1- Fish for in vivo treatment:
A total number of 50 sea bream, from a naturally infested farm with protozoan Trichodina sp. and monogenetic trematode Furnestinia with an average body weight of 150 ±20 gm and an average length of 14 ±2 cm, were collected from a private fish farm in Diba triangle in Damietta Governorate. Fish were acclimatized to laboratory conditions at a water temperature 25 ±1 C º for one week before launching the experimental infection.
Clinical and post-mortem examination:
External examination of fish samples were performed to record clinical abnormalities and post-mortem changes according to (Noga, 2010)
Parasitological examination and identification:
Fish were examined externally with naked eye and with aid of magnifying glass for detection of parasitism according to (Noga, 1996). Mucous smears were immediately prepared from the skin and fins with the aid of microscopic slides and covers then examined with stereo microscope at 10 ,40 and 100 X magnification for detection of external parasites .Fish were euthanized and gills were carefully removed and placed in separate petri dish containing sterilized marine water to remove any excess gill mucus, and they were examined for parasites under a stereo microscope. All detected parasites; were collected, examined, fixed and stained according to (Lucky, 1977, Paperna and Laurencin, 1979) then identified according to (Lom, 1995, Paperna, 1996)
Vaccium210 is a commercial preparation of herbal extracts origanum oil (Carvacrol and Thymol) and Capsicum oil, produced by Slant Kim Company, Germany.
Experiment 1 :In vitro effect of Vaccium210 on Trichodina sp.
The experiment was designed according to (Ling et al., 2012, Fridman et al., 2014) as Infested Fish with Trichodina sp. were anaesthetised in 0.025% clove oil (Kildea et al., 2004). Mucus scraps from body surface were collected in small petri dish that was examined under stereo microscope to confirm parasitic existence and parasite livability. Afterwards, 20 ±1 parasites were transferred in each well of 96 well plate containing 0.5 mL filtered marine water from same fish source filtered through a filter with a pore size of <20 micrometers (?m) prior to use in a test (EPA, 2016). Parasite movement was then evaluated using a stereo microscope to ensure parasite survival. Wells with parasites were exposed to the following set of Vaccium210 concentrations: 0, 2.5, 5, 7.5, 10, 12.5, 15 and 20 ppt. The control wells contained parasite in marine water without adding Vaccium210. The time was defined as zero and Parasites were observed every 5 min until all parasites were dead. The data were derived from the mean value of three replicates. Experiment 2: In vitro effect of Vaccium210 on monogenean Infected Fish with Furnestinia were anaesthetised in 0.025% clove oil (Kildea et al., 2004) followed by pithing. Areas of gill filaments with minimum of three parasites were cut off using a scalpel. Each clipped filaments were transferred to separate well of 96 well plate containing 0.5 mL filtered marine water. Parasites were observed for movement using a dissecting microscope to ensure parasite survival. Wells with parasites were exposed to the following set of Vaccium210 concentrations: 0, 2.5, 5, 7.5, 10, 12.5, 15and 20 ppt. The control well had parasite in only marine water .. The time was defined as zero and Parasite survival was observed every 5 min until complete eradication. Detached and Non-motile parasites that did not respond to gentle water current were considered dead. The data were derived from the mean value of three replicates. Experimental 3: Lethal concentration-50 of Vaccium210 to Sparus aurata: The half-lethal concentration (LC50) of drug was conducted to estimate the toxicity of the used drug for treated fish. Fish were divided into 5 groups (n=10 fish) and placed in 40 liter aquaria .The fish were exposed to different concentration of Vaccium210 (control , 30, 60 , 90 and 120 ppt). Aquarium water taken from same fish source should be filtered prior to use in a test .The water temperature were adjusted at 25±2C º in all aquarium and dissolved oxygen in water were measured during experiment with an oxygen meter (cole-parmer Instrument Co., Chicago) and every 24hr thereafter. Any abnormal fish behavior and mortality observations should be recorded at 6, 24, 48 and 96hr. All fish were not fed during the exposure ( The LC50 of Vaccium210 to Sparus aurata was calculated according to equation according to (Hamilton et al., 1977). LC 50 = LC100 - ? A×B N Where, LC50 = Median lethal dose LC100 = Least dose required to kill 100% A = Dose difference B = Mean mortality N = number of fish in group In vivo Bath treatment The protocol of in vivo experiment designed according to (Fridman et al., 2014) . Efficacy of Vaccium210 was tested using bath treatments of naturally parasitic infestation in fish. Fifty naturally infested sea bream with Trichodina spp and F. echeneis were divided into 5 groups (n = 10). Each group was placed in separate glass aquaria containing 60 L marine water equipped with aeration. Fish were examined for intensity of infestation before and after treatment. Vaccium210 was added at concentrations of control 0,2.5, 5, 7.5 and 10 ppt. Fish were examined after 1, 1.5 and 24 hours, as previously described (Fridman et al., 2014) parasite load determined according to (Alvarez- pellitero et al., 1995): with the range: (+) 1–5; (++) 6–10; (+++) 11–25; (++++) 26–50; (+++++) 51–100; (++++++) > 100 parasites /microscopic field.