JOURNAL OF THE ARABIAN AQUACULTURE SOCIETY Vol. 7 No 2 December 2012

Use of cina Against Gyrodactylus rysavyi Infecting Cyprinus carpio in Comparison with Praziquntel

Ramadan, R.A. M*, and Doaa , A. El Araby Mohammed

Fish Health Dep. Central Laboratory of Aquaculture Research, Abbassa, Agriculture Research Center, Ministry of Agriculture, Dokki- Giza – Egypt

*Corresponding Author

ABSTRACT

The present study analyzed recent trends of approved drugs derived from previously untapped species. on effective agents derived from natural sources against Gyrodactylus rysavyi infections in Cyprinus carpio. Praziquntel (4g/kg diet) was screened for their anthelmintic efficacy. Artemisia cina showed efficacy against infections by the monogenean in vitro and feeding trials. For challenge trials, natural agent (2.5g/kg diet) was effective in laboratory trials were also given to the fish in feed. Praziquntel prevented horizontal infection, furthermore, the survival of groups treated were significantly higher than the negative control(diet) and other groups additionally, the number of matured parasites on the branchial cavity, which occurred from the beginning of the challenge trials,decreased in the groups treated with Praziquantel. Artemisia cina was effective in the dislodgement and mortality of monogenean parasites, G. rysavyi of juvenile C. carpio at concentrations ranging from 50 to 200 mg/l. There were positive correlations between the number of parasites dislodged/killed concentration of A. cina extract, and the duration of exposure of affected fish to the substances. This led to the conclusion that A. cina contains substances that are effective against G. rysavyi and provide a knowledge can be explored in the aquaculture industry to eliminate the use of conventional synthetic organic drugs that may be detrimental to consumers of aquaculture products.

Keywords: blooded vertebrates, mainly the INTRODUCTION elasmobranches, bony fish and in some amphibians and reptiles. In fish, the Monogeneans are generally majority of monogenean is parasitic on found as parasites on or in cold- the gills or skin. These parasites are

© Copyright by the Arabian Aquaculture Society 2012 239 RAMADAN AND DOAA EL ARABY site- and host-specific, generally from A. annua have equally displayed occurring in relatively low numbers unique pharmacological activities (El-Naggar and Serag, 1987). against a wide range of bacteria (Bone However, monogeneans parasites can and Morgan, 1992). Effect of Artemisia easily multiply and disperse in annua L. leaves essential oil and confined areas, in a very high intensity ethanol extract on behavioral assays so, the establishment of a heavy (Perazzo et al.,2008), an opportunistic infection, particularly under pathogen which causes in AIDS and unfavorable culture conditions, may other immune-compromised patients. give rise to mass epizootics with severe artemisinin has also a therapeutic economic loss (Ramadan, 2000 and El- potential against Toxoplasma gondii Abbassy (2001). Skin and gills of (Jones-Brando et al., 2006 and affected host are damaged by the Ferreira,2009), Trypanosoma, and attachment hooks resulting in Schistosoma species (Mishina et al., secondary infection by bacteria or 2007), which cause toxoplasmosis that fungi. They cause severe economic is associated with behavioral losses among fish farms (Mohamed et abnormalities in patients, human al., 2010). trypanosomiasis or “sleeping sickness,” and schistosomiasis, Antiparasihc drugs for fish are respectively, as well as other lately being more widely studied due to pathogens responsible for the need to keep farmed fish free of cryptosporidiosis, amoebiasis, infestations supported by intensive giardiasis, leishmaniasis (Ma et al., farming. Praziquantel have been tested 2004). The chemotherapeutic agents on monogeneans other than currently used for the treatment of fish Gyrodactylus. The genus Artemisia monogenesis include mebendazole, belongs to the large family of organophosphate, praziquantel, , encompassing more than closantel, dichlorvos, formaldehyde,etc 300 species. Artemisia annua L. Keiser (2010) and Aberham et al., (: Asteracaea), commonly (2010). known as sweet wormwood or annual wormwood, grows widely in Europe Ferreira and Luthria (2010) and and America and is planted to a large Ferreira et al., (2010) assess the extent in China, Turkey, Vietnam, antioxidant capacity of Artemisia Afghanistan, and Australia (Alawa et annua L. leaves and their potential al., 2003 and Abdel-Hadi et al., 2008). synergism with artemisinin against They equally affect the early stage of malaria and cancer. gametocyte development, which reduces further retransmission of the Recently Ekanem and Brisibe parasites from humans to mosquitoes (2010); Albert and Ebiamadon (2010) ; in areas of low transmission. Many Elango and Rahuman (2011) and other bioactive compounds isolated Squires et al. (2011) try to evaluate 240

ARTEMISIA CINA AGAINST GYRODACTYLUS RYSAVYI IN COMPARISON WITH PRAZIQUNTEL IN CYPRINUS CARPIO effects of extract of Artemisia annua L. aeration and feeding rate for C. carpio against monogenean parasites of was employed for all the experiments. Heterobranchus longifilis and we take the same idea and try to screen our 500 individuals, one year old Egyptian A.cina on juvenile Cyprinus weight 100-150 g hatched at laboratory carpio to find an alternative means for were maintained in a2 ton tank. Gills, treatment of monogenean diseases of cultured fish using an extract of A. cina branchial cavity, wall and skin surface instead of chemical-based substances of 10 fish were randomly examined that may not be friendly to the under microscope to confirm that the environment. fish were free from parasites before each experiment. In the present paper, we try to make a screening experiment to Laboratory infections were discover new natural anthelmintic produced by placing infections agents derived from natural resources uninfected fish in a tank with 2-3 for oral administration with medicated infected fish for 1-3 days. Prevalence feed to be much more practical and to and intensity of were detected ensure comparable and realistic results seasonally and by size in specimens and asses of both prophylactic and taken monthly.50 fish were selected therapeutic effect and provide a randomly and the number of parasites knowledge can be explored in the presented on fins and body surfaces aquaculture industry to eliminate the were counted. 25 individuals,0ne year use of conventional synthetic organic old, weight 150-250g infected with drugs that may be detrimental to G.rysavyi hatched at our laboratory, consumers of aquaculture products. were maintained a 100L tank uninfected fish with no recorded of MATERIALS AND METHODS previous infection with parasites,were periodically mixed into the infected Parasites and hosts fish. Excised gills from these fish Cyprinus carpio were infected with parasites larvae 2-6 cm maintained in constantly aerated tanks long were used for in vitro studies. . containing 500 L water at 20°C. The The hatched larvae were used for pH of the water was 7.0± 0.05; its NO2 challenge trials. content was zero, and the NO3 content A whole gills infected with more was 10 mg/l. with regard to flow rate, than 5 parasites per gill arch were immersed with each 50 ml drug 241

RAMADAN AND DOAA EL ARABY solution in a tissue culture dish at 20°C Preparation of an ethanol extract according to Noritaka and Taro of Artemisia cina (2000). The behavior and release of the larvae from gills were observed every The ethanolic extract of A. cina hour for 10 h under a microscope to was used for the preparation of a stock determine the drug efficacy. Parasite solution from which the working infected gills immersed in filtered solution used for the efficacy testing water without the drug at the same was prepared. The stock solutions were temperature conditions acted as a obtained by dissolving 1 g of the control. extract in 5 ml of dimethyl sulfoxide (DMSO) and made up to 100 ml with Drugs de-ionized water. Four working solutions that were represented by Parziquantel were obtained from concentrations of 50, 100, 150, and 200 Sigma (Deisenhofen, Germany) mg/l, respectively were prepared from BILTRICIDE® (praziquantel) is a the stock solutions. A preliminary test trematodicide provided in tablet form. was carried out to guide in the Drug was dissolved in filtered water at selection of the concentration of the 20 mg/L and was made up to a volume test solutions. One-week-old fry of C. of 50 ml. carpio obtained by induced breeding were stored in were maintained in material a1ton tank for a period of one week. Artemisia cina leaves were Examination for the accumulation of washed thoroughly in running tap monogenean parasites was done from water to remove sand and debris; they the fourth day of stocking according to were dried by spreading under the sun Ekanem and Brisibe (2010). for 3 days and finally in a hot air oven Efficacy testing at 60°C for 8 hrs. The dried leaves were crushed to powder in a mortar One hundred 1-month-old with pestle and subjected to Soxhlet juvenile C.carpio were stocked in extraction with 70% ethanol as a1ton tank for 7 days to accumulate extracting solvent. The solvent was parasites. The approximate number of exhausted from the extract with the parasites per fish was confirmed by help of a rotary evaporator. The extract counting the number of parasites was stored in a refrigerator until attached to body surfaces and the gills required for use.

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ARTEMISIA CINA AGAINST GYRODACTYLUS RYSAVYI IN COMPARISON WITH PRAZIQUNTEL IN CYPRINUS CARPIO with stereo-microscope before being test solution and stocked with ten fish exposed to the extract under in vivo under aeration. The setup was conditions. Parasitized fish were also replicated three times with a set of placed in de-ionized water containing 5 controls under the same experimental ml of DMSO in plastic Petri dishes to conditions. Observation for fish serve as control. The bioactivity of the mortality and abnormal swimming extract was conducted in plastic Petri behavior was made for 96 hrs dishes with three replications and according to Albert and Ebiamadon controls. Parasites were merely (2010). dislodged from their attachment organs and killed some hours later in the same Anthelmintic activity concentration of A. cina. Fish were exposed for periods ranging from 30 The experiments were to120 min in both the test and the performed in glass aquaria with 8 L control treatments according to Albert capacity. Each aquarium contained 5 L and Ebiamadon (2010). They were re- aerated tap water and ten previously examined individually at the expiration infected fish. The water temperature of the exposure period for the presence was constant at 25±1°C and the water of parasites. The experiments with the pH ranged from 7.0 to 7.5. Fractions substances were replicated three times. of ethanol extract added in glass The homogeneity of the replicates was aquaria at a different series of checked by Kruskal–Wallis test before concentrations. The blank control the data of the replicates were pooled group with no extract was used under together (Albert and Ebiamadon 2010). the same experimental conditions. All treatment and control groups were Toxicity test conducted in triplicate. After 48 h, all fish were biopsied, and the lamella Toxicity of the extract of A. cina branchialis were placed on glass slides to C. carpio juveniles was tested for for counting the number of surviving 24, 48, 72, and 96 hrs, respectively, at parasites under a stereomicroscope. higher concentrations (250, 300, 350, The mortality of G. rysavyi and the 400, and 500 mg/l) to ascertain the mortality of fish were used to evaluate safety margin of the substance against the anthelmintic efficacy of each the fish host (Zibaee,2010). Glass treatment. The optimal anthelminthic aquaria of 10 L capacity were used, concentration was the concentration and each tank was filled with 3 L of the which led to the highest mortality of

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RAMADAN AND DOAA EL ARABY parasite with no intoxication of fish. tissue culture dish at 20°C. The The drug concentration which resulted behavior and release of the larvae from in less than 20% mortality of parasite gills were observed every hour for 1h was considered ineffective under a microscope to determine the concentration. No parasite or dead one efficacy according to Noritaka and on gills represented 100% mortality of Taro (2000). As a control, excised gills parasite. The mortality of parasite of infected with more than 30 parasites each treatment was calculated per gill arch were immersed with each according to the following formula 50 ml without natural agents in a tissue (Wang et al., 2010): culture dish at 20°C.

Feeding trials

Parziquantel which were Where MD is % of the mortality determinate to be the most effective in of Gyrodactylus rysavyi, B is the laboratory trials were evaluated for average number of surviving G. their efficacy in vivo challenge trials. rysavyi in the blank control, and T is Approximately, 200 hatched larvae that in the treatment. The mortality of (body length 100-200 um) were kept fish was also calculated by the follow into a 100L tank containing 24 L equation: freshwater with aeration., 10 uninfected fish weighting approximately 150 g were added . These fish exposed to the larvae for 2h at 20°C with no running water to Where MF is % of mortality of fish, B complete the infection. Then, fish is the average number of surviving divided into 5 groups and were fish in the beginning of test, and E transferred to 100L tank. is that at the end of test. Each group was fed diet Artemisia cina were tested by in supplemented with the drugs at 2 laboratory trials. The agent was doses, 2g drug/kg basal diet throughout dissolved in filtered water at 80 mg/L the trials. Each drug was mixed into and was made up to a volume of 50 ml. diet and the pellet was formed by Excised gills infected with more than 5 passing the past through a disc parasites per gill arch were immersed pellerter. The normal diet was dried at with each 50 ml drug solution in a 40°C for 3 h and was stored at 4°C, 244

ARTEMISIA CINA AGAINST GYRODACTYLUS RYSAVYI IN COMPARISON WITH PRAZIQUNTEL IN CYPRINUS CARPIO with an average of 20.9 °C, during the starting to feed the experiment diets 6 trials. The effectiveness of each fish infected with mature parasite treatment was determined by 114.6+8.5 parasites per fish from 10 comparing the number and growth fish randomly sampled were added to (body gain of the parasite=body each group. Each group was fed the length=growth) of the parasite on gills. experimental diets continuously and prevention of horizontal infection was Infection sources assessed together with assessment of the therapeutic effect on newly added Approximately 100 hatched infected individuals (Noritaka and Taro larvae were kept into a 100L tank 2000). The pectoral fins on the right containing 20 L water with aeration. side of initially infected fish were Forty uninfected 100g fish weighting removed to distinguish the second were added, and exposed to the larvae group. for 2h at 15°C with no running water and then were transferred to 500 L tank Challenge trials were made for where they maintained for 7weeks. more than 9 weeks. Water temperature Water temperature ranged between 17- ranged between 19-22°C, with an 20°C with an average 18.5°C. The fish average of 20°C during the trails. Four was fed the basal diet. The parasites fish of each group were randomly were matured 50 day later according to sampled and the number of infected Noritaka and Taro (2000). These parasites on gill was counted 30 days infected fish were used for the sources after beginning of the challenge. of infection and the assessment of the Mortalities were recorded daily and the therapeutic effect of the agents. parasites on the gills and branchial cavity wall of the dead fish were Forty parasites-free fish counted. At he end of the experiment, weighting approximately 100g were the number of the parasites on the divided into 4 groups and were survivor fish was counted. maintained in a 100L tank. Fish were fed the experimental diets before the Statistical analyses challenge trials. Each natural agent was adsorbed to the basal diet at 2.5 g agent The results were statistically / kg. The diet supplemented with 4g analyzed using analysis of variance (F- Parziquantel/ kg diet was used as test) and Duncan’s multiple range tests positive control and the diet alone as a to determine the differences in means negative control. Seven days after (6). 245

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RESULTS At the end of the study periods, examination of Cyprinus carpio Parasite morphology: Fig (A,B, C, D) demonstrated that, while some fish that and Table (1) previously harbored parasites were found to be free of some of the Bodies are elongate 521 (418– parasites, others were completely free. 590) µm long, 171 (138–226) µm Comparatively, the number of parasites wide. Mouth opening is 35 (31- 39) µm on the body surfaces of fish was the wide; Intestinal crura extending same throughout the test period. The beyond the anterior edge of the testes, concentration of A. cina in which 50% 1000 (900-1200) µm long. Haptor is of the parasites were killed was 100 roughly rectangular, 230 (210-250) µm mg/l within 60 min, and a significant long × 280 (260-290) µm wide. Total number (about 85%) were killed in 200 length of hamuli 89 (86–91) µm. Total mg/l Table (2). Interestingly, length of marginal hooks 70 (65-80) monogenean parasites were all µm; marginal hook blade 18 (16-19) dislodged from their attachment sites µm long. The gaffing action of the before the occurrence of mortality marginal hooklets is a predominant following treatment with A. cina. It feature in G. rysavyi. The haptor seems was also observed that the parasite to be formed of two functionally loads were reduced with increasing different compartments; the marginal concentrations of A. cina extract as hooklets are arranged in an amazing shown in Table (2). There was a manner, creating a paddle-like system positive correlation between the driving the parasite during migration number of parasites dislodged from the into the water column, whereas the body surfaces of fish and the time of hamuli are packed into an independent exposure of fish to the extract. In tegumantal envelope. The total length addition to all of these, an increased of the hamulus measured 89 to 100 μm. agility in the swimming of fish freed The opisthaptor was conspicuous with from parasites was also observed when 16 marginal (lateral) hooks with long compared to their counterparts in the shafts arranged in 3 groups. Two control with all parasites remaining groups consisting of 4 hooks each were intact. Some fish taken the upward located anterolaterally, and 8 hooks position in water but some else go formed a posterior group. The marginal down to bottom. hooks were flexible with long shafts, comprising proximal and distal parts.

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ARTEMISIA CINA AGAINST GYRODACTYLUS RYSAVYI IN COMPARISON WITH PRAZIQUNTEL IN CYPRINUS CARPIO

(A) (B)

(C) (D)

Table (3) showed that, extract of In the media containing A. cina was well tolerated by juveniles Paraziquantel our result show that the C.carpio. A minimal mortality larvae contracted immediately and observation was made throughout the 100% of larvae were released from the 96-h period of exposure of fish to the gills 1h after starting the immersion extract. A few fish showed weak (Fig A&B).in the media containing swimming activity in 350 to 500 mg/l Artemisia cina the larvae were drop off of the test solutions. The highest from the gills during the observation percentage mortality observed after 96 after1-3 minutes and 100% of larvae h in the highest concentration was 36 were released from the gills 1h too. %.

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Table (1): Gyrodactylus rysavyi measurements Feature Range (μm) Mean (n = 10)

Total length 418–590 521

Total width 138–226 171 31- 39 35 Width of suction disc 86–91 89 Hamulus\ Total length

Length of shaft 49–59 53 64–71 69 Deep root to curve of blade

Length of point 11–15 14

Length of deep root Length of 12–17 15 superficial

Root 34–42 39

Ratios 1:1.1–1.7 1: 1.4 Superficial root:shaft

Deep root:shaft 1:2.8–4.7 1:3.4

Length of transverse bar 23–29 25

Marginal hooks 63–75 71 Total length

Length of sickle 7–9 8

Proximal width of sickle 5–6 5.2

Distal width of sickle 8–10 8.4

Length of distal part of shaft 26–29 28

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ARTEMISIA CINA AGAINST GYRODACTYLUS RYSAVYI IN COMPARISON WITH PRAZIQUNTEL IN CYPRINUS CARPIO

Table (2): Parasite mortality against concentrations (milligrams per liter) of Artemisia cina at different time intervals

Concentrations (mg L) Time intervals/ mortalities(min) 50 100 150 200

30 7 15 19 28

60 16 22 28 31

90 26 31 32 31

120 32 35 39 35

Table (4) showed that, the group was significantly shorter number of parasites on the gills of the (1.06+0.80) than negative control fish fed on the diet containing 4g (3.46+6.80). No Statistical difference Paraziquantel / kg diet for 20 days was between 2gm Paraziquantel /kg diet significantly fewer (89.07+ 18.80)than group and negative control group, but negative control(298.07+ 123.76). The the length of the parasites was growth of the parasite was affected and significantly shorter (2.86+3.80). the length of the parasites from this

Table (3): Toxicity test of concentrations (mg per liter) of Artemisia cina against C. carpio mortality (%) Concentrations (milligrams per Fish mortality (%) liter) 24h 48h 72h 96h

250 0 0 0 17

300 0 12 14 16

350 8 21 18 31

400 7 28 25 28

500 24 31 33 36*

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Table (4): Determination of parasites number in 4g Praziquantel/ kg diet

Groups Mortalities Surviving fish

No Mature Larvae No Mature Larvae

Paraziquantel 2 0 55 5 0 22.6 +44.0

Artemisia cina 8 56.1 +50.9 841.3+1153.3 1 99 83

Negative control 5 132.7+53.1 286.1+ 866.9 0 -

After thirty days of the Examination of gills of dead fish challenge, the number of parasites was revealed that the infection of the significantly fewer on the gills of the parasites larvae was heavier in the fish examined to asses the prophylactic negative control groups and the groups effect on the groups treated with treated with A. cina compared with the Praziquantel than negative control infection in the groups treated with group No mature parasites were Praziquantel (Table, 4). The infection observed o the banchial cavity wall of of the surviving fish of the treated examined fish. groups with Artemisia cina was low compared with the dead fish, but the Fig (2) showed that, mortalities number of mature parasites in the of the fish in prophylactic group group treated with Praziquantel was occurred earliest in the negative control apparently fewer than those in the group when 100% of mortalities were groups treated with A. cina. The groups recorded by day 75 after starting the treated with Praziquantel was not trial. While the survival of the groups infected by any mature parasites and treated with Praziquantel was only few parasite were found significantly higher than negative control. However, the differences in As with prophylactic groups, the survival between treated with heavy infestation were observed in Artemisia cina and negative control dead negative control fish and in the group was statistically significant. groups treated with A. cina. The

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ARTEMISIA CINA AGAINST GYRODACTYLUS RYSAVYI IN COMPARISON WITH PRAZIQUNTEL IN CYPRINUS CARPIO

number of mature parasites in the the groups treated, A. cina and branchial cavity wall of the fish that Praziquantel was less than the negative survived in the group treated with control. Praziquantel decreased from the initial infections rates was the differences DISCUSSION between these groups and initial infection rates was statistically In this study, the Praziquantel significant, even through the survival anthelmintic efficacy against, G. of the groups treated essential oil was rysavyi in Cyprinus carpio was as low as the negative control, there established and it was used as a were few mature parasites on the positive control to compare the branchial cavity wall of the surviving efficacy of natural anathematic agents fish in these groups. The number of Artemisia cina. Praziquantel is widely larvae parasites on the surviving fish of used to control trematode and cestode infections in mammals (Martin et al., 251

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1997) and the recommended oral dose Praziquantel and Albendazole of Paraziquantel is 20-40 mg/kg pharmacokinetics in fish so it is BW/day in human (Kitahara,1995).In assumed that Praziquantel may reach fish, Paraziquantel has been used in the parasites more easier than experimental to treat monogean disease Albendazole via peripheral blood of by bath treatment (Kim et al.1998 who host and thus has anathematic efficacy, reported that the oral administration of this is coined with that reported by Paraziquantel by intubation into the (Hirazawa et al.,2000) on stomach of rockfish Sebastes schlegeli Heterobotherium okamoto in Tiger had an effect against Microcotyle puffer Takifugu rubripes in Japan. sebaste). The present study clearly showed that feeding a diet containing Hirazawa et al. (2000) reported Praziquantel at high dose to fish (40 that, Praziquntel (4gm/kg diet) is mg/kg BW/day) had an anathematic screened for their anthelmintic efficacy efficacy against G. rysavyi larvae on and so select the drug most suitable as gills and body in Cyprinus carpio. an effective positive control for the Also, showed that the growth of the trials of natural sources. Tubbs and parasite was affected in both groups of Tingle (2006) evaluate an oral PZQ high and low doses. Thus, the dosing strategy, the pharmacokinetics Praziquantel in feed was chosen for the of a dissolved and in feed oral PZQ effective positive control. The preparation (40 mg kg–1 body weight) recommended oral dose of were compared with an intravenous Albendazole is 20-40 mg/kg BW/day bolus in kingfish plasma and skin using in human to control gastrointestinal HPLC.Also,Leslie and Whittington nematode infections (Miller et al., (2002) who recorded that, Praziquantel 1978). In the previous studies is faster than the post-oncomiracidia application of Albendazole which and juveniles, which were attached hardly absorbed from GIT and act between the secondary lamellae of directly on nematode parasite, so we these tissues. This behavior apparently try to test levamisol at a high dose and improves parasite survival in found that the drug reach the target. treatments of short duration and high These results to some extent go in with concentration. Two 40-h baths (48 h the in vitro assay reported by Schmahl apart) in 5 mg/L Praziquantel in and Mehlhorn (1985) on rainbow trout seawater were required to remove all Oncorhynchus mykiss,for the best of parasites from branchial and nasal our knowledge no publication in tissues.

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Our result showed that the larvae vitro trials for A.cina were made for a treated with Praziquantel contracted short period compared with the trials immediately and 100% of larvae were for the anthlemintic drugs; fish survival released from the gills 1h after starting was as low as in the negative control the immersion .In the media containing group and clear effect in preventing Artemisia cina were the larvae treated horizontal infection in the challenge with contracted after 1-3 minutes and trials. However, the number of 100% of larvae drop off from the gills parasites on the surviving fish was during the observation 1h after starting fewer compared with those on the the immersion. negative control fish. Higher doses of A. cina may have a strong effect. But at Our results agreed with Ekanem doses 3g/kg BW/day, C.carpio lost and Brisibe (2010); Albert and their appetite. Ebiamadon (2010) ; Elango and Rahuman (2011) and Squires et al. Thus the incorporation of tested (2011)who reported that, the natural agents such as herbs with Mortality was best achieved by antiparasitic ability is hereby ethanolic extracts of Artemisia which, encouraged, as they will not only at 2 mg/mL, killed S. mansoni and E. reduce the challenge of toxicity if used caproni in 20 h or less (except for at recommended doses but will also wormwood), F. hepatica between 16 reduce the incidence of fish mortality and 23 h . Also, with Alawa et al., due to parasites. (2003).who found that result indicates that only A. senegalensis showed REFERENCES promising anthelmintic activity especially with use of ground whole Abdel-Hadi, Y.M.; Saleh, O.A. and plant materials as used by some A.M. Akar (2008): Study on the farmers. use of Artemisia cina L. (wormseed ) and Allium Artemisia cina had an effect sativum (garlic) in the control of against G. rysavyi larvae by in vitro Saprolegniosis in egg of Cyprinus trials. The parasite contracted quickly carpio (common carp) and and the degree of contraction was Hypophthalmichthys molitrix strong compared with the trials for (silver carp). Proceedings of the th anthlemintic drugs, possibly to the 3o Malaysian Symposium on higher dose of A.cina. Therefore, the in Microbiology (MSM), 16-19,

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Hyatt Regency Resort, Kuantan, Andrew J. Mitchella & Melissa S. Malaysia. Hobbsa (2007): The Acute Toxicity of Praziquantel to Grass Aberham A, Cicek SS, Schneider P, Carp and Golden Shiners North Stuppner H (2010) Analysis of American Journal of Aquaculture sesquiterpene lactones, lignans, Volume 69, Issue 3, pages 203- and flavonoids in wormwood 206 (Artemisia absinthium L.) using high-performance liquid Bone, K., Morgan, M. (1992(Clinical chromatography (HPLC)-mass Applications of Ayurvedic and spectrometry, reversed phase Chinese Herbs: Monographs for HPLC, and HPLC-solid phase the Western Herbal extraction-nuclear magnetic Practitioner .Phytotherapy Press, resonance. J Agric Food Chem Warwick, pp. 7-12. 58(20):10817–10823. Ekanem A. and Brisibe EA. (2010): Adams, J.G., (1978): Effects of ethanol extract Pharmacokinetics of Levamisole. of Artemisia annua; L. against J. Rheumatol. Suppl. 4, 137-142. monogenean parasites of Heterobranchus longifilis. Alawa,C.B., Adamu A.M., Gefu Parasitol Res 106(5):1135–1139 JO, Ajanusi O.J., Abdu P.A., Chiezey N.P., Alawa El-Abbassy, S. A. (2001): Biological J.N., Bowman, D.D.,,. (2003): In and histochemical studies on some vitro screening of two Nigerian monogenean parasites of the medicinal plants (Vernonia catfish Clarias gariepinus amygdalina and Annona inhabiting Nile Delta Waters. M. senegalensis) for anthelmintic Sc. Thesis, Faculty of Sciences, activity. Vet Parasitol 113:73–81 Mansoura University, Mansoura, Egypt. Albert, PE, and Ebiamadon, A.B. (2010( Effects of ethanol extract El-Naggar,M.M. and M.M of Artemisia annua L .Against Serag,(1987): The monogernean monogenean parasites Quadriacantus kearnin.sp and of Heterobranchus Quadriacantus claridis claridis longifilis . Parasitol Res , 106, Paperna 1979 reported on the 1135: 1139 gills of Clarias lazera in Nile

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أستخذام نبات الشيح ) أرتميسيا سينا ( ضذ طفيل الجيرودكتيلس ريسيفيي فى المبروك العادى المصاب بالمقارنة بعقار )البرازيكنتيل( رٍضاُ أّ٘ر ٍحَذ رٍضاُ ٗ دعاء أحَذ اىعزتٚ ٍحَذ اىَعَو اىَزمشٙ ىثح٘ز اىصزٗج اىسَنٞح تاىعثاسح- ٍزمش اىثح٘ز اىشراعٞح

فٚ ٕذا اىثحس ذٌ دراسح ذأشٞز ٍسرخيص ّثاخ اىشٞح عيٚ طفٞو اىعٞزٗدمرٞيس رٝسٞفٜٞ اىذٙ ٝصٞة اسَاك اىَثزٗك اىعادٙ. ٗقذ أظزٝد اىذراسح عيٚ أصثعٞاخ اىَثزٗك اىعادٙ. ذٌ ذقسٌٞ األسَاك اىٚ 6 ٍعَ٘عاخ فٚ شالز ٍنزراخ. ذٌ إسرخذاً ٍسرخيص ّثاخ اىشٞح ٗ تَعذالخ 1، 2، 3 % شالز ٍعاٍالخ ىنو ٍَْٖا ٍع اإلحرفاظ تَعَ٘عح تذُٗ ٍعاٍيح )اىَعَ٘عح اىخاٍسٔ( ىنو ٍنزر. ٗذٌ ذغذٝح األسَاك اىَخرثزج تَعذه 3% ٍِ ٗسّٖا اىحٚ ىَذج شٖز. ٗقذ أٗضحد اىْرائط اُ ٍسرخيص ّثاخ اىشٞح ىٔ أشز فعاه فٜ فصو اىطفٞيٞاخ ٍِ أّسعٔ االسَاك ّٗف٘قٖا تْسثح ذزمٞشاخ -55ٍِ 255ٍيٞعزاً ىيرز مذىل فقذ ٗظذ ارذثاط اٝعاتٚ تِٞ عذد اىطفٞيٞاخ اىَْفصئ ٗاىْافقٔ ٗمال ٍِ ذزمٞش اىَسرخيص ، ٗ ٍذج اىرعزض ىألسَاك اىَرأشزج ىٖذٓ اىَ٘اد ٗ اسرعَاه ٍسرخيص ّثاخ اىشٞح تَعذه أضافح1-3% قذ أعطٚ أعيٚ ّسة إعاشح ىيَثزٗك اىعادٙ. ٗال ٝرزك أشز ٗال رائحح فٚ عضالخ االسَاك الُ االسَاك ذْرقو تعذ اىعالض اىٍٞ ٚاج طثٞعٞٔ ىيرخيص ٍِ اىزائحح. ذٌ اسرخذاً عقار )اىثزاسٝنْرٞو( الظٖار اىنفاء ِج ض ّذ ٝزقاخ اىعٞزٗدمرٞيس رٝسٞفٜٞ ٗتاىراىٚ اّرخاب اىعقار اىَْرخة ٍْاسة االٝعاتٞح ف ّعاه ىيسٞطزج ىرعارب اىرحذٛ تاسرخذاً ٍصاد ِر اىطثٞعٞ ِح) ٍسرخيص ّثاخ اىشٞح( فٚ مال ٍِ اىرعارب اىَعَيٞٔ ٗذعارب االعطاء عِ طزٝق اىعيٞقٔ تَعذه )4ظزاً ىنو مٞي٘ ظزاً عيٞقٔ ( ٗاىرٚ اسرخذٍد ىيسٞطزج االٝعاتPositive control( ٔٞ ( فٚ ذعارب اىرحذٙ,عْذ شث٘خ مفأءج اىَصذر اىطثٞعٚ )2.5ظزاً ىنو مٞي٘ ظزاً عيٞقٔ ( فٚ اىرعارب اىَعَيٝ ٔٞرٌ اعطائح ىالسَاك فٚ اىعيٞقٔ. أٗضحد اىْرائط:- اُ )اىثزاسٝنْرٞو( ذَْع ذَاٍا اىعذٙٗ االفقٞٔ تِٞ االسَاك. 2-االحٞاء ٍِ االسَاك اىَعاىعٔ تٌٖ)اىثزاسٝنْرٞو( ٝنّ٘٘ا افضو ٍِ االسَاك غٞز اىَصاتٔ. 3- عذد االط٘ار اىثاىغٔ فٚ اىرعٝ٘ف اىخٞشٍٚ٘ ذقو عِ اىَعَ٘عاخ االخزٙ.

4- ذطثٞق ٍسرخيص ّثاخ اىشٞح االسرفادج ٍْٔ فٜ صْاعح ذزتٞح األحٞاء اىَائٞح ىيقضاء عيٚ اسرخذاً اىعقاقٞز اىرقيٞذٝح اىعضٝ٘ح االصطْاعٞح اىرٜ قذ ذنُ٘ ضارج ىيَسرٖينِٞ ٍِ ٍْرعاخ ذزتٞح األحٞاء اىَائٞح.

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