阅读说明：本技术 一种鸭疫里氏杆菌高效价阳性血清的快速简便制备方法 (Rapid and simple preparation method of high-titer positive serum of riemerella anatipestifer ) 是由 孟照洁 翁立雪 赵杰 赵情 于 2019-10-25 设计创作，主要内容包括：本发明涉及一种鸭疫里氏杆菌高效价阳性血清的快速简便制备方法,属于生物制品领域。包括以下步骤：1)培养基制备：培养基分为液体培养基和固体培养基；2)菌液制备；3)菌液灭活；4)免疫抗体制备；5)免疫；6)抗体滴度检测；7)特异性检测。该方法使用兔作为免疫对象,使用含有合适用量的鸭疫里氏杆菌的灭活抗原对其免疫,7天产生高免血清,试管凝集抗体可以达到1：16。(The invention relates to a rapid and simple preparation method of high-efficiency positive serum of riemerella anatipestifer, belonging to the field of biological products. The method comprises the following steps: 1) preparing a culture medium: the culture medium is divided into a liquid culture medium and a solid culture medium; 2) preparing a bacterial liquid; 3) inactivating bacterial liquid; 4) preparing immune antibodies; 5) immunization; 6) detecting the antibody titer; 7) and (4) detecting the specificity. The method uses a rabbit as an immune object, uses an inactivated antigen containing a proper amount of Riemerella anatipestifer to immunize the rabbit, generates high immune serum in 7 days, and can ensure that a test tube agglutination antibody reaches 1: 16.)

1. A rapid and simple preparation method of high-titer positive serum of Riemerella anatipestifer is characterized by comprising the following steps of: the method comprises the following steps:

1) preparation of the Medium

The culture medium is divided into a liquid culture medium and a solid culture medium;

2) preparation of bacterial liquid

Inoculating a strain prepared with serum into a solid culture medium;

3) inactivation of bacterial liquid

Adding 0.2% of formaldehyde into the mixed bacterial liquid obtained in the step 2), inactivating for 24 hours in a shaking table at 37 ℃, controlling the speed of the shaking table at 120r/min, performing inactivation inspection after inactivation, and obtaining an inactivated bacterial liquid after confirming aseptic growth;

4) immune antibody preparation

Centrifuging the inactivated bacteria liquid for 10min at 8000r/min, removing supernatant to obtain thallus, removing toxin and concentrating, and washing thallus with normal saline to obtain 1000 hundred million cfu/mL antigen liquid;

5) immunization

Inoculating the prepared antigen into a female rabbit through an ear edge vein, inoculating 0.5ml on the first day, inoculating 1ml on the third day, and inoculating 1.5ml on the 5 th day;

6) antibody titer detection

Collecting a small amount of serum on the next day after each immunization after the immunization, and detecting the serum antibody titer by using a test tube agglutination method, wherein the serum antibody titer is more than or equal to 1: collecting blood from heart at 16 hours, and collecting serum;

7) specificity detection

And (3) performing glass slide agglutination on the collected serum, escherichia coli, pseudomonas aeruginosa, pasteurella, salmonella and immune riemerella anatipestifer.

2. The method for rapidly and simply preparing the high-titer positive serum of the Riemerella anatipestifer according to claim 1, wherein the solid culture medium in the step 1) adopts a conventional mode: adding 5% of large bovine serum into a martin culture medium; the basic liquid of the liquid culture medium is Martin culture medium, 5% of Dairy cattle serum and B1 are added before inoculation, and 0.1g of B1 is added into 1 ten thousand milliliters of culture solution.

3. The method for rapidly and simply preparing the high-titer positive serum of the Riemerella anatipestifer according to claim 1, wherein the step 2) is carried out for 20 hours at 37 ℃ in a 5% carbon dioxide culture medium; preparing strains, preparing 100 hundred million cfu/ml of bacterial liquid, inoculating the bacterial liquid to a liquid culture medium, adding the bacterial liquid according to a proportion of 5%, and controlling the volume of the liquid culture medium and the volume of a container to be 1: 5, placing the mixture into a shaker for culturing at 37 ℃, controlling the speed at 120r/min, and culturing for 17 hours.

4. The method for rapidly and conveniently preparing the high titer positive serum for Riemerella anatipestifer according to claim 1, wherein the weight of the female rabbit inoculated in the step 5) is 2-3 kg.

5. The method for rapidly and easily preparing the Riemerella anatipestifer high-titer positive serum according to claim 1, wherein in the step 7), Escherichia coli, Pseudomonas aeruginosa, Pasteurella pasteurella and Salmonella are negative, and the Riemerella anatipestifer for immunization is positive.

Technical Field

The invention belongs to the technical field of biological products, and particularly relates to a rapid and simple preparation method of high-titer positive serum of riemerella anatipestifer.

Background

Riemerella anatipestifer is also called as infectious serositis of duck, and is a contact, acute or chronic infectious disease with septicemia. Is mainly characterized by cellulose pericarditis, cellulose perihepatitis, cellulose air sacculitis, caseous salpingitis, arthritis and paralysis.

The infectious serositis of duck is a chronic or acute septic infectious disease affecting ducks/geese caused by riemerella anatipestifer. Mainly infected ducks, geese, chickens and some wild birds can be infected. It is manifested as depression, anorexia, lacrimation, soft feet, thin and yellowish white or green feces, head-twisting and neck-circling, and intermittent convulsions until death. The pericardial fluid is increased or adhered to the epicardium, the liver is enlarged, or there are more cellulosic secretions on the surface. The morbidity mortality rate is generally 5% -80%, which often causes massive death of the ducklings/geese and leads to delayed development of the ducks/geese. It can be seen in four seasons, especially in autumn and winter. The traditional method for treating the infectious serositis of the duck is mainly based on medicines, seriously threatens the food safety and is released due to national banning of resistance. This has to be returned to vaccine prevention for the control of infectious serositis in ducks.

Currently, the method mainly depends on preparing Riemerella anatipestifer into oil emulsion vaccine, immunizing SPF chicken, and preparing hyperimmune serum through multiple immunizations; the defect is that the period is long, the experimental animal is homologous animal, and nonspecific agglutination is easy to occur; the emulsification and preparation of vaccines is relatively cumbersome.

Disclosure of Invention

In order to solve the technical problems, the invention provides a rapid and simple preparation method of high-titer positive serum of riemerella anatipestifer, and the invention provides a method for preparing the high-titer positive serum of the riemerella anatipestifer by preparing high-bacteria-content riemerella anatipestifer antigen and immunizing rabbits in a special mode to obtain the high-titer riemerella anatipestifer serum for 7 days.

The invention is realized by the following technical scheme, and on one hand, the invention provides a quick and simple preparation method of high-titer positive serum of riemerella anatipestifer, which comprises the following steps:

1) preparation of the Medium

The culture medium is divided into a liquid culture medium and a solid culture medium;

2) preparation of bacterial liquid

Inoculating a strain prepared with serum into a solid culture medium;

3) inactivation of bacterial liquid

Adding 0.2% of formaldehyde into the mixed bacterial liquid obtained in the step 2), inactivating for 24 hours in a shaking table at 37 ℃, controlling the speed of the shaking table at 120r/min, performing inactivation inspection after inactivation, and obtaining an inactivated bacterial liquid after confirming aseptic growth;

4) immune antibody preparation

Centrifuging the inactivated bacteria liquid for 10min at 8000r/min, removing supernatant to obtain thallus, removing toxin and concentrating, and washing thallus with normal saline to obtain 1000 hundred million cfu/mL antigen liquid;

5) immunization

Inoculating the prepared antigen into a female rabbit through an ear edge vein, inoculating 0.5ml on the first day, inoculating 1ml on the third day, and inoculating 1.5ml on the 5 th day;

6) antibody titer detection

Collecting a small amount of serum on the next day after each immunization after the immunization, and detecting the serum antibody titer by using a test tube agglutination method, wherein the serum antibody titer is more than or equal to 1: collecting blood from heart at 16 hours, and collecting serum;

7) specificity detection

And (3) performing glass slide agglutination on the collected serum, escherichia coli, pseudomonas aeruginosa, pasteurella, salmonella and immune riemerella anatipestifer.

Preferably, the solid medium of step 1) is prepared in a conventional manner: adding 5% of large bovine serum into a martin culture medium; the basic liquid of the liquid culture medium is Martin culture medium, 5% of Dairy cattle serum and B1 are added before inoculation, and 0.1g of B1 is added into 1 ten thousand milliliters of culture solution.

Preferably, step 2) is performed for 20h at 37 ℃ in 5% carbon dioxide medium; preparing strains, preparing 100 hundred million cfu/ml of bacterial liquid, inoculating the bacterial liquid to a liquid culture medium, adding the bacterial liquid according to a proportion of 5%, and controlling the volume of the liquid culture medium and the volume of a container to be 1: 5, placing the mixture into a shaker for culturing at 37 ℃, controlling the speed at 120r/min, and culturing for 17 hours.

Preferably, the female rabbits inoculated in step 5) weigh 2-3 kg.

Preferably, Escherichia coli, Pseudomonas aeruginosa, Pasteurella pasteurella and Salmonella are negative in step 7), and Riemerella anatipestifer is positive for immunization.

The invention has the beneficial effects that:

1 the preparation period of the original method is about 45 to 60 days, and the preparation period of the invention is 7 to 10 days, thus greatly shortening the preparation period.

2 the original method adopts SPF chicken and homologous animals, which are easy to have non-specific cross, and the method adopts heterologous animals, which avoids non-specific cross.

3 the original method adopts the vaccine emulsification preparation method to prepare the vaccine, which is relatively complicated and needs professional equipment.

4 the original antigen propagation method does not need a fermentation tank, the bacteria content is low, and a professional fermentation device is needed for fermentation in the fermentation tank.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

As introduced in the background art, currently, the duck plague Riemerella is mainly prepared into an oil emulsion vaccine to immunize SPF (specific pathogen free) chickens, and hyperimmune serum is prepared through multiple immunizations; the defect is that the period is long, the experimental animal is homologous animal, and nonspecific agglutination is easy to occur; the emulsification and preparation of vaccines is relatively cumbersome.