Application of tetracycline hydrochloride in preparation of microsporidia inhibitor and preparation of medicine for preventing or treating microsporidiosis

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of tetracycline hydrochloride to preparation of a microsporidia inhibitor and preparation of a medicine for preventing or treating microsporidiosis. According to the invention, tetracycline hydrochloride and most of compounds commonly used clinically at present are used for treating nosema bombycis capable of infecting bombyx mori, and nosema encephalitis and nosema helenii capable of infecting human beings and other mammals respectively; only tetracycline hydrochloride can inhibit proliferation of invertebrate microsporidia representative-nosema bombycis, and proliferation of vertebrate microsporidia representative-nosema helenii and nosema encephalitis can also be inhibited. The tetracycline hydrochloride can be used for inhibiting proliferation of microsporidia and preventing and / or treating microsporidiosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of tetracycline hydrochloride in the preparation of microsporidia inhibitors and drugs for preventing or treating microsporidiosis. Background Art

[0002] Microsporidia are a class of eukaryotic microorganisms that are obligate intracellular parasites. This type of pathogen has a wide host range and can infect almost all invertebrates and vertebrates. Microsporidia that infect silkworms, enterocytosis that infects shrimps, and microsporidia that infect honeybees are all serious threats to the livestock and aquaculture industries. Microsporidia can infect people with low immunity, such as AIDS patients, organ transplant recipients, and patients with congenital hereditary immunodeficiency diseases, and the proportion of infections in the elderly and children is increasing. Cases caused by microsporidia such as rabbit encephalitis microsporidia and Helen's encephalitis microsporidia are also being reported more and more frequently.

[0003] At present, the drugs that can be used clinically to treat human microsporidiosis are albendazole and fumagillin. However, fumagillin has a series of side effects such as thrombocytopenia and neutrophil granulocyte reduction. Although albendazole has been proven to inhibit the proliferation of microsporidia, there is often a phenomenon of recurrence after drug withdrawal, and the treatment effect on various microsporidia including Bixiong intestinal microsporidia is very poor. Carbendazim can be used for the prevention and treatment of microsporidia in the process of silkworm seed production, but the compound has liver and reproductive toxicity and is not suitable for human and animal husbandry production. Therefore, it is urgent to develop a safe and effective microsporidia antagonist for the treatment of human and animal microsporidiosis.

[0004] Tetracycline is a broad-spectrum antibiotic with a phenanthrene nucleus. It is a commonly used antibiotic for treating bacterial infections. Its hydrochloride, i.e., tetracycline hydrochloride, is generally used clinically. Tetracycline hydrochloride inhibits the synthesis of bacterial proteins by preventing the binding of aminoacyl-tRNA to the A site of the bacterial ribosome 30S subunit, thereby hindering the formation of nascent peptide chains and inhibiting protein synthesis. Tetracycline hydrochloride is active against gram-positive bacteria, gram-negative bacteria, aerobic bacteria, anaerobic bacteria, as well as chlamydia, mycoplasma and rickettsia, etc. It is generally believed that it has no antagonistic activity against fungal pathogens. In clinical practice, tetracycline hydrochloride is mainly used to treat typhus, mycoplasma pneumonia, urogenital system infections, trachoma, Brucella and rickettsia infections, etc. Summary of the invention

[0005] The object of the present invention is to provide the use of tetracycline hydrochloride in the preparation of microsporidia inhibitors and in the preparation of drugs for preventing or treating microsporidiosis, so as to inhibit microsporidia and effectively prevent and / or treat microsporidiosis.

[0006] To achieve the above object, the present invention provides the use of tetracycline hydrochloride in the preparation of a microsporidia inhibitor;

[0007] The microsporidia include invertebrate microsporidia and / or vertebrate microsporidia;

[0008] The invertebrate microsporidia include Nosema bombycis;

[0009] The vertebrate microsporidia include Encephalitozoon hellem and / or Encephalitozoon cuniculi.

[0010] Preferably, the microsporidia inhibitor includes a microsporidia proliferation inhibitor.

[0011] The present invention also provides the use of tetracycline hydrochloride in the preparation of a drug for preventing and / or treating microsporidiosis;

[0012] The microsporidiosis includes invertebrate microsporidiosis and / or vertebrate microsporidiosis;

[0013] The invertebrate microsporidiosis includes Nosema bombycis disease;

[0014] The vertebrate microsporidiosis includes Encephalitozoon hellem disease and / or Encephalitozoon cuniculi disease.

[0015] The present invention also provides a microsporidia inhibitor, the active ingredient of which includes tetracycline hydrochloride;

[0016] The microsporidia include invertebrate microsporidia and / or vertebrate microsporidia;

[0017] The invertebrate microsporidia include Nosema bombycis;

[0018] The vertebrate microsporidia include Encephalitozoon hellem and / or Encephalitozoon cuniculi.

[0019] Preferably, the effective concentration of tetracycline hydrochloride in the microsporidia inhibitor is ≥0.1 μg / mL.

[0020] The present invention also provides a feed for preventing and / or treating Nosema bombycis disease, the feed includes mulberry leaves; the mulberry leaves contain tetracycline hydrochloride and / or the microsporidia inhibitor described in the above technical solution.

[0021] The present invention also provides a preparation method of the feed described in the above technical solution, including the following steps: applying the microsporidia inhibitor to the mulberry leaves and then drying to obtain the feed.

[0022] Preferably, the mass-volume ratio of the mulberry leaves to the microsporidia inhibitor is 10 g:1 mL;

[0023] The concentration of tetracycline hydrochloride in the microsporidia inhibitor is 30-100 ppm.

[0024] The present invention also provides a drug for preventing and / or treating vertebrate microsporidiosis, the drug comprising the microsporidiosis inhibitor described in the above technical solution and a pharmaceutically acceptable excipient;

[0025] The vertebrate microsporidia include Encephalitis Helenii and / or Encephalitis Leptospira.

[0026] Preferably, the effective concentration of tetracycline hydrochloride in the drug is 0.1 to 25 μg / mL.

[0027] Beneficial effects:

[0028] The present invention finds that tetracycline hydrochloride can inhibit the proliferation of the representative of invertebrate microsporidia, namely, Microsporida bombycis, and can also inhibit the proliferation of the representative of vertebrate microsporidia, namely, Microsporida helenii and Microsporida foetida. Furthermore, by establishing a microsporidia infection model, it is found that tetracycline hydrochloride can prevent and / or treat microsporidia disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0030] Figure 1 This is a diagram showing the safety assessment results of tetracycline hydrochloride on Spodoptera frugiperda cells Sf9;

[0031] Figure 2 This is a graph showing the effect of adding tetracycline hydrochloride at the cellular level on the proliferation of Bombyx mori microspores;

[0032] Figure 3 This is a graph showing the effect of adding tetracycline hydrochloride at the individual level on the proliferation of Bombyx mori microspores;

[0033] Figure 4 This is a diagram showing the safety assessment results of tetracycline hydrochloride on rabbit kidney cells RK13;

[0034] Figure 5 This is a fluorescence microscopic observation result of the effect of tetracycline hydrochloride on the proliferation of rabbit encephalitis microsporidia at the cellular level;

[0035] Figures 6-7 The results show the effect of adding tetracycline hydrochloride on the proliferation of rabbit encephalitis microsporidia at the cellular level; Figure 6 The results of fluorescence counting after parasitic vacuole staining; Figure 7 The results of fluorescence quantitative PCR detection;

[0036] Figure 8This is a graph showing the dose-dependent inhibitory effect of tetracycline hydrochloride on rabbit encephalitis microsporidia;

[0037] Fig. 9 This is the result graph showing the effect of adding tetracycline hydrochloride at the individual level on the proliferation of rabbit encephalitis microsporidia;

[0038] Figures 10-11 The results show the effect of adding tetracycline hydrochloride on the proliferation of Helen's encephalitis microsporidia at the cellular level; Fig.10 The results of fluorescence counting after parasitic vacuole staining; Fig.11 The results of fluorescence quantitative PCR detection;

[0039] Fig.12 This is a graph showing the dose-dependent inhibitory effect of tetracycline hydrochloride on Helen's encephalitis microsporidia;

[0040] Fig.13 This is a graph showing the effect of adding tetracycline hydrochloride at the individual level on the proliferation of Helen's encephalitis microsporidia;

[0041] Fig.14 This is a graph showing the effect of adding tetracycline hydrochloride at the individual level on the proliferation of honey bee microsporidia;

[0042] Figures 1 to 13 In the table, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. DETAILED DESCRIPTION

[0043] The invention provides the use of tetracycline hydrochloride in preparing a microsporidian inhibitor; the microsporidian includes invertebrate microsporidian and / or vertebrate microsporidian; the invertebrate microsporidian includes Bombyx mori microsporidian; the vertebrate microsporidian includes Helenitis encephalitis microsporidian and / or Leptocephalus encephalitis microsporidian.

[0044] The present invention also provides the use of tetracycline hydrochloride in preparing a drug for preventing and / or treating microsporidiosis; the microsporidiosis includes invertebrate microsporidiosis and / or vertebrate microsporidiosis; the invertebrate microsporidiosis includes bombyx mori microsporidiosis; the vertebrate microsporidiosis includes Helen's encephalitis microsporidiosis and / or rabbit encephalitis microsporidiosis.

[0045] As an embodiment, the microsporidia inhibitor of the present invention includes a microsporidia proliferation inhibitor.

[0046] The present invention has no particular limitation on the source of tetracycline hydrochloride, and conventional commercially available tetracycline hydrochloride products known to those skilled in the art can be used. As an embodiment, the CAS number of the tetracycline hydrochloride described in the present invention is 64-75-5.

[0047] Tetracycline hydrochloride is a broad-spectrum antibacterial drug with a long clinical application time and a wide range of applications, low production cost and high safety. The present invention uses tetracycline hydrochloride to treat Bombyx mori microsporidia that can infect silkworms, rabbit encephalitis microsporidia and Helen's encephalitis microsporidia that can infect humans and other mammals. The results show that only tetracycline hydrochloride can inhibit the proliferation of Bombyx mori microsporidia, a representative of invertebrate microsporidia, and can also inhibit the proliferation of Helen's encephalitis microsporidia and rabbit encephalitis microsporidia, representatives of vertebrate microsporidia. Tetracycline hydrochloride can be used to inhibit the proliferation of microsporidia, prevent and / or treat microsporidiosis.

[0048] The present invention also provides a microsporidia inhibitor, the active ingredient of which includes tetracycline hydrochloride; the microsporidia include invertebrate microsporidia and / or vertebrate microsporidia; the invertebrate microsporidia include Bombyx mori microsporidia; the vertebrate microsporidia include Helenitis microsporidia and / or rabbit encephalitis microsporidia.

[0049] As an embodiment, the effective concentration of tetracycline hydrochloride in the microsporidia inhibitor of the present invention is 0.1-25 μg / mL; as another embodiment, the effective concentration of tetracycline hydrochloride in the microsporidia inhibitor of the present invention is 1-20 μg / mL; as another embodiment, the effective concentration of tetracycline hydrochloride in the microsporidia inhibitor of the present invention is 10 μg / mL. As an embodiment, when the microsporidia are Bombyx mori microsporidia, the effective concentration of tetracycline hydrochloride in the microsporidia inhibitor is 20 μg / mL. As an embodiment, when the microsporidia are Helen's encephalitis microsporidia and / or rabbit encephalitis microsporidia, the effective concentration of tetracycline hydrochloride in the microsporidia inhibitor is 10-25 μg / mL; as another embodiment, when the microsporidia are Helen's encephalitis microsporidia and / or rabbit encephalitis microsporidia, the effective concentration of tetracycline hydrochloride in the microsporidia inhibitor is 25 μg / mL. The present invention evaluates the safety of tetracycline hydrochloride and finds that tetracycline hydrochloride is safe to use within the effective concentration range defined by the present invention and does not affect cell viability.

[0050] The present invention also provides a feed for preventing and / or treating pyridiasis in silkworms, the feed comprising mulberry leaves; the mulberry leaves contain tetracycline hydrochloride and / or the microsporidia inhibitor described in the above technical solution.

[0051] As an embodiment, the present invention further provides a method for preparing the feed described in the above technical solution, comprising the following steps: applying the microsporidia inhibitor to mulberry leaves and then drying them to obtain the feed.

[0052] As an embodiment, the mass volume ratio of the mulberry leaf and the microsporidia inhibitor of the present invention is 10g:1mL. As an embodiment, the concentration of tetracycline hydrochloride in the microsporidia inhibitor of the present invention is 30-100ppm; as another embodiment, the concentration of tetracycline hydrochloride in the microsporidia inhibitor of the present invention is 40-80ppm; as another embodiment, the concentration of tetracycline hydrochloride in the microsporidia inhibitor of the present invention is 50-70ppm; as another embodiment, the concentration of tetracycline hydrochloride in the microsporidia inhibitor of the present invention is 60ppm.

[0053] The present invention also provides a medicine for preventing and / or treating vertebrate microsporidiosis, the medicine comprising the microsporidiosis inhibitor described in the above technical solution and pharmaceutically acceptable excipients; the vertebrate microsporidiosis comprises Helenitis microsporidia and / or rabbit encephalitis microsporidia.

[0054] As one embodiment, the effective concentration of tetracycline hydrochloride in the drug of the present invention is 0.1 to 25 μg / mL; as another embodiment, the effective concentration of tetracycline hydrochloride in the drug of the present invention is 1 to 25 μg / mL; as another embodiment, the effective concentration of tetracycline hydrochloride in the drug of the present invention is 10 to 25 μg / mL.

[0055] In order to further illustrate the present invention, the use of tetracycline hydrochloride provided by the present invention in the preparation of microsporidiosis inhibitors and the preparation of drugs for preventing or treating microsporidiosis is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1

[0057] 1. Sf9 cell subculture

[0058] (1) Microscopic observation confirmed that Spodoptera frugiperda cells Sf9 (Gibco TM Sf9 cells, catalog number: 12659017) are in good growth state and high cell confluence;

[0059] (2) Use a sterile glass pipette to gently blow up the attached cells;

[0060] (3) Transfer the cell suspension to a centrifuge tube, centrifuge at 1000 g for 5 min, and discard the supernatant;

[0061] (4) Add 1 mL of Gibco TM Sf-900 TM III SFM medium, gently blow off the cell pellet;

[0062] (5) Take two new cell culture flasks and add 2 mL of Gibco TM Sf-900 TM III SFM medium, then add 500 μL of cell suspension respectively, mix gently, let stand for 2 min, and discard the medium after the cells adhere to the wall;

[0063] (6) Add 3 mL of Gibco TM Sf-900 TM III SFM medium, mark it, and place it in a 28℃ constant temperature incubator for culture.

[0064] 2. Sf9 cell plating

[0065] (1) Open a 12-well cell culture plate and add 300 μL Gibco TM Sf-900 TM III SFM medium covers the bottom of the well;

[0066] (2) Add 3 mL of Gibco TM Sf-900 TM III SFM medium, use a sterilized glass pipette to gently blow down the cells, mix them evenly, take 10 μL of the suspension and drop it into a hemocytometer and count them directly under a microscope;

[0067] (3) Evenly distribute 3 mL of cell suspension into 12 sample wells, shake gently to make the cell suspension fill the bottom of the wells, let it stand for 3 min, remove the culture medium, and add 1 mL of new Gibco TM Sf-900 TM III SFM medium.

[0068] 3. Sf9 cell poisoning

[0069] (1) After Sf9 cells were plated in the manner of step 2, a certain amount of mature spore suspension of Bombyx mori was aspirated at a ratio of 1:10 between cells and Bombyx mori micropipette, and the suspension was centrifuged at 3500 g for 3 min, and the supernatant was discarded to obtain a spore precipitate;

[0070] (2) Treat the spore pellet with 0.1 M KOH solution (sterilized by filtration with a 0.22 μm sterile filter) for 2 min, then centrifuge at 2800 g for 2 min, discard the supernatant, and use Gibco TM Sf-900 TM III. Resuspend the spore pellet in SFM medium and add it to the sample well, then shake gently to mix evenly.

[0071] Example 2

[0072] Safety evaluation of tetracycline hydrochloride on Sf9 cells

[0073] (1) Dissolve tetracycline hydrochloride in dimethyl sulfoxide (DMSO) to a 20 mg / mL stock solution and add Gibco TM Sf-900 TM III SFM medium to a final concentration of 50, 30, 25, 20, 10, and 1 μg / mL to obtain drug solutions of different concentrations;

[0074] (2) Spodoptera frugiperda Sf9 cells were plated in a 12-well plate according to step 2 of Example 1. 12 h after plating, Gibco TM Sf-900 TM III The SFM medium was replaced with 1 mL of the drug solution of different concentrations in step (1), with the solvent containing DMSO added as a control;

[0075] (3) After 3 days of culture, discard the culture medium in the wells and add 1 mL of Gibco TM Sf-900 TM III SFM medium, add CCK-8 detection reagent according to the volume ratio of SFM medium to CCK-8 detection reagent of 1:10;

[0076] (4) Place the plate in a 28°C incubator and incubate for 30 min;

[0077] (5) Pipette 100 μL of the culture medium from each well into a 96-well plate, measure its absorbance at 450 nm using an ELISA reader, and calculate the cell survival rate. The results are as follows: Figure 1 And as shown in Table 1.

[0078] Table 1 Cell viability test results

[0079]

[0080] according to Figure 1 As can be seen from Table 1, when the concentration of tetracycline hydrochloride was 20 μg / mL, the cell survival rate was not significantly different from that of the control group without tetracycline hydrochloride, indicating that tetracycline hydrochloride at this concentration had almost no toxicity to Sf9 cells, that is, the subsequent drug inhibition experiment was carried out at a concentration of 20 μg / mL of tetracycline hydrochloride.

[0081] Example 3

[0082] Evaluation of the ability of tetracycline hydrochloride to inhibit the proliferation of Bombyx mori Nosema

[0083] (1) Cell plating and poisoning were performed according to steps 2 to 3 of Example 1;

[0084] (2) The subjects were randomly divided into four treatment groups and given the following treatments:

[0085] Control group: add DMSO of the same volume as that of the tetracycline hydrochloride group to the sample wells, mix well and culture in a cell culture incubator at 28°C;

[0086] Tetracycline hydrochloride group: Tetracycline hydrochloride was dissolved in dimethyl sulfoxide (DMSO) to a 20 mg / mL stock solution, added to the sample wells to a final concentration of 20 μg / mL, mixed evenly, and cultured in a 28°C cell culture incubator;

[0087] Albendazole group: Albendazole was dissolved in dimethyl sulfoxide (DMSO) to a 0.01 mg / mL stock solution, added to the sample wells to a final concentration of 0.01 μg / mL, mixed evenly, and cultured in a 28°C cell culture incubator;

[0088] Fumajillin group: Fumajillin was dissolved in dimethyl sulfoxide (DMSO) to a 2 mg / mL stock solution, added to the sample wells to a final concentration of 2 μg / mL, mixed evenly, and cultured in a 28°C cell culture incubator;

[0089] (3) Every three days, the old culture medium in the sample wells of different treatment groups was transferred to a sterile 1.5 mL centrifuge tube, centrifuged at 3500 g for 3 min, and the supernatant was discarded;

[0090] (4) Use fresh Gibco TM Sf-900 TM III After resuspending the precipitate in SFM medium, add it back to the corresponding sample well, then add tetracycline hydrochloride to a final concentration of 20 μg / mL, and continue to culture at 28°C;

[0091] (5) Extraction of total DNA from Sf9 cells infected with Bombyx mori microsporum: DNA was extracted using the OMEGA Tissue DNA Kit, and then the amount of pathogens in the sample was detected by fluorescence quantitative PCR. The results are shown in Table 2 and Figure 2 As shown. Among them, the reaction system of qPCR is: qPCR SYBR Green Master Mix 5.0 μL, upstream primer Nb-β-tubulin-qF 0.2 μL, downstream primer Nb-β-tubulin-qR 0.2 μL, template DNA 1.0 μL, deionized water to 10 μL; upstream primer Nb-β-tubulin-qF: 5'-AGAACCAGGAACAATGGACG-3' (SEQ ID NO: 1); downstream primer Nb-β-tubulin-qR: 5'-AGCCCAATTATTACCAGCACC-3' (SEQ ID NO: 2);

[0092] The reaction program of qPCR was as follows: 95°C for 5 min; 95°C for 10 s, 60°C for 30 s, for a total of 40 cycles;

[0093] Table 2 Number of copies of Bombyx mori microspores in different treatment groups (individuals)

[0094] Treatment Group 3d 6d 9d Control Group <![CDATA[4.48±0.12×10 5 ]]> <![CDATA[9.80±0.74×10 5 ]]> <![CDATA[1.97±0.14×10 6 ]]> Tetracycline Group <![CDATA[4.50±0.15×10 5 ]]> <![CDATA[6.25±0.20×10 5 ]]> <![CDATA[8.21±0.27×10 5 ]]> Albendazole group <![CDATA[4.37±0.12×10 5 ]]> <![CDATA[5.09±0.26×10 5 ]]> <![CDATA[6.42±0.34×10 5 ]]> Fumajillin group <![CDATA[4.29±0.07×10 5 ]]> <![CDATA[4.75±0.05×10 5 ]]> <![CDATA[4.90±0.22×10 5 ]]>

[0095] according to Figure 2 As can be seen from Table 2, on the sixth day after infection, the pathogen content in the tetracycline hydrochloride treatment group was significantly lower than that in the solvent control group to which only an equal amount of dimethyl sulfoxide was added, and there was no significant difference between the drug treatment groups. Tetracycline hydrochloride can inhibit the proliferation of Bombyx mori micropipette in infected cells.

[0096] Example 4

[0097] Evaluation of the ability of tetracycline hydrochloride to inhibit the proliferation of Bombyx mori Nosema in vivo

[0098] (1) Dissolve tetracycline hydrochloride in water to make a 100 ppm solution, apply the solution evenly on the mulberry leaves at a ratio of 1 mL of solution to 10 g of mulberry leaves, dry them in the air and set aside, and record it as mulberry leaves 1;

[0099] Dissolve tetracycline hydrochloride in water to make a 300 ppm solution, apply the solution evenly on the mulberry leaves at a ratio of 1 mL of solution to 10 g of mulberry leaves, dry them in the air and set aside, and record it as mulberry leaves 2;

[0100] Dissolve carbendazim in water to obtain a 3000 ppm solution, apply the solution evenly on the mulberry leaves at a ratio of 1 mL of solution to 10 g of mulberry leaves, dry them in the air and set aside, and record it as mulberry leaves 3;

[0101] According to the ratio of water to mulberry leaves 1mL:10g, evenly spread the drug solution on the mulberry leaves, dry them and set aside, recorded as Mulberry Leaves 4.

[0102] (2) Obtain fat bodies from silkworm pupae infected with Bombyx mori Nosema, grind them and perform density gradient centrifugation to obtain a clean single suspension of Bombyx mori Nosema; 4 After the poisoning was successful, the mice were randomly divided into 4 treatment groups and treated as follows:

[0103] Vehicle Control: Feed the mulberry leaves 4 in step (1) twice a day. After feeding the treated mulberry leaves 4 times in a row (i.e., after feeding for 2 days), feed the treated leaves once a day (i.e., feed the mulberry leaves 4 in step (1) once a day and feed the untreated mulberry leaves once a day) until the birds are clustered.

[0104] 30 ppm tetracycline hydrochloride group: same as the control group, except that mulberry leaf 4 was replaced by mulberry leaf 1 in step (1);

[0105] 100 ppm Tetracycline hydrochloride group: same as the control group, except that mulberry leaf 4 was replaced by mulberry leaf 2 in step (1);

[0106] Carbendazim group: same as the control group, except that mulberry leaf 4 was replaced by mulberry leaf 3 in step (1).

[0107] During the feeding process, each treatment group was sampled every two days, and total DNA was extracted according to the method of Example 1. The pathogen load was analyzed by quantitative PCR. The results are shown in Table 3 and Figure 3 shown.

[0108] Table 3 Number of copies of Bombyx mori microspores in different treatment groups (individuals)

[0109] Treatment Group 2d 4d 6d 8d 10d Control group <![CDATA[1.15±0.13×10 4 ]]> <![CDATA[1.49±0.22×10 4 ]]> <![CDATA[4.05±0.26×10 4 ]]> <![CDATA[5.75±0.34×10 5 ]]> <![CDATA[2.95±0.03×10 6 ]]> 30ppm Tetracycline Hydrochloride Group <![CDATA[9.95±1.25×10 3 ]]> <![CDATA[1.26±0.04×10 4 ]]> <![CDATA[2.49±0.36×10 4 ]]> <![CDATA[1.32±0.13×10 5 ]]> <![CDATA[7.85±0.33×10 5 ]]> 100ppm Tetracycline Hydrochloride Group <![CDATA[9.95±0.79×10 3 ]]> <![CDATA[1.25±0.29×10 4 ]]> <![CDATA[1.56±0.24×10 4 ]]> <![CDATA[2.68±0.24×10 4 ]]> <![CDATA[4.81±0.12×10 4 ]]> Carbendazim group <![CDATA[8.91±0.81×10 3 ]]> <![CDATA[1.09±0.22×10 4 ]]> <![CDATA[1.41±0.20×10 4 ]]> <![CDATA[1.56±0.23×10 4 ]]> <![CDATA[2.87±0.85×10 4 ]]>

[0110] According to Table 3 and Figure 3 It can be seen that in the tetracycline hydrochloride treatment group, the pathogen content on the 10th day was significantly lower than that in the solvent control group to which only an equal amount of dimethyl sulfoxide was added, while there was no significant difference between the carbendazim treatment group and the tetracycline hydrochloride treatment group, indicating that tetracycline hydrochloride can inhibit the proliferation of Bombyx mori micropipette in infected silkworms.

[0111] Example 5

[0112] RK13 cell recovery and subculture

[0113] (1) Take out the cryopreserved tube containing rabbit kidney cells RK13 from the liquid nitrogen storage tank and place it in a 37°C water bath to completely thaw;

[0114] (2) Centrifuge at 2000 g for 5 min and discard the cell freezing solution;

[0115] (3) Resuspend the cells in 3 mL of preheated MEM medium (added with 1% 5000 U / mL penicillin-streptomycin solution and 10% heat-inactivated fetal bovine serum), transfer the cells to a cell culture flask, and place at 37°C and 5% CO. 2 Culture overnight in a constant temperature incubator;

[0116] (4) Check the cell growth using an inverted microscope and replace the culture medium every 3 days;

[0117] (5) When the cell density in the culture flask reaches more than 90%, discard the culture medium and add 1 mL of preheated phosphate buffered saline (PBS) to the culture flask to remove the components in the culture medium;

[0118] (6) Discard PBS, add 1 mL of preheated trypsin cell digestion solution, and place in a 37°C incubator for digestion until all cells are separated from the wall of the culture flask. Add 1 mL of MEM medium to the culture flask to terminate the trypsin reaction;

[0119] (7) Remove the suspension, centrifuge at 2000 g for 5 min, discard the supernatant, resuspend the cells in 1 mL of preheated MEM medium, transfer an appropriate amount of cells to a new cell culture flask, and place at 37°C and 5% CO. 2 Culture in a constant temperature incubator.

[0120] Example 6

[0121] Safety evaluation of tetracycline hydrochloride on RK-13 ​​cells

[0122] (1) Tetracycline hydrochloride was dissolved in dimethyl sulfoxide (DMSO) to a 20 mg / mL stock solution, and added to MEM culture medium to a final concentration of 50, 30, 25, 20, 10, and 1 μg / mL to obtain drug solutions of different concentrations;

[0123] (2) Rabbit kidney RK13 cells were plated into a 24-well plate in the manner of step 1 of Example 5. After 12 h of plating, the MEM medium was replaced with 1 mL of the drug solution of different concentrations in step (1), and the solvent with DMSO added was used as a control;

[0124] (3) After 3 days of culture, discard the culture medium in the well, add 1 mL of serum-free MEM or 1640 culture medium, and add CCK-8 detection reagent at a volume ratio of 1:10;

[0125] (4) Place the plate in a 37°C incubator and incubate for 30 min;

[0126] (5) Pipette 100 μL of the culture medium from each well into a 96-well plate, measure its absorbance at a wavelength of 450 nm using an ELISA reader, and calculate the cell survival rate. The results are as follows: Figure 4 And as shown in Table 4.

[0127] Table 4 Cell viability test results

[0128] Tetracycline hydrochloride concentration (μg / mL) 50 25 10 1 0 Cell survival rate (%) 76.3 97.4 100.6 100.9 100.0

[0129] according to Figure 4 As can be seen from Table 4, there is no significant difference in cell survival rate when the concentration of tetracycline hydrochloride is 25 μg / mL, that is, the subsequent drug inhibition experiment is carried out at a concentration of 25 μg / mL of tetracycline hydrochloride.

[0130] Example 7

[0131] Evaluation of the ability of tetracycline hydrochloride to inhibit the proliferation of encephalitis microsporidia in rabbits

[0132] (1) Rabbit kidney cells RK13 were digested according to step 1 of Example 5, and 5×10 4 The cells were inoculated at a density of 100 cells / mL at 24-well cell culture plates, and 1 mL of MEM medium containing 10% fetal bovine serum was added to each well. The plates were incubated at 37°C and 5% CO. 2 Incubate overnight in an incubator;

[0133] (2) On the second day, the animals were randomly divided into two treatment groups and given the following treatments:

[0134] Vehicle Control: The culture medium in the culture plate was replaced with 1 mL of fresh MEM medium containing the same volume of solvent (DMOS) as that in the tetracycline hydrochloride group;

[0135] Tetracycline hydrochloride group: replace the culture medium in the culture plate with 1 mL of fresh MEM medium containing tetracycline hydrochloride at a final concentration of 25 μg / mL;

[0136] (3) After 8 hours of culture in the control group and the tetracycline hydrochloride group, mature spores of rabbit encephalitis microsporidia were added to the sample wells at a ratio of 5:1 between rabbit encephalitis microsporidia and cells, and gently shaken to mix;

[0137] (4) On the 3rd and 6th days after infection, the medium containing drugs or solvents was replaced with fresh medium, the microsporidia in the supernatant of the old medium were centrifuged at 8000 g for 5 min, resuspended in fresh medium, and added back to the corresponding sample wells;

[0138] (5) Parasitic vacuole staining and fluorescence counting: Take cell samples from the sample wells of the control group and the tetracycline hydrochloride group on the 3rd and 6th days, discard the culture medium in the wells; wash three times with 500 μL PBST (PBS buffer containing 0.01% Tween 20), and then fix the samples with 300 μL 4% paraformaldehyde for 15 min; wash the fixed samples three times with 500 μL PBST, each time for 5 min; add Fluorescent Brightener 28 at a ratio of 1:1000 and stain for 30 min; after staining, wash three times with 500 μL PBST, each time for 5 min; take pictures of 20 random fields of view of each sample well under an inverted fluorescence microscope, and count the stained parasitic vacuole. The results are shown in Figure 2. Figures 5-6 And as shown in Table 5.

[0139] Table 5 The number of encephalitis microsporidia in rabbits in different treatment groups (individuals)

[0140] Treatment Group 3d 6d VehicleControl Group 673 3316 Tetracycline Group 151 459

[0141] according to Figures 5-6 As shown in Table 5, when tetracycline hydrochloride was added to the culture medium at a final concentration of 25 μg / mL, observation under a fluorescence microscope on the sixth day after infection revealed that the blue fluorescent signal area representing the pathogen parasitic vesicles in the tetracycline hydrochloride group was significantly lower in both intensity and quantity than the control group that only added an equal amount of solvent ( Figure 5 ). By counting parasitic vacuoles, it can be seen that on the third day of infection, the pathogen content after tetracycline hydrochloride treatment was much lower than that of the control group with only an equal amount of solvent added ( Figure 6 and Table 5).

[0142] (6) Extraction of total DNA from RK13 cells infected with rabbit encephalitis microsporidia: DNA was extracted using the OMEGA Tissue DNA Kit, and then the amount of pathogens in the sample was detected by fluorescence quantitative PCR. The results are shown in Table 6 and Figure 7 As shown. Among them, the reaction system of qPCR is: qPCR SYBR Green Master Mix 5.0 μL, upstream primer EcunF1 0.2 μL, downstream primer EcunR2 0.2 μL, template DNA 1.0 μL, deionized water to 10 μL; upstream primer EcunF1: 5'-TCCTAGTAATAGCGGCTGACGAA-3' (SEQ ID NO: 3); downstream primer EcunR2: 5'-ACTCAGGACTCAGACCTTCCGA-3' (SEQ ID NO: 4);

[0143] The reaction program of qPCR was as follows: 95°C for 5 min; 95°C for 10 s, 60°C for 30 s, for a total of 40 cycles;

[0144] Table 6 Number of copies of encephalitis microsporidia in rabbits in different treatment groups (individuals)

[0145] Treatment Group 3d 6d VehicleControl Group <![CDATA[7.77±0.14×10 7 ]]> <![CDATA[1.67±0.08×10 9 ]]> Tetracycline Group <![CDATA[2.77±0.33×10 7 ]]> <![CDATA[1.53±0.03×10 8 ]]>

[0146] according to Figure 7 As can be seen from Table 6, on the sixth day after infection, the pathogen content in the tetracycline hydrochloride treatment group was significantly lower than that in the solvent control group to which only an equal amount of dimethyl sulfoxide was added, and tetracycline hydrochloride can inhibit the proliferation of rabbit encephalitis microsporidia.

[0147] Example 8

[0148] Dose-dependent inhibitory effect of tetracycline hydrochloride on encephalitis microsporidia in rabbits

[0149] According to the method of Example 7, different concentrations of tetracycline hydrochloride (25, 10, 1, 0.1 μg / mL) and solvent control DMSO were added to RK13 cells infected with rabbit encephalitis microsporidia; after 8 hours of culture, the mature spores of rabbit encephalitis microsporidia were added to the sample wells at a ratio of 5:1 between rabbit encephalitis microsporidia and cells, and gently shaken to mix; on the 3rd and 6th days after infection, fresh culture medium containing drugs or solvents was replaced, and the microsporidia in the supernatant of the old culture medium were centrifuged at 8000g for 5 minutes, resuspended in fresh culture medium and added back to the corresponding sample wells. On the 6th day after infection, the cells were counted according to the method of Example 7, and the results were as follows: Figure 8 And as shown in Table 7.

[0150] Table 7 The number of microsporidia in rabbits with encephalitis in different treatment groups (individuals)

[0151] Tetracycline hydrochloride concentration (μg / mL) 25 10 1 0.1 0 Loading capacity (pcs) 173 530 1834 2394 3706

[0152] according to Figure 8 As shown in Table 7, tetracycline hydrochloride still has a certain inhibitory effect at a lower dose (0.1 μg / mL), and the inhibitory effect on rabbit encephalitis microsporidia gradually increases with the increase of drug concentration, showing an overall dose-dependent effect.

[0153] Example 9

[0154] 1. Establishment of a wild-type mouse model of microsporidia infection

[0155] (1) First, C57BL / 6J female mice were divided into three groups: group a as a blank control group (no treatment), group b as a control group (Vehicle Control) (infected with rabbit encephalitis microsporidia and not treated with drugs), and group c as an experimental group (Tetracycline) (infected with rabbit encephalitis microsporidia and treated with drugs), and left for one week to adapt to the environment;

[0156] (2) Mice in the control and experimental groups were injected with dexamethasone sodium phosphate for 7 consecutive days to achieve immunosuppression, and then intraperitoneally injected with 1×10 7 A purified rabbit encephalitis microsporidia.

[0157] 2. Evaluation of the ability of tetracycline hydrochloride to inhibit the proliferation of rabbit encephalitis microsporidia at individual levels

[0158] Step 1 After the experimental group was infected with rabbit encephalitis microsporidia, the mice were treated by subcutaneous injection of tetracycline hydrochloride at 25 mg / kg b.w / day for 7 consecutive days; the control group was injected with an equal amount of solvent. The feces of the three groups of mice were collected for 7 days of drug administration and 7 days of drug withdrawal, and the pathogen quantity was detected by probe method absolute quantitative PCR. The results are shown in Table 8 and Fig. 9As shown. The PCR reaction system: fluorescent PCR enzyme reaction solution (probe method) 8.0 μL, upstream primer EcunF1 0.2 μL, downstream primer EcunR2 0.2 μL, Ec-ssu-Taqman 0.1 μL, template DNA 1.5 μL, deionized water to 10 μL; upstream primer EcunF1 (SEQ ID NO: 3); downstream primer EcunR2 (SEQ ID NO: 4); Ec-ssu-Taqman: 5'-TCGTCCAGTGCGTCATCTTAGATAGCG-3' (SEQ ID NO: 5)

[0159] The PCR reaction program was: 37°C for 2 min; 95°C for 30 s, 95°C for 10 s, and 60°C for 30 s, for a total of 40 cycles;

[0160] Table 8 Number of copies of encephalitis microsporidia in rabbits in different treatment groups (individuals)

[0161] Treatment Group 7d 14d VehicleControl Group <![CDATA[1.86±0.12×10 4 ]]> <![CDATA[5.56±1.08×10 4 ]]> Tetracycline Group <![CDATA[2.09±0.09×10 4 ]]> <![CDATA[2.42±0.19×10 4 ]]>

[0162] According to Table 8 and Fig. 9 It can be seen that in the experimental group treated with tetracycline hydrochloride, the pathogen content was much lower than that in the control group to which only an equal amount of solvent was added. Tetracycline hydrochloride can inhibit the proliferation of rabbit encephalitis microsporidia at the individual mouse level.

[0163] Example 10

[0164] Evaluation of the ability of tetracycline hydrochloride to inhibit Helen's encephalitis microsporidia

[0165] (1) Rabbit kidney cells RK13 were digested according to step 1 of Example 5, and 5×10 4 The cells were inoculated at a density of 100 cells / mL at 24-well cell culture plates, and 1 mL of MEM medium containing 10% fetal bovine serum was added to each well. The plates were incubated at 37°C and 5% CO. 2 Incubate overnight in an incubator;

[0166] (2) On the second day, the animals were randomly divided into two treatment groups and given the following treatments:

[0167] Vehicle Control: Replace the culture medium in the culture plate with 1 mL of fresh MEM medium containing the same volume of solvent (DMSO) as that in the tetracycline hydrochloride group;

[0168] Tetracycline hydrochloride group: replace the culture medium in the culture plate with 1 mL of fresh MEM medium containing tetracycline hydrochloride at a final concentration of 25 μg / mL;

[0169] (3) After 8 hours of culture in the control group and the tetracycline hydrochloride group, the mature spores of Helenitis microsporidia were added to the sample wells at a ratio of 5:1 between Helenitis microsporidia and cells, and gently shaken to mix;

[0170] (4) On the 3rd and 6th days after infection, the medium containing drugs or solvents was replaced with fresh medium, the microsporidia in the supernatant of the old medium were centrifuged at 8000 g for 5 min, resuspended in fresh medium, and added back to the corresponding sample wells;

[0171] (5) Parasitic vacuole staining and fluorescence counting: Take cell samples from the sample wells of the control group and the tetracycline hydrochloride group on the 3rd and 6th days, discard the culture medium in the wells; wash three times with 500 μL PBST (PBS buffer containing 0.01% Tween 20), and fix the samples with 300 μL 4% paraformaldehyde for 12 min; wash the fixed samples three times with 500 μL PBST, each time for 5 min; add Fluorescent Brightener 28 at a ratio of 1:1000 and stain for 30 min; after staining, wash three times with 500 μL PBST, each time for 5 min; take pictures of 20 random fields of view of each sample well under an inverted fluorescence microscope, and count the stained parasitic vacuole. The results are shown in Figure 2. Fig.10 and as shown in Table 9.

[0172] Table 9 Microsporidia load of Helen's encephalitis in different treatment groups (individuals)

[0173] Treatment Group 3d 6d VehicleControl Group 560 4971 Tetracycline Group 456 1413

[0174] according to Fig.10 As can be seen from Table 9, after RK13 cells were infected with Helenitis microsporidia, tetracycline hydrochloride was added to the culture medium at a final concentration of 25 μg / mL. The pathogen load on the sixth day after infection was much lower than that of the control group to which only an equal amount of solvent was added. Tetracycline hydrochloride can inhibit the proliferation of Helenitis microsporidia at the cellular level.

[0175] (6) Extraction of total DNA from RK13 cells infected with Helen's encephalitis microsporidia: DNA was extracted using the OMEGA TissueDNAKit kit, and then the amount of pathogens in the sample was detected by fluorescence quantitative PCR. The results are shown in Tables 10 and Fig.11 As shown. Among them, the reaction system of qPCR is: qPCR SYBR Green MasterMix 5.0 μL, upstream primer EhelF1 0.2 μL, downstream primer EhelR2 0.2 μL, template DNA 1.0 μL, deionized water to 10 μL; upstream primer EhelF1: 5'-GAATGATTGAACAAGTTATTTTGAATGTG-3' (SEQ ID NO: 6); downstream primer EhelR2: 5'-AACACGAAAGACTCAGACCTCTCA-3' (SEQ ID NO: 7);

[0176] The reaction program of qPCR was as follows: 95°C for 5 min; 95°C for 10 s, 60°C for 30 s, for a total of 40 cycles;

[0177] Table 10 The number of copies of Helen's encephalitis microsporidia in different treatment groups (individuals)

[0178] Treatment Group 3d 6d VehicleControl Group <![CDATA[1.39±0.15×10 8 ]]> <![CDATA[2.43±0.21×10 9 ]]> Tetracycline Group <![CDATA[4.22±0.62×10 7 ]]> <![CDATA[2.46±0.34×10 8 ]]>

[0179] according to Fig.11 As can be seen from Table 10, on the sixth day after infection, the pathogen content in the tetracycline hydrochloride treatment group was significantly lower than that in the solvent control group to which only an equal amount of dimethyl sulfoxide was added, and tetracycline hydrochloride can inhibit the proliferation of Helen's encephalitis microsporidia.

[0180] Embodiment 11

[0181] Dose-dependent inhibitory effect of tetracycline hydrochloride on Helen's encephalitis microsporidia

[0182] According to the method of Example 10, different concentrations of tetracycline hydrochloride (25, 10, 1, 0.1 μg / mL) and solvent control DMSO were added to RK13 cells infected with Helenitis microsporidia; after 8 hours of culture, mature spores of Helenitis microsporidia were added to the sample wells at a ratio of Helenitis microsporidia to cells of 5:1, and gently shaken to mix; on the 6th day after infection, the cells were counted according to the method of Example 10, and the results were as follows: Fig.12 As shown in Table 11.

[0183] Table 11 Microsporidia load of Helen's encephalitis in different treatment groups (individuals)

[0184]

[0185] according to Fig.12 As can be seen from Table 11, tetracycline hydrochloride still has a certain inhibitory effect at a lower dose (0.1 μg / mL), and the inhibitory effect on Helen's encephalitis microsporidia gradually increases with the increase of drug concentration, showing a dose-dependent effect overall.

[0186] Example 12

[0187] 1. Establishment of a wild-type mouse model of microsporidia infection

[0188] (1) First, C57BL / 6J female mice were divided into three groups: group a as a blank control group (no treatment), group b as a control group (Vehicle Control) (infected with Helen's encephalitis microsporidia and not treated with drugs), and group c as an experimental group (Tetracycline) (infected with Helen's encephalitis microsporidia and treated with drugs), and left for one week to adapt to the environment;

[0189] (2) After the mice in the control and experimental groups were injected with dexamethasone sodium phosphate for 7 consecutive days to achieve the immunosuppressive effect, 1×10 7 A purified Helen's encephalitis microsporidia.

[0190] 2. Evaluation of the ability of tetracycline hydrochloride to inhibit the proliferation of Helen's encephalitis microsporidia at individual levels

[0191] Step 1 After the experimental group was infected with Helen's encephalitis microsporidia, the mice were treated by subcutaneous injection of tetracycline hydrochloride at 25 mg / kg b.w / day for 7 consecutive days; the control group was injected with an equal amount of solvent. The feces of the three groups of mice were collected for 7 days of drug administration and 7 days of drug withdrawal, and the pathogen quantity was detected by probe method absolute quantitative PCR. The results are shown in Table 12 and Fig.13 As shown. The PCR reaction system: fluorescent PCR enzyme reaction solution (probe method) 8.0 μL, upstream primer EhelF1 0.2 μL, downstream primer EhelR2 0.2 μL, Eh-ssu-Taqman 0.1 μL, template DNA 1.5 μL, deionized water to 10 μL; upstream primer EhelF1 (SEQ ID NO: 6); downstream primer EhelR2 (SEQ ID NO: 7); Eh-ssu-Taqman: 5'-TCCTTTGTACACACCGCCCGTCGCTA-3' (SEQ ID NO: 8)

[0192] The PCR reaction program was: 37°C for 2 min; 95°C for 30 s, 95°C for 10 s, and 60°C for 30 s, for a total of 40 cycles;

[0193] Table 12 The number of copies of Helen's encephalitis microsporidia in different treatment groups (individuals)

[0194] Treatment Group 7d 14d VehicleControl Group <![CDATA[2.28±0.22×10 5 ]]> <![CDATA[1.03±0.17×10 6 ]]> Tetracycline Group <![CDATA[1.92±0.17×10 5 ]]> <![CDATA[1.42±0.29×10 5 ]]>

[0195] According to Table 12 and Fig.13 It can be seen that in the experimental group treated with tetracycline hydrochloride, the pathogen content was much lower than that in the control group to which only an equal amount of solvent was added. Tetracycline hydrochloride can inhibit the proliferation of Helen's encephalitis microsporidia at the individual mouse level.

[0196] Comparative Example 1

[0197] Evaluation of the ability of tetracycline hydrochloride to inhibit the proliferation of Nosema orientalis

[0198] (1) Preparation of antibiotic stock solution

[0199] Dissolve tetracycline hydrochloride in dimethyl sulfoxide (DMSO) to a 25 mg / mL stock solution;

[0200] (2) Preparation of bee feed

[0201] Sterile sucrose solution: add 50 μL of 50% sterile DMSO solution to 25 mL of sucrose solution;

[0202] Sterile sucrose solution of tetracycline hydrochloride: Use sterile sucrose solution to dissolve the tetracycline hydrochloride stock solution to make the final concentration of tetracycline hydrochloride 50μg / mL;

[0203] (3) Preparation of suspension of Nosema ceranae: 10 foraging bees infected with Nosema ceranae were captured, and the midgut of the bees was dissected in a clean bench. 1 mL of PBS was added for grinding, and the suspension was centrifuged at 1000 g for 10 min. After the supernatant was removed, the precipitate was resuspended with 1 mL of PBS, and 24 mL of 50% sterile sucrose solution was added for mixing. The Nosema ceranae was counted under a microscope using a hemocytometer, and the suspension was diluted with 50% sterile sugar water to a concentration of 5 × 10 4 Pieces / μL.

[0204] (4) Select Italian honey bees (Apis mellifera) from the same brood comb, and select the bees that have been out of the hive for less than 24 hours. Feed each bee with 2 μL of the suspension of Nosema orientalis prepared in step (3).

[0205] A total of 20 bees were fed and then divided into 2 treatment groups and treated as follows:

[0206] Control group (DMSO): fed with sterile sucrose solution from step (2);

[0207] Tetracycline hydrochloride group (TET): fed with the tetracycline hydrochloride sterile sucrose solution in step (2);

[0208] On the 6th day after the bees in the control group and tetracycline hydrochloride group were infected with microsporidia, all bees were killed and the midgut was removed to calculate the reproduction of microsporidia. After DNA was extracted using the OMEGATissue DNAKit kit, the amount of pathogens in the samples was detected by fluorescent quantitative PCR. The results are shown in Table 13 and Fig.14 As shown. Among them, the reaction system of qPCR is: qPCR SYBRGreen MasterMix 5.0 μL, upstream primer NcerF1 0.2 μL, downstream primer NcerR2 0.2 μL, template DNA 1.0 μL, deionized water to 10 μL; upstream primer NcerF1: 5'-TCGTGGTAAGCTTTGCTCCT-3' (SEQ ID NO: 9); downstream primer Ncer R2: 5'-ACCTCTAAACACGGCTGCTAC-3' (SEQ ID NO: 10);

[0209] The reaction program of qPCR was as follows: 95°C for 5 min; 95°C for 10 s, 60°C for 30 s, for a total of 40 cycles;

[0210] Table 13 Number of copies of encephalitis microsporidia in rabbits in different treatment groups (individuals)

[0211] Treatment Group 6d DMSO group <![CDATA[2.67±0.75×10 5 ]]> TET Group <![CDATA[3.58±1.2×10 5 ]]>

[0212] according to Fig.14 As can be seen from Table 13, the amount of oriental honey bee Nosema in the bees treated with tetracycline hydrochloride was slightly higher than that in the control group to which an equal amount of solvent was added, but there was no significant difference, indicating that tetracycline hydrochloride was not effective against oriental honey bee Nosema.

[0213] It can be seen from the above content that tetracycline hydrochloride can inhibit the proliferation of microsporidia and prevent and / or treat microsporidiosis.

[0214] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of tetracycline hydrochloride in the preparation of microsporidia inhibitors; The microsporidia include invertebrate microsporidia and / or vertebrate microsporidia; The invertebrate microsporidia include Nosema bombycis; The vertebrate microsporidia include Encephalitozoon hellem and / or Encephalitozoon cuniculi.

2. The application according to claim 1, characterized in that: The microsporidia inhibitors include microsporidia proliferation inhibitors.

3. Use of tetracycline hydrochloride in the preparation of drugs for preventing and / or treating microsporidiosis; The microsporidiosis includes invertebrate microsporidiosis and / or vertebrate microsporidiosis; The invertebrate microsporidiosis includes Bombyx mori microsporidia disease; The vertebrate microsporidiosis includes Helena encephalitis microsporidiosis and / or rabbit encephalitis microsporidiosis.

4. A microsporidia inhibitor, characterized in that: Active ingredients include tetracycline hydrochloride; The microsporidia include invertebrate microsporidia and / or vertebrate microsporidia; The invertebrate microsporidia include Bombyx mori microsporidia; The vertebrate microsporidia include Encephalitis Helenii and / or Encephalitis Leptospira.

5. The microsporidia inhibitor according to claim 4, characterized in that: The effective concentration of tetracycline hydrochloride in the microsporidia inhibitor is ≥0.1 μg / mL.

6. A feed for preventing and / or treating nematode disease in silkworms, characterized in that: The feed comprises mulberry leaves; the mulberry leaves contain tetracycline hydrochloride and / or the microsporidia inhibitor according to claim 4 or 5.

7. The method for preparing the feed according to claim 6, characterized in that: The method comprises the following steps: applying the microsporidia inhibitor to mulberry leaves and then drying them to obtain the feed.

8. The preparation method according to claim 7, characterized in that: The mass volume ratio of the mulberry leaf to the microsporidia inhibitor is 10 g:1 mL; The concentration of tetracycline hydrochloride in the microsporidia inhibitor is 30-100 ppm.

9. A drug for preventing and / or treating vertebrate microsporidiosis, characterized in that: The drug comprises the microsporidia inhibitor according to claim 4 or 5 and a pharmaceutically acceptable excipient; The vertebrate microsporidia include Encephalitis Helenii and / or Encephalitis Leptospira.

10. The drug according to claim 9, characterized in that The effective concentration of tetracycline hydrochloride in the medicine is 0.1-25 μg / mL.

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