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.     

Ionic Effects of Infection of Ichthyophthirius multifiliis in Goldfish

Abstract

Ichthyophthiriasis is a common parasitic disease of freshwater food and ornamental fish. This study examined the ionic effects of an induced infection of ichthyophthiriasis in goldfish Carassius auratus. The whole-animal net Na⁺ and Cl^– fluxes were measured from 12 goldfish each day for 5 d. The numbers of branchial mucous and chloride cells were also determined histologically. Two days of postexposure to 200 theronts/mL resulted in significant net losses of Na⁺ and Cl^–. Whole-body ionic effluxes were then reduced over the subsequent duration of the experiment. There was a general increase in the numbers of branchial mucous cells as reinfection progressed. Branchial chloride cells were elevated in infected fish, indicating some degree of compensation in response to infection. This suggested that acute ionic disturbances occurred in the host fish as a consequence of different stages of the life cycle of the parasite; the host exhibited a suite of physiological and morphological changes to compensate for parasitic infection.     

Production and preliminary characterization of murine monoclonal antibodies to Ichthyophthirius multifiliis, a protozoan parasite of fish

An initial panel of 34 hybridomas, each secreting antibodies reactive with an infective theront stage of an Ichthyophthirius multifiliis isolate, was produced. Three of these cell lines, each producing immunoglobulin M class antibodies, were cloned by limiting dilution and were expanded as ascites-producing tumors in syngeneic mice. Three monoclonal antibodies (MAB) reacted with intact whole theronts in an enzyme-linked immunosorbent assay to dilutions of 1:10,000 in ascitic fluids and had a similar pattern of surface and cytoplasmic staining in indirect immunofluorescent tests. Only antigen specified by MAB E6 could be characterized by acrylamide gel electrophoresis and immunoblotting; initial data indicated a molecular weight of 200,000. Physicochemical properties of the determinants recognized by the 3 MAB were tested by pronase digestion and periodate oxidation. Seemingly, a protein, glycoprotein, and carbohydrate were recognized by MAB E6, FE10, and AC8, respectively.     

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.     

Isolation of active compounds from methanol extracts of Toddalia asiatica against Ichthyophthirius multifiliis in goldfish (Carassius auratus)

The parasitic ciliate Ichthyophthirius multifiliis infests all species of freshwater fish and can cause severe economic losses in fish breeding. The present study aims to evaluate the antiparasitic activity of the active components from Toddalia asiatica against I. multifiliis. Bioassay-guided fractionation and isolation of compounds with antiparasitic activity were performed on the methanol extract of T. asiatica yielding two bioactive compounds: chelerythrine and chloroxylonine identified by comparing spectral data (NMR and ESI-MS) with literature values. Results from in vitro antiparasitic assays revealed that chelerythrine and chloroxylonine could be 100% effective against I. multifiliis at the concentration of 1.2 mg L^?1 and 3.5 mg L^?1, with the median effective concentration (EC₅₀) values of 0.55 mg L^?1 and 1.90 mg L^?1 respectively. In vivo experiments demonstrated that fish treated with chelerythrine and chloroxylonine at the concentrations of 1.8 and 8.0 mg L^?1 carried significantly fewer parasites than the control (P < 0.05). The acute toxicity (LC₅₀) of chelerythrine for goldfish was 3.3 mg L^?1.     

Values for Selected Serum Analytes during Experimental Ichthyophthirius multifiliis Infection of Channel Catfish

Abstract

Channel catfish Ictalurus punctatus were exposed to a sublethal dose of infectious Ichthyophthirius multifiliis theronts, and eight serum analytes were measured. Sequential serum samples from each fish were collected for serum immediately before exposure, 4 d postexposure (PE), and 11 d PE. All fish developed moderately heavy infections with the parasite as indicated by the development of cutaneous lesions 5 d PE to infective theronts. Small but statistically significant changes in total protein, creatinine, and alkaline phosphatase values occurred after acute infection with I. multifiliis. Serum concentrations of glucose, albumin, urea nitrogen, alanine aminotransferase, and aspartate aminotransferase remained constant, indicating little disturbance of liver or kidney functions detectable by a serum biochemical profile established during an acute infection with I. multifiliis.     

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.     

Prevention of Ichthyophthirius multifiliis Infestation in Channel Catfish Fingerlings by Copper Sulfate Treatment

Abstract

The ability of copper sulfate to control Ichthyophthirius multifiliis tomites was determined in pond water and in settled, decanted pond water at 23°C for 7 d. Results indicate that copper concentrations of 0.15 and 0.02 mg/L prevented tomites from infesting channel catfish Ictalurus punctatus in pond water (total alkalinity = 366 mg CaCO₃ /L) and in settled, decanted pond water (total alkalinity = 224 mg CaCO₃ /L), respectively.     

Ichthyophthirius multifiliis in Elvers and Small American Eels from the East River,Nova Scotia

Abstract

Elvers of American eels Anguilla rostrata entering the East River on the Atlantic coast of Nova Scotia between early May and the end of July were parasitized by Ichthyophthirius multifiliis during June of 1993 for the first time in 6 years of observation. Prevalence of infection in elvers (49.9–68.2 mm total length, TL) ranged from 7 to 30%; weekly mean prevalence (pooled sample) ranged from 13.0 to 23.3% over an infection period of 42 d. Weekly prevalence ranged from 8.3 to 12.2% for small eels (70–120 mm TL). Mean intensity of infection was 6.1 trophonts/host (range, 1–61) for elvers and 7.2 trophonts/host (range, 1–40) for small eels. Count distributions were right-skewed and leptokurtic for both elvers and small eels. No conclusion could be made (due to low statistical power) as to whether length or weight was related to mean intensity of parasitism in either elvers or small eels or whether mean intensity differed between elvers and small eels. High river discharge during June–July, but not water temperature (April–July), may have contributed to development of the epizootic of I. multifiliis in 1993.     

Parasitism by protozoan Ichthyophthirius multifiliis enhanced invasion of Aeromonas hydrophila in tissues of channel catfish

Protozoan Ichthyophthirius multifiliis Fouquet (Ich) and bacterium Aeromonas hydrophila are two common pathogens of cultured fish, which cause high fish mortality. Currently there is no information available for the effect of parasitism by Ich on survival of channel catfish and invasion of A. hydrophila in fish tissues following exposure to A. hydrophila. A trial was conducted in this study to: (1) determine whether A. hydrophila increased fish mortality in Ich-parasitized channel catfish; and (2) compare the bacterial quantity in different tissues between non-parasitized and Ich-parasitized catfish by real-time polymerase chain reaction (qPCR). The results demonstrated that the Ich-parasitized catfish showed significantly (P<0.05) higher mortality (80%) when exposed to A. hydrophila by immersion than non-parasitized fish (22%). Low mortality was observed in catfish exposed to Ich alone (35%) or A. hydrophila alone (22%). A. hydrophila in fish tissues were quantified by qPCR using a pair of gene-specific primers and reported as genome equivalents per mg of tissue (GEs/mg). Skin, gill, kidney, liver and spleen in Ich-parasitized fish showed significantly higher load of A. hydrophila (9400-188,300 GEs/mg) than non-parasitized fish (4700-42,100 GEs/mg) after exposure to A. hydrophila. This study provides evidence that parasite infections enhance bacterial invasion and cause high fish mortality.     

Antiparasitic effects of peracetic acid (PAA) against infective stages (theronts) of white spot disease, Ichthyophthirius multifiliis in vitro

White spot disease, caused by the protozoan parasite Ichthyophthirius multifiliis (I. multifiliis), invades nearly all fresh water fish species and causes huge economic losses. In Germany no protocide substance is legal for the treatment of I. multifilis. As an alternative substance the peracetic acid (PAA) was tested to treat the free invasive stage (theront) of the parasite. PAA concentrations of 0.3 ppm were able to kill all theronts in 120 min in our investigations. As a result of these investigations we recommend an interval-application of 0.3 to 0.5 ppm PAA for 30 to 150 min. This application should be prolonged for two life cycles of the parasite. Biotic parameters as e. g. fish species, and age as well as abiotic parameters as e. g. temperature, pH and organic load of the water could possibly influence the efficiency of the PAA application and should therefore be taken into account while picking the dosage and length of the PAA exposure.     

小瓜虫对草鱼肝脏和肾脏等组织器官线粒体的影响

本试验旨在了解小瓜虫(Ichthyophthirius multifiliis)感染草鱼后对草鱼肝脏、肾脏和肌肉等组织器官线粒体的影响.人工方法用小瓜虫感染健康草鱼,确定草鱼成功感染小瓜虫后,透射电镜观察肝脏、肾脏和肌肉组织中线粒体结构的变化,检测这些组织器官的过氧化物歧化酶(SOD)和丙二醛(MDA)含量.透射电镜观察结果显示,与正常组相比,感染组肾脏、肝脏和肌肉组织线粒体数量减少,线粒体肿胀、嵴断裂或消失.感染组肝脏、肾脏和肌肉组织中SOD含量平均值均高于正常组,其中肝脏、肾脏表现为差异显著(P <0.05),肌肉组织差异不显著(P >0.05);感染组肝脏、肾脏和肌肉组织中MDA平均含量均低于正常组,其中肝脏、肾脏表现为差异显著(P <0.05),肌肉组织差异不显著(P >0.05).结果表明,小瓜虫感染草鱼后,对草鱼的肝脏、肾脏和肌肉组织的线粒体结构及SOD和MDA活性均有影响.     

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.     

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.     

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.     

An outbreak of white spot disease (Ichthyophthirius multifiliis) in young fingerling rainbow trout (Salmo gairdneri Richardson)

An outbreak of white spot disease (‘Ich’) in fingerling rainbow trout is reported. Approximately 3,000 fish died or were killed in extremis in a period of 5 weeks. Post mortem examinations were carried out on 12 fish, all of which showed thickened whitish foci on the skin. One fish showed skin ulceration and another gill ulcerations. The gill infestation was more severe than the skin. Pathological results are described and discussed.     

Host responses to Cryptosporidium infection

Cryptosporidium is a clinically and economically important infection whose pathogenic effect begins with colonization of the intestinal epithelium. Despite intensive efforts, a consistently effective therapy for the infection has yet to be identified. Morbidity and mortality results from ongoing loss of absorptive epithelium, which leads to villous atrophy and malabsorption and release of inflammatory mediators that stimulate electrolyte secretion and diarrhea. With further clarification of the mechanisms underlying enterocyte malfunction in Cryptosporidium infection, it should be possible to design rational nutritional and pharmacologic therapies to enhance nutrient and water absorption, promote the clearance of infected enterocytes, and restore normal villus architecture and mucosal barrier function.     

Host effector mechanisms against parasites

The first part of this presentation considers some of the complexities of parasitic infections and parasite-specific effector mechanisms which have hampered the development of practical methods of immunisation against parasitic diseases. In the second part, an outline is given of the effector mechanisms involved in immunity of cattle to the protozoan parasite Theileria parva. Parasites are antigenically complex organisms which often have distinct developmental stages, sometimes with different predilection sites within the host. Antigenic polymorphism between strains is a common feature of parasites and sometimes results in strain-specific immunity. Certain parasites have also evolved mechanisms of modulating surface antigens which allow them to escape host effector mechanism. Effector mechanisms which control parasitic infections may operate by preventing establishment of the parasites, by eliminating the parasites once they have established or by affecting growth or fecundity of the parasites. In addition to specific antibody and cell-mediated immune responses, inflammatory or physiological responses play an important role in the control of some parasites. Current evidence suggests that effector mechanisms against T.parva parasites operate at two levels. First, antibodies produced against the infective stage of the parasite, the sporozoite, can, by neutralising infectivity, reduce the numbers of organisms which establish in the host. Second, cytotoxic T cells directed against parasitised lymphoblasts cause destruction of parasites following their establishment in the host. Moreover, in situations where immunity is parasite strain-specific, the cytotoxic T cell responses have also been found to be strain-specific. The elucidation of these effector mechanisms has indicated potential new strategies of immunisation against T.parva.