Traditional Chinese medicine composition for resisting giant frog blue eye disease pathogen Elizabeth mil and application of traditional Chinese medicine composition for resisting giant frog blue eye disease pathogen Elizabeth mil

Abstract

The invention discloses a traditional Chinese medicine composition for resisting giant frog blue eye disease pathogen Elizabeth mileli and application of the traditional Chinese medicine composition, and relates to the technical field of aquaculture disease prevention and treatment. It is found for the first time that the radix scutellariae has a good effect of inhibiting the Elizabeth mille, and the herba houttuyniae does not have the effect of inhibiting the Elizabeth mille but can enhance the inhibiting effect of the radix scutellariae on the Elizabeth mille, that is, the herba houttuyniae has a synergistic enhancement effect; the traditional Chinese medicine composition of the radix scutellariae and the herba houttuyniae is expected to be applied to treatment of the blue eye disease of the giant spiny frog, and the Chinese herbal medicine composition is used for replacing antibiotics in the breeding process.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture disease prevention and control, and in particular to a traditional Chinese medicine composition against Elizabethkingia miricola, the pathogen of the blue eye disease of the spiny frog, and its application. Background Art

[0002] Elizabethkingia is a Gram-negative, obligately aerobic, sporeless, non-fermenting, non-motile, slender and slightly curved bacillus. It can decompose fructose, glucose, lactose, maltose, mannitol and trehalose, but cannot decompose arabinose, raffinose, salicin, sucrose and xylose to produce acid. It produces H2S, is negative for malonate utilization, negative for nitrate reduction, grows well at 37 °C, does not grow at 5 °C and 42 °C, and can hydrolyze esculin.

[0003] Elizabethkingia miricola is an important pathogenic microorganism that infects both humans and frogs. In the aquaculture industry, this bacterium can infect a variety of farmed and wild frogs, with strong infectivity and high lethality, causing various symptoms in frogs such as meningitis (tilted head), cataracts, protruding eyeballs, blue eyes, spinning in the water, listlessness, loss of appetite, and abdominal distension.

[0004] Existing studies have shown that Elizabethkingia miricola may be naturally resistant to antibiotics commonly used to treat Gram-negative bacterial infections (such as aminoglycosides, β-lactam antibiotics, tetracyclines, and chloramphenicol, etc.). Therefore, empirical use of antibiotics not only is not conducive to disease control but may also cause its epidemic spread. In addition, all Elizabethkingia miricola strains isolated from the tilted head disease currently show high drug resistance, making the disease difficult to treat.

[0005] The existing treatment plan for the tilted head disease mainly uses antibiotics to inhibit Elizabethkingia miricola. However, the extensive use of antibiotics will pose a huge safety risk to frog products. The abuse of antibiotics has a negative impact on aspects such as environmental microbial drug resistance, the animal's own immunity, and intestinal microorganisms, and even passes from animals to humans through the food chain. Therefore, reducing or even avoiding the use of antibiotics in the breeding process of commercial frogs (such as spiny frogs) has become an urgent problem to be solved.

[0006] In order to overcome the defects of traditional antibacterial methods, traditional Chinese herbs have always attracted much attention. Through screening, traditional Chinese herbs or combinations of traditional Chinese herbs can not only achieve good disease prevention and control effects but also solve the drug resistance and drug residue problems brought by traditional antibiotics, and have good application prospects in the breeding process of spiny frogs. Summary of the Invention

[0007] The purpose of the present invention is to provide a traditional Chinese medicine composition against Elizabethkingia miricola, the pathogen of the blue eye disease of the spiny frog, and its application, so as to replace the application of traditional antibiotics in the breeding of commercial frogs and overcome the defects of traditional antibacterial methods.

[0008] To achieve the above object, on the one hand, the present invention provides a traditional Chinese medicine composition against Elizabethkingia miricola, the pathogen of blue eye disease in spiny-breasted frogs. The traditional Chinese medicine composition is a mixture of equal-weight extracts of Scutellaria baicalensis and Houttuynia cordata prepared by the water decoction method. Among them, the extract of Scutellaria baicalensis can directly inhibit Elizabethkingia miricola, and the extract of Houttuynia cordata does not have the effect of directly inhibiting Elizabethkingia miricola, but can enhance the inhibitory effect of the extract of Scutellaria baicalensis on Elizabethkingia miricola.

[0009] On the other hand, the present invention provides an application of the above traditional Chinese medicine composition in the preparation of a drug for inhibiting Elizabethkingia miricola.

[0010] On the other hand, the present invention provides an application of the above traditional Chinese medicine composition in the breeding of spiny-breasted frogs.

[0011] Therefore, a traditional Chinese medicine composition against Elizabethkingia miricola, the pathogen of blue eye disease in spiny-breasted frogs, and its application of the present invention have the following beneficial effects:

[0012] (1) In the present invention, Elizabethkingia miricola was obtained by isolating and culturing the eye bacteria of spiny-breasted frogs with blue eye disease; the drug sensitivity tests were carried out on 11 equal-weight extracts of Chinese herbal medicines together, and it was proved that the extract of Scutellaria baicalensis and the extract of Coptis chinensis have the ability to inhibit the growth of Elizabethkingia miricola, while the extracts of 9 Chinese herbal medicines such as Houttuynia cordata, Andrographis paniculata, Lonicera japonica, Isatis indigotica, Taraxacum mongolicum, Phellodendron amurense, Pulsatilla chinensis, Viola philippica, and Cortex fraxini do not have the effect of directly inhibiting Elizabethkingia miricola;

[0013] (2) In the present invention, it was first discovered that among the extracts of 10 Chinese herbal medicines, only the extract of Houttuynia cordata can enhance the inhibitory effect of the extract of Scutellaria baicalensis on Elizabethkingia miricola, but the extract of Houttuynia cordata cannot enhance the inhibitory effect of the extract of Coptis chinensis on Elizabethkingia miricola. A precipitate is formed after the extract of Coptis chinensis and the extract of Scutellaria baicalensis are mixed, and the mixture no longer has the effect of inhibiting Elizabethkingia miricola;

[0014] (3) It is expected to apply the mixture of the extract of Scutellaria baicalensis and the extract of Houttuynia cordata to the treatment of blue eye disease in spiny-breasted frogs and replace antibiotics to prepare a new drug against Elizabethkingia miricola.

[0015] The technical solution of the present invention will be further described in detail below through examples. Detailed implementation mode

[0016] The technical solution of the present invention will be further described below through examples.

[0017] It should be understood that the specific embodiments described herein are merely for explaining the embodiments of the present invention and are not intended to limit the embodiments of the present invention. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of this application.

[0018] Example 1

[0019] S1. Bacterial strain activation and identification.

[0020] S11. Streak: Take a typical frog with blue eye disease of R. spinosa, scrape the eyes to obtain bacteria, and then inoculate onto an antibiotic-free LB plate using the conventional streak method and incubate at 28°C for 20 hours.

[0021] The cornea of the frog with blue eye disease is cloudy and the pupil is light blue.

[0022] S12. Expansion: Pick a single clone from the solid LB plate and transfer it to liquid LB, and culture it at 28°C overnight.

[0023] S13. Bacterial Species Identification: PCR amplification was performed using the bacterial suspension as a template using the universal bacterial 16S rDNA primers 27F / 1522R, provided by Zhejiang Shangya Biotechnology Co., Ltd. The amplified product was sent to Zhejiang Shangya Biotechnology Co., Ltd. for gene sequencing. BLAST analysis of the sequencing results against the NCBI database revealed 100% identity with Elizabethkingia miricola, confirming that the isolated pathogen of blue eye disease in Rana spinosa was Elizabethkingia miricola.

[0024] S2. Drug sensitivity test.

[0025] S21. Bacterial propagation: transfer Elizabeth Millipore to 50 mL of antibiotic-free liquid LB at a ratio of 1:1000, culture on a shaker at 28°C for 14 h, and then harvest the bacteria. 600 Make it equal to 1.

[0026] S22. Plate preparation: Pour the sterilized LB solid culture medium into sterile culture dishes, about 20 mL per dish.

[0027] S23. Bacterial count: take OD 600 The bacterial solution was diluted to 10 by 10-fold gradient. -7 , take 100 μL of the diluted bacterial solution to plate and culture at 28℃ for about 24 h.

[0028] Count the specific number of colonies between 30 and 300, and then multiply it by the corresponding dilution factor to calculate the OD 600 The total number of viable bacteria was roughly calculated using a bacterial solution with a concentration of approximately 1.

[0029] The results showed that when diluted to 10 -6 , the number of colonies was 87. Multiplying this by the dilution factor of 10 6 and the volume of the bacterial suspension plated, which was 100 μL, the total viable count in the bacterial suspension with an OD 600 equal to 1 was approximately 8.7×10 8 CFU / mL.

[0030] According to the WS / T 650—2019 standard, the bacterial concentration used in the inhibition zone test was approximately 5.0×10 5 -5.0×10 6 CFU / mL. In subsequent experiments, the bacterial suspension with an OD 600 equal to 1 was diluted 200-fold before use.

[0031] S24, Bacterial spreading: Take a non-antibiotic plate, add 100 μL of the diluted bacterial suspension to the surface of the sterilized plate, spread it with a glass bead for spreading, place it upright in an incubator at 28 °C for 15 min, and set a negative control simultaneously.

[0032] S25, Punching holes in the plate: Use a hole punch to punch holes in the spread plate, one hole in the middle and 4 - 5 holes around it.

[0033] S26, Preparation of Chinese herbal medicine extracts by decoction method:

[0034] Weigh 8 times the volume of water of the Chinese medicine, soak the Chinese medicine in water for 30 min, boil it vigorously, continue to decoct it gently for 30 min, and pour out the Chinese medicine liquid; then add 6 times the volume of water of the Chinese medicine, boil it vigorously, continue to decoct it gently for 20 min, and pour out the liquid. Mix the two liquid portions and use a rotary evaporator to evaporate to 1 g of crude drug content per mL. Then conduct a drug sensitivity test to test whether 11 Chinese herbal medicines, namely Houttuynia cordata, Andrographis paniculata, Lonicera japonica, Isatis indigotica, Taraxacum mongolicum, Phellodendron amurense, Pulsatilla chinensis, Viola philippica, Cortex fraxini, Scutellaria baicalensis, and Coptis chinensis, and their combinations have inhibitory effects on Elizabethkingia miricola.

[0035] Drug sensitivity test of Chinese herbal medicine extracts: Take 100 μL of the diluted bacterial suspension, spread it on a sterilized plate with a glass bead for spreading and punch holes, add 18 μL of the Chinese herbal medicine extract to each hole around, and add 18 μL of sterile water to the middle hole as a negative control. Incubate at 28 °C for about 24 h. Observe the presence or absence of an inhibition zone and measure the diameter of the inhibition zone (unit: mm).

[0036] Drug sensitivity test of the Chinese herbal medicine extract composition: For every two combinations of Chinese herbal medicine extracts, explore their inhibitory effects on the Marteilia elizabethae circle. Take 100 μL of the diluted bacterial solution, coat it on a sterilized plate with glass beads, and punch holes. Add 18 μL of the Chinese herbal medicine extract composition (take 9 μL of each Chinese herbal medicine extract) to each surrounding hole, and add 18 μL of sterile water to the middle hole as a negative control. Incubate at 28 °C for about 24 h. Observe the presence or absence of the inhibition zone and measure the diameter of the inhibition zone (unit: mm).

[0037] The test results are shown in Table 1 as follows:

[0038] Table 1 Inhibition zone diameters of different Chinese herbal medicine extracts and their combinations (unit: mm)

[0039]

[0040] The results show that among the 11 Chinese herbal medicine extracts, only Scutellaria baicalensis Georgi extract and Coptis chinensis Franch extract have the ability to inhibit the growth of Marteilia elizabethae, while 9 Chinese herbal medicine extracts such as Houttuynia cordata Thunb., Andrographis paniculata (Burm. f.) Nees, Lonicera japonica Thunb., Isatis indigotica Fortune, Taraxacum mongolicum Hand.-Mazz., Phellodendron amurense Rupr., Pulsatilla chinensis (Bunge) Regel, Viola philippica Cav., and Cortex Fraxini do not have the direct inhibitory effect on Marteilia elizabethae.

[0041] For Scutellaria baicalensis Georgi extract or Coptis chinensis Franch extract, among the remaining 10 Chinese herbal medicine extracts, only Houttuynia cordata Thunb. extract can enhance the inhibitory effect of Scutellaria baicalensis Georgi extract on Marteilia elizabethae (has an enhancing effect), but Houttuynia cordata Thunb. extract cannot enhance the inhibitory effect of Coptis chinensis Franch extract on Marteilia elizabethae (does not have an enhancing effect). A precipitate is formed after mixing Coptis chinensis Franch extract and Scutellaria baicalensis Georgi extract, and the mixture no longer has the inhibitory effect on Marteilia elizabethae, and there is an antagonistic effect between them.

[0042] Therefore, for the antibacterial peptide Chinese medicine composition against the pathogen Marteilia elizabethae of the blue eye disease of Rana spinosa and its application of the present invention, it is first discovered that the mixture of Scutellaria baicalensis Georgi extract and Houttuynia cordata Thunb. extract has a good inhibitory effect on Marteilia elizabethae, and the inhibitory effect is better than that of the single Scutellaria baicalensis Georgi extract, and the single Houttuynia cordata Thunb. extract does not have the inhibitory effect on Marteilia elizabethae; it shows that Houttuynia cordata Thunb. extract has a synergistic enhancing effect on the inhibitory effect of Scutellaria baicalensis Georgi extract on Marteilia elizabethae, and it is expected to apply it to the treatment of the blue eye disease of Rana spinosa and replace antibiotics to prepare a new anti-Marteilia elizabethae drug.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A traditional Chinese medicine composition for resisting Mycobacterium elizabethae, the pathogen causing the blue eye disease of spiny frog, characterized in that: The traditional Chinese medicine composition is a mixture of equal parts by weight of Scutellaria baicalensis Georgi extract and Houttuynia cordata Thunb. extract prepared by the decoction method.

2. Use of the traditional Chinese medicine composition according to claim 1 in the preparation of a drug for inhibiting Elizabethkingia miricola.

3. Use of the traditional Chinese medicine composition according to claim 1 in the breeding of Paa spinosa.

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