CN106172260B - A kind of extracorporeal culturing method for fish monogentic trematode - Google Patents
A kind of extracorporeal culturing method for fish monogentic trematode Download PDFInfo
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- 241001262815 Dactylogyrus Species 0.000 claims description 13
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- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 7
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
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- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
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- Biodiversity & Conservation Biology (AREA)
- Farming Of Fish And Shellfish (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to technical field of aquatic organism, and in particular to a kind of extracorporeal culturing method for fish monogentic trematode.The extracorporeal culturing method includes the following steps: under (1) aseptic condition, cell through secondary culture is resuspended in the first culture medium, switching is in culture dish after adjusting cell density, it is placed under the first condition of culture and cultivates, after cell is adherent, first culture medium is discarded, the second culture medium is added, for use；(2) monogentic trematode removed from fish body is resuspended in the second culture medium, then transfers in step (1) described culture dish, is cultivated under the second condition of culture, obtain the fish monogentic trematode survived in vitro.The problem of present invention solves monogentic trematode and must colonize on the fish gill or juvenile fish fin ray, can not survive in vitro will not be limited as traditional monogentic trematode disease research by epidemic season, therefore have the function of shortening the monogentic trematode disease new drug innovative research period.
Description
Technical Field
The invention belongs to the technical field of aquatic organisms, and particularly relates to an in-vitro culture method for fish monozoites.
Background
Monozoiasis is one of the most serious parasitic diseases of farmed fish. The variety of the monozoites is various, the larvae directly develop into imagoes, no intermediate host exists, and the propagation speed is very high. They usually insert into fish tissues in the chitin structure of their post-attachment devices, destroy gill and skin tissues, and cause the clinical symptoms of "gill rot" of fish by secondary infection with pathogenic bacteria and fungi after parasitic infestation of monozoites, causing massive death of farmed fish.
At present, the main method for studying living samples for monozoites is to infect other healthy fish under natural reproduction by means of the monozoites parasitizing on gills and fin of young fish. The method has some disadvantages:
(1) the prevalence of the monozoic diseases is obvious seasonality, the prevalence time of one year is only about 3-4 months, living body samples carrying a large number of monozoic diseases are difficult to find except the period, and the monozoic diseases (including larvae and adults) die in several hours after leaving the fish body, so that the drug screening and pharmacological research are greatly limited;
(2) the fish body culture method is not favorable for experimental observation and data statistics, because the distribution of the monozoites on the gills is not uniform, the positions of the gills are easy to move and change in the experimental operation process, the observation of the monozoites is not favorable, the number difference of parasitic worms on the gills of different fishes is large, the detection results of different experimental technicians have large difference, human influence factors are large, and the experimental repeatability is poor;
(3) the method of culturing by fish body is inconvenient for the mechanism research of the monozoite, the water environment is very complex, and the method is difficult to control when the action of a single environmental factor on the monozoite is researched.
Therefore, the method for in vitro culture of the monozoites is feasible, and has important application value and significance for research on the monozoites and research on prevention and treatment technologies thereof.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and aims to provide an in-vitro culture method for fish monozoites.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
an in vitro culture method for fish monozoites comprises the following steps:
(1) under the aseptic condition, resuspending the subcultured cells in a first culture medium, adjusting the cell density, transferring the cells to a culture dish, culturing under the first culture condition, after the cells adhere to the wall, discarding the first culture medium, and adding a second culture medium for later use;
(2) and (3) resuspending the monozoites exfoliated from the fish body in a second culture medium, transferring the monozoites exfoliated from the fish body into the culture dish in the step (1), and culturing under a second culture condition to obtain the fish monozoites surviving in vitro.
In the above protocol, the cell density is 1.0X 106～3.0×106one/mL.
In the above scheme, the monozoite is dactylogyrus.
In the above scheme, the cell is a carp epithelial cell.
In the scheme, the first culture medium is a mixed solution of an M199 culture medium, Fetal Bovine Serum (FBS) and an antibiotic, the volume ratio of the M199 culture medium to the fetal bovine serum is 9:1, the antibiotic is a mixed solution of penicillin and streptomycin, the working concentration of the penicillin is 100U/mL, and the working concentration of the streptomycin is 100 mug/mL.
In the above embodiment, the first culture condition is: the culture temperature is 25 ℃, and CO with the volume concentration of 5 percent is introduced2A gas.
In the above scheme, the second culture medium is a mixed solution of the first culture medium and a cell isotonic solution, and the cell isotonic solution is a NaCl solution with a mass fraction of 0.65%.
In the scheme, the volume ratio of the first culture medium to the cell isotonic solution is 1: 9-7: 3. More preferably, the volume ratio of the first culture medium to the cell isotonic solution is 5: 5.
In the above embodiment, the second culture condition is: the incubation temperature was 25 ℃.
The method for culturing the fish monozoite in vitro can be widely popularized and applied to the in vitro culture of various fish parasites, and has better guiding significance for the in vitro research of different parasites.
The invention has the beneficial effects that: (1) the invention provides an in vitro culture method for fish monozoites, which solves the problem that the traditional fish monozoites must be parasitized on fish bodies and can not survive in vitro; (2) according to the invention, the carp epithelial cells are used as parasitic carriers, and the carp epithelial cells are fixedly attached to the bottom of the culture dish after adherent growth, so that the form of the monozoite can be better observed, culture dishes with different specifications can be selected according to research conditions, and a certain amount of monozoites are added, thereby facilitating data statistics. (3) The model of the in vitro culture method is a cell model, and the adopted parasitic carrier (carp epithelial cells) is very easy to obtain in large quantity and is not limited by time, so that the screening period of the monozoic innovative medicine can be greatly shortened, and meanwhile, the monozoic feeding research is more convenient.
Drawings
FIG. 1 is a photograph showing the observation of the in vitro culture of dactylogyrus under a light microscope at 80, wherein 1 is dactylogyrus, 11 is the anchorage disc of dactylogyrus, 12 is the head organ of dactylogyrus, and 2 is the epithelial cell of carp.
FIG. 2 is a cross-sectional view, x 80, of the in vitro culture of loopworm in the form of observation under a light mirror; wherein,
(A) observing the shape of the loopworm in vitro culture under a light mirror for 0 second of inchworm movement, wherein the time is multiplied by 80;
(B) observing the morphological observation picture under a light mirror when the loopworm moves for 1 second in vitro culture of the dactylogyrus, wherein the time is multiplied by 80;
(C) observing the shape of the loopworm in vitro under a light mirror when the loopworm moves for 2 seconds, wherein the observed value is multiplied by 80;
(D) observing the shape of the loopworm in vitro under a light mirror when the loopworm moves for 3 seconds, wherein the observed value is multiplied by 80;
(E) observing the shape of the loopworm in vitro under a light mirror when the loopworm moves for 4 seconds, wherein the observed value is multiplied by 80;
(F) observing the shape of the loopworm in vitro under a light mirror when the loopworm moves for 5 seconds, wherein the observed value is multiplied by 80;
(G) observing the morphological observation picture under a light mirror when the loopworm moves for 6 seconds in vitro culture of the dactylogyrus;
(H) observing the shape of the loopworm in vitro under a light mirror when the loopworm moves for 7 seconds, wherein the observed value is multiplied by 80;
(I) observing the shape of the loopworm in vitro under a light mirror when the loopworm moves for 8 seconds, wherein the observed value is multiplied by 80;
(J) observing the morphological observation picture under a light mirror when the loopworm moves for 9 seconds in vitro culture of the dactylogyrus;
(K) observing the shape of the loopworm in vitro under a light mirror when the loopworm moves for 10 seconds, wherein the observed value is multiplied by 80;
(L) a morphological observation picture under a light mirror, x 80, of the loopworm cultured in vitro and moved for 11 seconds.
Detailed Description
For better understanding of the present invention, the contents of the present invention will be further explained below with reference to the drawings and examples, but the contents of the present invention are not limited to the following examples.
FIG. 1 shows the observation of the morphology of the in vitro culture of the dactylogyrus of the invention under a light microscope, x 80, showing the morphology of the cells in which the monozoites of this patent parasitize.
The in vitro culture method for the fish monozoite comprises the following steps:
(1) under aseptic condition, re-suspending the carp epithelial cells after subculture in a first culture medium, and adjusting the cell density to 1.0 × 106～3.0×106Transferring the cells/mL into a culture dish, culturing under a first culture condition, removing the first culture medium after the cells adhere to the wall, and adding a second culture medium for later use;
(2) and (3) resuspending the monozoites exfoliated from the fish body in a second culture medium, transferring the monozoites exfoliated from the fish body into the culture dish in the step (1), and culturing under a second culture condition to obtain the fish monozoites surviving in vitro.
Observing the monozoites under a microscope, and observing that the monozoites are stably adhered to cells to move like inchworm and the movement mode of the monozoites on the gills and the fin strips is consistent with that of the monozoites. After the high-density cells in the step (1) adhere to the wall, a single-layer cell layer which is tightly arranged is formed, the purpose is to simulate the surface structure of gill filaments of the fish gills, parasitic carriers are provided for the monozoites, the cells are fixed on a cell flat dish, observation and counting of the monozoites are facilitated after the monozoites adhere to the surfaces of the cells, and the culture method can ensure that the monozoites survive for a long time under the in vitro condition after being separated from the parasitic hosts, and is convenient for research.
The monozoite is dactylogyrus. In the figure 1, a fixing disc 11 of the dactylogyrus 1 is adsorbed on carp epithelial cells 2, a head 12 stretches and retracts, in the figure 2, the dactylogyrus 1 is adhered on the carp epithelial cells 2 and moves in an inchworm manner, and a static diagram is observed from the 0 th second to the 10 th second and in a form under a light lens intercepted every 1 second, so that the activity of the dactylogus 1 is very good and is consistent with that of the dactylogus parasitized on gills of the carpi.
The cell is carp epithelial cell. The carp epithelial cells are anchorage-dependent growth cells, the optimal culture temperature is 25 ℃, and the optimal culture temperature of the monozoite is 25 ℃, so that the culture conditions are more appropriate.
The first culture medium is a mixed solution of an M199 culture medium, Fetal Bovine Serum (FBS) and antibiotics, the antibiotics are penicillin (working concentration is 100U/mL) and streptomycin (working concentration is 100 mu g/mL), and the volume ratio of the M199 culture medium to the fetal bovine serum is 9: 1. The M199 culture medium is a culture medium for culturing carp epithelial cells, is rich in relatively complete nutrients, has stronger activity than other culture media, and under the same experimental conditions, compared with a monozoite cultured by an RPMI1640 culture medium, penicillin and streptomycin are main antibiotics required by cell culture and have broad-spectrum bacteriostatic action, so the two antibiotics are selected as the antibiotics of the invention, but other antibiotics can also obtain the same effect, and are not limited to the above.
The first culture condition is that the culture temperature is 25 ℃, and CO with the volume concentration of 5 percent is introduced2A gas. The optimal culture temperature of the carp epithelial cells is 25 ℃, and the cells cultured at the optimal culture temperature have good shape and activity.
The second culture medium is the first culture medium: the volume fraction ratio of the cell isotonic solution is 1: 9-7: 3. When the first culture medium: when the volume fraction ratio of the cell isotonic solution is 1: 9-7: 3, the second culture medium can keep a sterile state in a short time, and the monozoites can survive in vitro in a short time.
The second culture medium is the first culture medium: the volume fraction ratio of cell isotonic solution was 5: 5. When the first culture medium: when the volume fraction ratio of the cell isotonic solution is 5:5, the second culture medium can keep a sterile state for a long time, and the monozoites can survive in vitro for a longer time.
The cell isotonic solution is NaCl solution with the mass fraction of 0.65%. Compared with water, the cell isotonic solution can better maintain the form and activity of the monozoite, and the cell isotonic solution is more suitable for amphibian physiological saline because the fish is a temperature-changing animal, but the cell isotonic solution not only refers to 0.65% NaCl solution, D-Hanks solution and PBS solution, but also can achieve the same effect.
The second culture conditions are: the incubation temperature was 25 ℃. Since 25 ℃ is the optimum culture temperature for carp epithelial cells and monozoites, CO with a volume concentration of 5% is not introduced2The gas does not change the cell morphology, and CO with the volume concentration of 5 percent is introduced2The gas causes the death of the monozoites, so 25 ℃ was chosen as the second culture condition.
The effectiveness of the present invention is further illustrated by the following experiments.
Experiment one
To investigate the effect of a mixture of M199 medium and Fetal Bovine Serum (FBS) mixed at a volume ratio of 9:1 on monozoites, the following experiments were designed: taking 25 monozoites stripped from fish gills, dividing the monozoites into 5 groups, placing 5 monozoites in each group, culturing in an antibiotic-free mixed solution with equal dilution (the dilution is cell isotonic solution, and all the dilutions in the first experiment to the fourth experiment are NaCl solution with the mass fraction of 0.65%), wherein the culture temperature is 25 ℃, and observing the monozoites once every 24 hours under a microscope.
TABLE 1 Effect of mixtures of M199 Medium and Fetal Bovine Serum (FBS) on Monozoites
According to the experimental results, the bacterial reproduction speed can be delayed after the dilution of the mixed solution (without antibiotics) of the M199 culture medium and the Fetal Bovine Serum (FBS), but the bacterial reproduction speed cannot be controlled finally, and antibiotics still need to be added; it is speculated that high concentrations of mixed liquor may not be suitable for growth of monozoites.
Experiment two
To determine the effect of high concentrations of a mixture of M199 medium and Fetal Bovine Serum (FBS) on the growth of monozoites, the following experiments were designed: taking 55 monozoites stripped from fish gills, dividing the monozoites into 11 groups, adding antibiotics into mixed liquor (M199 culture medium and Fetal Bovine Serum (FBS) are mixed according to a volume ratio of 9: 1) for 5 monozoites in each group, placing the monozoites into the mixed liquor with gradually diluted antibiotic concentration for culture at a culture temperature of 25 ℃, and observing the monozoites once every 24 hours under a microscope, wherein the concentration of the antibiotic mixed liquor of penicillin with working concentration of 100U/mL and streptomycin with working concentration of 100 mu g/mL is marked as 1 x; the mixed solution containing 1 × antibiotics is the first culture medium.
TABLE 2 Effect of a mixture of M199 Medium containing antibiotics and Fetal Bovine Serum (FBS) on the growth of Monozoites
According to the experimental results, the mixed solution (whether antibiotics are added or not) which is not diluted by the diluent is harmful to the monozoites; the working concentration of the antibiotic is 0.3X-1X, and the antibiotic has good bacteriostatic effect.
Experiment three
In order to further search for a suitable medium for the in vitro culture of monozoites and working concentrations of antibiotics, the following experiments were designed: 55 monozoites stripped from fish gills were divided into 11 groups of 5 monozoites per group and cultured in a first medium diluted stepwise with a diluent (0.65% by mass NaCl solution) at a temperature of 25 ℃ under a microscope every 24 hours.
TABLE 3 Effect of dilution of first Medium on growth of Monozoites
From the above experimental results, it was found that the suitable conditions for in vitro culture were the sixth and seventh groups of the experimental group, but considering that the high-concentration mixed solution and the high-concentration antibiotic in the first and second experiments have a bad influence on the monozoites, the sixth group was selected as the second medium for the subsequent in vitro culture of the monozoites.
Experiment four
In experiment three, the monozoites were able to survive in the second medium for two days but still not long enough compared to the dilution, and therefore the difficulties in ex vivo culture of monozoites were presumed to be not only a nutritional factor but also a cause of lack of parasitic vectors. To simulate gill surface layer, carp epithelial cells (EPC) cultured to form a monolayer were selected as parasitic vectors, and the following experiments were designed: 30 monozoites stripped from fish gills were divided into 6 groups, including 4 control groups and two experimental groups, each of which was 5 monozoites, and the culture temperature was 25 ℃ and the monozoites status was observed under a microscope every 24 hours.
TABLE 4 Effect of parasitic vector EPC cells on the growth of Monozoites
As can be seen from the above experimental results, in comparison with control 1 and control 2, we found that when EPC cells were not used as parasitic carriers, CO was introduced at a volume concentration of 5% even when the culture medium was the second medium2The gas still causes the death of the monozoites; comparison of control group 3 and control group 4 revealed the presence or absence of 5% CO2The morphology of the EPC cells can be maintained for a long time by the second culture medium; comparing experimental group 1 and experimental group 2, it was found that the monozoites can adhere to EPC cells, which can act as parasitic carriers for the in vitro culture of the monozoites, and in the absence of 5% CO2The attachment on the EPC cells in the environment survived for a longer time.
The invention solves the problem that the monozoic has to be parasitized on the gill or fin of the juvenile fish and can not survive in vitro, and the study of monozoic diseases is not limited by the epidemic season like the traditional study of monozoic diseases, so the invention has the function of shortening the new drug creating study period of monozoic diseases.
The invention uses the cell model to culture the monozoite in vitro, is convenient to transplant to the in vitro culture of other fish parasites, not only can ensure that the research on the fish parasites is not limited by living samples, but also can artificially control the density of inoculated parasites, and ensures that various experimental data are more accurate.
It is apparent that the above embodiments are only examples for clearly illustrating and do not limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications are therefore intended to be included within the scope of the invention as claimed.
Claims (3)
1. An in vitro culture method for fish monozoites is characterized by comprising the following steps:
(1) under the aseptic condition, re-suspending carp epithelial cells subjected to subculture in a first culture medium, adjusting the cell density, transferring the carp epithelial cells into a culture dish, culturing under the first culture condition, after the cells adhere to the wall, removing the first culture medium, and adding a second culture medium for later use; the first culture medium is a mixed solution of an M199 culture medium, fetal calf serum and an antibiotic, the volume ratio of the M199 culture medium to the fetal calf serum is 9:1, and the antibiotic is cyanMixed solution of streptomycin and penicillin, wherein the working concentration of penicillin is 100U/mL, and the working concentration of streptomycin is 100 mug/mL; the second culture medium is a mixed solution obtained by mixing the first culture medium and cell isotonic solution according to the volume ratio of 1: 9-7: 3; the cell isotonic solution is NaCl solution with the mass fraction of 0.65%; the monozoite is dactylogyrus; the first culture conditions are: the culture temperature is 25 ℃, and CO with the volume concentration of 5 percent is introduced2A gas;
(2) resuspending the monozoites exfoliated from the fish body in a second culture medium, then transferring the monozoites exfoliated from the fish body into the culture dish in the step (1), and culturing under a second culture condition to obtain fish monozoites surviving in vitro; the second culture conditions are: the incubation temperature was 25 ℃.
2. The method of claim 1, wherein the cell density is 1.0 x 106~3.0×106one/mL.
3. The method of claim 1, wherein the volume ratio of the first medium to the cell isotonic solution is 5: 5.
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| CN105052838A (en) * | 2015-08-14 | 2015-11-18 | 李阳 | Separating and culturing method for free-living marine nematodes |
| CN105132287A (en) * | 2015-10-19 | 2015-12-09 | 吉林省水产科学研究院 | Method for culturing myxobolus in vitro |
| CN105733948A (en) * | 2016-03-31 | 2016-07-06 | 吉林农业大学 | Cell culture method for myxobolus |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2193310C1 (en) * | 2001-03-30 | 2002-11-27 | Научно-исследовательский ветеринарный институт Нечерноземной зоны Российской Федерации | Method for cultivation of trematode eggs |
| CN101953322A (en) * | 2010-10-21 | 2011-01-26 | 武汉大学 | In-vitro schistosomulum co-culture method |
| CN103898217A (en) * | 2014-03-27 | 2014-07-02 | 中国科学院水生生物研究所 | Specific amplification primer pair for parasitic dactylogyrus lamellatus Achmerow of grass carp gill and application thereof |
| CN105052838A (en) * | 2015-08-14 | 2015-11-18 | 李阳 | Separating and culturing method for free-living marine nematodes |
| CN105132287A (en) * | 2015-10-19 | 2015-12-09 | 吉林省水产科学研究院 | Method for culturing myxobolus in vitro |
| CN105733948A (en) * | 2016-03-31 | 2016-07-06 | 吉林农业大学 | Cell culture method for myxobolus |
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