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.     

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.     

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.     

Olfactory Organ of Channel Catfish as a Site of Experimental Edwardsiella ictaluri Infection

Abstract

Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC), is one of the most important pathogens to infect channel catfish Ictalurus punctatus. Although the full pathogenesis of E. ictaluri is unclear, the olfactory organ is thought to be a site of entry. We have examined the effects of applying E. ictaluri directly into the olfactory capsule of channel catfish. Olfactory organs of 30 experimental fish were exposed to E. ictaluri for 1 h (1 mL, 1 × 10⁶ colony-forming units/mL). Live fish were sampled at 1, 24, 48, and 72 h, and days 5 and 14 postinfection, and their olfactory organs were examined by light and electron microscopy. Damage, including loss of sensory cilia and microvilli from the olfactory mucosal surface, was observed at 1 h postinfection. Degeneration of olfactory receptors and supporting cells was evident by 24 h postinfection. The nonsensory region also showed signs of degeneration, such as columnar cells lacking cilia. Electron microscopic immunocytochemistry confirmed the presence of E. ictaluri on the mucosal surface and within the epithelium. Host leukocytes responded to bacteria by migrating through the olfactory epithelium into the interlamellar lumen and phagocytosing organisms, but phagocytosed E. ictaluri did not appear to be destroyed. Our results indicate that during initial stages of infection channel catfish olfactory epithelium is vulnerable, and E. ictaluri can enter the host through the olfactory organ. It is also possible that host phagocytic cells serve as a vehicle for the systemic dissemination of E. ictaluri     

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.     

Electron Microscopy of Infection by Saprolegnia spp. in Channel Catfish

Abstract

Saprolegnia sp. isolated from channel catfish Ictalurus punctatus grew slower than S. parasitica on cornmeal agar (CMA). Oogonia in Saprolegnia sp. appeared frequently, whereas oogonia were rarely seen in S. parasitica on CMA. In experimental exposures of injured channel catfish to fungal spores, infections were apparent after 3–4 d, but were most common after 7–9 d. Multiple lesions were usually seen in naturally infected fish, whereas a single lesion appeared at the injured site of experimentally infected fish. No obvious differences were found between lesions caused by S. parasitica and those caused by Saprolegnia sp. Most of the epidermal cells in fungusinfected lesions were necrotic. In some lesions, the epidermis was completely sloughed and the dermis was exposed. Both Saprolegnia parasitica and Saprolegnia sp. penetrated the dermis, causing damage to fibroblasts and collagen lamellae.     

Experimental Induction of Proliferative Gill Disease in Specific-Pathogen-Free Channel Catfish

Abstract

Specific-pathogen-free channel catfish Ictalurus punctatus were exposed to sediment and mud from a pond containing channel catfish with proliferative gill disease. In one experiment, fish were to exposed to mud and sediment for 2 months in water maintained at 19°C. Fish were necropsied weekly, and certain tissues were examined histologically and ultrastructurally. Four trials were conducted with sediment samples from different epizootics of proliferative gill disease. In a second experiment, fish were exposed to sediment for 7 d in water maintained at 16, 19, or 26°C; the fish were then moved to clean water held at 16, 19, or 26°C. Fish were necropsied before transfer to clean water and weekly thereafter for 2 months. Channel catfish held at 19°C developed proliferative gill disease within 2 d of exposure to sediment. Primary cells of a uninucleate myxosporean parasite were present in the gills at the base of lamellae. These developed into plasmodia with numerous secondary cells, and some primary cells disintegrated, releasing their internal secondary cells. Similar development was observed in internal organs 1 week after appearance of the parasite in gills. Complete sporogony did not occur over the 2 months of this study. Plasmodia became necrotic and were not detected after 60 d. In fish exposed to sediment for 7 d at 16, 19, and 21°C, similar organisms were detected, but clinical disease occurred only at 19 and 26°C. Proliferative gill disease may be attributed to extrasporogonic stages of a myxosporean resembling Sphaerospora spp.     

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.     

Responses of Blue Catfish and Channel Catfish to Environmental Nitrite

Abstract

In nitrite-exposure experiments, percent methemoglobin, plasma nitrite concentration, and plasma chloride ion concentration were compared between channel catfish Ictalurus punctatus and blue catfish I. furcatus exposed to sublethal levels of nitrite for 48 h at 25°C. In nitrite-recovery experiments, fish exposed to elevated environmental nitrite for 12 h were transferred to freshwater, and blood characteristics were monitored during the 24-h recovery period. Blue catfish appeared to be more resistant to environmental nitrite than channel catfish. Methemoglobin levels (percent of hemoglobin in methemoglobin form) were significantly lower in blue catfish than in channel catfish. Maximum plasma nitrite concentrations were 137 mg NO₂-/L plasma in blue catfish and 164 mg NO₂-/L plasma in channel catfish. Percent methemoglobin and plasma nitrite concentration were closely correlated. Plasma chloride decreased initially with exposure to nitrite but quickly returned to control levels. Blue catfish exposed to nitrite at 10°C required 1 week to recover when placed in nitrite-free water. The methemoglobin reductase enzyme apparently functioned at a slow rate in fish acclimated to cold temperatures.     

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.     

Differences between Plasma and Serum Samples for the Evaluation of Blood Chemistry Values in Rainbow Trout,Channel Catfish,Hybrid Tilapias,and Hybrid Striped Bass

Abstract

Blood chemistry analytes are determined for fish from either serum or plasma samples. Serum and plasma are similar in that they both represent the fluid component of blood; however, plasma contains clotting factors that are not present in serum. This study was conducted to determine whether the type of sample—plasma or serum—had an effect on measured blood analytes in rainbow trout Oncorhynchus mykiss, channel catfish Ictalurus punctatus, hybrid tilapia Oreochromis spp., and hybrid striped bass (striped bass Morone saxatilis × white bass M. chrysops). Paired plasma and serum samples were analyzed for the following standard biochemical analytes: total protein, albumin, globulin, creatinine, total bilirubin, alkaline phosphatase, aspartate aminotransferase, sodium, potassium, chloride, calcium magnesium, phosphorus, glucose, and cholesterol. For all four taxa, values for potassium were lower in the serum and magnesium and phosphorus values were higher in the serum. Glucose values were lower in the serum from rainbow trout, hybrid striped bass, and channel catfish; whereas cholesterol values were higher in the serum of rainbow trout, channel catfish, and hybrid tilapias. The differences observed between serum and plasma were distinct from changes occurring with hemolysis and, therefore, do not represent release of erythrocyte constituents. The differences most likely represent metabolic utilization of blood constituents while the blood was clotting. This work indicates that plasma should be used preferentially to serum for biochemical analysis because analyte levels determined from serum may not accurately reflect those found in circulating blood.     

Decreased Resistance to Edwardsiella ictaluri Infection in Channel Catfish Intraperitoneally Administered an Oil Adjuvant

Abstract

The effects of intraperitoneal injection of squalene, an oil adjuvant, on nonspecific mortality of channel catfish Ictalurus punctatus and on their resistance to experimental Edwardsiella ictaluri infection were studied. Yearling channel catfish were assigned to control (N = 22) or squalene (N = 25) treatment groups, and mortality was monitored for 14 d following treatment. On day 14 both groups were infected with E. ictaluri, the causative agent of enteric septicemia of catfish, and mortality was monitored for an additional 11 d. Before infection, mortality did not differ between groups. After E. ictaluri infection, fish that received squalene were at a substantially higher risk of dying than control fish (relative risk after squalene treatment = 6.86). These results suggest that intraperitoneal administration of squalene, although not directly toxic, decreased resistance to E. ictaluri infection.     

Experimental Control of Metacercariae of the Yellow Grub Clinostomum marginatum in Channel Catfish

Abstract

Metacercariae of yellow grub Clinostomum marginatum in the flesh of farm-raised channel catfish Ictalurus punctatus pose a potential marketing problem to fish farmers. Infected fish may not be marketable. Three compounds were tested as possible control agents. Droncit (praziquantel) and Masoten (trichlorfon) were used separately as a bath treatment. Droncit and ivermectin were injected into the musculature to control yellow grub metacercariae. The two treatment rates were 0.65 mg/L plus 15 mg/kg offish as a bath treatment and 25 mg/kg offish as an injected treatment for Droncit. Masoten was treated at a rate of 2 mg/L as a bath treatment and ivermectin (1% solution) was injected at a rate of 0.022 mL/kg. Droncit significantly reduced metacercariae in both bath and injection treatments. Ivermectin also significantly reduced the number of metacercariae, but was not as effective as Droncit. Masoten was ineffective. More study is needed to obtain dosage rates for Droncit and ivermectin that would eliminate yellow grub metacercariae from the flesh of channel catfish.     

Dose Titration of Sarafloxacin (A-56620) against Edwardsiella ictaluri Infection in Channel Catfish

Abstract

Two dose-titration studies were performed with sarafloxacin (A-56620) on channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri. Fish were infected with E. ictaluri by water-bath exposure and subsequently fed rations for 5 d to equal 0, 2, 6, 10, or 14 mg sarafloxacin/kg body weight per day. In trial I, mortality was significantly reduced from 69% for fish receiving no medication to 17% for those receiving 10 or 14 mg sarafloxacin/kg per day (P ≤ 0.01). In trial II, mortality was reduced from 33% of the nonmedicated infected group to 5 and 10% offish receiving 10 and 14 mg sarafloxacin/kg per day, respectively. Fish receiving 2 or 6 mg sarafloxacin/kg per day had intermediate mortality rates.     

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.     

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.     

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.     

Longevity of Bolbophorus damnificus Infections in Channel Catfish

Abstract

The digenean Bolbophorus damnificus infects commercial channel catfish Ictalurus punctatus, causing mortality, lower feed consumption, and reduced growth in surviving fish. The purpose of this study was to determine the length of time for which B. damnificus prodiplostomulum metacercariae (juvenile trematode stage that infects fish) would remain viable (parasite appearing to be intact or exhibiting movement) in channel catfish. Fish (n = 210) were infected with molecularly confirmed B. damnificus cercariae harvested from naturally infected marsh rams-horn snails Planorbella trivolvis. During the first sampling (at 20 d postinfection), 8.3 ± 3.6 metacercariae/fish (mean ± SD) were found in the host muscle and visceral organs. The channel catfish were then acclimated to a water temperature of either 18°C or 28°C. After 11 months, 6.8 ± 3.5 and 5.9 ± 3.0 metacercariae/fish were found in groups held at 18°C and 28°C, respectively. The mean number of parasites per fish did not significantly differ between fish held at the two temperatures and did not significantly decline over time at either temperature. Fish examined from 13 to 30 months postinfection all contained viable metacercariae that were morphologically and molecularly identified as B. damnificus. At 18 months, 12 metacercariae (of which 11 were intact and 10 displayed movement) were found in the one fish sampled; at 30 months, the last fish sampled contained three intact metacercariae (one displayed slight movement). Our results indicate that B. damnificus metacercariae can remain viable in channel catfish for at least an 18–30-month production cycle during which they have the potential to affect fish growth; in addition, infected fish may serve as intermediate hosts for these metacercariae for at least 2.5 years postinfection.

Received July 14, 2010; accepted March 6, 2011