CN100423775C - Inactivated vaccine of cow chlamydia, its preparation and inspection method - Google Patents

Abstract

The present invention relates to an inactivated vaccine for preventing and treating the infectious diseases of cattle, a preparation method thereof and a relevant inspection method in the preparation processes. The preparation method comprises the following steps: diluting chlamydia psittaci SX5 germ poison or NX germ poison sourcing from cattle with a phosphate buffer or physiological saline, inoculating the germ poison in a healthy chick embryo without chlamydia psittaci infection incubated at a temperature of 37 DEG C for 6 to 7 days, and collecting the vitelline membrane and allantoic fluid of the dead chick embryo inoculated for 72 hours as antigens for preparing the vaccine; diluting the antigens after comminuted, and adding formaldehyde for inactivation; mixing the inactivated antigens with oil adjuvant according to the proportion of 1 to 1, uniformly shaking to obtain a mixture, and emulsifying the mixture by a high pressure homogenizing machine to obtain the vaccine.

Description

Inactivated vaccine of cow chlamydia and preparation thereof and the method for inspection

Technical field

The present invention relates to a kind of preparation method of preventing and treating the inactivated vaccine of cattle infectious disease, and the related check method in this vaccine and the preparation process.

Background technology

The chlamydia bovis disease is a kind of infectious disease of cattle, it to cattle-raising harm the most serious be the chlamydia bovis miscarriage, this disease is a kind of endemical contagious disease by chlamydia psitacci infection Niu Yinqi, is principal character with cow in calf miscarriage, premature labor, stillbirth or product debility calf.This sick alias has foothill abortion, the popular miscarriage in NIUDI side, the miscarriage of cattle Neorickettsia.The loss that causes to the cattle farm is not only that fetus dies young because the milk chlamydia bovis is miscarried, and more seriously milk yield is compared with the normal labor cattle, and the underproduction significantly causes feedstuff, manpower huge waste and the spending of huge medical fee simultaneously.External report, a pasture that on average has 60 through milking cow and 20 primiparity cows, annual milk yield decline, milk downgrade, miscarriage and the infertile economic loss that causes that causes because of chlamydia infection reaches 40,000 Euros.At present, about 1,000 ten thousand of China milch cow livestock on hands, the most conservative 10% the milk cattle infected chlamydiosis of pressing, the economic loss that causes every year (is amounted to 4,500,000,000 yuan of RMB) approximately up to 400,000,000 5 thousand ten thousand Euros.In addition, the chlamydia psitacci infection people causes people's ornithosis disease.Also whether cow-orign Bedsonia psittacosis harm humans health, also is the problem that people pay close attention to.

As far back as nineteen twenties, the American has at first reported the popular miscarriage in NIUDI side syndrome; By 1956, the German adopted serology experiment and cell culture technology, proved that the former federal republic of germany foothill abortion is by due to the chlamydia infection.Later European many countries, Canada, the former Soviet Union, Japan, India also report the chlamydia bovis miscarriage in succession.China is from the eighties, and some provinces and regions are reported this disease successively, and Hubei Province (1983) isolate chlamydia from abortus calf and the milk of the milk cows of trouble endemicity miscarriage; Check that with complement fixation test (CFT) 6 cows chlamydial antibody positive rates are 10%.1988, Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences found the miscarriage of yak chlamydia at home first, and detecting the chlamydial antibody positive rate with CFT is 29.03% (45/155), and is separated to 8 strain chlamydia psittacis from aborted fetus.The chlamydiosis serosurvey was carried out to 6 cattle farms, Xi'an in Shaanxi in 1991, and positive rate reaches 28.31%, and is separated to chlamydia from aborted fetus.1997-1998, the Lanzhou veterinary institute is with detecting chlamydial antibody with indirect hemagglutination test (IHAT) for 642 parts to stocker, meat labour dual-purpose cattle and the yak serum that picks up from 8 provinces and regions such as Shandong, Hebei, Henan, Shaanxi, Ningxia, Gansu, Qinghai and Sichuan, and the result: the average positive rate of stocker is 32.6%; Meat labour dual-purpose cattle 15.2%; Yak is 18.7%.Calendar year 2001, the Lanzhou veterinary institute detects 343 parts of Babalus bubalis L. serum I HAT that pick up from Guangxi, 12 counties in Anhui and Jiangsu, finds that all there is chlamydia infection in varying degrees in each county Babalus bubalis L. group, and what positive rate was minimum is 3.3%, is up to 39.3%.2002-2004, the conceived cattle miscarriage in some geographic cattle farms of Shaanxi and Ningxia problem is more serious, and the Lanzhou veterinary institute proves once more that with serology and nosetiology method chlamydia psittaci is one of important cause of disease that causes the milch cow miscarriage.Chlamydiosis can not be underestimated the harm of cattle-raising.

At present, carry out the prevention and the treatment of chlamydia bovis disease with antibiotic such as tetracycline, chlortetracycline, the expense costliness because a large amount of or life-time service antibiotic can cause producing new drug resistance strain, obviously reduces prevention effect or to no effect, the palindromia rate rises.Simultaneously, the intravital drug residue of the milch cow of taking medicine raises, the residual rising of milk Chinese medicine, and edible antibiotic high residue milk or meat are with the serious harm people health.Therefore, each state all forbids adding the bacterial infectious disease that all kinds of antibiosis are are usually prevented and treated domestic animal in feedstuff, and advocates the bacterial infectious disease that adopts based on the comprehensive measure control domestic animal of vaccine immunity.

Each state still by today, does not see the report that any relevant chlamydia bovis disease vaccine is succeeded in developing all at the vaccine research of being correlated with.

Summary of the invention

The object of the present invention is to provide the preparation method of the sick prevention of a kind of chlamydia bovis with inactivated vaccine, and the related check method in this vaccine and the preparation process.

The preparation method of inactivated vaccine of cow chlamydia of the present invention is with cow-orign Bedsonia psittacosis SX5 or NX kind malicious phosphate buffer or 10000 times of dilutions of normal saline with 0.1M pH7.2, be inoculated in 37 ℃ of 6～7 days and healthy Embryo Gallus domesticus that do not have chlamydia psitacci infection of hatching, the vitellinae membrana of results inoculation dead Embryo Gallus domesticus after 72 hours and allantoic fluid are as antigen for vaccine, smash antigen to pieces after-filtration with tissue mashing machine, then with filtrate 20 times of dilutions of phosphate buffer (PBS) with 0.1M pH7.2, the formaldehyde that adds concentration 37% by 0.2% of dilution back antigen amount, the container strict seal is good, fully shake evenly, in 4 ℃ of environment deactivations 72 hours, with disinfectant 206 adjuvants or No. 11 edible white oils as adjuvant, inactivation antigen and oily adjuvant are pressed 1: 1 mixed, shake evenly, use high pressure homogenizer emulsifying, get vaccine.

For cow-orign Bedsonia psittacosis SX5 or NX seedling bacterial strain (planting poison) in the ultra cold storage freezer long preservation, can be earlier when seedling with inoculation in no chlamydia infection breeder flock produced through the 6-7 of 37 ℃ of hatchings day instar chicken embryo, pass 1-2 for recovering, particularly adopt the recovery of 2 generations.Method for resuscitation is: with seedling bacterial strain SX5 or NX 10 times of dilutions of sterile saline of long-term ultralow temperature preservation, inoculation 6-7 day instar chicken embryo, under aseptic condition, collect the vitellinae membrana of the Embryo Gallus domesticus of inoculation death in 4-6 days, it is pulverized the back with after the phosphoric acid liquid buffer (PBS) or 1000 times of dilutions of isotonic saline solution of 0.1MpH7.2, again it is inoculated on the secondary Embryo Gallus domesticus by aforesaid method, under aseptic condition, collects the vitellinae membrana conduct of the second filial generation Embryo Gallus domesticus of inoculation death in 4-6 days again and produce the antigenic kind poison of seedling.

Inactivated vaccine of cow chlamydia of the present invention refers to the vaccine that aforementioned preparation method is prepared.

The method of inspection in the inactivated vaccine of cow chlamydia preparation of the present invention is:

A) seed poison check: the 6-7 day instar chicken embryo that the cow-orign Bedsonia psittacosis SX5 or the NX seedling inoculation of-70 ℃ of long preservation are not had 37 ℃ of hatchings that breeder flock produced of chlamydia infection, pass 1-2 generation, can make whole inoculated into chick embryo in inoculation death regularly later in 72 hours, get the vitellinae membrana smear Ji nurse Sa dyeing microscopic examination of dead Embryo Gallus domesticus, only see a large amount of mauve chlamydia elementary bodies (E), and without any other living contaminantses, and its toxicity evaluation is 10 ^-11-10 ^-13ELD ₅₀, this Embryo Gallus domesticus culture can be used as kind (son) poison of breeding vaccine antigen.

B) antigen breeding check: the vitellinae membrana of back 72 hours dead Embryo Gallus domesticus of results inoculation and allantoic fluid wherein contain a large amount of chlamydia elementary bodies, and bacterioscopy are negative, detection 〉=10 of tiring as antigen for vaccine ^-8ELD ₅₀For qualified;

C) antigen deactivation check: the antigen deactivation is after 72 hours, and aseptic draw samples is inoculated artificial two culture medium and 6-7 day instar chicken embryo respectively and cultivated, no any bacterial growth in the synthetic medium, chick embryo development does not normally have death, illustrates that the antigen deactivation is qualified, can be used for seedling;

D) antigen emulsifying check: emulsifying antigen is creamy white, and is not stratified.Oil-in-water type is a W/O/W, and it is circular at once to the water surface to drip emulsifying antigen, and cloud scatters for qualified;

E) vaccine test: randomization, the every bottle of Seedling of being quarantined is inoculated synthetic medium and white mice respectively, carry out steriling test, safety verification and efficacy test, 21 days counteracting toxic substances behind 5 these vaccines of mouse inoculation of test group, all can't check chlamydia from all mices, inoculation mice growth promoter is normal, simultaneously steriling test negative for vaccine imitate examine qualified.

This vaccine production and product quality inspection step comprise: the breeding of antigen Embryo Gallus domesticus and the results of cow-orign Bedsonia psittacosis seedling strain; The seedling antigen titre detects (〉=10 ^-8ELD ₅₀); Seedling antigen steriling test; Antigenic deactivation of seedling and check; The emulsifying of inactivation antigen; The vaccine packing; The steriling test of vaccine, safety verification and efficacy test.The inactivated vaccine of cow chlamydia of under the GMP condition, producing by this step, give suitable numerous cattle before breeding or breed back 1 month muscle or subcutaneous injection 5ml/ head can effectively prevent miscarriage, the stillbirth that causes by chlamydia psitacci infection; Give calf muscle or subcutaneous injection 2ml/ head, can effectively prevent the calf pneumonia-enteritis that causes by chlamydia psitacci infection, polyarthritis, conjunctivitis etc.Immunoprotection phase of this vaccine reached more than 10 months.The safety of a large amount of these vaccines of evidence is good.

Beneficial effect of the present invention is:

1) adopt the present invention can prepare inactivated vaccine of cow chlamydia, it can be effectively prevented the chlamydia bovis disease of various symptoms to the cows inoculation, can make and produce high-caliber chlamydia specific antibody in the immune cattle body, produce strong immunity, effectively resist chlamydia infection, corresponding test shows, inoculating once that its protection period reaches can 10 month, can guarantee substantially that pregnant cattle can normal labor and the arrival of high-yielding lactating phase; And effectively protect calf to avoid chlamydia infection.

2) adopt earlier with the healthy Embryo Gallus domesticus of inoculation during seedling of the present invention in 6～7 days no chlamydia psitacci infection of 37 ℃ of hatchings, pass 1-2 for recovering, particularly passing for 2 generations recovers, bacterial strain after the reuse recovery is the method for kind of malicious seedling, the cow-orign Bedsonia psittacosis SX5 or the NX bacterial strain virulence of ultra cold storage freezer long preservation are brought back to life, overcome cow-orign Bedsonia psittacosis SX5 or the NX bacterial strain seedling of direct use in the long-term preservation of ultralow temperature, produce because of its isolated strains virulence strong inadequately, or virulence hereditary character instability, pass its toxicity evaluation of several generations continuously at Embryo Gallus domesticus or cell and obviously reduce, and reduce the deficiency of tiring of vaccine.Experimental results show that adopting method of the present invention can make seedling bacterial strain SX5 or NX is 10 to the toxicity evaluation of 6-7 age in days SPF Embryo Gallus domesticus ^-11-10 ^-13ELD ₅₀, 20 generations of Embryo Gallus domesticus continuous passage with endogenous toxin try hard to keep hold constant.

3) application of this vaccine needn't be taken antibiotics chemical medicine prevention chlamydia infection again, and the milch cow drug disposition is residual to be reduced greatly thereby make, and can effectively guarantee the food safety in milch cow source, safeguards the safety of people's diet.Also can reduce the cost of cattle breeding on the other hand greatly, increase economic efficiency.

The specific embodiment

One embodiment of the present of invention below are provided:

A) plant the recovery and the check of poison: with the cow-orign Bedsonia psittacosis SX5 of-70 ℃ of long preservation or NX seedling inoculation in the 6-7 day instar chicken embryo of 37 ℃ of hatchings that breeder flock produced of no chlamydia infection, pass 1-2 generation, can make whole inoculated into chick embryo in inoculation death regularly later in 72 hours, get the vitellinae membrana smear Ji nurse Sa dyeing microscopic examination of dead Embryo Gallus domesticus, only see a large amount of mauve chlamydia elementary bodies (E), and without any other living contaminantses, this Embryo Gallus domesticus culture can be used as kind (son) poison of breeding vaccine antigen.

B) antigen breeding and check: the chlamydial antibody feminine gender is laid eggs after the hatching egg surface clean sterilization that breeder flock produced, put into 37 ℃ of hatchings of special-purpose hatch machine 6-7 days, phosphate buffer or the normal saline of kind of poison with 0.1MpH7.2 is diluted in the gnotobasis for 10000 times, through yolk sac approach inoculated into chick embryo, dosage of inoculation 0.4ml/ Embryo Gallus domesticus.The vitellinae membrana of back 72 hours dead Embryo Gallus domesticus of results inoculation and allantoic fluid wherein contain a large amount of chlamydia elementary bodies as antigen for vaccine.Bacterioscopy is negative, detection 〉=10 of tiring ^-8ELD ₅₀For qualified, can be used for seedling.

C) antigen deactivation and check: with b) antigen (vitellinae membrana) that is up to the standards smashs after-filtration to pieces with tissue mashing machine, then filtrate is diluted 20 times with 0.1M pH7.2PBS, add the commercially available formaldehyde of concentration more than 37% by 0.2% of dilution back antigen amount, fully shake evenly, the container strict seal is good, in 4 ℃ of environment deactivations 72 hours, will shake container 2-3 time every day, vibrated 5 minutes at every turn.After the antigen deactivation 72 hours, aseptic draw samples is inoculated synthetic medium and 6-7 day instar chicken embryo respectively and is cultivated, and no any antibacterial cattle is long in the synthetic medium, and chick embryo development does not normally have death, illustrates that the antigen deactivation is qualified, can be used for seedling.

D) antigen emulsifying and check: select mineral oil adjuvant (206 adjuvants or No. 11 edible white oils) autoclave sterilization for use.With inactivation antigen and oily adjuvant by 1: 1 mixed, shake evenly.Use high pressure homogenizer emulsifying, pressure is transferred to 35-40Pa during emulsifying, and emulsifying antigen is creamy white, and oil-in-water type is a Water-In-Oil bag oil, and it is circular at once to the water surface to drip emulsifying antigen, and cloud scatters.The vaccine branch vaccine bottle of packing into.

E) vaccine test: according to the regulation of national biological product check, sampling immediately, the every bottle of Seedling of being quarantined is inoculated synthetic medium and white mice respectively, carries out steriling test, safety verification and efficacy test.The used kind poison of efficacy test counteracting toxic substances is former seedling bacterial strain.21 days counteracting toxic substances behind 5 these vaccines of mouse inoculation of test group all can't check chlamydia (the organizational routine smear staining is checked or PCR checks negative) from all mices; 5 of matched groups are not inoculated this vaccine, and the same period, counteracting toxic substances all can detect chlamydia (the organizational routine smear staining is checked or PCR checks positive) from all mices.This is qualified for the inspection of vaccine effect.Growth promoter is normal behind 5 these vaccines of mouse inoculation of test group.Steriling test is negative simultaneously.This batch vaccine of producing is for being up to the standards.

Potency test data of the present invention see Table 1

The test of table 1 milch cow vaccine potency

Group	The vaccine lot number	For examination cattle number	The immunity time	Immunizing dose	The counteracting toxic substances time	Counteracting toxic substances dosage	Protective rate
								1	040113	3	04-04-08	5ml	04-05-08	2ml	3/3
2	040210	3	04-04-08	5ml	04-05-08	2ml	2/3
								3	040317	3	04-04-08	5ml	04-05-08	2ml	3/3
Contrast		2		0	04-05-08	2ml	0/2

Table 1 explanation: check negative milch cow with the immune respectively 3 groups of chlamydial antibodies of 3 batches of milch cow chlamydiosis inactivated vaccines of the present invention (the vaccine lot number is 040113,040210,040317), every group 3, immunizing dose 5ml/ head, back 21 days counteracting toxic substances of immunity, 3 test group have 1 hair disease as a result, its protective rate is respectively 3/3,2/3, and 3/3; Total protective rate of this test is 8/9 (88.9%).Matched group 2 cow heads are not inoculated this vaccine, 2/2 morbidity behind the counteracting toxic substances, and protective rate is 0/2.

The present invention is used for milch cow and carries out vaccine immunity phase test for data and see Table 2

The test of table 2 milch cow vaccine immunity phase

Group	The vaccine lot number	For examination cattle number	The immunity time	Immunizing dose	The counteracting toxic substances time	Counteracting toxic substances dosage	Protective rate
								1	030312	3	03-05-12	5ml	04-03-30	2ml	3/3
2	030312	3	03-05-12	5ml	04-03-30	2ml	3/3
								3	030312	3	03-05-12	5ml	04-03-30	2ml	3/3
Contrast		2		0	04-03-30	2ml	0/2

Table 2 explanation: check negative milch cow with the immune respectively 3 groups of chlamydial antibodies of 3 batches of milch cow chlamydiosis inactivated vaccines of the present invention (the vaccine lot number is 030312,030327,030403), every group 3, immunizing dose 5ml/ head, immunity back 10 wheat harvesting period counteracting toxic substances, 3 test group full guard as a result, protective rate is respectively 3/3,3/3, and 3/3; Matched group 2 cow heads are not inoculated this vaccine, 2/2 morbidity behind the counteracting toxic substances, and protective rate is 0/2.

Calf milch cow vaccine immunity safety testing, conceived milch cow vaccine immunity safety testing, and milch cow field immunity safety testing the results are shown in Table 3-1,3-2 and 3-3.

Table 3-1 calf milch cow vaccine immunity safety testing (30-40 age in days)

Group	The vaccine lot number	For examination cattle number	The immunity time	Immunizing dose	Injection site	Diet situation	Security evaluation
								1	030312	5	03-05-09	5ml	No swelling	Normally	Safety
2	030327	5	03-05-09	5ml	No swelling	Normally	Safety
								3	030403	5	03-05-05	5ml	No swelling	Normally	Safety

The conceived milch cow vaccine immunity of table 3-2 safety testing (conceived about 2 months)

Group	The vaccine lot number	For examination cattle number	The immunity time	Immunizing dose	Injection site	The fetus influence	Security evaluation
								1	030312	5	03-05-09	5ml	No swelling	Normal labor	Safety
2	030327	5	03-05-09	5ml	No swelling	Normal labor	Safety
								3	030403	5	03-05-09	5ml	No swelling	Normal labor	Safety

Table 3-3 milch cow field immunity safety testing

The area	Immunity milch cow number	Immunizing dose	Safety is observed	Security evaluation
					Shaanxi	100	5ml	Injection site and the no abnormal reaction of diet after the adult milch cow immunity	Safety
Ningxia	4350	2-5ml	Injection site and the no abnormal reaction of diet after calf, the milch cow immunity of growing up, lactogenic is normal substantially.	Safety
					Gansu	730	2-5ml	Injection site and the no abnormal reaction of diet after calf, the milch cow immunity of growing up, lactogenic is normal substantially.	Safety

Table 3 (1-3) illustrates: 3 batches of tests of laboratory show that milch cow chlamydiosis inactivated vaccine of the present invention is all fool proof to calf, about 2 months adult milch cow of pregnancy.1 death does not take place in field 5180 of milch cows of immunity, and injection site and diet Non Apparent Abnormality change, and after vaccinating, lactation amount is normal substantially.

Claims (5)

1. the preparation method of inactivated vaccine of cow chlamydia, it is characterized in that cow-orign Bedsonia psittacosis SX5 or NX kind malicious phosphate buffer or 10000 times of dilutions of normal saline with 0.1M pH7.2, be inoculated in 37 ℃ of 6～7 days and healthy Embryo Gallus domesticus that do not have chlamydia psitacci infection of hatching, the vitellinae membrana of results inoculation dead Embryo Gallus domesticus after 72 hours and allantoic fluid are as antigen for vaccine, smash antigen to pieces after-filtration with tissue mashing machine, then with filtrate 20 times of dilutions of phosphate buffer with 0.1M pH7.2, the formaldehyde that adds concentration 37% by 0.2% of dilution back antigen amount, the container strict seal is good, fully shake evenly, in 4 ℃ of environment deactivations 72 hours, with disinfectant 206 adjuvants or No. 11 edible white oils as adjuvant, inactivation antigen and oily adjuvant are pressed the 1:1 mixed, shake evenly, use high pressure homogenizer emulsifying, get vaccine.

2. the preparation method of inactivated vaccine of cow chlamydia according to claim 1, it is characterized in that the seedling bacterial strain, be inoculated in the healthy Embryo Gallus domesticus of 6～7 days no chlamydia psitacci infection of 37 ℃ of hatchings during use earlier, pass 1-2 for recovering, method for resuscitation is: with seedling bacterial strain SX5 or NX 10 times of dilutions of sterile saline of long-term ultralow temperature preservation, inoculation 6-7 day instar chicken embryo, under aseptic condition, collect the vitellinae membrana of the Embryo Gallus domesticus of inoculation death in 4-6 days and pulverize the back as first generation kind poison, phosphate buffer or the 1000 times of dilutions of isotonic saline solution with 0.1M pH7.2 of kind of poison pulverizing back are inoculated in it on secondary Embryo Gallus domesticus by aforesaid method again, under aseptic condition, collect the vitellinae membrana pulverizing back conduct of the second filial generation Embryo Gallus domesticus of inoculation death in 4-6 days again and produce the antigenic kind poison of seedling.

3. the preparation method of inactivated vaccine of cow chlamydia according to claim 2 is characterized in that the seedling bacterial strain, is inoculated in 6～7 days the healthy Embryo Gallus domesticus of no chlamydia psitacci infection of 37 ℃ of hatchings during use earlier, passes for 2 generations to recover.

4. according to the prepared vaccine of preparation method of claim 1 or 2 or 3 described inactivated vaccine of cow chlamydia.

5. the method for inspection during inactivated vaccine of cow chlamydia prepares is characterized in that:

A) seed poison check: the 6-7 day instar chicken embryo that the cow-orign Bedsonia psittacosis SX5 or the NX seedling inoculation of-70 ℃ of long preservation are not had 37 ℃ of hatchings that breeder flock produced of chlamydia infection, pass 1-2 generation, can make whole inoculated into chick embryo in inoculation death regularly later in 72 hours, get the vitellinae membrana smear Ji nurse Sa dyeing microscopic examination of dead Embryo Gallus domesticus, only see a large amount of mauve chlamydia elementary bodies, and without any other living contaminantses, and its toxicity evaluation is 10 ^-11-10 ^-13ELD ₅₀, this Embryo Gallus domesticus culture can be used as the seed poison of breeding vaccine antigen.

B) antigen breeding check: the vitellinae membrana of back 72 hours dead Embryo Gallus domesticus of results inoculation and allantoic fluid wherein contain a large amount of chlamydia elementary bodies, and bacterioscopy are negative, detection 〉=10 of tiring as antigen for vaccine ^-8ELD ₅₀For qualified;

C) antigen deactivation check: the antigen deactivation is after 72 hours, and aseptic draw samples is inoculated synthetic medium and 6-7 day instar chicken embryo respectively and cultivated, no any bacterial growth in the synthetic medium, and chick embryo development does not normally have death, illustrates that the antigen deactivation is qualified, can be used for seedling;

D) antigen emulsifying check: emulsifying antigen is creamy white, and is not stratified.Oil-in-water type is a W/O/W, and it is circular at once to the water surface to drip emulsifying antigen, and cloud scatters for qualified;

E) vaccine test: randomization, the every bottle of Seedling of being quarantined is inoculated synthetic medium and white mice respectively, carry out steriling test, safety verification and efficacy test, 21 days counteracting toxic substances behind 5 these vaccines of mouse inoculation of test group, all can't check chlamydia from all mices, inoculation mice growth promoter is normal, simultaneously steriling test negative for vaccine imitate examine qualified.