CN101612396A - A kind of canine distemper live vaccine and preparation method thereof - Google Patents
A kind of canine distemper live vaccine and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of canine distemper live vaccine and preparation method thereof.The present invention utilizes the inventor to be separated to such an extent that a strain has good immunogenic canine distemper virus nature attenuated strain CGMCC No.3201 as producing strain by the field, prepare safety, effectively, the canine distemper live vaccine of single composition.The present invention effectively reduces the risk of introducing other poison of living when immune canine distemper vaccine, for the diffusion of controlling the fur-bearing animal disease provides condition.
Description
Technical field
The present invention relates to a kind of canine distemper live vaccine and preparation method thereof, belong to the veterinary biological product technical field.
Background technology
Canine distemper (Canine Distemper; CD); be commonly called as the Canis familiaris L. pestilence; be by canine distemper virus (Canine Distemper virus; CDV) infecting a kind of acute, the deadly infectious diseases of rare animals such as fur-bearing animal such as the ermine cause, Nyctereutes procyonoides, fox and dog and panda, tiger, is at present dog industry, fur-bearing animal aquaculture and the conservation of wildlife to be supported by China already to endanger one of maximum eqpidemic disease.Mainly popular cold season in the Winter-Spring, be distribute, local popular or break out.Because the clinical manifestation of canine distemper is ever-changing, when clinical diagnosis, easily obscure mutually with canine distemper, brought certain difficulty to clinical diagnosis and experimentation.
Canine distemper virus belongs to Paramyxoviridae, and Morbillivirus is the ameristic RNA viruses of minus strand sub-thread of spiral shape symmetrical.Size 150～250nm, virion is most spherical in shape, and it is 15～17nm spiral type nucleocapsid that the center also has wide footpath, has shaft-like fibre prominent, only contains hemagglutinin, and the impassivity propylhomoserin.CDV is to warm and dry responsive, 50～60 ℃, gets final product deactivation in 30 minutes.Because CDV can not long-term surviving in hot season, so this disease is popular in Winter-Spring cold season.CDV is to ultraviolet and alkaline solution sensitivity, and visible light is easily with inactivation of virus, and available clinically 3% sodium hydroxide (Caustic soda), bleaching powder are as disinfectant.As far back as 1905, Carre with regard to the cause of disease that proposes primary disease be a kind of virus (Carr, Y H.Sur la maladie desjeunes chiens.Compt.Rend.Acad.d.Sc.1905,140:689-690).Nineteen fifty-one, Dedie has turned out CDV with the method for tissue culture first.Rockborn finds, CDV can form in syncytium, sternzellen and the nuclear on former generation Madin-Darby canine kidney(cell line) or intracytoplasmic inclusion (Xia Xianzhu. foster dog complete works. Changchun: the .1993.9:549-553 of the Jilin People's Press; Yin Zhen, Liu Jinghua. animal virology. second edition. Beijing: Science Press, 1997,10:789-801).China is the end of the seventies, priorities such as Hua Guoyin from the CDV attenuated vaccine of external import, be separated to the strain of many strain vaccines (Hua Guoyin, the research that the research I. of mink canine distemper immunity duplicates Embryo Gallus domesticus tissue culture attenuated vaccine. the poultry infectious disease, 1980,4:30-35).
Morbidity is anxious, characteristics such as be difficult to heal with medicine because canine distemper has; at present; the main canine distemper vaccine that relies on prevents; be used to have the vaccine product of multiple prevention canine distemper both at home and abroad; but in the practical application of disease prevention; because exist the immunogenicity of strain very different, problem such as antigen titre is not high, may there be the problem that can not produce effective immunoprotection in the vaccine of use and causes the situation of immuning failure to occur.Simultaneously, mostly multi-joint live vaccine in the canine distemper vaccine product both domestic and external, if plant is under the situation that disease beyond the canine distemper did not take place, use multi-joint live vaccine to prevent hastily, may exist and cause the canine distemper risk of disease in addition, this situation is particularly serious when using some to prepare nonstandard product.
The objective of the invention is to utilize the inventor to have good immunogenic canine distemper virus low virulent strain by the isolating strain in field, safety, canine distemper attenuated freeze dried vaccine are effectively prepared in the CDV-11 strain.Simultaneously, above-mentioned vaccine is a kind of single Seedling that can be used for canine distemper susceptible animals such as fox and mink, Nyctereutes procyonoides, dog, solved the problem that is not used in effective canine distemper list Seedling of animals such as fox on the market, fill up the blank on the market, and provide condition for the diffusion of control fur-bearing animal disease.
Summary of the invention
The objective of the invention is to utilize the inventor to be separated to such an extent that a strain has good immunogenic canine distemper virus nature attenuated strain as the Strain of producing vaccine by the field; be inoculated on the Vero cell; the cytopathy venom that results infect; add freeze drying protectant; carry out lyophilizing with the freeze-drying curve of suitable this strain, prepare safety, effectively, the canine distemper live vaccine of single composition.
The production strain of canine distemper live vaccine of the present invention is to be separated to such an extent that a strain has good immunogenic canine distemper virus nature attenuated strain from the field, therefore, those skilled in the art can expect that canine distemper virus nature attenuated strain of the present invention produces seed culture of viruses as live vaccine and can be used for producing the multi-joint live vaccine of canine distemper virus antigen preparation equally.
1 production of vaccine strain
(1) viral source
The inventor gathers the dog internal organs and the lymphoid tissue of body temperature one property crossed rising by in the doubtful sick dog of canine distemper, is separated to the doubtful virus of a strain canine distemper with the passage method, this virus has been carried out the virusology evaluation, and cell culture is inoculated pup.The virusology qualification result shows that PCR detects the CDV positive, and other physicochemical property and biological characteristics meet the characteristics of canine distemper virus; The pathological anatomy check result shows that the inoculation animal does not have the canine distemper clinical symptoms, does not all find the pathological change of canine distemper after the dissection yet, illustrates that the virus that is separated to is canine distemper attenuated, called after CDV-11 strain.It is that (this strain virus had been delivered China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation on 07 15th, 2009, was numbered: CGMCC No.3201) in strain canine distemper nature attenuation strain CDV-11 strain that the present invention prepares used production virus.
(2) Strain characteristic
1) Virus culture 5% is received Vero cell (the Vero cell that the inventor preserves with virus sample by what keep liquid measure, nutritional solution is that the MEM that contains the MEM growth-promoting media of 10% new-born calf serum or contain 2% new-born calf serum keeps liquid, cultivation according to a conventional method) on the monolayer, puts 37 ℃, 5%CO
2Leave standstill cultivation in the incubator, observe CPE every day, observed continuously 96～120 hours, when cell swelling occurs, becomes and justify, to merge, come off, the change of cell fusion sexually transmitted disease (STD) reaches 80% o'clock results, puts-20 ℃ of preservations.CDV11 strain C1 is made malicious valency for venom measure, detect altogether three times, the result is respectively 10
5.63TCID
50/ ml, 10
6.0TCID
50/ ml and 10
5.5TCID
50/ ml.
2) Bing Du morphologic observation is done the negative staining of Electronic Speculum phosphotungstic acid to the culture fluid in CDV-11 strain C1 generation and is observed and (see Fig. 1 electromicroscopic photograph, the arrow place is a virion), as seen have among Fig. 1 based on pleomorphic type virion circular or oval, that differ in size, come in every shape, the a plurality of virion that have are assembled in heaps, be also shown in virus nucleocapsid silk in heaps or wall scroll, also can see the regular helical form nucleocapsid of arranging in bigger virion inside, also can see baculovirus particle.
3) nucleic acid based evaluation routinely method measure, the viral growth metabolism of CDV-11 strain is not suppressed by BUDR, proves that CDV-11 strain C1 is the RNA type for the nucleic acid of virus, and is consistent with the nucleic acid type of canine distemper virus.
4) blood clotting routinely method carry out, CDV-11 strain C1 does not all have compendency for virus to 1% chicken, Cavia porcellus, rabbit, swine erythrocyte.
5) physicochemical property detects virus according to a conventional method to different pH value, temperature, time with to the tolerance test of chemical reagent such as ether, chloroform, formaldehyde.The physicochemical property qualification result shows, preserves viral malicious valency more than 0 ℃ and descends very soon, and 37 ℃, behind the 60min, malicious valency has reduced by 10
3.5TCID
50, 56 ℃, behind the 30min, malicious valency has reduced by 10
4.0TCID
50The CDV low virulent strain is to ether, chloroform, formaldehyde chemical reagent sensitivity; PH 7.0～9.0 virus activities are stable, and the following or 9.0 above activity of pH7.0 all are subjected to very big influence.
5) Molecular Virology is identified the F gene conserved region sequence of delivering CDV according to Genbank, designs 1 couple of primer: P1:5 '-GCTGGTTGGAGAATAAGG-3 ', P2:5 '-CCAACTCCCA TAGCATAA-3 '.For extracting RNA the venom, carry out the RT-PCR amplification with above primer from CDV-11 strain C1 after reverse transcription, the result amplifies the specific nucleic acid band of 585bp, as figure.
6) Serological testing CDV-11 strain C1 (is given by professor Zhu Ruiliang of Shandong Agricultural University for viral liquid and canine distemper positive serum, SN tires and is 1:355) neutralization back inoculation Vero cell, found that CDV-11 strain C1 is suppressed by canine distemper positive serum specificity for the pathological changes interaction energy to the Vero cell, shows that the CDV-11 strain is a canine distemper virus.
7) safety
1. animal returns test
The pathological material of disease inoculation test is numbered healthy susceptible dog (the SN antibody≤1: 4) 4 of pathological material of disease 2～3 monthly ages of inoculation of acquired original respectively, every while collunarium 2ml, abdominal cavity 8ml, and the inoculation back was observed 21 days, detected body temperature every day, observation spirit, diet, feces etc.Wherein 1 dog occurs once raising at body temperature on the 12nd, recovers normal again on 2nd, and other 3 the fervescence phenomenon do not occur; 4 dog diet, feces character are normal, spiritual no abnormality seen.4 test dogs are checked through pathological anatomy, all do not found the pathological change of canine distemper.
The virocyte inoculation test is inoculated healthy susceptible dog (SN antibody≤1: 4) 4 of 2～3 monthly ages with CDV-11 strain C1 for viral liquid, number respectively, every while collunarium 2ml, abdominal cavity 8ml, the inoculation back was observed 21 days, detect body temperature every day, observe spirit, diet, feces etc.After the inoculation, fervescence does not all appear in the inoculation dog in 21 days, the results are shown in Table 3; Diet, feces character are normal, spiritual no abnormality seen.4 test dogs are checked through pathological anatomy, all do not found the pathological change of canine distemper.
By isolating virus is carried out electron microscopic observation, specificity, nucleic acid based, blood clotting, physicochemical property, Molecular Virology, the evaluation of pure property confirms that the virus that we are separated to meets canine distemper paramyxovirus characteristic, and is and pure.Return test through animal, only have 1 dog body temperature to occur once raising behind the healthy susceptible dog of pathological material of disease poison inoculation, all the other are all normal; The pathological material of disease poison is inoculated dog connecting biography on the cell after 11 generations with viral cultures, does not cause the dog fervescence.Not finding that all canine distemper pathology changes after all test dogs are dissected, isolating virus is described to the dog had no pathogenicity, is the weak poison of a strain.
2. stable
The CDV-11 strain was passed for 15 generations in cell, every milliliter of viral level is all 10
5.5TCID
50More than, prove the CDV-11 strain 15 generations with interior be stable, can be used as production of vaccine with kind the poison.
By the CDV-11 strain dog, Vulpes, Nyctereutes procyonoides, mink are carried out heavy dose of test of inoculating, canine distemper clinical symptoms and pathological change all do not appear in the inoculation animal, and the result of the test of living together shows that nonimmune animal is not infected.
CDV-11 strain C5 is passed through animal body for poison passed for 3 generations continuously,, gather internal organs after slaughtering and detect per generation poison inoculation fox, when the 1st, 2 generations, can detect has CDV to exist in the animal body, repeat the 2nd generation and escalated dose inoculation 4,5 generations test again, does not all detect CDV.Result of the test shows that the CDV-11 strain is safe to dog, Vulpes, Nyctereutes procyonoides, mink, and does not have the anti-strong phenomenon of virulence, is the desirable strain that is used to prepare canine distemper attenuated Seedling.
8) immunogenicity
For the vaccine research of CDV-11 strain, the immunogenicity test has been carried out in the CDV-11 strain, animals such as immune fox, dog detect neutralizing antibody and carry out counteracting toxic substances, and the result shows that the CDV-11 strain has good immunogenicity.Relatively CDV11 strain C5, C15, C20 for immunogenicity after, the result there is no too big-difference, therefore chooses C5～C15 for planting poison as the basis, produces and is no more than for 3 generations with the seed subculture, can guarantee the immunogenicity of vaccine.
The production preparation of 2 canine distemper live vaccines
The production that (1) will prepare is with kind of a poison, adopts asynchronously to connect malicious method and be inoculated into Vero cell monolayer on the rolling bottle, then the rolling bottle cell put 37 ℃ of cultivations, and rotating speed is 8～10r/h.
(3) can gather in the crops viral liquid when cytopathy reaches 80% left and right sides, freeze thawing 1 time places-15 ℃ to freeze to preserve, and this is semi-finished product, and is to be checked standby.
(4) through the semi-finished product acceptance tests qualified after; with conventional freeze drying protectant (Chinese veterinary drug allusion quotation committee. People's Republic of China's veterinary drug allusion quotation; version was three ones in 2005. Chinese agriculture publishing house; 2006; to call " Chinese veterinary drug allusion quotation " in the following text) mix according to 1: 1 with the volume ratio of viral liquid, carry out lyophilizing.After lyophilizing is finished, labeling, warehouse-in is preserved.
The method of inspection of 3 canine distemper live vaccines
(1) steriling test is answered asepsis growth.Test by the regulation in " Chinese veterinary drug allusion quotation ".
(2) the mycoplasma check is tested by the regulation in " Chinese veterinary drug allusion quotation ", should not have the mycoplasma growth.
(3) diagnostic test does 10 with vaccine
-2Dilution is with equivalent 10
-1The canine distemper positive serum of dilution mixes, and in 37 ℃ and 30 minutes, 4 bottles in inoculation Vero cell is established 2 bottles of virus control groups simultaneously, puts 37 ℃ of cultivations, observes CPE96～120 hour continuously.
(4) the exogenous virus check is tested by the regulation in " Chinese veterinary drug allusion quotation ", should not have exogenous virus.
(5) after safety verification dissolves every part vaccine with the 1ml normal saline, 5 of 2～3 monthly age of intramuscular inoculation susceptible dogs (SN antibody titer≤1: 4), 10 parts/only, observed 21.
(6) efficacy test dissolves vaccine with the MEM that contains 2% new-born calf serum, makes every 1ml vaccine contain 1 part, does 10 times of dilutions of going forward one by one then, gets 10
-2, 10
-3, 10
-4, 10
-54 dilution factors, inoculation has grown up to the Vero cell 96 holes trace Tissue Culture Plate of monolayer respectively, each 8 hole of dilution factor inoculation, every hole 100 μ l, establish simultaneously and do not connect poison contrast, put in 37 ℃, 5% CO2 gas incubator and cultivate, observed 96～120 hours, record cytopathy (CPE) hole count is pressed the Reed-Muench method and is calculated TCID
50, every part vaccine virus content 〉=10
4.5TCID
50
(7) residual moisture is measured every batch of freeze-dried products and is appointed and take out 4 samples, and each sample residual moisture all should not surpass 4%.If have above the time, can heavily examine 1 time, heavily inspection back surpasses regulation if any 1 sample, this batch goods should be judged to defective (" Chinese veterinary drug allusion quotation ").
(8) vacuum is measured vacuum before measuring packing, and the goods of no vacuum should be rejected and be scrapped, and must not heavily find time to sell (" Chinese veterinary drug allusion quotation ").
Description of drawings
The normal Vero cell monolayer of Figure 11-1; The Vero cell monolayer showed cell pathological changes that the 1-2CDV-11 strain virus infects,
The electromicroscopic photograph of Fig. 2 virus, the arrow place is a CDV-11 strain virus particle.
Fig. 3 CDV11 strain virus pcr amplification product cleavage map 1 road M, 2 road CDV-11 strain virus, the specific nucleic acid band of the 585bp of demonstration amplified production
Advantage of the present invention
The present invention utilizes the inventor to be separated to such an extent that a strain has good immunogenic CDV and naturally subtracts by the field Strain is as producing strain, prepare safety, effectively, the canine distemper live vaccine of single composition. The present invention effectively reduces When immune canine distemper vaccine, introduce the risk of other poison of living, for the diffusion of controlling the fur-bearing animal disease provides condition.
Embodiment
Following examples further specify the present invention, but not as limitation of the present invention.
The pure property check of virus seed culture of viruses
1 steriling test is inoculated each 2 of T.G, G.P tubule and G.A slant mediums respectively with viral cultures, and every 0.2ml puts 37 ℃ of cultivations for one; Put 25 ℃ of cultivations for one, observed 3, T.G, G.P tubule and the G.A slant medium of the viral cultures inoculation after the transplanting are observed 5 average daily no antibacterials, fungus growth.Have or not antibacterial, fungus growth.
Viral cultures 5ml is got in the check of 2 mycoplasmas, is inoculated into the vial of liquid mycoplasma culture medium, gets 0.8ml again and be transplanted to 2 small test tube liquid mycoplasma culture mediums and 2 pairs of agar solid plates respectively from the bottle suspension.Liquid culture is placed 37 ℃ of cultivations, and solid culture is placed on 37 ℃ and contains 5%CO
2Environment is cultivated.Cultivated 5,10,15 days, and got 0.6～0.8ml culture respectively and transplant in to 2 small test tube fluid mediums (0.2ml/ props up) from bottle, 2 pairs of solid mediums (0.1～0.2ml/ pays) so repeat 3 times, and all transplanting cultures are all cultivated and observed 14 days.In the observation period, do not find that significant change appears in bottle and tubule culture color, the liquid culture of transplanting does not have " fried egg " shape mycoplasma bacterium colony on solid medium.
The check of 3 exogenous viruses
1) after cytopathogenic effect exogenous virus check neutralizes viral cultures and CDV positive serum fully, (every bottle of cell bottle floor space is 5cm * 7cm), and every bottle graft kind 1.0ml connects and passed for 2 generations to inoculate 3 bottles of F81, DK cell monolayer respectively, in per generation, observed 7, CPE all do not occur.
2) the fluorescent antibody inspection neutralizes viral cultures fully with the canine distemper positive serum, (every bottle of cell bottle floor space is 2cm * 3cm) to mixture inoculation Vero cell monolayer, cultivated 4, go down to posterity, with the 2nd generation culture through acetone fixed, detect the microscopy cell surface with fluorescence method and all do not find RV, BVDV fluorescent grain.
3) after hemadsorbing virus detected viral cultures and CDV positive serum neutralized fully, (every bottle of cell bottle floor space was that 2cm * 3cm), 37 ℃ of cultivations were tested on the 7th to inoculation Vero cell monolayer.Wash cell monolayer to be checked with the PBS buffer, rinsing 3 times, 0.2% guinea-pig red blood cell, people " O " type erythrocyte and the chicken red blood cell equivalent mixed liquor that add an amount of new preparation, jiggle, make it evenly to cover whole cell monolayer surface, select 2 cell monolayers, one bottle places 4 ℃, another bottle places 25 ℃ to cultivate 30min, wash with PBS, all do not adsorb chicken, Cavia porcellus, people " O " type erythrocyte with microscopic examination cell monolayer to be checked, show that the exogenous virus that does not have the erythrocyte characterization of adsorption pollutes.
The manufacturing of vaccine
The preparation of 1 cell nutrient solution
Adopt conventional cell growth medium (containing 10% nascent new-born calf serum) and keep liquid (containing 2% nascent new-born calf serum) and use as cell culture.
2 produce with kind of the preparation of poison
With the canine distemper that is separated to nature low virulent strain, the Vero cell monolayer on the inoculation rolling bottle, connect the poison amount for to keep 2% of liquid measure, when reaching 80%, cytopathy can gather in the crops, and in 1 generation of repeated transmission, preparation is produced with kind malicious.
The breeding of 3 viral liquid
With producing with kind of a poison, be inoculated into the Vero cell monolayer on the rolling bottle, connect the poison amount and be 2% of cell maintenance medium.Then the rolling bottle cell is put 37 ℃ of cultivations, rotating speed is 8～10r/h.
After the inoculation, every day, observation of cell pathological changes situation can be gathered in the crops when cytopathy reaches 80% left and right sides, and freeze thawing 1 time is put-15 ℃ and freezed to preserve, and this is semi-finished product.
5 lyophilizing
After the acceptance test of process semi-finished product is qualified, mixed according to 1: 1 with the volume ratio of viral liquid with conventional freeze drying protectant (" Chinese veterinary drug allusion quotation "), be distributed into the 2ml/ bottle, carry out lyophilizing according to the freeze-drying curve that designs, that is :-65 ℃, keep making the vaccine quick freezing in 2 hours, evacuation then is when vacuum reaches 13.3Pa, it is dry to begin to heat up, the sublimation stage product temperature is-20～-10 ℃, and shelf temperature is 8 ℃, and the distillation time is 10 hours; Resolution temperature is 30 ℃, 2 hours, all was freeze-drying time approximately? hour.
Warehouse-in is preserved after 6 labelings.
Embodiment 3
The product inspection of vaccine.
(1) steriling test is answered asepsis growth.Test by the regulation in " Chinese veterinary drug allusion quotation ".
(2) the mycoplasma check is tested by the regulation in " Chinese veterinary drug allusion quotation ", should not have the mycoplasma growth.
(3) diagnostic test does 10 with vaccine
-2Dilution is with equivalent 10
-1The canine distemper positive serum of dilution mixes, and in 37 ℃ and 30 minutes, 4 bottles in inoculation Vero cell is established 2 bottles of virus control groups simultaneously, puts 37 ℃ of cultivations, observes CPE96～120 hour continuously.
(4) the exogenous virus check is tested by the regulation in " Chinese veterinary drug allusion quotation ", should not have exogenous virus.
(5) after safety verification dissolves every part vaccine with the 1ml normal saline, 5 of 2～3 monthly age of intramuscular inoculation susceptible dogs (SN antibody titer≤1: 4), 10 parts/only, observed 21.0303001 batch, 0303002 batch of three batches of vaccine and 0303003 batch of equal safety verification of inventor's trial production are qualified.
(6) efficacy test dissolves vaccine with the MEM that contains 2% new-born calf serum, makes every 1ml vaccine contain 1 part, does 10 times of dilutions of going forward one by one then, gets 10
-2, 10
-3, 10
-4, 10
-54 dilution factors, inoculation has grown up to the Vero cell 96 holes trace Tissue Culture Plate of monolayer respectively, each 8 hole of dilution factor inoculation, every hole 100 μ l, establish simultaneously and do not connect poison contrast, put in 37 ℃, 5% CO2 gas incubator and cultivate, observed 96～120 hours, record cytopathy (CPE) hole count is pressed the Reed-Muench method and is calculated TCID
50
Tiring of three batches of vaccines of inventor's trial production: 0303001 batch is 10
5.19TCID
50/ head part, 0303002 batch be 10
5.0TCID
50/ head part, 0303003 batch be 10
5.12TCID
50/ head part.
(7) residual moisture is measured every batch of freeze-dried products and is appointed and take out 4 samples, and each sample residual moisture all should not surpass 4%.If have above the time, can heavily examine 1 time, heavily inspection back surpasses regulation if any 1 sample, this batch goods should be judged to defective (" Chinese veterinary drug allusion quotation ").
(8) vacuum is measured vacuum before measuring packing, and the goods of no vacuum should be rejected and be scrapped, and must not heavily find time to sell (" Chinese veterinary drug allusion quotation ").
Embodiment 4
The vaccine safety test
The 1 minimum safety test of using an age in days dog and a single dose inoculation of fox
Each 10 of two kinds of experimental animals (SN antibody≤1: 4 the 50 age in days left and right sides pups and back 1 all foxes of weaning) are divided into 2 groups again, 5 every group, 1 part of 0302003 batch of vaccine of the 1st group of intramuscular inoculation (inventor's manufacturing)/only, do not inoculate in contrast for the 2nd group.Isolated rearing detects body temperature, observes the mental status, diet and feces character.And in the time of 21 days every group extract 2 and catch and kill and do the pathology inspection.
After dog inoculates pup with canine distemper live vaccine, do not see any CDV symptom through clinical observation, body temperature is all in 38.2 ℃～39.2 ℃ normal range, and spirit, diet, feces character are all normal, and matched group is no abnormality seen also.Catch and kill 2 for every group in the time of the 21st day, through pathologic finding, no abnormality seen; After canine distemper live vaccine is inoculated young fox, do not see any CDV symptom through clinical observation, body temperature is all in 38.7 ℃～39.9 ℃ normal range, and spirit, diet, feces character are all normal, and matched group is no abnormality seen also.Catch and kill 2 with every group in the time of the 21st day, through pathologic finding, no abnormality seen.
Behind the dog and fox in single dose inoculation wean 1 week of back of canine distemper live vaccine (CDV-11 strain) freeze-dried vaccine, all dogs and fox are all normal.Illustrate that this vaccine is safe, the 2 week beginning immunity of animal wean back is safe and reliable in actual applications.
The safety test of the 2 pairs of dogs and fox single dose repeated inoculation
Each 10 of two kinds of experimental animals (dog and the foxes at 2～3 monthly ages of SN antibody≤1: 4) are divided into 2 groups, 5 every group, 0303003 batch of vaccine (10 of the 1st group of intramuscular inoculation
5.12TCID
50/ head part) 1 part/only, at interval 2 week back repeated inoculations 1 time, do not inoculate in contrast for the 2nd group.Isolated rearing detects body temperature, observes the mental status, diet and feces character.And when inoculating back 21 days the last time every group extract 2 and catch and kill and do the pathological anatomy inspection.
Canine distemper live vaccine is through 2 vaccinizations, and 10 pups do not see any CDV symptom through clinical observation, and body temperature is all in 38.2 ℃～39.4 ℃ normal range, and spirit, diet, feces character are all normal, and matched group is no abnormality seen also.In the time of the 21st day dog is catched and killed, through pathologic finding, no abnormality seen, instruction book dosage vaccinization vaccine is safe to pup.
Canine distemper live vaccine is through 2 vaccinizations, and 10 young Vulpes do not see any CDV symptom through clinical observation, and body temperature is all in 38.7 ℃～39.9 ℃ normal range.Spirit, diet, feces character are all normal, and matched group is no abnormality seen also.In the time of the 21st day fox is catched and killed, through pathologic finding, no abnormality seen.
By above evidence canine distemper live vaccine (CDV11 strain) freeze-dried vaccine single dose repeated inoculation dog and fox is safe.
The safety test of the 3 pairs of dogs and overdose of fox (10 part/only) inoculation
With each 20 of two kinds of experimental animals (dog and the foxes at 2～3 monthly ages of SN antibody≤1: 4), be divided into 4 groups, 5 every group.0303001 batch of vaccine (10 of the 1st group of intramuscular inoculation
5.19TCID
50/ head part) 10 parts/only, 0303002 batch of vaccine (10 of the 2nd group of intramuscular inoculation
5.0TCID
50/ head part) 10 parts/only, 0303003 batch of vaccine (10 of the 3rd group of intramuscular inoculation
5.12TCID
50/ head part) 10 parts/only, do not inoculate in contrast for the 4th group.4 group isolated rearings respectively detect body temperature, observe the mental status, diet and feces character.And every group of dog of inoculation randomly drawed 2 and catches and kills and do the pathology inspection in the time of 21 days.
All inoculation dogs were through 21 days clinical observation, and spirit, diet, feces are all normal, the canine distemper symptom do not occur.Body temperature is all in 38.2 ℃～39.4 ℃ normal range.In the time of the 21st day animal is catched and killed, do not seen any canine distemper pathological changes through dissecting.
All inoculation foxes were through 21 days clinical observation, and spirit, diet, feces are all normal, the canine distemper symptom do not occur.Body temperature is all in 38.7 ℃～39.9 ℃ normal range.In the time of the 21st day animal is catched and killed, do not seen any canine distemper pathological changes through dissecting.
The result shows: overdose of canine distemper live vaccine (CDV11 strain) (10 part/only) inoculation is safe to dog and fox.
The safety testing of the 4 pairs of conceived dogs and fox
Each 10 of conceived about 40 days dog of test usefulness and foxes.0303003 batch of vaccine (10 of the 1st group of 5 intramuscular inoculations
5.12TCID
50/ head part) 10 parts/only, do not inoculate in contrast for the 2nd group 5 are observed spirit on the 21st, diet, feces character, follow-up investigation farrowing situation (because pregnant animal may cause the machinery miscarriage in capture-process, so not detecting body temperature).
Through clinical observation on the 21st, inoculation dog spirit, diet, feces were all normal, and farrowing does not have obvious difference with matched group; Inoculation Vulpes spirit, diet, feces are all normal, and farrowing does not have obvious difference with matched group.Presentation of results canine distemper live vaccine (CDV11 strain) heavy dose of (10 parts) conceived dog of inoculation and fox are safe.
By canine distemper live vaccine to dog and fox carry out that minimum age in days single dose, single dose repeat, overdose inoculation and to the female beastly inoculation test of pregnancy, the result shows that canine distemper live vaccine (CDV-11 strain) is safe to dog and fox.Owing to may cause the machinery miscarriage in the capture-process, generally pregnant animal does not recommend to carry out vaccination in actual production for pregnant animal.
Embodiment 5
Canine distemper live vaccine (CDV-11 strain) potency test
Test is 3 batches of canine distemper (lyophilizing) live vaccine (CDV-11 strain) that the inventor prepares with vaccine: 0303001 (10
5.19TCID
50/ head part), 0303002 (10
5.0TCID
50/ head part), 0303003 (10
5.12TCID
50/ head part) tests.Before immunity, vaccine is diluted with normal saline, make every milliliter of vaccine contain 10
4.0TCID
50Canine distemper virus, carry out immunity after the dilution immediately.
With 45 young Vulpes of canine distemper SN antibody≤1: 4, observe through the health in a week, under all normal situation of body temperature, spirit, diet, feces, these young Vulpes are divided into 3 batches at random, 15 every batch, every batch is further divided into 3 groups, 5 every group.The 1st batch every (contains 2 * 10 through intramuscular injection 2ml respectively
4.0TCID
50) 0303001,0303002,0303003 batch of vaccine; The 2nd crowd every difference intramuscular injection 1ml (contains 10
4.0TCID
50) 0303001,0303002,0303003 batch of vaccine; The 3rd batch every respectively intramuscular injection 0.5ml (0.5 * contain 10
4.0TCID
50) 0303001,0303002,0303003 batch of vaccine; The 4th batch 5 are not in contrast immune.Isolated rearing, in back 21 days of inoculation, the SN antibody titer was measured in blood sampling respectively.
Immune 1ml (is contained 10
4.0TCID
50) fox and the matched group fox of dosage carried out counteracting toxic substances in back 21 days in immunity, attacks 100ID for every
50By dog preparation CDV/Q3 generation strong poison (to the ID of fox
50Be 10
-1.70/ ml), observed 21 behind the counteracting toxic substances.
At immune 0.5ml (0.5 * 10
4.0TCID
50) in the fox of canine distemper live vaccine, have at least 93.3% (toatl proportion 14/15) to reach more than 1: 50 at back 21 days SN antibody of immunity; Immunity 1ml (10
4.0TCID
50) and 2ml (2 * 10
4.0TCID
50) fox SN antibody 100% all more than 1: 50, wherein all obtain protection behind the fox counteracting toxic substances of immune 1ml.Immunity 0.5 * 10 is described
4.0TCID
50Above vaccine can make the protection immunity fox more than 90% resist the attack of strong poison.Concrete outcome sees Table 1.
Table 13 batch canine distemper live vaccine (CDV-11 strain) is to the potency test of fox
Above result shows: when immunity 0.5 * 10
4.0TCID
50/ only above canine distemper live vaccine (CDV-11 strain), the SN antibody of back 21 days 90% above foxes of immunity 〉=1: 50.This experimental result shows that the minimum immune dosage of vaccine is 0.5 * 10
4.0TCID
50/ only; Immunity 10
4.0TCID
50/ canine distemper live vaccine (CDV-11 strain) only, the counteracting toxic substances protective rate is 100%.Therefore the immunizing dose of fox is 10
4.0TCID
50/ only; The standard of dispatching from the factory of vaccine is decided to be: every part viral level 〉=10
4.5TCID
50
Sequence table
＜110〉Qilu Animal Health Products Co., Ltd.
＜120〉a kind of canine distemper live vaccine and preparation method thereof
<160>2
<170>
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: canine distemper virus PCR primer P1
<400>1
GCTGGTTGGA?GAATAAGG 18
<210>2
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: canine distemper virus PCR primer P1
<400>2
CCAACTCCCA?TAGCATAA 18
Claims (3)
1. a canine distemper live vaccine is characterized in that this vaccine contains the canine distemper live virus that preserving number is CGMCCNo.3201.
2. preparation method that is used for canine distemper live vaccine; it is characterized in that with preserving number being that the weak naturally viral disease strain of CGMCCNo.3201 is by inoculation Vero cell; 37 ℃ of cultivations; when connecing poison cell and have 80% pathological changes to occur; results virocyte culture fluid adds freeze drying protectant commonly used, makes freeze-dried live vaccine through lyophilization.
3. a preparation method that is used for canine distemper live vaccine is characterized in that with preserving number being that CGMCCNo.3201 can be used for producing the multi-joint live vaccine of canine distemper virus antigen preparation equally as live vaccine production seed culture of viruses.
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| US3080291A (en) * | 1960-06-10 | 1963-03-05 | Jensen Salsberg Lab Inc | Serial passage of distemper virus in tissue cultures of chick embryo and canine tissue and vaccine therefrom |
| US3138531A (en) * | 1961-07-12 | 1964-06-23 | Lilly Co Eli | Canine distemper vaccine |
| CN1117081A (en) * | 1995-03-16 | 1996-02-21 | 高云 | Triple live vaccine and toxin vaccine for distemper, rabies and pavovirus and its preparing method |
| TWI302570B (en) * | 2004-03-11 | 2008-11-01 | Komipharm Int Co Ltd | Canine distemper virus isolated in korea and recombinant vaccine using the same |
| CN1876181A (en) * | 2006-04-21 | 2006-12-13 | 中国人民解放军军事医学科学院军事兽医研究所 | Weak toxicity united vaccine formulation for canidae animal important disease series and preparation process |
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