Antiparasitic efficacy of flavonoids identified from Psoralea corylifolia against Tetrahymena piriformis in guppy (Poecilia reticulate).

Tetrahymena piriformis belongs to the ciliated protists (ciliates), causing severe economic losses in aquaculture. Chemical drugs currently used usually have toxic side effects, and there is no specific drug against Tetrahymena. Therefore, it is an urgent need to identify new antiparasitic lead compounds. In the present study, the in vitro parasiticidal activity of ethyl acetate (EtOAc) extracts and water extracts from 22 selected traditional Chinese medicines (TCMs) were evaluated against T. piriformis. The EtOAc extract of P. corylifolia turned out to be the most active with the minimum parasiticidal concentration of 100 mg/L within 3 h. Thus, it was separated into 12 fractions by the first-dimensional (D1) normal phase liquid chromatography (NPLC), meanwhile combining with in vitro antiparasitic tests for activity tracking. Subsequently, 8 flavonoids were identified in the active fractions by the second-dimensional (D2) reverse phase liquid chromatography (RPLC) tandem high-resolution mass spectrometry. According to the results, 5 flavonoids were selected for in vitro antiparasitic test, of which isobavachalcone showed the minimum parasiticidal concentration of 3.125 mg/L in 2 h. Bathing treatment of infected guppies with isobavachalcone could significantly reduce the burden of T. piriformis, obtaining a 24-h median effective concentration (24-h EC50) value of 1.916 mg/L. And the concentration of isobavachalcone causing guppies to die within 24 h is 39 times than that of 24-h EC50. The results demonstrated that isobavachalcone has the potential to be developed into a novel commercial fish drug against T. piriformis.

Silencing of heat shock protein 90 (hsp90): Effect on development and infectivity of Ichthyophthirius multifiliis.

BACKGROUND: Recently, an increasing number of ichthyophthiriasis outbreaks has been reported, leading to high economic losses in fisheries and aquaculture. Although several strategies, including chemotherapeutics and immunoprophylaxis, have been implemented to control the parasite, no effective method is available. Hence, it is crucial to discover novel drug targets and vaccine candidates against Ichthyophthirius multifiliis. For this reason, understanding the parasite stage biology, host-pathogen interactions, molecular factors, regulation of major aspects during the invasion, and signaling pathways of the parasite can promote further prospects for disease management. Unfortunately, functional studies have been hampered in this ciliate due to the lack of robust methods for efficient nucleic acid delivery and genetic manipulation. In the current study, we used antisense technology to investigate the effects of targeted gene knockdown on the development and infectivity of I. multifiliis. Antisense oligonucleotides (ASOs) and their gold nanoconjugates were used to silence the heat shock protein 90 (hsp90) of I. multifiliis. Parasite stages were monitored for motility and development. In addition, the ability of the treated parasites to infect fish and cause disease was evaluated. RESULTS: We demonstrated that ASOs were rapidly internalized by I. multifiliis and distributed diffusely throughout the cytosol. Knocking down of I. multifiliis hsp90 dramatically limited the growth and development of the parasite. In vivo exposure of common carp (Cyprinus carpio) showed reduced infectivity of ASO-treated theronts compared with the control group. No mortalities were recorded in the fish groups exposed to theronts pre-treated with ASOs compared with the 100% mortality observed in the non-treated control fish. CONCLUSION: This study presents a gene regulation approach for investigating gene function in I. multifiliis in vitro. In addition, we provide genetic evidence for the crucial role of hsp90 in the growth and development of the parasite, suggesting hsp90 as a novel therapeutic target for successful disease management. Further, this study introduces a useful tool and provides a significant contribution to the assessing and understanding of gene function in I. multifiliis.

Antiparasitical efficacy of sophoraflavanone G isolated from Sophora flavescens against parasitic protozoa Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis, a global distributed protozoan parasite, causes "White spot disease" and leads to serious mortality of freshwater fish in aquaculture. The present study was conducted to assess the anti-I. multifiliis efficacy of active compound isolated from Sophora flavescens. The isolated active compound was identified as sophoraflavanone G (SG) with ESI-MS and NMR. In vitro tests, SG at concentrations of 0.5 mg/L and 2 mg/L resulted in death of all theronts and tomonts, respectively; SG at concentrations of 0.125 mg/L and 0.25 mg/L notably decreased theronts infectivity (p < 0.05). Additionally, the in vivo test results showed that a cumulative delivery of SG at concentration of 2 mg/L for 7 days protected fish from I. multifiliis infection. The 96-h LC50 (median lethal concentration) and safety concentration of SG to grass carp were 46.6 mg/L and 11.3 mg/L, respectively. The present work indicated that SG was a potential safe and effectively therapeutic agent in treating I. multifiliis.

Molecular characterisation and biological activity of an antiparasitic peptide from Sciaenops ocellatus and its immune response to Cryptocaryon irritans.

Cryptocaryon irritans, a holotrichous ciliate parasitic protozoan, can trigger marine white spot disease and cause substantial economic losses in mariculture. However, methods of preventing and curing the disease have negatively affect fish, human, other organisms, and the natural environment. The antiparasitic activity of some antimicrobial peptides (AMPs) has garnered extensive attention of scholars. In this study, we identified and characterised a novel antiparasitic peptide, named So-pis, from Sciaenops ocellatus. The sequence analysis, structural features, and tissue distribution suggested that So-pis is genetically related to the piscidins family. However, So-pis showed a relatively low overall conservation compared with other known piscidins. So-pis is abound in glycine residues (22.7 %) and it has a neutral isoelectric point, weak amphipathicity, relatively long α-helix, and high hydrophobicity. These key elements are responsible for its biological activity. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicated that So-pis is a typically gill-expressed peptide. The expression of So-pis in the gill, skin, spleen, and head kidney could be regulated during C. irritans infection, thereby implicating a role of So-pis in immune defence against C. irritans. The synthetic So-pis had limited or no antimicrobial activity against bacterial and yeasts but exhibited potent antiparasitic activity against C. irritans in vitro. The activity of synthetic So-pis against erythrocytes was less potent than its antiparasitic activity against C. irritans. These results indicated that So-pis might be one of the crucial defence cytokines against C. irritans in the red drum. Cumulatively, our data suggested that So-pis might be a potential candidate for developing a novel, effective, and safe therapeutic agent against marine white spot disease.

Comparative proteomic analysis provides insight into the key proteins as potential targets underlying the effect of malachite green against Ichthyophthirius multifiliis.

Target identification is important for drug discovery. Unfortunately, no drug targets have been found in Ichthyophthirius multifiliis until now and further limited development of the novel drug for Ichthyophthiriasis. In this study, an iTRAQ-based quantitative proteomic analysis was used to find the target of malachite green (MG), exhibiting greater efficacy than the existing drugs, against I. multifiliis trophonts in situ. We also verified the proteomic results by RT-qPCR, TEM and cell apoptosis assay. Our results showed that major variations in protein abundance were found among many of the ribosome proteins, indicating ribosome might be a candidate target. Furthermore, GO and KEGG pathway analyses of differentially expressed proteins (DEPs) revealed that ribosome and PI3K-Akt signalling pathway were remarkably enriched. Taken together, the above DEPs were also verified by RT-qPCR and morphological observations. This study provides insights into the key proteins enriched in PI3K-Akt signal pathway and ribosome pathway as potential targets of MG killing I. multifiliis, which could be served as targets for other less toxic drugs and be tested as potential treatments for I. multifiliis.

Efficacy and antiparasitic mechanism of 10-gingerol isolated from ginger Zingiber officinale against Ichthyophthirius multifiliis in grass carp.

Ichthyophthirius multifiliis is a ciliate parasite of freshwater fish with a global distribution and results in severe economic losses in aquaculture. The present study aimed to investigate the efficacy and antiparasitic mechanism of active compounds isolated from Zingiber officinale against I. multifiliis. Three compounds were isolated from the Z. officinale extract and identified as 10-gingerol, 6-dehydroshogaol, and 6-dehydro-10-gingerol. 10-gingerol demonstrated the greatest antiparasitic efficacy in vitro. 10-gingerol resulted in 100% mortalities of theronts, nonencysted tomonts, and encysted tomonts at concentrations of 2, 8, and 16 mg/L, respectively. 10-gingerol significantly reduced theronts infectivity (p < 0.05) at a concentration of 1 mg/L, and it was effective in treating infected grass carp and protecting naïve fish from I. multifiliis infestation at a concentration of 4 mg/L. The antiparasitic mechanism might be attributed to the increase of intracellular osmotic pressure, accumulation of free radicals, and membrane damage of I. multifiliis post 10-gingerol treatment. The study demonstrated that 10-gingerol had the potential as a therapeutic agent against I. multifiliis.

Evaluation of salinomycin isolated from Streptomyces albus JSY-2 against the ciliate, Ichthyophthirius multifiliis.

The present study was undertaken to investigate the antiparasitic activity of extracellular products of Streptomyces albus. Bioactivity-guided isolation of chloroform extracts affording a compound showing potent activity. The structure of the compound was elucidated as salinomycin (SAL) by EI-MS, 1H NMR and 13C NMR. In vitro test showed that SAL has potent anti-parasitic efficacy against theronts of Ichthyophthirius multifiliis with 10 min, 1, 2, 3 and 4 h (effective concentration) EC50 (95% confidence intervals) of 2.12 (2.22-2.02), 1.93 (1.98-1.88), 1.42 (1.47-1.37), 1.35 (1.41-1.31) and 1.11 (1.21-1.01) mg L-1. In vitro antiparasitic assays revealed that SAL could be 100% effective against I. multifiliis encysted tomonts at a concentration of 8.0 mg L-1. In vivo test demonstrated that the number of I. multifiliis trophonts on Erythroculter ilishaeformis treated with SAL was markedly lower than that of control group at 10 days after exposed to theronts (P < 0.05). In the control group, 80% mortality was observed owing to heavy I. multifiliis infection at 10 days. On the other hand, only 30.0% mortality was recorded in the group treated with 8.0 mg L-1 SAL. The median lethal dose (LD50) of SAL for E. ilishaeformis was 32.9 mg L-1.

The in vitro and in vivo effect of tannic acid on Ichthyophthirius multifiliis in zebrafish (Danio rerio) to treat ichthyophthiriasis.

The in vitro antiparasitic effect of polyphenol tannic acid (TA) on Ichthyophthirius multifiliis theronts and tomonts was evaluated. In vitro antiparasitic assays revealed that TA in a dose- and time-dependent pattern through the damage of parasite plasma membrane could be 100% effective against I. multifiliis theronts at concentrations of 8 and 11 ppm during all the exposure times (45-270 min). The tomonts proliferation was completely inhibited by penetrating TA (at least 15 ppm for 22-hr exposure) into encysted tomont across the cyst wall. However, 10 ppm TA could result in a ninefold decrease in the population of live tomonts compared to the control group (p < 0.05). Although at theront concentrations of over 6,000 per zebrafish (Danio rerio), a 100% prevalence of ichthyophthiriasis during a 5-day exposure was recorded, results of in vivo tests showed that the parasite that pretreated up to 10 ppm TA for 70 min had not any capability to infect the studied zebrafish population. The acute toxicity (96 hr-LC50 ) of TA for zebrafish was 19.51 ppm. Thus, TA can be considered as a natural therapeutant to safely and efficiently improve the health of aquatic systems by controlling ichthyophthiriasis.

Melaleuca alternifolia essential oil prevents bioenergetics dysfunction in spleen of silver catfish naturally infected with Ichthyophthirius multifiliis.

Some evidence has demonstrated that Ichthyophthirius multifiliis, etiologic agent of "white spot disease", causes severe bioenergetics dysfunction in the spleen of naturally infected silver catfish (Rhamdia quelen), which contributes directly to disease pathogenesis. Recently, several studies have demonstrated the efficacy of Melaleuca alternifolia essential oil, popularly known as tea tree oil (TTO), in the treatment of freshwater fish naturally or experimentally infected with I. multifiliis. In this sense, the aim of this study was to evaluate whether TTO is capable of preventing or reducing splenic bioenergetics dysfunction in silver catfish naturally infected with I. multifiliis. Splenic cytosolic and mitochondrial creatine kinase (CK) and pyruvate kinase (PK) activities decreased in infected animals compared to uninfected animals, while adenylate kinase (AK) activity increased. Treatment with TTO was able to prevent the inhibition on splenic CK and PK activities but was not able to prevent the stimulation of AK activity. Based on this evidence, treatment with TTO prevents the impairment on energetic metabolism via improvement of enzymes belonging to the phosphotransfer network, such as CK and PK. In summary, this treatment can be considered an interesting approach to prevent the bioenergetics imbalance in spleen of silver catfish naturally infected with I. multifiliis.

Impact of Pseudomonas H6 surfactant on all external life cycle stages of the fish parasitic ciliate Ichthyophthirius multifiliis.

A bacterial biosurfactant isolated from Pseudomonas (strain H6) has previously been shown to have a lethal effect on the oomycete Saprolegnia diclina infecting fish eggs. The present work demonstrates that the same biosurfactant has a strong in vitro antiparasitic effect on the fish pathogenic ciliate Ichthyophthirius multifiliis. Three life cycle stages (the infective theront stage, the tomont and the tomocyst containing tomites) were all susceptible to the surfactant. Theronts were the most sensitive showing 100% mortality in as low concentrations as 10 and 13 µg/ml within 30 min. Tomonts were the most resistant but were killed in concentrations of 100 µg/ml. Tomocysts, which generally are considered resistant to chemical and medical treatment, due to the surrounding protective cyst wall, were also sensitive. The surfactant, in concentrations of 10 and 13 µg/ml, penetrated the cyst wall and killed the enclosed tomites within 60 min. Rainbow trout fingerlings exposed to the biosurfactant showed no adverse immediate or late signs following several hours incubation in concentrations effective for killing the parasite. This bacterial surfactant may be further developed for application as an antiparasitic control agent in aquaculture.

Evaluation of an antiparasitic compound extracted from Polygonum cuspidatum against Ichthyophthirius multifiliis in grass carp.

Ichthyophthirius multifiliis is a ciliated parasite that infests almost all freshwater fish species and causes great economic losses to the aquaculture industry. In this study, a compound with anti-I. multifiliis activity was isolated from Polygonum cuspidatum and identified as emodin. In vitro anti-I. multifiliis results showed that emodin at 1 mg/L killed all I. multifiliis theronts for 96.0 min, and at 0.5 mg/L or lower concentrations could not kill all I. multifiliis theronts, but could significantly reduce the infectivity of theronts after pretreatment with emodin at the low concentrations mentioned above for 2 h. Additionally, emodin at 1 mg/L and 2 mg/L completely terminated the reproduction of nonencysted and encysted tomonts, respectively. In vivo tests, emodin at 0.5 mg/L could cure infected grass carp and protect naive fish from I. multifiliis infection by continuous adding emodin for 10 days. The 96 h median lethal concentration (LC50) of emodin to grass carp was 3.15 mg/L, which were approximately 18 and 7 times the median effective concentration (EC50) of emodin for killing theronts (0.18 mg/L) and nonencysted tomonts (0.45 mg/L), respectively. On the basis of these results, emodin is an effective compound for the development of a new drug against I. multifiliis.

Essential oils of Varronia curassavica accessions have different activity against white spot disease in freshwater fish.

The aim of this study was to evaluate the antiprotozoal activity of essential oils from Varronia curassavica accessions against different stages of Ichthyophthirius multifiliis. Essential oils from each accession were tested in vitro at the concentrations 0, 10, 25, 50, 75, 100, and 200 mg/L. The VCUR-001, VCUR-202, VCUR-509, and VCUR-601 accessions presented the major compounds α-pinene, germacrene D-4-ol, (E)-caryophyllene and epiglobulol, and sabinene, respectively. These isolated compounds were tested in vitro at a concentration proportional to that found in the essential oil which caused 100% mortality of the parasite. The concentrations of 10 and 50 mg/L of the essential oil of accession VCUR-202 provided 100% mortality of trophonts and tomonts, respectively. For the accession VCUR-509, 100% mortality of trophonts and tomonts was observed at concentrations 75 and 200 mg/L of essential oil, respectively. The same mortality was observed at concentration 200 mg/L in both stages of the parasite for the other accessions. The major compounds α-pinene, sabinene, and the (E)-caryophyllene + epiglobulol mixture caused 100% mortality of trophonts and tomonts. The in vivo assay for white spot disease control was performed in a therapeutic bath of 1 h with the essential oil of accession VCUR-202 at concentrations of 0.5 and 2.0 mg/L. A significant reduction of about 30% of trophonts on infected fish was observed, independent of the oil concentration. The V. curassavica essential oil, especially the VCUR-202 accession, is a potential source of raw material for the formulation and commercialization of bioproducts to control freshwater white spot disease in fish.

Using video microscopy to improve quantitative estimates of protozoal motility and cell volume.

The objective of this study was to apply digital imaging to improve quantification of rumen protozoal biomass and distinguish treatment differences in cell motility and volume among ruminal protozoa. Observations of protozoa in rumen fluid treated with essential oils (CinnaGar, CIN; Provimi North America, Brookville, OH) or an ionophore (monensin, MON) indicated possible cell shrinkage. We hypothesized that MON would decrease protozoal motility and interact with CIN on cell volume. In addition, we hypothesized that analysis of still frames from video of swimming protozoa would improve volume prediction accuracy. Flocculated rumen fluid was incubated in batch culture dosed with N-free feed only (control), MON, CIN, or a combination of MON+CIN. Samples were taken at 0, 3, or 6 h post-treatment and wet-mounted on a microscope fitted with a high-definition camera. At 3 h post-inoculation, there was a treatment interaction for average speed such that CIN attenuated the effect of MON, with treatment means of 243, 138, 211, and 183 µm/s for control, MON, CIN, and MON+CIN, respectively. At 6 h post-inoculation, MON decreased average speed by 79.2 µm/s compared with the main effect mean without MON. We measured both minimum and maximum diameters (depth and width, respectively) perpendicular to the longitudinal axis of swimming protozoa, yielding a 3-dimensional estimate of protozoal volume. The ellipsoid formula (4/3)πabc, where a = 1/2 length, b = 1/2 width, and c = 1/2 depth, was compared with previously published volume estimations using genera-specific coefficients (genera-specific coefficient × length × width2). Residuals (genera-specific coefficients - ellipsoid) were plotted against predicted (ellipsoid) and centered to the mean (Xi-x¯) to evaluate both mean and slope biases. For Entodinium spp., Y = 0.248 (±0.037) (Xi - 7.98 × 104) + 1.97 × 104 (±1.48 × 103); n = 100; r2 [coefficient of determination (squared correlation coefficient)] = 0.31, with significant slope and mean biases. For family Isotrichidae, Y = -0.124 (±0.068) (Xi - 2.54 × 106) - 1.21 × 104 (±4.86 × 104); n = 32; r2 = 0.10, where slope tended to be different from zero but with no mean bias. For Epidinium spp., Y = 0.375 (±0.056) (Xi - 2.45 × 105) + 6.65 × 104 (±0.28 × 104); n = 64; r2 = 0.43, with both mean and slope biases. The present regression analyses demonstrate that the genera-specific coefficient-based method more likely overestimates volume for Entodinium and Epidinium than for the teardrop-shaped Isotrichidae. Based on simulations derived from previous literature reporting treatments that depress protozoal populations or among-animal changes in protozoal population structures, our proposed ellipsoid method offers potential to advance the prediction of treatment effects on protozoal volume and to shift focus from the number of cells present to the diversity, function, and biomass of protozoa under various treatment conditions.

The Essential Oil of Hyptis mutabilis in Ichthyophthirius multifiliis Infection and its Effect on Hematological, Biochemical, and Immunological Parameters in Silver Catfish, Rhamdia quelen.

This study evaluated the activity of leaf essential oil (EO) from Hyptis mutabilis as well as its major constituent, (-)-globulol, in infections by the parasite Ichthyophthirius multifiliis (ich). Effects on hematological, biochemical, and immunological parameters of silver catfish, Rhamdia quelen, exposed to the same samples also were evaluated. In the first experiment, naturally infected fish were treated with EO (0, 10, and 20 mg · L-1) and ethanol, using several methods of exposure. Fish mortality and the number of trophonts per fish were assessed after 48 and 96 hr. Hour-long daily baths resulted in optimal survival, so this methodology was used for the second experiment, in which infected animals were exposed to (-)-globulol at 2.5 and 5 mg · L-1. The most effective concentrations in Experiments 1 and 2 were chosen for Experiment 3, in which healthy animals were subjected to hour-long daily baths with EO (20 mg · L-1) or (-)-globulol (2.5 mg · L-1). Additionally, an in vitro experiment was performed with EO and globulol at the same concentrations of the in vivo test. EO and (-)-globulol increased the survival of fish infected with ich and altered certain hematological and biochemical parameters. After 4 days, levels of hematocrit, erythrocytes, and leukocytes increased significantly in healthy animals exposed to EO. Exposure to (-)-globulol increased leukocyte number alone. No significant differences in nonspecific immunological parameters were detected when treated groups were compared to controls, but the leukocytosis observed in EO- and globulol-treated healthy animals indicates that EO and (-)-globulol increased innate immunity in these fish. An in vitro antiparasitic effect was observed in both samples.

Combined effects of Chinese medicine feed and ginger extract bath on co-infection of Ichthyophthirius multifiliis and Dactylogyrus ctenopharyngodonid in grass carp.

Dactylogyrus ctenopharyngodonid and Ichthyophthirius multifiliis are two important ectoparasites of freshwater fish. Co-infection by the two parasites leads to high fish mortality and results in heavy economic losses. This study aimed to evaluate the efficacy of medicated feed and a ginger extract bath against D. ctenopharyngodonid and I. multifiliis on grass carp and investigate the hematological response of grass carp co-infected by the two parasites. These results demonstrated that red blood cell (RBC) and thrombocyte percentage among leucocytes significantly decreased after grass carp were co-infected by D. ctenopharyngodonid and I. multifiliis. The monocyte and neutrophil percentages significantly increased with the increment of parasite mean intensities, while the lymphocyte percentage decreased. The activities of serum acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM), and superoxide dismutase (SOD) significantly increased after co-infection. When grass carp treated with medicated feed containing 4% of Astragalus membranaceus, Allium sativum, Morus alba, and Glycyrrhiza uralensis, the activities of ACP, AKP, LZM, and SOD were significantly enhanced, and the mean intensities of D. ctenopharyngodonid and I. multifiliis were significantly decreased. When grass carp was treated with medicated feed and a 4-mg/L ginger extract bath, all parasites were eliminated during 28 days. The bath of ginger extract at a concentration of 4 mg/L kept a low mean intensity of I. multifiliis and D. ctenopharyngodonid, then the two parasites were eliminated by oral administration of the medicated feed with an immunostimulant (Chinese medicine compound).

Antiparasitic efficacy of curcumin from Curcuma longa against Ichthyophthirius multifiliis in grass carp.

Ichthyophthirius multifiliis is a ciliated parasite that elicits great economic losses in aquaculture. In the present study, a polyphenol compound, curcumin, was obtained from the rhizome of Curcuma longa by bioassay-guided isolation based on the efficacy of anti-I. multifiliis theronts. Anti-I. multifiliis efficacy of curcumin was evaluated in vitro and in vivo. Curcumin resulted in 100% mortality of I. multifiliis theronts at a concentration of 1mg/L within 21.7±1.2min and killed all tomonts at 8mg/L within 31.0±1.0min. Curcumin at 4mg/L for 16h exposure can completely terminate the reproduction of tomonts. The pretreatment with curcumin at concentrations of 0.5, 0.25, and 0.125mg/L for 2h significantly reduced the infectivity of I. multifiliis theronts. Curcumin at 4mg/L completely cured the infected grass carp and protected naive fish from I. multifiliis infection after 10days exposure. The 4h median effective concentration (EC50) of curcumin to I. multifiliis theronts and the 5h EC50 of curcumin to I. multifiliis tomonts were 0.303mg/L and 2.891mg/L, respectively. The 96h median lethal concentration (LC50) of curcumin to grass carp was 56.8mg/L, which was approximately 187.4 times EC50 of curcumin to theronts and 19.6 times EC50 of curcumin to tomonts. The results demonstrated that curcumin has the potential to be a safe and effective therapeutant for controlling ichthyophthiriasis in aquaculture.

Evaluation of medicated feeds with antiparasitical and immune-enhanced Chinese herbal medicines against Ichthyophthirius multifiliis in grass carp (Ctenopharyngodon idellus).

Since malachite green was banned for using in food fish due to its carcinogenic and teratogenic effects on human, the search of alternative drug to treat Ichthyophthirius multifiliis becomes urgent. This study aimed to (1) evaluate the ethanol extracts of medicinal plants Cynanchum atratum, Zingiber officinale, Cynanchum paniculatum, immunostimulant (A), and immunostimulant (B) for their efficacy against I. multifiliis, and (2) determine effects of medicated feeds with C. atratum, Z. officinale, C. paniculatum, and immunostimulant (A) to treat I. multifiliis in grass carp. The results in this study showed that the minimum concentrations of C. atratum, Z. officinale, and C. paniculatum extracts for killing all theronts were 16, 8, and 16 mg/L, respectively. In vivo experiments, fish fed with medicated feeds of C. atratum for 10 days, or Z. officinale for 3 days, or combination of three plants for 10 days resulted in a significant reduction in the I. multifiliis infective intensity on grass carp after theronts exposure. Grass carp fed with medicated feeds of immunostimulant (A) for 21 days showed no infection and 100 % of survival 15 days post theronts exposure. Therefore, immunostimulant (A) is a promising feed supplement to treated I. multifiliis with good antiparasitic efficacy.

Anti-parasitic effects of Leptomycin B isolated from Streptomyces sp. CJK17 on marine fish ciliate Cryptocaryon irritans.

The present study was conducted aiming to evaluate the in vitro and in vivo anti-parasitic efficacy of an isolated compound against the ciliate Cryptocaryon irritans. The compound was previously isolated from fermentation products of Streptomyces sp. CJK17 and designated as SFrD. Toxicity of the compound SFrD against the fish hosts (Larimichthys crocea) was also tested and its chemical structure was elucidated. The obtained results showed that the compound has potent anti-parasitic efficacy with the 10 min-, 1 h-, 2 h-, 3 h- and 4 h-LC50 (95% Confidence Intervals) of 6.8 (6.5-7.1), 3.9 (2.8-5.0), 3.3 (2.6-4.0), 2.7 (2.3-3.1) and 2.5 (2.2-2.8) mg L(-1) against theronts of C. irritans and the 6h-LC50 (95% CI) of 3.0 (2.8-3.2) mg L(-1) against the tomonts, respectively. Exposure of the compound SFrD remarkably reduced the mortality of fish infected with C. Irritans, from 100% in the control group to 61.7% and 38.3% in groups of 3.1 mg L(-1) and 6.3 mg L(-1), respectively. In the test of exposing fish to 40 mg L(-1) compound SFrD for 24h, no visible effects were observed affecting the normal behavior or any macroscopic changes. By spectrum analysis (EI-MS, (1)H NMR and (13)C NMR), the compound SFrD was identified as Leptomycin B. This study firstly demonstrated that Leptomycin B has potent anti-parasitic efficacy against ciliates in cultured marine fish.

Treatment of ichthyophthiriasis with photodynamically active chlorophyllin.

Water-soluble chlorophyll (chlorophyllin) exerts pronounced photodynamic activity on fish parasites. In order to determine its potential as a remedy against ectoparasites in fish carps were incubated in water with defined concentrations of chlorophyllin. The main focus of the experiments was on the ciliate Ichthyophthirius multifiliis (Fouquet) which is responsible for considerable losses in livestock in aquaculture. As malachite green, which in the past efficiently cured infected fishes, is banned because of its possible carcinogenicity; no effective remedy is presently available in aquaculture to treat ichthyophthiriasis. Using chlorophyllin, the number of trophonts was significantly reduced (more than 50 %) after 3 h incubation of infested fish at 2 and 4 mg/L and subsequent irradiation with simulated solar radiation. The lack of reinfection after light treatment indicates that also the remaining parasites have lost their multiplication capacity. In the controls (no chlorophyllin and no light, light but no chlorophyllin, or chlorophyllin but no light), no reduction of the I. multifiliis infection was observed. We propose that chlorophyllin (or other photodynamic substances) is a possible effective countermeasure against I. multifiliis and other ectoparasites in aquaculture.

In vitro and in vivo assessment of the effect of antiprotozoal compounds isolated from Psoralea corylifolia against Ichthyophthirius multifiliis in fish.

Ichthyophthirius multifiliis, an external fish parasite, often causes significant economic damage to the aquaculture industry. Since the use of malachite green was banned, the search of alternative substance to control I. multifiliis infections becomes stringent. In present study, in vitro and in vivo anti-ich efficacies of isopsoralen and psoralidin, two active compounds isolated from methanol extract of Psoralea corylifolia by bioassay-guided fractionation based on the efficacy of anti-ich encysted tomonts, were evaluated. In vitro antiprotozoal efficacy of psoralidin is much better than that of isopsoralen. Psoralidin can kill all theronts at concentrations of 0.8 mg/L or more during 4 h exposure; and terminate reproduction of I. multifiliis post 6 h exposure of protomonts to 0.9 mg/L and encysted tomonts to 1.2 mg/L. In vivo trials showed that 5 h exposure of infected fish to 2.5 mg/L of psoralidin significantly reduced the number of theronts released from tomonts. Furthermore, we observed that a part of protomonts, collected from infected fish post treatment, presented characteristic morphological changes of apoptosis after staining with Annexin V-EGFP/propidium iodide, indicating the possible mechanism of psoralidin against I. multifiliis trophont in situ. On the basis of these results, psoralidin can be used as a potential lead compound for the development of commercial drug against I. multifiliis.