CN110999825A

CN110999825A - A kind of method for measuring the infection rate of stimuli Cryptonucleus on fish

Info

Publication number: CN110999825A
Application number: CN201911143141.8A
Authority: CN
Inventors: 李安兴; 钟志鸿; 江飚
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2019-11-20
Filing date: 2019-11-20
Publication date: 2020-04-14

Abstract

The invention discloses a method for measuring the infection rate of cryptocaryon irritans to fish bodies. The method comprises the following steps: s1, infecting fishes with cryptocaryon irritans larvae; s2, assembling a collecting device; s3, respectively transferring the fish infected in the step S1 for 2h to the collecting devices prepared in the step S2 for feeding; then, replacing the collecting cloth liner 1 every day at the 2 nd to 12 th days after infection respectively, and recording the number of cysts collected on the collecting cloth liner 1; s4, calculating: infection rate (%) — total number of capsules collected/infectious dose 100. The invention finds out the linear relation between the number of cysts falling off from the fish body and the infection dosage of the cryptocaryon irritans through research, and the linear equation between the cysts and the infection dosage is that y is 192.5+3.9612x, r²The infection dose is 0.990, and the two are in good linear relation within the range of 0-2000 infection dose per tail, so that the infection rate of the cryptocaryon irritans can be accurately and rapidly calculated, the method can be effectively used for evaluating the infection intensity of the cryptocaryon irritans on the fish, and further evaluating the strength of the cryptocaryon irritans resistance of the fish.

Description

Method for measuring infection rate of cryptocaryon irritans on fish bodies

Technical Field

The invention relates to the technical field of fish disease prevention and control, in particular to a method for measuring the infection rate of cryptocaryon irritans to fish bodies.

Background

Cryptocaryon irritans is a parasitic disease caused by cryptocaryon irritans parasitizing on the body surface of marine teleostean fish, and a large number of macroscopic 'small white spots' can be formed on the body surfaces of skin, fin rays, gills and the like of the fish infected with the parasite, and the disease is also called 'white spot disease'. The life history of cryptocaryon irritans includes 4 stages of trophozoites, cysts precursors, cysts and larvae, wherein trophozoites are the stages of parasitizing fish bodies, cysts precursors and cysts are the stages of division and reproduction, and larvae are the stages of infection. The cryptocaryon irritans can almost infect all cultivated marine teleostean fishes in south China, is the parasitic disease with the most serious harm at present, and can cause hundreds of millions of economic losses every year. In recent years, a large amount of research works on cryptocaryon irritans prevention and treatment including immune prevention and treatment, drug prevention and treatment, ecological prevention and treatment and the like are carried out by scholars at home and abroad, but cryptocaryon irritans still seriously jeopardize the marine fish farming industry.

The evaluation of the prevention and treatment effect is a key link for disease prevention and control research. The relative protection rate and the infection rate can directly reflect the control effect of drugs, vaccines and the like on the cryptocaryon irritans disease resistance of the fish body, wherein the infection rate of the cryptocaryon irritans on the fish body (namely the ratio of the quantity of the cryptocaryon irritans to the infection dose of the infected fish body) is an important index for evaluating the infection intensity of the cryptocaryon irritans on the fish body. The intensity of infection of fish by cryptocaryon irritans is currently evaluated mainly against the parasitic stages of the parasite: the infection intensity of the fish is represented by the ratio of the number of trophozoites on the second gill or local body surface on the right side of the fish to the body weight of the fish. Although the method is short in time consumption and simple, the method causes large error of results due to large difference between fish bodies and difficulty in counting gill trophozoites. In addition, this method cannot directly evaluate the infection rate of cryptocaryon irritans to fish bodies. Therefore, a more scientific and accurate method for evaluating the infection intensity of cryptocaryon irritans to fish needs to be found.

Comparison of the infection rate of cryptocaryon irritans is the most direct and accurate method for evaluating the infection intensity of cryptocaryon irritans on fish, however, the total trophozoite number of fish is difficult to be counted. During the process from larva infection to cyst formation, a larva invading the fish body develops and matures through trophozoite, and finally an cyst is formed. Therefore, the capsule which is completely dropped by the collected fish body can directly respond to the infection rate of the cryptocaryon irritans to the fish, but the capsule which is directly collected at the bottom of the fish pond has large workload, large quantity of lost capsules and broken parts of capsules, and finally the infection rate is calculated inaccurately. There is therefore a need to provide a method for accurately collecting cysts and scientifically calculating the infection rate.

Disclosure of Invention

The invention aims to overcome the defect that no method for accurately measuring the cryptocaryon irritans infection rate exists in the prior art, and provides a method for measuring the cryptocaryon irritans infection rate of fish bodies. The invention accurately collects and counts cysts falling off from the fish body through a specific collecting device according to the life history characteristics of the cryptocaryon irritans.

In order to achieve the purpose, the invention is realized by the following scheme:

a method for measuring the infection rate of cryptocaryon irritans to fish bodies comprises the following steps:

s1, temporarily breeding uninfected fishes indoors, infecting the fishes by adopting a certain number of cryptocaryon irritans larvae, and recording the infection dose;

s2, a collecting device: laying the collecting cloth mat 1 at the bottom of an open container 2, then placing the fence 3 which is communicated up and down on the collecting cloth mat 1, and ensuring that the area of the bottom of the fence 3 is smaller than that of the collecting cloth mat 1; then adding water into the container 2 and the enclosure 3;

s3, respectively transferring the fish infected in the step S1 for 2h to the collecting devices prepared in the step S2 for feeding; then, replacing the collecting cloth liner 1 every day at the 2 nd to 12 th days after infection respectively, and recording the number of cysts collected on the collecting cloth liner 1;

s4, calculating: infection rate (%) — total number of cysts collected and/infectious dose 100.

The cyst precursor stimulating the cryptocaryon trophozoite to form sinks in the water bottom and slowly swims, slowly secretes substances within 2 hours to form a cyst wall on the surface of the cyst precursor, finally forms a cyst, and the cyst can be adhered to attachments in the process of forming the cyst wall; therefore, the collecting cloth gasket 1 is paved at the bottom of the container 2, and after the cysts are attached to the collecting cloth gasket 1, the quantity of all the cysts is counted, so that the infection rate of the cryptocaryon irritans to the fish body is effectively evaluated.

Preferably, the fish is marine teleost. Cryptocaryon irritans can infect almost all marine teleosts.

Preferably, the fish is trachinotus ovatus.

The host range of cryptocaryon irritans is wide, and the living environment of different species of fishes has certain difference, so that the life cycle of each stage of cryptocaryon irritans is different, and the specific cyst collecting days can be determined according to the determined species and living environment of the fishes. For example, when the fish is trachinotus ovatus, the temperature of the living water is 30 + -1 deg.C, the cysts can completely fall off 4 days after infection, and the cysts at 2d, 3d and 4d after infection can be collected, i.e. all the cysts on the fish body can be collected.

Preferably, the feeding conditions of the fish in step S1 and step S3 are: continuously aerating oxygen at the water temperature of 18-32 ℃, and culturing in still water; feeding the fish according to 6 percent of the weight of the fish every day, wherein the feeding is 1.5 percent every time, and the feeding is carried out for 4 times a day.

Preferably, the infection dose is 0-2000 per tail.

Preferably, when the fish is trachinotus ovatus, the linear equation of the total number of cysts collected by the 3d and the infection dose is that y is 192.5+3.9612x, r²＝0.990。

Preferably, the collecting cloth liner 1 is dark polyester rain awning cloth.

Preferably, the container 2 and the enclosure 3 can each be a tub-shaped container.

Preferably, the pen 3 is smaller in diameter and height than the container 2.

Preferably, the enclosure 3 is in the shape of a tapered barrel. When the pen 3 is in the shape of a cone-shaped tub, the capsule in the water can then sink completely onto the collecting mat 1 without adhering to the walls of the pen 3.

Preferably, the edge of the collecting cloth mat 1 is higher than the bottom end of the railing 3. In order to prevent the loss of the capsule from the space between the rail 3 and the collection sheet 1, the edge of the collection sheet 1 placed on the periphery of the rail 3 is too high or stuck to the rail 3, thereby preventing the loss of the capsule.

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

the invention finds the linearity between the number of cysts falling off from the fish body and the infection dose of cryptocaryon irritans through researchThe linear equation between the two is that y is 192.5+3.9612x, r²The infection dose is 0.990, and the two are in good linear relation within the range of 0-2000 infection dose per tail, so that the infection rate of the cryptocaryon irritans can be accurately and rapidly calculated, the method can be effectively used for evaluating the infection intensity of the cryptocaryon irritans on the fish, and further evaluating the strength of the cryptocaryon irritans resistance of the fish.

Drawings

Fig. 1 is a schematic view of the structure of the collecting apparatus in embodiment 1.

FIG. 2 is a schematic diagram of the collecting apparatus in example 1.

Fig. 3 is a diagram showing the capsules collected on the collecting cloth 1 after the collecting apparatus of example 1 is used.

FIG. 4 shows the number of capsules collected at 2d, 3d and 4d, respectively, for different doses of infection in example 1.

FIG. 5 is a linear equation between the infectious dose and the number of cysts per set of experiments in example 1.

FIG. 6 is a linear equation between the infectious dose and the number of cysts per fish in example 1.

FIG. 7 is the number of local epidermal trophoblasts counted at 2d, 3d and 4d, respectively, for different doses of infection in example 2.

FIG. 8 is a linear equation between the dose of infection at 2d and the number of local epidermal trophozoites in example 2.

FIG. 9 shows the number of right gill trophozoites counted at 2d, 3d and 4d, respectively, for different doses of infection in example 2.

FIG. 10 is a linear equation between the dose of infection at 2d and the number of right gill trophozoites in example 2.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1

1. Cryptocaryon irritans cyst collecting device

Wherein 1 is deep color terylene rain awning cloth, 2 is a white big barrel, 3 is a white small barrel, the bottom of which is removed and is communicated with the upper part and the lower part, and one end of the white small barrel is arranged on the collecting laying cloth 1; in use, infected fish is kept in the white keg 3, and then the fallen cysts are attached to the collecting cloth 1, and then the number of the collected cysts is counted.

2. Artificial infection animal model test

An indoor simulation experiment is carried out by taking trachinotus ovatus as an infected object, and the specific process is as follows:

(1) experimental trachinotus ovatus temporary culture

Temporarily culturing 10-20 g of 30-trachinotus ovatus in each culture barrel at the water temperature of 30 +/-1 ℃, continuously aerating and culturing in running water. Feeding according to 6% of fish weight every day, 1.5% every time, and 4 times a day (7: 30, 11: 30, 15:30, 18: 30). The bottom of the breeding barrel is brushed once every 3 days, so that the cryptocaryon irritans infection is avoided.

(2) Cryptocaryon irritans infection test

Larvae incubated for 2h were used to infect trachinotus ovatus at doses of 2000, 1000, 500, 250 and 0 larvae/tail, with a water volume of 100L. After 2h of still water infection, transferring the trachinotus ovatus to a larva-free collecting device, and temporarily culturing according to the conditions in the step (1); then, at 2d, 3d and 4d nights after infection, the collection pad 1 was cultured in still water and replaced with a new one, and the number of cysts on the replaced pad was counted. Each set of experiments was in triplicate.

(3) Data statistics

The number of shedding capsules in each group was counted and subjected to linear analysis using R²And (3) evaluating the fitting degree of the curve, and evaluating whether the trachinotus ovatus infected by the larvae/tail dose of 0-2000 can accurately reflect the degree of the trachinotus ovatus infected by the cryptocaryon irritans under the method.

The statistical and calculation results are shown in table 1 and fig. 4.

TABLE 1 encapsulation by collection of the pad at different infectious doses

From the above results, it was found that the number of collected cysts and the dose of infection in each test group showed a good linear relationship, with the linear equation of y being 192.5+3.9612x, r²0.990; at the same time, there is also a good linear relationship between the number of cysts collected per fish on average and the infectious dose, with the linear equation y being 6.41667+0.13204x, r²＝0.990。

The results show that when the infection dosage is 0-2000 larvae/tails, the total number of the fallen cysts and the average number of cysts generated by each fish have a good linear relation with the infection dosage, the infection rate of cryptocaryon irritans on trachinotus ovatus can be accurately calculated, and the infection degree of cryptocaryon irritans on fish bodies can be effectively evaluated.

Example 2 comparison of infection Rate determination methods

The measurement method of example 1, the method of counting the number of trophozoites on the skin above the body surface lateral line of the left side of the fish and below the dorsal fin, and the method of counting the number of trophozoites in the right two gills of the fish were compared.

1. Relationship between the amount of local epidermal trophoblasts and the amount of infectious agent

After infection, the number of trophozoites on the epidermis below the dorsal fin above the left lateral line of the fish body was counted, and then calculated, by counting the number of trophozoites on the epidermis after infection, and 2d, 3d and 4d after infection, respectively, with reference to the infection process in example 1.

As a result, as shown in FIG. 7, it was found by calculation that the number of trophozoites on the epidermis at the 2 nd position after infection had a certain linear relationship with the dose of the infection (R, as shown in FIG. 8)²0.987), the number of trophozoites on the topical epidermis at 3 and 4d post-infection was not linear with the dose of infection and did not differ significantly.

2. Relationship between right two gill trophozoite quantity and infection dose

Reference example 1 infection process after infection, the number of trophozoites on the right two gills of the fish was counted and calculated as 2d, 3d and 4d after infection, respectively.

The results are shown in FIG. 9, and it was found by calculation that the number of trophozoites in the right gill at 3d after infection had a certain linear relationship with the dose of infection (R is shown in FIG. 10)²0.861), the number of trophozoites in the right gill of the 2 nd and 4d fish after infection was not linear with the dose of infection and was not significantly different.

The number of shedding cysts counted in example 1 showed a better linear relationship to the infection dose compared to the method of counting the number of trophozoites on the local epidermis and the right two gills. The method for counting the number of trophozoites on local epidermis and right double branchia can only evaluate the degree of cryptocaryon irritans infecting trachinotus ovatus at a certain time point, and the method for counting the trophozoites on the right double branchia has the defects that the trophozoites are not easy to observe and sampled branchial filaments are incomplete; the method for counting the number of shedding cysts shown in example 1 allows complete calculation of the rate of infection of larvae of cryptocaryon irritans, and a good assessment of the extent to which cryptocaryon irritans infect fish.

Each embodiment is a complete technical solution.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. a kind of assay method that stimulates Cryptonucleus to fish infection rate, is characterized in that, comprises the steps:

S1. The uninfected fish are temporarily raised indoors, infected with a certain number of stimulated Cryptocaryon larvae, and the infection dose is recorded;

S2. Collection device: Lay the collection pad 1 at the bottom of an open container 2, then place the upper and lower fences 3 on the collection pad 1, and ensure that the bottom area of the fence 3 is smaller than the collection pad 1; then the container 2 and the Add water to fence 3;

S3. The fish infected in step S1 for 2 hours are respectively transferred to the collection device prepared in step S2 for rearing; then on the 2d to 12d after infection, the collection pads 1 are replaced every day, and the collected data on the collection pads 1 are recorded. the number of cysts;

S4. Calculation: infection rate (%)=sum of the number of cysts collected/infectious dose*100.

2. the assay method of stimulating Cryptocarpus according to claim 1 to fish infection rate, is characterized in that, described fish is seawater bony fish.

3. the assay method that stimulates Cryptonucleus according to claim 1 to fish infection rate, it is characterized in that, in step S1 and step S3, the rearing condition of fish is: 18～32 ℃ of water temperatures, continuous oxygen exposure, hydrostatic culture ; Feed 6% of fish body weight every day, 1.5% each time, 4 times a day.

4 . The method for measuring the infection rate of stimulated Cryptocaryon worms to fish according to claim 1 , wherein the infection dose is 0-2000/tail. 5 .

5. the assay method of stimulating Cryptonucleus according to claim 4 is characterized in that, when fish is oval pomfret, the total number of cysts that described 3d collects and the linear equation of infection dose For y=192.5+3.9612x, r ² =0.990.

6 . The method for measuring the infection rate of fish by stimulating Cryptonuclearia according to claim 1 , wherein the collection pad 1 is a dark polyester tarpaulin. 7 .

7 . The method for measuring the infection rate of fish by stimulating Cryptonucleosis according to claim 1 , wherein the container 2 and the fence 3 can both be barrel-shaped containers. 8 .

8 . The method for measuring the infection rate of fish by stimulating Cryptocaryon worm according to claim 7 , wherein the diameter and height of the fence 3 are smaller than the container 2 . 9 .

9 . The method for measuring the infection rate of fish by stimulating Cryptonucleus according to claim 7 , wherein the enclosure 3 is in the shape of a conical barrel. 10 .

10 . The method for measuring the infection rate of fish by stimulating Cryptonucleosis according to claim 6 , wherein the edge of the collection pad 1 is higher than the bottom end of the fence 3 . 11 .