CN108293925B - A kind of immune model construction method stimulating cryptonucleus insect local infection Epinephelus coioides gill tissue - Google Patents
A kind of immune model construction method stimulating cryptonucleus insect local infection Epinephelus coioides gill tissue Download PDFInfo
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- 208000015181 infectious disease Diseases 0.000 title claims abstract description 86
- 241000238631 Hexapoda Species 0.000 title claims abstract description 58
- 241000357444 Epinephelus coioides Species 0.000 title claims abstract description 25
- 230000004936 stimulating effect Effects 0.000 title claims abstract description 10
- 238000010276 construction Methods 0.000 title claims abstract description 6
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- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 9
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- 229920000153 Povidone-iodine Polymers 0.000 claims description 3
- 241000932075 Priacanthus hamrur Species 0.000 claims description 3
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- 206010042566 Superinfection Diseases 0.000 claims 1
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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
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The present invention is immunized using the stimulation azygobranchiate mode of cryptonucleus insect local infection Epinephelus coioides for the first time, using the infection concentration and infection duration of optimization, the immune model of stimulation cryptonucleus insect local infection Epinephelus coioides gill tissue is constructed for the first time, negative effect brought by " congenital sex differernce " during fish part gill tissue mucosal immune response is completely eliminated, provides ideal immune model to study local mucosal immune response rule.The construction method of immune model of the present invention mainly includes 5 Ge Walk rapid: (1) stimulating the collection and concentration calculation of cryptonucleus insect larva living；(2) it the grouping of Epinephelus coioides and raises and train；(3) local gill tissue is immune；(4) measurement of infection intensity；(5) after local infection fish body processing.
Description
Technical field
The invention belongs to fish immunity fields, specially fish mucosa-immune direction, and in particular to a kind of induction angled tape stone
The construction method of the immune model of Ban Yu gill tissue part mucosa-immune.
Background technique
Stimulating cryptonucleus insect (Cryptocaryon irritans) is a kind of primary infusorian, can infect nearly all seawater
Bony fish causes serious fatal disease --- stimulation cryptocaryoniosis or ichthyophthirius (Colorni A and Burgess
P., 1997).The history of life for stimulating cryptonucleus insect includes 4 stages such as trophozoite, packing precursor, packing and larva, wherein only growing
The obligatory parasitism life of body battalion is supported, is food with host body fluids, fragment of tissue and intact cell etc..In recent years, due to China's seawater
The excess load of cultivation develops, the deterioration of breeding environment, and stimulation cryptocaryoniosis has become the strong biography of a wide range of, regular outburst
It catches an illness, causes harm very serious to seawater fishery.2008, the Ministry of Agriculture will stimulate cryptocaryoniosis to be included in China's " two classes
Animal epidemic " register is unique a kind of aquatic products parasitic disease in China " one, two class animal epidemics " register, it is seen that it was endangered
Seriousness.
Different from the general infection of other microorganism cause of diseases such as virus, bacterium, stimulate cryptonucleus insect main parasitic in host's
On skin, fin ray and the gill, unique parasite property becomes research seawater fish local skin and gill mucosal immune response
Good model；Although we establish the thorn with egg-shaped pompano for host in addition, stimulation cryptonucleus insect cannot be cultivated in vitro
Swash cryptonucleus insect living body propagating method and low temperature protecting method, so that us is obtained a large amount of polypide at any time and carry out artificial quantitative sense
Dye experiment, this be most aquatic products helminths do not accomplish (Dan et al., 2006；Dan et al., 2009).
Currently, the research of the host part mucosal immune response infection induced about stimulation cryptonucleus insect, mainly passes through thorn
Swash that cryptonucleus insect whole body body surface infection host fish model carries out (Yambot et al., 2006；Luo et al., 2007；But it learns
It is bright etc., 2008；Misumi, et al., 2011,2012).After such method infection, the whole quilts simultaneously of skin, the gill and the fin ray of fish
Infection, then the difference by comparing mucosal immune response between infected group and non-infected group Different Individual fish disclose the anti-thorn of host
Swash the local mucous membrane response rule of cryptonucleus insect infection.However, since there are hereditary differences between fish Different Individual, it is more different
Between individual there are " birth defects " in the difference of immune response.In the recent period, there is researcher by part below grouper cloacal aperture
It is immersed in the worm liquid of stimulation cryptonucleus insect, carries out local tail portion infection, then acquire the skin of infection site and same fish is not felt
The skin of dye carries out transcript profile sequencing, infection site and non-infection site skin and mucosa immune response with more same tail fish
Difference (Hu et al., 2017).The process eliminate the difference of Different Individual genetic background, compared to comparing Different Individual
Between difference be improved.But this method can not rule out same tail fish tail portion and tail portion is exempted under normal circumstances with outer skin
The difference of epidemic disease response.Based on this, the present invention establishes a kind of stimulation cryptonucleus insect infection azygobranchiate model of fish body, for relatively more same
Infection and the difference for being uninfected by gill local immune response, keep result more accurate at left and right sides of one tail fish.
Summary of the invention
The present invention is directed to overcome fish influence caused by individual difference after stimulation cryptonucleus insect is immune, by same
The unilateral gill of tail fish implements local mucosa-immune, provides stable, reliable experimental material to study local mucosal immune response.
The present invention uses unique mode of infection, cultivates only unilateral gill local organization and completes immune fish.
Of the invention is unique in that, is immunized for the first time using the azygobranchiate mode of local infection, use is optimal
The infection concentration and infection duration of change, construct the immune model of the stimulation cryptonucleus insect local infection Epinephelus coioides gill for the first time, thorough
Bottom eliminates " congenital sex differernce " brought negative effect during fish local organization mucosal immune response, for research part
Mucosal immune response rule provides ideal immune model.Further, it is also possible to carry out multiple exempt to same gill local organization
Epidemic disease is to produce part repeatedly immune fish body, the research for facilitating follow-up immunization to remember.
It is an advantage of the present invention that solving during researching fish mucosa-immune, fish individual difference or tissue
The influence that difference generates, and ideal experimental material and immune model are provided for researching fish part mucosal immune response.
The present invention is achieved by the following technical solutions:
A kind of immune model construction method stimulating cryptonucleus insect local infection Epinephelus coioides gill tissue, including following step
It is rapid:
1, the collection and concentration calculation of stimulation cryptonucleus insect larva living
Stimulation cryptonucleus insect is passed on and collected using special stimulation cryptonucleus insect legacy system, passage host is ovum
Shape silvery pomfret Scad.Well-developed stimulation cryptonucleus insect packing is acquired, is placed in sterilized clean beaker and hatches, Spawning water is high pressure
Sterilizing, addition appropriate penicillin and streptomysin nature seawater: collecting the cryptocaryon irritans larva worm liquid hatched in 2 hours,
50 μ L are taken after mixing well, and are added 10 μ L formaldehyde to kill larva, are counted under the microscope, the polypide for calculating larva worm liquid is dense
Degree.
2, it the grouping of Epinephelus coioides and raises and train
Experimental fish is the Epinephelus coioides of 20g or so, which can cause after stimulating cryptonucleus insect to infect convenient for operation
Strong immune response.Epinephelus coioides are divided into 9 groups, including experimental group 8, control group 1, every group of 15 tail fishes.Fish body is tamed and dociled
It supports 2 weeks, until its feed, activity are normally.In entire breeding process, strainer filtering of the seawater through 600 mesh, salinity 30 ‰ or so,
PH8 or so, dissolved oxygen amount is in 5.6mg/L or more.
3, local gill tissue is immune
Experimental group carries out gill local infection with the cryptocaryon irritans larva for just hatching various concentration in 2 hours.Experimental group sets 4
A larva infects concentration: 1000,5000,10000,15000/ml, because having when 15000 larva/ml infection concentration
Part fish starts death occur, so highest infection concentration is set as this concentration；Two infection times of 30s and 60s are arranged in experimental group,
Environment-stress caused by water body is left in order to reduce fish body to the greatest extent, while guaranteeing that polypide can infect host.
Specific course of infection is as follows: will anaesthetize in the seawater of Epinephelus coioides merging eugenol containing 1ppm, completely fiber crops
It takes out after liquor-saturated and is organized with the seawater flushing gill portion of povidone iodine containing 2ppm.It will be lain against downward above a wet towel on the left of fish body,
Fish body head is fixed with hand and strutting the left gill cover with finger is completely exposed gill portion tissue；Setting timer is 30s or 60s, is taken
One disposable rubber head dropper with a scale draws the worm liquid that total amount is 1ml, press timer and starting turn left pleurobranchiae portion organize it is small
The heart instills worm liquid, drips in 15s, keeps the gill cover to be flared to after timer sounds, the fish body two sides gill cover is opened and uses nothing
Worm water rinses 10s, then fish body is transferred in fresh seawater and is normally cultivated.
Control group is infected with 0 larva/ml seawater, and infection time 60s, operation is same as above.
4, the measurement of infection intensity
After infection 48 hours, each group takes 5 tail fishes to be anaesthetized, and all gill tissues for obtaining the left and right sides, which are placed in, fills seawater
Culture dish in, under microscope count the right and left stimulation cryptonucleus insect trophozoite quantity, as a result indicated with mean value.
As a result, it has been found that: the left and right gill of the right gill and control group that worm liquid is not added dropwise finds no trophozoite, and worm liquid is added dropwise
The left gill infected has different degrees of stimulation cryptonucleus insect infection.With the hidden core of stimulation of higher concentration (15000 larvas/ml)
It is bigger to the destruction of the fish gill after insect infection, the breathing of fish is seriously affected, infection fish will appear different degrees of death；It is identical dense
The infection intensity of polypide, which is substantially less than, after the stimulation cryptonucleus insect local infection gill 30s of degree infects 60s.Therefore, higher to guarantee
Stimulation cryptonucleus insect infection intensity does not simultaneously impact the survival of fish body, and for the grouper of 20g or so, present invention recommendation makes
60s is contaminated with 10000 larva/ml worm liquid inductances.
5, after local infection fish body processing
Stimulation cryptonucleus insect will fall off after infecting fish body 3 days from fish body, and the packing reached maturity can hatch new larva
Fish body is infected again.The present invention be the larva that hatches of the stimulation cryptonucleus insect packing to fall off after avoiding grouper from being infected for the first time again
Infection influences that local infection is tested as a result, fish body to be transferred to clean culturing jar in every 2 days after stimulation cryptonucleus insect infects
It is interior, after rotation cultivates 3 times, it is placed in culturing jar and normally cultivates；Take obtain infection concentration and infection time be 10000/ml, 60s
Experimental group Epinephelus coioides, that is, obtain stimulation cryptonucleus insect local infection Epinephelus coioides gill tissue immune model.Remove fish
The fresh water of 30cm depth is added in culturing jar after body, impregnates 8 hours, can kill all stimulation cryptonucleus insect packets for being adhered to bottom of pond
Capsule empties fresh water after scrub, fills clean seawater and waits for using next time.
Advantages of the present invention and effect: unique gill local organization immunization ways are used, using the infection concentration of optimization
And infection time, making fish body only has the infection of trophozoite in the left gill, and the right gill is then without helminth.The immune model that the method obtains
Fish individual difference or histological difference bring, which can be greatly reduced, to be influenced, and is provided for researching fish gill part mucosal immune response
Ideal experimental material.
Specific embodiment
The specific embodiment of the invention is as follows:
A kind of immune model construction method stimulating cryptonucleus insect local infection Epinephelus coioides gill tissue, including following step
It is rapid:
1, the collection and concentration calculation of stimulation cryptonucleus insect larva living
Stimulation cryptonucleus insect is passed on and collected using special stimulation cryptonucleus insect legacy system, passage host is ovum
Shape silvery pomfret Scad.Well-developed stimulation cryptonucleus insect packing is acquired, is placed in sterilized clean beaker and hatches, Spawning water is high pressure
Sterilizing, addition appropriate penicillin and streptomysin nature seawater；The cryptocaryon irritans larva worm liquid hatched in 2 hours is collected,
50 μ L are taken after mixing well, and are added 10 μ L formaldehyde to kill larva, are counted under the microscope, the polypide for calculating larva worm liquid is dense
Degree.
2, it the grouping of Epinephelus coioides and raises and train
Experimental fish is the Epinephelus coioides of 20g or so, which can cause after stimulating cryptonucleus insect to infect convenient for operation
Strong immune response.Epinephelus coioides are divided into 9 groups, including experimental group 8, control group 1, every group of 15 tail fishes.Fish body is tamed and dociled
It supports 2 weeks, until its feed, activity are normally.In entire breeding process, strainer filtering of the seawater through 600 mesh, salinity 30 ‰ or so,
PH8 or so, dissolved oxygen amount is in 5.6mg/L or more.
3, local gill tissue is immune
Experimental group carries out gill local infection with the cryptocaryon irritans larva for just hatching various concentration in 2 hours.Experimental group sets 4
A larva infects concentration: 1000,5000,10000,15000/ml, because having when 15000 larva/ml infection concentration
Part fish starts death occur, so highest infection concentration is set as this concentration；Two infection times of 30s and 60s are arranged in experimental group,
Environment-stress caused by water body is left in order to reduce fish body to the greatest extent, while guaranteeing that polypide can infect host.
The larva infection concentration and infection time of 8 experimental groups are (table 1) as shown in the table:
| Group number | Larva concentration (a/ml) | Larva infects total duration (s) |
| Control group | 0 | 60 |
| 1 | 1000 | 30 |
| 2 | 5000 | 30 |
| 3 | 10000 | 30 |
| 4 | 15000 | 30 |
| 5 | 1000 | 60 |
| 6 | 5000 | 60 |
| 7 | 10000 | 60 |
| 8 | 15000 | 60 |
Table 1
Specific course of infection is as follows: will anaesthetize in the seawater of Epinephelus coioides merging eugenol containing 1ppm, completely fiber crops
It takes out after liquor-saturated and is organized with the seawater flushing gill portion of povidone iodine containing 2ppm.It will be lain against downward above a wet towel on the left of fish body,
Fish body head is fixed with hand and strutting the left gill cover with finger is completely exposed gill portion tissue；Setting timer is 30s or 60s, is taken
One disposable rubber head dropper with a scale draws the worm liquid that total amount is 1ml, press timer and starting turn left pleurobranchiae portion organize it is small
The heart instills worm liquid, drips in 15s, keeps the gill cover to be flared to after timer sounds, the fish body two sides gill cover is opened and uses nothing
Worm water rinses 10s, then fish body is transferred in fresh seawater and is normally cultivated.
Control group is infected with 0 larva/ml seawater, and infection time 60s, operation is same as above.
4, the measurement of infection intensity
After infection 48 hours, each group takes 5 tail fishes to be anaesthetized, and all gill tissues for obtaining the left and right sides, which are placed in, fills seawater
Culture dish in, under microscope count the right and left stimulation cryptonucleus insect trophozoite quantity, as a result indicated, be see the table below with mean value
(table 2):
| Group number | Left gill trophozoite quantity (a) | Right gill trophozoite quantity (a) |
| Control group | 0 | 0 |
| 1 | 30.56 | 0 |
| 2 | 80.84 | 0 |
| 3 | 140.63 | 0 |
| 4 | 200.82 | 0 |
| 5 | 70.68 | 0 |
| 6 | 126.98 | 0 |
| 7 | 280.76 | 0 |
| 8 | 670.52 | 0 |
Table 2
As a result, it has been found that: the left and right gill of the right gill and control group that worm liquid is not added dropwise finds no trophozoite, and worm liquid is added dropwise
The left gill infected has different degrees of stimulation cryptonucleus insect infection.With the hidden core of stimulation of higher concentration (15000 larvas/ml)
It is bigger to the destruction of the fish gill after insect infection, the breathing of fish is seriously affected, infection fish will appear different degrees of death；It is identical dense
The infection intensity of polypide, which is substantially less than, after the stimulation cryptonucleus insect local infection gill 30s of degree infects 60s.Therefore, higher to guarantee
Stimulation cryptonucleus insect infection intensity does not simultaneously impact the survival of fish body, and for the grouper of 20g or so, present invention recommendation makes
60s is contaminated with 10000 larva/ml worm liquid inductances.
5, after local infection fish body processing
Stimulation cryptonucleus insect will fall off after infecting fish body 3 days from fish body, and the packing reached maturity can hatch new larva
Fish body is infected again.The present invention be the larva that hatches of the stimulation cryptonucleus insect packing to fall off after avoiding grouper from being infected for the first time again
Infection influences that local infection is tested as a result, fish body to be transferred to clean culturing jar in every 2 days after stimulation cryptonucleus insect infects
It is interior, after rotation cultivates 3 times, it is placed in culturing jar and normally cultivates.The fresh water of 30cm depth, leaching is added in culturing jar after removing fish body
Bubble 8 hours, can kill all stimulation cryptonucleus insect packings for being adhered to bottom of pond, empty fresh water after scrub, fill clean seawater and wait for
It uses next time.
Claims (1)
1. a kind of immune model construction method for stimulating cryptonucleus insect local infection Epinephelus coioides gill tissue, which is characterized in that packet
Include following steps:
(1) collection and concentration calculation of stimulation cryptonucleus insect larva living
Stimulation cryptonucleus insect is passed on and collected using special stimulation cryptonucleus insect legacy system, passage host is oval silvery pomfret
Scad；Well-developed stimulation cryptonucleus insect packing is acquired, is placed in sterilized clean beaker and hatches, Spawning water is high pressure sterilization
, nature seawater that appropriate penicillin and streptomysin is added；The cryptocaryon irritans larva worm liquid hatched in 2 hours is collected, sufficiently
50 μ L are taken after mixing, are added 10 μ L formaldehyde to kill larva, are counted under the microscope, calculate the polypide concentration of larva worm liquid；
(2) it the grouping of Epinephelus coioides and raises and train
Experimental fish is the Epinephelus coioides of 20g or so；Epinephelus coioides are divided into 9 groups, including experimental group 8, control group 1
It is a, every group of 15 tail fishes；Fish body is raised and train 2 weeks, until its feed, activity are normally；In entire breeding process, filter of the seawater through 600 mesh
Net filtration, salinity 30 ‰ or so, pH8 or so, dissolved oxygen amount is in 5.6mg/L or more；
(3) local gill tissue is immune
Experimental group carries out local infection with the cryptocaryon irritans larva for just hatching various concentration in 2 hours, and experimental group sets 4 larvas
Infect concentration: 1000,5000,10000,15000/ml and two infection time: 30s and 60s；
Specific course of infection is as follows: will anaesthetize in the seawater of Epinephelus coioides merging eugenol containing 1ppm, after holonarcosis
It takes out and the seawater flushing gill portion of povidone iodine containing 2ppm is used to organize；It will be lain against downward above a wet towel on the left of fish body, use hand
It fixes fish body head and strutting the left gill cover with finger is completely exposed gill portion tissue；Setting timer is 30s or 60s, takes a band
The disposable rubber head dropper of scale draws the worm liquid that total amount is 1ml, presses timer and starts the careful drop of pleurobranchiae portion tissue of turning left
Enter worm liquid, dripped in 15s, the gill cover is kept to be flared to after timer sounds, the fish body two sides gill cover is opened and with no worm water
10s is rinsed, then fish body is transferred in fresh seawater and is normally cultivated；
Control group is infected with 0 larva/ml seawater, and infection time 60s, operation is same as above；
(4) measurement of infection intensity
After infection 48 hours, each group takes 5 tail fishes to be anaesthetized, and all gill tissues for obtaining the left and right sides are placed in the training for filling seawater
It supports in ware, the trophozoite quantity of left and right sides stimulation cryptonucleus insect is counted under microscope, is as a result indicated with mean value；According to measurement
Each group infection intensity guarantees higher stimulation cryptonucleus insect infection intensity as a result, the most suitable infection concentration of acquisition and infection time
The survival of fish body is not impacted simultaneously；The worm liquid inductance that most suitable infection concentration and infection time are 10000/ml contaminates
60s；
(5) after local infection fish body processing
To avoid superinfection, fish body was transferred in clean culturing jar in every 2 days after stimulation cryptonucleus insect infects, rotation cultivation
It after 3 times, is placed in clean culturing jar and normally cultivates, obtain infection concentration and infection time is the 10000/experimental group of ml, 60s
Epinephelus coioides obtain the immune model of stimulation cryptonucleus insect local infection Epinephelus coioides gill tissue；
The fresh water of 30cm depth is added in culturing jar after removing fish body, impregnates 8 hours, can kill all thorns for being adhered to cylinder bottom
Swash cryptonucleus insect packing, empties fresh water after scrub.
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