CN112274630A - Method for improving immunity and disease resistance of crustacean, application of phycoerythrin and composition thereof - Google Patents
Method for improving immunity and disease resistance of crustacean, application of phycoerythrin and composition thereof Download PDFInfo
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Abstract
The invention provides a method for improving the immunocompetence and the disease resistance of crustacean, the application of phycoerythrin and a composition thereof, and the method relates to applying a composition to the crustacean; wherein the composition comprises a phycoerythrin; the method provided by the invention can improve the immunocompetence of the crustacean, more specifically, the phagocytosis or the protophenol oxidase system capacity of the crustacean, and the disease resistance of the crustacean, especially the pathogen resistance of the crustacean; the invention further provides the application of the phycoerythrin serving as an immunostimulant and a feed composition thereof.
Description
Technical Field
The invention relates to the field of aquaculture, in particular to a method for improving the immunocompetence and disease resistance of crustacean, application of phycoerythrin and a composition thereof.
Background
The crustacean is a high-price aquatic food, has high market value, and is also a key artificial breeding object in all countries in the world. However, the immunity of crustaceans is easily reduced due to poor environment, and when the cultivation environment is poor, some pathogenic bacteria in water can multiply in a large amount and infect the crustaceans at random, so that infectious diseases are developed. In 1992, shrimp white spot disease was outbreaked in Fujian province, and the whole southeast Asia culture area was heavily created. In 2016, the same situation occurred in east coast australia, and a large scale of outbreaks of the shrimp white spot disease outbreak resulted in significant economic loss. The Global Aquaculture Alliance (GAA) has indicated in 2016 research reports that the biggest challenge facing the aquaculture industry today is the disease problem.
The current strategies for preventing or controlling the spread of disease in various countries still favor the use of antibiotics or chemicals, which can cause environmental damage and food safety problems in a non-trivial amount. Therefore, there is an urgent need in the aquaculture industry for an immunostimulant (immunostimulant) that can improve the disease resistance of crustaceans without causing environmental damage and food safety problems.
As can be seen from Table 1, since 2004, researchers began to propose immunostimulants that increase the disease resistance of crustaceans. The time and survival (survival rate) in the table indicates that there was a significant time difference from the survival at that time after challenge (challenge) with Vibrio alginolyticus (Vibrio algiryliticus) compared to the control group. As can be seen from Table 1, most natural immunostimulants require at least 3-4 days to significantly improve the disease resistance of crustaceans. Therefore, providing a natural immunostimulant capable of rapidly improving the disease resistance of crustaceans is another problem to be solved in the field of aquaculture.
TABLE 1 Effect of various immunostimulants on the disease resistance of crustaceans
| Immunostimulant | Time (day) | Survival rate (%) | Data source |
| Alginic acid (Sodium alginate) | 4 | 43.30 | Cheng et al.2004 |
| Copper sulfate (Copper sulfate) | 2 | 46.70 | Yeh et al.2004 |
| Carrageenans (Carrageenans) | 3 | 60 | Yeh and Chen 2008 |
| Gracilaria tenuistipitata | 3 | 63.63 | Hou and Chen 2004 |
| Gracilaria tenuistipitata | 3 | 63 | Yeh and Chen 2009 |
| Brown sugar glue (Fucoidan) | 3 | 60 | Suwaree et al.2013 |
Phycoerythrin (PE) is one of phycobiliproteins, which are water-soluble fluorescent proteins present in algae such as red algae, green algae, blue algae, cryptophyceae, and the like. Phycoerythrin has been widely used in various fields of research and development due to its many characteristics, including chemical and biological agents, tumor therapeutic drugs, in vitro diagnosis, etc., and is most often used in fluorescence research reagents. However, it is not clear whether phycoerythrin can be used as an immunostimulant to enhance the immunity and disease resistance of crustaceans.
Disclosure of Invention
The present invention is directed to a method for enhancing the immunocompetence and disease resistance of crustaceans, said method comprising applying a composition to the crustaceans; wherein the composition comprises phycoerythrin.
To achieve the above objects, the immunological competence is phagocytosis or pro-phenol oxidase system.
For the purpose of the invention, the disease resistance refers to the capability of resisting pathogens; wherein the pathogen comprises a virus or a bacterium.
In order to achieve the above object, the virus is a white spot disease virus.
In order to achieve the above object, the bacterium is Vibrio parahaemolyticus.
In order to achieve the above object, the crustacean comprises shrimp, crab, Charybdis, Limulus, shrimp pallidum or lobster.
In order to achieve the aim of the invention, the effective dose of the phycoerythrin is 0.45 mu g-2.4 mu g.
To achieve the above objects, the composition is applied by oral administration, soaking, spraying or injection.
The present invention further provides a use of phycoerythrin as an immunostimulant comprising applying a phycoerythrin to a crustacean; the phycoerythrin can improve the immunity or disease resistance of crustacean.
The invention also provides a feed composition, which comprises phycoerythrin, and the phycoerythrin can improve the immunity or disease resistance of the crustacean.
The invention provides a solution to the long-standing problem of diseases in the field. The phycoerythrin-containing composition provided by the present invention can enhance the immunocompetence of crustaceans, and more specifically, can enhance the phagocytosis of crustaceans and the nonspecific immunocompetence of protophenol oxidase systems and the like. The composition containing phycoerythrin provided by the invention is applied to the crustacean, so that the disease resistance of the crustacean after being attacked by pathogens can be improved. The composition provided by the invention takes phycoerythrin as an effective component, and has the advantages of nature, no food safety problem and low threat to environment.
Drawings
FIG. 1 is a flow chart of a test for the effect of phycoerythrin on the ability to combat pathogens;
FIG. 2 is a graph showing the results of a phagocytosis assay;
FIG. 3 is a graph showing the results of the assay of the crude phenol oxidizing enzyme system.
Detailed Description
All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs unless otherwise defined, and wherein the singular forms "a", "an", "the" and "the" refer to more than one of the plural forms unless otherwise specified, and further wherein the forms "comprise" and "comprise" are open-ended terms.
The term "effective amount" as used herein refers to an amount of an effective active agent sufficient to prevent or alleviate at least one symptom or condition of the disease in the grantor; the result is a reduction and/or alleviation of signs (sign), symptoms (symptoms), or causes of disease, or an intentional change in other physiological systems.
The term "crustacean" as used herein refers to arthropods belonging to the suborder crustacean (Crustacea), including but not limited to shrimp, crab, Charybdis, Limulus, Nippon albiflora, or lobster.
The term "pathogen" as used herein refers to a pathogenic substance that causes crustacean diseases, including but not limited to bacteria, fungi, viruses, or parasites.
The dosage form of the composition used in the present invention includes, but is not limited to, Solution (Solution), Emulsion (Emulsion), Suspension (Suspension), Powder (Powder), lozenge (Tablet), Pill (Pill), Tablet (Troche) or Capsule (Capsule) and other similar or applicable dosage forms of the present invention.
The composition of the present invention may further comprise an aquaculture supplement, which includes but is not limited to vaccines, adjuvants, immunostimulants (immunostimulants); wherein the immunostimulatory substance includes but is not limited to a chemical compound, a carbohydrate, or a carbohydrate derivative.
Suitable routes of administration for the compositions of the present invention include, but are not limited to, soaking, spraying, dipping, oral administration or injection.
Phycoerythrin (PE) is commercially available, and one skilled in the art can extract Phycoerythrin from algae including red algae. In addition, unless otherwise indicated, the materials used in the present invention are readily available commercially.
The present invention is illustrated by the following examples, but the present invention is not limited by the following examples.
EXAMPLE one test for the Effect of phycoerythrin on the immunological competence
The immune mechanism of crustaceans is mainly nonspecific (nonpepticific) immunity, which can be divided into humoral immunity and cellular immunity, and the cellular immunity includes phagocytosis (phagocytosis) reaction. The invention utilizes an in vitro test to test the influence of phycoerythrin on phagocytosis so as to be used as a basis for evaluating whether the phycoerythrin can improve the immunocompetence of crustaceans.
Phagocytosis assay: phagocytosis assays were performed using the phagocytosis reagent set (# P35361, Life Technologies), which was an improved assay according to the method proposed by Neaga a. (2011). 100 μ L of a quantified suspension of shrimp blood cells (1X 10)6cell/mL) or Shrimp salt solution buffer (SSS buffer) (instead of the hemocyte suspension as a negative control group), added to a 96-well plate with a black bottom, centrifuged at 800Xg at 4 ℃ for 20 minutes and the supernatant removed, rinsed once with SSS buffer, centrifuged once again under the same conditions, then added with 100. mu.L of phycoerythrin (0.013, 0.026, 0.052 and 0.116mg/mL) at different concentrations, added to the control group SSS buffer, incubated at room temperature for 60 minutes, centrifuged at 800Xg and 4 ℃ for 20 minutes and the supernatant removed, then added with 100. mu.L of K-12 BioParticle (fluorescence substance), incubated at room temperature for 20 minutes, and then detected with a fluorescence immunoassay Reader (ELISA Reader) for fluorescence at 480nm (scattered light) and then substituted into the following formula.
Phagocytosis activity (%). cndot. (experimental/control). times.100%
The results of the phagocytosis test are shown in fig. 2, the experimental group treated with phycoerythrin has better phagocytosis effect than the untreated control group, and the higher the concentration of the treated phycoerythrin is, the better the phagocytosis effect is. The results show that phycoerythrin has the efficacy of improving phagocytosis.
Example two test of the Effect of phycoerythrin on the immunological competence (two)
The prophenoloxidase system (proPO system) is an enzyme immune system similar to the complement system in crustaceans, and is also an important index factor for evaluating the immunological competence of crustaceans. The invention further utilizes an in vitro test to test the influence of the phycoerythrin on the protophenol oxidase system, and the influence is used as another basis for evaluating whether the phycoerythrin can improve the immunological competence of the crustacean.
And (3) carrying out a protophenol oxidase system test: modified according to the Hern' ndez-L Lopez (1996) method, shrimp blood cells are suspended in shrimp blood cell culture Medium (MCHBSS), and 500. mu.L of a well-quantified shrimp blood cell suspension (1X 10)6cell/mL), adding 500. mu.L of phycoerythrin (0.013, 0.026, 0.052 and 0.116mg/mL) with different concentrations, co-culturing at room temperature for 30 minutes, centrifuging at 800Xg and 4 ℃ for 10 minutes, removing the shrimp blood cell culture medium and phycoerythrin, adding 1mL of dimethylarsinate buffer (CA buffer) to suspend the shrimp blood cells, then crushing with ultrasound for 5 seconds for 4 times, centrifuging at 800Xg and 4 ℃ for 10 minutes, taking 50. mu.L of supernatant to a 96-well plate, adding 50. mu.L of 0.1% trypsin (trypsin) to stimulate the experimental group, adding 50. mu.L of dimethylarsinate buffer as a control to the control group, after 10 minutes of reaction, adding 50. mu. L L-Dopa (L-Dopa) as a substrate to each well (well), after 15 minutes of reaction, detecting the absorbance at 492nm with an enzyme immunoassay Reader (Reader), and the activity was calculated by the following formula.
Original phenol oxidase activity ═ (absorbance of experiment group) - (absorbance of control group)
The test results of the protophenol oxidase system are shown in fig. 3, the function of the protophenol oxidase system is better than that of an untreated control group in an experimental group for treating phycoerythrin, and the higher the concentration of the treated phycoerythrin is, the better the function of the protophenol oxidase system is. The results show that phycoerythrin has the function of improving the function of the protophenol oxidase system.
EXAMPLE III test of the Effect of phycoerythrin on disease resistance (I)
Phycoerythrin is added into the feed for oral administration or injection and then injected into shrimp bodies, and then the influence of the phycoerythrin on the pathogen resistance of the crustaceans is evaluated through a bacterial challenge test. The specific implementation is carried out according to the method proposed by Sirirustananun, n. (2011), after improvement. The experiment used Vibrio parahaemolyticus (Vibrio parahaemolyticus) as the challenge bacterium, as shown in FIG. 1, to confirmThe test group injected phycoerythrin (0.026, 0.052 and 0.116mg/mL) with different concentrations in 20 μ L each day before challenge, and injected Vibrio parahaemolyticus in 20 μ L each shrimp at an injection rate of 5 × 106A CFU; the control group is shrimps which are not treated by phycoerythrin and directly injected with vibrio parahaemolyticus; shrimp injected with only 20. mu.L of SSS buffer were additionally used as an unprovisioned control group; there were a total of five groups of 11 shrimps per group. Shrimp survival rates were observed at 12, 24, 36, 48, 60 and 72 hours post challenge.
As shown in Table 2, the survival rate of the shrimps without phycoerythrin treatment was only 27.27% after 1 day of Vibrio parahaemolyticus challenge, while the survival rate of the shrimps with phycoerythrin treatment was increased to more than 50%, and the higher the concentration of phycoerythrin, the higher the survival rate of the shrimps. The results prove that the phycoerythrin can effectively improve the capability of the shrimps on resisting bacteria.
TABLE 2 Effect of phycoerythrin prawn antibacterial ability
EXAMPLE four test of Effect of phycoerythrin on disease resistance (II)
Phycoerythrin is added into the feed for oral administration or injection and then injected into shrimp bodies, and then the influence of the phycoerythrin on the pathogen resistance of the crustaceans is evaluated through a virus challenge test. The specific implementation is carried out according to the method proposed by Sirirustananun, n. (2011), after improvement. As White Spot Syndrome Virus (WSSV) is used as Virus for challenge in the experiment, as shown in figure 1, 20 μ L phycoerythrin (0.026, 0.052 and 0.116mg/mL) with different concentrations is injected into the experimental group on the day before challenge, and 20 μ L White Spot Syndrome Virus is injected every other day, with the injection amount of 9.19 × 10 per shrimp4A copies number; the control group is not treated with phycoerythrin and injected with white spot virusShrimp; shrimps receiving only 20 μ L of SSS buffer were used as an unprovisioned control group; there were a total of five groups of 11 shrimps each. Shrimp survival was observed at 12, 24, 36, 48, 60, 72, 84 and 96 hours post challenge.
As shown in table 3, the shrimps without phycoerythrin treatment all died 3 days after White Spot Syndrome Virus (WSSV) challenge, while the survival rate and survival time of the shrimps treated with phycoerythrin were improved. The result proves that the phycoerythrin can effectively improve the antiviral ability of shrimps.
TABLE 3 Effect of phycoerythrin prawn antiviral Capacity
According to the results of the third and fourth embodiments, the effective dosage range of phycoerythrin for effectively improving the pathogen resistance of crustaceans is 0.45-2.4 μ g; wherein, the preferable effective dose range is 0.52 mu g-2.32 mu g; wherein, the more preferable effective dose range is 1.04 mug-2.32 mug; wherein the optimal effective dose is 2.32 μ g.
In summary, the present invention provides a method for enhancing the immunological and disease resistance of crustaceans using phycoerythrin as an immunostimulant. As can be seen from the examples of the present invention, the disease resistance of crustacean can be significantly improved by using phycoerythrin as an immunostimulant in only 1.5 days, and the survival rate of the experimental group (63.64%) treated with phycoerythrin is much higher than that of the control group (27.27%). Compared with the traditional immunostimulant (table 1), the immunostimulant using phycoerythrin as immunostimulant has the advantages of rapidly improving the disease resistance of crustaceans, and causing no food safety problem and no harm to the environment.
In the disclosure of the preferred embodiments of the present invention, it will be obvious to those skilled in the art that the foregoing embodiments are merely exemplary; those skilled in the art can implement the present invention by various modifications and substitutions without departing from the technical characteristics of the present invention. Many modifications and variations of the present invention are possible in light of the above teachings. The claims provided herein are for defining the scope of the invention to include both the methods and structures described above and their equivalents.
The technical solutions provided by the present application are introduced in detail, and the present application applies specific examples to explain the principles and embodiments of the present application, and the descriptions of the above examples are only used to help understand the method and the core ideas of the present application; meanwhile, for a person skilled in the art, according to the idea of the present application, there may be a change in the specific implementation and application scope, and as described above, the content of the present specification should not be construed as a limitation to the present application.
Claims (10)
1. A method for enhancing the immunological competence and disease resistance of crustaceans, comprising: applying a composition to the crustacean; wherein the composition comprises phycoerythrin.
2. The method of claim 1, wherein the immunological competence is phagocytosis or pro-phenolic oxidase system.
3. The method of claim 1, wherein the disease resistance is the ability to fight a pathogen; wherein the pathogen comprises a virus or a bacterium.
4. The method of claim 3, wherein the virus is a white spot virus.
5. The method of claim 3, wherein the bacterium is Vibrio parahaemolyticus.
6. The method of claim 1, wherein the crustacean comprises shrimp, crab, Charybdis, horseshoe crab, lobster, or lobster.
7. The method of claim 1, wherein the effective amount of phycoerythrin is between 0.45 μ g and 2.4 μ g.
8. The method of claim 1, wherein the composition is administered orally, by soaking, by spraying, or by injection.
9. Use of phycoerythrin as an immunostimulant, comprising applying to a crustacean a phycoerythrin; the phycoerythrin can improve the immunity or disease resistance of crustacean.
10. A feed composition characterized in that it is a phycoerythrin, said phycoerythrin being capable of enhancing the immunological competence or disease resistance of crustaceans.
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