CN104415353A - Toxin attack efficacy detection method for atrophic rhinitis inactivated vaccines of pigs - Google Patents
Toxin attack efficacy detection method for atrophic rhinitis inactivated vaccines of pigs Download PDFInfo
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Abstract
The invention provides a toxin attack efficacy detection method for the atrophic rhinitis inactivated vaccines of pigs. The method comprises a step of determining a toxigenic pasteurella multocida toxin attack dosage and a step of carrying out toxin attach efficacy on piglets after being immunized. According to the method, the problem that the toxin attack dosage cannot be accurately judged due to pasteurella multocida bacteria with different toxicities is solved, and the problem of post-immunization effect evaluation on the atrophic rhinitis inactivated vaccines of piglets and immunized pigs is also solved.
Description
Technical field
The present invention relates to a kind of vaccine counteracting toxic substances effect detecting method.
Background technology
Atrophic rhinitis (atrophic rhinitis, AR) be a kind of important respiratory diseases of swinery, it can cause the chronic rhinitis of pig, sneeze, drop tears, the symptom such as the degeneration of face portion and turbinates atrophy, the serious nose kiss that can also cause is out of shape and affects whole body skeleton development, thus cause pig growth retardation, make pig easily infect Secondary cases plyability pneumonia, cause huge loss to pig industry.This disease can infect the pig of any age level, and the pig infection rate wherein within 4 week age is the highest, and symptom generally appears at 8-12 age in week.Atrophic rhinitis is normally caused jointly by bordetella branchiseptica (Bb) and pasteurella multocida (Pm).Wherein, bordetella branchiseptica serotype is single, and virulence is relatively stable, plays a part to open nasal mucosa door in atrophic rhinitis's pathological changes, and then allows pasteurellosis bacillus enter in pig body, causes the generation of atrophic rhinitis.
Vaccine immunity is prevention progressive atrophic rhinitis of pig effective method, existing atrophic rhinitis vaccine effect detecting method is the inspection of counteracting toxic substances effect and alternative zoometry antibody, wherein counteracting toxic substances effect detecting method is that immune sow is to piglet counteracting toxic substances method, be specially the atrophic rhinitis vaccine counteracting toxic substances immunity sow containing bordetella branchiseptica (Bb) and pasteurella multocida (Pm) antigenic component, not immune sow matched group is set simultaneously, immunity sow litter is together with the first Bb counteracting toxic substances of not immune sow litter 7 age in days, 10 ages in days carry out Pm counteracting toxic substances, 42 age in days process piglet observed results.But not yet useful containing after the atrophic rhinitis vaccine immunity piglet of bordetella branchiseptica (Bb) and pasteurella multocida (Pm) antigenic component, detect the method that counteracting toxic substances effect is examined.
Further, the Pasteurella Multocida Strains virulence of separate sources is also incomplete same, adds that serotype is numerous, cause the dosage cannot judging counteracting toxic substances when the inspection of counteracting toxic substances effect accurately, so that counteracting toxic substances effect is examined unsuccessfully, counteracting toxic substances model cannot be set up, and then accurately cannot judge the effect of vaccine.
Summary of the invention
For solving the deficiencies in the prior art, the invention provides a kind of atrophic rhinitis inactivated vaccine counteracting toxic substances effect detecting method, described method not only solves the problem that different virulence pasteurella multocida antibacterial causes counteracting toxic substances dosage accurately to judge, also efficiently solves the problem of Efficacy evaluation after piglet immunological atrophic rhinitis inactivated vaccine.
Main purpose of the present invention is to provide a kind of atrophic rhinitis inactivated vaccine counteracting toxic substances to imitate detecting method, and described method comprises: the determining step of toxin producing pasteurellosis bacillus counteracting toxic substances dosage, and to counteracting toxic substances effect inspection step after piglet immunological.
Preferably, described method comprises the preparation process of counteracting toxic substances bacterium liquid further, the preparation process of described counteracting toxic substances bacterium liquid comprises and bordetella branchiseptica and pasteurella multocida strain being activated respectively at solid medium plane, bordetella branchiseptica and pasteurella multocida monoclonal described in picking are in culture medium culturing respectively, results bacterium liquid; To described bordetella branchiseptica and the count of bacteria of pasteurella multocida bacterium liquid, determine bacterium liquid viable bacteria content.
More preferably, described bordetella branchiseptica is bordetella branchiseptica HN8 strain, and described pasteurella multocida is pasteurella multocida D type HB4 strain.
Bordetella branchiseptica HN8 strain (Bordetella bronchiseptica HN8) preserving number is: CCTCC NO.M2011223, pasteurella multocida D type HB4 strain (Pasteurellamultocida D Type HB4) preserving number is: CCTCC NO.M2011221, preservation date is on 06 28th, 2011, preservation address is: Wuhan, China, Wuhan University; Depositary institution is China typical culture collection center (being called for short CCTCC).
More preferably, the culture medium of described cultivation bordetella branchiseptica and pasteurella multocida monoclonal bacterial strain is solid medium.
Use the preparation method of counteracting toxic substances bacterium liquid of the present invention to carry out the cultivation of bacterium liquid, the viable bacteria content accuracy estimated out is higher, has greatly improved to the accuracy of quantitative counteracting toxic substances dosage.
Preferably, the determining step of described pasteurella multocida counteracting toxic substances dosage comprises and determines that pasteurella multocida is to piglet every nostril counteracting toxic substances dosage by pasteurella multocida to mice median lethal dose(LD 50).
More preferably, wherein said pasteurella multocida is 1 ~ 2 × 10 to piglet every nostril counteracting toxic substances dosage/pasteurella multocida to mice median lethal dose(LD 50)
3~ 1 ~ 2 × 10
4.
Preferably, described counteracting toxic substances effect inspection step after piglet immunological to be comprised: after immune group piglet immunological two is exempted from, carry out intranasal counteracting toxic substances together with counteracting toxic substances matched group piglet, the counteracting toxic substances while of two nostrils with the viral mixed liquor of bordetella branchiseptica and killing property Pasteur bacterium more; Within after last counteracting toxic substances 35 days, dissect piglet, piglet turbinates atrophy situation is marked, counteracting toxic substances matched group at least 80% piglet turbinates atrophy mark 8 points and more than, immune group piglet turbinates atrophy average mark statistically significantly reduces than counteracting toxic substances matched group piglet turbinates atrophy average mark.
Describedly statistically significantly be reduced to immune group piglet turbinates atrophy average mark than counteracting toxic substances matched group piglet turbinates atrophy score by statistical analysis, P<0.05, significant difference.
More preferably, described piglet immunological two exempts from rear 21st day, the 24th day and the 25th day continuous counteracting toxic substances.
More preferably, B.bronchisepticai HN8 strain 2.05 × 10 is contained in described counteracting toxic substances bacterium liquid
9cFU/ nostril, containing pasteurella multocida D type HB4 strain 2.2 × 10
9~ 2.2 × 10
10cFU/ nostril.
More preferably, described piglet 7-10 age in days head exempts from, and head exempts to carry out two in latter 14 days and exempts from.
Detailed description of the invention
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The embodiment of the present invention illustrates the present invention for bordetella branchiseptica HN8 strain, pasteurella multocida LFB3 strain, P-2237 strain and HB4 strain.
The determination of the pasteurella multocida counteracting toxic substances dosage of embodiment 1, different virulence
1 method
1.1 strain
Bordetella branchiseptica HN8 strain (Bordetella bronchiseptica HN8) preserving number is: CCTCC NO.M2011223, pasteurella multocida D type HB4 strain (Pasteurellamultocida D Type HB4) preserving number is: CCTCC NO.M2011221, preservation date is on 06 28th, 2011, preservation address is: Wuhan, China, Wuhan University; Depositary institution is China typical culture collection center (being called for short CCTCC).Pasteurella multocida LFB3 strain is from doctor J.M.Rutter (Compton, UK), be disclosed in Evaluation ofvaccines for atrophic rhinitis-a comparison of three challenge models(Vaccine20 (2002) 1797 – 1802, T.Magyar etc.), pasteurella multocida P-2237 strain is disclosed in qualification (the Chinese animal and veterinary of Toxigenic Pasteurella multocida, 2010,37 (1): 149-151; Li Weijie etc.).
The preparation of 1.2 counteracting toxic substances bacterium liquid
The bordetella branchiseptica of preservation and pasteurella multocida strain are lined TSA(containing 10% chicken serum) dull and stereotyped 37 DEG C of cultivation 16h, use BHI(10% glycerol) wash lower rear mixing and be distributed into 5ml/ bottle, be stored in-70 DEG C, reply counting after melting and calculate every bottle of freeze-dried vaccine viable count (repeating to reply counting to take the mean for three times).Adjusting bacterial concentration according to experimental program is counteracting toxic substances desired concn.
The determination of the pasteurella multocida counteracting toxic substances dosage of 1.3 different virulence
1.3.1 the pasteurella multocida of different virulence is to mice half lethal dose
The pasteurella multocida LFB3 strain that three strain virulence are different, P-2237 strain and HB4 strain, use the bacterium liquid of the different gradient concentrations of above three strain bacterial strains to mice counteracting toxic substances respectively, calculate three strain strains to mice half lethal dose according to each dosage dead mouse situation.
1.3.2 the pasteurella multocida of different virulence is to piglet counteracting toxic substances
The pasteurella multocida LFB3 strain that three strain virulence are different, P-2237 strain and HB4 strain, together with Bb, counteracting toxic substances is carried out to 42 age in days piglets with three strain pasteurella multocida of above various dose respectively, every strain pasteurella multocida arranges three various dose groups, and often group experiment piglet number is 5.Counteracting toxic substances mode is as follows: piglet 42 age in days carries out the 1st counteracting toxic substances, and interval is carried out the 2nd time and the 3rd counteracting toxic substances afterwards for continuous 2 days on the 2nd, identical with dosage for the mode often organizing experiment piglet three counteracting toxic substances.The mode of collunarium is adopted to carry out during counteracting toxic substances, by B.bronchisepticai HN8 strain and the mixing of pig pasteurella multocida bacterium liquid, according to replying counting discreet value during subpackage containing B.bronchisepticai HN8 strain 2 × 10 in mixed bacterium liquid
9cFU/ml, pasteurella multocida LFB3 strain, P-2237 strain and HB4 strain bacterium liquid hold-up are respectively in table 1.1ml/ nostril during counteracting toxic substances, two nostril counteracting toxic substances simultaneously.Day by day Continuous Observation 35 days.Carry out count plate while counteracting toxic substances, determine actual counteracting toxic substances dosage, the actual counteracting toxic substances dosage of B.bronchisepticai HN8 strain is 2.05 × 10
9cFU/ml, pasteurella multocida LFB3 strain, P-2237 strain and the actual counteracting toxic substances dosage of HB4 strain bacterium liquid are respectively in table 1.
Table 1 respectively organizes pasteurella multocida and the dosage thereof of counteracting toxic substances
Estimate that counteracting toxic substances dosage is substantially identical by the visible actual counteracting toxic substances dosage of actual counteracting toxic substances Dose Results with experiment, bacterium solution preparation method of the present invention can Accurate Prediction viable bacteria content, and the accuracy of toxic agent amount of attacking against each other has greatly improved.
1.4 turbinates lesion examinings
After last counteracting toxic substances, 35 days process piglets, mark to turbinates atrophy situation, concrete dissect and standards of grading as follows:
By corpse accumbency, slightly raised by its lower jaw, removing nose epidermis, the line between the premolar teeth of pig upper jaw both sides first, cuts a transverse section with hacksaw, observes turbinates atrophy (TA) situation and deviation of nasal septum (NSD) from transverse section.
Turbinates atrophy (TA) judges that a point 0-4 divides
0: normal, without any pathological changes.
1: very little a part of atrophy (curl for and be no more than half atrophy).
2: slight atrophy: curl for and exceed half atrophy.
3 moderate atrophys: turbinates is tetanic.
4: severe atrophy: concha nasalis stock is whole curls disappearance.
Deviation of nasal septum (NSD) judges that a point 0-2 divides
0: normal
1: slight bending
2: severe bends
It is 18 points (pig has four turbinates, and severe atrophy 4 points of quadruplications add deviation of nasal septum 2 points totally 18 points) that turbinates atrophy (TA) situation and deviation of nasal septum (NSD) are added highest score.Each test group turbinates pathological changes mark is got its average mark (Mean nasallesion scores), then carries out statistical analysis.
2 results
The pasteurella multocida of 2.1 different virulence is to mice half lethal dose
The pasteurella multocida LFB3 strain that three strain virulence are different, P-2237 strain and HB4 strain are respectively 1.1 × 10 to mice half lethal dose
4cFU, 1.02 × 10
5cFU and 1.3 × 10
6cFU.
The pasteurella multocida of 2.2 different virulence is to piglet counteracting toxic substances
The pasteurella multocida LFB3 strain that three strain virulence are different, P-2237 strain and HB4 strain respectively with various dose to result after 42 age in days piglet counteracting toxic substances respectively in table 2, table 3 and table 4, blank group turbinates atrophy situation is in table 5.Have result visible, pasteurella multocida LFB3 strain counteracting toxic substances dosage is 2.1 × 10
7cFU/ml and 2.1 × 10
8during CFU/ml, piglet more than 4/5 falls ill, and the average atrophy mark of turbinates is 9.0-11.8.Pasteurella multocida P-2237 strain counteracting toxic substances dosage is 2.05 × 10
8cFU/ml and 2.05 × 10
9during CFU/ml, piglet more than 4/5 falls ill, and the average atrophy mark of turbinates is 9.4-11.2.Pasteurella multocida HB4 strain counteracting toxic substances dosage is 2.2 × 10
9cFU/ml and 2.2 × 10
10during CFU/ml, piglet more than 4/5 falls ill, and the average atrophy mark of turbinates is 9.6-11.6.
Piglet turbinates atrophy situation after table 2LFB3 strain counteracting toxic substances
Note: turbinates atrophy mark is at 8 points and be judged to morbidity above.
Piglet turbinates atrophy situation after table 3P-2237 strain counteracting toxic substances
Note: turbinates atrophy mark is at 8 points and be judged to morbidity above.
Piglet turbinates atrophy situation after table 4HB4 strain counteracting toxic substances
Note: turbinates atrophy mark is at 8 points and be judged to morbidity above.
Table 5 blank piglet group turbinates mark
The pasteurella multocida LFB3 strain that three strain virulence are different, P-2237 strain and HB4 strain, be respectively 1.1 × 10 to mice half lethal dose
4cFU, 1.02 × 10
5cFU and 1.3 × 10
6cFU, pasteurella multocida LFB3 strain, P-2237 strain and HB4 strain are respectively 2.1 × 10 to pig counteracting toxic substances bacterium liquid hold-up
7cFU/ml ~ 2.1 × 10
8cFU/ml, 2.05 × 10
8cFU/ml ~ 2.05 × 10
9cFU/ml and 2.2 × 10
9cFU/ml ~ 2.2 × 10
10during CFU/ml, the morbidity of piglet counteracting toxic substances group more than 4/5, has certain dependency by result visible different virulence pasteurella multocida to mice virulence with between piglet counteracting toxic substances dosage, namely to piglet every nostril counteracting toxic substances dosage/be about 1 ~ 2 × 10 to mice median lethal dose(LD 50)
3~ 1 ~ 2 × 10
4.
Embodiment 2, atrophic rhinitis inactivated vaccine Immunization effect detecting method
1 method
1.1 strain
Bordetella branchiseptica HN8 strain preserving number is: CCTCC NO.M2011223, and preservation date is on 06 28th, 2011; Pig pasteurella multocida serum D type HB4 strain preserving number is: CCTCC NO.M2011221, and preservation date is on 06 28th, 2011, and preservation address is: Wuhan, China, Wuhan University; Depositary institution is China typical culture collection center (being called for short CCTCC).
The preparation of 1.2 counteracting toxic substances bacterium liquid
Bb and the Pm-D strain of preservation is lined TSA(containing 10% chicken serum) dull and stereotyped 37 DEG C of cultivation 16h, use BHI(10% glycerol) wash lower rear mixing and be distributed into 5ml/ bottle, be stored in-70 DEG C, reply counting after melting and calculate every bottle of freeze-dried vaccine viable count (repeating to reply counting to take the mean for three times).Adjusting bacterial concentration according to experimental program is counteracting toxic substances desired concn.
1.3 piglet immunologicals, counteracting toxic substances
Be divided into three groups to 25 7-10 age in days piglets, immune group 10, immune group atrophic rhinitis inactivated vaccine (toxoid of the bordetella branchiseptica containing deactivation and PMT toxin), head exempts from 2ml/ head, and one exempts to carry out two in latter 14 days exempts from, 2ml/ head.Another 10 is non-Immunization matched group, and remaining 5 is blank group.Immune group piglet two carries out counteracting toxic substances together with non-Immunization matched group piglet in three weeks after exempting from, and counteracting toxic substances mode and program are with 1.3.2 in embodiment 1, and counteracting toxic substances dosage is respectively 2.2 × 10
9cFU/ nostril and 2.2 × 10
10cFU/ nostril.
1.4 turbinates lesion examinings
After last counteracting toxic substances, 35 days process piglets, mark to turbinates atrophy situation, concrete dissect and standards of grading as follows:
By corpse accumbency, slightly raised by its lower jaw, removing nose epidermis, the line between the premolar teeth of pig upper jaw both sides first, cuts a transverse section with hacksaw, observes turbinates atrophy (TA) situation and deviation of nasal septum (NSD) from transverse section.
Turbinates atrophy (TA) judges that a point 0-4 divides
0: normal, without any pathological changes.
1: very little a part of atrophy (curl for and be no more than half atrophy).
2: slight atrophy: curl for and exceed half atrophy.
3 moderate atrophys: turbinates is tetanic.
4: severe atrophy: concha nasalis stock is whole curls disappearance.
Deviation of nasal septum (NSD) judges that a point 0-2 divides
0: normal
1: slight bending
2: severe bends
It is 18 points that turbinates atrophy (TA) situation and deviation of nasal septum (NSD) are added highest score.Each test group turbinates pathological changes mark is got its average mark (Mean nasal lesionscores), then carries out statistical analysis.
2 results
Counteracting toxic substances result after 2.1 immunity
Within after immune group and non-immune group counteracting toxic substances 35 days, observe turbinates atrophy situation together with blank process, the results are shown in Table 6, table 7.Only there is slight turbinates atrophy in various degree in two kinds of counteracting toxic substances dose immunization group counteracting toxic substances pigs, sickness rate is all the nonimmune counteracting toxic substances group sickness rate of 0/5, two kinds of dosage is all 5/5.The remarkable reduction (P < 0.05) compared with nonimmune counteracting toxic substances matched group piglet turbinates atrophy average mark of Immunization group piglet turbinates atrophy average mark, immune piglet is effectively protected.
After table 6HB4 strain counteracting toxic substances, (dosage is 2.2 × 10
9cFU/ nostril) piglet turbinates atrophy situation
" * ": subscript difference represents that there were significant differences, P≤0.05.
After table 7HB4 strain counteracting toxic substances, (dosage is 2.2 × 10
10cFU/ nostril) piglet turbinates atrophy situation
" * ": subscript difference represents that there were significant differences, P≤0.05.
The above is only the preferred embodiments of the present invention, not any pro forma restriction is done to the present invention, although the present invention discloses as above with preferred embodiment, but and be not used to limit the present invention, any those skilled in the art, not departing from the scope of technical solution of the present invention, make a little change when the technology contents of above-mentioned announcement can be utilized or be modified to the Equivalent embodiments of equivalent variations, in every case be the content not departing from technical solution of the present invention, according to any simple modification that technical spirit of the present invention is done above embodiment, equivalent variations and modification, all still belong in the scope of technical solution of the present invention.
Claims (10)
1. an atrophic rhinitis inactivated vaccine counteracting toxic substances effect detecting method, described method comprises: the determining step of toxin producing pasteurellosis bacillus counteracting toxic substances dosage, and to counteracting toxic substances effect inspection step after piglet immunological.
2. method according to claim 1, wherein, described method comprises the preparation process of counteracting toxic substances bacterium liquid further, the preparation process of described counteracting toxic substances bacterium liquid comprises and bordetella branchiseptica and pasteurella multocida strain being activated respectively at solid medium plane, bordetella branchiseptica and pasteurella multocida monoclonal described in picking are in culture medium culturing respectively, results bacterium liquid; To described bordetella branchiseptica and the count of bacteria of pasteurella multocida bacterium liquid, determine bacterium liquid viable bacteria content.
3. method according to claim 2, wherein, described bordetella branchiseptica is bordetella branchiseptica HN8 strain, and described pasteurella multocida is pasteurella multocida D type HB4 strain.
4. method according to claim 2, wherein, the culture medium of described cultivation bordetella branchiseptica and pasteurella multocida monoclonal bacterial strain is solid medium.
5. method according to claim 1, wherein, to mice median lethal dose(LD 50), the determining step of described pasteurella multocida counteracting toxic substances dosage comprises determines that pasteurella multocida is to piglet every nostril counteracting toxic substances dosage by pasteurella multocida.
6. method according to claim 5, wherein said pasteurella multocida is 1 ~ 2 × 10 to piglet every nostril counteracting toxic substances dosage/pasteurella multocida to mice median lethal dose(LD 50)
3~ 1 ~ 2 × 10
4.
7. method according to claim 1, wherein, described counteracting toxic substances effect inspection step after piglet immunological to be comprised: after immune group piglet immunological two is exempted from, carry out intranasal counteracting toxic substances together with counteracting toxic substances matched group piglet, the counteracting toxic substances while of two nostrils with the viral mixed liquor of bordetella branchiseptica and killing property Pasteur bacterium more;
Within after last counteracting toxic substances 35 days, dissect piglet, piglet turbinates atrophy situation is marked, counteracting toxic substances matched group at least 80% piglet turbinates atrophy mark 8 points and more than, immune group piglet turbinates atrophy average mark statistically significantly reduces than counteracting toxic substances matched group piglet turbinates atrophy average mark.
8. method according to claim 7, wherein, described piglet immunological two exempts from rear 21st day, the 24th day and the 25th day continuous counteracting toxic substances.
9. method according to claim 7, wherein, containing B.bronchisepticai HN8 strain 2.05 × 10 in described counteracting toxic substances bacterium liquid
9cFU/ nostril, containing pasteurella multocida D type HB4 strain 2.2 × 10
9~ 2.2 × 10
10cFU/ nostril.
10. method according to claim 7, wherein, described piglet 7-10 age in days head exempts from, and head exempts to carry out two in latter 14 days and exempts from.
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Application publication date: 20150318 |