CN112011479A

CN112011479A - Streptococcus equi subsp equi HLJ2018D-LX strain and application thereof in preparation of streptococcus equi subsp equi inactivated vaccine

Info

Publication number: CN112011479A
Application number: CN202010780801.XA
Authority: CN
Inventors: 王晓钧; 郭巍; 胡哲; 戚亭; 刘荻荻
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2020-08-06
Filing date: 2020-08-06
Publication date: 2020-12-01
Anticipated expiration: 2040-08-06
Also published as: CN112011479B

Abstract

The invention discloses an donkey-derived streptococcus equi subsp equi HLJ2018D-LX strain and application thereof in preparation of streptococcus equi subsp equi inactivated vaccines, and belongs to the field of biological vaccines. The donkey-derived streptococcus equi subsp equi strain HLJ2018D-LX strain has the advantages of good immunogenicity, good culture characteristics and the like, and is inoculated into streptococcus enrichment broth, and inactivated by formaldehyde (with the final concentration of 0.2% v/v) after culture to prepare the inactivated vaccine. Experiments prove that after the streptococcus equi subsp equi inactivated vaccine prepared by the invention is used for immunizing a mouse, the mouse can be prevented from being attacked by virulent streptococcus equi subsp equi, the morbidity is greatly reduced in donkey body tests, the vaccine has the advantages of safety, low-dose immunization and the like, is the first streptococcus equi subsp equi inactivated vaccine for donkey, and can be used for preventing the plague caused by the streptococcus equi subsp equi.

Description

Streptococcus equi subsp equi HLJ2018D-LX strain and application thereof in preparation of streptococcus equi subsp equi inactivated vaccine

Technical Field

The invention relates to an application of an equine streptococcus equi subsp in preparation of an inactivated vaccine of the equine streptococcus equi subsp, in particular to an automatically separated equine streptococcus equi subsp HLJ2018D-LX strain and an inactivated vaccine prepared from the bacterial strain. Belongs to the field of biological vaccines.

Background

Adenitis (strengles) is an acute contact infectious disease with swelling of the mandibular lymph node of equine animals as a main symptom caused by the equine species Streptococcus equi (strepcoccus equi), wherein the equine species Streptococcus equi is a main pathogenic bacterium, is pathogenic only to equine animals, and has a plurality of antigens such as hemolysin S, phospholipase a2, surface antigen seM protein and the like. Except for the iceland, the epidemic situation of the glandular plague appears globally, and the streptococcus equi subspecies mainly infects horses and mules. Horses from 4 months to 4 years are susceptible, with foals under 2 years being the most ill, and foals within 2 months and horses over 5 years being less susceptible. The disease usually occurs in spring and autumn, generally in September to April of the next year, at the moment, the climate is cold, horses are concentrated, the disease is one of indirect reasons of the disease, and other seasons are sporadic and cause serious economic loss. As for the donkey-derived plague, the Chinese reports are relatively few. The previous researches on donkey gland plague are not concerned, and domestic and international related researches are rare and not deep, and are ignored infectious diseases. However, with the hot marketing of donkey products represented by donkey-hide gelatin in China and even the world, in recent years, China has developed a hot stream of donkey breeding, and the traditional way of breeding donkeys by farmers has not met the market demand, so that more and more donkey farms are produced, and the unique intensive donkey breeding way in China is gradually formed. With the development of intensive feeding, the traditional concept of "donkey is not sick" is broken, and more donkey farms begin to face the same epidemic disease spreading problem of intensive feeding farms such as chicken farms, pig farms and the like. Many donkey farms have the condition that one donkey is sick and one donkey is fallen to one place, donkey adenitis in areas such as Shandong and inner Mongolia of China has the morbidity of different degrees, and especially donkey colts have the morbidity as high as 80%. Bacteria separation and identification are carried out on the donkey disease sample with the adenitis by an equine infectious disease and lentivirus disease innovation team of Harbin veterinary institute, hemolytic bacteria are successfully separated, and the horse disease sample is determined to be an equine streptococcus equi subspecies by specific method detection (biochemical detection, gram staining, electron microscope observation and gene sequencing analysis). The occurrence of the disease causes serious indirect economic loss to the donkey industry, and the disease has a spreading trend, thereby generating potential threat and great influence on the sustainable development of the donkey industry in China and seriously hindering the development of the donkey industry in China.

The inventor of the invention separates a streptococcus equi subsp from a suspected adenitis donkey colt nasal swab in a donkey farm in 2018, and identifies the streptococcus equi subsp as a virulent strain through a laboratory, wherein the streptococcus equi subsp is named as HLJ 2018D-LX. The HLJ2018D-LX strain is utilized to develop the research and development work of the inactivated vaccine of the equine streptococcus equi subsp equi donkey source strain, the equine streptococcus equi virulent HLJ2018D-LX strain with good immunogenicity and stable titer is screened and cultured, and an inactivator, an adjuvant and other conditions which are in line with the vaccine manufacture are selected. The production process, safety, protection rate, immunization program, minimum dosage, antibody duration and storage period of the product are tested, and a large amount of test data are obtained to prove that the vaccine is safe and effective. On the basis, pilot production is carried out, sampling inspection is carried out, the pilot product completely meets the proposed quality standard, safety test and efficacy test are carried out on the pilot product, the result also proves that the vaccine can effectively prevent the plague caused by the equine streptococcus equi subspecies, on the basis of laboratory test and intermediate trial production, the inactivated vaccine of the equine streptococcus equi subspecies (HLJ2018D-LX strain) is developed, the inactivated vaccine of the equine streptococcus equi subspecies for donkey is prepared for the first time, the result also proves that the vaccine can effectively prevent the plague caused by the equine streptococcus equi subspecies, and further verifies each quality standard of the vaccine.

Disclosure of Invention

One of the purposes of the invention is to provide a streptococcus equi subsp equi donkey source strain with good immunogenicity and stable titer;

the second purpose of the invention is to provide the application of the streptococcus equi subsp equi strain in preparing vaccine;

the invention also aims to provide a vaccine prepared by the streptococcus equi subsp;

the fourth object of the invention is to provide the use of said vaccine for the preparation of a biological preparation of diseases caused by Streptococcus equi subsp.

In order to achieve the purpose, the invention adopts the following technical means:

the inventor of the invention separates a streptococcus equi subsp equi donkey source strain from a nasal swab of an adenitis donkey colt in a certain donkey farm, and the streptococcus equi subsp equi donkey source strain is identified as a virulent strain and is named as HLJ 2018D-LX. The HLJ2018D-LX strain of the invention is isolated from a nasal swab which is infected by a typical streptococcus equi subsp equi and causes a horse colt to be stained with pus. After separation by Columbia blood plate culture medium and identification in laboratory, the strain can be used as vaccine virus seed, and the strain is 1 x 10³CFU/0.2ml of 18-21g Balb/c mice aged 6 weeks can cause 100% of the mice to die within 9 days.

The donkey source strain of the equine Streptococcus equi subsp, identified by a laboratory, is named as HLJ2018D-LX, and is classified and named as equine Streptococcus equi subsp.equi, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and is addressed to the institute of microbiology, academy of sciences in Sichuan No.1 institute, North Cheng, the rising area of Beijing, and the strain preservation numbers are as follows: CGMCC No.19353, with preservation date of 2020, 1 month and 14 days.

The invention also provides application of the streptococcus equi subsp equi donkey source strain in preparation of streptococcus equi subsp equi inactivated vaccines.

The invention relates to an inactivated streptococcus equi subsp equi vaccine which is characterized by comprising an inactivated streptococcus equi subsp equi donkey source strain HLJ 2018D-LX.

In the inactivated vaccine for equine streptococcus equi subsp, preferably, the donkey-derived strain HLJ2018D-LX of the equine streptococcus equi subsp is inactivated by the following method: inactivating 0.2% (v/v) formaldehyde (HCHO) at 28 deg.C and 130rpm/min for 48 hr, coating Columbia blood plate, culturing at 37 deg.C for 3 days, sterilizing, inactivating for 12 hr, washing with PBS for 2 times, and removing formaldehyde.

In the present invention, preferably, the inactivated vaccine of the equine streptococcus equi subsp.sp.is obtained by emulsifying 5% (v/v) of commercial MONTANIDE PET GEL A adjuvant of Spiraceae and 0.2% (v/v) of formaldehyde (HCHO) inactivated equine streptococcus equi subsp.equi (HLJ2018D-LX strain).

In the invention, the emulsification process adopts commercial MONTANIDE PET GEL A adjuvant of French Saibox company, and the sterilization can be directly used for the emulsification preparation of vaccines; before emulsification, the inactivated streptococcus equi subsp equi bacterial liquid of the same batch is mixed, and the adjuvant is mixed according to the proportion of 5 percent of the total volume.

Furthermore, the invention also provides the application of the inactivated vaccine in preparing a biological product for preventing diseases caused by streptococcus equi subsp.

Preferably, the disease is an adenitis caused by the equine subspecies streptococcus equi.

Compared with the prior art, the invention has the beneficial effects that:

the equine streptococcus equi subsp equi source strain HLJ2018D-LX disclosed by the invention has the advantages of good immunogenicity, good culture characteristics and the like, and is inoculated to streptococcus enrichment broth, and after culture, the streptococcus equi subsp equi source strain HLJ2018D-LX is inactivated by formaldehyde (with the final concentration of 0.2%) to prepare an inactivated vaccine. Experiments prove that after the inactivated vaccine of the streptococcus equi subsp equi source strain prepared by the invention is used for immunizing a mouse, the mouse can be prevented from being attacked by virulent streptococcus equi subsp equi, the morbidity is greatly reduced in donkey body tests, the vaccine has the advantages of safety, low-dose immunity and the like, is the first inactivated vaccine of the streptococcus equi subsp equi for donkey, and can be used for preventing the plague caused by the streptococcus equi subsp equi.

Drawings

FIG. 1 shows the isolation and identification of equine subspecies of Streptococcus equi;

FIG. 2 shows PCR amplification of the 16S rRNA gene;

FIG. 3 is a HLJ2018D-LX growth curve;

FIG. 4 is a survival curve of mice after challenge with HLJ 2018D-LX.

Detailed Description

The present invention is further illustrated by the following experiments in conjunction with examples, it being understood that these examples are for illustrative purposes only and in no way limit the scope of the present invention.

Example 1 isolation and culture identification of Streptococcus equi Malaysia Asso-derived Strain HLJ2018D-LX

According to the requirements of new biological product declaration and in combination with a large amount of test data obtained by the invention, the virus seeds for vaccine preparation are identified by referring to the Chinese pharmacopoeia (2005 edition), and the standard of the virus seeds in the trial practice (draft) is explained below.

1 source of virulent seed

The HLJ2018D-LX strain of the invention is isolated from mucopurulent nasal fluid resulting from foal shedding caused by infection with a subspecies equisimilis. Nasal swabs of adenitis donkey colt were streaked on columbian blood plating medium to develop typical beta hemolysis, as shown in figure 1.

Inoculating the separated and purified beta hemolytic bacteria strain into a blood agar culture medium, culturing at 37 ℃ for 12h, selecting a single colony by using an aseptic inoculating loop, inoculating the single colony into a biochemical identification microtube, and referring to the standard of the agricultural industry of the people's republic of China, namely Malayan disease diagnosis technology (NY/T571-2018), carrying out 12 biological characteristics identification on a pyrrolidone test (PYR), a Voges-Proskauer test (V-P), arginine, Diphenylphosphine (DPP), Phosphoglucomutase (PMG), escin, myelin-related glycoprotein (MAG), Temozolomide (TMZ), mycose, sucrose, sorbitol and hemolytic characteristics. The result of the inquiry and identification according to the streptococcus biochemical coding identification manual is 1005, the identification index of the equine streptococcus equi subsp.equi is 96.8 percent, and the isolate can be determined to be the equine streptococcus equi subsp.equi (Table 1).

TABLE 1 Biochemical identification of S.equi subsp

Note: positive and negative

The HLJ2018D-LX strain is subjected to PCR amplification (figure 2) and sequencing by using the equine streptococcus equi 16S rRNA primer, and the sequencing result proves that the strain is the equine streptococcus equi.

Measuring the growth curve of the HLJ2018D-LX bacteria in the streptococcus enrichment liquid, and counting the bacteria every hour; the result shows that the time is 0-1h and is in the equilibrium period; 1-5h in logarithmic growth phase and 5h later in equilibrium phase, the results are shown in FIG. 3.

The streptococcus equi subsp strain HLJ2018D-LX is respectively added with 1 × 10²CFU/0.2ml、1*10³CFU/0.2ml、1*10⁴CFU/0.2ml、1*10⁵CFU/0.2ml、1*10⁶CFU/0.2ml, i.p. mice, strain HLJ2018D-LX at a concentration of 1 x 10³CFU/0.2ml was able to kill all mice within 9 days, as shown in FIG. 4. Thus, the HLJ2018D-LX strain belongs to a virulent strain.

The HLJ2018D-LX strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the microbial research institute of Zhongkoyao institute of Xilu No.1 Hospital, North Cheng, the rising area, Beijing, and the culture preservation numbers are as follows: CGMCC No.19353, with preservation date of 2020, 1 month and 14 days.

2 seed Virus (HLJ2018D-LX Strain) Standard

2.1 virulence of

The bacterial generation is 2-10 generations of a streptococcus equi subsp equi HLJ2018D-LX strain. The dose of the drug is 1 x 10³CFU/0.2ml, wherein the procedure is that 8 SPF grade Balb/C female mice with the age of 6 weeks are subjected to intraperitoneal injection, the mice are observed for about two weeks after injection, and the death condition of the mice is recorded every day; the judgment standard is that after the virus is attacked, all mice die, and the average weight of the mice in the group can be judged to be increased back to be not lethal.

2.2 immunogenicity

Inoculating the virulent strain to streptococcus enrichment liquid for culturing, diluting by using a centrifuge tube, counting by adopting a plate coating method, inactivating by using formaldehyde solution, adding MONTANIDE PET GEL A adjuvant, mixing and emulsifying to prepare an oil-in-water inactivated vaccine, injecting 8 Balb/C female mice with the SPF grade of 6 weeks old into the peritoneal cavity, injecting bacterins 1 x 10^7CFU/0.2ml into the peritoneal cavity, after 18 days, injecting 5 minimum complete lethal doses of 5 virulent strains C77-1 into the peritoneal cavity/0.2 ml MLD into the control mice with the same conditions, observing for 15 days after challenge, completely killing the control group within 7 days, and killing no more than 2 mice in the immune group within 15 days.

2.3 replacement of the seed with the poison

By using a chromogenic culture medium, streaking and serial passage are carried out, the passage is transmitted to 40 generations, 20 generations, 30 generations and 40 generations are used, the MLD with the minimum complete lethal dose is used for attacking 8 mice, and the death rate is respectively 87.5 percent, 75 percent and 75 percent after 15 days of observation. Preparing inactivated vaccine, immunizing 8 mice with the lowest immunizing dose, and killing live bacteria and 5MLD on the 19 th day. Each group of mice died 1, the immune protection rate of the mice was 87.5%, and the control group died 100%. In order to consider the virulence of the strain and the immunogenicity of the vaccine, the strain is designated for 20 generations at the highest.

EXAMPLE 2 preparation of inactivated vaccine

1 inactivation process

The formaldehyde solution with final concentration of 0.1% (v/v), 0.15% (v/v) and 0.2% (v/v) is selected to inactivate at 37 ℃ and 4 ℃ and 28 ℃, and the result shows that 0.2% (v/v) formaldehyde inactivates at 28 ℃ for 48 hours, and the inactivation effect is complete. Therefore, finally, 0.2% formaldehyde is inactivated for 48 hours at 28 ℃, coated on a Columbia blood plate, cultured for 3 days at 37 ℃, and is sterilized, so that the inactivation is qualified, and then the inactivation is continued for 12 hours. Washing with PBS 2 times to remove formaldehyde.

2 emulsification process

The commercial MONTANIDE PET GEL A adjuvant of French Seebeck is adopted for mixing and emulsifying to prepare the oil-in-water type inactivated vaccine, the inactivated same batch of streptococcus equi subsp equi bacterial liquid is mixed before emulsification, and the adjuvant is mixed according to the proportion of 5 percent of the total volume. The volume was fixed with PBS. Adjusting the pH value to 7.40 +/-0.02.

3 semi-finished product inspection quality standard

3.1 sterility testing

The bacteria-free growth is carried out according to the examination of the appendix of the current Chinese veterinary pharmacopoeia.

3.2 inactivation assay

The inactivated original bacterial liquid is taken and directly coated on a Columbia blood plate with the volume of 100 mu l, observed at 37 ℃ for 3 days, and then aseptically grown.

4 quality standard for finished product inspection

4.1 safety inspection Standard

In order to ensure the safety of the vaccine, the safety test of 'safety test on mice' and 'single-dose vaccination on donkey without glandular disease' are sequentially carried out on 3 batches of vaccine prepared in a laboratory. The test result shows that: the single dose inoculation and the single dose repeated inoculation of each immune animal are good, the donkey state is good, the body temperature and the drinking water are normal, the appetite of individual donkey is reduced or the donkey is not eaten for 1 to 3 days, the swelling condition of the immune part of individual donkey appears, and the donkey disappears gradually within 15 days. The above tests all prove that the vaccine is safe.

4.2 preparation of efficacy test standards

4.2.1 mice minimal immunization dose and minimal use dose test

3 batches of inactivated vaccines qualified in the safety test are treated according to different dosages of 10⁶CFU/0.2ml、10⁷CFU/0.2ml、10⁸CFU/0.2ml respectively immunizes 8 balb/C mice with the age of 6 months, live bacteria attack 5MLD dose is carried out for 19 days, and the results show that the protection effect of the three groups of mice is 100 percent, the control group is not immunized, and 100 percent of the mice die. 10⁶CFU/0.2ml can make mice resist 5MLD lethal dose, and meets the established standard, the control group should die completely within 7 days, and the immune group should die no more than 2 mice within 15 days. The immunoprotection effect of the mice is determined by 10 doses⁷CFU/0.2ml。

4.2.2 determination of the highest challenge dose at the lowest immunization dose

8 immunizations were carried out at the lowest immunization dose, and after 19 days, C77-1 was attacked at 5, 10, 15 and 20MLD, and the control group was attacked at 5MLD, and the observation was carried out for 15 days. 100% of the control group died within 7 days, and the number of the mice died in the immune group within 15 days was 0, 1 and 1 respectively. The death rate is not more than 2, which meets the requirement.

5 determination of immunization program

According to the onset characteristics of donkey plague and previous reports, the immune injection is generally born for 2 months. Inactivated vaccines are typically given 4 times per 1 year, and are given every 4 months.

6 shelf life of vaccine

The invention carries out experimental study on the storage life of the inactivated vaccine prepared in a laboratory on a mouse, stores a batch of vaccines for 2, 6 and 8 months at the temperature of 2-8 ℃, and samples are respectively taken in each time period to detect the characters, the safety and the immune efficacy (immunization with the minimum immune dose). The result shows that the characters of the vaccine do not change obviously after being stored for 6 months at the temperature of 2-8 ℃, and the sterility test and the safety meet the requirements of quality standards. At the lowest immunization dose, the death rate of 5MLD live strains is not more than 2, and 100% of the control group died within 7 days. Considering titer loss caused by the vaccine in the transportation and use processes, the storage period of the vaccine is temporarily set to be 2-8 ℃ for 6 months.

The foregoing is merely a preferred embodiment of this invention, which is intended to be illustrative, not limiting; those skilled in the art will appreciate that many variations, modifications, and even equivalent variations are possible within the spirit and scope of the invention as defined in the appended claims.

Claims

1. The streptococcus equi subspecies equi source strain is named as HLJ2018D-LX, is preserved in the China general microbiological culture Collection center, and has the strain preservation number as follows: CGMCC No. 19353.

2. Use of the donkey-derived strain of streptococcus equi subsp equi as claimed in claim 1 for the preparation of inactivated streptococcus equi subsp equi vaccine.

3. An inactivated vaccine of equine streptococcus subspecies characterized by comprising the inactivated equine streptococcus subspecies donkey-derived strain of donkey according to claim 1.

4. The inactivated vaccine according to claim 3, wherein the inactivation is to inactivate the source strain of donkey of the Streptococcus equi subsp equi species according to claim 1 with 0.2% v/v formaldehyde at 28 ℃ for 48 hours, coat Columbia blood plates, culture the cells at 37 ℃ for 3 days, if the cells are sterile, then inactivate the cells for 12 hours, wash the cells with PBS for 2 times, and remove the formaldehyde.

5. Use of the inactivated vaccine of claim 3 or 4 for the preparation of a biological product for the prevention of diseases caused by Streptococcus equi subsp.

6. The use according to claim 5, wherein the disease is an adenitis.