Sensitivities of Vibrio vulnificus to Chinese Herbs and Effect of Medicinal Bath on Vibrio vulnificus

Guisheng GAO,Qiumei SHI*,Yanying ZHANG,Guangping GAO,Hongsheng HAN,Xiaoyuan HUANG,Baofeng JIAO,Jianhua DOU

1.Hebei Provincial Key Laboratory of Preventive Veterinary Medicine,Hebei Normal University,Qinhuangdao 066600,China;2.Qinhuangdao Liangfeng Marine Ecological Technology Development Co.,Ltd.,Qinhuangdao 066600,China

Vibrio vulnificusis a major kind of Vibrio greatly threatening marine fish.Currently,it is reported thatV.vulnificusmainly harmsNibea albiflora,Trachinotus ovatus,Scophthalmus maximus,Kareius bicoloratus,AnguillajaponicaandRachycentron canadum[1-4],resulting in huge economic losses to the aquaculture industry every year.Chinese herbs are natural medicines,and they attract much attention due to their no drug resistance,no residue,no side effects,no drug-induced diseases and obvious bacteriostatic and bactericidal effects[5-9].In this experiment,the antibacterial activities of total 18 kinds of Chinese herbs,such as stone calamus,were determinedin vitro.From the 18 kinds of Chinese herbs,some herbs with better inhibition effect onV.vulnificuswere screened,providing certain reference for prevention and control ofV.vulnificusin aquaculture,as well as non-specific immunity tests.

Materials and Methods

Materials

Test animal and strainThe test animal wasCynoglossus semilaevis,which was purchased from certain coastal farm of Changli.TheV.vulnificusstrain was provided by the Hebei Provincial Key Laboratory of Preventive Veterinary Medicine.

Medium and Chinese herbsThe common agar medium was purchased from the Beijing Luqiao Technology Co.,Ltd.,while the serum agar medium was prepared by the Hebei Provincial Key Laboratory of Preventive VeterinaryMedicine.Total18 kinds of Chinese herbs were selected in this study, including Chinese rhubarb,clove,houttuynia,amur corktree bark,citrus,stone calamus,redknees herb,purslane,herba andrographitis, licorice, sappan wood,pomegranate bark, Chinese gall,goldthread root,eucalyptus leaves,sea buckthorn and dark plum,and they were all purchased from the Anguo City Shenhe Medicine Herb Pieces Co.,Ltd.

Equipment and instrumentsIn this study,the used equipment and instrumentsincluded double-sided clean bench (SW-CJ-2FD,Suzhou Antai Purification Equipment Co.,Ltd.),thermostaticincubator(HPX-9162MBE,Shanghai Boxun Industrial Co.,Ltd.),and so on.

Methods

Preparation of Chinese herb extractA certain amount(100 g)of each Chinese herb was soaked in a certain volume(500 ml)of water for 1 h.Subsequently,the Chinese herbs were decocted to boiling and then simmered for 1 h.The concentrates,with Chinese herb concentration of 1 g/ml,were collected and packed in sterile vials.After sterilized,the solutions were preserved at 4℃.

Preparation of susceptibility paperTotal 100 sheets of paper were prepared for each Chinese herb.They were sterilized and soaked in a certain amount(1 ml)of concentrate of each Chinese herb.Subsequently,the paper was expanded and dried in an oven.

Determination of bacteriostasis effectTheV.vulnificuscolonies were inoculated in nutrient broth and cultured at 180 r/min and 28℃for 24 h.The broth was dipped with a cotton swab and smeared evenly on solid medium.After the broth was dried,susceptibility paper was pasted on the nutrient agar.Total 6 kinds of susceptibility paper were pasted in a petri dish,and they were well marked.The petri dishes were placed in an incubator at 28℃for 24 h.And then,the diameters ofinhibition zones were measured using a vernier caliper.If the diameter of certain inhibition zone was less than 10 mm,V.vulnificuswas judged to be insensitive to certain Chinese herb,which was represented by"-";if the diameter of certain inhibition zone ranged from 10 to 15 mm,V.vulnificuswas judged to be moderately sensitive to certain Chinese herb,which was represented by"+";if the diameter of certain inhibition zone ranged from 15 to 20 mm,V.vulnificuswas judged to be highly sensitive to certain Chinese herb,which was represented by"++";if the diameter of certain inhibition zone ranged was higher than 20 mm,V.vulnificuswas judged to be extremely highly sensitive to certain Chinese herb,which was represented by"+++"[10-11].

Sensitivity test of V.vulnificus to medicinal bathCertain Chinese herbs with significant control effects onV.vulnificuswere selected to make up prescriptions.The prescription I was made up of eucalyptus leaves,Chinese gall,dark plum,Chinese rhubarb and mentha;the prescription II was made up of Chinese gall,dark plum,pomegranate bark and mentha;the prescription III was made up of Chinese rhubarb,sea buckthorn,sappan wood and clove.In each prescription,the Chinese herbs were mixed together according to certain proportions.Each prescription was soaked in certain amount of water(m/v,1/5)for 1 h.The solutions were decocted to boiling and then simmered for about 30 min.There was amount 300 ml of concentrate remained for each prescription.The concentrates were packed for use(1 g of original prescription was equivalent to 1 ml of concentrate).

Control effect of medicinal bath in Cynoglossus semilaevis artificially infected with V.vulnificusTotal 25 tongue soleswereselected.They were first bred for 15 d,and then distributed randomly in five groups(five for each group),including health control group(no infection),infection control group (infection,but no administration),Prescription I group(infection and administration),Prescription II group (infection and administration)and Prescription III group(infection and administration).TheV.vulnificuswas inoculated in ordinary broth,andcultured at 28℃for 8-12 h.A certain amount (0.5 ml)of bacterial broth,with bacterial concentration of 2×106colonies/ml,was injected into abdominal cavity of each tongue sole with one disposable syringe.Thus the tongue soles were infected withV.vulnificus.The experimental grouping and treatments were shown in Table 1.

Results and Analysis

Bacteriostasis effect of susceptibility paper made of from single Chinese herb

As shown in Fig.1,Fig.2,Fig.3 and Table 2,the bacteriostasis effects of the Chinese herbs were different.Among the 18 kinds of Chinese herbs,the bacteriostasis effects of Chinese gall,dark plum,sea buckthorn and eucalyptus leaves were greater,and the diameters of their inhibition zones were 26,24,22 and 20 mm,respectively;the bacteriostasis effects of pomegranate bark,Chinese rhubarb,sappan wood,mentha and clove were moderate,and the diameters of their inhibition zones were 19,15,19,12 and 16 mm,respectively;the remaining Chinese herbs showed no bacteriostasis effects.

Bacteriostasis effect of medicinal bath composed of several Chinese herbs

The results showed that the diameter of inhibition zone of Prescription III was largest(21 mm),indicating strongest bacteriostasis effect;the diameter of inhibition zone of Prescription I ranked second (20 mm),indicating moderate bacteriostasis effect;the diameter of inhibition zone of Prescription II was smallest(18 mm),indicating weakest bacteriostasis effect.

Control effect of medicinal bath against V.vulnificus

Among the three prescriptions,the bacteriostasis effect of Prescription III was greatest.The three prescriptions were sprayed in water bodies of the three administration groups according to the dilution of 1∶150.The water and dressing were changed once a day.In the other two control groups,the water was changed,and the feeding was carried out regularly.The changes in tongue soles in the five groups were observed.

Once tongue soles were infected withV.vulnificus,their responses became slow,and their feed intake became smaller.Under severe conditions,some tongue soles even rejected feed intake.In the early infective stage,partial tongue soles began to move slowly,and localized infection appeared on their mouths.In addition,some tongue soles were unresponsive to outside stimuli.Evisceration phenomenon was observed in most tongue soles.In the middle infective stage,the bodies of tongue soles become stiff;small-area ulcers were first shown in the backs of tongue soles,and they further formed into small white spots.In the late infective stage,the lesions in tongue soles become larger,the ulcers were increased,and large area of skin began to fester,leading to the death of tongue soles.The effects of medicinal bath of the three prescriptions on tongue soles were shown in Table 3.

Conclusions and Discussion

Chinese herbs contain alkaloids,glycosides,saponin,trace elements and other ingredients.Different Chinese herbs have different therapeutic functions.In this study,some Chinese herbs,with functions of detoxifying,preventing bleeding and clearing heat,were selected for sensitivity test.The results showed that under the same test conditions,the bacteriostasis effects of Chinese gall,dark plum,sea buckthorn and eucalyptus leaves were greatest,and the diameters of their inhibition zones were all higher than 20 mm;Chinese rhubarb and some other Chinese herbs had certain bacteriostasis and bactericidal effect,and the diameters of their inhibition zones ranged from 10 to 20 mm.According to the results of sensitivity test,different prescriptions ofmedicinalbath were prepared.The results showed that under the concentration of 1∶150,the vigor of tongue soles could be better restored after illness;the three prescriptions of Chinese herbs all showed certain control effects onV.vulnificus.The result of susceptibility test showed that Chinese gall had greater inhibitory effect onV.vulnificus.Related reports[12-14]also showed that Chinese gall also had inhibitory effect onV.anguillarum,which might be related to lung-constraining,sweating-arresting, intestine-astringing, bleedingstopping and detoxifying effects of tannin,as well as a variety of glucoses,such as galloyl-β-D-glucose in Chinese gall.

Table 3 Effects of different prescriptions of medicinal bath on V.vulnificus

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