CN111297884B - Application of adenosine in prevention and treatment of bee virus infection - Google Patents
Application of adenosine in prevention and treatment of bee virus infection Download PDFInfo
- Publication number
- CN111297884B CN111297884B CN202010255841.2A CN202010255841A CN111297884B CN 111297884 B CN111297884 B CN 111297884B CN 202010255841 A CN202010255841 A CN 202010255841A CN 111297884 B CN111297884 B CN 111297884B
- Authority
- CN
- China
- Prior art keywords
- bee
- adenosine
- larvae
- group
- antibacterial peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/153—Nucleic acids; Hydrolysis products or derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/90—Feeding-stuffs specially adapted for particular animals for insects, e.g. bees or silkworms
-
- 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
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
Abstract
The invention relates to the technical field of bee breeding, in particular to application of adenosine in prevention and treatment of bee virus infection. The invention discovers that adenosine can obviously improve the survival rate of Chinese bee larvae infected with Chinese sacbrood virus (CSBV) and reduce the death rate of the Chinese bee larvae. Adenosine can inhibit CSBV proliferation in bee body, and reduce virus copy number; meanwhile, the expression of endogenous antibacterial peptide can be induced, and the innate immunity defense capacity of bees is improved. Adenosine can be used for preparing medicines or feeds for preventing and treating CSBV infection, has important significance for preventing and treating Chinese bee sacbrood, and has good market application value.
Description
Technical Field
The invention relates to the technical field of bee breeding, in particular to application of adenosine in prevention and treatment of bee virus infection.
Background
Bees are important pollination insects in nature, about one third of crops in the world need bee pollination to a certain extent, and some fruits, vegetables and the like completely depend on bee pollination. Bee pollination can significantly improve the yield and quality of crops, thus having important economic value in the aspect of agricultural production. In addition, the bee can also produce various peak products, such as honey, royal jelly, propolis, bee pollen and the like. The quality of bee products is closely related to the health state of bees, potential safety hazards exist in the bee products produced by susceptible bees, and medicines for treating bee diseases can also be remained in the bee products to further cause the safety problems of the bee products.
The Chinese honeybee (Apis cerana) has the advantages of strong anti-mite capability, low temperature resistance and good collection of sporadic honey powder source, plays an important role in the bee-keeping industry and plays an extremely important role in maintaining the balance of a natural ecosystem. However, apis cerana are often harmed by a variety of pathogens, of which apis cerana are most seriously harmed by middle bee sacvirus (CSBV). CSBV mainly infects 1 ~ 3 day old larva, causes the larva in the bee colony to die in a large amount, even when luck to survive can't pupate, leads to the worker bee quantity of new room not enough, and then leads to the well bee colony trend to weaken, and bee colony resistance descends, is more easily invaded by other virus to show the phenomenon of many virus coinfects. CSBV can be transmitted within bee colonies by means of food mutual feeding, faecal contamination, mating behaviour and reproduction of gametes. The drone can act as a viral vector to transmit the virus to offspring through the reproductive gametes. The non-group-boundary property of the male bees can directly determine the genetic diversity of the bee colony, so that the disease resistance of the bee colony can be greatly influenced. At present, the Chinese bee sacbrood disease is mainly prevented and controlled by comprehensive prevention and control measures of changing the prince-ova, strictly disinfecting, strengthening management, preventing secondary infection by using antiviral drugs and antibiotics, but the prevention and control effect is not ideal, the harm and the spread of the virus are difficult to be effectively controlled by the common antiviral drugs, and drug residues in bee products can be caused.
Endogenous antimicrobial peptides play a key role in immune regulation and pathogen defense as an important component of innate immunity. Endogenous antimicrobial peptides have immunoregulatory activity, participate in regulating innate and adaptive immune responses, are important mediators for the body to defend against foreign substance invasion, and are also immunocompetent molecules generated by the body to adapt to the environment.
When bees are infected by microorganisms or other exogenous substances, hemolymph produces a certain amount of antibacterial peptides including hymenoptera antibacterial peptide (hymenoptera antibacterial peptide), bee Defensin (Defensin), bee antibacterial peptide (Apidaecin) and bee moth antibacterial peptide (abeecin). These peptides are synthesized from fat bodies and secreted into haemolymph, which is a rapid and effective defense mechanism and can be used to rapidly kill or eliminate exogenous microorganisms. The defense mechanism is different from the immune response of animals, insects have no strict immune response mechanism, the antibacterial peptides produced by insect hemolymph can be invaded by one or more microorganisms and have no specificity, and the induction products are not only produced by specific invaded microorganisms but also have resistance to non-invaded microorganisms generally. The antibacterial peptides have different molecular weights, chemical structures and antibacterial mechanism effects, and different antibacterial peptides can resist different bacteria, fungi, viruses, tumor cells and the like, wherein the bee antibacterial peptide is the main antibacterial peptide of bees, and the hymenoptera antibacterial peptide is the most important supplementary peptide in the bee antibacterial peptide and mainly plays an inhibiting role on part of gram-negative bacteria generating resistance to the bee antibacterial peptide; the bee moth antibacterial peptide is used as a backup peptide of the bee antibacterial peptide and can only play a role when the bactericidal capability of the bee antibacterial peptide and the hymenoptera antibacterial peptide is lost. The bee defensin has the least expression amount and is the only antibacterial peptide for inhibiting gram-positive bacteria in bee hemolymph. The content of the bee antibacterial peptide is influenced by different factors, including bacteria, fungi, baby bugs, viruses, bee mites, medicaments and the like. The antibacterial peptide plays a role in resisting bee viruses, and researchers find that: when the bee is inoculated with Acute Bee Paralysis Virus (ABPV), the expression level of the antibacterial peptide gene is obviously increased; in the midgut epithelial tissue of the bee, the infection level of the bee residual wing virus (DWV) and the expression level of the bee antibacterial peptide gene are linearly related; after the bee is infected by the virus, the expression levels of the bee antibacterial peptide, the hymenoptera antibacterial peptide and the bee moth antibacterial peptide in the bee body are obviously reduced. Bee mites can also affect the expression of bee antimicrobial peptides. In the aspect of medicine, researchers find that the acaricide can significantly influence the expression of the bee antibacterial peptide gene, after bees contact the flumethrin vinegar, the expression level of the hymenoptera antibacterial peptide is significantly up-regulated, and the coumaphos can down-regulate the expression of the hymenoptera antibacterial peptide and the bee moth antibacterial peptide gene. In conclusion, the bee antibacterial peptide is a key part of humoral immunity of bees, and plays a role in coordination to resist invasion of various pathogenic microorganisms.
Chinese patent CN103524602A discloses the treatment of bee sacbrood by administering exogenous antibacterial peptide, but the administration of exogenous antibacterial peptide still faces the potential safety risk of antibacterial peptide drugs, the mass production of antibacterial peptide is limited, the production efficiency is low and the production cost is high (linchengde, penhongjuan, wang derived sea. the application and existing problems of antibacterial peptide, tropics medical journal 2007,1 (7): 86-90.). Therefore, the development of a high-efficiency prevention and treatment method for the Chinese sacbrood disease is of great significance to the bee breeding industry.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention aims to provide the application of adenosine in preventing and treating bee virus infection.
Adenosine of the present invention is a compound known in the art, which is a compound formed by connecting N-9 of adenine and C-1 of D-ribose via a beta glycosidic bond, and the phosphate ester thereof is adenylic acid. Adenosine, a metabolite of adenosine glance sideways at, exerts important physiological and biochemical effects in vivo, and is an important intermediate for the synthesis of Adenosine Triphosphate (ATP), adenine, adenylate, and vidarabine. Adenosine has the following structural formula:
the invention provides the use of adenosine or a derivative thereof in the manufacture of a medicament for the treatment or prevention of viral infection in bees.
The invention provides the use of adenosine or a derivative thereof in the preparation of a feed for the treatment or prevention of viral infection in bees.
The invention provides the use of adenosine or a derivative thereof in the inhibition of bee virus proliferation.
The application of the adenosine or the derivative thereof in inhibiting the bee virus proliferation can be used for inhibiting the virus proliferation in apparently healthy bees and can also be used for inhibiting the virus proliferation in virus disease bees.
The virus of the invention is preferably a Chinese bee sacbrood virus. Experiments prove that the survival rate of Chinese bee larvae infected with Chinese vesicular viruses can be remarkably improved by the administration of adenosine, on one hand, adenosine can directly act on viruses to reduce the copy number of the viruses, and on the other hand, adenosine can also induce the expression of endogenous antibacterial peptides to enhance the innate immunity defense of organisms to resist the viruses and reduce the morbidity of infected larvae.
In terms of the effect of adenosine on bee virus infection, substances containing adenosine structures, which are capable of being metabolized in the body to produce adenosine (including adenylic acid, adenylate, etc.), or other adenosine derivatives should have the same effect.
The derivatives of adenosine comprise adenylic acid and adenylate.
Experiments prove that adenosine can induce the transcription and translation of endogenous antibacterial peptide genes, improve the expression level of endogenous antibacterial peptides (such as hymenoptera antibacterial peptide, bee defensin, bee antibacterial peptide and bee moth antibacterial peptide), and further improve the immunity of organisms.
Therefore, the invention provides the application of adenosine or derivatives thereof in preparing the medicine for improving the immunity of bees. The invention also provides application of adenosine or derivatives thereof in preparing feed for improving bee immunity.
The improvement of the bee immunity is realized by improving the expression quantity of endogenous antibacterial peptide of the bee.
The endogenous antibacterial peptide can be any one or more of hymenoptera antibacterial peptide, bee defensin, bee antibacterial peptide and bee moth antibacterial peptide.
The active ingredient of the medicament or feed comprises one or more selected from adenosine and derivatives thereof.
The invention also provides a product for the treatment or prevention of a viral infection of honey bees, the active ingredient of which comprises one or more selected from adenosine and derivatives thereof. The product is a medicament, feed or feed additive.
The active ingredient of the medicament of the present invention may comprise adenosine or a derivative thereof alone; may also comprise a plurality selected from adenosine and derivatives thereof; other active ingredients besides adenosine and its derivatives may also be included (e.g., other active ingredients that can enhance the immunity or antiviral ability of bees).
The medicine of the invention can also contain auxiliary materials allowed in the field of pharmacy; the dosage form of the medicament can be any dosage form allowed in the field of pharmacy.
The feed of the present invention may comprise adenosine or a derivative thereof, and may also comprise a plurality selected from adenosine and a derivative thereof; other active ingredients besides adenosine and its derivatives (e.g., other active ingredients capable of enhancing immunity or antiviral ability of bees) may also be included; in addition to the above active ingredients, the feed according to the invention may also contain nutrients required by the bees, such as: pollen, royal jelly, fructose, glucose, sucrose, soybean flour, yeast powder, skimmed milk powder, inorganic salt, vitamins, water, etc.
The feed of the invention can be solid feed, semi-solid feed or liquid feed.
In the application of the adenosine provided by the invention, the dosage of the adenosine administered to bees is preferably more than or equal to 15 ng/bee/time. More preferably 0.1 to 10. mu.g/piece. The time interval of each adenosine administration is preferably 22-24 h.
Specifically, for 3-day-old bees, 15 ng-1.5 mug/bee/time; 22.5 ng-2.25 mug of bees at the age of 4 days per bee; 37.5 ng-3.75 mug of bees in 5 days old per bee per time; 6-day-old bees 60 ng-6 mug/bee/time.
The invention has the beneficial effects that: the invention discovers for the first time that adenosine can obviously improve the survival rate of Chinese bee larvae infected with CSBV and reduce the death rate. On one hand, adenosine can inhibit the proliferation of CSBV and reduce the copy number of virus; on the other hand, adenosine can also induce the expression of endogenous antibacterial peptide, and improve the innate immunity defense of bees. The experimental result of the invention shows that the death rate of Chinese bee larvae infected with CSBV is greatly reduced after adenosine intervention, the survival rate is obviously improved (the highest survival rate can be improved by 1.32 times), and the survival larvae can pupate and emerge into adult bees; the virus copy number in the bee larvae infected with CSBV is greatly reduced after adenosine is dried (the virus copy number infected with CSBV can be up to 121000, no CSBV is detected after adenosine is given, and an amount-effect relationship is presented, meanwhile, the virus infection degree of apparent healthy larvae can be improved by adenosine intervention, the normal growth and development of the larvae are facilitated, and the expression quantities of hymenoptera antimicrobial peptide, bee defensin and bee antimicrobial peptide in the bee larvae infected with CSBV are obviously increased after adenosine is dried.
The new function of the adenosine provided by the invention on bee virus infection provides a basis for the research of bee immune defense mechanism. The adenosine can be applied to preparation of medicines or feeding materials for preventing and treating Chinese bee sacbrood or improving the immunity of bees, provides a new method for preventing and treating the Chinese bee sacbrood, and has good market application prospect.
Drawings
FIG. 1 is a graph showing the statistical results of the survival rate of 6-day-old larvae after adenosine is dried in Experimental example 1; wherein CK represents the control group, 0.75 represents the low concentration group of the preparation group, 7.5 represents the medium concentration group of the preparation group, 75 represents the high concentration group of the preparation group, CSBV represents the infection group, CSBV +0.75 represents the low concentration group of the intervention group, CSBV +7.5 represents the medium concentration group of the intervention group, and CSBV +75 represents the high concentration group of the intervention group.
FIG. 2 is a statistical result of mortality of larvae of 4-6 days old after adenosine is dried in Experimental example 1; wherein CK represents the control group, 0.75 represents the low concentration group of the preparation group, 7.5 represents the medium concentration group of the preparation group, 75 represents the high concentration group of the preparation group, CSBV represents the infection group, CSBV +0.75 represents the low concentration group of the intervention group, CSBV +7.5 represents the medium concentration group of the intervention group, and CSBV +75 represents the high concentration group of the intervention group.
FIG. 3 shows the results of the detection of the viral copy number of 4-6 day-old larvae after adenosine desiccation in Experimental example 2; wherein CK represents the control group, 0.75 represents the low concentration group of the preparation group, 7.5 represents the medium concentration group of the preparation group, 75 represents the high concentration group of the preparation group, CSBV represents the infection group, CSBV +0.75 represents the low concentration group of the intervention group, CSBV +7.5 represents the medium concentration group of the intervention group, and CSBV +75 represents the high concentration group of the intervention group.
FIG. 4 shows the pupation and eclosion of healthy larvae and post-contamination adenosine-mediated larvae in Experimental example 3; wherein A is adenosine dry-control group (7.5 μ g/ml); b is a Control (CK) group.
FIG. 5 shows the amount of bee antimicrobial peptide (Apidaecin) expression after contamination and adenosine depletion in Experimental example 4; wherein CK represents a control group, low represents a low concentration group of the preparation group, medium represents a medium concentration group of the preparation group, high represents a high concentration group of the preparation group, CSBV represents an infection group, CSBV + low represents a low concentration group of the intervention group, CSBV + medium represents a medium concentration group of the intervention group, and CSBV + high represents a high concentration group of the intervention group.
FIG. 6 shows the expression level of Hymenoptera antimicrobial peptide (Hymenoptaecin) after contamination and adenosine drying in Experimental example 4 of the present invention; wherein CK represents a control group, low represents a low concentration group of the preparation group, medium represents a medium concentration group of the preparation group, high represents a high concentration group of the preparation group, CSBV represents an infection group, CSBV + low represents a low concentration group of the intervention group, CSBV + medium represents a medium concentration group of the intervention group, and CSBV + high represents a high concentration group of the intervention group.
FIG. 7 shows the expression level of the bee moth antibacterial peptide (Abaecin) for post-contamination and adenosine depletion in Experimental example 4; wherein CK represents a control group, low represents a low concentration group of the preparation group, medium represents a medium concentration group of the preparation group, high represents a high concentration group of the preparation group, CSBV represents an infection group, CSBV + low represents a low concentration group of the intervention group, CSBV + medium represents a medium concentration group of the intervention group, and CSBV + high represents a high concentration group of the intervention group.
FIG. 8 shows the amount of bee Defensin (Defensin) expression after infection with poison and adenosine in Experimental example 4; wherein CK represents a control group, low represents a low concentration group of the preparation group, medium represents a medium concentration group of the preparation group, high represents a high concentration group of the preparation group, CSBV represents an infection group, CSBV + low represents a low concentration group of the intervention group, CSBV + medium represents a medium concentration group of the intervention group, and CSBV + high represents a high concentration group of the intervention group.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Statistical analysis of the data in the following examples was performed using SPSS 22.0 (SPSS corporation, chicago, usa). The effect of adenosine on Apis cerana larvae is expressed as "Mean ± SE". And comparing the change of the virus copy number infected with CSBV in the Chinese bee larva by adopting an analysis of variance and an LSD multiple analysis of variance method.
Example 1 intervention of healthy Apis cerana larvae and Poison larvae with adenosine
1. Sample supply book
The Apis cerana Fabricius is from Apis cerana Fabricius research institute of China academy of agricultural sciences. In order to obtain 2-day-old larvae, after queen bees are closed on the son spleens to lay eggs for 96 hours, the son spleens containing the 2-day-old larvae are taken out from the bee colony and put into a 24-well plate, and the plate is placed in a constant temperature and humidity incubator with the temperature of 32 +/-1 ℃ and the relative humidity of 75 +/-5% RH for pre-culture.
The presence or absence of CSBV and other viral infections (including BQCV, DWV, IAPV, KBV, ABPV, CBPV) in the larvae was determined by RT-PCR, and healthy larvae without viral infections were used as samples in this example.
The primer sequences provided in the above-mentioned detection primer references for various viruses are as follows: CSBV detection primers were referenced to Grabenstein, E., W.Ritter, M.J.Carter, S.Davison, H.Pechhalker, J.Kolodziejek, O.Boeckking, I.Derakshifar, R.Moosbeckhofer, E.Licek, and N.Nowotny.Sacbrood virus of the honeybee (Apis mellea): rapid identification and phylogenetic analysis conversion-PCR.clin.Diagen. Lab.Immunol.2001,8: 93-104; BQCV detection primer references M, Benjeddou, N, Leat, M, Allsop, S, Davison.detection of acid bean analysis virus and black queen cell virus from the host genes by reverse transcription transcriptional pcr.applied and environmental microbiology.2001,67(5): 2384-; DWV detection primer refer to Tentcheva, D., L.Gauthier, S.Jouvee, L.Canaday-Rochelle, B.Dainat, F.Counters, M.E.Colin, B.V.Ball, and M.Berginin.Polymeraschen reaction detection of formed with virus (DWV) in Apis melifera and Varroa detector. idiology.2004, 35: 431-; IAPV detection primers are referred to Eyal, Maori, Shai, Lavi, Rita, Mozes-Koch, Yulia, Gantman, Yuv al, Peretz, Orit, Edelbaum, Edna, Tanne, Ilan, Sela. isolation and characterization of analysis resources, a direct infection of genes in an array, identification for direction product to intra-and inter-site recognition, the Journal of general vision, 2007,88(Pt 12): 3428-38; KBV detection primers were Stoltz, D., X.R.Shen, C.Boggis, and G.Sisson.molecular diagnostics of Kashmir bean virus infection.J.apic.Res.1995,34: 153-; ABPV detection primer refer to Bakonyi, T., R.Farkas, A.Szendroi, M.Dobos-Kovacs, and M.Rusvai.detection of acid bean analysis virus by RT-PCR in the gene bean and Varroa derivative field samples, screening of sensing human diagnosis experiments.Apidiologue.2002, 33: 63-74; CBPV detection primers are referred to Ribie're, M., C.Triboultot, L.Mathieu, C.Aurie ' res, J.P.Faucon, and M.Pe ' pin.molecular diagnostic of molecular be medical science in Apidiologie, 2002,33: 339-.
The detection method specifically comprises the following steps: RNA was extracted using RNeasy mini kit (edley) according to the product instructions (the method includes a step of removal of DNA, thus ensuring that only RNA is present in the final extract). Total RNA was eluted in 30. mu.L of elution buffer and used directly for RT-PCR. First strand cDNA was generated immediately using the extracted RNA using the Quantitec reverse transcription kit (Takara) (according to the product instructions) and the cDNA was stored at-20 ℃. The PCR amplification reaction is a 50 mu L system; PCR reaction conditions included pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 15 seconds, and 35 cycles; extension for 5min at 72 ℃. The results showed that no CSBV and other viruses were detected.
2. Preparation of syrup
Preparation and feeding of the syrups are carried out according to the Standard for feeding Apis mellifera in 2016 and adjusted on this basis (Karl Crailsheim, Robert Brodschneider, Pierrick Aupinel, Dieter Behrens, Elke Genersch, Jutta Vollmann & Ulrike Riessberger-Gall. Standard methods for identifying the real area re-addressing of Apis mellea. journal of Apimulturral Research,2013,52(1): 1-15.). The formula, ratio and dosage of the syrup fed to the larvae of different ages of days are shown in table 1.
TABLE 1 syrup formulation, ratio and dosage to feed
3. Preparation of virus liquid
Chinese bee sacbrood with typical sacbrood morphology is selected from another Chinese bee farm of the bee institute of Chinese agricultural academy of sciences, and the existence of CSBV in the Chinese bee body is confirmed by RT-PCR. To obtain CSBV, 210 larvae infected with CSBV were taken, placed in a sterile triturator and 2100 μ L sterile Phosphate Buffered Saline (PBS) was added and triturated. Grinding, centrifuging at 8000rpm at 4 deg.C for 30min, repeating for 2 times, collecting supernatant, performing RT-PCR detection with cDNA reverse transcribed after RNA extraction as template, and storing the supernatant at-80 deg.C as original venom without other viruses.
Viruses were detected using the absolute quantitative PCR method (K.M.Hong, H.Najjar, M.Hawley, R.D.Press.quantitative real-time PCR with automatic sample preparation for diagnosis and monitoring of cytological infection in bone matrix transfer protocols. clinical chemistry.2004,50(5):846 856; HU Zhi Gang, CHEN Ke Ping, YAO Qin, GAO Gui Tian, XU Jia-Ping, CHEN Hui-Qing.cloning and Characterification of Borumbx movement PP-BP, Gene Induced by Viral infection. acta.acta.acta.2005, En 48: 875).
The PCR reaction is carried out in a BIOER lineGene9600 real-time PCR system, and the upstream and downstream primers are respectively: 5'-ccttggagtttgctatttacg-3', and 5'-cctacatccttgggtcag-3'. The real-time fluorescent quantitative PCR reaction system is 15 mu L, comprises 7.5 mu L of reaction solution of 0.3 mu L, SYBR of each forward primer and reverse primer, 1 mu L of template and water till 15 mu L. The qPCR reaction conditions adopt a two-step method: the first step comprises a constant temperature section (denaturation at 95 ℃ for 3min) and a circulation section (denaturation at 95 ℃ for 5 s; 6)0 ℃ annealing for 30s, 40 cycles); the second step is a melting section (denaturation at 95 ℃ for 15 s; annealing at 60 ℃ for 5 s; denaturation at 95 ℃ for 15 s.). The concentration of the original venom was 6.74X 10 by qPCR detection4copies/. mu.L, this original venom was used for subsequent vaccination experiments.
Preparing the toxic syrup: IC based on Pre-test Virus infection50And (3) detecting results, selecting the feed venom according to the proportion of the original venom: syrup (syrup composition see table 1) ═ 1: 3, preparing a mixture of 5 μ L virus stock solution and 15 μ L syrup, i.e. CSBV of 1.685 × 104copies/. mu.L of toxic syrup. The toxicant-containing syrup was fed to 3-day-old test larvae.
4. Preparation of adenosine preparations
Purchasing adenosine standard (source leaf), preparing 1mg/mL mother liquor with high-purity water, filtering and sterilizing for later use. In the test, the test was carried out by dilution in multiple proportions as required using the day-old syrups shown in Table 1 to give adenosine preparations, and a low concentration group (0.75. mu.g/mL), a medium concentration group (7.5. mu.g/mL) and a high concentration group (75. mu.g/mL) were set, respectively.
Preparation of the toxin-containing preparation: the original venom was mixed with adenosine preparations (syrup composition see table 1) at a ratio of 1: 3 (for example, 20. mu.L of virus-containing preparation is prepared by mixing 5. mu.L of virus stock solution with 15. mu.L of adenosine preparation), namely CSBV is 1.685 multiplied by 104copies/. mu.L of the toxin-containing preparation. The toxicant-containing formulation was fed to 3-day-old test larvae.
5. Adenosine intervention test
The larvae after preculture for 1 day (3 days old) were taken out of the incubator, and the larvae with good health status were placed in 48-well plates, and randomly divided into a control group, an infected group, a preparation group and an intervention group, each group consisting of 24 samples, and the steps were repeated three times. Grouping and feeding are shown in table 2, and the composition of the syrup in table 2 is shown in table 1 (control day age).
TABLE 2 adenosine intervention test groups
The optimal time for bee larvae to infect the CSBV is 2-3 days old, so that at 3 days old of the larvae, CSBV infection and adenosine intervention treatment are carried out to analyze the prevention and treatment effects of adenosine on the CSBV infection of the bees. Feeding the infection group with the syrup containing toxin (preparation method is described in the above 3), and feeding the intervention group with the preparation containing toxin (preparation method is described in the above 4); the larvae aged 4-6 days were fed with syrup or adenosine preparations, respectively, and only the larvae fed with syrup were used as CK control (the feeding amount is shown in Table 1). The above fed food is placed on one side of the bottom of the culture plate to avoid contacting with larvae. Feeding was done every 24h and feeding and mortality of larvae were observed and recorded.
Forceps were sterilized with 75% ethanol (prepared with DEPC-treated water) for 5min and rinsed three times with DEPC-treated water, 5 each of the differently treated live larvae were removed each day, immediately treated with liquid nitrogen for freezing, and then stored in a-80 ℃ freezer for subsequent viral copy number detection.
Experimental example 1 survival and mortality analysis after adenosine intervention in healthy and infected larvae
1. Survival rate
Survival rate statistics is carried out on each group of bee larvae in example 1, the results are shown in fig. 1, and the results show that the survival rate of larvae of CK group and preparation group fed conventionally can reach over 86%, which indicates that adenosine intervention does not cause abnormal death of healthy larvae and does not affect growth and development of healthy larvae.
The larvae of the infected group die in large quantity, the survival rate is only 43 percent, the survival rate of the larvae of the intervention group is greatly improved to more than 76 percent, wherein the larvae with low concentration (0.75 mu g/mL) intervene the infected larvae to survive for 6 days, and the result shows that about 50 percent of the infected larvae can be prevented from dying due to illness by adopting adenosine intervention, so that the infected bee colony is prevented from collapsing, and the method plays an important role in recovering and breeding the bee colony.
2. Mortality rate
Mortality statistics were performed on each group of bee larvae in example 1, and the mortality of the larvae was determined as follows: the death numbers of the larvae in the pre-culture (2 days old) and the virus inoculation day (3 days old) are not counted and analyzed to eliminate the influence of mechanical death of the larvae in the experimental process, the death numbers of the larvae are counted at regular time every day from the next day (4 days old) of virus inoculation, the death larvae are removed, and the steps are repeated until the 6 days old is finished.
Larval mortality per day-number of larvae dead per day/number of larvae alive per day x 100%;
overall larval mortality rate-number of larval deaths/total number of samples x 100%.
The statistical results of the mortality of all groups are shown in fig. 2, and the results show that the mortality of 4-6-day-old larvae in CK groups fed conventionally is lower than 3% every day, and the mortality of 4-6-day-old larvae after adenosine at all concentrations is not greatly increased every day, which indicates that adenosine intervention does not affect the growth and development of healthy larvae and can be used for auxiliary feeding of larvae.
While CSBV is a typical induction source, the death rate of larvae at the next day (4 days old) after 3 days old larvae are infected with virus is greatly increased and is up to 25 percent, the death rate at 5 days old and 6 days old is reduced, but the death rate per day is more than 20 percent, and only 43 percent of experimental larvae survive by 6 days old (figure 1). The study of Liang Qin et al (Liang Qin, Chendafu, bee protection science [ M ]. China agricultural Press, 2009.2-3.) found that CSBV is most susceptible to 2-3 days old larvae, the infected larvae cannot pupate, and the larvae die largely in the later stage of the larvae, which is consistent with the experimental results of the present invention.
Compared with the infected group, the death rate of the larvae at each day age of the adenosine intervention group is greatly reduced, the death rate of the larvae at 4 days age is about 15%, the larvae are reduced day by day, the survival rate of the larvae at each concentration group reaches over 86% when the larvae are at 6 days age, wherein the low-concentration group (0.75 mug/mL) intervenes in the survival of the infected larvae at all 6 days age (figure 1), and the result shows that the death rate of the infected larvae can be greatly reduced by adopting the adenosine intervention, so that the infected bee colony is prevented from collapsing, and the adenosine intervention plays an important role in the recovery and reproduction of the bee colony.
Experimental example 2 detection of viral copy number following adenosine intervention in healthy and infected larvae
CSBV test was performed on the 4-6 day old Apis cerana larva (CK group) with healthy appearance in example 1, and the number of Virus copies per day old larva was found to be 66-70, which indicated that apparently healthy larva also had Virus Infection but showed no obvious symptoms, indicating that CSBV has a phenomenon of hidden Infection in bee colony, and that the larva could live with Virus in a certain concentration, which is consistent with the results of study on Liu Shan, L., Liuhao, W., Jun, G., Yujie, T., Yanping, C., Jie, W., Jilian, L., Chinese Sacbroud infestations in Asian Honey Bees (iris Apis cerana cerana cerana) and Host response to the Virus infestations Impection, Journal of neurology.2017, and dot htx/357.006.09. The virus copy number of the internal bodies (preparation groups) of the apparent healthy 4-6-day-old Chinese bee larvae is reduced to below 6 after adenosine intervention, which shows that the virus infection degree of the apparent healthy larvae can be improved by adenosine intervention, and the normal growth and development of the larvae are facilitated.
The virus copy number in the larvae of the infected group grows in an explosive manner, and the virus copy number in the larvae of 4 days old is as high as 12.1 multiplied by 104Is 1700 times of CK group, so that the multiplication of a large amount of virus causes infected larvae to be died, the death rate is up to 25 percent, the virus copy number in bodies of 5 days old and 6 days old is reduced along with the growth and development, but the virus copy number is still maintained at 2.4 multiplied by 104Above, it was shown that the single-day larval mortality rate was still above 20% (fig. 3). The copy number of the virus in 4-6-day-old larvae in each concentration adenosine intervention group is reduced to below 10, which can be regarded as no CSBV detection, thus showing that strong antagonistic virus reaction is generated in the contaminated larvae after adenosine is fed, obviously reducing the infection degree of the virus to the larvae, and further reducing the fatality rate of the contaminated larvae.
EXAMPLE 3 morphological analysis of adenosine after intervention in healthy and infected larvae
On the 18 th day (22 th day from the egg stage) of the experiment of example 1, the states of the larvae of each group in example 1 were observed with a stereomicroscope and recorded by photographing.
Morphological observation results As shown in FIG. 4, although the larvae in the infected group did not pupate even though they did not die, the larvae in both the CK group and the low concentration group (0.75. mu.g/mL) dried group were alive and both pupated and had emerged as adult bees. The CSBV infection is characterized in that 1-3 day old larvae are infected, so that the larvae in bee colonies die in large quantity and cannot pupate, healthy larvae pupate and emerge into adult bees, which is the normal development process of honey, and infected CSBV larvae can pupate and emerge into adult bees, which shows that proline intervention plays a role in antagonizing viruses, so that infected larvae can develop normally.
Experimental example 4 influence of CSBV infection and adenosine intervention on the expression level of genes involved in larva immunity
To analyze the effect of CSBV Infection and adenosine intervention on the Immune system of bee larvae, the expression levels of the antimicrobial peptide genes in each group of bee larvae in example 1 were examined by real-time fluorescent quantitative PCR using β -actin as an internal reference gene (Shan, L., Liuhao, W., Jun, G., Yujie, T., Yanping, C., Jie, W., Jilian, L., Chinese Sacbrood Virus Infection in Asian Honey Bees (Apis cerana) and Host Immune Responses to the Virus Infection, Journal of Invertebrate Pathology.2017, doi: http:// dx. doi.org/10.1016/j.jj.2017.09.006). The detection result is specifically as follows:
1. expression detection of bee antibacterial peptide (Apidaecin) gene
The bee antibacterial peptide (Apidaecin) is the main antibacterial peptide of bees, the detection result is shown in fig. 5, the relative expression quantity of the bee antibacterial peptide of the larvae of the Control (CK) group is only 1.24-1.55, the relative expression quantity of the bee antibacterial peptide is increased in the larvae 24 hours after CSBV infection, and the relative expression quantity of the bee antibacterial peptide of the larvae of 4-6 days old is maintained at about 20.
The adenosine intervention (preparation group) on healthy larvae can cause the expression quantity of the bee antibacterial peptide to be greatly up-regulated, the highest expression quantity is 6 days old, and the relative expression quantity of a low-concentration group (0.75 mu g/ml) is up-regulated to about 1000; the adenosine intervention (intervention group) on the infected larvae can also greatly up-regulate the expression quantity of the bee antibacterial peptide, the 6-day-old bee antibacterial peptide is the highest, and the relative expression quantity reaches about 650 which is 32 times of the expression quantity of the infected group.
The result shows that the adenosine intervention strongly induces the antibacterial peptide expression capacity of the infected larva, enhances the innate immunity defense capacity of the larva to resist CSBV invasion, and reduces the fatality rate. It is demonstrated that adenosine has the ability to strongly induce the expression of the antibacterial peptide in healthy larvae and CSBV infected larvae bees.
2. Hymenoptera antibacterial peptide (Hymenoptaecin)
The hymenoptera antimicrobial peptide is the most important supplementary peptide in the bee antimicrobial peptide, and the detection result is shown in fig. 6, the relative expression quantity of the hymenoptera antimicrobial peptide of the larvae of a Control (CK) group is only 1.31-1.56, after the larvae are infected with CSBV for 24 hours, the expression quantity of the hymenoptera antimicrobial peptide in the larvae is increased, the relative expression quantity of the larvae of 4-6 days old is not obviously changed, and the relative expression quantity is maintained at about 20.
Adenosine intervention (preparation group) on healthy larvae can cause the expression level of hymenoptera antibacterial peptide to be obviously up-regulated, and the relative expression level is up-regulated to 97.24; the adenosine intervention (intervention group) is carried out on the infected larvae, the expression quantity of the hymenoptera antibacterial peptide is greatly increased, the highest relative expression quantity per day can reach 342.14, and is 17 times of the expression quantity of the infected larvae.
The result shows that the expression capacity of hymenoptera antibacterial peptide is strongly induced through adenosine intervention, the innate immunity defense of larvae is enhanced to resist CSBV invasion, and the fatality rate is reduced. The adenosine has the capacity of strongly inducing the expression of hymenoptera antibacterial peptide of healthy larvae and CSBV infected larvae.
3. Bee moth antibacterial peptide (Abaecin)
The bee moth antibacterial peptide is used as a backup peptide of the bee antibacterial peptide, and only plays a role when the bactericidal capability of the bee antibacterial peptide and the hymenoptera antibacterial peptide is lost. The detection result of the bee moth antibacterial peptide is shown in fig. 7, the relative expression quantity of the bee moth antibacterial peptide of the larvae of the Control (CK) group is only 1.52-3.68, after the larvae are infected with CSBV for 24 hours, the expression quantity of the bee moth antibacterial peptide in the larvae is obviously increased to 73.25, but the expression quantity of 5-6 days old falls back to 25.4-26.97.
Adenosine intervention (preparation group) is given to healthy larvae to cause the expression level of the bee moth antibacterial peptide to be up-regulated to about 35, and the expression level of the single-day bee moth antibacterial peptide of a low-concentration group (0.75 mu g/ml) is improved to about 100; the adenosine intervention (intervention group) is given to the infected larvae, so that the expression quantity of the bee moth antibacterial peptide of 6-day-old larvae can be greatly increased to 50.81 at most and is increased by 88.4 percent compared with the infected group, and the effect of the bee moth antibacterial peptide as the backup peptide of the bee antibacterial peptide is fully exerted.
The result shows that the ability of expressing the bee moth antibacterial peptide is strongly induced through adenosine intervention, the innate immunity defense of the larva is enhanced to resist CSBV invasion, and the fatality rate is reduced. The adenosine has the capacity of strongly inducing the expression of the antibacterial peptide of the healthy larva and the CSBV infected larva bee moth.
4. Bee Defensin (Defensin)
The detection result of the bee defensin is shown in figure 8, the relative expression quantity of the bee defensin of the larva in the Control (CK) group is only 1.32-1.62, the expression quantity of the bee defensin in the larva is increased to 46.26 after the larva is infected with CSBV for 24 hours, the maximum expression quantity of 5-day-old bee defensin is 52.59, and the 6-day-old bee defensin falls back to 22.28.
The adenosine intervention (preparation group) on healthy larvae can cause the defensin expression of bees to be greatly up-regulated, the highest expression is realized in 5-day-old larvae, and the low-concentration group (0.75 mu g/ml) is up-regulated to more than 160; the adenosine intervention (intervention group) is given to the infected larvae, so that the expression quantity of the bee defensin is greatly increased, the highest expression quantity is reached at the age of 5 days, and the highest relative expression quantity can reach more than 210 and is 4 times of the expression quantity of the infected group.
The bee defensin is the only antibacterial peptide in bee hemolymph for inhibiting gram-positive bacteria, but the expression amount is the least. The invention strongly induces the expression capacity of endogenous bee defensins by interfering the exogenous way with adenosine, enhances the innate immunity defense of larvae to resist CSBV invasion, and reduces the fatality rate. It is demonstrated that adenosine has the ability to strongly induce the expression of endogenous bee defensins in healthy and infested larvae.
According to the invention, the dynamic change of the expression of the Immune related genes in larvae of 4-6 days old of Chinese Bees infected with CSBV is analyzed, and the expression level of the antibacterial peptide genes in the infected larvae is higher than that of healthy larvae on the whole, which indicates that the infected CSBV can promote the expression of the Immune related genes in the larvae of the Bees, and the result is matched with the research result of Liu-san (Shan, L., Liuhao, W., Jun, G., Yujie, T., Yanping, C., Jie, W., Jiian, L., Chinese Sacbrood Virus Infection in Asian Home Bees (Apis cerana cerana) and Host Immune Responses to the Virus Infection, Journal of Inverteby Pathology.2017, and phi: http:// dx.doi.org/10.1016/j.j.006).
The invention takes Chinese bee infected with CSBV and healthy Chinese bee as research objects, and utilizes a fluorescent quantitative PCR method to quantitatively detect and compare the expression conditions of 4 antibacterial peptide genes in the disease-sensitive and healthy bee larva development stages, thereby defining the immune response condition of the Chinese bee after CSBV infection; and quantitatively detecting and comparing the expression of 4 antibacterial peptide genes after adenosine is given to sick and healthy bee larvae:
whether the larvae are healthy or infected larvae, adenosine intervention is given, so that the expression of hymenoptera antibacterial peptide Hymenoptaecin of the larvae of 4-6 days old can be integrally adjusted upwards; greatly up-regulating the expression quantity of Defensin of 5-day-old larva bees; greatly up-regulating the expression quantity of 6-day-old larva antibacterial peptide Apidacin and bee moth antibacterial peptide Abaecin. In summary, the ability to express the endogenous antimicrobial peptides hymenoptera (hymenopteracin), bee Defensin (Defensin), bee (Apidaecin) and bee (Apidaecin) of the infected larvae was strongly induced by adenosine intervention. The high expression of the four antibacterial peptides for one day or more successfully resists the invasion of CSBV to larvae, reduces the fatality rate of the larvae, promotes the larvae to pupate and emerge into adult bees, and is an effective method for enhancing the immunity of the larvae of the Chinese bees and resisting CSBV infection; meanwhile, adenosine intervention can also up-regulate the expression level of the healthy Chinese bee larva antibacterial peptide to form a rapid and effective defense mechanism for rapidly killing or eliminating exogenous pathogenic microorganisms and protecting the growth and development of the larva.
In conclusion, adenosine can inhibit the proliferation of CSBV and reduce the copy number of CSBV in bee larvae; can also induce the expression of endogenous antibacterial peptide of bee larva, improve the innate immunity defense of bee, resist the invasion of CSBV to larva and prevent the further infection of CSBV and other pathogenic microorganisms, thus being used for preventing and treating the bee sacbrood disease in bee.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> bee institute of Chinese academy of agricultural sciences
Application of <120> adenosine in prevention and treatment of bee virus infection
<130> KHP201110404.3
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ccttggagtt tgctatttac g 21
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cctacatcct tgggtcag 18
Claims (7)
1. Use of adenosine in the manufacture of a medicament for the treatment or prevention of bee sacbrood virus in a bee infection.
2. Use of adenosine in the manufacture of a feed for the treatment or prevention of bee sacbrood virus infection in bees.
3. Use of adenosine in the manufacture of a medicament or feed for inhibiting the proliferation of vesicular virus in a bee infection.
4. The application of adenosine in preparing a medicament for improving the expression level of endogenous antibacterial peptide after honeybee is infected with the vesicular virus, wherein the endogenous antibacterial peptide is any one or more of hymenoptera antibacterial peptide, honeybee defensin, honeybee antibacterial peptide and honeybee moth antibacterial peptide.
5. The application of adenosine in preparing the feed for improving the expression quantity of endogenous antibacterial peptide after honeybee is infected with the vesicular virus, wherein the endogenous antibacterial peptide is any one or more of hymenoptera antibacterial peptide, honeybee defensin, honeybee antibacterial peptide and honeybee moth antibacterial peptide.
6. The use according to claim 1, 3 or 4, wherein the active ingredient of the medicament comprises adenosine.
7. Use according to claim 2,3 or 5, wherein the active ingredient of the feed comprises adenosine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010255841.2A CN111297884B (en) | 2020-04-02 | 2020-04-02 | Application of adenosine in prevention and treatment of bee virus infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010255841.2A CN111297884B (en) | 2020-04-02 | 2020-04-02 | Application of adenosine in prevention and treatment of bee virus infection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111297884A CN111297884A (en) | 2020-06-19 |
| CN111297884B true CN111297884B (en) | 2021-03-30 |
Family
ID=71155461
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010255841.2A Active CN111297884B (en) | 2020-04-02 | 2020-04-02 | Application of adenosine in prevention and treatment of bee virus infection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111297884B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992007948A1 (en) * | 1990-11-06 | 1992-05-14 | The Lubrizol Corporation | Compositions and methods for analyzing genomic variation |
| US20160237455A1 (en) * | 2013-09-27 | 2016-08-18 | Editas Medicine, Inc. | Crispr-related methods and compositions |
| WO2019236673A1 (en) * | 2018-06-06 | 2019-12-12 | Massachusetts Institute Of Technology | Circular rna for translation in eukaryotic cells |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8217163B2 (en) * | 2010-09-20 | 2012-07-10 | Biomics Biotechnologies Co., Ltd. | Application of highly conserved domain sequences from viral genome as template to design therapeutic slirnas |
| CA3020400A1 (en) * | 2016-04-12 | 2017-10-19 | Illustris Pharmaceuticals, Inc. | Compositions for topical application of compounds |
-
2020
- 2020-04-02 CN CN202010255841.2A patent/CN111297884B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992007948A1 (en) * | 1990-11-06 | 1992-05-14 | The Lubrizol Corporation | Compositions and methods for analyzing genomic variation |
| US20160237455A1 (en) * | 2013-09-27 | 2016-08-18 | Editas Medicine, Inc. | Crispr-related methods and compositions |
| WO2019236673A1 (en) * | 2018-06-06 | 2019-12-12 | Massachusetts Institute Of Technology | Circular rna for translation in eukaryotic cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111297884A (en) | 2020-06-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Palmer-Young et al. | Nectar and pollen phytochemicals stimulate honey bee (Hymenoptera: Apidae) immunity to viral infection | |
| Yoshiyama et al. | Inhibition of Paenibacillus larvae by lactic acid bacteria isolated from fermented materials | |
| Maruščáková et al. | Effect of application of probiotic pollen suspension on immune response and gut microbiota of honey bees (Apis mellifera) | |
| CN105980399B (en) | Compositions and methods for virus control in varroa mites and bees | |
| Defer et al. | Screening for antibacterial and antiviral activities in three bivalve and two gastropod marine molluscs | |
| US9609875B2 (en) | Methods for the prevention and control of pathogenic infections in bees and relative composition | |
| Dang et al. | Variation in the antiviral and antibacterial activity of abalone Haliotis laevigata, H. rubra and their hybrid in South Australia | |
| Royan | Mechanisms of probiotic action in the honeybee | |
| Al-Ghamdi et al. | Immune investigation of the honeybee Apis mellifera jemenitica broods: A step toward production of a bee-derived antibiotic against the American foulbrood | |
| US10925909B2 (en) | Bacteriophage and composition comprising same | |
| Huang et al. | A potential probiotic Leuconostoc mesenteroides TBE-8 for honey bee | |
| Pluta et al. | Changes in the expression of antimicrobial peptide genes in honey bees (Apis mellifera) under the influence of various pathogens | |
| CN111297884B (en) | Application of adenosine in prevention and treatment of bee virus infection | |
| CN111481541B (en) | Application of proline in preventing and treating bee virus infection | |
| CN111297851B (en) | Application of coumarin in resisting virus infection of bees | |
| US11178895B2 (en) | Composition for enhancing immunity of insects and method thereof | |
| Usta et al. | Investigation of deformed wing virus, black queen cell virus, and acute bee paralysis virus infections in honey bees using reverse transcriptase-polymerase chain reaction (RT-PCR) method | |
| CN111265596B (en) | Traditional Chinese medicine composition for preventing and treating middle capsule disease | |
| Cebotari et al. | The use of biologically active substances for strengthening of resistance to diseases of honeybee colonies Apis mellifera. | |
| Vung et al. | Controlling sacbrood virus disease in Apis cerana colonies with biological methods in Korea | |
| CN103524602A (en) | Application of antibacterial peptide in pharmacy | |
| Li et al. | Gut microbiota of honey bees. | |
| KR101600983B1 (en) | Therapeutic and preventive agent for sacbrood of bees | |
| CN114569592B (en) | Application of meclofenamic acid in preventing and treating Chinese bee sacbrood | |
| KR102023380B1 (en) | Composition comprising bee venom for treating or preventing Coccidiosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |