CN109619044B

CN109619044B - Method for rapidly preparing schistosoma japonicum unisexual polypide

Info

Publication number: CN109619044B
Application number: CN201811639058.5A
Authority: CN
Inventors: 孙军
Original assignee: Tongji University
Current assignee: Tongji University
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2020-11-27
Anticipated expiration: 2038-12-29
Also published as: CN109619044A

Abstract

The invention relates to a method for quickly preparing schistosoma japonicum monosomy bodies, which is characterized in that after a plurality of schistosoma japonicum miracidiums are adopted to infect a single oncomelania, all schistosoma japonicum cercaria released by the single oncomelania are used to infect only one animal, and after the infection, at a specific time, the sex of the schistosoma japonicum or adult in the animal body is identified and screened to obtain the monosomy bodies. Compared with the prior art, the method of the invention omits the complicated process of infecting oncomelania one by one with miracidium and the problem of overlong culture time, and the whole process starts from the acquisition of oncomelania, and a large amount of single sex polypide, single female worm or single male worm is rapidly obtained. The method of the invention greatly simplifies and shortens the process of obtaining the solitary cercariae because the complexity of 'one-to-one' infection operation is avoided. The single sex polypide obtained by the method is a multi-genotype mixed population, is convenient for omics research, can avoid individual difference interference of polypides, and in a one-to-one infected group, although the single sex polypide is obtained, the genotypes are consistent.

Description

Method for rapidly preparing schistosoma japonicum unisexual polypide

Technical Field

The invention relates to the technical field of schistosoma japonicum katsurada research, in particular to a rapid preparation method of a schistosoma japonicum katsurada unisexual polypide.

Background

Schistosomiasis japonica is a parasitic disease which can be infected by both human beings and animals, has wide epidemic range and causes serious harm to both human beings and livestock. Although China has achieved great success in preventing and treating schistosomiasis japonica, the prospect of comprehensively controlling the spread of schistosomiasis japonica is still unattractive due to the difficulty in killing the oncomelania as the intermediate host and the serious phenomenon of schistosoma japonicum repeated infection.

In the schistosoma japonicum research, it is often necessary to obtain cercariae, trichuris or imago of a single sex. The preparation method is commonly used for infecting oncomelania in a one-to-one way mode by using single metacercaria, and the oncomelania is obtained after 2-half and half-month culture. The problems with this preparation method are: the oncomelania needs 2 and a half months of culture time, and the culture time is too long; secondly, the "one-to-one" infected oncomelania is complicated to operate.

Chinese patent CN102177858A discloses a method for preparing positive Oncomelania hupensis Gredler of Schistosoma japonicum, which comprises the following steps: cocultivation of the miracidium incubated with the eggs of Schistosoma japonicum with sf9 cells to obtain the mother miracidium developed by the miracidium; microinjecting the metacercaria into negative oncomelania bodies; and (4) continuously feeding the oncomelania injected with the maternal miracidium, and detecting whether the oncomelania is positive to obtain the schistosoma japonicum positive oncomelania. The cultured cercaria released by the positive oncomelania has the same infectivity as the cercaria released by the wild positive oncomelania and has the same biological characteristics as the schistosoma japonicum.

However, the positive Oncomelania japonica is not equivalent to the Schistosoma japonicum, and in the related research of Schistosoma japonicum, the positive Oncomelania japonica is usually used as a monocysticercus, a monogamic trichuris or a monogamic adult. The preparation by the conventional method needs to start from the infection of oncomelania by single miracidium, and is time-consuming and labor-consuming.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for quickly preparing schistosoma japonicum unisexual polypide.

The method can quickly obtain the polypide with single sex, saves the whole time of the research process, and can be used for the schistosomiasis japonica omics research.

The purpose of the invention can be realized by the following technical scheme:

a method for quickly preparing the monogamic schistosome of Japanese blood fluke features that the whole cercaria of Japanese blood fluke released by single positive Oncomelania snail is used to infect only one animal, and at a certain time after infection, the sex of schistosome or adult in animal is screened to obtain the monogamic schistosome.

Further, the animal includes a mouse or a rabbit.

Further, the specific time is at least 3 weeks, at which time the sex of the worm body can be discriminated.

Furthermore, the obtained polypide with single sex is a polygenic mixed population, which is convenient for omics research and avoids the interference of individual difference of polypides.

Further, the positive oncomelania may be a commercially available standardized positive oncomelania infected with schistosoma japonicum.

Further, the single positive oncomelania can also be replaced by oncomelania after a plurality of schistosoma japonicum miracidia infect a single oncomelania.

Furthermore, the method of the invention can adopt a plurality of schistosoma japonicum miracidium to infect a single oncomelania, then infect only one animal with all schistosoma japonicum cercaria released by the single oncomelania, and obtain the single sex worm body by screening the sex of the schistosoma japonicum or adult worm in the animal body at a specific time after infection.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the method of the invention saves the culture time of the oncomelania which is too long (2.5 months), and a large amount of single sex polypide, single female polypide or single male polypide can be rapidly obtained in the whole process.

2. The method of the invention avoids the complexity of 'one-to-one' infection operation, and the 'many-to-one' infection operation of a plurality of schistosoma japonicum miracidia infected single oncomelania is convenient.

3. The single-sex polypide obtained by the method is a multi-genotype mixed population, is convenient for omics research, avoids the individual difference interference of polypides, and has the defects that the proportion of different genotypes in the mixed polypide can not be controlled and the method is not suitable for genome sequencing research. In the "one-to-one" infection group, although a parthenocarpic insect body was also obtained, the genotypes were consistent.

4. The method of the invention can infect oncomelania with a plurality of metacercaria, is different from the prior one-to-one treatment, and is convenient and easy to operate. When collecting schistosoma japonicum cercaria or collecting schistosoma japonicum schistosomulum or imago after infecting animal with cercaria, screening to obtain monosomysoma.

5. The method is a rapid and ideal method for researchers to obtain the unisexual polypore at present, and is suitable for schistosomiasis japonica drug and vaccine research and development institutions, universities, research institutes and pharmaceutical companies.

Drawings

FIG. 1 shows sex observation and STR gene polymorphism analysis of Schistosoma japonicum.

A: the flow of single schistosoma japonicum miracidium infecting single oncomelania and obtaining schistosoma japonicum cercaria is shown schematically, namely, the single schistosoma japonicum miracidium infecting one-to-one infection;

b: in the "one-to-one" infected group, the single oncomelania release was all female worms;

b1, B2: STR analysis showed no difference between females; b3, B4: STR analysis shows that there are differences between females;

c: in the "one-to-one" infection group, the single oncomelania releases were all males;

c1, C2: STR analysis showed no difference between males; c3, C4: STR analysis shows differences between males;

d: showing the life history of the single oncomelania infected by the metacercaria, namely the 'many-to-one' infection;

e: in the many-to-one infection group, the single oncomelania release is all female worms;

e1, E2: STR analysis showed no difference between females; e3, E4: STR analysis shows that there are differences between females;

f: in the "many-to-one" infection group, all the single oncomelania released was males;

f1, F2: STR analysis showed no difference between males; f3, F4: STR analysis shows differences between males;

g: in the many-to-one infection group, a single oncomelania releases both males and females;

g1, G2: STR analysis showed no significant differences between worms; g3, G4: STR analysis showed differences between worms.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

Example 1

Performing sex observation and STR gene polymorphism analysis of schistosoma japonicum

The process of infecting a single oncomelania with a single schistosoma japonicum miracidium and obtaining schistosoma japonicum cercaria, namely 'one-to-one' infection, is schematically shown as A in figure 1,

b indicates that in the "one-to-one" infection group, single oncomelania release was all female worms, B1, B2: STR analysis showed no difference between females; b3, B4: STR analysis shows that there are differences between females;

c means that in the "one-to-one" infection group, single oncomelania releases were all males, C1, C2: STR analysis showed no difference between males; c3, C4: STR analysis shows differences between males;

schistosoma japonicum miracidium infects the oncomelania group one by one, and the released cercaria only has a single sex, namely male or female. The oncomelania were infected by 4 oncomelania, one female cercaria was released, three male cercaria were released, and the coexistence of male and female was 0, and the data are shown in table 1.

After a plurality of schistosoma japonicum miracidiums are adopted to infect a single oncomelania, namely 'many-to-one' infection, the process of infecting only one mouse by using all schistosoma japonicum cercaria released by the single oncomelania is shown as D in figure 1, the insects are taken 3 weeks after the mice are infected, and the sex of the bodies of the insects can be distinguished.

E represents that in the 'many-to-one' infection group, after only one mouse is infected by all schistosoma japonicum cercaria released by a single oncomelania, all the schistosoma japonicum cercaria are released to be female worms; e1, E2: STR analysis showed no difference between females; e3, E4: STR analysis shows that there are differences between females;

f represents that in the 'many-to-one' infection group, after only one mouse is infected by all schistosoma japonicum cercaria released by a single oncomelania, all the schistosoma japonicum cercaria are released to be male worms; f1, F2: STR analysis showed no difference between males; f3, F4: STR analysis shows differences between males;

g represents that in a multi-to-one infection group, after only one mouse is infected by all Schistosoma japonicum cercaria released by a single oncomelania, male and female are released and coexist; g1, G2: STR analysis showed no significant differences between worms; g3, G4: STR analysis showed differences between worms.

The sex of the worms released from 43 mice infected with "many-to-one" was seen, 17 mice in which the worms were mixed sex, 19 were all female, and 7 were all male, and the data are shown in table 1.

TABLE 1 sex ratio of cercaria released from Oncomelania hupensis or mice individually

The experimental result shows that the method can quickly obtain a large amount of single sex polypide, single female polypide or single male polypide. The genes in the group are diversified through microsatellite analysis. In the "one-to-one" infection group, although a parthenocarpic insect body was also obtained, the genotypes were consistent.

The analysis shows that the method can obtain the single insect body quickly and saves labor. Moreover, the obtained polypide is suitable for omics research, and can effectively avoid the polypide individual difference.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims

1. A method for rapidly preparing schistosoma japonicum unisexual bodies is characterized in that all schistosoma japonicum cercaria released by a single positive oncomelania are used for infecting only one animal, and the single-sex bodies are obtained by screening the sex of schistosomulum in the animal body at a specific time after infection; the single positive oncomelania is oncomelania after a plurality of schistosoma japonicum miracidiums are adopted to infect the single oncomelania; the obtained polypide with single sex is a multigenotype mixed population.

2. The method for rapidly preparing schistosoma japonicum unisexual bodies according to claim 1, wherein the animal comprises a mouse or a rabbit.

3. The method for rapidly preparing schistosoma japonicum monozoa according to claim 1, wherein the specific time is at least 3 weeks.