Application of peracetic acid for the treatment of juvenile sander (Sander lucioperca) during Ichthyophthirius multifiliis infestation

White spot disease caused by the ciliate protozoan parasite Ichthyophthirius multifiliis (I. m.), is one of the most dangerous diseases in aquaculture and ornamental fish breeding worldwide. The parasite is characterized by three developmental stages: a reproductive tomont, an infective theront and a parasitic trophont. In sander (Sander lucioperca) breeding I. m. causes serious economic losses. After banning of the traditional therapeutic agent malachite green we have to face a state of emergency for the treatment of the ichthyophthiriasis in Germany. The peracetic acid (PAA), characterized by positive therapeutical properties, might close this gap. The purpose of our investigations was the determination of the toxicity of PES to juvenile sander as well as the evaluation of the therapeutic effectiveness of the substance to combat I. m. For juvenile sander (length 3 cm) we determined a 24-h-LC50 of 1.14 (0.97; 1.3) ppm PES. In two investigations PAA was applied in daily intervals of 0.5; 1, 3, 5 and 24 h and concentrations of 0.5; 1; 1.5 and 2 ppm to treat I.-m.-infection in sander. In test I all sander (length 9 cm) died as a result of the I.-m.-infection. However, the PAA exposed fish survived longer than the PES-free controls! This might be due to a disinfection of other pathogens by PAA. In test II, the fish (length 12 cm) were less infected than fish in test I. Four of six fish died in the group exposed with 2 ppm PAA for 24 hours. The abundance of I.-m.-trophonts was determined in mucus, fin and gill tissues of all fish. Significant differences could not be observed between test I and test II because of dissimilar: 1. exposure in time and concentration, 2. age and condition of the fish and 3. homogenity of the variances. Therefore, no therapy strategy was successful while fighting the parasitic trophonts protected by the overlaying fish tissues. We speculate that a successful therapy of I. m. with PAA is, as known e.g. for malachite green, only possible while fighting the free living stages theronts and tomonts. This will be part of our subsequent investigations.     

Antiparasitic efficacy of dihydrosanguinarine and dihydrochelerythrine from Macleaya microcarpa against Ichthyophthirius multifiliis in richadsin (Squaliobarbus curriculus)

Ichthyophthirius multifiliis is a holotrichous protozoan that invades the gills and skin surfaces of fish and can cause morbidity and high mortality in most species of freshwater fish worldwide. The present study was undertaken to investigate the antiparasitic activity of crude extracts and pure compounds from the leaves of Macleaya microcarpa. The chloroform extract showed a promising antiparasitic activity against I. multifiliis. Based on these finding, the chloroform extract was fractionated on silica gel column chromatography in a bioactivity-guided isolation affording two compounds showing potent activity. The structures of the two compounds were elucidated as dihydrosanguinarine and dihydrochelerythrine by hydrogen and carbon-13 nuclear magnetic resonance spectrum and electron ionization mass spectrometry. The in vivo tests revealed that dihydrosanguinarine and dihydrochelerythrine were effective against I. multifiliis with median effective concentration (EC(50)) values of 5.18 and 9.43 mg/l, respectively. The acute toxicities (LC(50)) of dihydrosanguinarine and dihydrochelerythrine for richadsin were 13.3 and 18.2mg/l, respectively. The overall results provided important information for the potential application of dihydrosanguinarine and dihydrochelerythrine in the therapy of serious infection caused by I. multifiliis.     

Effects of long duration, low dose bronopol exposure on the control of Ichthyophthirius multifiliis (Ciliophora), parasitising rainbow trout (Oncorhynchus mykiss Walbaum)

Ichthyophthirius multifiliis Fouquet, 1876 infections on intensively reared fish stocks can increase rapidly, which if left unmanaged, can result in the heavy loss of stock. The present study explores the efficacy of long duration, low dose (1, 2 and 5 mg L(-1)) treatments of bronopol (marketed as Pyceze?, Novartis Ltd.) in reducing the number of trophonts establishing on juvenile Oncorhynchus mykiss held under small scale culture conditions. The effect of bronopol on the colonisation success of infective theronts was also investigated by adding 2 mg L(-1) bronopol to the water prior and during the infection process. The number of parasites surviving on fish treated this way was compared to groups of fish that only received treatment after infection had occurred. The effect of bronopol on exiting trophonts throughout their external development to the point of theront release was also assessed through the delivery of 1 mg L(-1), 2 mg L(-1) and 5 mg L(-1) bronopol for up to 27 days consecutively (days 9-36 post-infection). The trial showed that a nominal dose of 2 mg L(-1) bronopol administered prior to infection significantly reduced the number of theronts surviving in the water column at the time of the initial challenge by 35-40% (P<0.05). Similarly, doses of 2 and 5 mg L(-1) bronopol administered as the first wave of mature I. multifiliis trophonts exited fish (i.e. day 11 onwards) to develop externally, reduced the number of trophonts establishing on fish as the second cycle of infection by 52-83%. Continuous application of 2 and 5 mg L(-1) bronopol throughout the second and third cycles of I. multifiliis infection gave further reductions of between 90 and 98%. The number of trophonts on the fish in the control tanks and those treated with 1 mg L(-1) and the 2 mg L(-1) dose at the time of initial infection, by comparison, were observed to increase with successive cycles of infection. From these small scale tank trials, this study demonstrates that the strategic, long duration, low dose delivery of drugs like bronopol can significantly reduce the number of trophonts establishing on fish suggesting the potential of this drug at managing I. multifiliis infections.     

Mobilization and activation of nonspecific cytotoxic cells (NCC) in the channel catfish (Ictalurus punctatus) infected with Ichthyophthirius multifiliis

Channel catfish demonstrate a shift in the tissue distribution of nonspecific cytotoxic cells (NCC) when infected with the protozoan parasite, Ichthyophthirius multifiliis. NCC, isolated from head kidney (HK) tissues (hemopoietic organ) or peripheral blood leukocytes, were assessed for cytotoxic activity against NC-37 (a transformed mammalian cell line). NCC activity from HK tissue of moribund I. multifiliis-infected fish was depressed compared to HK-NCC activity in uninfected or I. multifillis-immune fish. The activity of NCC, isolated from the peripheral blood of moribund I. multifiliis-infected fish was significantly greater than the NCC activity in peripheral blood from either immune or uninfected fish. Chromium-51 release assays were combined with effector and target conjugate assays to determine killing capacity (Vmax) and affinity (Km) for target cells of peripheral blood NCC from moribund I. multifiliis-infected and uninfected fish. These experiments indicated that the peripheral blood from the moribund infected fish contained an increased percentage of active NCC with increased killing capacity and target cell affinity compared to peripheral blood NCC activity of uninfected fish.     

Role of Ichthyophthirius multifiliis in the Infection of Aeromonas hydrophila

Aeromonas hydrophila and Ichthyophthirius multifiliis are important fish pathogens. To clarify their relationship, two host models, goldfish with trauma and those infected with I. multifiliis, were used to study infection kinetics of A. hydrophila 4332 tagged with plasmid pGFPuv (Ah4332(GFP)). The results showed that in fish suffering from ichthyophthiriosis, the bacterial infection process was accelerated and more persistent than in the other groups. It is suggested that I. multifiliis plays an important role in the infection of A. hydrophila. Mechanical trauma caused by I. multifiliis might act as a portal of entry for A. hydrophila present in the water, thereby facilitating the invasion of bacteria. The ectoparasite might also serve as a vector and reservoir of pathogenic bacteria.     

Enhanced susceptibility of channel catfish to the bacterium Edwardsiella ictaluri after parasitism by Ichthyophthirius multifiliis

Bacterium Edwardsiella ictaluri and parasite Ichthyophthirius multifiliis (Ich) are two common pathogens of cultured fish. The objective of this study was to evaluate the susceptibility of channel catfish Ictalurus punctatus to E. ictaluri and determine bacterial loads in different fish organs after parasitism by Ich. Fish received the following treatments: (1) infected by I. multifiliis at 5000 theronts/fish and exposed to E. ictaluri; (2) infected by I. multifiliis alone; (3) exposed to E. ictaluri alone; and (4) non-infected control. E. ictaluri in fish organs were quantified by quantitative real-time polymerase chain reaction and reported as genome equivalents per mg of tissue (GEs/mg). The results demonstrated that the Ich-parasitized catfish showed significantly (P<0.05) higher mortality (91.7%) when exposed to E. ictaluri than non-parasitized fish (10%). The bacterial loads in fish infected by 5000 theronts/fish ranged from 6497 to 163,898 GEs/mg which was between 40 and 2000 fold higher than non-parasitized fish (49-141 GEs/mg). Ich infection enhanced the susceptibility of channel catfish to bacterial invasion and increased fish mortality.     

Oxidative stress parameters in silver catfish (Rhamdia quelen) juveniles infected with Ichthyophthirius multifiliis and maintained at different levels of water pH

The aim of this study was to determine oxidative stress parameters in the liver, gill and muscle of silver catfish juveniles infected with Ichthyophthirius multifiliis and maintained at pH 5.0 or 7.0 for three days. Juveniles were infected by adding one I. multifiliis-infected juvenile and water containing theronts to tanks. After the appearance of white spots on the skin, infected juveniles exposed to pH 5.0 and 7.0 showed significantly higher thiobarbituric acid reactive substances (TBARS) levels in the liver and gills compared to uninfected juveniles. Liver of infected juveniles exposed to pH 7.0 showed higher catalase (CAT) and lower glutathione-S-transferase (GST) activities, but those maintained at pH 5.0 showed significantly higher GST activity than uninfected juveniles. The gills of infected juveniles showed significantly higher CAT (day two) and GST activity at both pH 5.0 and 7.0 compared to uninfected juveniles. Muscle of infected juveniles showed significantly lower CAT and GST activity and TBARS levels (at day three) when maintained at both pH 5.0 and 7.0 compared to uninfected juveniles. In conclusion, I. multifiliis infection induces liver and gill damage via lipid peroxidation products in silver catfish, but higher antioxidant enzyme activity could indicate a greater degree of protection against this parasite.     

Treatment of ichthyophthiriasis in rainbow trout and common carp with common and alternative therapeutics

The goal of this laboratory study was to provide better knowledge about the treatment of ichthyophthiriasis (causative agent: Ichthyophthirius multifiliis, a ciliate bacteria) in rainbow trout Oncorhynchus mykiss and common carp Cyprinus carpio. The following questions were investigated: (1) the effectiveness of different chemicals (formalin, sodium chloride, hydrogen peroxide, Perotan, Virkon, Aquahumin, Baycox, and Ivomec) and at different concentrations and durations of application, (2) the number of treatments and the time intervals between treatments that were necessary to remove the parasite, and (3) how treatment effectiveness differed between the two species. The most effective treatment was a 37% stock solution of formalin at 110 microL/L of bath water for 1 h in rainbow trout and for 2 h in common carp. Aquahumin (150 microL/L for 2 h) was effective in slightly or moderately infected rainbow trout and at low water temperatures, but it was not effective for common carp. All other tested chemicals were ineffective. With formalin and Aquahumin, five treatments were necessary to remove I. multifiliis infestation. At 10 +/- 1 degrees C, the parasites were eradicated when the treatment was performed at 48-h intervals. At 18 +/- 1 degrees C the infestation was eliminated when treatment was performed at 24-h intervals but not at 48-h intervals. At 25 +/- 1 degrees C, treatment at 24-h intervals was ineffective; however, shorter intervals between treatments might improve treatment efficacy at this temperature. In contrast, the number of treatment repetitions played a minor role, and parasites were eliminated with five treatments in all experiments when the type of chemical and treatment interval were optimal.     

The formaldehyde content in fish in relation to anemia in mink (author's transl)]

The fish-induced anemia in mink is an alimentary disease produced by feeding high amounts of some raw marine fishes. The anemiogenic properties of the fish has been related mainly to its content of the iron binding agent-trimethylaminoxide. The aim of the present investigation was to examine how far formaldehyde could also play a part as an anemiogenic factor. The content of formaldehyde has been analysed in all species of raw, cold stored fish known to be used in mink food and in a few samples of ready made food (Table II). The content of formaldehyde varied within wide limits from 12 to 105 ppm, but none of the measured contents reached the high values obtained by Costly (1970). The mean values of formaldehyde in gutted coalfish, fillet waste of coal fish, cod and haddock prepared for the feeding experiments, were all close to 50 ppm. 175 female mink and 632 mits were tested during the whole of the breeding period from 15.2-30.6, 80 per cent of the diet (page 1) was fish products with and without supplements of formaldehyde. Amounts from 200 to 50 ppm were tried (Table I). The supplement of 200 ppm formaldehyde had an appetite-decreasing and anemiogenic effect, but the supplement of 50 ppm, i.e. a formaldehyde content up to the highest value observed in fillet waste, had no effect on appetite or hemoglobin synthesis neither in females nor in kits. This content of formaldehyde did not counteract the anti-anemiogenic effect of iron glutamate. The fish-induced anemia occurred in mink thus appears unaffected by the quantities of formaldehyde found in fish diets to fur bearing animals. Triox must be regarded as the dominant anemiogenic factor in raw fish diets.     

Isolation of antibacteriologically active compounds from species of the Cottidae family

Antibacteriologically active compounds were isolated from the skin of several species of the fish family Cottidae. Suitable samples were obtained from species living in the Pacific, lakes in North America and the Fenno-Scandinavian peninsula as well as the Baltic sea. The compounds isolated from the skin of Triglops quadricornis (fourhorn sculpin) from the Baltic sea were particularly studied. The activity was partially characterised by chemical and biochemical investigations and the susceptibility of several human and fish pathogens described. The substance or substance(s) are of a high molecular mass, resist proteolytic and glycolytic enzymes, are heat and pH sensitive and water soluble.     

Health status of salmonids in river systems in Natal. I. Collection of fish and parasitological examination

A detailed microbiological and parasitological survey of salmonids and indigenous fish in the upper reaches of selected river systems in Natal was undertaken. Fish were collected from the rivers by electro-fishing, from dams by gill netting and from hatcheries along the rivers. A total of 678 fish from 26 different sites along 8 river systems were collected. No parasites could be detected on fish from four of the rivers tested. Parasites were, however, detected on fish from large production sites in Natal and in the rivers feeding these sites. Parasites which were detected were Ichthyopthirius multifiliis, various Trichodina spp., Apiosoma sp. and Gyrodactylus sp. The heads of all fish estimated to be less than one year of age were collected and examined for the spores of Myxosoma cerebralis. All of the samples were found to be free of this parasite.     

The anti-protozoal activity of bronopol on the key life-stages of Ichthyophthirius multifiliis Fouquet, 1876 (Ciliophora)

Pyceze? (Novartis Animal Vaccines Ltd.) is licensed as a veterinary medicine to treat fungal infections in salmon, trout and their eggs. The active ingredient is bronopol, which due to its broad-spectrum activity has the potential to be an effective treatment against other important aquatic pathogens. In this study the efficacy of bronopol against Ichthyophthirius multifiliis was tested both in vitro and in vivo. In vitro trials demonstrated a 30 min exposure to 100 mg L(-1) bronopol killed 51.7% of the infective theronts. In vitro exposure of the protomonts to bronopol (0, 20, 50 and 100 mg L(-1)) for 30 min was observed to kill 0%, 76.2%, 97.2% and 100% respectively. Protomonts surviving treatment, demonstrated delayed development with the time taken from protomont until the release of theronts ranging from 28.3h for 0 mg L(-1) exposure, to 70 h for parasites in 20 and 50 mg L(-1) exposure groups. These concentrations also caused asymmetric cell division of the encysted tomonts. Exposure of encysted tomonts (min. 8 cell stage) to 100 mg L(-1) bronopol for 30 min, killed 50% within this period, with the remainder dying within the subsequent 42 h post exposure. Lower doses of bronopol were less effective in killing encysted tomonts than the higher doses (3.3% of parasites were killed in 20 mg L(-1); 10% in 50 mg L(-1)), but they still delayed theront release significantly (25.7 h for 0 mg L(-1) to 46.2h for parasites exposed to 20-50 mg L(-1)). Long, low dose (1 mg L(-1)) exposure to bronopol was also efficacious against theronts. Survival after 12h was 29% (c.f. 100% in control parasites), and <1% after 24 h exposure (c.f. 74% in control parasites). Theronts surviving these exposures demonstrated reduced infection success compared to control theronts. The findings of this study demonstrate that bronopol (Pyceze?) affects the survival of all free-living stages of I. multifiliis (protomonts, tomont and theronts), thus suggesting that bronopol may serve a useful role in the control of I. multifiliis infections.     

Nematode infection in fish from Cartagena Bay, North of Colombia

Parasitic nematode infection indices were recorded in different fish species collected at Cartagena Bay, North of Colombia. Among 19 studied species, the Mugil genus presented the highest prevalence (83.9-100%), although Sciades herzbergii, Caranx hippos and Centropomus undecimalis were also found infected with nematodes. Parasites were found in the liver, intestinal mesenteries and encysted near the intervertebral joints, with an average parasite abundance of 4.0 ± 0.3 nematodes per fish. Morphological analysis allowed the identification of these nematodes as Contracaecum sp. A small, but positive correlation was found between parasite abundance and length (R=0.294, P<0.001) and weight (R=0.244, P<0.001). In contrast, the correlation between parasite abundance and condition factor was negative (R=-0.191, P<0.001). These results are the first describing the presence of nematodes in several fish species of this ecosystem, and it highlights the need for monitoring parasitism in Mugil species in order to avoid parasite ingestion during fish consumption.     

Lack of Protection following Re-Exposure of Chinook Salmon to Ceratonova shasta (Myxozoa)

The recent identification of multiple genotypes of the salmonid parasite Ceratonova shasta with different virulence levels in Chinook Salmon Oncorhynchus tshawytscha suggests that it is possible to immunize fish against subsequent infection and disease. We hypothesized that exposure of Chinook Salmon to the less-virulent parasite genotype (II) prior to the more virulent parasite genotype (I) would decrease disease and/or result in fewer mature parasites compared with fish only infected with the more virulent genotype. To test this hypothesis, fish were challenged in a combination of field and laboratory exposures, and we measured infection prevalence, percent morbidity, and mature parasite production. Neither mortality nor mature parasite production were reduced when fish were exposed to genotype II prior to genotype I compared with fish exposed only to genotype I, suggesting that protection against C. shasta using a less-virulent genotype of the parasite does not occur.

Received June 9, 2014; accepted September 19, 2014     

Effects of herbicides (Gramoxone and Defenuron) on the development and protein levels of quail (Coturix coturix japonica)]

The herbicides Gramoxone (active substance paraquat dichloride) and Defenuron (active principle N-phenyl-N'-methylurea) were studied as influencing the development of the Japanese quail (Coturnix coturnix japonica) and the proteins in their blood serum. The aqueous solution of Gramoxone (500 ppm), applied to the egg surface, reduces the hatchability percentage and delays hatching. Under similar conditions, Defenuron has no inhibitory effect on embryonal development. Gramoxone in drinking water (100 ppm) causes 100% mortality of young quail within a week's time whereas the same amount of Defenuron does not affect the vitality of the birds. Sublethal concentrations of Gramoxone in drinking water (40 ppm) cause great quantitative changes in the proteinogram of the blood serum of the birds. The herbicide Defenuron does not influence the proteinogram even at a concentration of 100 ppm of the active substance in drinking water.     

Predicted Redistribution of Ceratomyxa shasta Genotypes with Salmonid Passage in the Deschutes River,Oregon

Abstract

A series of dams on the Deschutes River, Oregon, act as migration barriers that segregate the river system into upper and lower basins. Proposed fish passage between basins would reunite populations of native potamodromous fish and allow anadromous fish of Deschutes River origin access to the upper basin. We assessed the potential redistribution of host-species-specific genotypes (O, I, II, III) of the myxozoan parasite Ceratomyxa shasta that could occur with fish passage and examined the influence of nonnative fish on genotype composition. To determine the present distribution of the parasite genotypes, we exposed eight salmonid species—three native and five stocked for sport fishing—in present and predicted anadromous salmonid habitats. We monitored fish for infection by C. shasta and sequenced a section of the parasite ribosomal DNA gene from fish and water samples to determine parasite genotype. Genotype O was present in both upper and lower basins and detected only in steelhead Oncorhynchus mykiss. Genotype I was spatially limited to the lower basin, isolated predominately from Chinook salmon O. tshawytscha, and lethal for this species only. Genotype II was detected in both basins and in multiple species, but only as a minor component of the infection. Genotype III was also present in both basins, had a wide host range, and caused mortality in native steelhead and multiple nonnative species. Atlantic salmon Salmo salar and kokanee O. nerka were the least susceptible to infection by any genotype of C. shasta. Our findings confirmed the host-specific patterns of C. shasta infections and indicated that passage of Chinook salmon would probably spread genotype I into the upper Deschutes River basin, but with little risk to native salmonid populations.

Received April 20, 2012; accepted July 19, 2012     

Clinical Anesthesia and Analgesia in Fish

Fish have become a popular experimental model and companion animal, and are also farmed and caught for food. Thus, surgical and invasive procedures in this animal group are common, and this review will focus on the anesthesia and analgesia of fish. A variety of anesthetic agents are commonly applied to fish via immersion. Correct dosing can result in effective anesthesia for acute procedures as well as loss of consciousness for surgical interventions. Dose and anesthetic agent vary between species of fish and are further confounded by a variety of physiological parameters (e.g., body weight, physiological stress) as well as environmental conditions (e.g., water temperature). Combination anesthesia, where 2 anesthetic agents are used, has been effective for fish but is not routinely used because of a lack of experimental validation. Analgesia is a relatively underexplored issue in regards to fish medicine. However, recent studies have investigated opioid agents, nonsteroidal antiinflammatory drugs, and local anesthetics to determine their efficacy in minimizing pain and discomfort. The opioid morphine and the local anesthetic lidocaine do have significant effectiveness in reducing pain-related responses in rainbow trout (Oncorhynchus mykiss). Studies aimed at developing reliable analgesic protocols should explore a wide range of analgesic drug classes in several fish species.     

Comparison of the susceptibility of brown trout (Salmo trutta) and four rainbow trout (Oncorhynchus mykiss) strains to the myxozoan Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD)

Differences in susceptibility to the myxozoan parasite Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD), between four strains of rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) were evaluated. Fish were exposed to water enzootic for the parasite in the field for 5 days and were subsequently transferred to the laboratory. Relative parasite load was determined after 2, 3 and 4 weeks post-exposure (wpe) by quantitative real-time PCR (qPCR) of kidney samples and number of parasite stages was determined in immunohistochemical stained sections of kidney, liver and spleen tissues. According to qPCR results, the highest amount of parasite DNA per equal amount of host tissue at all time points was measured in brown trout. Two of the rainbow trout strains showed lower relative parasite load than all other groups at the beginning of the experiment, but the parasite multiplied faster in these strains resulting in an equal level of relative parasite load for all rainbow trout strains at 4 wpe. A weak negative correlation of fish size and parasite load was detected. Only in samples of a few fish, single stages of T. bryosalmonae were found in sections stained by immunohistochemistry impeding quantitative evaluation of parasite numbers by this method. The results indicate a differential resistance to T. bryosalmonae between the rainbow trout strains investigated and between rainbow trout and brown trout.     

Activity of the Pinus elliottii resin compounds against Lernaea cyprinacea in vitro

Lernaea cyprinacea infestation is a major problem for fishing culture in Brazil, which was introduced in the mid-eighties. To attempt controlling this parasite, an evaluation of the activity of the Pinus elliottii resin constituents against it was undertaken. To run the bioassay, fish infested with L. cyprinaceae were transferred to aquaria and kept at room temperature and ventilation for 15 days for adaptation. Afterwards, fish were sacrificed and the parasites were kept under water to run the experiments by evaluating the activities of the steamed oil and the chloroform fraction of the resin at concentrations of 0.5, 1.0, 2.0 and 5.0 ppm. The crude resin was also evaluated at concentrations of 1.0, 5.0 and 10 ppm, as well as the major components of its steamed oil, alpha and beta-pinenes, both at concentrations of 0.5 and 1.0 ppm. The results showed that both fractions from the resin were effective at 0.5 ppm concentration, while the pure compounds were less active. Moreover, the acute toxicity (DL(50)) of the crude resin for the fish Leptorinus piau, aged 1 month, was established at 200.0 ppm, which ensures its safe use.