusions and discussion
4.1 result of the test displays, two test group and matched group T lymphocyte percentage significant difference (P < 0.05), prove that Nephropathogenic infectious bronchitis specific transfer factor solution is to the effect of being significantly improved of T cell value tool.Containing bioactive little peptide in Nephropathogenic infectious bronchitis transfer factor solution, immune function can be improved when it enters in body, and T lymphocyte is as the one of immunocyte, also raising to a certain extent, therefore comparatively matched group is high for test group T cell percentage rate, and significant difference.
4.2 injection groups and oral group of T cell ratio difference not significantly, prove that injection is identical with the effect of oral Nephropathogenic infectious bronchitis specific transfer factor solution.Because this solution contains little peptide, by injecting pathway, it is acted on body by the speed that body absorbs, and therefore injection group can promote that T cell ratio raises.Little peptide molecular weight less (lower than 10000 dalton), can not by decomposition such as trypsin, Chymotrypsin, pepsin, DNA enzymatic and RNA enzymes, therefore when it enters in body, meeting can fast by intestinal absorption, enter blood circulation, thus act on immune system, so oral group of T cell ratio also can raise.The speed that injection and oral route absorb effective ingredient is all very fast, and the effect therefore showing as both Promote immunities is clinically suitable, and difference is not remarkable, and therefore this immunostimulant uses more for convenience.
4.3 Nephropathogenic infectious bronchitis specific transfer factor solution have comparatively high bioactivity, and its mechanism of action and chemical classes immunostimulant (such as levamisole hydrochloride etc.) exist larger difference.Nephropathogenic infectious bronchitis transfer factor solution is the material of normal presence in animal body, under normal condition, its concentration is lower, after concentration improves, the performance of its biological activity is quite remarkable, intensity and the speed of immune response reaction can be improved within a short period of time, therefore comparatively traditional chemical para-immunity reinforcing agent is very remarkable for biological activity advantage, is the biological immunopotentiator very with development prospect.
two, Nephropathogenic infectious bronchitis specific transfer factor is to the Experiment on therapy of Nephropathogenic infectious bronchitis
1 materials and methods
1.1 material
1.1.1 Nephropathogenic infectious bronchitis specific transfer factor, prepared by this research department, freezen protective.
1.1.2 chicken transferfactor, is purchased from market, and Tianjin Rui Pu animal health-care product company limited produces, freezen protective.
1.1.3 doxycycline hydrochloride soluble powder, oresol, interferon etc. are purchased from market.
1.1.4 supply examination chicken to select the commodity fowl disease chicken of natural occurrence second day chicken group respectively for the selection trying chicken, the sick chicken of Stochastic choice, dead chicken, the chicken group that dissection and laboratory diagnosis all meet pathological change feature is for examination chicken.Be divided into 3 groups, often group is 500, and feeding and management environmental condition is similar.
1.2 method
1.2.1 Nephropathogenic infectious bronchitis specific transfer factor and chicken transferfactor are taken out, according to 10mg/ml concentration dilution, aseptic subpackaged, 4 DEG C save backup.
1.2.2 diagnose: according to clinical symptoms and pathological anatomical change, and the diagnosis of Binding experiment room, can confirm that this disease is Nephropathogenic infectious bronchitis.
1.2.3 therapeutic test: test divides 3 groups,
1st group of therapeutic scheme is treated for selecting poultry genetic engineering interferon, controls secondary infection with the use of antibiotic medicines such as doxycycline hydrochloride soluble powders.Meanwhile, use compound oral fluid infusion salt to supplement sodium in body, potassium loss and eliminate in renal inflammation or drinking-water and add compound various vitamins etc., improve a fowl resistance, be used in conjunction 3 days.
2nd group be on the basis of the 1st prescription case+Nephropathogenic infectious bronchitis specific transfer factor, Nephropathogenic infectious bronchitis specific transfer factor using dosage is that every 1000 chickens use 10ml.
3rd group be on the basis of the 1st prescription case+chicken transferfactor, every 1000 chickens of chicken transferfactor use 10ml.
2 results
As can be seen from the test results, each test group has good therapeutical effect to Nephropathogenic infectious bronchitis, effective with regard to energy second day of medication, in 3rd day with regard to basic controlling the state of an illness, sick chicken stops dead, wherein second group of survival rate 93.6%, 3rd group of survival rate 90.2%, first group of survival rate is 82.2%, and significant difference (P<0.05), in table 2.
the therapeutic effect of table 2 pair Nephropathogenic infectious bronchitis
Group |
Test chicken |
Morbidity age in days |
To fall ill death toll in latter 5 days |
Survival number |
Cure rate |
1st group |
500 plumages |
16 days |
89 plumages |
411 |
82.2% |
2nd group |
500 plumages |
16 days |
32 plumages |
468 |
93.6% |
3rd group |
500 plumages |
16 days |
49 plumages |
451 |
90.2% |
3 clinical expansion therapeutic effect
According to result of the test, adopt Nephropathogenic infectious bronchitis specific transfer factor scheme to carry out expanding test to the clinical Nephropathogenic infectious bronchitis broken out, treat 32126 according to statistics, therapy rehabilitation 29681, cure rate reaches more than 92%.Within after medication second day, just can control death, within the 3rd day, just can take a turn for the better, after the 4th day, gradually can recover appetite, substantially can rehabilitation after one week.
4 analysis and thinking
4.1 Nephropathogenic infectious bronchitis are a kind of inhibitive ability of immunity, high degree in contact sexually transmitted disease, annoying poultry husbandry for many years always, carry out mortality harm to poultry industrial belt.When the kidney of chickling is subject to encroaching on, body produces decline or the disappearance of immune antibody, metabolite discharges existing problems, thus cause immunosuppressant, young chicken group is declined 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 huge economic loss to poultry husbandry.So, the prevention and corntrol to this disease must be strengthened at ordinary times.
4.2 according to primary disease cause a disease reason be because infringement kidney, so matter of utmost importance be will from alleviation kidney weak, dissolve, get rid of tangible material, dredging urinary tract, control death set about.Because chicken Nephropathogenic infectious bronchitis specific transfer factor not only has renewable reservoir effect to pathological tissue, the omnibearing immunity of body can also be improved, sick chicken death is controlled rapidly so have after use said preparation, the course of disease is short, recover the feature such as fast, fully demonstrates the advantage that Animal diseases prevented and treated by biogenetic products.
4.3 chicken Nephropathogenic infectious bronchitis specific transfer factors have enhancing human body immunity effect, have selectivity and act on targetedly the symptom of Nephropathogenic infectious bronchitis.Pharmaceutical research proves, chicken Nephropathogenic infectious bronchitis specific transfer factor is a kind of immunologic active material, immunogen performance can be improved, there is the phagocytic function of enhancing body reticuloendothelial system, in addition, the weight of immune organ can also be improved, improve total white blood cells and lymphocyte number, strengthen antibody forming capacity.Therefore, in production reality, good application value is had.
three, bin stability experiment
The stability of product ensures primary condition safe and effective for medication, especially polypeptide amino acid series products, and in water, effective content declines very fast, and therefore stability test is an important topic of this subject study.Affect the factor of preparation stability, except its factor such as prescription, technique, also have the factors such as temperature, light, humidity.Therefore, to this product carried out influence factor's test, accelerated test, room temperature keep sample for a long time investigate test.
1.1 samples: selection meets the sample of three batch samples as stability of Nephropathogenic infectious bronchitis specific transfer factor solution quality standard; Reference substance is mannitol: maltose: the Nephropathogenic infectious bronchitis specific transfer factor sample that chitosan (weight ratio 2:3:1) is protected.
1.2 periods of storage and condition
1) influence factor's test:
A. high temperature and humidity test: above-mentioned sample and reference substance are placed in respectively the mid-temperature of commercially available back 40 ± 2 DEG C, lower 3 months of relative humidity 75% condition, each moon detects once.
B. exposure experiments to light: above-mentioned sample and reference substance are positioned over respectively in lighting box, arrange illuminance 4500 ± 500Lx, temperature is lower 10 days of 25 DEG C 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 in respectively the mid-temperature of commercially available back 25 ± 2 DEG C, lower 8 months of relative humidity 60% condition, respectively at 0,2,4,6,7,8 month pick test once, to determine EDD.
1.3 testing indexs and method:
Inspection item: outward appearance; Folin phenol method measures content of peptides.
1.4, result of the test:
1) influence factor's result of the test:
Table 3 stores result statistical table under hot and humid experiment condition
Table 4 stores result statistical table under illumination experiment condition
2) long-term test results
Table 5 is long storage periods result statistical table under room temperature experiment condition
1.5 conclusion (of pressure testing)s:
Sample and reference substance are tested through influence factor, accelerated test, room temperature keep sample investigation experimental observation for a long time, and sample quality is stablized, and appearance character, content all meet the requirements, and reference substance quality stability is slightly poor.