CN109157546B - Compound microecological preparation for preventing and controlling acute hepatopancreas necrosis syndrome of penaeus vannamei boone and application thereof - Google Patents

Compound microecological preparation for preventing and controlling acute hepatopancreas necrosis syndrome of penaeus vannamei boone and application thereof Download PDF

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CN109157546B
CN109157546B CN201810844204.1A CN201810844204A CN109157546B CN 109157546 B CN109157546 B CN 109157546B CN 201810844204 A CN201810844204 A CN 201810844204A CN 109157546 B CN109157546 B CN 109157546B
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penaeus vannamei
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providencia
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沈辉
甄晓然
万夕和
蒋葛
乔毅
王李宝
史文军
黎慧
范贤平
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JIANGSU MARINE FISHERIES RESEARCH INSTITUTE
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Abstract

The invention relates to a compound microecological preparation for preventing and controlling acute hepatopancreas necrosis syndrome (AHPNS) of penaeus vannamei boone and application thereof, wherein the compound microecological preparation comprises providencia and enterobacter. The compound microecological preparation is added into feed for feeding. The composite microecological preparation has a strong effect of inhibiting vibrio parahaemolyticus, has a remarkable application effect, is one of important ways for realizing green culture of penaeus vannamei boone, and has a good application prospect.

Description

Compound microecological preparation for preventing and controlling acute hepatopancreas necrosis syndrome of penaeus vannamei boone and application thereof
Technical Field
The invention belongs to the field of disease prevention and control of aquatic products, and particularly relates to a compound microecological preparation for preventing and controlling acute hepatopancreas necrosis syndrome of penaeus vannamei boone and application thereof.
Background
The acute hepatopancreas necrosis syndrome (AHPNS) of the penaeus vannamei is a disease which is mainly characterized in that the hepatopancreas of the penaeus vannamei is necrotic so as to cause sudden death of the penaeus vannamei, and is one of the most serious diseases affecting the culture of the penaeus vannamei in recent years. The Early stage of the disease mainly occurs in the seedling stage of prawns and is called Early Mortality Syndrome (EMS). It has been found that some Vibrio bacteria carrying a specific pathogenic gene are causative agents of the disease, and among them, Vibrio parahaemolyticus is mainly used. Tran et al isolated 1 Vibrio harveyi (Vibrio harveyi) from shrimp ponds with Vietnam AHPNS morbidity, and the mortality rate of the shrimps after soaking infection reached 100%. Joshi et al isolated several strains of Vibrio parahaemolyticus from Thailand shrimp, and infected the shrimp intramuscularly, wherein one group died 100% within 24h, and the strain detection was AHPNS positive. Liu isolated 1 strain of Vibrio owens in the sea AHPNS pathogenic shrimp pond, and the strain was considered as the AHPNS pathogenic strain. Xuan et al suggested that 1 strain of Vibrio campylobacter isolated from shrimp could lead to the development of AHPNS.
PirvpThe binary toxin coded by AB gene is the main virulence factor of pathogenic vibrio, the produced binary toxin has a structure similar to the parasporal crystal structure produced by bacillus thuringiensis, the virulence effect is similar to the virulence effect of bacillus thuringiensis Cry protein, and the binary toxin is inserted into the cell membrane of a host to form a die-through hole, so that the osmotic pressure inside and outside the cell is changed, and the target cell is damaged or even killed.
Some researchers begin to pay attention to the research and development of the microecological preparation, but the microecological preparation specially aiming at the acute hepatopancreas necrosis syndrome of the penaeus vannamei has not been published in any documents at present.
Disclosure of Invention
The invention aims to solve the technical problem of providing a compound microecological preparation for preventing and controlling acute hepatopancreas necrosis syndrome of penaeus vannamei and application thereof, and overcomes the defect that no microecological preparation specially aiming at the acute hepatopancreas necrosis syndrome of penaeus vannamei exists in the existing product.
The invention provides a compound microecological preparation for preventing and controlling acute hepatopancreas necrosis syndrome of penaeus vannamei, which comprises Providencia sp and Enterobacter sp; wherein the preservation number of providencia is CGMCC No.15415, and the preservation number of enterobacter is CGMCC No. 15416.
Providencia sp (Providencia sp.) used in the present invention, the name of the depository: CGMCC (China general microbiological culture Collection center) general microbiological culture Collection center, the preservation address is as follows: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, has a preservation number of CGMCC NO.15415 and a preservation date of 2018, 3 months and 6 days. The proposed classification is named: providencia (Providencia sp.).
Enterobacter (Enterobacter sp.) used in the present invention, the name of the depository unit: CGMCC (China general microbiological culture Collection center) general microbiological culture Collection center, the preservation address is as follows: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, has a preservation number of CGMCC NO.15416 and a preservation date of 2018, 3 months and 6 days. The proposed classification is named: enterobacter (Enterobacter sp.).
The providencia and the enterobacteria are respectively cultured until the concentration is 1.0 multiplied by 108CFU/mL and mixing for later use.
The formula of the adopted culture medium is as follows: 5g of peptone, 1g of yeast extract, 0.01g of ferric phosphate, 3g of sodium acetate, 1g of dipotassium phosphate, 1000mL of old seawater and 7.6-7.8 of pH value.
The preparation ratio of the providencia and the enterobacter after liquid fermentation is 1: 0.5-2.
The providencia and the enterobacter account for 2-3% of the composite microecological preparation.
The invention also provides application of the compound microecological preparation for preventing and controlling the acute hepatopancreas necrosis syndrome of the penaeus vannamei, and the compound microecological preparation is added into feed for feeding. Can not be mixed with antibiotics for use, and the dosage is as follows: and adding 20-40 ml of the compound microecologics into each kilogram of feed, and feeding for 3-5 days.
The invention is mainly used for preventing and controlling the acute hepatopancreatic necrosis syndrome of the penaeus vannamei boone, inhibiting the growth of the vibrio necrotizing to the acute hepatopancreatic necrosis and reducing the high death rate caused by the acute hepatopancreatic necrosis syndrome.
The providencia CGMCC No.15415 has the ability of synthesizing or semi-synthesizing lactam antibiotic active substances, can produce lipopeptide antibiotic substances, and can strongly inhibit pathogenic bacteria of penaeus vannamei.
The enterobacter CGMCC No.15416 can regulate intestinal flora balance, produce bacteriocin and lactic acid, inhibit pathogenic bacteria growth, and improve immunity and disease resistance of Penaeus vannamei Boone.
The inhibition performance of the pathogenic vibrio parahaemolyticus causing acute hepatopancreas necrosis of the penaeus vannamei can be further enhanced by combining the two according to the proportion of the invention, and in addition, the combination of the two can inhibit the pathogenic vibrio parahaemolyticus causing acute hepatopancreas necrosis of the penaeus vannamei within the range of Jiangsu province in 10 bacterial strains. However, the existing combination of bacillus subtilis and lactic acid bacteria in the market can only inhibit conventional vibrio parahaemolyticus to a certain extent, but cannot inhibit the pathogenic parahaemolyticus arc of acute hepatopancreas necrosis of penaeus vannamei of multiple bacterial strain types. The combination of the two strains of bacteria has definite specificity to the pathogenic vibrio parahaemolyticus of acute hepatopancreatic necrosis of 10 strains of penaeus vannamei in Jiangsu province.
Advantageous effects
The composite microecological preparation has a strong effect of inhibiting vibrio parahaemolyticus, has a remarkable application effect, is one of important ways for realizing green culture of penaeus vannamei boone, and has a good application prospect.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
This example provides a complex probiotic comprising Providencia sp and Enterobacter sp; wherein the preservation number of providencia is CGMCC No.15415, and the preservation number of enterobacter is CGMCC No. 15416. The two kinds of bacteria are respectively prepared according to the proportion of 1:1 after liquid fermentation. The two kinds of bacteria are cultured in advance to the concentration of 1.0X 108CFU/mL and mixing for later use. Wherein, the formula of the culture medium is as follows: 5g of peptone, 1g of yeast extract, 0.01g of ferric phosphate, 3g of sodium acetate, 1g of dipotassium phosphate, 1000mL of old seawater and 7.6-7.8 of pH value.
The use method of the composite microecological preparation comprises the following steps:
adding the culture mixed solution of providencia and enterobacter into the granulated feed for the penaeus vannamei, mixing the feed in a ratio of 1:50, feeding the mixed feed to the penaeus vannamei, and observing the eating condition of a bait table. Feeding for the first time every day on the day of 8: 00-9: 00 in the morning, feeding once every day for 2 hours, and feeding for 7 days.
The effect experiment of the composite microecological preparation is as follows:
test for inhibiting acute hepatopancreatic necrosis lethal vibrio
The method comprises the steps of selecting a prawn culture greenhouse with 9 prawn seedlings in a certain region of Jiangsu and consistent in growth state, infecting and outbreak of acute hepatopancreatic necrosis syndrome (about 0.7 mu/greenhouse), randomly dividing the prawn culture greenhouse into 3 groups, feeding the prawn culture greenhouse according to a common mode of farmers according to a control group (no addition of any microecological preparation), adding bacillus subtilis and lactic acid bacteria into feeds of the control group, adding a providencia and enterobacter composite microecological preparation into feeds of the test group, taking diseased prawn samples in ponds (each pond takes prawn samples at 3 different positions, takes hepatopancreatic organs in the prawn samples, mixing the prawn samples uniformly) on days 0, 1, 3, 5 and 7, qualitatively analyzing acute hepatopancreatic necrosis and lethal vibrio in hepatopancreatic tissue by adopting a molecular biological method, and observing the death condition of the prawns.
TABLE 1 changes in the number of Vibrio in Water
Figure BDA0001746274900000031
Note: different letters indicate significant difference (P <0.05), same letters indicate insignificant difference (P >0.05)
As can be seen from Table 1, in the control group without any probiotics, the acute hepatopancreatic necrosis syndrome pathogen exists in the shrimp body all the time, and the number of dead individuals is remarkably increased until all the shrimps die at day 5. In the control group, bacillus subtilis and lactic acid bacteria are used as a composite microecological preparation, the acute hepatopancreatic necrosis comprehensive disease pathogen in the shrimp bodies exists all the time, the number of dead individuals is remarkably increased, all the shrimps die in the 5 th day, and the control group has no remarkable difference with the control group. After 3 days of feeding the compound microecologics, the detection of the acute hepatopancreatic necrosis comprehensive disease pathogen is weak positive, and is negative after 5 days, dead individuals are remarkably reduced from 3 days later, and no dead individual is found at 7 days, so that the difference is remarkable compared with that before the use (P is less than 0.05). It can be seen that the use effect of the present example is significantly better than that of the first and second control groups.
Second, inhibition experiment of pathogenic vibrio plate
3 strains of acute hepatopancreatic necrosis syndrome lethal vibrio are selected in the test, and antagonism experiments are set by vibrio phagicus, bacillus, lactobacillus and the composite bacterium fermentation liquid. The test group is divided into four groups randomly, the first control group is bacteriophage, the second control group is bacillus, the third control group is lactic acid bacteria, and the test group is the composite bacteria. 3 pathogenic bacteria are separated and purified from the pathogenic shrimps in Jiangsu province, and the pathogenic bacteria contain acute hepatopancreatic necrosis syndrome virulence genes pirA and pirB which are numbered HYS-AHPND-1, HYS-AHPND-2 and HYS-AHPND-3 through molecular biological identification, and the concentration of the three pathogenic bacteria is 106CFU/ml is coated on an LB flat plate, four kinds of microorganism fermentation liquor (vibrio phagostii, bacillus, lactobacillus and the composite bacteria) are used as drug sensitive paper sheets, the culture is carried out for 48 hours at 28 ℃, the diameter of a bacteriostasis ring is measured, and the experimental result is shown in table 2. The results show that the antibacterial effect of the compound preparation of the invention on the lethal vibrio of acute hepatopancreatic necrosis syndrome of three south America white shrimps in Jiangsu is remarkably higher than that of vibrio phagostimuli, bacillus and lactobacillus (P)<0.01)。
TABLE 2 bacteriostatic activity of the strain on lethal strain of acute hepatopancreas necrosis syndrome of Penaeus vannamei Boone
Figure BDA0001746274900000041
Note: different letters indicate significant difference (P <0.05) and the same letters indicate insignificant difference (P > 0.05).

Claims (6)

1. The compound microecological preparation for preventing and controlling acute hepatopancreas necrosis syndrome of penaeus vannamei boone is characterized by comprising the following components in parts by weight: the composite ecological preparation comprises Providencia sp and Enterobacter sp; wherein the preservation number of providencia is CGMCC No.15415, and the preservation number of enterobacter is CGMCC No. 15416.
2. The compound microecological preparation for preventing and controlling acute hepatopancreas necrosis syndrome of penaeus vannamei according to claim 1, is characterized in that: the providencia and the enterobacteria are respectively cultured until the concentration is 1.0 multiplied by 108CFU/mL and mixing for later use.
3. The compound microecological preparation for preventing and controlling acute hepatopancreas necrosis syndrome of penaeus vannamei according to claim 2, is characterized in that: the formula of the adopted culture medium is as follows: 5g of peptone, 1g of yeast extract, 0.01g of ferric phosphate, 3g of sodium acetate, 1g of dipotassium phosphate, 1000mL of old seawater and 7.6-7.8 of pH value.
4. The compound microecological preparation for preventing and controlling acute hepatopancreas necrosis syndrome of penaeus vannamei according to claim 1, is characterized in that: the preparation ratio of the providencia and the enterobacter after liquid fermentation is 1: 0.5-2.
5. The compound microecological preparation for preventing and controlling acute hepatopancreas necrosis syndrome of penaeus vannamei according to claim 1, is characterized in that: the providencia and the enterobacter account for 2-3% of the composite microecological preparation.
6. The application of the compound microecological preparation for preventing and controlling acute hepatopancreas necrosis syndrome of penaeus vannamei according to claim 1, which is characterized in that: the compound microecological preparation is added into feed for feeding.
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