CN110468069B - Lactobacillus casei YFI-5 and application thereof in resisting carp herpesvirus II - Google Patents
Lactobacillus casei YFI-5 and application thereof in resisting carp herpesvirus II Download PDFInfo
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Abstract
The invention relates to the technical field of aquatic product antiviral microecological preparations, and in particular relates to lactobacillus casei YFI-5 and application thereof in resisting carp herpesvirus type 2. The preservation number of the lactobacillus casei is CCTCC NO: m2019654 Lactobacillus casei YFI-5 fermentation supernatant in the invention can reduce the activity of cyprinid herpesvirus II (CyHV-2) on infecting host cells and inhibit CyHV-2 virus invasion from infecting cells. Has no toxic and side effects on cells. The feed added with Lactobacillus casei YFI-5 thallus can effectively reduce death rate of crucian infected with CyHV-2, and can be used for preventing and treating crucian hematopoietic organ necrosis.
Description
Technical Field
The invention relates to the technical field of aquatic product antiviral microecological preparations, and in particular relates to lactobacillus casei YFI-5 and application thereof in resisting carp herpesvirus type 2.
Background
Crucian carp is one of important species of freshwater fish in China, the annual total yield of the crucian carp exceeds 300 million tons, and the crucian carp occupies an important position in freshwater fish culture. In recent years, hemorrhagic diseases which cause the sudden death of cultured carassius auratus gibelio occur in successive years to cause great economic loss. The superthin section of spleen and kidney tissues of the diseased Crucian is observed by an electron microscope, a large number of typical herpesvirus-like particles are found, and further research confirms that the disease is Crucian hematopoietic necrosis (Crucian Carp hepatosis Necrosis) and the pathogeny thereof is Carp herpesvirus II (Cyprinid herpesvirus2, CyHV-2). Typical clinical symptoms of carassius auratus gibelio infected with CyHV-2 are anorexia, outlier independent swimming, accelerated respiratory rate, poor vitality and sinking to the water bottom. Bleeding on the body surface, fin base and gill of the diseased fish, abdominal distension and exophthalmos. The gill threads are pale, the muscles bleed a bit, the spleen and the kidney are red and swollen, the bladder bleeds punctate, the liver is swollen, the bleeding is caused, and the intestinal tract is empty. The crucian hematopoietic organ necrosis is a highly infectious disease, but the spreading range is limited, and researches show that the disease only infects goldfish, crucian and common varieties thereof at present. Researchers found that hybrids of goldfish and carp are also susceptible to CyHV-2 and that fish of various specifications can be infected.
The prevention and treatment of diseases in aquaculture has long been an important problem that plagues the development of aquaculture. Currently, there are two main methods for controlling diseases of aquaculture animals, drug control and vaccine immunization. However, a series of problems such as side effects, drug residues, drug resistance, water pollution and the like caused by drug control are gradually paid attention by all social circles, and in addition, the treatment effect of the drug control on viral diseases is not very obvious; the development of the vaccine for fishing is influenced by the lack of sensitive cell lines, virus variation and inconvenient immune mode. Therefore, there is a continuing need for effective methods of controlling viral diseases, and probiotics are receiving widespread attention for their non-toxic, non-drug resistant, residue-free, antibacterial, antiviral, growth-promoting, green and safe advantages.
In recent years, a plurality of researchers think that lactic acid bacteria in the aquaculture process are expected to become a substitute of medicaments to a certain extent for preventing and treating various diseases, and a new strategy is provided for a disease prevention and control scheme. Yang Yong et al (2006) prove that the lactic acid bacteria metabolite has a very significant inhibition effect on the growth of Vibrio anguillarum, and the inhibition efficiency is over 90%. Gildberg et al (1998) feed cod fry with a feed containing lactic acid bacteria extracted from the viscera of Atlantic cod fry, and after 3 months, the fry was kept in an environment with strong pathogenic vibrio bacteria, and the disease resistance was improved. The existing data show that lactic acid bacteria not only have antibacterial activity, but also have antiviral activity. Ang et al (2016) found that Lactobacillus casei was able to control hand-foot-and-mouth disease by inhibiting infection by Coxsackie virus. At present, lactic acid bacteria are widely used in aquaculture processes, but research on inhibition of aquatic pathogenic bacteria and viruses by lactic acid bacteria is less.
The lactobacillus casei screened out for the first time can inhibit the carp herpesvirus type 2, and a new idea is provided for prevention and treatment of the virus.
Disclosure of Invention
The invention aims to provide lactobacillus casei YFI-5, wherein the preservation number of the strain is as follows: CCTCC NO: and M2019654.
Another object of the present invention is to provide the use of Lactobacillus casei YFI-5.
In order to achieve the purpose, the invention adopts the following technical measures:
the lactobacillus casei YFI-5 is obtained by separating a pond water sample in an aquaculture area, and specifically comprises the steps of diluting the water sample in the aquaculture pond with physiological saline, coating the diluted water sample on a BHI solid flat plate, finally inverting the flat plate, and culturing for 24 hours in a constant temperature incubator at 30 ℃. Selecting colonies with different forms, inoculating the colonies on a common broth plate for separation and purification, and determining antiviral functions of different bacteria to finally obtain a carp herpesvirus II resistant strain which is named as YFI-5, wherein the strain YFI-5 is identified as lactobacillus casei through physiological and biochemical characteristic determination and 16S rDNA sequence homology analysis.
Lactobacillus casei YFI-5 belongs to Lactobacillus, and is suitable for growth at 37 deg.C, and forms white, round, smooth and moist surface, and regular and convex edge colony after culturing in lactobacillus culture Medium (MRS) for 48 hr; can ferment fructose, galactose and glucose, cannot utilize melibiose, raffinose and xylose, and cannot decompose arginine to produce ammonia.
The strain is delivered to China center for type culture Collection in 2019, 8, 19 and is classified and named: lactobacillus casei (Lactobacillus casei) YFI-5, accession number: CCTCC NO: m2019654, address: wuhan university in Wuhan, China.
The application of lactobacillus casei YFI-5 comprises preparing medicine for treating or preventing crucian hematopoietic necrosis by lactobacillus casei, or preparing medicine for treating or preventing diseases caused by carp herpesvirus II (CyHV-2) infection, or preparing carp herpesvirus II antiviral agent, or preparing aquatic animal feed additive.
Compared with the prior art, the invention has the following advantages:
in the invention, the probiotic lactobacillus casei YFI-5 is used for resisting the carp herpes virus II (CyHV-2) to infect cells, and the lactobacillus casei YFI-5 as a potential antiviral microecological preparation has the following advantages compared with the traditional antiviral chemical medicine:
1. no toxic side effect, no residue, antibiosis, antivirus, growth promotion, green and safety.
2. Has the advantages of convenient and safe use, no immune stress, high economic benefit and the like
3. Enters the fish body by an oral administration mode, inhibits the invasion and proliferation of carp herpes virus II (CyHV-2), and effectively prevents and treats crucian hematopoietic organ necrosis. According to the invention, the lactobacillus casei YFI-5 fermentation supernatant is added firstly, then CyHV-2 diluent is added, and simultaneously the fermentation liquor and the CyHV-2 diluent are added, so that the virus can be directly inhibited, and the inhibition rates are 34% and 27% respectively. The lactobacillus casei YFI-5pH7.0 fermentation supernatant has no toxic and side effects on cells.
4. The feed is added with lactobacillus casei YFI-5 thallus, so that the death rate of crucian infected by CyHV-2 can be effectively reduced.
Drawings
Fig. 1 is a schematic diagram of the level of the respiratory explosive force of each group of crucian carps.
Fig. 2 is a schematic diagram of the serum lysozyme levels of various groups of crucian carps.
Fig. 3 is a schematic diagram of the levels of complement C3 of various crucian carp groups.
Detailed Description
Unless otherwise specified, the test methods and conditions in the examples of the present invention are conventional methods. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples. The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.
Example 1:
strain YFI-5 isolation and identification
1. Strain YFI-5 isolation and identification
The lactobacillus casei is obtained by separating from a pond water sample in an aquaculture area, and specifically, the water sample in the aquaculture pond is continuously diluted by 10 times by using 0.85 percent sterile normal saline for 6 times, 100 mu L of solution is respectively absorbed in each concentration gradient diluent by using a pipette gun to be coated on a BHI solid plate, and the coating rod is used for coating, numbering and repeating for 3 times. And after the uniform coating, placing the mixture in a super-clean workbench for 5-10 min to ensure that the bacteria liquid on the surface of the culture medium is fully absorbed. Finally, the plate was inverted and incubated in a constant temperature incubator at 30 ℃ for 24 hours. Selecting colonies with different forms, inoculating the colonies on a common broth plate, separating and purifying, and measuring the antiviral function of different bacteria to finally obtain a carp herpesvirus II resistant strain which is named YFI-5.
2. YFI-5 Strain identification
1) Physiological and biochemical characteristics
Taking a pure cultured strain YFI-5 by using an MRS solid culture medium, streaking and inoculating a single colony on a BUG identification plate, culturing for 16-24 h at 30 ℃, taking an inoculation liquid of a Biolog bacteria identification kit IF-A when the colony size is proper, wiping the outer wall of a tube, and putting the tube into a Biolog turbidity meter to adjust the reading to be 100% T; a proper amount of single colonies were dipped into the inoculum using a sterile cotton swab to read between 92% T and 98% T by a turbidimeter, the mixture was transferred to GEN III plates in a volume of 100. mu.L per well using an 8-well pipette, and the plates were loaded into a Biolog system for culture, which automatically read and output the results. The physiological and biochemical characteristics are shown in Table 1.
TABLE 1 physiological and biochemical characteristics of Strain YFI-5
2) Molecular biological identification of Strain YFI-5
The gene of the amplified strain 16SrRNA was amplified using the universal primer, 16SF (27F): AGAGTTTGATCMTGGCTCAG, 16SR (1492R): ggttactctgttacgaactt, synthesized by shanghai biotechnology services ltd. The strain YFI-5 is identified as lactobacillus casei by physiological and biochemical characteristic determination and 16S rDNA sequence homology analysis, and the strain is sent to China center for type culture Collection for collection at 19/8 in 2019, and is classified and named: lactobacillus casei (Lactobacillus casei) YFI-5, accession number: CCTCC NO: m2019654, address: wuhan university in Wuhan, China.
Example 2: antiviral spectrum detection method for lactobacillus casei YFI-5 fermentation supernatant
Selecting a single colony of the flat rejuvenated lactobacillus casei YFI-5, inoculating the single colony in 150ml of MRS liquid culture medium, culturing for 24h at 37 ℃, centrifuging for 10min at 5000rpm, taking supernatant, filtering with a 0.22 mu m filter membrane, and storing in a sterile centrifuge tube at 4 ℃ for later use. The inhibition effect of lactobacillus casei YFI-5 fermentation supernatant on carp herpesvirus II (CyHV-2), Giant Salamander Iridovirus (GSIV), Grass Carp Reovirus (GCRV) and carp spring viremia virus (SVCV) is respectively detected.
The cells used in this example were a Carassius auratus brain tissue cell line (GiCB, CCTCC NO: C2013179), a giant salamander muscle cell line (GSM), a grass carp kidney cell line (CIK), and a carp epithelial cell line (EPC).
The volume of the fermentation supernatant of Lactobacillus casei YFI-5 added in this example was 100. mu.L per well, and 100TCID was added500.1ml of virus solution was the same volume as the fermentation supernatant.
Respectively inoculating lactobacillus casei YFI-5 fermentation supernatant and CyHV-2 into GiCB cells in a 96-well culture plate growing into a monolayer simultaneously, inoculating lactobacillus casei YFI-5 fermentation supernatant and GSIV into GSM cells in the 96-well culture plate growing into the monolayer, inoculating lactobacillus casei YFI-5 fermentation supernatant and GCRV into CIK cells in the 96-well culture plate growing into the monolayer, inoculating lactobacillus casei YFI-5 fermentation supernatant and SVCV into EPC cells in the 96-well culture plate growing into the monolayer, inoculating only the same amount of virus to a control group, culturing for 90min, abandoning and washing with PBS, and adding mixed liquid cell maintenance liquid to continue culturing. Cytopathic effect (CPE) was observed under an optical inverted microscope, and the antiviral effect of the fermentation supernatant of Lactobacillus casei YFI-5 was judged based on the amount of CPE, as shown in Table 2, wherein + indicates resistance and-indicates no resistance.
TABLE 2 antiviral Spectrum of Lactobacillus casei YFI-5
Example 3:
application of lactobacillus casei YFI-5 fermentation supernatant in resisting carp herpesvirus II (CyHV-2):
a single colony of the rejuvenated Lactobacillus casei YFI-5 was picked and inoculated into 150ml MRS liquid medium, cultured at 37 ℃ for 24h, centrifuged at 5000rpm for 10min, the supernatant was filtered through a 0.22 μm filter and stored in a sterile centrifuge tube at 4 ℃ for further use in this example and example 4.
The cells used in this example were the crucian brain tissue cell line (gibb), CCTCC NO: C2013179. the cell maintenance solution used was M199 medium of 10% FBS;
the volume of the fermentation supernatant of Lactobacillus casei YFI-5 added in this example was 100. mu.L per well, and 100TCID was added500.1ml of CyHV-2 virus liquid and the volume of the fermentation supernatant are the same;
and (3) virus titer detection:add 1X 10 density to 96 well cell culture plates4GiCB cells per well are cultured for 16-24 hours at 25 ℃ in 100. mu.L. When the cells grow to 80-90%, inoculating the cells with a dilution of 101～1010The virus solution of (2) was cultured in an incubator at 25 ℃ for 2 hours, with 8 parallel wells for each dilution, at 100. mu.L/well. After the incubation was completed, the virus solution was recovered, the cells in the wells were rinsed 2 times with M199 medium, and 100. mu.L of cell maintenance medium was added to continue the culture for 96 hours. Experiment sets 3 groups are parallel, CPE phenomenon of each dilution monolayer cell is observed and recorded every 24h, corresponding lesion hole number is recorded, and half tissue cell infection amount (TCID 50) of CyHV-2 is calculated according to a Reed-Muench method.
The experimental groups were as follows:
group 1: the group was pretreated with lactobacillus casei YFI-5 fermentation supernatant and inoculated with virus: inoculating fermentation supernatant of Lactobacillus casei YFI-5 into cells of 96-well culture plate grown as monolayer, culturing for 2 hr, washing, and adding 100TCID500.1ml of CyHV-2 infects monolayer cells, put in an incubator to adsorb for 90min, add cell maintenance liquid after washing, and continue culturing.
And 2, group: the lactobacillus casei YFI-5 fermentation supernatant and the CyHV-2 are inoculated into the cells at the same time: and 100TCID500.1ml CyHV-2, mixed in equal volume, added to cells grown in a monolayer 96-well culture plate, cultured for 90min, discarded the mixed solution and washed with PBS, and added with cell maintenance solution to continue culturing.
And 3, group: firstly, adding virus to infect cells, and then inoculating lactobacillus casei fermentation supernatant: at 100TCID500.1ml of CyHV-2 infects cells growing into a monolayer of 96-well culture plate, the cells are adsorbed in an incubator for 90min and then washed, lactobacillus casei fermentation supernatant is added, the incubator is placed for 90min and then washed by PBS, and cell maintenance solution is added for continuous culture.
4 groups are as follows: adding the lactobacillus casei supernatant and the cells of the 96-well culture plate growing into a monolayer for co-culture for 2h, and then adding the cell maintenance liquid for continuous culture.
And 5, group: 100TCID500.1ml CyHV-2 infects cells growing into a monolayer 96-well culture plate, the cells are placed in a warm box for adsorption for 90min and washed by PBS, and cell maintenance liquid is added to continue culture
6 groups are as follows: normal cell control.
Indirectly measuring the inhibition rate of the fermented supernatant of the lactobacillus casei YFI-5 on CyHV-2 by an MTT method after 48 hours,
viral inhibitory rate (lactobacillus treatment group OD)490Viral control group OD490) V (cell control OD)490Viral control group OD490)×100％
Specific inhibition rates are shown in table 3:
TABLE 3 inhibition of CyHV-2 by Lactobacillus casei YFI-5 fermentation supernatant
Example 4:
the application of lactobacillus casei YFI-5 in preparing carp herpesvirus II preparation comprises:
1) temporarily culturing crucian (20 +/-2 g) for 2 weeks under laboratory conditions before experiment, feeding at 8 and 18 points per day, wherein the feeding amount is 1% of the weight of the crucian, and continuously oxygenating; the experimental water is aerated tap water, the water temperature is 25 +/-1 ℃, and the dissolved oxygen is more than 5mgL-1pH 7.3 ± 0.5 (chengjie 2011); experimental crucian carp was tested to be free of virus and bacterial infection.
The average was divided into 8 groups of 60 tails each. The following treatments are respectively carried out:
group 1: 100 mul CyHV-2 (10) for crucian abdominal cavity injection7TCID50) And injecting 100 mul of lactobacillus casei YFI-5 fermentation supernatant into abdominal cavity after 48 hours;
and 2, group: intraperitoneal injection of 100 mul of Lactobacillus casei YFI-5 fermentation supernatant to crucian carp, and intraperitoneal injection of 100 mul of CyHV-2 (10. mu.l) after 48 hours7TCID50)；
And 3, group: simultaneously injecting 100 mul CyHV-2 (10) into abdominal cavity of crucian7TCID50) And 100. mu.l of Lactobacillus casei YFI-5 fermentation supernatant;
4 groups are as follows: feeding feed containing Lactobacillus casei YFI-5 for crucian for 2 days (10 days)7cfu/g) was fed at 1% of fish weight and then 100. mu.l CyHV-2 (10. mu.l) was intraperitoneally injected7TCID50)；
And 5, group: 100 mul CyHV-2 (10) for crucian abdominal cavity injection7TCID50) Feeding feed containing Lactobacillus casei YFI-5 (10) 48h later7cfu/g) the feeding amount is 1 percent of the weight of the fish.
6 groups are as follows: 100 mul CyHV-2 (10) for crucian abdominal cavity injection7TCID50) Simultaneously, feeding feed (10) containing Lactobacillus casei YFI-57cfu/g) the feeding amount is 1 percent of the weight of the fish.
7 groups of: 100 mul CyHV-2 (10) for crucian abdominal cavity injection7TCID50)；
And 8 groups: and (3) performing intraperitoneal injection on the crucian by using 100 mul of lactobacillus casei YFI-5 fermentation supernatant for treatment.
9 groups of: the crucian is injected with 100 mul sterile PBS.
The mortality of each crucian group was recorded within 14 days of the group treatment day (table 4). And detected on day 0 and day 14
The related immunity indexes of various groups of crucian: respiratory burst activity (fig. 1), serum lysozyme activity (fig. 2), serum complement C3 levels (fig. 3).
Protection rate calculation formula:
the protection ratio was (V '-V)/V' x 100%
V', the death rate of crucian after direct injection of CyHV-2 (7 groups);
v, mortality of lactobacillus treatment group.
A respiratory burst activity detection method comprises the following steps:
the determination of the respiratory burst force is carried out according to the method described by Anderson: NBT reduction (Anderson, Brubacher et al 1998).
Determination of serum lysozyme Activity
Measurement of serum lysozyme activity was determined by the turbidity method described by Ellis (Ellis 1988). One lysozyme activity unit is defined as: an amount of lysozyme that could decrease the absorbance by 0.001 at a wavelength of 530nm for 1 min.
Determination of serum complement C3
The level of serum complement C3 is determined by Nanjing as-built complement C3 determination kit (Nanjing as-built bioengineering institute). Results are expressed in mg/mL.
TABLE 4 Lactobacillus casei YFI-5 protection ratio for crucian carp
Claims (5)
1. An isolated Lactobacillus casei (I)Lactobacillus casei) The preservation number of the lactobacillus casei is CCTCC NO: and M2019654.
2. The use of lactobacillus casei as claimed in claim 1 in the preparation of a medicament for treating or preventing crucian haematopoietic necrosis.
3. Use of lactobacillus casei according to claim 1 in the manufacture of a medicament for the treatment or prophylaxis of a disease caused by herpes carp virus type II (CyHV-2) infection.
4. Use of lactobacillus casei as claimed in claim 1 in the manufacture of a carp herpesvirus II antiviral agent.
5. Use of lactobacillus casei as claimed in claim 1 in the manufacture of an aquatic animal feed additive.
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