CN105820972B - Mink pseudomonas aeruginosa serum C-type strain and inactivated vaccine and application thereof - Google Patents
Mink pseudomonas aeruginosa serum C-type strain and inactivated vaccine and application thereof Download PDFInfo
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Abstract
The invention discloses a mink pseudomonas aeruginosa serum C-type strain and application thereof, and belongs to the field of separation and application of mink pseudomonas aeruginosa strains. According to the invention, a mink pseudomonas aeruginosa serum C type WD013 strain with strong pathogenicity and high stability is finally obtained from a plurality of mink lungs suspected of suffering from hemorrhagic pneumonia of minks by methods such as separation, identification and screening, and the microbial preservation numbers are as follows: CGMCC No. 12145. The invention also discloses a method for preparing inactivated vaccine by using the strain, which comprises the following steps: culturing the strain to obtain a bacterial liquid; adding an inactivating agent to inactivate the bacterial liquid; and (4) uniformly mixing the adjuvant and the bacterial liquid, and preparing the vaccine to obtain the vaccine. Safety and efficacy tests prove that the inactivated vaccine prepared by the separated mink pseudomonas aeruginosa serum C type WD013 strain has higher safety and good immune efficacy, and can provide good clinical protection for mink hemorrhagic pneumonia caused by pseudomonas aeruginosa.
Description
Technical Field
The invention relates to mink pseudomonas aeruginosa, in particular to a mink pseudomonas aeruginosa serum C-type strain, further relates to an inactivated vaccine prepared from the strain and used for preventing hemorrhagic pneumonia of minks, and belongs to the field of separation and application of the mink pseudomonas aeruginosa.
Background
Hemorrhagic pneumonia of minks is a highly lethal infectious disease caused by infection with Pseudomonas aeruginosa (also known as Pseudomonas aeruginosa), which is frequently transmitted in 9 to 11 months per year by droplets and aerosols formed by sick minks. The pneumonia of pseudomonas aeruginosa is characterized by dyspnea, oral and nasal hemorrhage and sudden death, typical symptoms after death are foam of oral and nasal blood samples, and diffuse bleeding of the lung after autopsy.
Pseudomonas aeruginosa belongs to the family Pseudomonas of the gamma-denaturing bacteria, and is a gram-negative bacterium of aerobic or facultative anaerobic bacteria. Currently, the common method for typing pseudomonas aeruginosa is serological typing, and an international serological typing system classifies pseudomonas aeruginosa into 20 different serotypes according to a thallus O antigen. The Japanese scientist Homma classifies Pseudomonas aeruginosa into A-N groups according to O antigen, and a kit formed according to the system is widely applied to the international society.
Pseudomonas aeruginosa is a conditional pathogen and widely exists in the environment, and vaccination is an effective method for preventing the disease, and meanwhile, drug resistance of strains caused by using antibiotics is avoided. Cases of hemorrhagic pneumonia of minks are firstly reported in China in the early 80 s, particularly, hemorrhagic pneumonia of minks is continuously developed in many places such as Shandong, Hebei, Liaoning, Shanxi and Jilin in China since 2000, the death rate is 6% -54%, investigation shows that the C type hemorrhagic pneumonia prevalence rate of minks in China is 30% -40%, and the prepared single seedlings have pertinence due to the fact that cross protection does not exist among serotypes. Therefore, the development of the inactivated vaccine with good safety and high protection effect for preventing and treating the hemorrhagic pneumonia of the mink has important practical significance.
Disclosure of Invention
One of the purposes of the invention is to provide a C-type strain of the pseudomonas aeruginosa serum of mink with strong pathogenicity and high stability;
the other purpose of the invention is to apply the separated mink pseudomonas aeruginosa serum C type strain to preventing and treating the hemorrhagic pneumonia of minks.
The invention also aims to provide a method for preparing the inactivated vaccine for preventing and treating the hemorrhagic pneumonia of minks.
The above object of the present invention is achieved by the following technical solutions:
the invention finally obtains 14 serum C-type strains from a plurality of cases of lungs of minks suspected to be dead of mink hemorrhagic pneumonia through separation, identification, screening and detection, screens the pathogenicity of the 14 strains to obtain 5 serum C-type strains with better pathogenicity, screens the stability of the 5 strains to finally obtain a pseudomonas aeruginosa serum C-type WD013 strain with high stability, and submits the separated strains to a patent approved organization for preservation, wherein the microorganism preservation numbers are as follows: CGMCC No. 12145; the classification is named as: pseudomonas aeruginosa. The preservation unit: china general microbiological Collection center; the preservation time is as follows: 2016, 2 months and 24 days; and (4) storage address: west road No.1 institute 3, north kingdom rising area, china academy of sciences, and the institute of microbiology.
The method comprises the steps of aseptically collecting lungs of minks suspected to be dead and infected with hemorrhagic pneumonia of the minks, inoculating 2% bovine serum broth agar plates, carrying out overnight culture at 37 ℃, selecting suspected single colonies, streaking and inoculating a culture medium (PDP) for determination of pyocyanic acid, carrying out culture at 37 ℃ for 24 hours, selecting positive colonies, inoculating 5ml of 2% bovine serum broth culture medium, carrying out overnight culture at 37 ℃, and marking as F0 generation. Through PCR identification and 16S rRNA gene sequence analysis, the result shows that: the PCR was identified as positive, the sequencing result was in agreement with the expectation, and the fragment size was found to be 956 bp. Then the morphology, biochemical characteristics and culture characteristics of the pseudomonas aeruginosa are analyzed, and the result shows that the isolate accords with the morphology, biochemical characteristics and culture characteristics of the pseudomonas aeruginosa and is preliminarily identified as positive bacteria liquid. And (4) performing serotype identification detection on the bacterial liquid which is preliminarily identified to be positive, and screening 14 serogroup C strains. The serovar C strain was then purified to give strains numbers DL001, DL002, SD003, DL004, SD005, DL006, PL007, DL008, LN009, DL010, LN011, SD012, WD013, and SD 014. Then, the pathogenicity of the 14 serum C-type strains is screened to obtain 5C-type strains with better pathogenicity: DL001, SD005, PL007, LN009, and WD 013. Further, stability of pseudomonas aeruginosa serum C type DL001 strain, SD005 strain, PL007 strain, LN009 strain and WD013 strain was screened, and finally, pseudomonas aeruginosa serum C type WD013 strain with high stability was obtained.
In order to verify the immune efficacy of the pseudomonas aeruginosa serum C type WD013 strain, the toxicity and immunogenicity of the pseudomonas aeruginosa serum C type WD013 strain are measured, and the toxicity measurement shows that: LD of Pseudomonas aeruginosa serum C type WD013 strain on mice50Is 8.11X 105A CFU; for mink LD of 2-5 months old50Is 2.53X 106And (4) CFU. Immunogenicity assays showed: 5 minks of 2-5 months old were immunized with the vaccine prepared from the isolate F1 generation 21 days after immunization at 10LD50The challenge was performed, the protection rate of the immune group was 5/5, and the protection rate of the control group was 0/5. The above results indicate that the isolate F1 of the C WD013 strain of Pseudomonas aeruginosa was excellent in immunogenicity.
Furthermore, the invention prepares the pseudomonas aeruginosa serum C type WD013 strain into three batches of inactivated vaccines, and safety tests are carried out on single dose and overdose of non-target animals and target animals, and the results show that: the clinical manifestations of non-target animals and target animals are normal, no adverse reaction exists on local injection and whole body, and the animals survive completely. The efficacy of the inactivated vaccine is tested, and the result shows that: for minks: after 21 days of C-type inactivated vaccine immunization, the protection rate of the three batches of vaccines on minks in an immunization group is not less than 80%, and the minks in a control group are 100% infected; for mice: after 21 days of immunization, the protection rate of the three batches of vaccines on mice in an immunization group is not less than 90 percent, and the minks in a control group all have 100 percent of disease. The duration of the immunization was tested and the results showed that: the protection period of the C-type inactivated vaccine on mink anti-pseudomonas aeruginosa WD013 strain is 6 months, and the immune protection rate is not less than 80%. The above shows that the serum type C WD013 strain is relatively safe and can provide good clinical protection.
The invention also provides application of the serum C type WD013 strain in preparation of drugs for preventing and treating hemorrhagic pneumonia of minks.
Therefore, the invention provides a vaccine composition for preventing and treating hemorrhagic pneumonia of minks, which comprises the following components: a prophylactic or therapeutic effective amount of the isolated pseudomonas aeruginosa serum C type WD013 strain of the present invention and a pharmaceutically acceptable adjuvant.
The invention further provides a method for preparing the vaccine composition for preventing and treating hemorrhagic pneumonia of minks, which comprises the following steps:
(1) culturing a C-type WD013 strain of pseudomonas aeruginosa serum to obtain a bacterial liquid;
(2) adding an inactivating agent into the bacterial liquid to inactivate the bacterial liquid;
(3) and (3) uniformly mixing the inactivated bacteria liquid and an adjuvant, and preparing the seedling to obtain the vaccine.
Inoculating a serum C type WD013 strain to an agar plate containing 2% bovine serum broth, culturing at 37 ℃ overnight, selecting a single typical colony for amplification culture, inoculating a TSB culture medium accounting for 1% of the total volume of the culture medium, and culturing at 37 ℃ under aeration and stirring;
and (3) adding formaldehyde into the bacterial liquid in the step (2) until the final concentration of the formaldehyde is 0.1 percent of the total volume of the bacterial liquid, and inactivating for 24 hours.
In the step (3), the adjuvant is aluminum hydroxide gel, wherein the aluminum hydroxide gel is 30% by volume of the aluminum hydroxide gel.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
the separated C-type WD013 strain of the pseudomonas aeruginosa serum disclosed by the invention is strong in pathogenicity and high in stability, and the prepared inactivated vaccine has higher safety and good immune efficacy, can provide good clinical protection for the hemorrhagic pneumonia of the minks caused by the pseudomonas aeruginosa, and can effectively prevent the hemorrhagic pneumonia of the minks.
Definitions of terms to which the invention relates
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term "vaccine" refers to an autoimmune preparation for preventing infectious diseases, which is prepared from pathogenic microorganisms (such as bacteria, rickettsia, viruses, etc.) and metabolites thereof by artificial attenuation, inactivation, or genetic engineering.
The term "adjuvant" means a non-specific immunopotentiator that, when injected or pre-injected into a body with an antigen, enhances the body's immune response to the antigen or alters the type of immune response.
The term "immunization" means a function in which the body's immune system recognizes self and foreign substances and excludes antigenic foreign substances by an immune response to maintain the physiological balance of the body.
The term "immunogenicity" refers to the ability of an organism to stimulate the formation of specific antibodies or sensitized lymphocytes, i.e., the property of an antigen to stimulate specific immune cells, activate, proliferate, differentiate, and ultimately produce antibodies to immune effectors and sensitized lymphocytes, and also refers to the specific immune response of the immune system of the organism to form antibodies or sensitized T lymphocytes following stimulation of the organism by the antigen.
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FIG. 1 shows the result of PCR amplification of the 16S rRNA gene fragment of the isolate, in which: m: DL2000 Marker; 1: separating the plant; 2: a positive control; 3: negative control;
FIG. 2 shows the change of the cell content of Pseudomonas aeruginosa serum C-type DL001, SD005, PL007, LN009 and F1 generation of WD013 during passage.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
1. Test materials
1.1 Primary reagents
rTaq, dNTP, DL2000DNA Marker, pMD18-T vector and DH5 alpha are all purchased from Takara Bio-engineering (Dalian) Co., Ltd; the gel recovery kit and the small-amount plasmid extraction kit are purchased from Tiangen Biotechnology (Beijing) products of Ltd; agarose was purchased from bio-engineering (shanghai) ltd; ampicillin (Amp)+) Purchased from Sigma company; glycerol, formalin and NaCl were purchased from the national pharmaceutical group.
1.2 culture Medium
LB medium, LB agar plate, 2% bovine serum broth medium, 2% bovine serum broth agar plate, and TSB medium.
1.3 Positive sera
Pseudomonas aeruginosa was typed using a standard serum series purchased from Nippon gazette Co.
1.4 test animals
18-20 g clean-grade Kunming mice were purchased from Harbin group Sanzhiyao GmbH; minks of 2-5 months of age were purchased from the professional marten co-culture (negative for agglutination test antibody of pseudomonas aeruginosa B, C and G type slides) in Shangzhi, Heilongjiang province.
1.5 pathological material
89 lungs of minks suspected to be dead of hemorrhagic pneumonia of minks are collected aseptically and marked 1#～89#And storing at 4 ℃ for later use.
Experimental example 1 isolation, identification and purification of C-type strain of Pseudomonas aeruginosa serum
1. Experimental methods
1.1 preliminary isolation of Pseudomonas aeruginosa strains
Respectively and aseptically dipping 89 diseased material sections, streaking and inoculating 2% bovine serum broth agar plates, culturing at 37 ℃ overnight, picking suspected single colony, streaking and inoculating a culture medium (PDP) for measuring pyocyanin, culturing at 37 ℃ for 24 hours, and if the green pigment is produced, the periphery of the colony is light green and is positive to pyocyaneus. Positive colonies were picked, inoculated into 5ml of 2% bovine serum broth and cultured overnight at 37 ℃ and recorded as F0 passages.
1.2 Positive Strain 16S rRNA Gene identification
1.2.1PCR identification
According to the GenBank reference strain sequence, a pair of primers specific to the pseudomonas aeruginosa gene are designed by Primer 5.0 software and are used for amplifying the partial sequence of the pseudomonas aeruginosa gene. Taking the DNA of the extracted isolate as a template, and the primers are as follows: the upstream primer is 5'-GGAATCGTCGGACTAC-3'; the downstream primer was 5'-TCCCTAGTTCCATCC-3' (primer synthesized by Shanghai bioengineering, Inc.). The expected target fragment size of the amplified product is 956 bp. The reaction system was 25. mu.l: buffer 2.5. mu.l, dNTP 2. mu.l, upstream and downstream primers 1. mu.l each, template 1. mu.l, rTaq 0.5. mu.l, and deionized water. The reaction program is 95 ℃ for 5 min; circulating for 35 times at 95 deg.C for 1min, 55 deg.C for 1min, and 72 deg.C for 1 min; 10min at 72 ℃. PCR amplification and electrophoresis in 1% agarose gel were carried out to obtain 16S rRNA gene fragment.
1.2.216S rRNA Gene sequence analysis
The amplified fragment is connected and transformed to extract recombinant plasmid, and the positive recombinant is named as pPA 16S-1. The size of the 16S rRNA gene fragment was analyzed as a result of the sequencing.
1.3 Positive isolates morphology, Biochemical Properties and culture Properties
Performing further genus identification on the positive isolate, taking a typical colony which is subjected to streak culture of F0 generation and identified as positive by PCR, performing gram staining, and observing that the bacteria are gram-negative bacilli under an oil lens; meanwhile, the bacterial colony is subjected to an oxidase test to be positive; growing on a 2% bovine serum broth culture medium which is uniformly turbid and is in yellow green; on sheep fresh blood agar plates, significant beta hemolysis should occur; growing on the PDP slant should produce green pigment, and the periphery of the colony should be light green.
1.4 serotype identification
And (3) carrying out serotype detection on the F0 bacterial liquid identified as positive by using the standard serum for Pseudomonas aeruginosa typing, and further screening different serotypes of Pseudomonas aeruginosa. The procedure was carried out according to the instructions for Pseudomonas aeruginosa typing using standard serum series. The mixed disease material was not screened further.
1.5 purification of serum type C isolates
Purifying the F0 generation of the initially screened serum C-type positive isolate, carrying out streak culture, selecting a single typical colony for amplification culture, and storing the obtained F1 generation in glycerol at-70 ℃.
2. Results of the experiment
2.1 results of bacterial isolation
89 parts of the disease material is subjected to bacteria isolation culture, 83 parts of bacterial colonies are suspected, the morphology basically accords with the morphology of a target bacterial colony, and the culture medium turns yellow and green. Colonies were not cultured in 6 specimens or the morphology of the colonies did not match, and the specimens were judged negative.
2.2PCR identification and sequence analysis
2.2.1 PCR amplification results of 16S rRNA Gene fragments of isolates
Designing a primer according to a GenBank reference strain sequence, and carrying out PCR amplification on a16S rRNA gene segment by taking DNA of an isolated strain as a template. The agarose gel electrophoresis result of one of the isolates is shown in figure 1, 1 fragment is amplified at 1000bp, and the result is consistent with the expected result, which indicates that the isolate is positive to 16S rRNA gene.
2.2.216S rRNA Gene fragment sequencing results
The sequencing results were expected to be consistent, with a fragment size of 956 bp.
2.3 morphological, biochemical and cultural Properties
Culturing the separated strain identified as positive by PCR, wherein the gram staining result is gram negative bacilli; positive in oxidase test; growing on a 2% bovine serum broth culture medium, wherein the culture medium is uniformly turbid and is in yellow green; hemolytic rings appear around the bacterial colonies on the sheep fresh blood agar culture medium; growing on the PDP slant, green pigment is produced, and the periphery of the colony is light green. The results show that the isolated strain conforms to the morphological, biochemical and culture characteristics of pseudomonas aeruginosa.
2.4 serotype identification results
83 positive bacteria liquids are identified primarily, serotype detection is carried out by using standard serum (Nibunsheng research corporation) for Pseudomonas aeruginosa typing, and 14 serum C type strains are screened out.
2.5 purification of serum type C isolates
Purifying the 14 serum C-type F0 generation to obtain F1 generation, keeping part of fresh bacterial liquid at 4 ℃, and storing the rest in glycerol at-70 ℃. The numbers are DL001, DL002, SD003, DL004, SD005, DL006, PL007, DL008, LN009, DL010, LN011, SD012, WD013 and SD 014.
Experimental example 2 screening of C-type Pseudomonas aeruginosa serum Strain
1. Experimental methods
1.1 pathogenicity screening
Each serum type C isolate F1 which meets the requirements was diluted to 107CFU/mL and 108CFU/mL, each titer was nasally inoculated to 10 mice, 0.1 mL/mouse, with 10 saline controls; the death of the mice was recorded and serum C-type candidates were selected that killed half and more of the mice within 48 hours.
1.2 passage stability assay
The serological type C candidate bacterium meeting the standard is continuously passaged from the F1 generation to the F25 generation. And carrying out shake culture for 18 hours after each passage, respectively carrying out viable bacteria counting and morphological observation on F1, F3, F6, F9, F12, F15, F18, F21 and F24 generations, and finally screening a stable strain with high bacterial content as a strain for preparing seedlings and checking.
2. Results of the experiment
2.1 pathogenicity screening
The results of the pathogenicity of the serum C-type vaccine strain are shown in tables 1 and 2, and it can be seen from tables 1 and 2 that the control mice are healthy and alive, and 14 serum C-type pseudomonas aeruginosa 10 strains8CFU/mL concentration 5 of mice that died within 48 hours in half and above: DL001, SD005, PL007, LN009, and WD 013.
TABLE 1 serum C type vaccine strain pathogenicity screening results
TABLE 2 serum C type vaccine strain pathogenicity screening results
2.2 stability screening
The culture of serum C-type pseudomonas aeruginosa DL001, SD005, PL007, LN009 and WD013 strains is continuously transmitted for 25 generations, viable bacteria counting and bacterial morphology observation are carried out for every three generations, the bacterial content is shown in figure 2, 5 strains are stable in the bacterial content within 25 generations, morphological characteristics are not obviously changed, but the WD013 strain is relatively more stable and the CFU number is higher, so the strain is selected as a strain for preparing seedlings and testing and is named as WD013 strain. The invention submits a serum C-type pseudomonas aeruginosa WD013 strain to China general microbiological culture collection center for preservation, and the microbiological preservation numbers are as follows: CGMCC No. 12145; the classification is named as: pseudomonas aeruginosa; the preservation time is as follows: 2016, 2 months and 24 days; and (4) storage address: west road No.1 institute 3, north kingdom rising area, china academy of sciences, and the institute of microbiology.
Experimental example 3 determination of virulence and immunogenicity of serum C-type Pseudomonas aeruginosa WD013 Strain
1. Experimental methods
1.1 virulence assay
1.1.1 virulence determination in mice
Selecting 40 healthy mice, dividing into 4 groups, each group containing 10 mice, performing 10-fold serial dilution on WD013 strain bacterial liquid, and taking 10-1～10-4Titer, nasal drip inoculation of mice, 0.1 mL/mouse, observation for 7 days, recording death status of each group of mice, calculating half Lethal Dose (LD) of the isolate to mice according to Reed-Muench method50)。
1.1.2 determination of the virulence of minks
Selecting 20 healthy minks, dividing into 4 groups, diluting 5 healthy minks in series by 10 times with WD013 strain bacterial liquid, and taking 10 healthy minks0～10-3Titer, nasal dripInoculating mink, 0.2 mL/mink, observing for 7 days, recording death condition of each group of mink, and calculating LD of the isolate for mink according to Reed-Muench method50。
1.2 immunogenicity assays
The strain F1 WD013 was adjusted to 3.0X 108CFU/ml is inactivated by 0.1% formaldehyde solution, 5 healthy minks of 2-5 months old are inoculated subcutaneously, 1.0 ml/mink, and the other 5 minks are used as controls. Day 21 after immunization, with 10LD50The mink was challenged and 0.2 ml/mouse was inoculated nasally. At 7 days post challenge, the immune group should be protected at least 4/5 and the control group should be totally dead.
2 results of the experiment
2.1 determination of virulence of serum C type WD013 Strain
2.1.1 virulence in mice
Performing LD on mice50The results are shown in Table 3, and LD of serum C type WD013 strain for mouse50Is 8.11X 105CFU。
TABLE 3 serum C type WD013 strain LD50Results
2.1.2 virulence of minks
Performing LD on mink50The results are shown in Table 4, and the results of the measurement are shown in the table 4, wherein the serum C type WD013 strain is used for LD of minks at 2-5 months of age50Is 2.53X 106CFU。
TABLE 4 serum C type WD013 strain to LD of mink of 2-5 months age50Results
2.2 immunogenicity
The immunogenicity results of the serum type C WD013 strain are shown in table 5. 5 minks of 2-5 months old were immunized with the vaccine prepared from the isolate F1 generation 21 days after immunization at 10LD50The toxic materials are removed by the air purifier,the protection rate of the immune group is 5/5, and the protection rate of the control group is 0/5. The results showed that isolate F1 was highly immunogenic.
TABLE 5 immunogenicity of serum C type WD013 strain
Experimental example 4 preparation and final product inspection of serum C type WD013 strain inactivated vaccine
1. Experimental methods
1.1 preparation of vaccines
Streaking a serum C type WD013 strain to a broth agar plate containing 2% of bovine serum, carrying out overnight culture at 37 ℃, selecting a single typical colony for amplification culture, inoculating a TSB culture medium according to 1% of the total volume of the culture medium, and carrying out aeration and stirring culture at 37 ℃; adding formaldehyde solution according to 0.1 percent of the total volume of the bacteria liquid, inactivating for 24 hours, and stirring for 3 times; adding 30 percent by volume of aluminum hydroxide gel into the obtained inactivated bacterial liquid for seedling preparation. In this way, 3 batches of vaccine were prepared.
1.2 safety test
The 3 batches of vaccines prepared were individually subjected to safety tests for non-target animals, single dose and overdose safety tests for target animals.
1.2.1 safety tests on non-target animals
3 batches of the prepared vaccine are respectively inoculated to 18-20 g of mice, 0.3 ml/mouse and 10 mice per batch in an abdominal cavity, and the mice are observed for 14 days after inoculation. Mice local and systemic responses were recorded.
1.2.2 Single and overdose immune safety test in target animals
Selecting 30 minks of 2 months old, randomly dividing the minks into 6 groups and 5 groups, and performing single-dose (1-3 groups) and super-dose (4-6 groups) subcutaneous immunization on the prepared 3 batches of vaccines respectively, wherein the single dose is 1.0ml per mink and the super-dose is 2.0ml per mink. After immunization, the mink was observed for 14 days and the local and systemic reactions were recorded.
1.3 efficacy test
1.3.1 efficacy test on minks
The three batches of trial-produced vaccines are all subjected to the following operations, and 2-5 months old vaccines are taken to be healthy and easy to prepare10 minks were felt and randomly divided into 2 groups of 5 minks each. 1.0ml of vaccine is injected subcutaneously into the minks of the group 1; minks in group 2 were not immunized as controls. After 21 days of immunization, the two groups of minks are respectively inoculated with 10LD by nasal drip500.2ml of strain WD013, observed for 7 days after challenge. The minks in the control group should all die and the minks in the immunization group should protect at least 4 minks.
1.3.2 efficacy testing in mice
The three batches of trial-produced vaccines are subjected to the following operations, 20 Kunming mice with the weight of 18-20 g and the clean level are taken and randomly divided into 2 groups, and each group comprises 10 mice. 0.3ml of vaccine is injected into the abdominal cavity of each mouse in the group 1; group 2 mice were not immunized as a control. 21 days after immunization, two groups of mice were inoculated with 10LD by nasal drip respectively500.1ml of strain WD013, observed for 7 days after challenge. The control mice should die entirely and the immunized mice should protect at least 8 mice.
1.4 immune phase potency assay
The three batches of trial vaccines are subjected to the following operations, 40 minks with the age of 2-5 months are selected, 20 minks are immunized, and 20 minks are used as controls. Avoid nasal drip attack on 30, 90, 180 and 210 days after the treatment of 10LD500.2ml of strain WD013, 5 cells each for the immune group and the control group. After 14 days of observation after immunization, the minks in the control group should die completely, and the minks in the immune group should be protected by at least 4 minks.
2. Results of the experiment
2.1 producing seedlings and fruiting
A total of 3 batches of vaccine were prepared, batch number 01-C, batch number 02-C and batch number 03-C, 2000ml each, 20ml per bottle.
2.2 safety test results of vaccines
2.2.1 safety test results for vaccines against non-target animals
The safety test results of the vaccine on the mice are shown in tables 6 and 7, and the results show that after 3 batches of the vaccine are inoculated on the mice, the mice are all healthy and alive in the observation period, the clinical manifestation is normal, and the immune local part and the whole body have no adverse reaction.
TABLE 6 clinical observations of safety tests on mice
Note: decrease in mink feed intake only on the day of immunization
Table 7 safety test of vaccines against mice local and systemic response results
2.2.2 safety test results of Single dose immunization with minimum day-old target animals
The results of the single dose immune safety test of the vaccine against the target animals of the minimum use day age are shown in table 8 and table 9. As can be seen from the results, after the minks of 2 months old are inoculated with the 3 batches of vaccines in a single dose, the minks are all healthy and alive in the observation period, the clinical manifestations are normal, no adverse reaction exists on the local injection part and the whole body, and the minks are all alive.
TABLE 8 clinical observations of the Single dose immune safety test for mink minimum day of use
Note: decrease in feed intake and depression of minks only on the day of immunization
TABLE 9 Single dose immune safety test local and systemic response to mink minimum day of use
2.2.3 one-time overdose immune safety test results for target animals
The safety test results of one-time overdose immunization of the vaccine on the target animals are shown in tables 10 and 11. The results show that after the minks of 2 months old are inoculated with the 3 batches of vaccines in an overdose mode, the minks are all healthy and alive in the observation period, the clinical manifestations are normal, no adverse reaction exists on the part of immunity and the whole body, and the minks all survive.
TABLE 10 clinical observations of one-time overdose immune safety tests on target animals
Note: decrease in mink feed intake only on the day of immunization
TABLE 11 results of local and systemic reactions in one-time overdose immune safety test of target animals
2.3 efficacy testing of three batches of vaccine
2.3.1 results of efficacy test on minks
The results are shown in table 12, the protection rate of minks in three vaccine immunization groups is not less than 80% when the minks are attacked 21 days after the C-type inactivated vaccine is immunized, and the minks in the control group are 100% attacked.
TABLE 12 results of the Immunopotential test on minks
2.3.2 efficacy test results on mice
The results are shown in Table 13, the protection rate of mice in three vaccine immunization groups is not less than 90% after 21 days of immunization, and the disease of minks in a control group is 100%.
TABLE 13 results of the Immunopotentiality test for mice
2.4 duration of immunization test results
The results are shown in table 14, which indicates that the C-type inactivated vaccine has a protection period of 6 months for mink against pseudomonas aeruginosa WD013 strain, and the immune protection rate is not less than 80%.
TABLE 14 duration of immunization test results
Claims (10)
1. A mink Pseudomonas aeruginosa (Pseudomonas aeruginosa) serous type C strain is characterized in that the microbial preservation number is as follows: CGMCC No. 12145.
2. Use of the mink pseudomonas aeruginosa serum type C strain of claim 1 in the preparation of a vaccine for preventing and treating hemorrhagic pneumonia in minks.
3. A vaccine composition for preventing and treating hemorrhagic pneumonia of minks is characterized by comprising the following components: a prophylactically effective amount of the mink pseudomonas aeruginosa serotype C strain of claim 1 and a pharmaceutically acceptable adjuvant.
4. A method for preparing a mink pseudomonas aeruginosa serum C type strain inactivated vaccine is characterized by comprising the following steps:
(1) culturing the mink pseudomonas aeruginosa serum type C strain of claim 1 to obtain a bacterial solution;
(2) adding an inactivating agent into the bacterial liquid to inactivate the bacterial liquid;
(3) and (3) uniformly mixing the inactivated bacteria liquid and an adjuvant, and preparing the seedling to obtain the vaccine.
5. The method of claim 4, wherein: the culturing in the step (1) comprises the following steps: the mink pseudomonas aeruginosa serum type C strain of claim 1 is inoculated to bovine serum broth agar plates, cultured overnight, a single typical colony is picked and inoculated to TSB medium, and cultured under aeration and agitation to obtain a bacterial solution.
6. The method of claim 5, wherein: the temperature of the overnight culture and the temperature of the aeration-agitation culture were both 37 ℃.
7. The method of claim 4, wherein: in the step (2), the inactivator is formaldehyde.
8. The method of claim 4, wherein: adding formaldehyde into the bacterial liquid in the step (2) until the final concentration of the formaldehyde is 0.1 percent of the total volume of the bacterial liquid; the inactivation time is 24 hours.
9. The method of claim 4, wherein: in the step (3), the adjuvant is 30% of aluminum hydroxide gel by volume percentage.
10. The inactivated vaccine for preventing hemorrhagic pneumonia of mink, which is prepared by the method of any one of claims 4-9.
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Non-Patent Citations (6)
| Title |
|---|
| Comparison of Pseudomonas aeruginosa isolates from mink by serotyping and pulsed-field gel electrophoresis;AS Hammer等;《Veterinary Microbiology》;20030717;第94卷(第3期);第237-243页 * |
| 水貂出血性肺炎二价灭活疫苗的候选株评价及疫苗制备;韩明明等;《黑龙江畜牧兽医》;20140531(第5期);第151-156页 * |
| 水貂绿脓杆菌的分离与鉴定;马俊等;《中国动物传染病学报》;20140228;第22卷(第1期);第44-49页 * |
| 绿脓杆菌比色LAMP检测法的建立及水貂出血性肺炎绿脓杆菌三价灭活疫苗的研制;韩明明;《中国优秀硕士学位论文全文数据库 农业科技辑》;20141015(第10期);第D050-137页 * |
| 貂源C型绿脓杆菌的分离鉴定及其药敏试验;刘佳卉等;《山东畜牧兽医》;20140131;第35卷(第01期);第14-15页 * |
| 马俊等.水貂绿脓杆菌的分离与鉴定.《中国动物传染病学报》.2014,第22卷(第1期),第44-49页. * |
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