JP7670943B2 - Schistosomiasis agent and method for eliminating schistosomiasis - Google Patents

Description

The present invention relates to an antischistosomal agent and an antischistosomal method.
This application claims priority based on Japanese Patent Application No. 2022-143599, filed on September 9, 2022, the contents of which are incorporated herein by reference.

Schistosomiasis is a parasitic disease that infects more than 200 million people worldwide and is included in the list of neglected tropical diseases. Schistosomiasis is found in tropical and subtropical regions of Africa and Asia, and in Africa, epidemics caused by Schistosoma mansoni and Schistosoma bilharzia are common. All schistosomiasis use snails, which are intermediate hosts, as vectors, and cercariae that infect humans are produced in the snails. These cercariae infect humans or animals through the skin, develop into adults in the portal vein or veins near the bladder, lay eggs, and release eggs into the outside world through feces or urine. Miracidium hatched from the eggs infect the snails, which are intermediate hosts, and develop into infectious larvae called cercariae, thus circulating the parasitic life cycle.

As shown in Figure 1, eggs (1) released from the definitive host hatch when they come into contact with freshwater, and miracidia begin to swim out (2). The miracidia infect freshwater snails through their body surface (3) and transform into sac-like sporocysts in the tissues (4). Within these mother sporocysts, daughter sporocysts form through larval reproduction. The daughter sporocysts migrate to the midgut gland, where they again form numerous cercariae through larval reproduction. These cercariae escape in accordance with the circadian rhythm of the host snail and swim in the water (5). When the cercariae reach the skin of a mammal, they secrete proteolytic enzymes and penetrate the skin (6), and only their heads transform into cystosomulae (7). The cystosomulae stay on the skin for about two days and then travel to the lungs via the venules in the bloodstream (8). After passing through the lungs, the schistosomes invade the vena cava and use the systemic circulation to reach the portal vein in about 8 to 10 days after infection, where they develop oral suckers to prey on red blood cells and mature into adults (9). Adult schistosomes are about 10 mm long, and form pairs in which the female is sandwiched between the somewhat shorter brood tubes of the male, then move to the final parasitic site and lay eggs (10). It takes 5 to 7 weeks from infection with cercariae to maturity and start laying eggs, and the parasite continues for several years. The laid eggs are excreted from the host's body through the digestive tract or urinary tract. Cercariae are transmitted when the host comes into contact with water, such as when swimming, bathing, or washing clothes. Cercariae can also be transmitted when the host is exposed to water during activities such as fishing or rice paddy cultivation. For this reason, the anti-trematode drug praziquantel is used to treat schistosomiasis. Praziquantel is safe, inexpensive, and easy to treat, but only 14% of patients who need treatment receive it. On the other hand, the WHO currently recommends the use of the chemical pesticide niclosamide for the purpose of killing shellfish. However, after 20 years of use, there have been reports of niclosamide-resistant snails. Furthermore, there are concerns about its biocidal activity, genotoxicity, carcinogenicity, and other effects on non-target organisms such as plants and animals in the environment, as well as its high cost.

Therefore, in order to effectively prevent such schistosomiasis, the objective of the present invention is to provide an inexpensive, low-toxicity schistosomiasis repellent and a method for eliminating schistosomiasis using said schistosomiasis repellent.

That is, as a result of intensive research, the inventors focused on Spirulina, a type of cyanobacteria that lives in highly alkaline saltwater lakes where there are no intermediate host shellfish. They discovered that Spirulina is consumed by the intermediate host shellfish of schistosomes and has the effect of specifically reducing schistosomes, while not affecting shellfish or other organisms, and thus completed the present invention.

The present invention includes the following aspects.
[1] A schistosomicidal agent comprising spirulina.
[2] The schistosomiasis repellent according to [1], wherein the spirulina content is 10 mass% or more in terms of dry weight of spirulina.
[3] The schistosomiasis repellent according to [1] or [2], which is food for snails that are intermediate hosts of schistosomiasis.
[4] A method for eliminating schistosomiasis, comprising spraying the schistosomiasis elimination agent according to any one of [1] to [3] into a water area inhabited by snail shells that are intermediate hosts of schistosomiasis.

According to the present invention, there is provided an inexpensive, low-toxicity schistosomiasis agent and a method for eliminating schistosomiasis using said schistosomiasis agent.

Schematic diagram showing the life cycle of schistosomes: (A) Schistosoma japonicum; (B) Schistosoma mansoni; (C) Schistosoma haematobium; (1) Eggs are excreted from the host through feces and urine; (2) Eggs hatch and become miladium; (3) Miladium infects snails; (4) Inside the snail, they become sporocysts and live for two generations; (5) Cercariae swim out of the snail into the water; (6) Transcutaneous infection; (7) Cercariae lose their tails during infection and become juvenile schistosomes; (8) They travel in the bloodstream; (9) They travel to the hepatic portal vein and become adults; (10) Gynandrous adults: (A) S. japonicum and (B) S. (C) S. mansoni migrates to the mesenteric venules of the small intestine/rectum (where the eggs are laid and passed to the liver and excreted in the feces), and (D) S. haematobium migrates to the vesical venous plexus. The experimental schedule for the pre-experiment using a 1.4 L aquarium is shown. 1 is a graph showing the results of a preliminary experiment comparing the growth inhibition of cercariae between a Spirulina preparation and a competitor's feed. 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 a spirulina-containing feed and a base feed on the growth of cercariae in Experiment 1. 1 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 3 L aquarium is shown. 1 is a graph showing the results of comparing the effects of a spirulina-containing feed and a base feed on the growth of cercariae in Experiment 2. 13 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 1.4 L aquarium is shown. 13 is a graph showing the results of evaluating the effect on cercariae growth by changing the blending rate of spirulina in the feed in Experiment 3. The experimental schedule for Experiment 4 is shown. 1 is a graph showing the results of evaluating the effect of Spirulina on cercariae in vitro in Experiment 4.

A numerical range expressed using "to" means a range that includes the numerical values before and after "to" as the lower and upper limits.
The term "comprise" means that it may contain components other than the target components. The term "consist of" means that it does not contain components other than the target components. The term "consist essentially of" means that it does not contain components other than the target components in a form that exerts a special function (such as a form that completely loses the effect of the invention). In this specification, when it is written "comprise", it includes an embodiment that "consists of" and an embodiment that "consists essentially of".

<Schistosomidicide>
A first aspect of the present invention is a schistosomicidal agent comprising spirulina.

(Spirulina)
Spirulina is a microscopic spiral algae belonging to the genus Spirulina in the family Oscillatory family of the order Nostocaceae. Among the species that were previously classified in the genus Spirulina, species that are cultivated for industrial purposes may be classified in the genus Arthrospira in recent years. In one embodiment, spirulina is a cyanobacterium belonging to the genus Spirulina or the genus Arthrospira.

Spirulina is a spiral-shaped organism measuring 0.3 to 0.5 mm in size that grows naturally in saltwater lakes in Africa and is known as a food ingredient that has been eaten by the people living in those areas for many years as a valuable food source. Currently, there are approximately 35 types of spirulina known in the world.

Specific examples of Spirulina include Arthrospira platensis, Arthrospira maxima, Arthrospira geitleri, Arthrospira siamese, Spirulina major, Spirulina subsalsa, etc. Among them, Arthrospira platensis, Arthrospira maxima, Arthrospira geitleri, and Arthrospira siamese are preferred because they can be artificially cultivated and are easily available.

Algae are used as food for snail shells. Spirulina is found mainly in brackish waters in tropical and subtropical regions of Africa and Central and South America, and compared to algae that live in freshwater, it can thrive in harsh environments of high temperature, high alkalinity and high salinity. Like ordinary plants, it absorbs carbon dioxide and grows by photosynthesis, but unlike plants, it does not produce starch during the process, but instead produces glycogen (an excitatory carbohydrate) which is effective in energy conversion. For this reason, it has attracted attention as a source of nutrition. However, it is not known as an extermination agent for schistosomes.

The spirulina used may be commercially available or may be cultured. The spirulina may be isolated.
The cultivation method for Spirulina can be the same as that used for culturing blue-green algae. For example, Spirulina can be cultivated and grown outdoors under alkaline conditions.
The cultured spirulina may be used as it is, or the cultured spirulina may be collected on a filter cloth or filter paper, washed with water, and then suspended in water to form a suspension. Spirulina may be in the form of wet algae obtained by concentrating the culture solution or suspension, or may be in the form of dried algae obtained by drying the wet algae by freeze-drying or spray-drying (spray drying). The dried algae may be powdered, or the powder may be mixed with an excipient and formed into tablets. Spirulina is preferably used as a dry powder or tablets.

The content of spirulina in the schistosomicidal agent is not particularly limited. When used with dried spirulina algae, dried powder or tablets, the content of spirulina in the schistosomicidal agent is preferably 5% by mass or more, more preferably 10% by mass or more, even more preferably 15% by mass or more, and particularly preferably 20% by mass or more, in terms of the dry weight of spirulina, relative to the total mass of the schistosomicidal agent. The content of spirulina in the schistosomicidal agent is preferably 5 to 100% by mass, more preferably 10 to 100% by mass, even more preferably 15 to 100% by mass, and particularly preferably 20 to 100% by mass, relative to the total mass of the schistosomicidal agent.

(Optional ingredients)
In addition to spirulina, the schistosomicidal agent may contain other ingredients, including ingredients commonly used in snail diets.

For example, powder raw materials include grains (corn, wheat, flour, wheat germ, wheat bran, rice, bread crumbs, barley, oats, rye, etc.), potatoes (sweet potatoes, potatoes, etc.), beans (whole soybeans, etc.), starches (wheat starch, corn starch, rice starch, potato starch, tapioca starch, sweet potato starch, sago starch, modified starch, etc.), vegetable proteins (corn gluten meal, wheat protein, bean protein, rice protein, etc.), and Examples of such ingredients include: fish proteins, potato proteins, etc.), seafood (fish such as tuna, bonito, horse mackerel, etc.; crustaceans such as shrimp and crab; mollusks such as octopus and squid; shellfish such as scallops and turban shells; fish extracts; dried bonito flakes, meal (fish meal), etc.); algae other than spirulina; additives (vitamins, minerals, amino acids, flavor ingredients, powdered palatability enhancers, fiber, colorants, phosphates, pH adjusters, seasonings, antioxidants, trehalose, etc.). Meal refers to a powder made by compressing and finely crushing seafood.

The schistosomicidal agent may be raw spirulina or spirulina powder. Alternatively, spirulina may be mixed with a conventional excipient to form a tablet, or may be prepared by incorporating spirulina into fish feed or naturally occurring snail feed. The fish feed or snail feed may be an algae other than spirulina.

The schistosomiasis targeted for extermination by the schistosomiasis repellent of this embodiment is a parasite belonging to the Schistosoma genus. Examples of schistosomiasis include Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mekongi, and Schistosoma intercalatum.

The schistosome repellent of this embodiment contains spirulina, which has the effect of reducing the number of cercariae in snail shells. Spirulina can be produced inexpensively and is highly safe. Therefore, a low-cost and highly safe schistosome repellent can be realized.

<Schistosoma extermination method>
A second aspect of the present invention is a method for eliminating schistosomiasis, which comprises spraying the schistosomiasis repellent according to the first aspect into a water area inhabited by snail shells that are intermediate hosts of schistosomiasis (hereinafter also referred to as the "spraying step").

(Spraying process)
"Schistosomidicide" means inhibiting the reproduction of schistosomes. More specifically, schistosomidicide of schistosomes includes killing cercariae and reducing the number of cercariae. The killing of cercariae by a schistosomiatic agent may occur within the snail, which is an intermediate host, or may occur outside the snail in the water environment.

Snails that are intermediate hosts for schistosomes are snails into whose bodies the miracidia, the first stage larvae of schistosomes, invade and grow into cercariae inside. Examples of snails that are intermediate hosts for schistosomes include the genus Bulinus for Schistosoma haematobium and Schistosoma intercolorum, the genus Biomphalaria for Schistosoma mansoni, the genus Oncomelania for Schistosoma japonicum, and Tricula aperta for Schistosoma mekongense. These are freshwater snails that live in rivers, lakes, and rice paddies.

The method of feeding the schistosomiasis agent to the snail shells is not particularly limited. For example, the schistosomiasis agent may be sprayed in the water area where the snail shells, which are intermediate hosts, live. For example, the schistosomiasis agent may be sprayed in freshwater areas (rivers, lakes, rice fields, etc.) where snail shells live in areas where schistosomiasis has occurred. The spraying method is not particularly limited, and may be done manually or using a spraying device, etc.

The number of times of spraying is not particularly limited, and may be, for example, a single spray or repeated spraying. In the case of repeated spraying, 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.

It is expected that the schistosomicidal agent sprayed in the waters inhabited by snails, which are intermediate hosts, will be ingested by the snails and taken up into the snails' bodies. The spirulina in the schistosomicidal agent taken up into the snails' bodies kills the cercariae living inside the snails' bodies. This reduces the number of cercariae parasitizing the snails. As a result, the number of cercariae in the waters inhabited by the snails is reduced, making it possible to suppress schistosomiasis infection in humans. The killing of cercariae by the schistosomicidal agent may occur not only inside the snails' bodies, but also in the aquatic environment in which the snails live.

(Optional step)
The method of the present embodiment may include other steps in addition to the spraying step, such as a step of determining a spraying area for the schistosomiasis agent (a spraying area determining step).

≪Spraying area determination process≫
The determination of the area to which the schistosomicid is to be sprayed may include, for example, investigating the inhabitation of snails, which are intermediate hosts of schistosomiasis, and/or the inhabitation of cercariae in the target water area. The target water area may be a freshwater area in an area where schistosomiasis occurs. Examples of the freshwater area include rivers, lakes, marshes, and rice paddies.
Surveys of snail habitats can be carried out through visual observation.
The habitat of cercariae can be investigated by collecting water from the surveyed water area, killing the cercariae with a potassium iodide solution or the like, and observing the water with an inverted microscope, etc. Alternatively, snails, which are intermediate hosts, can be collected from the surveyed water area, placed in a container of water, exposed to light for about an hour, the snails removed, and potassium iodide solution or the like added to the remaining water to kill the cercariae, after which the water can be observed with an inverted microscope, etc.
By conducting such surveys, it is possible to determine areas for spraying schistosomicidal agents, such as water bodies inhabited by intermediate snail hosts or water bodies inhabited by confirmed inhabitants of cercariae.

The method of this embodiment may include a step of investigating the presence of cercariae in the sprayed water area after spraying of the schistosomicidal agent.

<Other Aspects>
In one embodiment, the present invention provides the use of Spirulina to combat schistosomiasis.
In one embodiment, the present invention provides the use of Spirulina for the manufacture of a schistosomicidal agent.
In one embodiment, the present invention provides Spirulina for combating schistosomiasis.
In the above, the spirulina may be in the form of wet algae, dried body, dry powder, or tablets.

The present invention will now be described in further detail with reference to the following examples, but the present invention is not limited to these examples.

(Preparation of Schistosomicidal Agents)
Spirulina-containing tablets were prepared by mixing 10%, 20%, 40% or 80% by weight of dry spirulina powder with Kyorin base feed (fish meal, wheat germ, soybean meal, wheat flour). Also, a tablet containing 100% by weight of dry spirulina was prepared.

(Pre-experiment) Search for the effect of reducing schistosomes in various feeds 1. Experimental conditions Snails used: Biomphalaria glabrata Puerto Rico strain albino strain Infection conditions: Eggs were isolated from the liver of mice infected with Schistosoma mansoni 8 weeks ago by artificial digestion. The eggs were left in water for 15 minutes or more to hatch the miracidia, and the miracidia were collected by light stimulation. Eight miracidia were collected in a 24-hole plate using a Pasteur pipette. Each snail of about 1 cm was placed there, and water was added to completely submerge the snail. The snail was left to stand overnight to complete the infection. After that, all snails were once collected in one tank, and 24 snails were distributed and raised in each tank divided into groups.

Breeding conditions: The fish were bred in a 95 mm x 155 mm x 95 mm plastic tank (water volume approximately 1.4 L) in an artificial climate chamber (closed type) (Japan Medical Equipment Manufacturing Co., Ltd.) with the temperature set at 28°C and no humidity setting. The amount of water in the tank was periodically replenished whenever it became insufficient.
Feeding schedule: 60 mg of each feed (200 mg of Spirulina tablets only) was administered three times a week. Measurement method: After 6 weeks of breeding, snails were removed from the tank and placed one by one in a 24-well plate. After 1 hour of light exposure, the snails were removed and 2 drops of potassium iodide solution were added to the remaining solution to kill and fix the cercariae. The cercariae were counted using an inverted microscope.

The experimental schedule is shown in Figure 2A. Company A's product: Tetra Corydoras, Company B's product: #1 Kyorin Hikari Corydoras, Company B's product (different lot): #2 Kyorin Hikari Corydoras, Company B's product-specific feed (for carp): Kyorin carp feed, base feed: Kyorin base feed (fish meal, wheat germ, soybean meal, wheat flour), Spirulina tablets: 100% Spirulina tablets.

2. Results The results are shown in Figure 2B. Figure 2B shows the results of counting the number of cercariae emerging from each of the 24 snails. Box plots were created from the number of cercariae in each well. X indicates the average value. The top of the box is 75% and the bottom is 25%, the center line is the median, and the whiskers indicate the maximum and minimum values.
From this graph, it was confirmed that the 100% Spirulina tablet showed a cercaria death rate of 88%, and thus had a higher cercaria eradication rate than the other feeds.

(Experiment 1) Confirmation of significant growth inhibition of cercariae by Spirulina 1. Experimental conditions:
The experiment was conducted in the same manner as in the pre-experiment, except that spirulina tablets and base feed were used as feed, and the feeding amount of the base feed was 200 mg. The experimental schedule for Experiment 1 is shown in Figure 3A.

2. Results The results are shown in Figures 3B and 3C. Cercariae were counted in the same manner as in the pre-experiment, box plots were created, and a two-sample t-test assuming unequal variances was performed (Figure 3B). As a result, a significant difference was observed between the Spirulina tablets and the base feed (p<0.0005). Cercariae were reduced by 79% with the Spirulina tablets, but no reduction was observed with the base feed.

The results of the survival rate of the snails are shown in Figure 3C. Six weeks after infection, when the snails were transferred from the tank to the plate, only the surviving snails were identified, and the survival rate of the 24 snails was calculated. Most of the snails in the spirulina tablets survived, but the survival rate was slightly reduced in the base feed.

(Experiment 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.

The results are shown in Figures 4B and 4C. It was confirmed that the number of cercariae had decreased (Figure 4B) and the survival rate of the shellfish had decreased (Figure 4C). It is possible that larger aquariums were more prone to environmental deterioration, lowering the survival rate of the shellfish, and that poor growth of the shellfish may have influenced the number of cercariae. However, the spirulina tablets significantly reduced the number of cercariae compared to the base feed. Furthermore, the survival rate of the shellfish was higher with the spirulina tablets compared to the base feed.

(Experiment 3) Verification that Spirulina Concentration-Dependent Inhibits Cercaria Growth 1. Experimental Conditions: The experiment 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.

The results are shown in Figure 5B. When spirulina was mixed into the feed, inhibition of cercariae was confirmed when the spirulina content was 10% or more. The growth inhibition effect on cercariae tended to increase with increasing amounts of spirulina, with the effect being highest at amounts of 80% or more. From these results, it was confirmed that the amount of spirulina to be mixed is preferably 10% by mass or more, and more preferably 80% by mass or more.

(Experiment 4) Verification of Spirulina's direct killing of cercariae in vitro 1. Experimental conditions: snails infected with Schistosoma mansoni miracidia were raised for 8 weeks. After that, snails were collected in one 100 ml beaker, water was added, and the snails were exposed to light for 1 hour to release cercariae. Spirulina powder was dissolved in water and diluted from 4 mg/ml to prepare Spirulina powder suspensions at concentrations of 2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml, or 0.0625 mg/ml. 100 μL of these Spirulina powder suspensions were placed in each well of a 96-well plate.

100 μL of the solution containing cercariae was added to the wells to make the total volume 200 μL. After 16 hours, the total number of cercariae was counted, and the number of live and dead worms was calculated. The experimental schedule for Experiment 4 is shown in Figure 6A.
Judgment of viability: Insects that moved under an inverted microscope and had a three-dimensional swelling in the body were considered viable. Insects that were not moving, had broken membranes, disintegrated bodies, or had no swelling in the body were considered dead.

The results are shown in Figure 6B. The vertical axis indicates survival rate, and the horizontal axis indicates spirulina concentration. The effective spirulina concentrations were EC50 = 0.039 mg/ml and EC90 = 0.087 mg/ml. A spirulina concentration of 0.09 mg/ml or higher is considered to be a preferable effective concentration.

From the above experimental results,
1) It has been confirmed that spirulina inhibits the growth of schistosomes.
2) It was confirmed that administration of Spirulina alone significantly reduced the number of cercariae formed in snails.
3) It was confirmed that the inhibitory effect on cercaria formation was dependent on the concentration of spirulina.
4) It has been confirmed that spirulina is not toxic to snails.
5) It was confirmed that spirulina has a direct killing effect on cercariae.
Therefore, it has been demonstrated that spirulina can be used as an effective schistosomicidal agent for eliminating schistosomiasis, and that an effective method for eliminating schistosomiasis can be established using spirulina.

According to the present invention, an inexpensive and low-toxicity schistosome repellent and a method for eliminating schistosomes using the schistosome repellent are provided. In one embodiment, the schistosome repellent uses a feed consisting of spirulina, which is effective as a feed for snail shells, or a feed containing spirulina, which is eaten by snail shells and does not affect the snail shells themselves, and can specifically reduce the number of schistosomes in the snail shells, which are intermediate hosts. Existing schistosome repellents are toxic to snail shells, whereas spirulina is not toxic to snail shells. According to the present invention, a composition containing 10% or more of spirulina by dry weight in fish feed, or spirulina itself, is sprayed as a schistosome repellent in a water area inhabited by snail shells infected with miracidia, for example, at a concentration of 0.03 mg/ml to 2 mg/ml, at least twice a week, to reduce the population of infected larval cercariae.

Although preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments. Addition, omission, substitution, and other modifications of the configuration are possible without departing from the spirit of the present invention. The present invention is not limited by the above description, but is limited only by the scope of the appended claims.

Claims (3)

A schistosomicidal agent that contains spirulina and is food for snails, which are intermediate hosts for schistosomes . The schistosomicidal agent according to claim 1, wherein the spirulina content is 10% by mass or more as the dry weight of spirulina. A method for exterminating schistosomiasis, comprising spraying the schistosomiasis repellent according to claim 1 or 2 into a water area inhabited by snail shells that are intermediate hosts of schistosomiasis.

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