CN112029696B - Edwardsiella piscicola derived from turbot and application thereof - Google Patents

Edwardsiella piscicola derived from turbot and application thereof Download PDF

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CN112029696B
CN112029696B CN202011220240.4A CN202011220240A CN112029696B CN 112029696 B CN112029696 B CN 112029696B CN 202011220240 A CN202011220240 A CN 202011220240A CN 112029696 B CN112029696 B CN 112029696B
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edwardsiella
turbot
ichthyophthirius
derived
piscicola
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CN112029696A (en
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黄华
王鹤
张建柏
王力勇
徐晓莹
张岚
王晓飞
史文凯
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Yantai Institute Of Marine Economy Yantai Institute Of Marine Science And Technology Yantai Fishery Technology Promotion Station Yantai Aquatic Animal Disease Prevention And Control Center
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Yantai Institute Of Marine Economy Yantai Institute Of Marine Science And Technology Yantai Fishery Technology Promotion Station Yantai Aquatic Animal Disease Prevention And Control Center
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine

Abstract

The invention provides an Edwardsiella piscicola derived from turbot and application thereof; the Edwardsiella ichthyophthirius derived from the turbot is an Edwardsiella ichthyophthirius H4-S18 strain, the gene sequences of the Edwardsiella ichthyophthirius are shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, and the Edwardsiella ichthyophthirius H4-S18 strain is preserved with the preservation number of CCTCC NO: M2020450. The invention also relates to application of the Edwardsiella piscicola derived from turbot in preparation of vaccines. The Edwardsiella ichthyophthirius is obtained by screening diseased turbots, is a turbot source Edwardsiella ichthyophthirius carrying a drug-resistant gene screened for the first time in China, and is used for preparing a vaccine by using the turbot source Edwardsiella ichthyophthirius carrying the drug-resistant gene for the first time, so that the immune protection rate of the turbot on the drug-resistant Edwardsiella ichthyophthirius is effectively improved.

Description

Edwardsiella piscicola derived from turbot and application thereof
Technical Field
The invention belongs to the field of pathogenic microorganism screening; in particular to Edwardsiella piscicola derived from turbot and application thereof.
Background
Turbot is rich in nutrition and high in economic value, is cold water benthic seawater fish, is originally produced in Europe, is introduced into China in 1992, and becomes important economically cultured fish in northern areas of China. But with the scaleThe development is interrupted, the culture intensification degree is improved, turbot diseases frequently occur during the culture period, the economic loss caused by the turbot diseases is huge, and the occurrence frequency of bacterial diseases is the highest and the loss is serious in the reported turbot diseases. The infection rate and the fatality rate of the turbot to the Edwardsiella are both high, and the Edwardsiella is a main disease damaging the healthy culture of the turbot. Edwardsiella piscioides of Edwardsiella is pathogenic bacteria of turbot, and Edwardsiella tarda (Edwardsiella tarda) in 2013Edwardsiella tarda) The strain is separated out and classified as a new species of Edwardsiella and can cause diseases of the turbot such as ascites disease, reddish body disease, white feces disease and the like.
The frequent occurrence of the Edwardsiella turbot diseases, the gradual appearance of drug-resistant strains along with the time lapse and environmental change and the long-term use of antibacterial drugs, and the strengthening of the research on the drug-resistant strains is particularly important.
The use of the vaccine can improve the specific immunity level of the fish, so that the fish body can generate immunity for resisting infection of specific pathogenic microorganisms, and the vaccine has no pollution and residue after injection, and is the main research direction for preventing and controlling fish diseases. The inactivated vaccine is prepared aiming at the drug-resistant bacterial strain, so that the immunity level of the fish to the drug-resistant bacterial strain can be effectively improved, the drug administration can be effectively reduced, and the vicious circle problems of drug-resistant bacterial strain formation, drug residue and the like in drug prevention and treatment are solved. Therefore, the method has important significance for developing vaccine research and development aiming at drug-resistant strains of turbots.
Disclosure of Invention
The invention aims to provide an Edwardsiella piscicola derived from turbot and application thereof. The invention is realized by the following technical scheme:
the invention relates to Edwardsiella ichthyophthirius derived from turbot, wherein the Edwardsiella ichthyophthirius derived from turbot is an Edwardsiella ichthyophthirius H4-S18 strain, the gene sequence of the Edwardsiella ichthyophthirius is shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, (Edwardsiella ichthyophthirius H4-S18) provided by the inventionEdwardsiella piscicidaH4-S18) is preserved in China Center for Type Culture Collection (CCTCC) at 26.8.2020, with the preservation address of Wuhan, China and the preservation number of CCTCC NO: M2020450.
The invention also relates to application of the Edwardsiella piscioides from the turbot in preparing a vaccine, wherein the vaccine is an inactivated vaccine.
The invention has the following advantages:
(1) the Edwardsiella ichthyophthirius is obtained by screening diseased turbots, is a turbot source Edwardsiella ichthyophthirius carrying a drug-resistant gene screened for the first time in China, and is used for preparing a vaccine by using the turbot source Edwardsiella ichthyophthirius carrying the drug-resistant gene for the first time, so that the immune protection rate of the turbot on the drug-resistant Edwardsiella ichthyophthirius is effectively improved.
(2) The Edwardsiella piscicola provided by the invention can be used for preparing inactivated vaccines.
Drawings
FIG. 1 is a diagram showing the effect of the PCR detection result of the drug-resistant gene of Edwardsiella piscicola H4-S18 strain;
FIG. 2 is a diagram showing the effect of the virulence gene PCR detection of Edwardsiella piscicida H4-S18 strain.
Detailed Description
The present invention will be described in detail with reference to specific examples. It should be noted that the following examples are only illustrative of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
The embodiment relates to separation and identification of Edwardsiella ichthyophthirius, wherein the Edwardsiella ichthyophthirius derived from the turbot is an Edwardsiella ichthyophthirius H4-S18 strain, and the gene sequence of the Edwardsiella ichthyophthirius is shown as SEQ ID No. 1.
The method comprises the following specific steps:
the diseased turbot sample is collected from a turbot farm on a Shandong tobacco bench, the individual mass is between 205g and 518 g, the average weight is 436 +/-5 g, and the average body length is 29 +/-2 cm. The diseased turbot is characterized by impaired feeding ability, slow movement, abdominal distension of fish, red and swollen anus, redness and redness of the snout, fin and basal part, and the like. The abdominal cavity has yellowish ascites, intestinal enlargement, yellowish liquid in the thin intestinal wall, and hepatic congestion.
Taking an individual dying with typical symptoms, carrying out sterile dissection on internal organs, taking tissues such as liver, kidney, spleen, heart and the like, washing with sterile water, grinding, streaking on a BHI agar plate, carrying out inverted culture at 30 ℃ for 24-48 h, and selecting dominant bacterial colonies with consistent forms for purified culture until a pure culture is obtained.
Performing scale-up culture on the pure culture obtained by separation on BHI plate, eluting bacterial colony with 0.9% sterile physiological saline to obtain bacterial suspension, and determining the strain concentration to be 7.8 × 10 by turbidimetry9CFU/mL, intraperitoneal injection.
Selecting healthy turbot with similar specification to the diseased turbot as experimental fish for artificial infection. After one week of temporary rearing of experimental fish, the experimental fish were randomly divided into 4 groups, 3 of which were injected with 0.1mL of bacterial suspension, and 1 was injected with sterile physiological saline as a control. The water temperature of the culture is 18 +/-1 ℃, the air is filled all day long, the water is changed by 30 percent every day, and the death number and the disease symptoms are observed and recorded every day. Pathogenic bacteria are separated again from the diseased individuals.
The effect of the detection of the drug resistance gene of the isolated strain is shown in figure 1: the TEM is carried by the screening,tet(A)、mcr-1、ant(3') -I, and 4 drug resistance genes H4-S18.
Toxicity analysis was performed on the H4-S18 strain. The effect is shown in figure 2, strains which can cause 90 percent of death of experimental turbots are separated, and the death individuals have clinical symptoms consistent with the diseased turbots. The separated strain is proved to be a pathogen causing turbot red body and ascites, and the strain can be separated again in an individual infected with diseases. 16S rRNA detection and housekeeping generpoB (the gene sequence is shown in SEQ ID No. 2) andgyrb (the gene sequence is shown in SEQ ID No. 3), BLAST comparison analysis and specific primer PCR detection, and the separated strain is identified to be Edwardsiella piscicola.
Detecting the bacterial carryoverfimA、fimB、fimC、fimD、sodB、citC、esrB、godB、mukF、katB, 10 virulence genes. Is obviously different from the reported Edwardsiella piscicola.
Example 2
The embodiment relates to the preparation of an inactivated vaccine by using Edwardsiella piscicola, wherein the Edwardsiella piscicola derived from turbot is an Edwardsiella piscicola H4-S18 strain, and the gene sequence of the Edwardsiella piscicola is shown as SEQ ID No. 1.
The method comprises the following specific steps:
inoculating the Edwardsiella piscicola H4-S18 strain to a nutrient agar medium (NA) plate, and culturing at 37 ℃ for 24-48H for resuscitation; selecting the above pathogenic bacteria, culturing in NB culture medium at 37 deg.C under shaking for 24h, collecting culture solution, centrifuging at 4 deg.C under 5000rpm for 10min, washing the centrifuged deposited bacteria with PBS buffer solution with pH =7.2 for 3 times, and preparing into 10% concentration with sterile physiological saline (containing 2% NaCl)9cfu/mL of a stock solution of the bacterial suspension; adding 37% formalin solution until the final concentration of formaldehyde is 0.2%, inactivating at 37 deg.C and 140r/min, respectively taking the inactivated bacteria solution for 12h and 24h, detecting the inactivation condition, and observing the inactivation condition. Centrifuging the inactivated bacteria solution at 4 deg.C and 5000rpm for 10min, washing the centrifuged deposited bacteria with PBS buffer solution with pH =7.2 for 3 times, and making into 10% concentration with sterile physiological saline (containing 2% NaCl)9cfu/mL of inactivated bacteria suspension; mixing the inactivated bacteria suspension of Edwardsiella piscicida H4-S18, cecropin (North China pharmaceutical division Co., Ltd.), and Freund' S incomplete adjuvant (SIGMA Co., Ltd.) at a ratio of 2:1:3 to obtain inactivated vaccine;
example 3
The embodiment relates to the effect detection of the vaccine, and the Edwardsiella ichthyophthirius derived from the turbot is the Edwardsiella ichthyophthirius H4-S18 strain.
The gene sequence is shown in SEQ ID No. 1.
According to the step of preparing the vaccine in the step 2, the vaccine is prepared by taking the Edwardsiella piscicida (ET 883) as an antigen.
Adjusting the concentration of 2 inactivated vaccines to OD6000.1 +/-0.01, injecting 0.1mL of immune turbot into the abdominal cavity, and injecting sterile normal saline with the same dosage into a control group. 20 animals were immunized per group, and 3 replicates, 1 control, were set. The water temperature of the cultivation is 18 +/-1 ℃, air is filled all day long, and the water is changed by 30% every day; 30 days after immunization, the Edwardsiella piscicola is used for counteracting the toxinThe injection concentration of each tail is 8.9 multiplied by 10100.1mL of CFU/mL bacterial liquid; the death number of each experimental group is observed and recorded every day, and the immune protection rate of the 2 inactivated vaccines to the Edwardsiella piscicola is calculated.
The result shows that the fish bodies injected with the normal saline begin to die in 6 days after the toxin attack, and die peak appears in 8-10 days, and the death rate reaches 93.5% in 12 days after the toxin attack; the virus-attacked fish body has typical symptoms of redness, redness and swelling of fin rays and the like, and the bacteria can be isolated from infected dead fish bodies again. While the mortality rate of the 2 vaccine immunization groups is significantly lower than that of the control group, the mortality rate of the inactivated vaccine prepared by using the H4-S18 strain in the immunization group for the challenge H4-S18 strain in example 2 is significantly lower than that of the vaccine prepared by the ET883 strain in Table 1, and the table 1 is the immune protection test result table of the vaccines prepared by 2 strains.
TABLE 1
Figure 288342DEST_PATH_IMAGE002
Note: indicates survival/challenge
The results show that the strain obtained by screening has variation compared with the reported Edwardsiella multocida, and the inactivated vaccine prepared based on the strain has higher immune protection rate and can provide immune protection to a certain degree for the turbot to kill the diseases caused by the Edwardsiella multocida.
The Edwardsiella ichthyophthirius is obtained by screening diseased turbots, is a turbot source Edwardsiella ichthyophthirius carrying a drug-resistant gene screened for the first time in China, and is used for preparing a vaccine by using the turbot source Edwardsiella ichthyophthirius carrying the drug-resistant gene for the first time, so that the immune protection rate of the turbot on the drug-resistant Edwardsiella ichthyophthirius is effectively improved.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Sequence listing
<110> ocean economic research institute of cigarette Tai City (ocean science and technology institute of cigarette Tai City, fishery technology promotion station of cigarette Tai City, aquatic animal epidemic disease prevention and control center of cigarette Tai City)
<120> Edwardsiella ichthyophthirius derived from turbot and application thereof
<141> 2020-08-31
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1163
<212> DNA
<213> Edwardsiella piscicola (H4-S18Edwardsiella pisciida H4-S18)
<400> 1
cgcaggccgg gcaggcaacc agcaagcgag cggagcaggg agaaagcgcc ccgcgacgag 60
cggcggacgg ggagaagcgg ggacgccgag gagggggaaa cacggaaacg gagcaaaccg 120
caaacgcgca agaccaaagg ggggacccgg gcccagccac agagaaccca gagggaagca 180
gaggggggaa ggccaccagg cgacgaccca gcggcgagag gagaccagcc acacggaacg 240
agacacggcc agacccacgg gaggcagcag ggggaaagca caagggcgca agccgagcag 300
ccagccgcgg agaagaaggc ccggggaaag accagaggga ggaaggggaa cgaaagcgcc 360
acaagacgac cacagaagaa gcaccggcaa cccggccagc agccgcggaa acggaggggc 420
aagcgaacgg aaacgggcga aagcgcacgc aggcgggaag ggaggaaacc ccgggcaacc 480
gggaacgcac caagacggca agcagagccg agagggagga gaaccaggga gcgggaaagc 540
gagagacgga ggaaaccggg gcgaaggcgg ccccggacga agacgacgcc agggcgaaag 600
cgggggagca aacaggaaga acccggagcc acgcgaaacg agcgaggagg ggcccgaggc 660
gggcccgaag caacgcgaaa cgaccgccgg ggagacggcc gcaaggaaaa ccaaagaaga 720
cgggggcccg cacaagcggg gagcagggaa cgagcaacgc gaagaaccac caccgacacc 780
agcgaaccga gagaacggga ggcccgggaa cgcgagacag ggcgcaggcg cgcagccggg 840
gaaaggggaa gcccgcaacg agcgcaaccc accggccagc ggcggccggg aaccaaagga 900
gacgccagga aaacggagga aggggggaga cgcaagcaca ggcccacgag agggcacaca 960
cggcacaagg cgaacaaaga gaagcgaacc gcgagagcaa gcggacccaa aagacgcgag 1020
ccggaggagc gcaaccgacc cagaagcgga acgcagaacg ggacagaagc cacgggaaac 1080
gcccgggccg acacaccgcc cgcacaccag ggagggggca aaagaagagg agcaacccgg 1140
gagggccgca cccaccgagc cgg 1163
<210> 2
<211> 1202
<212> DNA
<213> Edwardsiella piscicola (H4-S18Edwardsiella pisciida H4-S18)
<400> 2
ccgggggtgt tgacgatact cctataaggt ctccggcggc ctgcacggcg tgggggtttc 60
cgtggtgaac gccctgtctg aaaagctgga gctggtgatc cgtcgcgacg gccatgtcca 120
tgagcaaatc taccgacacg gtgtgccggc ggcgccgctg aaagtggttg gcgacaccga 180
gcagaccggt actcgcgtgc gtttctggcc gagcatggag accttcagca atgtggtcga 240
attccagtat gacattctgg ctaagcgcct gcgcgaactc tctttcctga actccggcgt 300
ctccatccgc ctgcgcgata aacgcaacga tcgcgaagat catttccact atgagggtgg 360
gattaaggcg tttgtcgagt atctgaacaa gaacaaaacg ccaatccatc cgaacgtgtt 420
ttacttttcg accatgaagg atgatatcgg cgtagaagtg gcgctgcagt ggaacgatgg 480
cttccaggag aatatctact gctttaccaa taacatcccg cagcgcgacg gcgggaccca 540
tttggccggt ttccgtgcgg cgatgacccg tacgctgaac agctacatgg aaaacgaagg 600
ctataccaag aagagcaaaa tctccgctac cggtgacgac gcgcgtgagg ggctgattgc 660
cgtggtgtcc gtgaaggtgc cggaccccaa gttctcctct cagaccaagg ataagctggt 720
ctcttcggag gtgaaatccg ccgttgagtc gctgatgaac gagcgtctgg ctgaatacct 780
gctggaaaac cccagtgatg ccaaaatcgt cgtcggcaaa atcatcgacg cggcccgcgc 840
acgtgaagcc gcgcgtaagg cccgtgagat gacccgccgc aagggtgcgc tggatctggc 900
cggcctgccg ggcaagctgg cggactgcca ggagcgcgac cccgcccact cagagcttta 960
cttagtggaa ggggactccg cgggcggctc tgccaagcag ggacgtaacc gtaagaatca 1020
ggccatcctg ccgctgaagg ggaaaatcct caacgtcgag aaggcgcgct tcgacaagat 1080
gctctcctcg caggaggtcg cgaccctgat caccgcgctg ggctgcggca ttggccggga 1140
tgagtacaac ccggataagc tgcgctacca cagcatatca tcatgaccga tgccatagcc 1200
cc 1202
<210> 3
<211> 950
<212> DNA
<213> Edwardsiella piscicola (H4-S18Edwardsiella pisciida H4-S18)
<400> 3
caggtcgcgc attttgatcg cgtcaactct tcggtcttag tttgcgcttt cagaaacttc 60
agtaagtctt tcacggatta gctcccgtcg gagtgagaac tcttaaggcg cccgccgtaa 120
cgggcgccct gtgatcggta cggattcgag tgcttactcg tcttccagct cgatgttgat 180
gcccagcgag cggatttctt tcaacagtac gttgaaggat tccggcatgc ctggttccat 240
ctgatggctg ccatccacga tgttcttata catcttggta cggccgttca cgtcatcaga 300
cttaacggtg agcatttcct gcagggtgta ggcggcgccg taggcttcca gcgcccacac 360
ttccatctcc ccgaagcgct gaccaccgaa ctgcgcctta ccacccagcg gctgctgggt 420
aaccaggctg taggagccgg tggaacgcgc atgcatcttg tcgtcgacca agtggttcag 480
cttcagcatg tacatgtaac ccacggttac ctgacgctcg aaacgctcac cggtacgacc 540
atcatacagg gtgatctgac cggaggtcgg gataccaccc agctccagca ggcccttgat 600
ttcgcgctct ttagcaccgt cgaacaccgg cgttgcaacc ggcagacctt tcttcaggtt 660
ttcggccaga cgcaggactt cttcgtcggt gaaggtgttc agatccacct gctgacgggt 720
gtgatcaccc agatcgtagg ctttctggat aaactcacgc agcttggcca cttcctgctg 780
ctgcttaagc atggcgttga tcttatcgcc gatacctttc gccgccatac ccagatgggt 840
ttccaggatc tgaccaatgt tcatacgcga cggtacgccc agcgggttca gtacgatgtc 900
gaccggagtc ccgttctcat cgtaaggcat atctcgatcg ggtgatctaa 950

Claims (3)

1. The Edwardsiella ichthyophthirius derived from turbot is an Edwardsiella ichthyophthirius H4-S18 strain which is classified and named as Edwardsiella ichthyophthirius H4-S18 (Edwardsiella ichthyophthirius H4-S18)Edwardsiella piscicida H4-S18) and is preserved in China center for type culture Collection (CCTCC NO: M2020450) at 26 months 8 and 2020.
2. The use of the Edwardsiella ichthyophthirius derived from claim 1 in the preparation of a vaccine against Edwardsiella ichthyophthirius.
3. The use of the Edwardsiella ichthyophthirius derived from turbot in the preparation of the Edwardsiella ichthyophthirius vaccine according to claim 2, wherein the vaccine is an inactivated vaccine.
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CN114807057A (en) * 2022-04-01 2022-07-29 中国科学院大学 Edwardsiella piscicida phage EPP-1 and application thereof
CN114736877B (en) * 2022-05-20 2023-07-07 青岛诺安百特生物技术有限公司 Edwardsiella fish-killing phage, phage composition and application thereof
CN117305191B (en) * 2023-11-30 2024-03-15 烟台市海洋经济研究院(烟台市渔业技术推广站、烟台市海洋捕捞增殖管理站) Drug-resistant Edwardsiella tarda for killing turbot

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CN110548021A (en) * 2018-05-31 2019-12-10 华东理工大学 Application of long-chain unsaturated fatty acid in preparation of composition for preventing Edwardsiella

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CN115216453A (en) * 2021-12-31 2022-10-21 华东理工大学 Edwardsiella specific phage, composition thereof and application thereof
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