Immune responses and host protection of channel catfish, Ictalurus punctatus (Rafinesque), against Ichthyophthirius multifiliis after immunization with live theronts and sonicated trophonts

The humoral immune responses and host protection of channel catfish, Ictalurus punctatus (Rafinesque), against Ichthyophthirius multifiliis (Ich) were determined after immunization with live theronts and sonicated trophonts. Immunizations with live theronts or sonicated trophonts were carried out by both bath immersion and intraperitoneal (i.p.) injection. Cutaneous and serum immunoglobulin (Ig) levels and anti-Ich antibodies were measured 12 and 21 days post-immunization. The level of Ich infection and survival of catfish were determined after theront challenge. Cutaneous and serum anti-Ich antibodies were significantly higher (P < 0.05) in fish immunized with live theronts by immersion or i.p. injection, or with sonicated trophonts administered by i.p. injection, than in fish immunized with sonicated trophonts by immersion, with bovine serum albumin by i.p. injection, or non-immunized controls. Host protection was noted only in fish immunized with live theronts by immersion or i.p. injection or with sonicated trophonts by i.p. injection. There was a positive correlation between higher levels of anti-Ich antibodies and host survival in the immunized fish.     

Antibody mediated immune response against Ichthyophthirius multifiliis using excised skin from channel catfish, Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius

This study investigated antibody mediated immune response against Ichthyophthirius multifiliis (Ich) by determining whether theronts would retain the potential for reinfection, both in vitro and in vivo, after treatment with the culture fluid of excised skin from channel catfish, Ictalurus punctatus , immune to Ich. The invasion was reduced significantly ( P  < 0.05) for theronts treated with the immune culture fluid compared with those treated with the culture fluid from naive fish. The treatment of theronts with the immune culture fluid greatly reduced the size and survival of trophonts compared with those treated with the culture fluid from naive fish. Fewer fish were infected and the infection density was less for fish exposed to theronts treated with immune culture fluid. The infection was severe for fish invaded by theronts treated with the culture fluid from naive fish, with a high number of infected fish and heavy density of trophonts per fish. All fish were infected by Ich when exposed to the theronts treated with the immunoadsorbed culture fluid. In summary, results of this study show that cutaneous antibodies in the culture fluid of excised skin from immune fish significantly reduces theront infectivity by immobilizing or weakening theronts.     

Protective effect of cutaneous antibody produced by channel catfish,Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius multifiliis Fouquet on cohabited non-immune catfish

Fish which survive a sublethal ichthyophthiriasis acquire protective immunity against Ichthyophthirius multifiliis Fouquet (Ich). This study evaluated the protective effect of cutaneous antibody secreted by channel catfish, Ictalurus punctatus (Rafinesque), immune to Ich on cohabited non-immune catfish. Non-immune and immune fish controls were separately maintained and infected with theronts. The Ich infection was assessed by scoring 0, < 50, 50-100, and > 100 trophonts fish(-1) at 5 days post-infection. The results of infection showed that cohabited fish at the ratio of 15 non-immune to two immune fish had < 50 trophonts fish(-1). Eighty per cent of the cohabited fish at the ratio of 10 non-immune to two immune fish showed 0 or < 50 trophonts fish(-1). The 76% of control non-immune fish had more than 100 trophonts fish(-1). The control immune fish had 0 trophonts fish(-1). Anti-Ich antibody was detected using enzyme-linked immunosorbent assay in water samples taken from tanks containing immune fish after the water samples were concentrated 40-fold. The study suggests that immune fish cohabited with non-immune fish may protect non-immune fish against Ich infection.     

Cutaneous antibodies from channel catfish, Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius multifiliis (Ich) may induce apoptosis of Ich theronts

This study explored the existence of apoptosis (programmed cell death) in Ichthyophthirius multifiliis Fouquet (Ich) theronts and determined the effect of cutaneous antibodies in skin culture fluid from fish immune to Ich on theront apoptosis. Apoptosis was detected in theronts and was clearly distinguished by fluorescent microscopy after staining with acridine orange and propidium iodide. The apoptotic theronts showed characteristic chromatin condensation and nuclear fragments containing chromatin pieces. The externalization of phosphatidylserine on the plasma membrane of apoptotic theronts was detected with fluorescein isothiocyanate-conjugated annexin using flow cytometry. Theront apoptosis was induced using the skin culture fluid from fish immune to Ich, which contained cutaneous antibodies against Ich. The highest apoptosis appeared in theronts exposed to immune skin culture fluid at a 1:10 dilution, compared with those at 1:20 and 1:40 dilutions. A direct correlation was noted between the percentage of apoptotic theronts and exposure duration to immune skin culture fluid. The study indicated that antibody reaction with theronts (immobilization) played an important role in theront apoptosis, but it could not be excluded that other components released from the excised skin had effects on theronts.     

Cutaneous antibodies in excised skin from channel catfish, Ictalurus punctatus Rafinesque, immune to Ichthyophthirius multifiliis

This study determined whether cutaneous antibodies were present in the excised skin of channel catfish, Ictalurus punctatus Rafinesque, immune to Ichthyophthirius multifiliis Fouquet (Ich). Theronts were immobilized on or near the excised skin from immune fish. The survival of immobilized theronts was significantly reduced after exposure for 8 h to the culture of excised skin from immune fish. Culture fluids from excised skin of immune fish immobilized theronts with a peak in the immobilization titre at 24 h post-exposure. Immobility of theronts in the culture fluid from immune skin was removed after immunoabsorption with theronts. Indirect immunofluorescent staining of theronts treated with culture fluid from excised skin of immune fish revealed strong and uniform fluorescence on the cilia and cell surface of theronts. Western blot analysis of the culture fluid from immune fish revealed a 70-kDa band which corresponded to the molecular weight of catfish immunoglobulin heavy chain. The results of this study show that cutaneous antibodies to Ich theronts were present in and released from the excised skin from fish immune to Ich. Immobilization and killing of the theronts are two characteristics of the antibody response that appear to prevent the successful invasion of theronts into excised skin.     

Susceptibility of three strains of blue catfish,Ictalurus furcatus (Valenciennes), to Ichthyophthirius multifiliis

This study compared the susceptibility of three blue catfish strains (D&B, USDA 101 and USDA 102) to the parasite Ichthyophthirius multifiliis (Ich). In Trial I, a cohabitation study (all strains stocked communally) was conducted and fish were exposed to theronts at 0, 200, 1000, 5000 or 25 000 theronts fish^?1, respectively. All fish died when exposed to theronts at 5000 or 25 000 theronts fish^?1. When exposed to 1000 theronts fish^?1, USDA 102 strain of blue catfish showed significantly lower mortality (78.5%) compared to USDA 101 and D&B strains (92.7% and 100%). In Trial II, the same three strains of blue fish were evaluated for their susceptibility to Ich with strains challenged in separate tanks by adding Ich theronts at 0, 200 and 1000 theronts fish^?1, respectively. All D&B and USDA 101 blue catfish died; however, 42.3% of USDA 102 strain survived the infection when exposed to 1000 theronts per fish. The results indicate that there are differences among strains of blue catfish for susceptibility to Ich, and these differences will be useful in the development of improved catfish germplasm for commercial aquaculture.     

Immunization of channel catfish, Ictalurus punctatus Rafinesque, against Ichthyophthirius multifiliis (Fouquet): killed versus live vaccines

Abstract. Fish surviving infection with the pathogenic ciliated protozoan, Ichthyophthirius multifiliis (Fouquet, 1876), become resistant to subsequent infection by the parasite. The acquired immunity suggests that development of a vaccine against the parasite may be possible. Because of the advantages of immunoprophylaxis for treatment of the disease, an effort has been made to determine whether fish exposed to killed parasite preparations can resist subsequent lethal challenge. Both the route of administration and the effects of stage specific antigens have been examined. Channel catfish vaccinated by intraperitoneal (i.p.) injection or bath immersion with killed I multifiliis tomites show 100% mortality following a standard challenge protocol. Similarly, 100% mortality was observed in test groups injected with tomite cilia. In both cases, a consistent difference in days to death between control and test group animals was observed. Although complete mortality was seen with fish injected with tomite preparations, fish vaccinated with killed trophonts (the feeding stage of the parasite) had a much greater degree of protection with approximately 50% of fish surviving an otherwise lethal challenge. Finally, animals injected intraperitoneally with live tomites showed nearly complete immunity and were identical in their response to fish which survive natural infection. The response of fish vaccinated with live parasites indicates that animals injected intraperitoneally can develop surface immunity and that i.p. injection is a suitable route of administration for potential I. multifiliis vaccines.     

Cross-protection elicited in channel catfish (Ictalurus punctatus Rafinesque) immunized with a low dose of virulent Edwardsiella ictaluri strains

Cross-protection of channel catfish ( Ictalurus punctatus Rafinesque) immunized with a low dose of virulent Edwardsiella ictaluri and challenged with six E. ictaluri strains was examined in four trials. The relative per cent survival among low-dose immunized and then challenged fish ranged from 27.7% to 100%. Significant protection ( P <0.05), with the exception of strain ATCC-33202, was conferred by immunization with a given E. ictaluri strain challenged either with a homologous or a heterologous strain. Antibody titres of pooled serum collected on day 22 from surviving fish examined by enzyme-linked immunosorbent assay (ELISA) ranged from 1:40 to 1:320, but no differences were apparent among different vaccinated groups. The protein profiles of six E. ictaluri strains examined by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a relatively homogeneous pattern. Immuno-blots probed with pooled serum from immunized and challenged fish showed a pattern similar to LPS-reaction patterns observed with E. ictaluri in other studies. Since the present studies further corroborate that E. ictaluri is a clonal bacterial species with no apparent antigenic differences, it is possible that immunization with a single E. ictaluri field strain should confer protection against any other strain.     

Evaluation of diquat against an acute experimental infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus (Rafinesque)

A trial was performed to evaluate the efficacy of diquat (6,7-dihydrodipyrido[1,2-a:2',1'-c]pyrazinediium dibromide) against an acute experimental infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus . Diquat is an Environmental Protection Agency-approved herbicide and has the potential to be legally and practically used against columnaris. Channel catfish were challenged, by cutaneous abrasion, and waterborne exposure to F. columnare and treated once at 22-h post-challenge with 2.5, 5.0, 10.0 and 15 mg L⁻¹ of diquat active ingredient for 6 h. At the conclusion of the trial, 21-day post-challenge, diquat at 5.0, 10.0 and 15 mg L⁻¹ significantly ( P  < 0.05) reduced the mortality of infected fish from 95% in the challenged non-treated fish to 68%, 59% and 49%, respectively. In vitro , the minimum inhibitory concentration (MIC) of 23 isolates of F. columnare was assayed. The majority of the isolates had an MIC value of 5 μg mL⁻¹ (15 of the 23 isolates). Infected fish exhibited acute clinical signs similar to a natural infection. The skin had severe ulcerative necrotizing dermatitis and the muscles had severe necrotizing myositis. The gills had severe multifocal necrotizing branchitis. The results demonstrate that diquat would reduce mortalities caused by an acute columnaris infection.     

Evidence that channel catfish, Ictalurus punctatus (Rafinesque), mortality is not linked to ingestion of the hepatotoxin microcystin-LR

Catfish farms located in the south-eastern USA using brackish (3–5 g NaCl L⁻¹) well water experience sporadic fish kills sometimes with high mortality. An investigation of three catastrophic losses occurring in this region identified no involvement of infectious diseases or traditional water quality problems, including oxygen, ammonia or nitrite. The high mortality and time course of the problem was indicative of exposure to a toxin. Attempts by other workers to explain the cause of this unique syndrome (high chloride associated toxicosis of catfish, HCTC), suggested that the losses might be because of microcystin-producing blooms of Microcystis aeruginosa , but our investigations failed to support this conclusion . We found that (1) the liver histology of catfish experimentally exposed to pure microcystin-LR is very different from that of catfish sampled during outbreaks of HCTC; (2) measurements of microcystin-LR concentrations in the three cases were far lower than the concentration required to kill catfish by experimental immersion; (3) the HCTC toxin appears to have a short half-life, whereas microcystin-LR does not; (4) experimental gavage of catfish with massive amounts of microcystin-LR does not cause the acute mortality typical of HCTC; (5) outbreaks of HCTC appear to be associated with heavy blooms of Anacystis marina , a halophytic cyanobacteria, not with blooms of M. aeruginosa .     

Kaolinitic clay protects against Flavobacterium columnare infection in channel catfish Ictalurus punctatus (Rafinesque)

Columnaris disease, caused by the bacterial pathogen Flavobacterium columnare, continues to be a major problem worldwide in both wild and cultured freshwater finfish. Despite the far-reaching negative impacts of columnaris disease, safe and efficacious preventatives and curatives for this disease remain limited. In this study, we evaluated the potential of kaolin (Al₂Si₂0₅(OH)₄), a type of clay, for the prevention of columnaris disease. Channel catfish, Ictalurus punctatus (Rafinesque), fingerlings were experimentally challenged with Flavobacterium columnare in untreated water or with water containing kaolin (1 g L⁻¹). Over the 7-day course of study, kaolin treatment led to significantly (P < 0.001) improved survival (96%) as compared to untreated fish (78% survival). Histological examination of the gills revealed that kaolin-treated fish had substantially less gill damage than untreated controls. Quantitative PCR analysis of gill tissue revealed that kaolin significantly reduced F. columnare adhesion (measured at 1 h post-challenge) and colonization (24 h post-challenge). Incubation of kaolin with F. columnare in vitro demonstrated that kaolin reduced the number of F. columnare cells in culture supernatants, presumably through the formation of physical complexes through adsorption. In summary, kaolin can improve survival, reduce gill pathologies and reduce bacterial attachment to key tissues associated with columnaris disease in channel catfish by binding to F. columnare.     

Evaluation of potassium permanganate against an experimental subacute infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus (Rafinesque)

An experiment was performed to evaluate the efficacy of potassium permanganate (KMnO₄) as a prophylactic and therapeutic treatment of an experimental subacute infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus . Fish were cutaneously abraded and divided into five treatment groups: (i) challenged by waterborne exposure to F. columnare and not treated with KMnO₄ (positive control), (ii) challenged and simultaneously treated with KMnO_4, (iii) challenged and treated with KMnO₄ at 1, 6 and 9 days post-challenge, (iv) not challenged and treated with KMnO₄ at 1, 6 and 9 days post-challenge (first negative control) and (v) not challenged and not treated (second negative control). The dosing of KMnO₄ was 2.0 mg L⁻¹ above the potassium permanganate demand for 2 h duration. The survival of the group challenged and simultaneously treated with KMnO₄ (99%) was significantly higher than the positive control (78%) and was not significantly different from the negative control groups. The challenged fish treated with KMnO₄ post-challenge had 7% higher survival than the positive control (85% compared with 78%), but that difference was not statistically significant. The results demonstrate that KMnO₄ has a clear prophylactic value but probably a marginal therapeutic value once the infection has established.