WO2024053739A1 - Répulsif contre les schistosomes et procédé de répulsion des schistosomes - Google Patents

Répulsif contre les schistosomes et procédé de répulsion des schistosomes Download PDF

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WO2024053739A1
WO2024053739A1 PCT/JP2023/032891 JP2023032891W WO2024053739A1 WO 2024053739 A1 WO2024053739 A1 WO 2024053739A1 JP 2023032891 W JP2023032891 W JP 2023032891W WO 2024053739 A1 WO2024053739 A1 WO 2024053739A1
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spirulina
schistosoma
cercariae
repellent
snails
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PCT/JP2023/032891
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English (en)
Japanese (ja)
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貴 熊谷
雅彰 宮本
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国立大学法人東京医科歯科大学
株式会社キョーリン
Dic株式会社
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Priority to JP2024545728A priority Critical patent/JPWO2024053739A1/ja
Publication of WO2024053739A1 publication Critical patent/WO2024053739A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/03—Algae
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P15/00—Biocides for specific purposes not provided for in groups A01P1/00 - A01P13/00
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs

Definitions

  • the present invention relates to a schistosoma repellent and a schistosoma repellent method.
  • This application claims priority based on Japanese Patent Application No. 2022-143599 filed in Japan on September 9, 2022, the contents of which are incorporated herein.
  • Schistosomiasis is a parasitic disease that infects more than 200 million people worldwide, and is also included in neglected tropical diseases. This schistosomiasis is seen in tropical and subtropical regions of Africa and Asia, and in Africa, epidemics caused by Schistosoma mansoni and Schistosoma bilharzia are frequently observed. All schistosomes use intermediate hosts, snails, in which they produce cercariae that can infect humans. These cercariae infect humans or animals through the skin, develop into adult worms in the portal vein or veins near the bladder, lay eggs, and the eggs are released into the outside world through feces or urine. As a result, the miracidium that hatches from the insect egg infects the intermediate host, the snail, and develops into an infected larva, the cercariae, leading to a cycle of parasitic life.
  • the eggs (1) expelled from the definitive host hatch when they come into contact with fresh water, and the miracidium swims out (2).
  • Miracidium infects freshwater snails through the body surface (3) and metamorphoses into sac-like sporocysts within the tissues (4).
  • Daughter sporocysts are formed within this mother sporocyst through larval reproduction.
  • Daughter sporocysts migrate to the midgut gland and again form numerous cercariae through larval reproduction. These cercariae escape and swim in water according to the circadian rhythm of the host shellfish (5).
  • the cercariae reach the mammalian skin, they secrete proteolytic enzymes and invade the skin (6), and only their heads transform into cystosomulae (7).
  • Cystosomula stays in the skin for about two days and then travels through the bloodstream from the venules to the lungs (8). After passing through the lungs, it invades the vena cava and uses the systemic circulation to reach the portal vein about 8 to 10 days after infection, develops oral suckers, preys on red blood cells, and matures into an adult (9).
  • Adult schistosomes are approximately 10 mm long, and they pair up so that the female is wedged in the female duct of the slightly shorter male, and move to the final parasitic site where they lay eggs (10). It takes 5 to 7 weeks for the cercariae to mature and start laying eggs after infection, and the parasite continues for several years.
  • the eggs are excreted from the host body through the gastrointestinal tract or urinary tract. Cercariae infections occur when people come into contact with water, such as when swimming, bathing, or washing clothes. You can also become infected with cercariae when you come into contact with water while fishing or cultivating rice fields. Therefore, the anti-fluke drug praziquantel is used as a countermeasure against schistosomiasis. Praziquantel is safe, inexpensive, and easy to treat, but it only reaches 14% of patients who need treatment. On the other hand, the WHO currently recommends the use of the chemical pesticide niclosamide for shellfish killing purposes. However, there are now reports of niclosamide-resistant snails after 20 years of use. Furthermore, there are concerns about biocidal activity, genotoxicity, carcinogenicity, and other effects on non-target organisms such as animals and plants in the environment, as well as high costs.
  • Spirulina is a type of blue-green algae that originally lives in highly alkaline saline lakes with no distribution of intermediate host shellfish.
  • the present invention was completed based on the discovery that spirulina is preyed on by shellfish, an intermediate host for schistosomes, and has the effect of specifically reducing schistosomes, while having no effect on shellfish or other organisms.
  • the present invention includes the following aspects.
  • [1] A schistosoma repellent containing spirulina.
  • [2] The schistosoma repellent according to [1], wherein the content of spirulina is 10% by mass or more as the dry weight of spirulina.
  • [3] The schistosoma repellent according to [1] or [2], which is food for snails that are intermediate hosts of schistosoma.
  • a method for exterminating schistosomas which comprises spraying the schistosoma repellent according to any one of [1] to [3] into a water area inhabited by snails, which are intermediate hosts of schistosomas.
  • an inexpensive and low-toxicity schistosoma repellent and a method for exterminating schistosoma using the schistosoma repellent are provided.
  • FIG. 2 is an explanatory diagram showing the life cycle of a schistosome.
  • A Schistosoma japonicum;
  • B Schistosoma mansoni;
  • C Schistosoma haematobium; (1) Eggs are excreted from the host through stool and urine; (2) Eggs hatch into miradium; (3) Miradium Infects the snail; (4) It becomes a sporochist in the snail's body and spends two generations; (5) Cercariae swim out of the snail into the water; (6) Percutaneous infection; (7) Cercariae lose their tails during infection.
  • the experimental schedule for Experiment 1 using a 1.4 liter aquarium is shown.
  • 1 is a graph showing the results of comparing the effects of spirulina-containing feed and base feed on the growth of cercariae in Experiment 1.
  • 2 is a graph showing the results of comparing shellfish survival rates between Spirulina feed and base feed in Experiment 1.
  • the experimental schedule for Experiment 2 using a 3L aquarium is shown.
  • 2 is a graph showing the results of comparing the effects of Spirulina-containing feed and base feed on the growth of cercariae in Experiment 2.
  • 2 is a graph showing the results of comparing shellfish survival rates between Spirulina feed and base feed in Experiment 2.
  • the experimental schedule for Experiment 3 using a 1.4L aquarium is shown.
  • 3 is a graph showing the results of evaluating the influence on the growth of cercariae by changing the blending ratio of spirulina in the feed in Experiment 3.
  • the experimental schedule for Experiment 4 is shown.
  • 2 is a graph showing the results of in vitro evaluation of the influence of spirulina on cercariae in Experiment 4.
  • a numerical range expressed using “ ⁇ ” means a range that includes the numerical values written before and after " ⁇ ” as lower and upper limits.
  • the term “comprise” means that components other than the target component may be included.
  • the term “consist of” means that there are no components other than the component in question.
  • the term “consist essentially of” does not include components other than the target component in a manner that exerts a special function (such as a manner in which the effect of the invention is completely lost). means.
  • the term “comprise” includes embodiments “consisting of” and embodiments “consisting essentially of.”
  • the first aspect of the present invention is a schistosoma repellent.
  • the schistosomicidal agent contains spirulina.
  • Spirulina is a microscopic spiral alga that belongs to the genus Spirulina, which is a blue-green algae and belongs to the order Nostocidae and the family Prunidae.
  • Spirulina is a blue-green alga belonging to the genus Spirulina or the genus Arthrospira.
  • Spirulina has a spiral shape with a size of 0.3 to 0.5 mm, and grows naturally in saltwater lakes in Africa, and is known as a food material with many years of experience as a valuable food for the ethnic groups living in those areas. It is being Currently, about 35 types of spirulina are known in the world.
  • spirulina include, for example, Arthrospira platensis, Arthrospira maxima, Arthrospira geitleri, Arthrospira siamise, and Arthrospira siamise.
  • ospira siamese Spirulina major, Spirulina ⁇ Spirulina subsalsa and the like can be mentioned.
  • Arthrospira platensis, Arthrospira maxima, Arthrospira gaitoreri, and Arthrospira siamise are preferred because they can be artificially cultured and are easily available.
  • Algae are used as food for snails.
  • Spirulina is mainly found in brackish waters in tropical to subtropical regions of Africa, Central and South America, and can thrive in harsh environments of high temperature, high alkalinity, and high salinity compared to algae that live in freshwater. .
  • it is not known as an exterminator for schistosomes.
  • Spirulina may be commercially available or cultured. Spirulina can be isolated.
  • the culture method for culturing Spirulina can be carried out according to the usual method used for culturing blue-green algae.
  • Spirulina can be cultured and grown outdoors under basic conditions.
  • Spirulina obtained by culturing can be used as it is, or the cultured Spirulina can be collected with a filter cloth or paper, washed with water, and then suspended in water to be used as a suspension.
  • Spirulina may be obtained as a wet algae obtained by concentrating a culture solution or suspension, or as a dried algae obtained by drying the wet algae by freeze-drying, spray drying, or the like.
  • the dried algal bodies may be powdered, or the powder may be mixed with excipients and the like to form tablets.
  • Spirulina is preferably used as a dry powder or tablet.
  • the content of spirulina in the schistosoma repellent is not particularly limited.
  • the content of spirulina in the schistosomiasis agent is preferably 5% by mass or more, based on the total mass of the spirulina dry weight, and 10% by mass or more.
  • the content is more preferably at least 15% by mass, even more preferably at least 15% by mass, and particularly preferably at least 20% by mass.
  • the content range of spirulina in the schistosomiasis agent is preferably 5 to 100% by mass, more preferably 10 to 100% by mass, and even more preferably 15 to 100% by mass, based on the total mass of the schistosomiasis agent. Preferably, 20 to 100% by weight is particularly preferable.
  • the schistosomicidal agent may contain other ingredients in addition to spirulina.
  • Other ingredients include ingredients commonly used in snail feed.
  • powder raw materials include grains (corn, wheat, flour, wheat germ, wheat bran, rice, bread crumbs, barley, oats, rye, etc.), potatoes (sweet potatoes, potatoes, etc.), legumes (round soybeans, etc.), and starch.
  • raw spirulina or spirulina powder may be used.
  • tablets may be prepared by mixing Spirulina with common excipients, or may be prepared by blending Spirulina with fish feed or naturally-derived snail feed.
  • the fish feed or snail feed may be algae other than Spirulina.
  • the schistosoma to be exterminated by the schistosoma repellent of the present embodiment is a parasite belonging to the genus Schistosoma.
  • Examples of schistosomiasis include Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, and Schistosoma japonicum. osoma mekongi), and Schistosomiasis intercalatum ( Schistosoma intercalatum).
  • the schistosoma repellent of this embodiment has the effect of reducing the number of cercariae in snails by containing spirulina.
  • Spirulina can be produced at low cost and is highly safe. Therefore, a low-cost and highly safe schistosoma repellent can be realized.
  • the second aspect of the present invention is a method for exterminating schistosomes.
  • the schistosome exterminating method includes spraying the schistosome repellent according to the first aspect into a water area inhabited by snails, which are intermediate hosts of schistosomes (hereinafter also referred to as "spraying step").
  • schistosome control of schistosomes includes killing cercariae and reducing the number of cercariae. Killing of cercariae by a schistosomicidal agent may occur within the snail, which is an intermediate host, or may occur outside the body of the snail, in an aqueous environment.
  • Snails which are intermediate hosts for schistosomes, are snails in which miracidium, the first stage larva of schistosomes, invades the body and grows into cercariae within the body.
  • Snails that are intermediate hosts for schistosomes include the genus Bulinus for Schistosoma bilharzum and Schistosoma intercolorum, the genus Biomphalaria for Schistosoma mansoni, the genus Oncomelania for Schistosoma japonicum, and the genus Tricula aperta for Schistosoma japonica. . These are freshwater snails that live in rivers, lakes, rice fields, etc.
  • the method of feeding the schistosoma repellent to the snails is not particularly limited.
  • the spraying method is not particularly limited, and may be manually sprayed or a spraying device or the like may be used.
  • the number of times of spraying is not particularly limited, and for example, it may be sprayed once or repeatedly.
  • the spraying interval may be, for example, 1 to 3 times a day, once every 2 to 10 days, once every 10 to 30 days, etc., but is not limited thereto.
  • the schistosoma repellent sprayed in the habitat of snails which are intermediate hosts, will be ingested by the snails and taken into their bodies.
  • the spirulina contained in the schistosoma repellent taken into the snail's body kills the cercariae that live inside the snail's body. This reduces the number of cercariae parasitic on the snail. As a result, the number of cercariae in the habitat of snails is reduced, and it is possible to suppress the infection of schistosomes to humans. Killing of cercariae by a schistosomicidal agent may occur not only within the body of the snail, but also in the aquatic environment where the snail inhabits.
  • the method of this embodiment may include other steps in addition to the above-mentioned spraying step.
  • Other steps include a step of determining the spraying area of the schistosoma repellent (spraying area determining step).
  • Determining the area to be sprayed with the schistosomiasis agent may include, for example, investigating the habitat status of snails, which are intermediate hosts of schistosomiasis, and/or the habitat status of cercariae in the target water area.
  • Target waters include freshwater bodies in areas where schistosomiasis occurs. Examples of freshwater areas include rivers, lakes, rice fields, and the like.
  • the habitat status of snails can be investigated by visual observation.
  • the habitat status of cercariae can be investigated by collecting water from the survey water area, killing the cercariae with an iodopotassium solution, etc., and then observing the sample using an inverted microscope or the like.
  • snails which are intermediate hosts, were collected from the research area, placed in water in a container, exposed to light for about an hour, then removed, and an iodopotassium solution was added to the remaining water to kill the cercariae. Afterwards, it may be observed using an inverted microscope or the like. Through the above-mentioned investigation, it is possible to determine the areas where the schistosomiasis repellent should be sprayed, such as water areas where snails, which are intermediate hosts, inhabit or where cercariae have been confirmed to inhabit.
  • the method of this embodiment may include the step of investigating the habitat status of cercariae in the sprayed water area after spraying the schistosoma repellent.
  • the invention provides the use of spirulina to combat schistosomes. In one embodiment, the invention provides the use of spirulina to produce a schistosomicidal agent. In one embodiment, the invention provides spirulina for combating schistosomes.
  • Spirulina may be a wet algal body, a dried body, a dry powder, or a tablet.
  • Spirulina dry powder is added to Kyorin's base feed (fish meal, wheat germ, soybean meal, wheat flour) so that the dry weight of Spirulina is 10% by mass, 20% by mass, 40% by mass, or 80% by mass.
  • Kyorin's base feed fish meal, wheat germ, soybean meal, wheat flour
  • Spirulina combination tablets were prepared.
  • spirulina-containing tablets containing 100% by mass of spirulina on a dry basis were prepared.
  • breeding conditions The animals were bred in a 95 mm x 155 mm x 95 mm plastic aquarium (water volume: 1.4 L), an artificial weather machine (closed type) (Nippon Medical Instruments Manufacturing Co., Ltd.) with a temperature setting of 28°C and no humidity setting. The amount of water in the tank was added periodically whenever it became insufficient.
  • Feed administration schedule 60 mg of each type of feed (200 mg for Spirulina tablets only) was administered three times a week
  • Measurement method After 6 weeks of rearing, the snails were taken out of the aquarium and placed one by one in a 24-well plate. After 1 hour of light exposure, the snails were removed and two drops of potassium iodo solution were added to the remaining solution to kill and fix the cercariae. All cercariae were counted using an inverted microscope.
  • Figure 2A shows the experimental schedule.
  • Company A product Tetra corydoras
  • Company B product #1 Kyorin Hikari Corydoras
  • Company B product (separate lot): #2 Kyorin Hikari Corydoras
  • Company B product Separate feed for carp: Kyorin carp bait
  • base Feed Kyorin base feed (fish meal, wheat germ, soybean meal, wheat flour), Spirulina tablets: 100% Spirulina tablets.
  • FIG. 2B shows the results of counting the number of cercariae that emerged from each of the 24 snails.
  • a boxplot was created from the number of cercariae in each well.
  • X indicates the average value.
  • the top side of the box is 75%, the bottom side is 25%, the center line is the median value, and the maximum and minimum values are represented by whiskers. From this graph, it was confirmed that Spirulina 100% tablets showed a cercariae killing rate of 88%, and that the cercariae killing rate was higher than that of other feeds.
  • Example 1 Confirmation of significant growth inhibition of cercariae by Spirulina 1.
  • Experimental conditions The experiment was conducted in the same manner as the pre-experiment, except that Spirulina tablets and base food were used as the food, and the amount of base food fed was 200 mg.
  • the experimental schedule for Experiment 1 is shown in Figure 3A.
  • Example 2 Verification that Spirulina inhibits the growth of cercariae regardless of the size of the aquarium 1.
  • Experimental conditions The experiment was conducted in the same manner as in Experiment 1 except that the amount of water was changed from 1.4 liters to 3 liters.
  • the experimental schedule for Experiment 2 is shown in Figure 4A.
  • FIGS. 4B and 4C The results are shown in FIGS. 4B and 4C. It was confirmed that the number of cercariae decreased (Figure 4B) and the survival rate of shellfish decreased (Figure 4C). Larger aquariums may have been more susceptible to environmental deterioration, lowering the survival rate of shellfish, and it is possible that poor growth of shellfish may have affected the number of cercariae. However, spirulina tablets significantly reduced cercariae compared to the base diet. Additionally, shellfish survival rates were higher with Spirulina tablets compared to the base feed.
  • Example 3 Verification that Spirulina inhibits the growth of cercariae in a concentration-dependent manner 1.
  • Experimental conditions Experiment 2 was conducted in the same manner as in Experiment 2, except that feed containing different amounts of Spirulina was used.
  • the experimental schedule for Experiment 3 is shown in Figure 5A.
  • Example 4 Verification that spirulina directly kills cercariae in vitro 1.
  • Experimental conditions Snails infected with Schistosoma mansoni miracidium were reared for 8 weeks. The snails were then collected in one 100 ml beaker, water was added, and the cercariae were extracted by exposing to light for 1 hour. Dissolve spirulina powder in water and dilute from 4mg/ml to a concentration of 2mg/ml, lmg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, or 0.0625mg/ml. A spirulina powder suspension was prepared. 100 ⁇ L of these Spirulina powder suspensions were placed in each well of a 96-well plate.
  • the results are shown in Figure 6B.
  • the vertical axis shows survival rate, and the horizontal axis shows spirulina concentration.
  • a Spirulina concentration of 0.09 mg/ml or higher is considered to be a preferred effective concentration.
  • an inexpensive and low-toxicity schistosoma repellent and a method for exterminating schistosoma using the schistosoma repellent are provided.
  • the schistosoma repellent uses a bait made of spirulina or a bait containing spirulina, which is effective as a bait for snails, so that it is preyed on by the snails, does not affect the snails themselves, and is contained within the snails, which are intermediate hosts. Schistosoma can be specifically reduced. Existing schistosomicidal agents are toxic to snails, but spirulina is not toxic to snails.
  • a composition comprising 10% or more of spirulina by dry weight in fish feed, or spirulina itself as a schistosoma repellent, is sprayed in the water area where miracidium-infected snails live, for example, 0.03 mg.
  • the population of infected cercariae larvae can be reduced by spraying at a concentration of 2 mg/ml or more and 2 mg/ml or more twice a week.

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Abstract

L'invention concerne un répulsif contre les schistosomes contenant de la spiruline. L'invention concerne également un procédé de répulsion des schistosomes, ledit procédé comprenant la dispersion du répulsif contre les schistosomes dans un habitat aquatique des escargots qui sont des hôtes intermédiaires des schistosomes.
PCT/JP2023/032891 2022-09-09 2023-09-08 Répulsif contre les schistosomes et procédé de répulsion des schistosomes WO2024053739A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4238484A (en) * 1979-04-27 1980-12-09 Abbott Laboratories Molluscicide compositions and methods of use
CN106538471A (zh) * 2016-10-28 2017-03-29 韦忠 一种羊的养殖方法
CN111066828A (zh) * 2019-11-21 2020-04-28 吴杨 一种养殖场专用植物萃取杀菌消毒组合物及其制备方法
CN111202164A (zh) * 2019-06-13 2020-05-29 杭州海渤源生物科技有限公司 一种宠物驱虫复合软胶囊及其制备方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4238484A (en) * 1979-04-27 1980-12-09 Abbott Laboratories Molluscicide compositions and methods of use
CN106538471A (zh) * 2016-10-28 2017-03-29 韦忠 一种羊的养殖方法
CN111202164A (zh) * 2019-06-13 2020-05-29 杭州海渤源生物科技有限公司 一种宠物驱虫复合软胶囊及其制备方法
CN111066828A (zh) * 2019-11-21 2020-04-28 吴杨 一种养殖场专用植物萃取杀菌消毒组合物及其制备方法

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