JP4976006B2 - Parasitic disease prevention and treatment method for marine fish - Google Patents

Description

  The present invention relates to a method for preventing and treating parasitic diseases occurring in marine fish.

Parasitic diseases that occur in fish cause growth inhibition and can lead to death. In the aquaculture industry, damage caused by parasitic diseases in cultured fish is a serious problem, and various countermeasures are being investigated. Corresponding methods are roughly divided into a method in which an ingredient effective for parasitic diseases is added to the feed and orally administered, and a method in which fish is immersed in water in which the active ingredient is dissolved. Examples of the latter include formalin baths, high salt water baths, fresh water baths, medicinal baths and the like (see Patent Documents 1 to 5).
A fresh water bath can control parasites, but it is also a burden for fish. For example, in the case of yellowtails, if a fresh water bath is used for more than 5 minutes, the fish may bleed and drown. Usually limited to about 5 to 10 minutes.

JP-A-6-284835 JP-A-9-94036 JP-A-10-72344 JP 2000-16937 JP 2000-23587 A

  It is an object of the present invention to provide a method for preventing and treating parasitic diseases that does not cause concern that a medicinal component remains in fish, does not contaminate environmental water, and has little influence on fish.

  Freshwater baths are used to control marine fish parasites. Although it has been thought that the mechanism of action of a fresh water bath may be due to a difference in osmotic pressure, the present inventors have now confirmed that the effect of a fresh water bath is not due to a difference in osmotic pressure, but an essential component for parasites. The inventors have found that this is due to the absence of certain metals and have completed the present invention. That is, even if it did not use fresh water, it was confirmed by removing a specific ion among the metal ions contained in seawater that it has the same effect on the parasite as fresh water.

The present invention removes at least one of potassium ions and calcium ions, which are metal ions essential for the survival of the parasite , or halves at least two of potassium ions, calcium ions, and magnesium ions. The gist is a method for preventing and / or treating parasitic diseases of marine fish characterized by bathing in the removed seawater or artificial seawater. The method for removing metal ions from seawater is to produce artificial seawater that does not contain the metal ions, chelate the metal ions with a chelating agent, or add a substance that insolubilizes the metal ions to precipitate it as a metal salt, etc. The method can be used. As metal ions essential for the survival of parasites to be removed from seawater or artificial seawater, it is preferable to remove two kinds of metal ions, calcium ions and magnesium ions. Moreover, it is preferable to correct the osmotic pressure, which fluctuates due to the removal of metal ions, so as to return to the same level as the original seawater.

  Compared to conventional fresh water baths, it has less effect on fish and can provide the same effect as fresh water baths in preventing and treating parasitic diseases. Since no special chemicals are used, there is no fear of chemical accumulation in fish meat or environmental impact.

In the present invention, water bathing is performed using seawater or artificial seawater from which specific metal ions have been removed, instead of a freshwater bath used to combat parasitic diseases. Known fresh water bath methods and equipment can be used as they are. Compared to fresh water baths, the burden on fish is less, so it is possible to bathe for a longer time than conventional fresh water baths.
The marine fishes that are the subject of the present invention are the pufferfish family (Traphugu), the Thai family (Madai, Kurodai), the horse mackerel family (Buri, Hiramasa, Kampachi, Hiragakanpachi), the grouper family (Que, Chiromarata, Sarasahata, Mahata), salmon There are a wide variety of fishes such as coho salmon (coho salmon, atlantic salmon, trout, king salmon) and flounder (flounder, hoshigurai). Any fish that has parasites on its body surface or shark can be the subject of the present invention. Moreover, it can be targeted from fry to adult fish.

The fish parasite that is the subject of the present invention is a parasite that parasitizes on the body surface and the coral. In particular, parasites belonging to monoprotozoa such as neobenedenia and heterobotulium shown in the examples, for example, Heteraxine, Bivagina, Hererobothrium, Benedenia, Benedenia serilae; Neobenedenia), Gyrodactylus, etc., and parasites belonging to protozoa known not to proliferate when bred in 50% seawater, for example, Cryptocaryon sp, Ichthyophithirius sp, Trichodina sp ), Scuticociliatida, Ichthyobodo sp, Pseudodactylogyrus and the like.
In the present invention, examples of metal ions essential for the survival of parasites include sodium ions (Na ⁺ ), potassium ions (K ⁺ ), calcium ions (Ca ²⁺ ), and magnesium ions (Mg ²⁺ ). In particular, the removal of both calcium ions and magnesium ions is preferable because the survival rate of the parasite can be reduced to the same extent as in a fresh water bath.

In the present invention, seawater or artificial seawater from which metal ions have been removed is obtained by removing specific metal ions from seawater with an ion exchange resin or the like, or by precipitating metal ions with a metal ion chelating agent, etc. Can be used. Artificial seawater in which necessary salt is blended and seawater components other than specific metal ions are blended can also be used.
As a chelating agent that can be used in the present invention, a chelating agent that can be used as a food additive such as citric acid may be directly added, or a chelating agent having a high metal salt blocking ability such as EDTA may be used. . When EDTA or the like is used, it is preferable that the EDTA is recovered as a precipitate after being used by being bound to a polymer or the like so that EDTA can be recovered. The amount of the chelating agent used (g / liter) is calculated based on “metal salt (mg / liter) / amount of metal salt sequestered with 1 g of chelating agent (mg)”.
In the present invention, metal ions can also be removed by introducing a substance that reacts with a metal salt and makes the metal salt a precipitate. For example, by adding sodium carbonate to seawater, the calcium salt becomes calcium carbonate precipitate and is insolubilized. Calcium and magnesium become insoluble salts by making carbonates, phosphates, and the like.

Although the component composition of artificial seawater is variously known, the main components are sodium chloride, potassium chloride, calcium chloride, magnesium chloride, magnesium sulfate, and a pH buffer, and other trace components are added thereto. In order to obtain the effects of the present invention, it is only necessary to remove metal ions essential to the parasite, and therefore any artificial seawater having any component composition can be applied. When a specific metal ion is removed, the osmotic pressure and pH are preferably adjusted using salts such as sodium chloride because the burden on fish is less when it is closer to the original artificial seawater.
In the present invention, to bathe is to transfer the fish for a certain period of time to normal seawater in which the metal ions of the present invention are removed. Usually, fish are put in a net with a net and returned to the original breeding water after a certain period of time. Any method may be used as long as it is a method used in a fresh water bath.
Since the water bath liquid of the present invention has less burden on fish than fresh water, the fish species that have been used in the conventional fresh water bath are bathed under the same conditions (water bath time, etc.) as those used in the fresh water bath. Should be implemented. Further, the water bath time can be made longer than that of the fresh water bath.

By bathing fish in seawater from which specific metal ions of the present invention have been removed, it is possible not only to combat adult parasites as shown in the Examples, but also to reduce the hatching rate of parasite eggs. . Both these effects can prevent and / or treat fish parasitic diseases.
The element of artificial seawater from which metal ions essential for the survival of the parasites of the present invention are removed is a mixture of salts sold as elements of artificial seawater, excluding specific metal ions. What remove | excluded magnesium ion and calcium ion is preferable. What adjusted osmotic pressure, pH, etc. to the same extent as normal artificial seawater using salts, such as sodium chloride, is preferred. Parasitic diseases can be prevented and / or treated by dissolving the artificial seawater in a certain amount of water to an appropriate concentration and bathing the fish.

  Examples of the present invention will be described below, but the present invention is not limited thereto.

<Anti-Neo Benedenia Activity of Artificial Seawater from which Na ⁺ , K ⁺ , Ca ²⁺ and Mg ²⁺ have been removed alone (in vitro test)>
Artificial seawater from which Na ⁺ , K ⁺ , Ca ^2+, and Mg ²⁺ were removed alone was prepared, and the lethal effect of these seawaters on neobenedenia (Neobenedenia girellae) was examined.

Materials and methods Test zone: Artificial seawater zone (control zone), Na ⁺ removal artificial seawater zone, K ⁺ removal artificial seawater zone, Ca2 ⁺ removal artificial seawater zone, Mg2 ⁺ removal artificial seawater zone, distilled water zone ( It was set as 6 districts of freshwater district).
Artificial seawater: The composition of artificial seawater in the control zone is 0.4638M sodium chloride, 0.01M potassium chloride, 0.0092M calcium chloride dihydrate, 0.0359M magnesium chloride hexahydrate, 0.0175M magnesium sulfate heptahydrate Hepes 0.01M (pH 8.0). Na ⁺ , K ⁺ , Ca ²⁺ and Mg ²⁺ removed artificial seawater was prepared based on this. Also, mannitol (sugar) is used as an osmotic pressure regulator for Na ⁺ -removed artificial seawater, and sodium chloride is used as an osmotic pressure regulator for K ⁺ , Ca ²⁺ and Mg ²⁺ -removed artificial seawater. Supplemented the minute.
Exposure of artificial seawater to Neo Benedenia: Twenty individual adults of Neo Benedenia 12-15 days after hatching were placed in a 12-well dish containing artificial sea water. After each hole was washed twice with artificial seawater, artificial seawater, distilled water, and artificial seawater from which each metal ion was removed independently were added and cultured at 25 ° C. for 2 hours. The viability of the worms was examined 5, 10, 15, 20, 25, 30, 60 and 120 minutes after the start of culture.

The results and discussion results are shown in FIG. The survival rate of the artificial seawater area (control area) was 100%. In the distilled water section, all the worms died 5 minutes after the start of the culture. Therefore, the effectiveness of the fresh water bath carried out at the aquaculture site was shown. On the other hand, in the metal ion-removed artificial seawater area, a clear decrease in the survival rate was observed in the Na ⁺ removed artificial seawater area, the Ca ²⁺ removed artificial seawater area, and the K ⁺ removed artificial seawater area. Among them, the survival rate of the Na ⁺ -removed artificial seawater zone was the lowest, and the survival rate was 6.7% after 60 minutes and 4.2% after 120 minutes. The survival rate of the Ca ²⁺ removed artificial seawater zone and the K ⁺ removed artificial seawater zone was about 50% after 120 minutes. Therefore, these metal ions are essential for maintaining the homeostasis of the worms, and seawater from which any of Na ⁺ , K ⁺ and Ca ²⁺ is removed or artificial seawater which does not contain has an anthelmintic effect on fish parasites. It became clear.

<Anti-Neo-Benedenia activity of artificial seawater from which Ca ²⁺ and Mg ²⁺ are removed alone or in combination (in vitro test)>
Seawater from which Ca ²⁺ and Mg ²⁺ were removed alone or in combination was prepared, and the lethal effect of these seawaters against adult neobenedenia girellae was examined.

Materials and Methods <br/> test group: artificial seawater District (control group), ^{Ca 2+} removed artificial seawater ku, ^{Mg 2+} removed artificial seawater ku, ^{Ca 2+} and ^{Mg 2+ Ca 2+} was 1/2 the amount of artificial sea water, mg ²⁺ 1/2 removing seawater ku, ^{Ca 2+} and 1/4 amount of ^{mg 2+} artificial seawater and were ^{Ca 2+, mg 2+} 3/4 removing seawater ku, ^{Ca 2+} and ^{mg 2+} does not contain ^{Ca 2+, mg 2+} removed Seven zones, a seawater zone and a distilled water zone (freshwater zone), were used.
Artificial seawater: The composition of artificial seawater in the control plot is: sodium chloride 0.4638M, potassium chloride 0.01M, calcium chloride dihydrate 0.0092M, magnesium chloride hexahydrate 0.0359M, magnesium sulfate heptahydrate 0.0175 M, Hepes 0.01M (pH 8.0). Ca ²⁺ and Mg ²⁺ removed artificial seawater was prepared based on this. Moreover, sodium chloride was used as the osmotic pressure regulator of the Ca ²⁺ and Mg ²⁺ removed artificial seawater, and the lack of osmotic pressure was compensated for by adding it.
Artificial seawater exposure to Neo Benedenia: 20 neobenedenia adults 12-15 days after hatching were each placed in a 12-well dish containing artificial seawater. After each hole was washed twice with artificial seawater, normal artificial seawater, distilled water, and respective ion-removed artificial seawater were added and cultured at 25 ° C. for 2 hours. The viability of the worms was examined 5, 10, 15, 20, 25, 30, 60 and 120 minutes after the start of culture. In addition, in order to examine histologically what kind of influence each artificial seawater has on the worm body, the main insects raised in each seawater were collected and observed using a transmission electron microscope.

Results and Discussion FIG. 2 shows the results of the present worm survival rate. The survival rate of the worms in the artificial seawater area (control area) was 100%. In the ion-removed artificial seawater test section, a clear decrease in the worm survival rate was observed in all ^sections except for the Mg 2 ⁺ -removed seawater section, and the results of Example 1 were reproduced. Among them, the Ca ²⁺ and Mg ²⁺ removed seawater ^sections had the lowest survival rate of the worms, and the survival rate was 24.2% after 5 minutes and 3.7% after 10 minutes. In this test group, death of all worms was observed 20 minutes after the start of culture. The time required to kill all parasites in the Ca ²⁺ , Mg ²⁺ removed seawater section is the same value as that in the distilled water section, and the lethal effect of the insects in the Ca ²⁺ , Mg ²⁺ removed seawater (artificial seawater) ) Was considered equivalent to a fresh water bath. Ca ^{2+, Mg 2+} 1/2 removing seawater ku, ^{Ca 2+, Mg 2+} 3/4 removing seawater Ward showed similarly lethal effect (antiparasitic effect), the effect is ^{Ca 2+,} and ^{Mg 2+} removed seawater Zone The value was lower than that of the Ca 2 ⁺ -removed artificial seawater area. The above results indicate that the removal of metal ions in combination is more effective in reducing the survival rate of the worms than the removal of metal ions alone.
The results of tissue observation are shown in FIG. When the tissue behind the body of the dead worm was exposed to Ca ²⁺ -removed artificial seawater and Ca ²⁺ , Mg ²⁺ -removed seawater, many large gaps were formed between the cells. On the other hand, such a gap was not observed in the control artificial seawater-exposed individuals. Ca ²⁺ and Mg ²⁺ are known to be involved in cell adhesion. Cadherin, one of the cell adhesion factors, has been shown to be Ca ²⁺ dependent. From these, it was considered that Ca 2 ⁺ -removed seawater, Ca ²⁺ and Mg ²⁺ combined-removed seawater weaken the binding force between cells of the worms and cause the worms to die.

<Anti-Neo-Benedenia Activity / In Vivo Test of Artificial Seawater from which Ca ²⁺ and Mg ²⁺ are Removed Single or Combined>
Seawater from which Ca ²⁺ and Mg ²⁺ were removed singly or in combination was prepared, and the lethal effect on adult neobenedenia girellae (parasitoid) parasitic on the seawater host was examined.

Materials and methods Test zone: Natural seawater zone, artificial seawater zone (control zone), Ca2 ⁺ -removed artificial seawater zone, Mg2 ⁺ -removed artificial seawater zone, Ca2 ⁺ and Mg2 ⁺ as 1/2 amount of artificial seawater the ^{Ca 2+, Mg 2+} 1/2 removing artificial seawater ku, ^{Ca 2+} was 1/4 the amount of artificial seawater ^{Ca 2+} and ^{Mg 2+, Mg 2+} 3/4 removing artificial seawater ku, without ^{Ca 2+} and ^{Mg 2+} Seven ^{sections of} Ca ²⁺ and Mg ²⁺ removal artificial seawater ^sections were used.
Artificial seawater: The composition of artificial seawater in the control zone is 0.4638M sodium chloride, 0.01M potassium chloride, 0.0092M calcium chloride dihydrate, 0.0359M magnesium chloride hexahydrate, 0.0175M magnesium sulfate heptahydrate Hepes 0.01M (pH 8.0). Ca ²⁺ and Mg ²⁺ removed artificial seawater was prepared based on this. Moreover, sodium chloride was used as the osmotic pressure regulator of the Ca ²⁺ and Mg ²⁺ removed artificial seawater, and the lack of osmotic pressure was compensated for by adding it.
Exposure of seawater to neobenedenia parasitizing host: Hoshigarei infested with about 200 adult neobenedenia 12-12 days after hatching was transferred to a 10 liter aquarium containing 4 liters of each test seawater and bred for 1 hour. One hour later, the presence or absence of the worm was examined and the survival rate was calculated. The same test was conducted three times.

The results and discussion results are shown in FIG. Ca ²⁺ removed artificial seawater Ward and ^{Ca 2+, Mg 2+} removed artificial seawater District of the insect survival, respectively 67.4%, was 14.7% ^{Ca 2+} removed artificial seawater Zone and ^{Ca 2+, Mg 2+} removed artificial seawater A clear anthelmintic effect was observed in the ward. Also, under these seawater conditions, no abnormal appearance such as abnormal swimming was observed in the flounder. In the case of seawater from which metal ions have been removed, there is less influence on fish compared to freshwater, and the same effect as freshwater baths can be obtained. It was shown that it can be used as an agent.

<Inhibition of hatching of artificial seawater from which Na ⁺ , K ⁺ , Ca ^2+, and Mg ²⁺ have been removed alone against heterobotulium eggs (in vitro test)>
Seawater from which Na ⁺ , K ⁺ , Ca ^2+, and Mg ²⁺ were removed alone was prepared, and the hatching-inhibiting action of these seawaters on heterobotlium (Heterobothrium okamotoi) was examined.

Materials and methods Test zone: Natural seawater zone, artificial seawater zone (control zone), Na ⁺ removal artificial seawater zone, K ⁺ removal artificial seawater zone, Ca2 ⁺ removal artificial seawater zone, Mg2 ⁺ removal artificial seawater zone It was set as 6 wards.
Artificial seawater: The composition of the artificial seawater in the control area was that of Van't Hoff. The composition per liter was 26.75 g sodium chloride, 0.75 g potassium chloride, 0.51 g calcium chloride, 3.42 g magnesium chloride, 2.1 g magnesium sulfate and 0.21 g bicarbonate. Na ⁺ , K ⁺ , Ca ²⁺ and Mg ²⁺ ion-removed artificial seawater was prepared based on this.
Artificial seawater exposure to heterobotulium eggs: Eggs within 12 hours of egg laying were collected, transferred to a plastic beaker containing 300 ml of each test seawater, and cultured at 25 ° C. for 14 days. The number of eggs used in each section was about 500. Every day, the eggs were observed under a microscope and the test seawater was changed. The hatching rate was calculated 14 days after the start of culture.

Results and discussion results are shown in FIG. The hatching rate of the artificial seawater zone (control zone) was 93.0%, indicating a hatching rate equivalent to that of natural seawater. On the other hand, the hatching rate of the metal ion-removed seawater area was clearly lower in all areas than in the control area. Therefore, it has been clarified that the seawater from which metal ions have been removed has not only an anthelmintic action on fish parasites but also an action to inhibit hatching of parasite eggs.
From the above results, the seawater from which metal ions have been removed, particularly Ca2 ⁺ and Mg2 ⁺ combined seawater, has an anthelmintic effect equivalent to that of a freshwater bath and has a small effect on the host, and thus is considered to be effective as a bathing solution. In particular, it is very effective in combating parasites against juvenile fish that are susceptible to the outside world. In addition, these seawaters have a hatching-inhibiting action, and can be used for breeding water for the prevention and treatment of parasites during marine fish culturing on land, which are bred for a long time.

<Effect study on the effect of artificial seawater from which Ca ²⁺ and Mg ²⁺ have been removed either alone or in combination>
Seawater from which Ca ²⁺ and Mg ²⁺ were removed alone or in combination was prepared, and the influence of these seawaters on yellowtail was examined.

Materials and methods Test zone: natural seawater zone, artificial seawater zone (control zone), Ca2 ⁺ -removed artificial seawater zone, Mg2 ⁺ -removed artificial seawater zone, Ca2 ⁺ and Mg2 ⁺ as 1/4 amount of artificial seawater the ^Ca 2+ ^{· Mg} 2+ 3/4 removing artificial seawater ku, ^{Ca 2+} and ^{Mg 2+} does not contain a ^Ca 2+ · ^{Mg 2+} removed artificial seawater Ward, was 7 ° distilled water gu (freshwater bath Ward).
Artificial seawater: The composition of artificial seawater in the control zone is sodium chloride 0.4638M, potassium chloride 0.01M, calcium chloride dihydrate 0.0092M, magnesium chloride hexahydrate 0.0359M, magnesium sulfate heptahydrate 0.0175M Hepes 0.01M (pH 8.0). Ca ²⁺ and Mg ²⁺ removed artificial seawater was prepared based on this. Further, sodium chloride was used as an osmotic pressure adjusting agent for Ca ²⁺ and Mg ²⁺ removed artificial seawater, and the lack of osmotic pressure was compensated for by adding it.
Each test seawater and fresh water exposure to yellowtail: Three juveniles of yellowtail were transferred to a 100 liter water tank containing 20 liters of each test seawater and reared for 1 hour. The yellowtail was observed during the breeding time, and the survival rate was calculated after 1 hour. In addition, in order to examine histologically what effect each artificial seawater has on the yellowtail, we collected the coral of the yellowtail raised in each seawater and performed histological observations.

Results and discussion The results of the survival rate of yellowtail are shown in FIG. The survival rate of yellowtail in the artificial seawater area (control area) was 100%. In the distilled water section (freshwater section), all yellowtails bleed and died 7-10 minutes after the start of breeding. On the other hand, in the artificial seawater area from which metal ions were removed, all the test fish were alive in all sections in the treatment for 1 hour. Neobenedenia (Neobenedenia girellae, Hadamushi) appearance abnormalities such as even abnormal swimming in Ca 2+ ^· Mg ²⁺ removed artificial seawater ku lethal effect was highest for adult was observed. From this, it was found that a long-time fresh water bath causes great damage to the host, and an artificial sea water bath with metal ion removal has much less influence on the host than the fresh water bath.
The results of tissue observation are shown in FIG. No abnormalities were observed in the coral tissue in the natural seawater area. In the distilled water section, secondary epithelial cells, secondary intercostal cells, and wall column cells were observed to swell, and in some cases, detachment of these cells from the secondary sputum was also observed. Although expansion of some cells was observed in the Ca 2 ⁺ · Mg ²⁺ -removed artificial seawater area, no significant difference was observed from the natural seawater area.
From the above, it was shown that the metal ion-removed seawater has a significantly smaller effect on yellowtail than fresh water, and can be used as a bath salt for controlling parasites.

  The metal ion-removed seawater (artificial seawater) of the present invention can be used as a bath solution for controlling parasites of marine fish including cultured fish and ornamental fish. The metal ion-removed seawater (artificial seawater) of the present invention can be used for the control of parasites as breeding water for onshore circulation culture of marine fish.

It is a figure which shows the anti-neo-benedenia activity (in vitro test) of artificial seawater from which various metal ions were removed one by one. It is a figure which shows the anti-neo-benedenia activity (in vitro test) of the artificial seawater from which one or two kinds of various metal ions are removed. It is the photograph which observed the structure | tissue of the parasite cultured by the artificial seawater from which the metal ion was removed with the transmission electron microscope. It is a figure which shows the anti-neo-benedenia activity (in vivo test) with respect to the parasite which parasitized the host of the artificial seawater from which one or two kinds of various metal ions were removed. It is a figure which shows the hatching inhibitory effect (in vitro test) with respect to the heterobotium egg of the artificial seawater from which various metal ions were removed one by one. It is a figure which shows the survival rate at the time of raising yellowtail young fish for 1 hour in the artificial seawater from which one or two kinds of various metal ions were removed. It is the photograph of the structure | tissue of the yellowtail of the yellowtail reared for 1 hour by natural seawater, distilled water, and Ca2 ⁺ · Mg2 ⁺ removal artificial seawater.

Claims (4)

Any removal of one or more potassium ion, calcium ion is an essential metal ions to the survival of the parasite, or potassium ion, calcium ion, sea water or to remove any two or more half or more magnesium ions A method for preventing and / or treating parasite diseases of marine fish characterized by bathing in artificial seawater.   The method for preventing and / or treating parasitosis of marine fish according to claim 1, wherein the method for removing metal ions from seawater is by adding a substance that chelates or insolubilizes metal ions. The method for preventing and / or treating marine fish parasitic diseases according to claim 1 or 2 , wherein the metal ions essential for the survival of the parasite to be removed from the seawater or the artificial seawater by 1/2 or more are calcium ions and magnesium ions. Removing the metal ions is essential for the survival of the parasite, of claims 1 to 3 parasitic diseases of any marine fish using seawater or artificial seawater was correct for variations in osmotic pressure due to the removal of metal ions prevention and / Or a treatment method.