CN110819549B - Hippocampus kelloggi pathogenic strain and enteritis inactivated vaccine - Google Patents
Hippocampus kelloggi pathogenic strain and enteritis inactivated vaccine Download PDFInfo
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Abstract
The invention discloses a preparation method of a hippocampus kelloggi pathogenic strain and an enteritis inactivated vaccine, and aims to provide an inactivated vaccine capable of effectively preventing hippocampus kelloggi enteritis, which is characterized in that the inactivated vaccine is an inactivated thallus of vibrio parahaemolyticus, and the concentration of the inactivated vaccine thallus is not less than 109CFU/ml. The preparation process of the hippocampal enteritis inactivated vaccine is simple, the yield is stable, the cost is low, and the inactivated vaccine with higher immune effect can be obtained in large quantity. The immune protection rate of the inactivated vaccine prepared by the method can reach 60%, the vaccine is a pure biological preparation, has the characteristics of safety and environmental protection, does not generate drug residues, does not pollute the environment, can fundamentally inhibit the occurrence of hippocampal enteritis, and provides technical support for large-scale preparation of the hippocampal enteritis inactivated vaccine.
Description
Technical Field
The invention belongs to the field of microbial vaccine preparation and biological prevention and treatment, and particularly relates to a hippocampus kelloggi pathogenic strain and enteritis inactivated vaccine.
Background
Hippocampus kelloggi (Hippocampus erectus), native to the sea in the Atlantic sea, inhabits mainly in the shallow sea and coral reef areas, is a precious species with medicinal and ornamental values. In recent years, the number of wild hippocampus has been drastically reduced due to destruction and over-fishing of wild habitats, and the like. Since 2004, all hippocampal species have been listed in the red catalogue of the Washington convention and the world conservation of Nature. The sea horse cultivation in China has been developed sufficiently in recent years, but the sea horse is extremely easy to be infected due to high density pressure in the cultivation process, and in addition, the sea horse catches live copepods carrying a large number of pathogenic bacteria and artemia as food, so that the disease problem is prominent day by day, and the healthy development of the sea horse cultivation industry is severely restricted.
Since 2016, in the process of culturing hippocampus kelloggi in northern China, more than 30% of deaths are caused by frequent enteritis outbreak, so that disastrous economic losses are caused, and the continuous development of the industry is severely restricted. The affected hippocampus has weakened swimming ability, whitish and swollen anus, whitish and ulcerated abdomen, and has anatomical characteristics of whitish intestinal tract accompanied by a large amount of mucus, accumulated ascites, dark color of liver and gill tissues and the like.
At present, chemical medicines are generally used in a method for controlling the hippocampus kelloggi enteritis, and although the method has a certain effect, the disease cannot be controlled fundamentally, and the drug resistance of an organism is easily caused, so that the drug residues in the body and the environment are caused, the ecological balance is damaged, and the health and the safety of a human body are threatened. Therefore, the search for a safe and effective way to treat the disease is a problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a hippocampus kelloggi enteritis pathogenic bacterium and a preparation method and application of an inactivated vaccine thereof, wherein the inactivated vaccine has a prevention and treatment effect on enteritis of hippocampus kelloggi and hippocampus japonicus, and is used for solving the problems of drug residue and drug resistance generated when antibiotics are used for treating the enteritis of hippocampus kelloggi and hippocampus japonicus in the past so as to make up for the defects of the prior art.
The Vibrio parahaemolyticus (Vibrio parahaemolyticus) is preserved in the China general microbiological culture Collection center (CGMCC) in 2018, 03 and 12 months, and the preservation number is CGMCC No. 15441. The inactivated thallus of vibrio parahaemolyticus CGMCC No.15441 is used for preparing inactivated vaccine, and the prepared inactivated vaccine can prevent enteritis of hippocampus kelloggi and hippocampus japonicus.
The inactivated thallus of vibrio parahaemolyticus CGMCC No.15441 is used for preparing inactivated vaccine, and the prepared inactivated vaccine can prevent enteritis of hippocampus kelloggi and hippocampus japonicus.
For obtaining the best control effect, the concentration of the inactivated bacteria of the inactivated vaccine is 107-1010CFU/ml。
The preparation method of the hippocampal enteritis inactivated vaccine is simple in preparation process, stable in yield and low in cost, and the inactivated vaccine with a high immune effect can be obtained in a large quantity. The immune protection rate of the inactivated vaccine prepared by the method on the hippocampus striatus reaches 50%, the immune protection rate on the Japanese hippocampus reaches 60%, the inactivated vaccine is a pure biological preparation, and has the characteristics of safety and environmental protection, no drug residue and no environmental pollution are generated, and the method can fundamentally inhibit the occurrence of hippocampus enteritis.
Detailed Description
Example 1 information of Vibrio parahaemolyticus CGMCC No.15441 screened by the present invention
1) Screening of Vibrio parahaemolyticus
The method comprises the steps of flushing the body surface of a diseased hippocampus kelloggi (with the body length of 2-3cm and the body weight of 0.7 +/-0.16 g) with 0.01mol/L sterile Phosphate Buffered Saline (PBS) for three times, dissecting the diseased hippocampus, collecting ulcerated muscle and intestinal tissues in a centrifuge tube filled with sterile PBS buffer solution by using sterile scissors, cutting the tissues into pieces, suspending the tissues in the PBS buffer solution, diluting a supernatant, coating the diluted supernatant on a 2216E culture plate, culturing for 24 hours in a 28 ℃ culture box, picking out dominant bacterial colonies with consistent morphological characteristics, separating and purifying until a pure cultured dominant bacterial strain is obtained, performing routine physiological and biochemical treatment and 16S rDNA gene sequence determination, and transferring the dominant bacterial strains into a slant culture medium for storage at 4 ℃.
2) Culture conditions of Vibrio parahaemolyticus
The strain is spread on a 2216E solid medium plate and cultured for 16-18 hours at 28 ℃ to grow a single colony.
3) Morphological characteristics
The vibrio parahaemolyticus is negatively dyed with phosphotungstic acid for 3min, and is observed under a transmission electron microscope after being dried, the strain under the electron microscope is in a long rod shape, is provided with periflagellum, no capsule, no spore and periflagellum, the extreme of the strain is provided with main flagellum, and the size of the strain is about 10 multiplied by 15 microns.
4) Physiological and biochemical characteristics
The vibrio parahaemolyticus is gram-negative bacteria and can grow in 1-10% NaCl peptone water; fermentable glucose, mannose, sucrose, arabinose and maltose, non-fermentable xylose and lactose; mannitol positive, inositol negative; arginine decarboxylase, lysine decarboxylase, and ornithine decarboxylase positive.
5) Pathogenicity detection of strains
The healthy Hippocampus kelloggi is provided by Qingdao Qingyuan ocean biology technology Co., Ltd, and is cultured in a laboratory storage box under the conditions of salinity of 31 + -0.5, temperature of 25 + -1 deg.C, pH7.4, and continuous oxygenation, wherein the water change amount is 50% per day. Injecting purified Vibrio parahaemolyticus, injecting bacterial liquid with different concentrations into abdominal cavity of experimental group, injecting PBS buffer solution with same amount into control group, injecting Hippocampus kelloggi, and infecting with the injection concentration of 6.32 × 108In the group, the average incidence of Hippocampus kelloggi reached 100%, the mortality rate was 66%, the concentration was 3.16X 108Group, 1.58X 108Group sum of 0.39X 108The average morbidity rate in the group was 62%, 32% and 0%, and the average mortality rate was 43%, 12% and 0%, respectively, while the hippocampus kelloggi in the control group did not develop disease or die. Intestinal tissues of the diseased hippocampus kelloggi were collected again for re-isolation of bacteria, and the resulting bacteria were identified by 16SrDNA as the bacteria isolated for the first time.
Example 2 preparation of inactivated vaccine of Vibrio parahaemolyticus CGMCC No.15441
1) Firstly, inoculating the screened vibrio parahaemolyticus in 3000ml of high-salt common LB liquid culture medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 30g/L), shaking and culturing at 28 ℃ for 12 hours, and detecting the concentration of the bacterial liquid to 10 by a blood counting plate9Terminating the culture at CFU/ml to obtain an expanded culture solution of the vibrio parahaemolyticus;
2) adding 150ml of formaldehyde solution according to the volume ratio of 0.5%, inactivating the expanded culture solution of the strain at room temperature for 12 hours, and shaking for 5 times; obtaining an inactivated bacterial liquid of vibrio parahaemolyticus;
3) finally, the inactivated bacteria liquid of the strain is centrifugated for 15 minutes at 8000g and 4 ℃, supernatant is discarded, the sediment is washed for 2 times by sterile normal saline, thalli are collected, the bacteria liquid is re-suspended by the sterile normal saline, and the concentration of the inactivated thalli in the bacteria liquid is 107-1010CFU/ml。
4) 0.1ml of inactivated vaccine is taken and spread on 2216E solid medium, and after 48 hours of culture at 28 ℃, a sterile colony grows out, which indicates that the bacteria are completely inactivated. The 10-tailed hippocampus kelloggi is injected into the abdominal cavity at a rate of 0.1 ml/tail, the 10-tailed sterile normal saline with the same dose is injected into a control group, and then the inactivated vaccine is normally fed and observed for one week, and is completely alive, normal in feeding and activity, and free of any abnormality in visceral autopsy, so that the prepared inactivated vaccine is safe and reliable. Storing at 4 deg.C for use.
Example 3 immunoprotection test of vaccine 1
The prepared inactivated vaccine is used for carrying out immune effect experiments on the Japanese sea horse. 100 healthy Japanese hippocampus, which is about 2cm in body length, were randomly extracted and divided into two treatment groups, two in parallel per treatment group, and 25 in parallel per treatment group. The inactivated vaccine prepared in example 2 was used for immersion immunization test, in which group 1 was a control group and group 2 was an immunization group, and group 2 of Japanese hippocampus was immersed in 8% NaC1 solution for 2 minutes and immediately transferred to 1O-fold diluted inactivated bacterial solution (2X 10)8CFU/m1) for 3min, performing challenge test on 2 groups of Japanese sea horses after 15 days, and observing and recording morbidity and mortality of each group regularly. The hippocampus of the control group started to attack and have dead individuals appeared at 12h of challenge, the cumulative morbidity rate of 60h was 80%, the cumulative mortality rate of 64%, the cumulative morbidity rate of 96h was 100%, and the cumulative mortality rate of 76%, the hippocampus of the vaccine treatment group of the group 2 started to attack and have no dead individuals appeared at 24h of challenge, the cumulative morbidity rate of 60h was 32%, the cumulative mortality rate of 20%, the cumulative morbidity rate of 96h was 40%, and the cumulative mortality rate of 30%.
EXAMPLE 4 immunoprotection test of vaccine 2
The immune effect of the prepared inactivated vaccine is detected by adopting a soaking immune method: randomly selecting 200 tails of healthy Hippocampus kelloggi with body length of 2cm, randomly dividing into a control group and an immune group, soaking the immune group in 8% NaC1 solution for 2 minutes, and immediately transferring to inactivated bacterial liquid (2 × 10) diluted by 10 times8CFU/m1) for 3 min. After 7 days, the immunization is strengthened once by using the same method, and other feeding conditions are consistent with those of the control group of uninfected hippocampus. On day 14 post immunization, challenge was performed. The hippocampus kelloggi died from the control group the next day, with a cumulative morbidity of 100% and a cumulative mortality of 58% on the fourth day, and a cumulative morbidity of 50% and a cumulative mortality of 32% on the fourth day in the immune group.
The experimental results show that the screened hippocampal enteritis inactivated vaccine has a high immune protection effect, can effectively prevent the occurrence of hippocampus kelloggi and hippocampal japonicus enteritis, and has a good market popularization prospect.
Claims (5)
1. A pathogenic strain of Hippocampus is Vibrio parahaemolyticus (Vibrio parahaemolyticus) with preservation number of CGMCC No. 15441.
2. An inactivated hippocampal vaccine, characterized in that the vaccine comprises the inactivated thallus of vibrio parahaemolyticus CGMCC No.15441 of claim 1.
3. The inactivated vaccine of claim 2, wherein the inactivated bacteria is present in the vaccine at a concentration of 10%7-1010CFU/ml。
4. The inactivated vaccine of claim 3, wherein the inactivated bacteria is present in the vaccine at a concentration of 10%9CFU/ml。
5. The inactivated vaccine of claim 3, which is prepared by the following method:
first, the separated and purified secondary hemolysis arcInoculating the strain into LB liquid culture medium containing 3% NaCl, shake culturing at 28 deg.C for 16-24 hr until the concentration of the strain liquid reaches 109Stopping culturing when OD600 is detected to be above 0.6 by CFU/ml or spectrophotometer to obtain an enlarged culture solution of the strain;
then adding formaldehyde solution according to the volume ratio of 5 per mill, inactivating for 12-16 hours at room temperature, and shaking for 5-10 times; obtaining an inactivated bacterial liquid of the strain;
then, the inactivated bacterial liquid of the strain was centrifuged at 8000g and 4 ℃ for 15 minutes, the supernatant was discarded, the precipitate was washed with sterile physiological saline 2 times, the cells were collected, and the bacterial liquid was resuspended with sterile physiological saline at a concentration of 10%9CFU/mL, and storing at 4 ℃ for later use;
and finally, coating 0.1ml of the inactivated vaccine on a 2216E solid culture medium, and culturing at 28 ℃ for 48h to obtain a sterile colony, namely the qualified inactivated vaccine.
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