Multifiliis Theronts Research Articles

Single-cell transcriptome profiles and E−64 inhibitor data reveal the essential role of cysteine proteases in the ontogeny of Ichthyophthirius multifiliis

Ichthyophthiriasis (Ich), also known as white spot disease, causes significant economic losses to fish farmers once an outbreak occurs. For fish survival, it is therefore crucial to understand the pathogenic mechanism and find effective prevention methods. In this study, we obtained data for four stages (theront, trophont, protomont and tomont) of Ichthyophthirius multifiliis by single-cell RNA sequencing (scRNA-seq). We found that the invasion-related proteins encoded by highly expressed genes in the theront stage mainly belong to the leishmanolysin family proteins, heat shock proteins, transmembrane proteins and cysteine proteases (CPs). Additionally, the exosome pathway appears to play a significant role in the invasion process of the theront. Since cysteine proteases are expressed at all stages of the I. multifiliis, and five CP-related genes were significantly upregulated at the theront stage of its life cycle — two of which are enriched in the exosome pathway — we incubated I. multifiliis theronts and protomonts with cysteine protease inhibitor (E−64). Our findings revealed that E−64 could kill both stages of the parasite in vitro and affected tomont division and subsequent release. Furthermore, infection experiment showed that E−64 could significantly inhibit the invasion of theronts. Based on our preliminary analysis from the transcriptomic and E−64 experiments, we have confirmed that CPs play a crucial role in I. multifiliis. This research establishes a foundation for future strategies in the prevention and control of Ich.

Can Chilodonella uncinata induce cross-protection in koi carp (Cyprinus carpio) against Ichthyophthirius multifiliis? Evidence from immune response and challenge experiments

The intraperitoneal injection of koi carp with inactivated Ichthyophthirius multifiliis or Chilodonella uncinata combined with Freund's adjuvant was evaluated for its capacity to protect the fish from I. multifiliis infection. Our results showed that the level of Ig M in serum significantly increased after vaccination, reaching up to 4.09 μg/mL on day 14 in fish vaccinated with C. uncinata, while C3 content also generally increased. In addition, genes related to immunity, including Ig M, IL-1β, TNF-α, TLR 9, TLR 20 and TLR 21, exhibited differential expression, which suggests that the koi carp did indeed produce immune responses after vaccination. In challenge experiments, each fish specimen was challenged with 30,000 theronts on day 14 post vaccination. The relative survival rate of fish vaccinated with 10,000 I. multifiliis theronts was 93.3%, while the survival rate of fish vaccinated with 10,000C. uncinata or with phosphate buffer saline (the latter being the control) was 0% in both cases. However, as the dose of C. uncinata was increased, the survival rate of the fish also increased, with fish vaccinated with 60,000C. uncinata cells achieving a higher survival rate than those vaccinated with 30,000 cells. Above all, we demonstrated that Ig M does not play a major role in protection against ichthyophthiriasis and the protection conferred by inactivated C. uncinata vaccination is related to the dose.

In Vitro Assessment of Berberine against Ichthyophthirius multifiliis in Goldfish.

Ichthyophthirius multifiliis is a pathogenic ciliate parasite, which infects almost all freshwater teleost fish and leads to significant economic losses. The present study aimed to evaluate the acute toxicity of berberine to the free-living stages of I. multifiliis, that is, theronts and tomonts. Our results indicated that 99.30% of I. multifiliis theronts were killed by a concentration of 15 mg/L berberine during the 4 h exposure time, while berberine had no effect on protomonts. Nevertheless, berberine at a concentration of 5 mg/L could effectively reduce the release of theronts from tomonts treated for 4 h. Additionally, according to the transmission electron microscopy results, berberine at 15 mg/L could strongly change the shape of protomonts, destroy their organelles, and significantly decrease the number of ribosomes. The median lethal concentration (LC50) of berberine for goldfish at 96 h was 528.44 mg/L, which was almost 67 times the median effective concentration (EC50) of berberine for killing theronts (7.86 mg/L). The results demonstrated that berberine could be an effective and safe potential parasiticide for killing I. multifiliis.

Parasiticidal effects of Eclipta alba and Arctium lappa extracts against Ichthyophthirius multifiliis.

Ichthyophthiriasis, commonly known as white spot disease, occurs in both wild and cultured fish and is responsible for heavy economic losses to the aquaculture industry. In past decade, several chemical therapeutants were used to treat ichthyophthiriasis, but the effective drugs, such as malachite green, have been banned for use in food fish due to its genotoxic and carcinogenic properties. To find efficacious drugs to control Ichthyophthirius multifiliis (Ich), whole Eclipta alba plants and dried root of Arctium lappa were evaluated for their antiprotozoal activity. E. alba and A. lappa extracts significantly reduced the survival of Ich trophonts and theronts. In vitro, the E. alba and A. lappa methanol extracts killed all trophonts at 3200 mg l-1. All trophonts were killed after exposure to E. alba aqueous extract at 3200 mg l-1. The methanol extracts of E. alba and A. lappa killed 100% of I. multifiliis theronts at 400 mg l-1 and 800 mg l-1, respectively. The aqueous extract of E. alba and A. lappa killed 100% of I. mulitifiliis theronts at 1600 mg l-1 and 3200 mg l-1, respectively. E. alba and A. lappa extracts may be new and efficacious drugs for the control of ichthyophthiriasis.

Double trouble: could Ichthyophthirius multifiliis be a vehicle for the bacterium associated with red mark syndrome in rainbow trout, Oncorhynchus mykiss?

Red mark syndrome (RMS) is a disease of farmed rainbow trout, Oncorhynchus mykiss (Walbaum, 1792), one of the most widespread freshwater farmed species in Europe. The disease emerges at water temperatures below 16 °C and consists of one or more bright red skin lesions on the fish body. Mortality due to RMS is reportedly rare, but the disease leads to downgrading of the product and subsequent economic losses. Despite the disease impact, the causative agent for RMS is still formally undetermined although increasing evidence points to a bacterium ascribed to the Midichloriaceae family (order Rickettsiales), hereafter referred to as RMS-Midichloria like organism (RMS-MLO).Intriguingly, recently deposited sequences revealed the presence of RMS-MLO-like bacteria associated with Ichthyophthirius multifiliis, a common protozoan skin parasite of freshwater fish frequently harboring bacterial endosymbionts. Therefore, we hypothesized that I. multifiliis could be a vehicle for RMS-MLO. This was tested by infecting RMS-diseased rainbow trout with I. multifiliis theronts, and subsequently investigating the presence of RMS-MLO in tomonts detached from the fish.Real time PCR analyses showed clearly that I. multifiliis previously exposed to RMS-affected fish become positive to RMS-MLO suggesting that this bacterium can be at least transiently acquired and carried by the protozoan. Moreover, statistical analyses suggested a possible level of vertical transmission in I. multifiliis from one trophic stage to the next one. Further studies will be necessary to prove whether I. multifiliis has a role in the horizontal transfer of RMS-MLO bacteria from diseased RMS fish to healthy ones.

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

The invitro 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 11ppm during all the exposure times (45-270min). The tomonts proliferation was completely inhibited by penetrating TA (at least 15ppm for 22-hr exposure) into encysted tomont across the cyst wall. However, 10ppm 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 invivo tests showed that the parasite that pretreated up to 10ppm TA for 70min had not any capability to infect the studied zebrafish population. The acute toxicity (96hr-LC50 ) of TA for zebrafish was 19.51ppm. Thus, TA can be considered as a natural therapeutant to safely and efficiently improve the health of aquatic systems by controlling ichthyophthiriasis.

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.

Antiparasitic efficacy and safety assessment of magnolol against Ichthyophthirius multifiliis in goldfish

Ichthyophthirius multifiliis causes high mortality rate of freshwater fish. It is necessary to find the effective alternatives that may both be affordable and environmentally acceptable since malachite green is banned to use. In this work, magnolol exhibited great anti-parasitic activity against all stages of I. multifiliis. Magnolol at concentrations of 0.6mg/L or more resulted in 100% mortality of I. multifiliis theronts within 4h exposure; and terminated reproduction of I. multifiliis post 6h exposure of protomonts and encysted tomonts to 0.8 and 1.0mg/L, respectively. Moreover, some of tomonts presented morphological characteristic changes and a larger part of tomonts were unable to reproduce. Magnolol possibly had a directly effect on the cell membrane, and more importantly, it could permeate into an encysted tomont across the cyst wall and then impact on the tomont proliferation. In vivo trials showed that 5h exposure of infected fish to 1.5mg/L of magnolol significantly reduced the number of theronts released from tomonts. In addition to determination of anti-Ichthyophthirius activity, we also systematically evaluated the safety of magnolol against fish. First of all, the results of acute toxicity experiment showed that the 96h median lethal concentration (LC50) of magnolol to goldfish was 6.02mg/L. Furthermore, gene expression (cyp1a, hsp70, gst and sod) and enzyme activity (GSH, SOD and T-AOC) in liver, spleen and kidney could recover to the same level as the control group up to 48h, though magnolol induced goldfish stress response during 6–24h exposure. More than that, magnolol can be easily synthesized with low cost, and further embellished and modified for higher bioactivity and lower toxicity because of its simple and symmetry chemical structure. Therefore, magnolol has the potential to be a safe and effective therapeutant, and it also can be used as a probable leading compound for the development of commercial drug to control ichthyophthiriasis in aquaculture.

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.

Identification and effect of two flavonoids from root bark of Morus alba against Ichthyophthirius multifiliis in grass carp.

Ichthyophthirius multifiliis (Ich) is an important ciliate that parasitizes gills and skin of freshwater fish and causes massive fish mortality. In this study, two flavonoids (kuwanons G and O) with anti-Ich activity were isolated by bioassay-guided fractionation from the root bark of Morus alba, an important plant for sericulture. The chemical structures of kuwanons G and O were elucidated by spectroscopic analyses. Kuwanons G and O caused 100% mortality of I. multifiliis theronts at the concentration of 2 mg/L and possessed a median effective concentration (EC50) of 0.8 ± 0.04 mg/L against the theronts. In addition, kuwanons G and O significantly reduced the infectivity of I. multifiliis theronts at concentrations of 0.125, 0.25, 0.5, and 1 mg/L. The median lethal concentrations (LC50) of kuwanons G and O to grass carp were 38.0 ± 0.82 and 26.9 ± 0.51 mg/L, which were approximately 50 and 35 times the EC50 for killing theronts. The results indicate that kuwanons G and O have the potential to become safe and effective drugs to control ichthyophthiriasis.

Evaluation of nystatin isolated from Streptomyces griseus SDX-4 against the ciliate, Ichthyophthirius multifiliis.

The present study was conducted to evaluate the in vitro and in vivo antiparasitic efficacy of active compounds from the bacterial extracellular products of Streptomyces griseus SDX-4 against Ichthyophthirius multifiliis. Bioassay-guided fractionation and isolation of compounds with antiparasitic activity were performed on n-butanol extract of S. griseus yielding a pure bioactive compound, nystatin (Nys), identified by comparing spectral data (EI-MS, (1)H NMR, and (13)C NMR) with literature values. Results from in vitro antiparasitic assays revealed that Nys could be 100% effective against I. multifiliis theronts and encysted tomonts at the concentration of 6.0 mg L(-1), with the median effective concentration (EC50) values of 3.1 and 2.8 mg L(-1) for theronts and encysted tomonts (4 h), respectively. Results of in vivo test demonstrated that the number of I. multifiliis trophonts on the gold fish treated with Nys was markedly lower than the control group at 10 days after exposed to theronts (p < 0.05). In the control group, 85.7% mortality was observed owing to heavy I. multifiliis infection at 10 days after the exposure. On the other hand, only 23.8% mortality owing to parasite infection was recorded in the groups treated with the Nys (4.0 and 6.0 mg L(-1)). In addition, our results showed that the survival and reproduction of I. multifiliis tomont exited from the fish were significantly reduced after treated with the 6.0 mg L(-1) Nys. The median lethal dose (LD50) of Nys for goldfish was 16.8 mg L(-1). This study firstly demonstrated that Nys has potent antiparasitic efficacy against I. multifiliis, and it can be a good candidate drug for chemotherapy and control of I. multifiliis infections.

Antiprotozoal screening of traditional medicinal plants: evaluation of crude extract of Psoralea corylifolia against Ichthyophthirius multifiliis in goldfish

Ichthyophthirius multifiliis (also called "ich") is an external protozoan parasite that may infest almost all freshwater fish species and caused significant economic damage to the aquaculture industry. Since the use of malachite green was banned, there have been relatively few effective alternative strategies for controlling I. multifiliis infections. The present study was designed to screen potential antiparasitic medicinal plants based on our previous studies, and comprehensively evaluate in vitro and in vivo anti-ich activity of selected plant extracts. The screening results showed that the methanol extract of Psoralea corylifolia had the highest activity against I. multifiliis theronts. In vivo theront trials demonstrated that 1.25mg/L or more concentrations of P. corylifolia methanol extract caused 100% mortality during the 4-h exposure period, and the subsequent in vitro trials indicated that the minimum concentration of P. corylifolia methanol extract that prevented the initial infestation was 2.50mg/L. Protomonts and encysted tomonts surviving trials suggested that encysted tomonts were less susceptible to P. corylifolia methanol extract than protomonts, and the methanol extract of P. corylifolia at a concentration of 5.00mg/L could kill 100% of protomonts and 88.89% of encysted tomonts. It was also observed that after 12-h exposure of protomonts or encysted tomonts to 2.50mg/L of P. corylifolia methanol extract, the theronts emerged from encysted tomonts led to more infection level than the ones in the other groups. The results suggested that whether the protomonts finish encystment is crucial to the survival, reproduction, and theronts infectivity. In addition, our results showed that long duration (24h) and high concentration (5.00mg/L) significantly reduced the survival and reproduction of I. multifiliis tomont exited from the fish after in-bath treatment, and it is indicated that P. corylifolia methanol extract had a potential detrimental effect on I. multifiliis trophont in situ.

Effects of aqueous extract of Capsicum frutescens (Solanaceae) against the fish ectoparasite Ichthyophthirius multifiliis

Ichthyophthirius multifiliis is an important fish ectoparasite that often results in significant economic losses to freshwater aquaculture. The search of alternative substances to control infections of I. multifiliis became stringent after malachite green, an effective and widely used chemotherapeutant, is banned on fish farms because of its carcinogenicity and teratogenicity. In this study, the effects of the aqueous extract of Capsicum frutescens, which is readily available and affordable, were evaluated under in vitro and in vivo conditions. The results in the in vitro conditions showed that the aqueous extracts of C. frutescens with the ratios (V (SS)/V (T)-V (SS), the volume of stock solution; V (T), the volume of total solution) of 1:32 and 1:64 led to more than 70 % mortality of I. multifiliis theronts during 4 h of exposure and significantly reduced the survival of the tomonts and the total number of theronts released by the tomonts within 22 h (P < 0.05). A 96-h bioassay was carried out to determine the acute toxicity of the aqueous extract of C. frutescens to goldfish. No visible effect was observed in the treatments with the aqueous extracts of C. frutescens with the ratios (V (SS)/V (T)) of 1:32, 1:64 and 1:128, while in the other treatments, the erratic behaviour of fish was noted. In addition, in vitro tests demonstrated that the aqueous extract of C. frutescens had an adverse effect on I. multifiliis trophonts in situ. Fish treated with the aqueous extracts of C. frutescens in ratios V (SS)/V (T) of 1:32 and 1:64 carried significantly fewer parasites than the control and the other treatments (P < 0.05). These results suggest, therefore, that aqueous extracts of C. frutescens have potential for the control of ichthyophthiriasis in the aquaculture industry, though further phytochemical studies will need to be performed for isolation and identification of the active compounds.

Temperature effects on immune response and hematological parameters of channel catfish Ictalurus punctatus vaccinated with live theronts of Ichthyophthirius multifiliis

This study evaluated the influence of temperature on the immune responses and hematological parameters in channel catfish Ictalurus punctatus immunized via intraperitoneal injection with live theronts of Ichthyophthirius multifiliis. Fish were distributed in 18 aquaria and received 9 treatments: 4 groups of fish were vaccinated with live theronts and maintained at constant temperature 15 °C, 20 °C, 25 °C and 30 °C; 3 groups of fish vaccinated and subjected to cycling temperature regime from 15–25 °C, 20–25 °C and 20–30 °C, changed 5 °C each day; 2 groups of fish were not vaccinated and served as controls at 25 °C, one with Ich challenge and the other without challenge. Non vaccinated fish and those vaccinated at 15 °C or 15–25 °C did not show anti-Ich antibodies in the serum 14 and 21 days post-immunization. The antibody levels were significantly higher from fish vaccinated at 25 °C, 30 °C, 20–25 °C and 20–30 °C compared to fish at 15 °C, 20 °C and 15–25 °C both 14 and 21 days post-immunization. At constant water temperature, fish vaccinated at 15 °C showed significantly higher mortality rate (67.8%, P < 0.05) than those vaccinated at 20 °C, 25 °C, and 30 °C (0–10.7% mortalities). At cycling water temperature, fish vaccinated at 15–25 °C showed significantly higher mortality rate (67.8%) than those vaccinated at 20–25 °C and 20–30 °C ( P < 0.05). Twenty days after immunization fish vaccinated at 30 °C and 20–30 °C showed significant increase in the red blood cells, white blood cells, thrombocytes and monocytes. Six days after challenge with I. multifiliis theronts the fish showed decreased white blood cells, thrombocytes and monocytes. This study suggests that vaccinated catfish were severely impacted by low temperature, either at 15 °C constant temperature or at 15–25 °C cycling temperature. The fish showed no anti-Ich antibodies and suffered high mortality similar to non vaccinated control fish.

Effect of potassium ferrate(VI) on survival and reproduction of Ichthyophthirius multifiliis tomonts

Ichthyophthirius multifiliis is an important freshwater teleost pathogen that can infect most species of freshwater fish worldwide and often leads to significant economic losses to the aquaculture industry. Potassium ferrate(VI), as a potential therapeutic agent for external protozoan parasite infections, has been proven to kill I. multifiliis theronts effectively; however, no information is available on effects of potassium ferrate(VI) exposure to I. multifiliis tomonts. This study evaluated the effects of potassium ferrate(VI) on the survival and reproduction of I. multifiliis tomonts. The results of experiment 1 showed that potassium ferrate(VI) at concentrations of 2.4, 4.8, 9.6, and 19.2 mg/L resulted in tomont survival rates of 82.2%, 34.2%, 14.6%, and 0, respectively, and significant differences were noted in tomont reproduction between the treatments and the control (P < 0.05). Additionally, this study was designed to determine the effect of potassium ferrate(VI) toxicity on age of the tomont. The results indicated that encysted I. multifiliis was more resistant to potassium ferrate(VI) treatments. In addition, this study was designed to investigate I. multifiliis tomont survival and reproduction when collected from infested goldfish in potassium ferrate(VI) solutions at different concentrations (0, 2.4, 4.8, 9.6, and 19.2 mg/L) for 30 min. It was found that the tomont survivals in the treatments were significantly lower than those in the control (P < 0.05). This study demonstrated that a bath with potassium ferrate(VI) possibly was an effective method to treat ichthyophthiriasis.

Cellular and humoral factors involved in the response of rainbow trout gills to Ichthyophthirius multifiliis infections: Molecular and immunohistochemical studies

The parasitic ciliate Ichthyophthirius multifiliis infecting skin, fins and gills of fish induces a protective immune response in rainbow trout ( Oncorhynchus mykiss) surviving the infection and a similar protection can be conferred by i.p. injection of live theronts. A combined molecular and immunohistochemical approach has been used in this work for pinpointing cellular and humoral immune factors in gill tissue involved in the response and indicating interactions between the systemic and local responses. Fish were immunized by intra-peritoneal injection of live I. multifiliis theronts, control fish were injected with PBS and subgroups were treated with the immuno-suppressant hydrocortisone before fish were challenged with live theronts. Significant up-regulations of genes encoding IgM, IgT, C3, SAA, IL-8, IL-22 and IFN-γ were induced by immunization and challenge. Hydrocortisone treatment had a significant down-regulating effect on genes incoding IgT, IgM, CD4, CD8, IFN-γ, IL-8 and IL-22 in all groups. Immunohistochemistry, using monoclonal antibodies to detect cellular markers, demonstrated active involvement of CD8, MHC II, IgT and IgM positive cells in gill tissue. Putative T-cells (CD8 positive cells) were detected in the intraepithelial lymphoid tissue located at the base of gill filaments and in hyperplastic gill tissue but following infection a clear efflux of these cells was detected. MHC II positive cells were distributed across the gill filaments and accumulated in hyperplastic tissue but hydrocortisone treatment affected their density negatively in both immunized and non-immunized fish. IgT positive cells were present in the epithelial lining of the gill lamellae (suggesting a primary role of this protein in the mucosal defence against the ciliate) whereas IgM positive cells were found only in gill arterioles and the lamellar capillaries. The present work indicates an intensive activity and specialized function of immune cells (B-cells, T-cells and macrophages) and humoral elements such as immunoglobulins IgT and IgM which are orchestrated by cytokines in gill tissue reacting against I. multifiliis.

Effects of Short- and Long-Term Glucan Feeding of Rainbow Trout (Salmonidae) on the Susceptibility to &lt;I&gt;Ichthyophthirius Multifiliis&lt;/I&gt; Infections

Background. Various compounds are being used as immuno-stimulants in various aquaculture feed types but the importance of their prolonged use is at present unknown. It is also true for those intended to modify trout susceptibility to white spot disease caused by Ichthyophthirius multifiliis. The immuno-modulating effects on rainbow trout of short- and long-term feeding using dry feed with inclusion of beta-glucan (0.2%) have therefore been evaluated. Materials and methods. The immune reactivity was assessed by measuring susceptibility to I.multifiliisinfections and plasma lysozyme in the host, factors which are considered innate response parameters. Rainbow trout, Oncorhynchus mykiss(Walbaum, 1792) (Actinopterygii: Salmoniformes: Salmonidae),were fed (control feed or glucan-containing feed) for 14 or 35 days before challenge (exposure to infective I. multifiliis theronts) and the resulting infection (number of trophonts in the skin) was recorded on day 11 post-exposure to theronts. Results. All fish obtained infection following parasite exposure but glucan-fed fish showed a lower infection at day 46 compared to fish fed control-feed. The lysozyme activity of fish plasma was evaluated throughout the experimental period and found slightly but significantly increased in association with glucan-feeding at day 14, 17, and 46. Conclusion. No evidence for an exhaustion of these selected innate response parameters due to prolonged glucan-feeding could be detected within the experimental period of 46 days.

Prevention of Ichthyophthirius multifiliis infestation in goldfish ( Carassius auratus) by potassium ferrate(VI) treatment

Ichthyophthirius multifiliis is an important freshwater teleost pathogen that often leads to significant economic losses to the aquaculture industry. The purpose of this study was to assess the acute toxicity of potassium ferrate(VI) to I. multifiliis theront and the concentration needed to prevent I. multifiliis infestation in goldfish, Carassius auratus. Five hundred theronts were exposed to concentrations of potassium ferrate(VI) in each well of a 96-well microtiter plate and observed for 4 h to determine the acute toxicity. Results showed that the exposure of I. multifiliis theronts to potassium ferrate(VI) at concentrations of 4.80 mg/L or more resulted in 100% mortality by 4 h; the LC 50 value was estimated to be 1.71 mg/L. Aqueous static renewal 96-h bioassays were carried out to determine the acute toxicity of potassium ferrate(VI) to goldfish. The LC 50 value for potassium ferrate(VI) in goldfish was 42.51 mg/L. Goldfish were exposed to 4000 theronts/fish in aerated tap water (a dose previously shown to result in consistent infestation) and treated with a single dose of potassium ferrate(VI) after 30 min contact with theronts. Infection level and prevalence were recorded everyday after exposure. The results revealed that potassium ferrate(VI) at the 4.80 mg/L or more concentrations can significantly reduce not only the number of trophonts on the fin of goldfish on day 3 ( P < 0.05), but also the prevalence of ichthyophthiriasis ( P < 0.05). Potassium ferrate(VI) at a concentration of 4.80 mg/L was considered to be the lowest effective dose to prevent infestation of I. multifiliis in goldfish.

Evaluation of continuous 4-day exposure to peracetic acid as a treatment for Ichthyophthirius multifiliis

The parasitic ciliate Ichthyophthirius multifiliis infests all species of freshwater fish and can cause severe economic losses in fish breeding. The most effective treatment, malachite green, has been banned in Europe and North America for use in food fish production. Peracetic acid (PAA) was found to be toxic to I. multifiliis theronts at low concentrations. I. multifiliis-infested carp were exposed to 1 mg/l PAA in a dynamic exposure by means of peristaltic pumps. Five days after infestation, gills, tail fins, and skin below the dorsal fin were observed microscopically for I. multifiliis abundance. After PAA exposure, PAA-treated fish showed lower infestation of I. multifiliis in all investigated tissues than the unexposed control fish. The infestation increased in the control group whereas the infestation in the PAA-exposed groups significantly decreased (p = 0.0083, Bonferroni correction). The fish in the two exposure groups showed a slight reinfestation with I. multifiliis. This might be caused by a peroxide degradation (hydrolysis) and/or reduction of the delivered PAA concentration. Thus, PAA concentrations were possibly too low to be effective on the released trophonts and/or the infective theronts. This hypothesis is corroborated by the fact that the I. multifiliis in the gills, skin, and fins of the PAA-exposed carp were in an early developmental stage.

Acute toxicity of peracetic acid (PAA) formulations to Ichthyophthirius multifiliis theronts

Peracetic acid (PAA) is an antimicrobial disinfectant used in agriculture, food processing, and medical facilities. It has recently been suggested as a means to control infestations of Ichthyophthirius multifiliis. The purpose of this study was to determine the acute toxicity of two products containing 4.5% and 40% PAA to I. multifiliis theronts from two geographically separate isolates. Theronts were exposed to concentrations of PAA in 96-well plates containing groundwater at 23 degrees C. Acute toxicity was observed over a 4-h period. No significant difference in the median lethal concentration (LC(50)) estimates was evident between the two isolates at 4 h with the 4.5% PAA product (0.146 versus 0.108 mg/l PAA), while there was a statistical difference between the 4 h LC(50) with the 40% PAA product (0.274 versus 0.158 mg/l PAA). These results suggest that PAA is toxic to I. multifiliis theronts at low concentrations and that one of the isolates was more resistant to this compound.