CN103536586B - A kind of method of preventing and treating saprolegniasis of aquatic animals - Google Patents

A kind of method of preventing and treating saprolegniasis of aquatic animals Download PDF

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Publication number
CN103536586B
CN103536586B CN201310442736.XA CN201310442736A CN103536586B CN 103536586 B CN103536586 B CN 103536586B CN 201310442736 A CN201310442736 A CN 201310442736A CN 103536586 B CN103536586 B CN 103536586B
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China
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water
rieter
mould
compound recipe
saprolegnia
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CN201310442736.XA
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CN103536586A (en
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李浩然
杨先乐
胡鲲
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上海海洋大学
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Abstract

The present invention relates to a kind of method of preventing and treating saprolegniasis of aquatic animals, described method is mainly used in the fish molds of the control germ cell such as Fish, shrimps and adult fish, it is characterized in that, described method adopts Drug therapy water mo(u)ld; Described method adopts splashes and soaks two kinds of mode medications respectively the water mo(u)ld of prevention and therapy Ctenopharyngodon idellus and Carassius auratus wound, and described water mo(u)ld is Saprolegnia parasitica bacterium.Advantage of the present invention: medication is simple, prevention and therapy Be very effective.

Description

A kind of method of preventing and treating saprolegniasis of aquatic animals

[technical field]

The present invention relates to microbial technology field, specifically, is a kind of method of preventing and treating saprolegniasis of aquatic animals.

[background technology]

Water mo(u)ld is extensively stored in fresh water all over the world or brackish water waters and slowly drained soil, saprophytic on the Organic substance of death, for a kind of mycete of being everlasting, mainly contain Saprolegniales (Saprolegniales), Peronosporales (peronosporales) and Leptomitales (Leptomitales) etc., the most common with water mo(u)ld (Saprolegnia) again, the most applicable growth in 15 ~ 25 DEG C time, trip spore (zoospore) reproductive capacity in each when more than 25 DEG C weakens, more not easy infection.

The generation of fish molds is main because the urgent Secondary infection caused, fish body is because of impact that is crowded, mobile or other poor environment factors, cause body surface organization injured, namely fish molds trip spore in water waits for an opportunity to adhere to, and slough starting germinate forms mycelia, and mycelia is except parasitizing slough, the normal structure still can stretched near invading, secretion zymigen decomposes surrounding tissue, more runs through corium and gos deep into muscle, make skin and muscular death disintegrate.The mycelia on top layer then stretches out, and is formed as byssaceous covering, and forms Sporangium in end, releases trip spore in water, propagates everywhere via water.Symptom is mainly infection site and forms canescence flocculence covering; The diseased region initial stage is rounded, and stage is irregular speckle, and time serious, skin injury muscle is exposed; Gill tissue also can be infected by infringement, causes death.

Chinese patent literature CN102293831A discloses a kind of compounding powder and preparation method of preventing and treating saprolegniasis of aquatic animals, is made up of the raw material of Galla Chinensis, Radix Et Rhizoma Rhei, bronopol, dispersant constant weight percentage ratio.Chinese patent literature CN101708187A discloses a kind of for dry powder formulations preventing and treating saprolegniasis of aquatic animals and preparation method thereof, and component consists of: 71 ~ 79% biochemical fulvic acids, 10 ~ 15% sodium metabisulfite, 10 ~ 15% bronopols.But yet there are no report about the patent of preventing and treating saprolegniasis of aquatic animals for key agents with " Rieter is mould ".

[summary of the invention]

The object of the invention is for deficiency of the prior art, a kind of method that medication is simple, therapeutic effect significantly prevents and treats saprolegniasis of aquatic animals is provided.

For achieving the above object, the technical scheme that the present invention takes is:

Prevent and treat a method for saprolegniasis of aquatic animals, described method is mainly used in the fish molds of the control germ cell such as Fish, shrimps and adult fish, and described method adopts Drug therapy water mo(u)ld; Described method adopts splashes and soaks two kinds of mode medications respectively the water mo(u)ld of prevention and therapy Ctenopharyngodon idellus and Carassius auratus wound, and described water mo(u)ld is Saprolegnia parasitica bacterium.

Described medicine is that Rieter is mould.

Described medicine is the mould powder of compound recipe Rieter after the mould processing of Rieter.

The described mould powder of compound recipe Rieter is solid particulate medicament.

The described mould powder of compound recipe Rieter is mould by 45% Rieter, the kieselguhr of 10%, the anhydrous sodium sulfate of 20%, the ferrous sulfate of 24.5% and 0.5% anticaking agents form.

Described medicine adopts the optium concentration of splashing with immersion way prevention and therapy water mo(u)ld effect to be 8mg/L and 20mg/L respectively.

Advantage of the present invention:

Medication is simple, prevention and therapy Be very effective.

[detailed description of the invention]

embodiment 1

The preparation of the mould powder medicine of compound recipe Rieter

Adopt following weight proportion: the Rieter getting 45% mouldly (has another name called metalaxyl, english common name metalaxyl, molecular formula C 15h 21nO 4, CAS 57837-19-1), the kieselguhr of 10%, 20% anhydrous sodium sulfate, 24.5% ferrous sulfate, 0.5% anticaking agents (gas-phase silica) mixing after make anti-saprolegnia preparation " the mould powder of compound recipe Rieter ".

Production technology is as follows:

Mix under 1 anti-saprolegnia preparation " the mould powder of compound recipe Rieter " each component room temperature:

1) first kieselguhr, ferrous sulfate and anticaking agents are dropped into premix 10 ~ 15 min, rotating speed 50 ~ 60 r/min in mixing machine;

2) again anhydrous sodium sulfate is dropped into mixing machine and said mixture is mixed together 10 ~ 15 min, rotating speed 50 ~ 60 r/min;

3) finally mould for Rieter input mixing machine and said mixture are mixed together 30 ~ 40 min.Rotating speed 50 ~ 60 r/min.

Pack under 2 anti-saprolegnia preparations " the mould powder of compound recipe Rieter " room temperature:

1) said mixture is carried out subpackage, accurately take 200 g every part, in the inner packing band that loading can be sealed, seal;

2) then packing inside bag is put into outer packaging bag, and print the date of manufacture;

3) the mould powder of compound recipe Rieter every 50 good for above-mentioned production is packaged into a case.

embodiment 2

1 materials and methods

1.1 trial drugs and reagent

" the mould powder of compound recipe Rieter ", special biotechnology institute company limited is visitd in Changsha

1.2 bacterium source

Fish molds bacterial strain ATCC200013 for Shanghai Ocean University country aquatic animal cause of disease storehouse provide.

1.3 to fish molds interaction in vitro effect experimental

1.3.1 prepared by fish molds spore suspension

The saprolegnia hypha that picking is preserved in test tube is transferred on potato dextrose agar (potato ball 200 ~ 300g, glucose 20g, agar 20g, water 1000mL) flat board, flat board drips appropriate amounts of sterilized water 25 DEG C and cultivates 48h, period increases sterilized water in right amount.After being paved with whole culture dish, pour out the sterilized water inducing spore, then with sterilized water gently washing plate 1 time merge, through 8 layers of filtered through gauze, adjusting spore concentration is respectively 10 2individual/mL, 10 3individual/mL, 10 4individual/mL treats that experiment is used.

1.3.2 treat reagent thing mother liquor and gradient dilution

Anti-saprolegnia preparation " the mould powder of compound recipe Rieter " is mixed with the mother liquid medicine of 1000mg/L with distilled water.By doubly interpretation of the law, this mother solution is diluted ten gradients successively: 1000,500,250,125,62,31,16,8,4mg/L.

1.3.3 fish molds spore Inhibition test

Doubly release liquid 2mL with the medicine of different gradient concentration and add to the PDA(potato dextrose agar of 18mL through melting 56 DEG C: potato ball 200 ~ 300g, glucose 20g, agar 20g, water 1000mL) make it even, be poured into rapidly in culture dish to be solidified for experiment, namely the dull and stereotyped concentration of medicine PDA is 1/10 of original content.

Get 0.1mL fish molds pityrosporion ovale suspension to coat in the culture medium of solidifying.If blank group, cultivate 5d for 25 DEG C.Incubation period every 24h observes once, and naked eyes judge that drug level without the minimum medicine culture dish of colony growth is as MIC.Each concentration establishes two contrasts, sets the blank PDA culture medium not adding medicinal liquid as contrasting simultaneously.

1.3.4 saprolegnia hypha killing test

Will through potato dextrose agar (potato ball 200 ~ 300g, glucose 20g, agar 20g, water 1000mL), 25 DEG C cultivate 48h after, be paved with dull and stereotyped fish molds sterile razor blade be cut into about 1,2,3 cm 2fritter.The fish molds fritter cut is put into centrifuge tube that the medicine filling above-mentioned each concentration doubly releases liquid soak 0.17 respectively, 0.5,1,2h. aseptic nipper takes out the fish molds fritter soaked and is placed on potato dextrose agar, cultivate for 25 DEG C and observe 5d.Incubation period every 24h observes once, and naked eyes judge to be born without water mo(u)ld long, represents that this kind of medicine has killing action to saprolegnia hypha under this concentration this action time.Each concentration establishes two contrasts, establishes the fish molds block blank crossed through distilled water immersion simultaneously.

2 results

2.1 anti-saprolegnia preparations " the mould powder of compound recipe Rieter " affect (table 1) to fish molds spore MIC

Table 1 anti-saprolegnia preparation " the mould powder of the compound recipe Rieter " MIC to fish molds spore Bacterium amount (individual/mL) MIC(mg/L) 10 2 3 10 3 3 10 4 3

Table 1 experimental result shows: anti-saprolegnia preparation " the mould powder of the compound recipe Rieter " inhibition to the water mo(u)ld of mycelia number different under same volume is consistent, and MIC has concordance under different bacterium amount, illustrate that anti-saprolegnia preparation " the mould powder of compound recipe Rieter " has good inhibition to fish molds spore.

2.2 anti-saprolegnia preparations " the mould powder of the compound recipe Rieter " killing action (table 2) to saprolegnia hypha

Table 2 pair saprolegnia hypha effectively kill concentration and action time thereof

Note :+represent fish molds growth; – represents fish molds no growth

Table 2 experimental result shows: anti-saprolegnia preparation " the mould powder of compound recipe Rieter " acts on 1h to saprolegnia hypha and starts there is killing action under 20mg/L concentration, along with the increase of concentration, it is effectively killed the time and also can shorten, under 80mg/L concentration, only must act on 0.17h namely start there is killing action to saprolegnia hypha, and three kinds of different bacterium amount results have concordance, illustrate that anti-saprolegnia preparation " the mould powder of compound recipe Rieter " has good bactericidal action to saprolegnia hypha.

3 discuss

Final bacterium amount due to filamentous fungi drug sensitive experiment is determined so far still without quantitatively standardized, with reference to the final bacterium amount of other funguses, as: dermatophytosis 10 3individual/mL, Aspergillus conidia number 5 × 10 4individual/mL.The final bacterium amount of this experiment to fish molds has established three to contrast concentration, i.e. spore count 10 respectively 2individual/mL, 10 3individual/mL, 10 4individual/mL, result shows, and three bacterium measure MIC has concordance, does not change because of the change of bacterium amount.Illustrate, fish molds spore amount does not directly affect the determination of experiment MIC terminal in finite concentration.Equally, in the Inhibition test of saprolegnia hypha, the amount for saprolegnia hypha has established three matched groups, i.e. saprolegnia hypha area: 1,2,3 cm 2.Result has concordance equally.

It is generally acknowledged that fish molds is S, is a kind of secondary infection to aquatic animal.Cultivated animals is injured or because of antibacterial, viral infection, causing fish molds spore to attach to wound, and then be subject to the invasion and attack of saprolegnia hypha, is the important step that fish molds infects.Therefore, medical treatment must destroy this link, namely all has good suppression or killing action to fish molds zoospore and mycelia.Experimental result display anti-saprolegnia preparation " the mould powder of compound recipe Rieter " all has suppression or killing action preferably to the spore of fish molds and mycelia, is 3mg/L to the MIC of fish molds spore three bacterium amounts.Under 20mg/L concentration, 1h is acted on to three bacterium amounts of saprolegnia hypha and namely has killing action, and along with the rising of concentration, the time of effectively killing also decreases, and namely 0.17h has killing action under 80mg/L concentration.Therefore, only from the interaction in vitro effect to fish molds spore and mycelia, it has good anti-saprolegnia effect.

embodiment 3

1 materials and methods

1.1 trial drugs and reagent

" the mould powder of compound recipe Rieter ", special biotechnology institute company limited is visitd in Changsha

1.2 Experimental fish

Experiment Ctenopharyngodon idellus, crucian carp are purchased from state farm, Nantong, and specification is crucian carp: the long 12 ~ 13cm of body weight 24 ~ 29g/ tail, body; Ctenopharyngodon idellus: the long 14 ~ 16cm/ tail of body weight 30 ~ 37g/ tail, body.Body surface and anatomic observation health, support one week temporarily.

1.3 bacterium source

Fish molds bacterial strain ATCC200013 for Shanghai Ocean University country aquatic animal cause of disease storehouse provide.

1.4 feeding and management

Raise with 200 L volume plastic boxes, automatic aerator 24 h oxygen supplement; Water source is the tap water after aeration, sterilization and dechlorination process, pH 6.5 ~ 6.8, average temperature 26 DEG C, and dissolved oxygen 9 ~ 12 mg/L, other indexs meet water quality standard for fishery.Feed formulation is with reference to NRC trophic level.

1.5 Ctenopharyngodon idellus, crucian carp fish molds artificial challenge

Picking is preserved in invisible spectro saprolegnia hypha and transfers on potato dextrose agar (potato ball 200 ~ 300g, glucose 20g, agar 20g, water 1000mL) culture dish, cultivates 72h for 25 DEG C and makes it be paved with whole culture dish.Get appropriate cultured fish molds mortar to grind in glass jar (20L) water poured into for infecting.Some prior niblets with decocting in water cracking are put into this glass jar water body and observes 72h, observing 90% niblet has fish molds to grow, and namely judging has fish molds to exist in glass jar water body, is ready to use in infection experiment

Removed by both sides scale below Ctenopharyngodon idellus, crucian carp dorsal fin with knife blade, scrape the osculum of a cm at muscle place off, the Ctenopharyngodon idellus after scratch and Carassius auratus are placed with respectively in the glass jar water being used for infecting of fish molds existence, 10 tail Ctenopharyngodon idellus or crucian carps put into by each infection cylinder.Temperature of cultivation drops to 18 ± 1 DEG C (temporary vatting water temperature 26 DEG C infects cylinder water temperature 18 DEG C ± 1) by 26 DEG C, carries out fish molds artificial challenge.

1.6 Ctenopharyngodon idellus, crucian carp fish molds prevention experiment

1.6.1 mode of splashing medication prevention of water mildew

Before above-mentioned Ctenopharyngodon idellus, crucian carp carry out artificial challenge, to Ctenopharyngodon idellus and crucian carp splash respectively mode medication prevention of water mildew experiment.2,4,8mg/L arrange 3 drug level of splashing according to Vitro Experimental Results:, each drug level establishes three parallel control cylinders, arranges three blank parallel control cylinders simultaneously.Each contrast cylinder puts the Ctenopharyngodon idellus or crucian carp 10 tail that are ready to use in infection.Observe one week.

1.6.2 immersion way medication prevention of water mildew

Before above-mentioned Ctenopharyngodon idellus, crucian carp carry out artificial challenge, the experiment of immersion way medication prevention of water mildew is carried out respectively to Ctenopharyngodon idellus and crucian carp.10,20,40mg/L arrange 3 drug immersion concentration according to Vitro Experimental Results:, soak 1h, every day soaks twice at every turn.Each drug level establishes three parallel control cylinders, arranges three blank parallel control cylinders simultaneously, and each contrast cylinder puts the Ctenopharyngodon idellus or crucian carp 10 tail that are ready to use in infection.Observe one week.

1.7 Ctenopharyngodon idellus, crucian carp fish molds Experiment on therapy

1.7.1 mode of splashing medication treatment fish molds

Choose and infect successful Ctenopharyngodon idellus, crucian carp and carry out Drug therapy experiment of splashing respectively.According to Vitro Experimental Results arrange 3 drug level be respectively 2,4,8mg/L.Each drug level arranges three parallel control cylinders, arranges three blank parallel control cylinders simultaneously, and each contrast cylinder is put and infected successful Ctenopharyngodon idellus or crucian carp 10 tail.Observe 1 week.

1.7.2 immersion way medication treatment fish molds

Choose and infect successful Ctenopharyngodon idellus, crucian carp and carry out drug immersion Experiment on therapy respectively.According to Vitro Experimental Results arrange 3 drug level be respectively 10,20,40mg/L, soak 1h, every day soaks twice at every turn.Each drug level establishes three parallel control cylinders, arranges three blank parallel control cylinders simultaneously, and each contrast cylinder is put and infected successful Ctenopharyngodon idellus or crucian carp 10 tail.Observe 1 week.

1.8 data statistic analysis

Drug effect (protective rate) evaluation is carried out according to Ministry of Agriculture's " fisheries drug clinical experiment technical specification ".Protective rate formula is as follows:

2 results

Table 3 is splashed the preventive effect of medicine to Ctenopharyngodon idellus, crucian carp fish molds

Table 4 drug immersion is to the preventive effect of Ctenopharyngodon idellus, crucian carp fish molds

Table 3 and 4 experimental result show: in preventing and treating, no matter two kinds of application methods of anti-saprolegnia preparation " the mould powder of compound recipe Rieter " are splashed or soaked all has good preventive effect to fish molds and also increases along with the rising protective rate of concentration, and mode of splashing Drug level is respectively 90%, 96.7% at the average protective rate of the fish molds of 8mg/L to Ctenopharyngodon idellus, crucian carp; Immersion way medication all reaches 76.7% when 20mg/L concentration to the average protective rate of Ctenopharyngodon idellus, crucian carp fish molds, illustrates that application method effect of splashing in preventing and treating is much better than to flood application method effect (being not suitable for using owing to increasing the murder by poisoning of fish during high concentration).

Table 5 is splashed the therapeutic effect of medicine to Ctenopharyngodon idellus, crucian carp fish molds

Table 6 drug immersion is to the therapeutic effect of Ctenopharyngodon idellus, crucian carp fish molds

Table 5 and 6 experimental result show: anti-saprolegnia preparation " the mould powder of compound recipe Rieter " in treatment experimental result display; mode of splashing only has 6.67% and 13.3% when 8mg/L concentration to the average protective rate of Ctenopharyngodon idellus and Carassius auratus; and immersion way all can reach 43.3% when 20mg/L concentration to the average protective rate of Ctenopharyngodon idellus and Carassius auratus, illustrate that in treatment, soak application method effect is much better than mode effect of splashing.

3 discuss

Medicine is splashed and drug immersion is two kinds of mainstay methods of aquatic animal disease control.Two kinds of methods are splashed or dipping bath experimental result all shows, anti-saprolegnia preparation " the mould powder of compound recipe Rieter " has good protective rate for prevention of water mildew, and mode of wherein splashing can reach 90%, 96.7% when drug level is 8mg/L respectively to the average protective rate of Ctenopharyngodon idellus, crucian carp; Immersion way all can reach 76.7% when concentration is 20mg/L to the protective rate of Ctenopharyngodon idellus, crucian carp.

Interaction in vitro effect experimental shows, and anti-saprolegnia preparation " the mould powder of compound recipe Rieter " acts on 1h and starts there is killing action to saprolegnia hypha under 20mg/L concentration, and the concentration of splashing arranges maximum concentration and is only 8mg/L.The different-effect of Resistance mechanism performance also illustrates on disease control of aquatic animal, important to therapeutic effect of correct application method, and is better than medicine for the obvious drug immersion effect of control of fish molds and splashes.

The clinical effect experiment of anti-saprolegnia preparation " the mould powder of compound recipe Rieter " shows, it is good to the preventive effect of fish molds, also has certain fish molds therapeutic effect simultaneously.

The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.

Claims (1)

1. Rieter is mould is characterized in that preparing the application prevented and/or treated in saprolegniasis of aquatic animals medicine, and described medicine adopts splashes and soaks the water mo(u)ld that two kinds of modes prevent and/or treat Ctenopharyngodon idellus and Carassius auratus wound respectively; Described water mo(u)ld is Saprolegnia parasitica bacterium; Described medicine is the mould powder of compound recipe Rieter after the mould processing of Rieter; The described mould powder of compound recipe Rieter is mould by the Rieter of 45%, the kieselguhr of 10%, 20% anhydrous sodium sulfate, the ferrous sulfate of 24.5%, the anticaking agents of 0.5% form; The described mould powder of compound recipe Rieter is solid particulate medicament; Described medicine employing is splashed and immersion way is 8mg/L and 20mg/L in the optium concentration of prevention and therapy Ctenopharyngodon idellus and Carassius auratus water mo(u)ld effect respectively.
CN201310442736.XA 2013-09-26 2013-09-26 A kind of method of preventing and treating saprolegniasis of aquatic animals CN103536586B (en)

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CN104988204A (en) * 2015-07-07 2015-10-21 杭州银江环保科技有限公司 Saprolegnia Evans Blue intravital staining method
CN105018563B (en) * 2015-07-07 2018-07-06 杭州银江环保科技有限公司 A kind of method of simple and fast screening antifungal drug
CN105461588A (en) * 2015-12-17 2016-04-06 上海海洋大学 Method for compounding metabolites 6 in ridomil
CN105853460A (en) * 2016-06-17 2016-08-17 上海海洋大学 Application of ferrous sulfate to promoting grass-carp wound healing
CN106431962B (en) * 2016-06-23 2018-04-06 上海海洋大学 It is vertical to reach the mould metabolin in grass carp body
CN106135279A (en) * 2016-06-30 2016-11-23 上海海洋大学 Compound recipe founds the production method reaching mould powder
CN106561512B (en) * 2016-11-08 2019-08-16 中国水产科学研究院长江水产研究所 A kind of water mold preventing control method of Songpan naked carp
CN107028928A (en) * 2017-06-12 2017-08-11 江苏宝灵化工股份有限公司 A kind of medicine and its preparation and application for being used to prevent and treat saprolegniasis

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EP1982717A1 (en) * 2007-04-20 2008-10-22 Bayer CropScience AG Use of fungicides for treating fish mycoses
CN101708187B (en) * 2009-02-27 2011-05-18 上海海洋大学 Dry powder preparation for preventing and controlling saprolegniasis of aquatic animals and preparation method thereof

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