CN115369059B

CN115369059B - Lactococcus with effect of resisting aeromonas hydrophila infection of aquatic animals and application thereof

Info

Publication number: CN115369059B
Application number: CN202211008762.7A
Authority: CN
Inventors: 陈德芳; 杨飞; 张鑫; 耿毅; 黄小丽; 欧阳萍; 李志琼
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2022-08-22
Filing date: 2022-08-22
Publication date: 2023-05-05
Anticipated expiration: 2042-08-22
Also published as: CN115369059A

Abstract

The invention provides a lactococcus with the function of resisting the infection of aeromonas hydrophila of aquatic animals, which comprises the following steps: the honey bag lactococcus petasites Lactococcus petauri JLlg PX is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M2022927. The strain is acid-resistant and cholate-resistant, and has the ability of survival in the digestive tract in severe environment. The honey bag lactococcus jilg 08PX disclosed by the invention has no pathogenicity, and the co-infection can relieve the tissue pathological damage of sturgeon liver and valve intestines caused by aeromonas hydrophila, reduce the loading amount of aeromonas hydrophila in the hybrid sturgeon liver after the aeromonas hydrophila infection, and reduce the morbidity and mortality caused by the aeromonas hydrophila infection. The cell-free supernatant has an inhibiting effect on the growth of fish pathogenic bacteria aeromonas hydrophila, aeromonas veronii and yersinia ruckeri; has potential application value in preparing microecological preparation for preventing and treating fish septicemia caused by aeromonas hydrophila.

Description

Lactococcus with effect of resisting aeromonas hydrophila infection of aquatic animals and application thereof

Technical Field

The invention belongs to the field of probiotics, and particularly relates to a lactococcus capable of resisting aeromonas hydrophila infection of aquatic animals and application thereof.

Background

Chinese is the first large sturgeon culture country in the world, and hybrid sturgeon bred by using Siberian sturgeon (Acipenser bailii) as a female parent is the main culture variety of the sturgeon industry in the southwest region at present. In the artificial reproduction of parent fish, bacterial infection is easy to occur after the operation, and about 30% of mortality rate can be caused, so that huge economic loss is caused, and the reserve of the parent of the sturgeon in Siberia and the supply of offspring seed are directly influenced. Sturgeon sepsis is a typical bacterial disease associated with an aeromonas bacterial infection. Aeromonas caviae infection has been reported to cause sepsis in hybrid sturgeons and Huso dauricus. Infection with aeromonas veronii can cause sepsis in chinese sturgeon (a.sinensis). Aeromonas hydrophila infection can cause sepsis including Acipenser sinensis, acipenser sinensis (A. Schrenckii) and Siberian sturgeon. In addition to single bacterial infections, mixed infections are also common phenomena in aquaculture, however, the role and effects of non-pathogenic strains in the development of mixed infection disease processes are rarely reported.

Lactococcus is a gram-positive unpowered spherical or oval bacterium of the genus Lactococcus (Lactobacillus) of the family Streptococcaceae, belonging to the order Lactobacillus, belonging to the order Firmides, bacillus, and Lactobacillus. Bacteria of the genus lactococcus have been continuously discovered for many years, currently, 23 species and 6 subspecies of bacteria of the genus lactococcus are counted according to the prokaryotic standard naming list (List of Prokaryotic names with Standing in Nomenclature, LPSN) database listing information and related reports, and as the genome data of bacteria of the genus lactococcus are continuously enriched, the number of bacteria of the genus lactococcus is continuously increased, and related classification research is also faster.

Currently, bacteria of the genus lactococcus include both species having probiotic properties, such as lactococcus lactis, lactococcus centralis; also included are pathogenic bacteria such as lactococcus garvieae. Although related experiments showed that some strains of lactococcus garvieae have probiotic properties, the mainstream study still considered lactococcus garvieae as a fish pathogen. As a main pathogen of fish lactococcus disease, the lactococcus garvieae is susceptible to rainbow trout, and the disease is popular at the water temperature of more than 14 ℃. The infection with lactococcus garvieae can cause rainbow trout paneyeball inflammation, pericarditis, epicarditis, myocarditis, peritonitis, gastritis and enteritis.

Goodman et al in 2017 isolated a strain of catalase negative gram-positive coccus from facial abscess lesions of bagged mammalian honey bag-like peter, analyzed the strain by morphology, physiological characteristics and phylogenetic analysis as lactococcus, and subsequently determined that the strain was different from lactococcus garvieae by 16S rRNA and rpoB gene sequence similarity and ANI and dDDH analysis, named L.petaluri, chinese name translated as lactococcus nectar bag-like. In fact, the close relatedness of M.necoxins to M.griseus, the inability of the 16S rRNA gene sequence to distinguish it from M.griseus, resulted in inaccurate identification of many species in the early stages, e.g., as early as 2007, savvdis et al isolated a species from the onset of the disease, rainbow trout, in the Greek farm, identified early as M.griseus, and identified as M.necoxins by whole genome sequencing analysis until 2020.

The honeyed-bag lactococcus petasites is a newly discovered strain, the number of the reported honeyed-bag lactococcus petasites strains is small, the functional research of the honeyed-bag lactococcus petasites is very small, and the research of the honeyed-bag lactococcus petasites with probiotics or pathogenic characteristics is relatively lack.

Disclosure of Invention

The invention aims to provide a probiotic strain of Siberian sturgeon source: the honey bag lactococcus pensis Lactococcus petauri JLlg PX is preserved in China Center for Type Culture Collection (CCTCC), the address is eight paths of Lopa nationality mountain in Wuchang district of Wuhan, hubei province, the preservation number is CCTCC M2022927, and the preservation date is 2022, 6 months and 20 days.

The invention firstly provides a honeyed-bag lactococcus jilike Lactococcus petauri JLlg PX which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M2022927.

The invention also provides a culture of the honeyed-bag lactococcus jisii, which is obtained by culturing the honeyed-bag lactococcus jisii Lactococcus petauri JLlg PX in a culture medium; preferably, the medium is TBS medium or MRS medium; and/or the culture conditions are: culturing at 37 ℃ for 24 hours under 180 r/min.

The invention also provides application of the honeyed-bag lactococcus and/or the culture thereof in preparing medicines for resisting pathogenic bacteria of aquatic animals.

Further, the aquatic pathogenic bacteria include aeromonas and/or yersinia ruckeri.

Still further, the aeromonas comprises aeromonas hydrophila and/or aeromonas veronii.

Further, the above-mentioned drugs are drugs for controlling diseases caused by pathogenic bacteria infection of the aquatic animals.

Further, the medicine is a medicine for preventing and treating septicemia of aquatic animals.

Further, the medicine is a medicine for preventing and treating septicemia of aquatic animals caused by aeromonas hydrophila infection.

Further, the drug is a drug for reducing the aeromonas hydrophila load in the liver of the aquatic animal and inhibiting the growth of aeromonas hydrophila.

Further, the aquatic animal is a fish, preferably sturgeon.

Still further, the sturgeons include Acipenser sinensis, siberian sturgeon and/or hybrid sturgeon.

In the product obtained by culturing the honeyed-bag lactococcus jis in the culture medium, the product is cell-free supernatant obtained by culturing the honeyed-bag lactococcus jis in the culture medium or a mixture of the cell-free supernatant obtained by culturing the honeyed-bag lactococcus jis and the honeyed-bag lactococcus jis.

The invention has the beneficial effects that: the invention identifies a honeybag lactococcus vaginalis Lactococcus petauri JLlg PX, which is acid-resistant and cholate-resistant and has the ability of surviving in the digestive tract in a severe environment. The breast-shaped streptococcus agaricus hancei Lactococcus petauri JLlg PX has no pathogenicity, and the co-infection can relieve the tissue pathological damage of the liver and intestinal tracts of sturgeons caused by aeromonas hydrophila and reduce the morbidity and mortality. The cell-free supernatant has an inhibiting effect on the growth of pathogenic bacteria such as aeromonas hydrophila, yersinia ruckeri and aeromonas veronii of fish sources, and can reduce the capacity of aeromonas hydrophila in the liver of hybrid sturgeon after the aeromonas hydrophila infection; the strain has potential application value in preparing medicines for preventing and treating fish septicemia.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 is a JLlg08PX 16S rRNA phylogenetic tree.

FIG. 2 shows the growth of JLlg08PX in various media. A, BHIA culture medium; b, MRS medium.

Fig. 3 is a jlg 08PX scanning electron microscope image.

Fig. 4 shows the growth of jlg 08PX at various bile salt concentrations. TSB,0% bile salt TSB medium; 0.2%,0.2% bile salt TSB medium; 0.3%,0.3% bile salt TSB medium; 0.4%,0.4% bile salt TSB medium; * P <0.05.

Fig. 5 shows the antibacterial effect of jlg 08PX on aquatic pathogens (experimental result of inhibition zone).

FIG. 6 is an effect of JLlg08PX CFS on growth of pathogenic bacteria in fish. * P <0.05.

FIG. 7 shows the cumulative mortality after infection of the hybrid sturgeon with the isolated strain.

Fig. 8 shows pathological changes of liver tissue. A. B, PBS control group; C. d, a honey bag galactococcus wufenensis virus-killing group; E. f, aeromonas hydrophila challenge group; G. h, a co-infection group of the M.nectar and the Aeromonas hydrophila.

Fig. 9 shows pathological changes of the valve intestine tissue. A, PBS control group; b, a honey bag galactococcus petasites virus-killing group; c, aeromonas hydrophila challenge group; d, a co-infection group of the M.nectar and the Aeromonas hydrophila.

Figure 10 is the aeromonas load in the liver.

Detailed Description

The Aeromonas hydrophila (A.hydrophila) CW strain used in the invention is separated, identified and stored by the university of Sichuan agriculture fish disease research center, the L.meliloti Lactococcus petauri JLlg PX strain, the Aeromonas hydrophila JLAH08PH strain, the Aeromonas verrucosa, the Yersinia ruckeri, the Vibrio parahaemolyticus (V.paramolyticus), the Elizabeth (Elizabethkingia miricola), the Citrobacter freundii (Citrobacter freundii), the Streptococcus agalactiae and the Streptococcus iniae (S.iniae) are all separated, identified and stored in a laboratory where the inventor is located.

62.28+ -14.65 g of 48 healthy hybrid sturgeons (Siberian sturgeons x Acipenser schneiderian) and 68.98 + -12.51 g of 147 healthy Siberian sturgeons are from Sichuan-Ten fishery Co.

The materials and equipment used in the present invention are known products and are obtained by purchasing commercially available products, unless otherwise specified.

EXAMPLE 1 identification of the honeyed-bag lactococcus vaginalis JLlg08PX of the invention

The inventor of the invention separates and obtains a probiotic strain of Siberian sturgeon source: the method comprises the steps of obtaining the complete genome sequence of the strain JLlg08PX by combining Illumina Hiseq second generation sequencing and PacBio RS II third generation sequencing through the honey bag lactococcus penus Lactococcus petauri JLlg PX (hereinafter referred to as JLlg08 PX), wherein the JLlg08PX has a chromosome with the size of 2113250bp and a plasmid with the size of 45868bp, the genome size is 2159118bp, and the GC content is 38.01%. The phylogenetic tree of the complete sequence of the 16S rRNA gene of the strain JLlg08PX is shown in FIG. 1, and JLlg08PX is clustered with the M.nectaricum, the M.griseus and subspecies thereof, and Fulmor Sha Ru cocci. The 16S rRNA gene of the strain JLlg08PX has 100 percent of similarity with the lactococcus jingyasii 159469 and 100 percent of similarity with the lactococcus garvieae M14 and the FLG2 respectively and 99.87 percent; similarity to lactococcus garvieae subspecies FDAARGOS929 and subspecies BSN307 was 99.93% and 99.71%, respectively; the similarity with Fulmor Sha Ru coccus 516 is 99.05% and is greater than 98.65. Therefore, the strain jlg 08PX cannot be classified at the seed level, and further analysis of ANI and dDDH is required to determine the classification at the seed level.

The analytical statistics of the JLlg08PX strain ANI and dDDH are shown in Table 2. ANI values of JLlg08PX and honeybag lactococcus pekinensis 159469, LZys1 and CF11 are all greater than 96%, 98.55%, 98.32% and 98.15%, respectively; ANI values with lactococcus garvieae Lg2, ATCC49156 and FDAARGO S929 were less than 95%, 92.52%, 92.48% and 92.46%, respectively; ANI value with lactococcus garvieae subspecies CCUG32208T is 92.47%; ANI values with Fulmor Sha Ru coccus NBRC109475 and its subspecies LMG30663 were 92.35% and 92.81%, respectively. dDDH analysis found that dDDH values were greater than 70% for jlg 08PX and honeybag lactococcus pennii 159469, LZys1 and CF11, 86.00%, 84.30% and 83.80%, respectively; the dDDH values of the strain and the lactococcus garvieae Lg2, ATCC49156 and FDAARGOS929 are less than 70 percent, namely 51.50 percent, 51.40 percent and 51.10 percent respectively; the dDDH value with lactococcus garvieae subspecies garvieae CCUG32208T is 50.80%; ANI values with Fulmor Sha Ru coccus NBRC109475 and its subspecies LMG30663 were 50.00% and 51.50%, respectively. The results show that JLlg08PX is the honeyed-bag lactococcus pennii.

TABLE 2

The honey bag type lactococcus pensis Lactococcus petauri JLlg PX is preserved in China Center for Type Culture Collection (CCTCC), the address is eight paths of Lopa nationality mountain in Wuchang district of Wuhan, hubei province, the preservation number is CCTCC M2022927, and the preservation date is 2022, 6 months and 20 days.

The following experiments prove the beneficial effects of the invention.

Experimental example 1 morphological observation and physicochemical Properties of the honeybag of the invention, lactococcus vaginalis JLlg08PX

1. Experimental method

1.1 morphological observations

1.1.1 colony morphology observations

JLlg08PX streaked BHIA and MRSA medium were cultured at 28℃for 24 hours, and then colony growth was observed.

1.1.2 bacterial morphology observations

Colonies cultured on TSA medium for 18h are collected by flushing with sterile PBS, centrifuging for 1min at 12000r/min, discarding supernatant, sucking 4% paraformaldehyde solution by a pipette, blowing and sucking uniformly, and fixing at 4 ℃. After the fixation was completed, the precipitate was collected by centrifugation, washed with ultrapure water 2 times for 5min each, dehydrated with a series of gradient alcohols, 30%, 50%, 70%, 80%, 90%, 95%, 100%, 10min per gradient. After 100% alcohol is added for resuspension, a small amount of suspension is sucked and dripped on a slide, the slide is lightly adhered on conductive adhesive, ion sputtering spraying is carried out, and finally a proper position and a proper multiple are selected under a mirror for observation.

1.2 Observation of JLlg08PX tolerance to temperature, pH and salinity

1.2.1 temperature tolerance observations

The overnight cultured JLlg08PX bacterial liquid is inoculated into TSB culture medium at 1% (v/v), and cultured for 72 hours under shaking at 18 ℃,28 ℃, 40 ℃ and 45 ℃ 180r/min respectively. JLlg08PX streaks TSA medium, and is cultured for 72 hours at 4 ℃ to observe whether the medium is turbid or colony growth.

1.2.2 pH tolerance observations

JLlg08PX bacterial liquid is inoculated in TSB culture media with different pH values of 3, 4, 7, 10 and 11 at 1% (v/v), and the culture media are subjected to shaking culture for 24 hours at the temperature of 28 ℃ under the condition of 180r/min to check whether the culture media are turbid.

1.2.3 NaCl tolerance observation

The overnight cultured JLlg08PX bacterial liquid is inoculated with 1% (v/v) of TSB culture media with different NaCl contents, namely 2%, 4%, 6%, 8% and 10% (w/v), and the culture media are cultured for 24 hours under the condition of 180r/min at 28 ℃ in a shaking way and are observed whether the culture media are turbid or not.

1.3 JLlg08PX biochemical analysis

JLlg08PX is inoculated in TSA medium and cultured at 28 ℃. Preparation of bacterial suspension by taking out colonies using sterile inoculating loop according to instruction, and standing

2 gram positive identification card (GP), and 43 biochemical tests including carbon source utilization, enzyme activity and drug resistance are carried out on JLlg08PX by using a VITEK 2COMPACT full-automatic microorganism identifier.

1.4 acid resistance test

Overnight cultured JLlg08PX bacteriumThe solution was centrifuged at 12000r/min and resuspended in PBS, the bacterial concentration calculated using the formula y= 0.4903ln (x) +9.3062 and adjusted to 2×10 ⁹ CFU/mL was ready for use. The adjusted bacterial liquid is inoculated to TSB culture with pH value of 4 according to inoculum size of 1% (v/v) and is cultured based on 180r/min shaking at 28 ℃,0, 1, 2 and 3 hours of culture liquid is taken, samples are diluted in a gradient mode and then coated on a TSA plate to be cultured for 24 hours at 28 ℃, the count is carried out, the bacterial quantity is compared with the bacterial quantity of 0 hour, and the survival rate after acid treatment is calculated.

1.5 bile salt resistance test

The concentration was set at 2X 10 ⁹ CFU/mL of JLlg08PX bacterial liquid is inoculated into TSB culture medium containing 0.2 percent, 0.3 percent and 0.4 percent of ox gall salt and no ox gall salt according to the inoculation amount of 10 percent (v/v), and is subjected to shaking culture at the temperature of 28 ℃ for 180r/min, and absorbance at 600nm is measured by using an enzyme-labeling instrument in 200uL to 96-well plates of culture liquid of 1, 2, 3 and 4 hours.

1.6 CFS (computational fluid dynamics) antibacterial effect determination for aquatic pathogenic bacteria

The overnight cultured JLlg08PX bacterial liquid is inoculated with MRS for culturing for 24 hours based on 180r/min at 37 ℃, bacterial liquid is taken out for 12000r/min, centrifugation is carried out for 1min, supernatant is filtered by a 0.22 mu m sterilizing needle filter, and filtrate is Cell-Free Supernatant (CFS) for standby at 4 ℃.

Culturing test strain Aeromonas hydrophila JLAH08PH strain, aeromonas veronii, yersinia ruckeri, vibrio parahaemolyticus, aerosol aesculus, citrobacter freundii, streptococcus agalactiae and Streptococcus ragmitis with TSB or BHI culture medium at 28deg.C 180r/min for 24 hr, and diluting with PBS to 10 ⁶ ～10 ⁷ CFU/mL, 100 mu L of each strain to be tested is coated on TSB or BHI culture medium, a sterilized 6mm puncher is used for punching on the culture medium, 2% agar is used for bottom filling at the bottom of the hole, 100 mu L of CFS is added into each hole, and an equivalent MRS liquid culture medium is used as a blank control. After culturing for 12 hours at 28 ℃, observing the formation condition of the inhibition zone, and measuring the diameter of the inhibition zone.

1.7 measurement of the influence of JLlg08PX on the growth of pathogenic bacteria

Culturing overnight JLlg08PX inoculated TSB at 37deg.C 180r/min for 24 hr, centrifuging 12000r/min for 2min, filtering supernatant with 0.22 μm sterilizing needle filter, and standing at 4deg.C.

The indicator strains Aeromonas hydrophila JLAH08PH, aeromonas veronii, yersinia ruckeri and Streptococcus agalactiae were cultured at 28℃180r/min for 24h respectively using TSB or BHI medium. Under aseptic condition, collecting bacterial liquid, centrifuging at high speed, re-suspending with PBS for 2 times, and diluting bacterial liquid to 1.0X10 ³ CFU/mL was used as an indicator strain for backup. mu.L of the indicated strain broth was added to TSB/BHI+CFS (9 mL TSB/BHI+1mL CFS) medium, and the same amount of the indicated strain broth was added to TSB/BHI (9 mL TSB/BHI+1mL MRS) medium as a control, and incubated at 28℃for 24 hours at 180r/min, and OD600 values were measured by an enzyme-labeled instrument.

1.8 data analysis

Data were statistically analyzed using SPSS 27.0 software and tested for significance using T-test, P <0.05 representing data with significance, data expressed as mean ± standard error.

2. Experimental results

2.1 morphological observations

2.1.1 colony morphology

JLlg08PX is activated and streaked on BHIA and MRSA culture medium, and is subjected to static culture for 24 hours at 28 ℃, and colony morphology is shown in figure 2.JLlg08PX grows as a translucent, well-defined, round-raised, moist, milky white colony on BHIA medium (FIG. 2A). The colony morphology of JLlg08PX grown in MRSA medium was consistent with BHIA medium (fig. 2B).

2.1.2 bacterial morphology

The result of the scanning electron microscope of the strain JLlg08PX is shown in FIG. 3, and the bacterial morphology is consistent with that observed by gram staining, and the bacterial cells of the strain JLlg08PX are oval and distributed in a chain shape. 10 cells were selected and measured for length and width using imageJ 1.51k (FIG. 3B), the cell length and width were between 0.80 and 1.27 μm and 0.57 and 0.67 μm, respectively, and the average cell length and width were 1.00.+ -. 0.13 μm and 0.63.+ -. 0.03 μm, respectively.

2.2 Observations of JLlg08PX tolerance to temperature, pH and salinity

The culture results of the strain JLlg08PX under different conditions are shown in Table 3. JLlg08PX does not grow at 4deg.C, grows at 18, 28, 40 and 45deg.C, wherein the growth rate is slower than 40deg.C at 45deg.C, and is flocculent. JLlg08PX was cultured in TSB medium at different pH at 28deg.C 180r/min, and the results showed that it did not grow at

pH

3 and 4 and grown at

pH

7, 10 and 11. Meanwhile, JLlg08PX grows in TSB medium containing 2%, 4% and 6% NaCl (w/v) and does not grow in TSB medium containing 8% and 10% NaCl (w/v) at 28℃180 r/min.

TABLE 3 Table 3

2.3 JLlg08PX biochemical analysis

Full-automatic microorganism identifier by using VITEK 2COMPACT

2 gram-Positive identification card (GP) the JLlg08PX was identified, including 43 biochemical tests for carbon source utilization, enzyme activity and resistance, the results are shown in Table 4.

TABLE 4 Table 4

2.4 acid resistance

The statistics of survival of jlg 08PX inoculated in TSB medium at pH 4 are shown in table 5. When JLlg08PX is inoculated for 1h, the survival rate is 113.66% +/-11.90%, when the JLlg08PX is inoculated for 2h, the survival rate is reduced to 61.86% +/-1.39%, and when the JLlg08PX is inoculated for 3h, the JLlg08PX is inoculated for 56.65% +/-11.10%. The JLlg08PX is shown to survive under the condition of pH 4 and has certain acid tolerance.

TABLE 5

2.5 bile salts resistance

The growth of jlg08 PX under different bile salts is shown in fig. 4. Jlg08 PX grew normally in TSB medium without bile salts. At 0.2% bile salts, the measured value of OD600 of JLlg08PX in the previous 3h is on the rise, with 3h significantly higher than 1h (P < 0.05); at 0.3% bile salts, the measured value of OD600 of JLlg08PX in the first 4 hours is on the rise, with 4 hours significantly higher than 1 hour (P < 0.05); under the condition of 0.4% bile salt, the measured value of OD600 of JLlg08PX in the previous 3h is in an ascending trend, and the measured value is obviously higher than 1h (P < 0.05). The JLlg08PX can grow in 0.2-0.4% of bile salt environment and has bile salt tolerance.

2.6 Effect of JLlg08PX on pathogenic growth

2.6.1 Antibacterial effect of CFS on aquatic pathogenic bacteria

The antibacterial effect of JLlg08PX on 8 aquatic pathogenic bacteria is shown in figure 5, and the diameter statistical result of the antibacterial zone is shown in table 6. After CFS treatment of the lactococcus necator JLlg08PX strain, obvious inhibition zones can be observed around culture medium holes of the test strains aeromonas hydrophila, aeromonas verrucosa and yersinia ruckeri, wherein the diameter of the inhibition zone of the yersinia ruckeri is maximum and is 13.87+/-0.29 mm, the diameter of the inhibition zone of the second aeromonas hydrophila is 11.93+/-0.09 mm, and the diameter of the inhibition zone of the aeromonas verruckeri is 9.57+/-0.85 mm. No obvious inhibition zone was observed around the holes of the test strains Vibrio parahaemolyticus, elizabeth, citrobacter freundii, streptococcus agalactiae and Streptococcus ragmitis medium. The honeybag lactococcus peter JLlg08PX strain has an inhibiting effect on the growth of aeromonas hydrophila, aeromonas verrucosa and yersinia ruckeri, but has no inhibiting effect on the growth of vibrio parahaemolyticus, illicina miehei, citrobacter freundii, streptococcus agalactiae and streptococcus ragus.

TABLE 6

2.6.2 Effect on growth of pathogenic bacteria in fish

The effect of the strain JLlg08PX on the growth of aquatic pathogens is shown in FIG. 6. After aeromonas hydrophila JLAH08PH inoculation of TSB medium for 24h, the average OD600 value was 1.18, and after CFS addition the average OD600 value was significantly reduced (P < 0.05); after aeromonas veronii is inoculated with TSB medium for 24 hours, the average OD600 value is 1.24, and the average OD600 value is obviously reduced (P < 0.05) after CFS is added; after 24h inoculation of TSB medium with yersinia ruckeri, the average OD600 value was 0.99, and after CFS addition the average OD600 value was significantly reduced (P < 0.05). After 24h inoculation of streptococcus agalactiae with BHI medium, the average OD600 value was 0.97, and there was no significant difference (P > 0.05) in average OD600 after CFS addition. The CFS of the lactococcus necator JLlg08PX strain has an inhibiting effect on the growth of aeromonas hydrophila, aeromonas veronii and yersinia ruckeri.

Experimental example 2, animal infection experiment

1. Experimental method

1.1 Pediococcus jingjingjingjingjingjingjingjingjingjingjingjingjingjingjingjing

144 Siberian sturgeons are randomly divided into 6 groups, the infection is carried out by three modes of intraperitoneal injection, soaking and gastric lavage respectively, and a PBS control group which is treated correspondingly is arranged, wherein each group is provided with 3 parallels, and 8 parallels are respectively provided with 8 fish. Intraperitoneal injection treatment group injected 0.1mL 1X 10 ⁹ CFU/mL of the honeybag lactococcus vaginalis JLlg08PX, and the control group is injected with 0.1mL of PBS; soaking treatment group at final concentration of 5.7X10 ⁷ After being soaked for 30min, the CFU/mL honeybag lactococcus jilg 08PX is transferred into a water body without bacteria liquid for feeding, and a control group is not subjected to bacteria liquid soaking treatment; the lavage treatment group was perfused with 0.1mL 1X 10 by using a lavage needle ⁹ CFU/mL lactococcus JLlg08PX, control group perfused with 0.1mL PBS. Symptoms and death were recorded every 12h after challenge, and each group of siberian sturgeons was anesthetized and sacrificed by MS-222 after 14d, the liver and middle kidney were bacteria isolated using TSA medium and morphological observations of the isolated bacteria were performed, and isolation of the challenge strain from at least one tissue was considered positive.

1.2 Coinfection test of M.nectar JLlg08PX and Aeromonas hydrophila

The 45 healthy hybrid sturgeons are randomly divided into A-E5 treatment groups, and 9 fish in each group. Group A is a control group, and 0.2mL of 0.01mol/L PBS solution is injected into the abdominal cavity; group B intraperitoneal injection of 0.2mL 2.0X10 ⁸ CFU/mL of honey bag Pediococcus jilg 08PX; group C intraperitoneal injection of 0.2mL 2.0X10 ⁸ CFU/mL water-vapor-hydrophilic sheetThe cytopenicillium JLAH08PH; group D intraperitoneal injection 0.2mL 5.0X10 ⁷ CFU/mL Aeromonas hydrophila JLAH08PH; group E was co-infected, and was intraperitoneally injected with 0.1mL of 2.0X10 ⁸ CFU/mL honeybag Pediococcus jilg 08PX and 0.1mL 2.0X10 ⁸ CFU/mL Aeromonas hydrophila JLAH08PH. After the challenge was completed, clinical symptoms were observed every 12 hours and the 7d cumulative mortality was counted. Bacteriological detection and tissue sampling of moribund fish. After 7d challenge, all experimental fish were sacrificed using MS-222 anesthesia and bacterial isolation and tissue sampling were performed.

1.3 histopathological observations

The moribund or fresh dead sturgeon in the A, B, C and E groups of 1.2 is subjected to anesthesia and sacrifice, and then the liver and the valve intestinal tissues are taken out and placed in normal saline for rapid cleaning and blood stain removal, and are placed in 10% neutral formalin for fixation for 48 hours. The procedure is described with reference to He Yang (He Yang. Pathological research of spleen of Nile-tilapia infection [ D ] Sichuan university of agriculture, 2017), tissue is trimmed, washed with running water, dehydrated, transparent, embedded, the tissue embedded block is sectioned with a paraffin microtome to a thickness of 4 μm, and the tissue section is H & E stained after drying. After the preparation of the sections, the sections are placed under an optical microscope to observe the tissue lesions.

1.4 bacterial load determination of aeromonas

36 healthy hybrid sturgeons were randomly divided into 4 groups of 9 fish. The control group was injected with 0.2mL of 0.01mol/L PBS solution; the remaining 3 groups were injected with 0.2mL of 1.0X10, respectively ⁸ CFU/mL Aeromonas hydrophila JLAH08PH, 0.1mL 2.0X10 ⁸ CFU/mL Aeromonas hydrophila JLAH08PH and 0.1mL 2.0X10 ⁷ CFU/mL honeybag Pediococcus jilg 08PX, 0.1mL 2.0X10 ⁸ CFU/mL Aeromonas hydrophila JLAH08PH and 0.1mL 2.0X10 ⁸ CFU/mL of the honey bag is the enterococcus faecalis JLlg08PX, and the injection mode is intraperitoneal injection. At 24h of challenge, all experimental fish were sacrificed using MS-222 anesthesia, and liver tissue of 3 fish per group was collected for detection of aeromonas load. After homogenization of liver tissue, the tissue was resuspended to 1g/mL using sterile PBS, 30. Mu.L of the tissue suspension was taken and template DNA extraction was performed using TIANamp Genomic DNA Kit. Reference to Khan et al [ Khan I U, loughborough A, edge T A. DNA-based real-time detection and quantification of aeromonads from fresh water beaches on Lake Ontario[J]The tissue load of Aeromonas was tested by the J Water Health,2009,7 (2): 312-323.

2. Experimental results

2.1 mortality and detection rate of lactococcus jilg 08PX of honeyed bag

The JLlg08PX strain is respectively used for infecting the Siberian sturgeon by three modes of intraperitoneal injection, soaking and gastric lavage, and the toxin attacking group and the contrast group of Siberian sturgeon have no death and clinical symptoms during the whole experiment period. Bacteria were not isolated from each control group, and after the stomach and immersion treatment, bacteria were not isolated from the liver and middle kidney of the Siberian sturgeon, and bacteria were isolated from the liver and middle kidney tissue of the 4-tail Siberian sturgeon in the intraperitoneal injection group, with a detection rate of 16.67% and were confirmed to be morphological by staining (Table 7).

TABLE 7

2.2 clinical symptoms and death Condition in infected fish

The control hybrid sturgeon had no clinical symptoms and death during the trial (fig. 7). After the honey bag and the galactococcus petasites group attack the poison, the anus is engorged but gradually disappears only for 2 days, and the spleen is enlarged and whitened in color and luster by anatomy, and no death occurs within 7 days (figure 7). At 1.0X10 ⁷ CFU/fish and 4.0X10 ⁷ After the aeromonas hydrophila of the CFU/fish attacks the toxin, the hybrid sturgeons have weakened vitality, unbalanced swimming and red and swollen anus and have mucous. Dissecting and showing ascites due to blood sample in abdominal cavity, round and enlarged edges of liver and spleen, bleeding spleen, intestinal congestion, and effusion in part of moribund stomach, wherein 4.0X10 ⁷ The death time of the aeromonas hydrophila virus challenge group of CFU/fish is earlier, the accumulated death rate is higher, the death rate is 55.56% in 1d, the accumulated death rate is 88.89% in 7d, and the accumulated death rate is 1.0x10 ⁷ The total death rate of the air-hydrophila virus challenge group of CFU/fish after virus challenge is 0% in 3d, death begins to occur in 4d, and the total death rate is 77.78% in 7d (FIG. 7). Honey bag type lactococcus peter and water-vapor-permeable sheetAfter co-infection with the cytobacteria, the hybrid sturgeons had slow swimming, reduced vitality and red and swollen anus, and the dissections showed a rounded swelling of the liver and spleen, congestion in the intestine, a cumulative mortality rate of 22.22 d, no mortality occurring in 2-4 d, and a cumulative mortality rate of 33.33 d (fig. 7).

2.3 pathological changes in tissue

Liver: the hepatic chordae of the control group are closely arranged, and the blood vessel and blood sinus structure are clear; the hematoxylin staining of the nuclei of hepatocytes was well defined and the cytoplasmic eosinophil staining was reticulocyte-like (FIGS. 8A-B). The honeybag lactococcus peter group is stained with strong eosinophilia, the tissue structure is still present but the intercellular boundary is fuzzy, and a large amount of ferrioxazine is accumulated; the nuclei of hepatocytes were well defined and the cytoplasm was homogenously red stained (FIGS. 8C-D). Aeromonas hydrophila tissue structure collapses, a small amount of cell outline remains, and a large amount of liver cell nuclei chromatin is concentrated, deeply stained, disintegrated and dissolved, with inflammatory cell infiltration and liver blood Dou Yuxie (fig. 8E-F); the co-infected group had a clear tissue structure, a concentrated and disappeared nucleus of the liver cells, a large vacuole-like cytoplasm, and red-stained drip-like particles in the blood vessel and blood sinus (fig. 8G-H).

Valve sausage: the control group had clear valve intestine structure and compact cell arrangement (fig. 9A); the structure of the honeybag lactococcus peter group is clear, and has no obvious difference from the control group (figure 9B); aeromonas hydrophila group valve intestinal serosa layer expansion is accompanied by inflammatory cell infiltration and vasodilation, and the muscular layer and submucosal structure is loose and edematous, and the mucosal layer is severely necrotic and shed (fig. 9C); the co-infected group of valve intestinal serosa and serosa lower layer cells swelled and thickened, inflammatory cells infiltrated, and there were small numbers of exfoliated epithelial cells in the intestinal lumen (fig. 9D).

2.4 aeromonas load variation

After single challenge with aeromonas hydrophila, the average aeromonas loading in liver was 103.23 fold higher than that of PBS control group (fig. 2-7); at the same time of same dose of aeromonas hydrophila infection, respectively injecting 2.0X10 ⁶ CFU/fish and 2.0X10 ⁷ The average value of aeromonas load in the liver was reduced by 30.28-fold and 101.36-fold respectively for CFU/fish honeybag lactococcus jlg 08PX (fig. 10).

In conclusion, the invention identifies the lactococcus vaginalis JLlg08PX, and the strain is acid-resistant and cholate-resistant, and has the ability of surviving in the digestive tract in a severe environment. The honey bag lactococcus jilg 08PX has no pathogenicity, and the co-infection can relieve the tissue pathological damage of sturgeon liver and intestinal tract caused by aeromonas hydrophila, reduce the morbidity and mortality, and simultaneously reduce the loading capacity of aeromonas hydrophila in hybrid sturgeon liver after the aeromonas hydrophila infection; the cell-free supernatant has an inhibition effect on the growth of fish pathogenic bacteria aeromonas hydrophila, yersinia ruckeri and aeromonas verrucosa, and has potential application value in preparing medicaments for preventing and treating fish septicemia and red mouth disease.

Claims

1. A honeyed-bag lactococcus peter is characterized by being preserved in China Center for Type Culture Collection (CCTCC), and the honeyed-bag lactococcus peter with the preservation number of CCTCC M2022927 is preservedLactococcus petauri)JLlg08PX。

2. A culture of the honeyed-bag lactococcus pennii, which is the product obtained by culturing the honeyed-bag lactococcus pennii according to claim 1 in a culture medium.

3. The culture of claim 2, wherein the culture medium is TBS medium or MRS medium; and/or the conditions of the culture are: culturing at 37 ℃ for 24 hours.

4. Use of the honeybag of lactococcus vaginalis and/or its culture in the manufacture of a medicament against pathogenic bacteria of aquatic animals as defined in claim 1; the pathogenic bacteria of aquatic animals comprise aeromonas hydrophilaAeromonas hydrophil) And/or Aeromonas Vickers [ ]Aeromonas veronii) And/or Yersinia ruckeriYersinia ruckeri)。

5. The use according to claim 4, wherein the culture of lactococcus honeypack is the culture of claim 2 or 3.

6. The use of claim 4, wherein the medicament is a medicament for controlling a disease caused by pathogenic bacterial infection in the aquatic animal.

7. The use according to any one of claims 4 to 6, wherein the medicament is a medicament for the control of sepsis in aquatic animals.

8. The use according to claim 7, wherein the medicament is a medicament for controlling sepsis in aquatic animals caused by aeromonas hydrophila infection.

9. The use according to claim 7, wherein the medicament is a medicament for reducing aeromonas hydrophila load in the liver of an aquatic animal and inhibiting aeromonas hydrophila growth.

10. The use of claim 7, wherein the aquatic animal sepsis is fish sepsis.

11. The use according to claim 10, wherein the fish sepsis is sturgeon sepsis.

12. The use according to claim 10, wherein the sturgeons comprise Acipenser sinensis, siberian sturgeon and/or hybrid sturgeon.