In vitro activity of chemicals and commercial products against Saprolegnia parasitica and Saprolegnia delica strains

Oomycetes of the genus Saprolegnia are responsible for severe economic losses in freshwater aquaculture. Following the ban of malachite green in food fish production, the demand for new treatments pushes towards the selection of more safe and environment‐friendly products. In the present work, in vitro activity of ten chemicals and three commercial products was tested on different strains of Saprolegnia, using malachite green as reference compound. The compounds were screened in agar and in water to assess the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC), respectively. Two strains of Saprolegnia parasitica and one isolate of Saprolegnia delica were tested in triplicate per each concentration. Among tested chemicals, benzoic acid showed the lowest MIC (100 ppm) followed by acetic acid, iodoacetic acid and copper sulphate (250 ppm). Sodium percarbonate was not effective at any tested concentration. Among commercial products, Virkon^?S was effective in inhibiting the growth of the mycelium (MIC = MLC = 1,000 ppm). Actidrox® and Detarox® AP showed MIC = 5,000 and 1,000 ppm, respectively, while MLCs were 10‐fold lower than MICs, possibly due to a higher activity of these products in water. Similarly, a higher effectiveness in water was observed also for iodoacetic acid.     

In vitro Antifungal activity of essential oils from Tymbra,Origanum, Satureja species and some pure compounds on the fish pathogenic fungus,Saprolegnia parasitica

In the present study, the chemical composition and antifungal activity of the essential oils of the three Lamiaceae species, black tyme (Thymbra spicata L.), oregano (Origanum onites L.) and savory (Satureja tymbra L.) against Saprolegnia parasitica Coker strains (3) were evaluated. The composition of oils was analysed by gas chromatography/mass spectrometry (GC/MS). The major constituent of the oils was carvacrol (82.68% in Thymbra spicata, 82.24% in Origanum onites and 52.38% in Satureja tymbra). The antifungal activity of essential oils was determined by a tube dilution and disc diffusion method. All essential oils inhibited fungal strains at concentration of 100 μL mL^?1. The essential oil of O. onites and T. spicata was the most active (MIC 10 and 50 μL mL^?1, respectively). Thymol did not show inhibiting activity. The essential oils exhibited stronger inhibition than pure carvacrol. Essential oil derived from Tymbra, Origanum and Satureja species has the potential to be used as health control of rainbow trout (Oncorhyncus mykiss) against S. parasitica infection. This study describes the antifungal activity of three essential oils for developing alternative and native control methods against Saprolegniasis.     

In vitro and in vivo antifungal activity of Satureja cuneifolia ten essential oil on Saprolegnia parasitica strains isolated from rainbow trout (Oncorhynchus mykiss,Walbaum) eggs

In the present study, the chemical composition and the antifungal properties against Saprolegnia parasitica (in vitro and in vivo) of the essential oils of thyme (Satureja cuneifolia) from Mediterranean region of Turkey were evaluated for the first time. The composition of oils was analysed using gas chromatography/mass spectrometry (GC/MS). The major constituents of oil of S. cuneifolia were cavracrol (46,84%) and cymene (16.90%). Antifungal effects of S. cuneifolia essential oil against S. parasitica strains (A1 and E1) were detected by disc diffusion and tube dilution assays. The antifungal effect of S. cuneifolia was determined to be stronger against S. parasitica E1 isolate (MIC 50 μL mL^?1, MLC 250 μL mL^?1) compared with S. parasitica A1 isolate (MIC 50 μL mL^?1, MLC 500 μL mL^?1). Following in vitro assays, effective doses of S. cuneifolia for disease control in rainbow trout eggs experimentally infected with S. parasitica were investigated. For this aim, infected eggs were treated with the essential oil (0, 5, 10, 20 and 50 ppm) during incubation period (21 days) after fertilization. Formalin (5 mL L^?1) was used as positive control. Hatching rate of eggs at the end of incubation period were calculated. The highest hatching rates were recorded in S. parasitica E1 strain at 5 and 10 ppm concentrations of S. cuneifolia and in S. parasitica A1 strain at 10 and 20 ppm (P < 0.05).     

Biocontrol of saprolegniosis in rainbow trout (Oncorhynchus mykiss Walbaum) using two bacterial isolates (LE89 and LE141) of Pseudomonas fluorescens

The probiotic activity of 15 bacterial isolates that inhibit Saprolegnia parasitica in vitro was tested for the biocontrol of saprolegniosis in rainbow trout (Oncorhynchus mykiss Walbaum), adding the bacteria to tank water for 14 days at a concentration of 10⁶ bacteria ml^?1 water. Pseudomonas fluorescens LE89 and Pseudomonas fluorescens LE141 were effective in controlling experimental infection with S. parasitica since of the fish treated with LE89, 24.5% ± 16.27% (p < 0.05) became infected, as did 42.8% ± 8.41% (p < 0.05) of those treated with LE141. Given their protective effect when administered in water, their effect was also studied when administered in feed before and after experimental infection. Both bacterial isolates survived low pH levels and the action of bile, grew in skin and intestinal mucus, were resistant to several antibiotics and survived in feed; however, neither of the two isolates prevented S. parasitica infection when administered in feed.     

The Efficacy of Copper Sulfate in Controlling Infection of Saprolegnia parasitica

Saprolegnia parasitica is a severe fish pathogen that causes important economic losses worldwide. Copper is an important additive in the aquaculture industry for control of algal growth, ectoparasites, and fungal disease. However, at present no data is available on the specific interaction of copper sulfate with oomycete. In our study, the efficacy of copper sulfate on the mycelium and zoospore of S. parasitica was assessed in vitro, and S. parasitica infection experiment was conducted to assess its performance in vivo. The results indicated that copper sulfate at ≥0.5 mg/L inhibited the growth of mycelium, no primary zoospores were released at ≥1.0 mg/L. Additionally, 0.5 mg/L copper sulfate could reduce the infection rate of S. parasitica in the grass carp, Ctenopharyngodon idellus. This study demonstrates the good efficacy of copper sulfate on the control of S. parasitica infection in grass carps, and suggests that copper sulfate could be used as a drug additive to control the S. parasitica infection in the aquaculture industry.     

Saprolegnia diclina IIIA and S. parasitica employ different infection strategies when colonizing eggs of Atlantic salmon,Salmo salar L.

Here, we address the morphological changes of eyed eggs of Atlantic salmon, Salmo salar L. infected with Saprolegnia from a commercial hatchery and after experimental infection. Eyed eggs infected with Saprolegnia spp. from 10 Atlantic salmon females were obtained. Egg pathology was investigated by light and scanning electron microscopy. Eggs from six of ten females were infected with S. parasitica, and two females had infections with S. diclina clade IIIA; two Saprolegnia isolates remained unidentified. Light microscopy showed S. diclina infection resulted in the chorion in some areas being completely destroyed, whereas eggs infected with S. parasitica had an apparently intact chorion with hyphae growing within or beneath the chorion. The same contrasting pathology was found in experimentally infected eggs. Scanning electron microscopy revealed that S. parasitica grew on the egg surface and hyphae were found penetrating the chorion of the egg, and re‐emerging on the surface away from the infection site. The two Saprolegnia species employ different infection strategies when colonizing salmon eggs. Saprolegnia diclina infection results in chorion destruction, while S. parasitica penetrates intact chorion. We discuss the possibility these infection mechanisms representing a necrotrophic (S. diclina) vs. a facultative biotrophic strategy (S. parasitica).     

Recovery of Aliphatic Fatty Acids from Red Seaweed Champia parvula (C. Agardh) and Its Antifungal Action

The aim of the present study was to evaluate the antifungal activity of seaweed Champia parvula and identification of active compounds for this action. In this regard, C. parvula crude extracts were obtained from n-hexane, ethyl acetate, chloroform, methanol, and water used to study the antifungal action. Results showed that methanolic extract had potent action against C. albicans, C. tropicalis, T. mentagrophytes, and A.flavus, with the highest inhibition of 16.7 mm found against C. tropicalis. Minimum inhibition concentration at 12.5 and minimum fungal concentration at 25 μg/mL were found in the methanolic extract. Therefore, to identify the bioactive compound for this antifungal action in the methanolic extract, thin-layer chromatography (TLC) and reverse-phase high-performance liquid chromatography (RP-HPLC) were used. The RP-HPLC had three peaks with retention times at 3.30, 3.86, and 4.73 min, showing the presence of ester fatty acid compound. Further, these compounds were characterized by nuclear magnetic resonance (NMR), gas chromatography (GC), and liquid chromatography-mass spectrometry (LC-MS), which showed the presence of long-chain aliphatic fatty acid like eicosanoic acid with formation of oct-1-en-3-ol compounds attached.     

Saprolegnia molecular phylogeny among farmed teleosts in Nova Scotia,Canada

To identify the pathogens causing saprolegniosis among farmed fish in Nova Scotia, 172 infected tissues and 23 water samples were collected from six species of teleosts: Atlantic salmon (Salmo salar), brown trout (Salmo trutta), Arctic charr (Salvelinus alpinus), brook trout (Salvelinus fontinalis), striped bass (Morone saxatilis) and rainbow trout (Oncorhynchus mykiss) at nine facilities over a 600 km range. Following laboratory culture, 132 isolates were recovered. Six species of oomycetes were identified from analysis of the internal transcribed spacer (ITS) sequence of the nrDNA: Saprolegnia parasitica, Saprolegnia ferax, Saprolegnia diclina, Saprolegnia aenigmatica, Saprolegnia torulosa, Saprolegnia sp. and Pythiopsis cymosa. Further phylogenetic analyses of the ITS and cytochrome c oxidase subunit 1 (Cox1) regions revealed four strains of Saprolegnia parasitica (named here as S1, S2, S3 and S4), of which S1 and S2 were common (37% and 42% of the isolates), and two strains of S. ferax. Among S. parasitica, S2 and S3 are more closely related to each other than to S1 based on the phylogenetic analyses and predicted RNA secondary structure of the ITS region. Sexual structures with a similar morphology were formed by S1 and S3 in vitro, but were not formed by S2.     

Reduction of in vitro growth in Flavobacterium columnare and Saprolegnia parasitica by products containing peracetic acid

Commercial products containing peracetic acid (PAA) are strong disinfectants with a wide spectrum of antimicrobial activity and have been suggested as potential therapeutic agents in aquaculture. The aim of this study was to compare the in vitro reduction of growth on two fish pathogens, Flavobacterium columnare and Saprolegnia parasitica, by seven commercial PAA‐containing products. Flavobacterium columnare was exposed to 1, 2, 4, 6, 8 and 10 mg L^?1 PAA and S. parasitica was exposed to 0.5, 1, 2, 4, 6, 8 and 10 mg L^?1 PAA in petri dishes for 24 h incubation. The reduction of growth was measured in comparison to a PAA‐free control. A reduction of the growth was observed for both pathogens with increasing PAA concentration. Hydrogen peroxide (H₂O₂) possibly has a role in the effectiveness of the products, since products with lower PAA concentrations had a higher concentration of H₂O₂. The commercial products with a low concentration of PAA and a low PAA:H₂O₂‐ratio were generally more effective against pathogens. The practical application of the products with low PAA concentration should be prioritized.     

Physicochemical Properties and Bioactivity of Extracts from the Roe of New Zealand Hoki and Southern Blue Whiting

ABSTRACT

Various physicochemical properties of New Zealand hoki and southern blue whiting (SBW) roes and the biological activities of their extracts were investigated. Protein, moisture, and ash contents in both roes were similar; however, the lipid content of hoki roe was higher (p < 0.05) than SBW roe (11.0 ± 0.9 and 2.5 ± 0.1, respectively). Both fish roe extracts contained active protease inhibitors toward trypsin and papain. Neither of the roe extracts agglutinated erythrocytes, indicating the absence of lectins. Both fish roe extracts at 5 mg/mL concentration exhibited slight inhibitory effects on the proliferation of breast cancer cells, but neither of the roe extracts exhibited RNase or antifungal activity.     

In Vitro Screening of Fungicidal Chemicals for Antifungal Activity against Saprolegnia

Saprolegnia is an important fish fungal pathogen that often results in significant economic losses to freshwater aquaculture. To find effective drugs to control saprolegniasis, 30 fungicidal chemicals used in agriculture were screened, in which kresoxim‐methyl and azoxystrobin, with minimum inhibitory concentration (MIC) values of 1.0 and 0.5 mg/L, respectively, showed good in vitro antifungal activities against Saprolegnia. Azoxystrobin has the most promising anti‐Saprolegnia activity with 50% effective concentration (EC₅₀) value of 0.212 mg/L against mycelial growth and minimum fungicidal concentration (MFC) value of 0.13 mg/L against spores, while EC₅₀ and MFC values to kresoxim‐methyl are 0.240 and 0.25 mg/L, respectively. Through the acute toxicity assay using goldfish, Carassius auratus, azoxystrobin exhibited wider margin of safety with a safe concentration (SC) value of 0.553 mg/L than kresoxim‐methyl with an SC value of 0.131 mg/L. These findings demonstrated that azoxystrobin has the potential for the development of therapy for the control of Saprolegnia in aquaculture. Both kresoxim‐methyl and azoxystrobin were tested with a post‐antifungal effects (PAFE) assay and the results revealed that the two chemicals had no significant effect on fungal growth inhibition after a 1‐hour exposure, indicating that the treatment needs to be carried out over an extended period.     

Use of Onion (Allium cepa) and Garlic (Allium sativum) Wastes for the Prevention of Fungal Disease (Saprolegnia parasitica) on Eggs of Rainbow Trout (Oncorhynchus mykiss)

This study was designed to prevent fungal disease (Saprolegnia parasitica) that occurs on rainbow trout eggs (Oncorhynchus mykiss) by using wastes of onion (Allium cepa) and garlic (Allium sativum) plants. For this purpose, fertilized rainbow trout eggs were exposed to garlic skin, garlic stem and onion skin aqueous methanolic extracts by bathing in concentrations of 0.4, 0.8, 1.6 and 3.2 g/L, whereas the control group was left untreated. The larvae in all groups were monitored until they become free-swimming larvae, the number of eggs died due to fungus was recorded, and the data obtained from experimental groups and control group were compared. As a result, it was determined that onion skin had no effect on the number of eggs that died due to fungus (p > .05), while garlic skin and garlic stem extracts significantly reduced the number of fungal infestations without affecting the number of live larvae or the embryological development of the eggs (p < .05). In light of these data, we conclude that garlic skin (0.4, 0.8, and 1.6 g/L) and garlic stem (0.8 and 1.6 g/L) aqueous methanolic extracts are effective at preventing Saprolegnia parasitica infestation on rainbow trout eggs and may be used in aquaculture.     

Effect of miconazole nitrate on immunological response and its preventive efficacy in Labeo rohita fingerlings against oomycetes Saprolegnia parasitica

This study evaluated the effect of sublethal doses of antifungal drug miconazole nitrate (MCZ) on immunological responses and its role as a prophylactic drug against S. parasitica in Labeo rohita fingerlings. Fish were fed with sublethal doses of MCZ, that is, T1—6.30 mgMCZ kgBW^?1, T2—12.61 mgMCZ kgBW^?1 and T3—25.22 mgMCZ kgBW^?1, and sampling was done at different time intervals for 240 hr. Immunological parameters viz. lysozyme activity, oxygen radical production and plasma antiprotease activity showed significant enhancement (p < 0.05) in fish fed with T2 and T3 doses. Expression of immune‐relevant genes such as TLR‐22 and β2‐M showed significantly higher expression at 6 hr and 24 hr of sampling in both liver and head kidney. However, these genes showed a downregulation after 120 hr of sampling in both the tissues. Preventive efficacy study showed that single dose of MCZ provides protection against oomycetes up to the fourth day of infection. Significantly higher mortality was observed in control diet‐fed fish as compared to fish fed with MCZ medicated diet. Thus, it can be concluded that the MCZ can act as a potent antifungal agent for preventing oomycetes infection as well as to enhance the immune response.     

Assessment of the Nutritive,Biochemical, Antioxidant and Antibacterial Potential of Eight Tropical Macro algae Along Kachchh Coast,India as Human Food Supplements

Eight tropical marine macro algae were investigated for in-vitro antioxidant, antibacterial, and biochemical properties. The moisture content [% DW (dry weight)] ranged from 7.21 to 14.72%; ash, 24.92 to 47.04%; lipids, 0.73 to 2.67%; protein, 4.56 to 12.59%; and carbohydrate, 30.1 to 48.51%. The % sum of polyunsaturated fatty acids (PUFAs) ranged from 21.71 to 78.22%, while omega-6/omega-3 (ω6/ω3) ratio was from 0 to 2.08, which remained within the prescribed World Health Organization (WHO) standards (<10). The % sum of essential amino acids (EAAs) ranged from 86.18 to 204.66, and Na/K ratio ranged from 0.37 to 2.85. The extracts exhibited significant (P < 0.05) values for total phenol (7.25–16.0 µg PGE mg^–1 DW) and total flavonoid (4.06–15.63 µg QE mg^–1 DW) at 200 µg/ml. Antibacterial activity was tested against the selected food spoilage and pathogenic bacteria. The principal component analysis (PCA) confirmed a positive correlation between total phenolic content, 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) radical-scavenging activities, and ferric reducing antioxidant power (FRAP), which supports the integration of seaweeds as ingredients in functional foods.     

The inhibitory activities and antiviral mechanism of Viola philippica aqueous extracts against grouper iridovirus infection in vitro and in vivo

Grouper iridovirus (GIV) is one of the most serious pathogens in mariculture and causes high mortality rates in cultured groupers; then, effective medicines for controlling GIV infections are urgently needed. Viola philippica is a well‐known medicinal plant, and the application of V. philippica aqueous extracts against GIV infection was assessed by different methods in this study. The results showed that the working concentration of V. philippica aqueous extracts was 10 mg/ml. V. philippica aqueous extracts below 10 mg/ml have no significant cytotoxic effects on cell viability, while extracts over 15 mg/ml decreased cell viability and showed cytotoxic activity. V. philippica aqueous extracts had excellent inhibitory effects against GIV infection in vitro and in vivo. The possible antiviral mechanism of V. philippica was further analysed, which indicated that V. philippica did no damages to GIV particles, but it could disturb GIV binding, entry and replication in host cells. V. philippica had the best inhibitory effects against GIV during viral infection stage of binding and replication in host cells. Overall, the results suggest that appropriate concentration of V. philippica aqueous extracts has great antiviral effects, making it an interesting candidate for developing effective medicines for preventing and controlling GIV infection in farmed groupers.     

Antioxidant Properties of Seven Cultivated and Natural Edible Seaweed Extracts from Taiwan

Seven species of cultivated and edible seaweeds found in Southern Taiwan—including Enteromorpha intestinalis, Monostorma nitidum, Ulva lactuca, Dictyopteris undulata, Sargassum hemiphyllum var. chinense, Laurencia intermedia, and Porphyra dentate—were used to determine their antioxidant activity by different extraction methods. All seaweeds were extracted using water at 90°C, sterilization at 121°C, homogenization, or enzymatic hydrolysis. Extracts were evaluated for their free radical-scavenging capacity using 1,1-diphenyl-2-pricrylhydrazyl (DPPH), their reducing power, and their superoxide anion scavenging activity. The DPPH-scavenging capacity of S. hemiphyllum var. chinense extracts produced by sterilization (98.59%), homogenization (94.72%), and carbohydrate-degrading enzyme treatment was higher than that of protease extracts, except for U. lactuca. The reducing power of S. hemiphyllum var. chinense water extracts and M. nitidum Viscozyme extracts was better than that of a commercial antioxidant. S. hemiphyllum var. chinense exhibited higher superoxide anion scavenging activity than the other species. The results demonstrate that different extracts of some of the seaweeds showed remarkable antioxidant activity in different assays. The results also provide important information for the marine food industry and suggest that seaweed might be a valuable source of natural antioxidants.     

Effect of different diets on growth and digestive enzyme activity in Litopenaeus vannamei (Boone, 1931) early post-larvae

Growth rate, soluble-protein content and digestive-enzyme activities were studied in Litopenaeus vannamei (Boone, 1931) early post-larvae under six feeding regimens, which included combinations of freshly hatched Artemia nauplii, an artificial diet and algae. Growth (0.11 mg DW day⁻¹) and soluble-protein content (61.8 μg protein larvae⁻¹ at PL₁₀) of post-larvae fed mixed diets were significantly higher (P < 0.05). An artificial diet used alone or co-fed with algae caused the lowest growth (0.03–0.05 mg DW day⁻¹) and soluble-protein content (13.7–15.5 μg protein larvae⁻¹ at PL₁₀). Trypsin-like activity was higher (up to 10 times) in post-larvae fed Artemia nauplii and an artificial diet alone or plus algae. The artificial diet stimulated chymotrypsin activity, apparently in response to squid meal present in this diet. Amylase activity increased when post-larvae were fed the artificial diet. This was apparently related more to the origin of the starch than to the total carbohydrate level of the diet. No obvious relationship was found between enzyme activity and growth in any feed combination. Based on growth and soluble-protein content, we determined that partial substitution (50%) of Artemia nauplii by artificial diet and the use of algae co-fed beyond the first post-larval stage benefits growth and the nutritional state of L. vannamei post-larvae.     

Pharmacodynamics of amoxicillin against field isolates of Streptococcus parauberis from olive flounder (Paralichthys olivaceus)

Olive flounder is the most important species for the Northeast Asian fish farming industry. However, this species is substantially affected by multiple infectious agents, including Streptococcus parauberis. Evaluation of antibiotics before their application is critical to treat infections and prevent drug resistance. Therefore, in this study, the pharmacodynamics of amoxicillin (AMX) and other antimicrobials against the planktonic‐ and biofilm‐forming bacteria were assessed. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and Time–kill curve assay were analysed using micro‐dilution method. The minimum biofilm eradicating concentration (MBEC) was determined using the Calgary Biofilm device. The effects of temperature, pH, hardness and salinity were detected for both planktonic‐ and biofilm‐forming bacteria. The MIC of AMX ranged from 0.015 to 2 μg/ml, whereas that of cephalexin (CEP), enrofloxacin (ENR) and oxytetracycline (OTC) ranged from 0.125 to 256, 0.125 to >512 and 0.25 to >512 μg/ml respectively. No bacteria were resistant against AMX, while the percentage of resistance to CEP, OTC and ENR were 68.7%, 52.6% and 11.1% respectively. The IC₅₀ of AMX, CEP, ENR and OTC was 0.03, 0.091, 0.015 and 0.213 μg/ml respectively. The MBEC of amoxicillin against S. parauberis ranged from 0.5 to 16 μg/ml. Higher rates of bacterial growth were obtained at 30°C, pH = 8 and salinity of 7.5–10 ppt. The hardness of the media suppressed the bacterial growth. In conclusion, AMX was found to be effective against both the planktonic and the biofilm forms of the prominent fish pathogen, S. parauberis.     

Protective effects of a synbiotic against experimental Saprolegnia parasitica infection in rainbow trout (Oncorhynchus mykiss)

A combination of probiotics and prebiotics as synbiotics allows assessing their synergistic effects. This study evaluated the effects of a synbiotic supplement on growth performance, haematological parameters and resistance to Saprolegnia parasitica in rainbow trout, Oncorhynchus mykiss (Walbaum) fingerlings. Fish fed a dietary synbiotic in three levels of 0.5, 1.0 and 1.5 g kg^?1 thrice a day. The fingerlings were challenged with Saprolegnia parasitica after 60 days post feeding and their mortalities recorded up to 15 days. The fingerlings at all three experimental treatments showed significant (P < 0.05) increases in final mean weights and specific growth rates (SGR). The best feed conversion ratio (FCR), feed conversion efficiency (FCE) and maximum survival rate were also obtained by the fish fed 1.0 g synbiotic kg^?1 diet. Furthermore, supplementation with synbiotic significantly increased blood factors at all treatments. After challenges with Saprolegnia parasitica, the synbiotic‐fed groups showed significantly higher survival rates compared with the control group. These results reveal that a dietary synbiotic of 1.0 g kg^?1 fed for 60 days leads to increased growth performance and survival rate as well as improved feeding efficiency in rainbow trout fingerling, rendering them more resistant against infection by Saprolegnia parasitica.     

Extracts of Oliviera decumbens and Satureja khuzestanica as immunostimulants affect some innate immunity indices of Cyprinus carpio against Aeromonas hydrophila infection

In this study, the effect of extracts of two herbs (Oliviera decumbens and Satureja khuzestanica) on immune response of carp was investigated. At the beginning of experiment, fish were divided to two groups including vaccinated (using vaccine developed against A. hydrophila) and non‐vaccinated. Fish in both groups were fed diets containing O. decumbens and S. khuzestanica and combination of two herbal extracts for 5 weeks. Control fish (negative control) and fish vaccinated only (positive control) were fed basal diets without supplements of herbal extracts. Lysozyme activity, antibody titre, complement activity and bactericidal activity in serum were measured. After 5 weeks feeding, fish were infected with A. hydrophila and mortalities were recorded. In both experimental groups, no significant differences were found in terms of alternative haemolytic complement (ACH50) activity and antibody titres of Serum. In non‐vaccinated fish group, lysozyme and bactericidal activity of fish fed S. khuzestanica or combination of O. decumbens and S. khuzestanica was higher compared with control and other experimental treatments. In vaccinated fish group, the lysozyme and bactericidal activity was not significant in all treatments compared with control group. The results of this study showed that feeding non‐vaccinated and vaccinated carp with only S. khuzestanica or in combination with O. decumbens enhance only some immunity indices including lysozyme and bactericidal activity in non‐vaccinated fish and these extracts have no immunological stimulatory role on vaccinated individuals.