CN111088182B

CN111088182B - Mannheimia haemolytica and application thereof

Info

Publication number: CN111088182B
Application number: CN201911322657.9A
Authority: CN
Inventors: 赵�卓; 商云鹏; 胡义彬; 高荣荣; 李晓亮; 尚川川; 王力; 王秀敏; 江厚生; 李三鹏; 周瑞杰; 魏树阁; 王顺山
Original assignee: Beijing Centre Biology Co ltd; Beijing Huaxia Xingyang Biological Science & Technology Co ltd
Current assignee: Beijing Centre Biology Co ltd; Beijing Huaxia Xingyang Biological Science & Technology Co ltd
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2021-09-10
Anticipated expiration: 2039-12-20
Also published as: CN111088182A

Abstract

The invention discloses a haemolytic mannheimia and application thereof, wherein the haemolytic mannheimia A1 type M164 strain is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is as follows: CGMCC No.18873, preservation date: 11/15/2019, deposit address: western road No.1, north chen west city of township, 3, institute for microbiology, china academy of sciences, common microbiology center of the committee for preservation and management of microbial cultures, china, and zip code 100101. The vaccine preparation obtained by the invention is safe to apply, has good immune protection effect on pneumonia caused by mannheimia haemolytica, and can protect cattle for at least 6 months.

Description

Mannheimia haemolytica and application thereof

Technical Field

The invention relates to the field of biological products for livestock, in particular to mannheimia haemolytica and application thereof.

Background

Mannheimia haemolytica (Mannheimia haemolytica) belonging to the family Pasteurellaceae, the genus Mannheimia. Based on the results of DNA-DNA hybridization and 16srRNA sequence analysis, serotypes were classified into A1, A2, A5, A6, A7, A8, A9, A12, A13, A14, A16, and A17.

Mannheimia haemolytica is a weakly haemolytic, gram-negative coccobacillus, which is similar in morphology to Pasteurella multocida, is 2.5 μm long and 0.5 μm wide and is spherical or rod-shaped; no spore, no flagellum, no movement, capsulized, and bipolar coloration and gram negative staining. The nutrient requirement is not strict, the bacterial colony can grow well on a common culture medium, and the bacterial colony is round, smooth, moist and translucent after being cultured for 24 hours. The newly separated colony is weakly beta-hemolytic on blood agar, after culturing for 48 hours, hemolytic rings can be seen after removing the colony, and the hemolysis is weakened or disappeared after continuous subculture. Among the serotypes, serotypes a1 and a2 are the dominant strains. The strain of serotype A1 is the cause of Mannheimia (also known as "shipping fever") in cattle. Mannheimia haemolytica is ubiquitous in antibiotic resistance. This antibiotic resistance occurs in part as a result of the extensive use of antibiotics either as feed additives for prophylactic administration or as growth promoters. The resistance genes are present on plasmids, and some on chromosomes. The most common types of resistance are beta-lactams, tetracyclines, streptomycins, sulfadiazines, macrocyclic lipids and sulfadimidine.

At present, the few literature in the field discloses inactivated vaccine for bovine mannheimia disease, and whether the bovine mannheimia can be used in the field of vaccine is a technical problem to be solved in the field.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide mannheimia haemolytica and application thereof.

In order to solve the problems, the technical scheme adopted by the invention is as follows:

the haemolytic Mannheimia, belonging to the Pasteurellaceae, the Mannheimia haemolytica, which is Mannheimia haemolytica type A1M 164 strain, is preserved in the China general microbiological culture Collection center (CGMCC), and has the preservation number: CGMCC No.18873, preservation date: 11/15/2019, deposit address: western road No.1, north chen west city of township, 3, institute for microbiology, china academy of sciences, common microbiology center of the committee for preservation and management of microbial cultures, china, and zip code 100101.

Preferably, the 16S rRNA gene sequence of the mannheimia haemolytica is the sequence shown in SEQ ID.1.

The invention also comprises an application of the mannheimia haemolytica, which is used for preparing a vaccine preparation for preventing the mannheimia bovis caused by the mannheimia haemolytica.

Preferably, the vaccine formulation comprises inactivated mannheimia haemolytica, a leukotoxin and a vaccine adjuvant.

Preferably, the vaccine adjuvant is any one or a combination of 603 adjuvants, 605 adjuvants, aluminum hydroxide gel adjuvants or mineral oil adjuvants.

Preferably, the vaccine adjuvant is 605 adjuvant.

Preferably, the vaccine formulation is administered to the bovine in two doses per immune cycle, the immunizing dose being 2ml per head, wherein the second dose is administered during the 14-28 days after the first administration.

Preferably, the vaccine preparation contains 1.0X 10 inactivated by formaldehyde in each head dose⁸～1.0×10¹⁰The bacillus mansoni haemolyticus of each colony forming unit and the leukotoxin of 640-5120 toxin concentration units.

Compared with the prior art, the invention has the beneficial effects that: the vaccine preparation obtained by the invention is safe to apply, has good immune protection effect on pneumonia caused by mannheimia haemolytica, and can protect cattle for at least 6 months.

Drawings

FIG. 1 shows the result of PCR for typing and identifying Mannheimia haemolytica;

wherein M: DL2000 marker; 1: negative control; 2: m164 strain; 3. a positive control;

FIG. 2 shows the growth and development rules of the antibody of the calf immunized by the inactivated vaccine against Mannheimia bovis.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments.

The following examples are illustrative only and not intended to limit the scope of the present invention in any way, and modifications or substitutions in the details and form of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these modifications and substitutions fall within the scope of the present invention.

In the following examples, the 605 adjuvant is a patent adjuvant (patent publication No. CN103083659A) applied by Beijing Huaxia Xingyang Biotechnology Co., Ltd, and has been applied to the research and production of vaccines, and the 605 adjuvant has been used to develop and register a new veterinary drug product, Newcastle disease inactivated vaccine, and has obtained a new veterinary drug certificate and a production license; the 603 adjuvant is a patent adjuvant (patent publication No. CN101428145A) applied by Beijing Sheng Tai science and technology Co., Ltd, and has been applied to the research and production of vaccines, and the 603 adjuvant is used for researching and registering new veterinary drug products, namely, the inactivated vaccine of mycoplasma suis, the inactivated vaccine of haemophilus parasuis and the inactivated vaccine of infectious rhinotracheitis of cattle, and new veterinary drug certificates and production license letters are obtained; the mineral oil adjuvant and the aluminum hydroxide gel adjuvant are prepared according to the typical methods of veterinary medicine, and the technical means used in the examples are conventional means well known to those skilled in the art, and other raw materials are commercially available, if not specified.

Example 1 isolation and identification of M164 Strain of Mannheimia haemolytica type A1

1.1 isolation of Mannheimia haemolytica Lung lesion site of cattle infected with Mannheimia haemolytica was picked up with an inoculating loop, streaked and inoculated on a TSA plate containing 5% sheep blood, cultured at 37 ℃ for 16 hours, and a single colony was picked up to obtain a pure culture, which was then transferred to a liquid medium for culture and expanded, and preserved. On 5% sheep blood TSA medium, the colony morphology is smooth, convex, gray, and β -hemolytic ring can be seen.

1.2 pure test 0.2ml of the bacterial liquid was inoculated into 2 5% sheep blood TSA plates, incubated at 37 ℃ for 16 hours, and the colony morphology was observed, showing that the colony morphology was uniform.

1.3 Biochemical identification the bacterial colony growing on 5% sheep blood TSA plate is purely cultured, then inoculated in a glucose, lactose, maltose, xylose, mannitol, indole, inositol, urease and other micro bacteria biochemical test tube, cultured in an incubator at 37 ℃ for 24-72 hours, and the result shows that the bacterial colony is consistent with the haemolytic mannheimia in Bergey handbook.

1.4 typing identification, the serotype A1, the serotype A2 and the serotype A6 standard positive serum and the haemolytic mannheimia separating strain act on a glass slide for 1-2 minutes respectively, and the result shows that the haemolytic mannheimia separating strain and the serotype A1 standard positive serum have precipitation reaction, which indicates that the haemolytic mannheimia separating strain serotype is A1.

1.5 extracting the haemolytic mannheimia isolate genome, amplifying 16SrRNA by using a universal primer, and performing BLAST analysis on the amplified gene sequence and the sequence in Genbank as shown in figure 1, wherein the similarity with the haemolytic mannheimia 16SrRNA reaches 96-99%.

1.6 animal regression experiment the pure culture of Mannheimia haemolytica was inoculated into liquid medium for scale-up culture, and fresh bacterial liquid 1X 10 was inoculated via pulmonary injection¹⁰CFU, observed 4 days after challenge. Animal regression tests can reproduce the symptoms of the bovine mannheimia, the clinical manifestations of the bovine mannheimia are depression, lying difficulty, dyspnea, death and the like, and pathogens can be recovered in the lung.

1.7 based on the bacterial morphology and culture characteristics, and the sequencing result of the 16SrRNA gene, the strain is judged to be mannheimia haemolytica A1 type, and the strain is named as M164 strain.

EXAMPLE 2 preparation of inactivated vaccine against bovine Mannheimia

2.1 taking X generation basic seeds of haemolytic mannheimia A1M 164 strain, inoculating the X +1 generation bacterial liquid in a liquid culture medium preheated at 37 ℃ according to 2%, culturing at the constant temperature of 37 +/-1 ℃, harvesting at 100r/min for 48 hours to obtain X +1 generation bacterial liquid, inoculating the X +1 generation bacterial liquid in the liquid culture medium preheated at 37 ℃ according to the proportion of 5%, culturing at the constant temperature of 37 +/-1 ℃, harvesting at 100r/min for 24 hours to obtain X +2 generation, inoculating the X +2 generation culture in the liquid culture medium preheated at 37 ℃ according to the proportion, culturing at the constant temperature of 37 +/-1 ℃, culturing at 100r/min for 16 hours to obtain X +3 generation bacterial liquid which is used as a vaccine-making bacterial liquid.

2.2 taking 1.0ml of bacterial liquid, diluting the bacterial liquid by 10 times in series by using sterile normal saline, taking 10^-7And (3) diluting, coating and inoculating 5% sheep blood TSA plates, wherein each plate is 0.1ml, dispersing bacterial liquid, airing in a constant-temperature incubator at 37 ℃, turning the plates, culturing for 24 hours, counting the average value of bacterial colonies on the 3 plates, and calculating CFU. The results showed that 1.0X 10 bacteria solution per 1.0ml of the bacteria solution⁸～1.0×10¹⁰Viable count per colony forming unit. Filtering the bacterial liquid through a 0.22-micron filter membrane, and measuring the concentration of the leukotoxin in the filtrate by using BL-3 cells, wherein the result shows that each 1.0ml of the bacterial liquid contains the leukotoxin with the concentration unit of 640-5120 toxin.

2.3 adding formaldehyde solution with the concentration of 37% in 0.3% of the total bacteria liquid, and inactivating the mixture for 24 hours at the temperature of 37 ℃ and at the speed of 100 r/min. After inactivation is finished, sampling is carried out for aseptic inspection and inactivation inspection, and the inactivated vaccine can be used for vaccine preparation after the sample is qualified.

2.4, centrifugally concentrating the qualified inactivated antigen to ensure that the antigen content of the vaccines prepared by different adjuvants is the same. Mixing the inactivated antigen and the 605 adjuvant according to the proportion of 5:1 to obtain a patent vaccine preparation 1; the inactivated antigen is mixed with 603 adjuvant in a ratio of 2:1 to obtain a patent vaccine preparation 2; the inactivated antigen and the mineral oil adjuvant are mixed and emulsified according to the proportion of 1:2, and the inactivated antigen is a patent vaccine preparation 3; the inactivated antigen and the aluminum hydroxide gel adjuvant are mixed according to the proportion of 5:1 to form the patent vaccine preparation 4, and the mixture is subpackaged and then is hermetically stored.

Example 3 Studies of the efficacy of inactivated vaccine against Mannheimia bovis

3.1 the experimental animals used in the study were 8-12 weeks old Mannheimia bovis antibody-negative calves, which were divided into 5 groups as shown in Table 1. Group of1-4 are vaccine preparation immunization groups, wherein 2ml is inoculated on the 0 th day of the research, the immunization is strengthened once in the same dosage and mode 21 days after the immunization, and the virus is attacked 14 days after the strengthening of the immunization; group 5 was a control, not immunized, challenged in the same manner and dose as the previous group, and was injected intrapulmonary with fresh Mannheimia haemolytica 1X 10¹⁰CFU。

TABLE 1 Immunity group for the study of the effectiveness of inactivated vaccine against Mannheimia bovis

And 3.2, continuously observing for 4 days after the toxin attacking, observing and recording the clinical symptoms and death conditions of the toxin attacking cattle every day, performing a caesarean examination on all test cattle on the 4 th day, observing lung lesions, measuring the size of a focus, and counting the difference between the immunization group and the control group by using a Mann-Whitney U test method.

3.3 results

And continuously observing 8-12 weeks old calves for 4 days after toxin attack, and recording clinical symptoms. After 4 days, all the test cattle were subjected to a necropsy, intact lungs were removed, lesions characteristic to the infection caused by mannheimia haemolytica were collectively estimated, and the lesion size was measured. The results are shown in Table 2.

TABLE 2 results of clinical observations and dissection

Note: since the sum of the maximum lesion volumes was 453.25, the size of the lesion in the experimental animal that died within 4 days was determined to be 500.

The man-Whitney U test was used to analyze the toxicity attacking protective effect according to the measured lesion size of the lung injection site, and the results of the immunization group were differentially compared with those of the control group, and the results are shown in tables 3 to 6.

TABLE 3 comparison of vaccine 1 immunization group with control Mann-Whitney U test

TABLE 4 comparison of vaccine 2 immunization groups with control Mann-Whitney U test

TABLE 5 comparison of vaccine 3 immunization groups with control Mann-Whitney U test

TABLE 6 comparison of vaccine 4 immunization groups with control Mann-Whitney U test

As can be seen from the results, the challenge protection results of the group administered with the vaccine of the present invention showed significant differences compared to the control group.

Example 4 Bos-Mandarin's disease inactivated vaccine antibody duration Studies

4.1 the experimental animals used in the study were 8-12 weeks old Mannheimia bovis antibody-negative calves, and were divided into 5 groups as shown in Table 7. Groups 1-4 are the vaccine immunization groups of the invention, vaccinated on day 0 of the study, 2ml subcutaneously, and boosted once at the same dose and manner 21 days after immunization.

TABLE 7 Immunity groups for antibody duration study of inactivated vaccine against bovine Mannheimia

4.2 blood collection is carried out 14 days after primary immunization, 21 days after primary immunization, 14 days after secondary immunization, 28 days after secondary immunization, 2 months after secondary immunization, 4 months after secondary immunization, 6 months after secondary immunization and 8 months after secondary immunization respectively to determine the antibody titer. The results are shown in FIG. 2, and show that Mannheimia haemolytica antibody was produced 14 days after the first immunization, and the antibody titer was increased 21 days after the first immunization, but the antibody level was low. At 14 days after the boost, the antibody levels increased rapidly, with the antibody levels reaching a maximum 2 months after the second immunization, with the highest vaccine antibody formulated with adjuvant 605 and the longest antibody duration, and the antibody levels remained high up to 8 months after the second immunization.

Sequence listing

<110> Beijing Huaxia xing ocean Biotech Co., Ltd

BEIJING CENTRE BIOLOGY Co.,Ltd.

<120> Mannheimia haemolytica and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 490

<212> DNA

<213> Mannheimia haemolytica (Mannheimia haemolytica16S rRNA gene sequence)

<400> 1

atgagcgaga acccaaaaca tattacagta ttattgcacg aagcggttga cggcttggca 60

attaagccta acggtatcta tattgacggt actttcgggc gtggcgggca ttctcgcttg 120

attttatcca aactgggcga aaatgggcga ttaatcgcaa ctgatcgtga cccacgagcg 180

atagccgaag ctcaaacgat tagtgaccct cgttttcaaa ttgaacatac ggcattttct 240

gccattcccg aactgtgtga acgaatgggg ttggtgggca aaattgacgg tattttattg 300

gatttaggtg tgtcttcccc gcagcttgac gaagccgaac gaggctttag ttttatgcgt 360

gacgggccgc ttgatatgcg aatggatacc accaaaggct tatcggcagc cgagtggctt 420

gcccaagttt cggcagaaga tttagcttgg gtattgaaag aatttggcga agagcgtttt 480

gccaaacgga 490

Claims

1. The mannheimia haemolytica is mannheimia haemolytica type A1M 164 strain, is preserved in China general microbiological culture Collection center (CGMCC), and has the preservation number as follows: CGMCC No. 18873.

2. The mannheimia haemolytica of claim 1, wherein: the 16S rRNA gene sequence of the mannheimia haemolytica is a sequence shown in SEQ ID No. 1.

3. Use of mannheimia haemolytica according to claim 1, wherein: is used for preparing a vaccine preparation for preventing the bovine mansoniosis caused by haemolytic mansonii.

4. Use according to claim 3, characterized in that: the vaccine formulation comprises inactivated mannheimia haemolytica, a leukotoxin and a vaccine adjuvant.

5. The use of claim 4, wherein: the vaccine adjuvant is any one or a combination of a plurality of 603 adjuvants, 605 adjuvants, aluminum hydroxide gel adjuvants or mineral oil adjuvants.

6. The use of claim 5, wherein: the vaccine adjuvant is 605 adjuvant.

7. Use according to claim 3, characterized in that: the vaccine formulation is administered to cattle in two doses per immune cycle, the immunizing dose being 2ml per head, wherein the second dose is 14-28 days after the first administration.

8. The use of claim 7, wherein: the vaccine preparation contains inactivated formaldehyde 1.0 × 10 in each dose⁸～1.0×10¹⁰The bacillus mansoni haemolyticus of each colony forming unit and the leukotoxin of 640-5120 toxin concentration units.